WO2021013693A1 - Antibody drug conjugates (adcs) with nampt inhibitors - Google Patents

Antibody drug conjugates (adcs) with nampt inhibitors Download PDF

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Publication number
WO2021013693A1
WO2021013693A1 PCT/EP2020/070149 EP2020070149W WO2021013693A1 WO 2021013693 A1 WO2021013693 A1 WO 2021013693A1 EP 2020070149 W EP2020070149 W EP 2020070149W WO 2021013693 A1 WO2021013693 A1 WO 2021013693A1
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group
phenyl
alkyl
dihydro
oxo
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PCT/EP2020/070149
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French (fr)
Inventor
Niels Böhnke
Nils Griebenow
Anette Sommer
Stefanie Hammer
Sandra Berndt
Beatrix Stelte-Ludwig
Rudolf Beier
Christoph Mahlert
Simone Greven
Anja Giese
Judith GÜNTHER
Naomi BARAK
Ulf Bömer
Lisa Dietz
Hannah JÖRIßEN
Michael Erkelenz
Antje Rottmann
Antje Margret Wengner
Amaury Ernesto FERNANDEZ-MONTALVAN
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Bayer Pharma Aktiengesellschaft
Bayer Aktiengesellschaft
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Publication of WO2021013693A1 publication Critical patent/WO2021013693A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to novel conjugates of a binder or a derivative thereof with one or more molecules of an active component, wherein the active component is a NAMPT inhibitor, which is conjugated to the binder via a linker Z’ as described and defined herein, and methods for their preparation, their use for the treatment and/or prophylaxis of disorders, in particular of hyper-proliferative disorders.
  • Nicotinamide adenine dinucleotide is a biologically important coenzyme that plays a critical role in many cell metabolism-related transformations and in cell signaling [Lin, S-J.; Guarente L. Current Opinion Cell Biol. 2003, 15, 241–146; Ziegler M. Eur. J. Biochem. 2000, 267, 1550–1564].
  • NAD nicotinamide
  • nicotinamide pathway - is the most efficient process compared to the de novo synthesis of NAD + from the essential amino acid L-tryptophan which takes mainly place in the liver [Schramm V. L. et al.
  • NAMPT (nicotinamide phosphoribosyltransferase also known as pre-B-cell-colony-enhancing factor (PBEF) and visfatin, NMPRT, NMPETase or NAmPRTase, international nomenclature E.C.2.4.2.12) catalyzes the first step of this process, the phosphoribosylation of NAM to NMN (nicotinamide mononucleotide) which is further converted to NAD + by NMNAT (nicotinamide mononucleotide adenylyltransferase).
  • PBEF pre-B-cell-colony-enhancing factor
  • NMN nicotinamide mononucleotide
  • NMNAT nicotinamide mononucleotide adenylyltransferase
  • NAMPT is the rate-limiting enzyme in the production of NAD + and its inhibition leads to a rapid depletion of NAD + [Deng Y. et al. Bioanalysis 2014, 6, 1145–1457].
  • an altered cell metabolism is one of the basic characteristics of cancer cells as hypothesized by Otto Heinrich Warburg [Warburg, O. Automat den Stoffunci der Carcinomzelle. Klin. Schuschr.4, 534–536 (1925)].
  • NAD + is used as electron carrier in glycolysis, which is up-regulated in cancer cells due to the Warburg effect, as well as in mitochondrial oxidative phosphorylation.
  • NAD + serves as a substrate for several enzymes, for example poly-ADP-ribose polymerases (PARPs) and sirtuins (SIRTs) which are involved in DNA repair and gene expression, processes often aberrantly regulated in cancer cells and leading to consumption of NAD + [Berger F et al. 2004 Trends Biochem. Sci. 29, 111–118].
  • Phosphorylated forms of NAD + /NADH also exist and are often employed for biosynthetic and/or cell protection purposes in addition to energy generation.
  • NAMPT is implicated in the regulation of cell viability during genotoxic or oxidative stress and that NAMPT inhibitors are potentially useful for the treatment of e.g. inflammation, metabolic disorders and cancer [Tong L. et al. Expert Opin. Ther. Targets 2007, 11, 695–705; Galli, M. et al. Cancer Res.2010, 70, 8–11, J. Med. Chem 2013, 56, 6279– 6296].
  • Daporinad also known as APO866, FK866, WK175 or WK22 ((E)-N-[4-(I-benzoylpiperidin-4- yl)butyl]-3-(pyrldine-3-yl)-acrylamide) is a highly potent and selective inhibitor of NAMPT which interferes with NAD biosynthesis, ATP generation and induces cell death.
  • RENCA murine renal cell carcinoma model RENCA [Drevs J. et al. Anticancer Res 2003, 23, 4853-4858].
  • CHS-828 also known as GMX1778 (N-[6-(4-chlorophenoxy)hexyl]-N'-cyano-N''-4-pyridinyl- guanidine), an inhibitor of NAMPT as well as an inhibitor of NF- ⁇ B pathway activity [Hassan S. B. et al.
  • Anticancer Res 2006, 26, 4431-4436 showed highly cytotoxic effects in vitro and in vivo in human breast and lung cancer cell line-derived in vivo models [Hijarnaa PJ et al. Cancer Res. 1999, 59, 5751–5757].
  • a Phase I study for this compound in patients with solid tumors was published in the year 2002 [Hovstadius P et al. ClinCancerRes 2002, 9, 2843–2850]. Best observed responses in the clinical trials were stable disease. Therefore, it has been assumed that the lack of significant activity in clinical trials may result from the inability to dose NAMPT inhibitors to higher drug exposures due to dose-limiting toxicities [Sampath D. et al. Pharmacology and Therapeutics 2015, 151, 16–31].
  • the present invention relates to novel conjugates of a binder or a derivative thereof with one or more molecules of an active component, wherein the active component is a NAMPT inhibitor, which is conjugated to the binder via a linker.
  • NAMPT inhibitors A number of chemical compounds have been shown to act as NAMPT inhibitors.
  • Bioorganic & Medicinal Chemistry Letters (2013), 23, 4875–4885; WO 2014111871 and WO 2013067710 discloses 1,3-dihydro-2H-isoindoles as NAMPT inhibitors.
  • WO9206087 and WO2006064189 disclose 1-alkyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl derivatives which may be useful for the treatment of anemia, cardiovascular and diglyceride acyltransferase (DGAT) mediated disorders (e.g. diabetes), respectively.
  • DGAT diglyceride acyltransferase
  • WO2012067965 discloses 4-oxo-3,4-dihydrophthalazine phenyl cyclic urea derivatives which may be useful as NAMPT and ROCK inhibitors.
  • the invention provides conjugates of a binder or derivatives thereof with one or more active compound molecules, the active compound molecule being a NAMPT inhibitor attached to the binder via a linker Z’.
  • the binder is preferably a binder protein or peptide, particularly preferably a human, humanized or chimeric monoclonal antibody or an antigen-binding fragment thereof.
  • the conjugate according to the invention can be represented by the general formula:
  • the active component may have the Formula (I) below:
  • R 1 represents, independently of each other, halogen, hydroxy, C 1 -C 3 -alkyl, C 1 -C 3 - haloalkyl, C 1 -C 3 -alkoxy, C 1 -C 3 -haloalkoxy, -N(H)R 6 , -N(R 6 )R 7 or -NH 2 ;
  • R 2 represents H, C 1 -C 6 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 4 -haloalkyl or phenyl,
  • phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • R 5 represents H, C 1 -C 3 -alkyl or C 1 -C 3 -haloalkyl
  • n 0, 1, 2 or 3
  • the inventors have found a number of methods to attach the binder to the NAMPT inhibitor in order to achieve the object mentioned above.
  • the NAMPT inhibitor may be attached to the binder via a linker Z’ at position # in formula (I).
  • the conjugates according to the invention can have chemically labile linkers, enzymatically labile linkers or stable linkers.
  • the linker–Z’- may represent one of the following general structures (i) to (iii):
  • ⁇ represents the attachment point to D
  • ⁇ represents the attachment point to AB
  • SG represents an in vivo cleavable group
  • L1 represents an in vivo non-cleavable organic group
  • L2 represents an attachment group.
  • SG may represent a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal.
  • L1, L1’ may represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -S-, -SO-, SO 2 , -NH-, -CO-, -NMe-, -NHNH-, -SO 2 NHNH-, -NHCO-, -CONH-, - CONHNH-, arylene groups, heteroarylene groups, straight C 1 -C 6 -alkylene groups, branched C 1 -C 6 -alkylene groups, C 3 -C 7 -cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the group consisting of
  • L2 may represent:
  • # 1 represents the attachment point to the binder
  • # 2 represents the attachment point to the group L1, L1’ or SG.
  • the invention furthermore provides processes for preparing the conjugates according to the invention, and also precursors and intermediates or salts thereof useful for their preparation.
  • the preparation of the conjugates according to the invention regularly comprises the following steps: (i) Preparation of a linker precursor which optionally carries protective groups; (ii) Conjugation of the linker precursor to the derivative, which optionally carries protective groups, of a low-molecular weight NAMPT inhibitor (preferably a NAMPT inhibitor having Formula (I), giving a NAMPT inhibitor/linker conjugate which optionally carries protective groups; (iii) Attachment of a reactive group to the NAMPT inhibitor/linker conjugate; (iv) Removal of any protective groups present in the NAMPT inhibitor/linker conjugate and (v) Conjugation of the binder to the NAMPT inhibitor/linker conjugate, giving the binder/NAMPT inhibitor conjugate according to the invention.
  • a linker precursor which optionally carries protective groups
  • Attachment of the reactive group may also take place during preparation of the linker precursor (e.g. during step (i) above)) rather than after the construction of an optionally protected NAMPT inhibitor/linker precursor conjugate.
  • succinimide-linked ADCs may, after conjugation, be converted according to Scheme A into the open-chain succinamides, which have an advantageous stability profile.
  • conjugation of the linker precursor to a low-molecular weight NAMPT inhibitor may take place at position # in formula (I).
  • any functional groups present may also be present in protected form. Prior to the conjugation step, these protective groups are removed by known methods of peptide chemistry. Conjugation can take place chemically by various routes.
  • linker Z it is optionally possible to modify the low-molecular weight NAMPT inhibitor for conjugation to the linker, for example by introduction of protective groups or leaving groups to facilitate substitution.
  • position # 1 of group L2 preferably reacts with an amino or thiol group on binder AB to form a covalent bond, preferably with a cysteine or a lysine residue in a protein of AB.
  • the cysteine residue in a protein may of course be present naturally in the protein, may be introduced by biochemical methods or, preferably, may be generated by prior reduction of disulphides of the binder.
  • Constituents that are optionally substituted as stated herein may be substituted, unless otherwise noted, one or more times, independently from one another at any possible position.
  • each definition is independent.
  • each definition of R 1 , R 6 , R 7 , R 8 , R 9 , R 10 and/or R 11 is independent.
  • the position of a possible substituent can be at any of these parts at any suitable position.
  • a hyphen at the beginning or at the end of the constituent marks the point of attachment to the rest of the molecule. Should a ring be substituted, the substitutent(s) could be at any suitable position of the ring, also on a ring nitrogen atom if suitable.
  • the terms as mentioned in the present text have preferably the following meanings:
  • the term“halogen atom”,“halo-” or“Hal-” is to be understood as meaning a fluorine, chlorine, bromine or iodine atom.
  • the term“C 1 -C 6 -alkyl” is to be understood as meaning a linear or branched, saturated, monovalent hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms, e.g.
  • said group has 1, 2, 3 or 4 carbon atoms (“C 1 -C 4 -alkyl”), e.g. a methyl, ethyl, propyl, butyl, iso-propyl, iso-butyl, sec-butyl, tert- butyl group, more particularly 1, 2 or 3 carbon atoms (“C 1 -C 3 -alkyl”), e.g. a methyl, ethyl, n- propyl- or iso-propyl group.
  • C 1 -C 4 -alkyl e.g. a methyl, ethyl, propyl, butyl, iso-propyl, iso-butyl, sec-butyl, tert- butyl group, more particularly 1, 2 or 3 carbon atoms (“C 1 -C 3 -alkyl”), e.g. a methyl, ethyl, n- propyl- or iso-propy
  • C 1 -C 3 -haloalkyl is to be understood as meaning a linear or branched, saturated, monovalent hydrocarbon group in which the term“C 1 -C 3 -alkyl” is defined supra, and in which one or more hydrogen atoms is replaced by a halogen atom, identically or differently, i.e. one halogen atom being independent from another. Particularly, said halogen atom is F. Said C 1 -
  • C 3 -haloalkyl group is, for example,–CF 3 , -CHF 2 , -CH 2 F, -CF 2 CF 3 , -CH 2 CH 2 F, -CH 2 CHF 2 , - CH 2 CF 3 , -CH 2 CH 2 CF 3 , or -CH(CH 2 F) 2 .
  • said group has 1, 2 or 3 carbon atoms.
  • the term“C 1 -C 3 -alkoxy” is to be understood as meaning a linear or branched, saturated, monovalent, hydrocarbon group of formula–O-(C 1 -C 3 -alkyl), in which the term“C 1 -C 3 -alkyl” is defined supra, e.g.
  • C 1 -C 3 -haloalkoxy is to be understood as meaning a linear or branched, saturated, monovalent C 1 -C 3 -alkoxy group, as defined supra, in which one or more of the hydrogen atoms is replaced, identically or differently, by a halogen atom.
  • said halogen atom is F.
  • Said C 1 -C 3 -haloalkoxy group is, for example,–OCF 3 , -OCHF 2 , -OCH 2 F, -OCF 2 CF 3 , or - OCH 2 CF 3 . Particularly, said group has 1, 2 or 3 carbon atoms.
  • the term“C 3 -C 6 -cycloalkyl” is to be understood as meaning a saturated, monovalent, monocyclic hydrocarbon ring which contains 3, 4, 5 or 6 carbon atoms (“C 3 -C 6 -cycloalkyl”).
  • Said C 3 -C 6 -cycloalkyl group is for example, a monocyclic hydrocarbon ring, e.g.
  • a cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl ring Particularly, said group has 3 carbon atoms (“C 3 - cycloalkyl”), i.e. a cyclopropyl group.
  • said heterocycloalkyl can be a 5-membered ring, such as, but not limited to, tetrahydrofuranyl, pyrrolidinyl or pyrrolinyl, or a 6-membered ring, such as, but not limited to, tetrahydropyranyl, piperidinyl, morpholinyl or piperazinyl, or a 7- membered ring, such as, but not limited to, an azepanyl ring, for example.
  • said heterocycloalkyl can be benzo fused.
  • said 5- to 7-membered heterocycloalkyl can be partially unsaturated, i.e. it can contain one or more double bonds, such as, without being limited thereto, a 2,5-dihydro- 1H-pyrrolyl, for example, or, it may be benzofused, such as, without being limited thereto, a dihydroisoquinolinyl ring, for example.
  • Said fused heterocycloalkyl group is, for example, azabicyclo[3.3.0]octylene, azabicyclo[4.3.0]nonylene, diazabicyclo[4.3.0]nonylene, oxazabicyclo[4.3.0]nonylene, thiazabicyclo[4.3.0]nonylene, 5,6,7,8-tetrahydrophthalazinylene, 1,2,5,6,7,8- hexahydrophthalazinylene, 1,2-dihydrophthalazinylene, 1,2-dihydroisoquinolinylene, 1,2- dihydropyrrolo[1,2-d][1,2,4]triazinylene, isoindolinylene, 4,5-dihydro-3H-2,3- benzodiazepinylene or azabicyclo[4.4.0]decylene.
  • heteroarylene group is understood as meaning a bivalent, monocyclic, or bicyclic aromatic ring having 5, 6, 8, 9, 10, 11, 12, 13 or 14 ring atoms (a“5- to 14-membered heteroarylene” group), particularly 5, 6, 9 or 10 ring atoms (a“5-, 6-, 9- or 10-membered heteroaryl” group), more particularly 5 or 6 ring atoms (a“5- to 6-membered heteroarylene” group), which contains at least one ring heteroatom and optionally one, two or three further ring heteroatoms from the series: N, O and/or S. It is understood that any heteroarylene group is attached to the rest of the molecule via carbon atoms of the heteroarylenic ring or, if present, a nitrogen atom.
  • said heteroarylene group can be a 5-membered ring, such as, but not limited to, thienylene, furanylene, pyrrolylene, oxazolylene, thiazolylene, imidazolylene, pyrazolylene, isoxazolylene, isothiazolylene, thia-4H-pyrazolylene, furylene, triazolylene (1,2,4-triazolylene, 1,3,4-triazolylene or 1,2,3-triazolylene), thiadiazolylene (1,3,4- thiadiazolylene, 1,2,5-thiadiazolylene, 1,2,3-thiadiazolylene or 1,2,4-thiadiazolylene) and oxadiazolylene (1,3,4-oxadiazolylene, 1,2,5-oxadiazolylene, 1,2,3-oxadiazolylene or 1,2,4- oxadiazolylene), etc., and benzo derivatives thereof, such as, for example, benzo
  • the heteroarylenic radicals include all the possible isomeric forms thereof, e.g. the positional isomers thereof.
  • pyridinylene includes pyridin-2-ylene, pyridin-3-ylene and pyridin- 4-ylene; or the term thienylene includes thien-2-ylene and thien-3-ylene.
  • any heteroatom of a heteroarylic ring with unsatisfied valences mentioned herein is assumed to have the hydrogen atom(s) to satisfy the valences.
  • “C 3 -C 6 -cycloalkyl” in the context of the definition of“C 3 -C 6 -cycloalkyl”, is to be understood as meaning a cycloalkyl group having a finite number of carbon atoms of 3 to 6, i.e.3, 4, 5 or 6 carbon atoms. It is to be understood further that said term“C 3 -C 6 ” is to be interpreted as any sub-range comprised therein, e.g. C 3 - C 6 , C 4 -C 5 , C 3 -C 5 , C 3 -C 4 , C 4 -C 6 , C 5 -C 6 ; particularly C 3 -C 6 .
  • substituted means that one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
  • optionally substituted means optional substitution with the specified groups, radicals or moieties.
  • Ring system substituent means a substituent attached to an aromatic or nonaromatic ring system which, for example, replaces an available hydrogen on the ring system.
  • the term“one or more”, e.g. in the definition of the substituents of the compounds of the general formulae of the present invention, is understood as meaning“one, two, three, four or five, particularly one, two, three or four, more particularly one, two or three, even more particularly one or two”.
  • the invention also includes all suitable isotopic variations of a compound of the invention.
  • An isotopic variation of a compound of the invention is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually or predominantly found in nature.
  • isotopes that can be incorporated into a compound of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine, chlorine, bromine and iodine, such as 2 H (deuterium), 3 H (tritium), 11 C, 13 C, 14 C, 15 N, 17 O, 18 O, 32 P, 33 P, 33 S, 34 S, 35 S, 36 S, 18 F, 36 Cl, 82 Br, 123 I, 124 I, 125 I, 129 I and 131 I, respectively.
  • isotopic variations of a compound of the invention are useful in drug and/or substrate tissue distribution studies.
  • Tritiated and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability.
  • substitution with isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence is preferred in some circumstances.
  • Isotopic variations of a compound of the invention can generally be prepared by conventional procedures known by a person skilled in the art such as by the illustrative methods or by the preparations described in the examples hereafter using appropriate isotopic variations of suitable reagents. Where the plural form of the word compounds, salts, polymorphs, hydrates, solvates and the like, is used herein, this is taken to mean also a single compound, salt, polymorph, isomer, hydrate, solvate or the like.
  • stable compound' is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
  • the compounds of this invention optionally contain one or more asymmetric centres, depending upon the location and nature of the various substituents desired.
  • Asymmetric carbon atoms are present in the (R) or (S) configuration, resulting in racemic mixtures in the case of a single asymmetric centre, and diastereomeric mixtures in the case of multiple asymmetric centres.
  • asymmetry may also be present due to restricted
  • the compounds of the present invention optionally contain sulphur atoms which are asymmetric, such as an asymmetric sulfoxide, of structure:
  • Substituents on a ring may also be present in either cis or trans form. It is intended that all such configurations (including enantiomers and diastereomers), are included within the scope of the present invention. Preferred compounds are those which produce the more desirable biological activity. Separated, pure or partially purified isomers and stereoisomers or racemic or diastereomeric mixtures of the compounds of this invention are also included within the scope of the present invention. The purification and the separation of such materials can be accomplished by the technicques provided herein or by (other) standard techniques known in the art.
  • the optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example, by the formation of diastereoisomeric salts using an optically active acid or base or formation of covalent diastereomers.
  • appropriate acids are tartaric, diacetyltartaric, ditoluoyltartaric and camphorsulfonic acid.
  • Mixtures of diastereoisomers can be separated into their individual diastereomers on the basis of their physical and/or chemical differences by methods known in the art, for example, by chromatography or fractional crystallisation.
  • the optically active bases or acids are then liberated from the separated diastereomeric salts.
  • a different process for separation of optical isomers involves the use of chiral chromatography (e.g., chiral HPLC columns), with or without conventional derivatisation, optimally chosen to maximise the separation of the enantiomers.
  • Suitable chiral HPLC columns are manufactured by Daicel, e.g., Chiracel OD and Chiracel OJ among many others, all routinely selectable.
  • Enzymatic separations, with or without derivatisation are also useful.
  • the optically active compounds of this invention can likewise be obtained by chiral syntheses utilizing optically active starting materials. In order to limit different types of isomers from each other reference is made to IUPAC Rules Section E (Pure Appl Chem 45, 11-30, 1976).
  • the present invention includes all possible stereoisomers of the compounds of the present invention as single stereoisomers, or as any mixture of said stereoisomers, e.g. R- or S- isomers, or E- or Z-isomers, in any ratio.
  • Isolation of a single stereoisomer, e.g. a single enantiomer or a single diastereomer, of a compound of the present invention is achieved by any suitable state of the art method, such as chromatography, especially chiral chromatography, for example.
  • the compounds of the present invention may exist as tautomers.
  • the present invention includes all possible tautomers of the compounds of the present invention as single tautomers, or as any mixture of said tautomers, in any ratio.
  • the compounds of the present invention can exist as N-oxides, which are defined in that at least one nitrogen of the compounds of the present invention is oxidised.
  • the present invention includes all such possible N-oxides.
  • the present invention includes all possible salts, polymorphs, metabolites, hydrates, solvates, prodrugs (e.g.: esters) thereof, and diastereoisomeric forms of the NAMPT inhibtors or precursors (including intermediates) thereof as single salt, polymorph, metabolite, hydrate, solvate, prodrug (e.g.: esters) thereof, or diastereoisomeric form, or as mixture of more than one salt, polymorph, metabolite, hydrate, solvate, prodrug (e.g.: esters) thereof, or diastereoisomeric form in any ratio.
  • the compounds of the present invention can exist as a hydrate, or as a solvate, wherein the compounds of the present invention contain polar solvents, in particular water, methanol or ethanol for example as structural element of the crystal lattice of the compounds.
  • polar solvents in particular water, methanol or ethanol for example as structural element of the crystal lattice of the compounds.
  • the amount of polar solvents, in particular water may exist in a stoichiometric or non-stoichiometric ratio.
  • stoichiometric solvates e.g. a hydrate, hemi-, (semi-), mono-, sesqui-, di-, tri-, tetra-, penta- etc. solvates or hydrates, respectively, are possible.
  • the present invention includes all such hydrates or solvates.
  • the compounds of the present invention can exist in free form, e.g. as a free base, or as a free acid, or as a zwitterion, or can exist in the form of a salt.
  • Said salt may be any salt, either an organic or inorganic addition salt, particularly any pharmaceutically acceptable organic or inorganic addition salt, customarily used in pharmacy.
  • “pharmaceutically acceptable salt” refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present invention.
  • pharmaceutically acceptable salt refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present invention.
  • a suitable pharmaceutically acceptable salt of the compounds of the present invention may be, for example, an acid-addition salt of a compound of the present invention bearing a nitrogen atom, in a chain or in a ring, for example, which is sufficiently basic, such as an acid-addition salt with an inorganic acid, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, bisulfuric, phosphoric, or nitric acid, for example, or with an organic acid, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3-hydroxy-2-naphthoic, nicotinic, pamoic, pectinic
  • an alkali metal salt for example a sodium or potassium salt
  • an alkaline earth metal salt for example a calcium or magnesium salt
  • an ammonium salt or a salt with an organic base which affords a physiologically acceptable cation, for example a salt with N-methyl-glucamine, dimethyl-glucamine, ethyl-glucamine, lysine, dicyclohexylamine, 1,6-hexadiamine, ethanolamine, glucosamine, sarcosine, serinol, tris-hydroxy-methyl- aminomethane, aminopropandiol, sovak-base, 1-amino-2,3,4-butantriol.
  • basic nitrogen containing groups may be quaternised with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, and dibutyl sulfate; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and strearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates like dimethyl, diethyl, and dibutyl sulfate
  • diamyl sulfates long chain halides such as decyl, lauryl
  • acid addition salts of the claimed compounds may be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods.
  • alkali and alkaline earth metal salts of acidic compounds of the invention are prepared by reacting the compounds of the invention with the appropriate base via a variety of known methods.
  • the present invention includes all possible salts of the compounds of the present invention as single salts, or as any mixture of said salts, in any ratio.
  • the present text in particular in the Experimental Section, for the synthesis of intermediates and of examples of the present invention, when a compound is mentioned as a salt form with the corresponding base or acid, the exact stoichiometric composition of said salt form, as obtained by the respective preparation and/or purification process, is, in most cases, unknown.
  • salts include water-insoluble and, particularly, water-soluble salts. This applies analogously to cases in which synthesis intermediates or example compounds or salts thereof have been obtained, by the preparation and/or purification processes described, as solvates, such as hydrates with (if defined) unknown stoichiometric composition.
  • conjugates described herein and the salts thereof which are converted into a conjugate as described herein or a salt thereof in a biological system are covered by the invention.
  • Said biological system is e.g. a mammalian organism, particularly a human subject.
  • the bioprecursor is, for example, converted into a conjugate as described herein or a salt thereof by metabolic processes.
  • the present invention includes all possible crystalline forms, or polymorphs, of the conjugates of the present invention, either as single polymorph, or as a mixture of more than one polymorph, in any ratio.
  • pharmacokinetic profile means one single parameter or a combination thereof including permeability, bioavailability, exposure, and pharmacodynamic parameters such as duration, or magnitude of pharmacological effect, as measured in a suitable experiment.
  • Conjugates with improved pharmacokinetic profiles can, for example, be used in lower doses to achieve the same effect, may achieve a longer duration of action, or a may achieve a combination of both effects.
  • A“fixed combination” in the present invention is used as known to persons skilled in the art and may be present as a fixed combination, a non-fixed combination or kit-of-parts.
  • A“fixed combination” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein the said first active ingredient and the said second active ingredient are present together in one unit dosage or in a single entity.
  • a“fixed combination” is a pharmaceutical composition wherein the said first active ingredient and the said second active ingredient are present in admixture for simultaneous administration, such as in a formulation.
  • Another example of a“fixed combination” is a pharmaceutical combination wherein the said first active ingredient and the said second active ingredient are present in one unit without being in admixture.
  • a non-fixed combination or“kit-of-parts” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein the said first active ingredient and the said second active ingredient are present in more than one unit.
  • a non-fixed combination or kit-of-parts is a combination wherein the said first active ingredient and the said second active ingredient are present separately.
  • the components of the non-fixed combination or kit-of-parts may be administered separately, sequentially, simultaneously, concurrently or chronologically staggered. Any such combination of a compound of formula (I) of the present invention with an anti-cancer agent as defined below is an embodiment of the invention.
  • the term“(chemotherapeutic) anti-cancer agents” includes but is not limited to:
  • 131I-chTNT abarelix, abiraterone, aclarubicin, adalimumab, ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alectinib, alemtuzumab, alendronic acid, alitretinoin, altretamine, amifostine, aminoglutethimide, hexyl aminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anethole dithiolethione, anetumab ravtansine, angiotensin II, antithrombin III, aprepitant, arcitumomab, arglabin, arsenic trioxide, asparaginase, atezolizumab, axitinib, azacitidine, basiliximab, belotecan, bendamustine,
  • decitabine degarelix, denileukin diftitox, denosumab, depreotide, deslorelin, dianhydrogalactitol, dexrazoxane, dibrospidium chloride, dianhydrogalactitol, diclofenac, dinutuximab, docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin + estrone, dronabinol, eculizumab, edrecolomab, elliptinium acetate, elotuzumab, eltrombopag, endostatin, enocitabine, enzalutamide, epirubicin, epitiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib,
  • ramucirumab ranimustine, rasburicase, razoxane, refametinib , regorafenib, risedronic acid, rhenium-186 etidronate, rituximab, rolapitant, romidepsin, romiplostim, romurtide, roniciclib , samarium (153Sm) lexidronam, sargramostim, satumomab, secretin, siltuximab, sipuleucel-T, sizofiran, sobuzoxane, sodium glycididazole, sonidegib, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, talimogene laherparepvec, tamibarotene, tamoxifen, tapentadol, tasonermin
  • the invention provides conjugates of a binder or derivative thereof with one or more active compound molecules, the active compound molecule being a NAMPT inhibitor attached to the binder via a linker Z’.
  • the invention relates to a conjugate of a binder or a derivative thereof with one or more molecules of an active compound that has the formula: n wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8, and D stands for an active component having Formula (I) described herein.
  • the binder is preferably a binder peptide or protein such as, for example, an antibody.
  • the linker is preferably attached to different amino acids of the same chemical nature of the binder peptide or protein or derivative thereof. Particular preference is given to binding to different cysteine or lysine residues of the binder, even more preferable is binding to different cysteine residues of the binder.
  • Binders which can be used according to the invention, NAMPT inhibitors which can be used according to the invention and linkers which can be used according to the invention which can be used in combination without any limitation are described below.
  • the binders represented in each case as preferred or particularly preferred can be employed in combination
  • NAMPT inhibitors represented in each case as preferred or particularly preferred, optionally in combination with the linkers represented in each case as preferred or particularly preferred.
  • the NAMPT inhibitors used in the binder drug conjugates according to the invention preferably show anti-proliferative activity in tumor cell lines, such as THP-1, U251 MG, MV-4-11, MDA- MB-453, NCI-N87 or SK-OV-3, for example.
  • the NAMPT inhibitors (D) are described by Formula (I):
  • R 1 represents, independently of each other, halogen, hydroxy, C 1 -C 3 -alkyl, C 1 -C 3 - haloalkyl, C 1 -C 3 -alkoxy, C 1 -C 3 -haloalkoxy, -N(H)R 6 , -N(R 6 )R 7 or -NH 2 ;
  • R 2 represents H, C 1 -C 6 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 4 -haloalkyl or phenyl, wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • n 0, 1, 2 or 3
  • the invention relates to a conjugate as described supra, wherein:
  • A represents:
  • R 1 represents, independently of each other, halogen, hydroxy, C 1 -C 3 -alkyl, C 1 -C 3 - haloalkyl, C 1 -C 3 -alkoxy, C 1 -C 3 -haloalkoxy, -N(H)R 6 , -N(R 6 )R 7 or -NH 2 ;
  • R 2 represents H, C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 3 -haloalkyl or phenyl,
  • phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • R 3 represents H, C 1 -C 3 -alkyl or C 1 -C 3 -haloalkyl
  • R 4 represents C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 3 -haloalkyl or phenyl;
  • R 5 represents H, C 1 -C 3 -alkyl or C 1 -C 3 -haloalkyl
  • n 0, 1, 2 or 3
  • the invention relates to a conjugate as described supra,
  • A represents:
  • R 1 represents, independently of each other, halogen, hydroxy, C 1 -C 3 -alkyl, C 1 -C 3 - haloalkyl, C 1 -C 3 -alkoxy, C 1 -C 3 -haloalkoxy, -N(H)R 6 , -N(R 6 )R 7 or -NH 2 ;
  • R 2 represents H, C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 3 -haloalkyl or phenyl,
  • phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • R 3 represents H or C 1 -C 3 -alkyl
  • R 2 and R 3 together with the carbon to which they are attached form a C 3 -C 6 -cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR 8 ;
  • R 4 represents C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 3 -haloalkyl or phenyl;
  • R 5 represents H or C 1 -C 3 -alkyl
  • R 4 and R 5 together with the carbon to which they are attached form a C 3 -C 6 -cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR 8 ; q is 0, 1, 2 or 3,
  • n 0, 1, 2 or 3
  • R 6 , R 7 represent, independently of each other C 1 -alkyl or C 3 -cycloalkyl;
  • R 8 represents, independently of each other hydrogen, C 1 -C 3 -alkyl or C 3- C 6 -cycloalkyl; or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer.
  • the invention relates to a conjugate as described supra,
  • A represents:
  • R 1 represents, independently of each other, halogen, hydroxy, C 1 -C 3 -alkyl, C 1 -C 3 - haloalkyl, C 1 -C 3 -alkoxy or -NH 2 , -N(H)R 6 ;
  • R 2 represents H, C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl or phenyl,
  • phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • R 3 represents H or C 1 -C 3 -alkyl
  • R 2 and R 3 together with the carbon to which they are attached form a C 3 -C 6 -cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR 8 ;
  • R 4 represents C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 3 -haloalkyl or phenyl;
  • R 5 represents H or C 1 -C 3 -alkyl
  • R 4 and R 5 together with the carbon to which they are attached form a C 3 -C 6 -cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR 8 ;
  • * represents the point of attachment of said group with the rest of the compound of formula (I) ; represents a group which is selected from : in which * and # represent the points of attachment of said group with the rest of the compound of formula (I),
  • R 6 represents, independently of each other, C 1 -alkyl or C 3 -cycloalkyl
  • R 8 represents, independently of each other, hydrogen, or C 1 -C 3 -alkyl; or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer.
  • the invention relates to a conjugate as described supra,
  • A represents:
  • R 1 represents, independently of each other, F, Cl, Br, hydroxy, methyl, CF 3 , methoxy, - NH 2 , or -N(H)(CH 3 );
  • t is 0, 1 or 2, preferably 0;
  • R 2 represents H, methyl, propan-2-yl or phenyl,
  • R 3 represents H, or methyl
  • R 4 represents methyl
  • R 5 represents H or methyl
  • the invention relates to a conjugate as described supra, wherein: A represents:
  • R 1 represents, independently of each other, halogen, hydroxy, C 1 -C 3 -alkyl, C 1 -C 3 - haloalkyl, C 1 -C 3 -alkoxy, C 1 -C 3 -haloalkoxy, -N(H)R 6 , -N(R 6 )R 7 or -NH 2 ;
  • R 2 represents H, C 1 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 4 -haloalkyl or phenyl,
  • phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • R 4 represents C 1 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 4 -haloalkyl or phenyl;
  • n 1;
  • the invention relates to a conjugate as described supra, wherein:
  • A represents:
  • R 1 represents, independently of each other, halogen, hydroxy, C 1 -C 3 -alkyl, C 1 -C 3 - haloalkyl, C 1 -C 3 -alkoxy, C 1 -C 3 -haloalkoxy, -N(H)R 6 , -N(R 6 )R 7 or -NH 2 ;
  • R 2 represents H, C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 3 -haloalkyl or phenyl,
  • phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • R 3 represents H, C 1 -C 3 -alkyl or C 1 -C 3 -haloalkyl
  • R 4 represents C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 3 -haloalkyl or phenyl;
  • R 5 represents H, C 1 -C 3 -alkyl or C 1 -C 3 -haloalkyl
  • n 1;
  • the invention relates to a conjugate as described supra, wherein:
  • A represents:
  • R 1 represents, independently of each other, halogen, hydroxy, C 1 -C 3 -alkyl, C 1 -C 3 - haloalkyl, C 1 -C 3 -alkoxy, C 1 -C 3 -haloalkoxy, -N(H)R 6 , -N(R 6 )R 7 or -NH 2 ;
  • R 2 represents H, C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 3 -haloalkyl or phenyl,
  • phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • R 3 represents H or C 1 -C 3 -alkyl
  • R 2 and R 3 together with the carbon to which they are attached form a C 3 -C 6 -cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR 8 ;
  • R 4 represents C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 3 -haloalkyl or phenyl;
  • R 5 represents H or C 1 -C 3 -alkyl
  • R 4 and R 5 together with the carbon to which they are attached form a C 3 -C 6 -cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR 8 ; q is 1, m is 1;
  • R 6 , R 7 represent, independently of each other C 1 -alkyl or C 3 -cycloalkyl
  • R 8 represents, independently of each other hydrogen, C 1 -C 3 -alkyl or C 3- C 6 -cycloalkyl; or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer.
  • the invention relates to a conjugate as described supra, wherein:
  • A represents:
  • R 1 represents, independently of each other, halogen, hydroxy, C 1 -C 3 -alkyl, C 1 -C 3 - haloalkyl, C 1 -C 3 -alkoxy or -NH 2 , -N(H)R 6 ;
  • R 2 represents H, C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl or phenyl,
  • phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • R 3 represents H or C 1 -C 3 -alkyl
  • R 2 and R 3 together with the carbon to which they are attached form a C 3 -C 6 -cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR 8 ;
  • R 4 represents C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 3 -haloalkyl or phenyl;
  • R 5 represents H or C 1 -C 3 -alkyl; or
  • R 4 and R 5 together with the carbon to which they are attached form a C 3 -C 6 -cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR 8 ;
  • R 6 represents, independently of each other, C 1 -alkyl or C 3 -cycloalkyl
  • R 8 represents, independently of each other, hydrogen, or C 1 -C 3 -alkyl
  • the invention relates to a conjugate as described supra, wherein:
  • A represents:
  • R 1 represents, independently of each other, F, Cl, Br, hydroxy, methyl, CF 3 , methoxy, - NH 2 , or -N(H)(CH 3 );
  • R 2 represents H, methyl, propan-2-yl or phenyl
  • R 3 represents H, or methyl
  • R 4 represents methyl
  • R 5 represents H or methyl; represents a group ,
  • the invention relates to a conjugate as described supra, wherein: A represents:
  • R 1 represents, independently of each other, halogen, C 1 -C 3 -alkoxy, or -NH 2 ;
  • t is 0, or 1, preferably 0;
  • R 2 represents C 1 -C 6 -alkyl, or phenyl,
  • R 3 represents H, R 4 represents C 1 -C 6 -alkyl
  • R 5 represents H, or C 1 -C 3 -alkyl; q is 1,
  • n 1;
  • the invention relates to a conjugate as described supra, wherein: A represents:
  • the invention relates to a conjugate as described supra, wherein: R 1 represents, independently of each other, halogen, hydroxy, C 1 -C 3 -alkyl, C 1 -C 3 - haloalkyl, C 1 -C 3 -alkoxy, C 1 -C 3 -haloalkoxy, -N(H)R 6 , -N(R 6 )R 7 or -NH 2 .
  • R 6 and R 7 are as defined herein for the compound of formula (I).
  • the invention relates to a conjugate as described supra, wherein: R 1 represents, independently of each other, halogen, hydroxy, C 1 -C 3 -alkyl, C 1 -C 3 - haloalkyl, C 1 -C 3 -alkoxy or -NH 2 , -N(H)R 6 ,
  • R 6 is as defined herein for the compound of formula (I).
  • the invention relates to a conjugate as described supra, wherein: R 1 represents, independently of each other, F, Cl, Br, hydroxy, methyl, CF 3 , methoxy, - NH 2 , or -N(H)(CH 3 ).
  • the invention relates to a conjugate as described supra, wherein: R 1 represents, independently of each other, halogen, C 1 -C 3 -alkyl, C 1 -C 3 -alkoxy, C 1 -C 3 - haloalkoxy, -N(H)R 6 , -N(R 6 )R 7 or -NH 2 ,
  • R 6 and R 7 are as defined herein for the compound of formula (I).
  • the invention relates to a conjugate as described supra, wherein: R 1 represents halogen, C 1 -C 3 -alkoxy or -NH 2 . In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R 1 represents F, Br, methoxy or -NH 2 . In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R 1 represents F, Cl, Br, Me, or NH 2 . In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R 1 represents F, Cl, Me, or NH 2 , and
  • t is 0 or 1.
  • the invention relates to a conjugate as described supra, wherein: t is 0, 1, 2 or 3.
  • the invention relates to a conjugate as described supra, wherein: t is 0, 1 or 2.
  • the invention relates to a conjugate as described supra, wherein: t is 0, or 1.
  • the invention relates to a conjugate as described supra, wherein: t is 0.
  • the invention relates to a conjugate as described supra, wherein: t is 1. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: t is 2. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R 2 represents H, methyl, propan-2-yl or phenyl.
  • the invention relates to a conjugate as described supra, wherein: R 2 represents H, C 1 -C 6 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 4 -haloalkyl or phenyl,
  • phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • the invention relates to a conjugate as described supra, wherein: R 2 represents H, C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 3 -haloalkyl or phenyl,
  • phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • the invention relates to a conjugate as described supra, wherein: R 2 represents H, C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl or phenyl,
  • phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • the invention relates to a conjugate as described supra, wherein: R 2 represents methyl, propan-2-yl or phenyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R 3 represents H, or methyl.
  • the invention relates to a conjugate as described supra, wherein: R 3 represents H, C 1 -C 3 -alkyl or C 1 -C 3 -haloalkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R 3 represents H or C 1 -C 3 -alkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R 3 represents H.
  • the invention relates to a conjugate as described supra, wherein:
  • the invention relates to a conjugate as described supra, wherein: R 2 and R 3 together with the carbon to which they are attached form a C 3 -C 6 -cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR 8 ; wherein R 8 is as defined herein for the compound of formula (I).
  • the invention relates to a conjugate as described supra, wherein: R 4 represents C 1 -C 6 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 4 -haloalkyl or phenyl.
  • the invention relates to a conjugate as described supra, wherein: R 4 represents C 1 -C 4 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 3 -haloalkyl or phenyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R 4 represents methyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R 5 represents H, C 1 -C 3 -alkyl or C 1 -C 3 -haloalkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R 5 represents H or C 1 -C 3 -alkyl.
  • the invention relates to a conjugate as described supra, wherein: R 5 represents H or methyl.
  • the invention relates to a conjugate as described supra, wherein: R 4 and R 5 together with the carbon to which they are attached form a C 3 -C 6 -cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR 8 ; wherein R 8 is as defined herein for the compound of formula (I).
  • the invention relates to a conjugate as described supra, wherein: q is 0, 1, 2 or 3,
  • n 0, 1, 2 or 3
  • the invention relates to a conjugate as described supra, wherein:
  • n 1 or 2
  • the invention relates to a conjugate as described supra, wherein:
  • the invention relates to a conjugate as described supra, wherein: represents a group .
  • the invention relates to a conjugate as described supra, wherein:
  • the invention relates to a conjugate as described supra, wherein:
  • the invention relates to a conjugate as described supra, wherein: R 6 , R 7 represent, independently of each other C 1 -alkyl or C 3 -cycloalkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:
  • the invention relates to a conjugate as described supra, wherein:
  • the invention relates to a conjugate as described supra, wherein: R 6 represents, independently of each other, C 1 -alkyl or C 3 -cycloalkyl.
  • the invention relates to a conjugate as described supra, wherein: R 7 represents, independently of each other, C 1 -alkyl or C 3 -cycloalkyl.
  • R 8 represents, independently of each other hydrogen, C 1 -C 3 -alkyl or C 3- C 6 -cycloalkyl.
  • the invention relates to a conjugate as described supra, wherein: R 8 represents, independently of each other, hydrogen, or C 1 -C 3 -alkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:
  • R 8 represents, independently of each other, hydrogen, or methyl.
  • a further aspect of the invention are conjugates as described supra, which are present as their salts.
  • Yet another aspect of the invention are conjugates as described supra in which the salt is a pharmaceutically acceptable salt. It is to be understood that the present invention relates to any sub-combination within any embodiment or aspect of the present invention of conjugates as described supra. More particularly still, the present invention covers conjugates that are disclosed in the Example section of this text, infra. In accordance with another aspect, the present invention covers methods of preparing conjugates of the present invention, said methods comprising the steps as described in the Experimental Section herein. Linkers
  • the literature discloses various options for covalently coupling (conjugating) organic molecules to binders such as, for example antibodies (see, for example, K. Lang and J. W. Chin. Chem. Rev. 2014, 114, 4764-4806, M. Rashidian et al. Bioconjugate Chem. 2013, 24, 1277-1294).
  • Preference according to the invention is given to conjugation of the NAMPT inhibitors to an antibody via one or more sulphur atoms of cysteine residues of the antibody which are either already present as free thiols or generated by reduction of disulphide bridges, and/or via one or more NH groups of lysine residues of the antibody.
  • linkers can be categorized into the group of the linkers which can be cleaved in vivo and the group of the linkers which are stable in vivo (see L. Ducry and B. Stump, Bioconjugate Chem.21, 5-13 (2010)).
  • the linkers which can be cleaved in vivo have a group which can be cleaved in vivo, where, in turn, a distinction may be made between groups which are chemically cleavable in vivo and groups which are enzymatically cleavable in vivo.
  • “Chemically cleavable in vivo” and “enzymatically cleavable in vivo” means that the linkers or groups are cleaved at or in the target cell by the chemically or enzymatically different environment therein (e.g. lower pH; elevated glutathione concentration; presence of lysosomal
  • enzymes such as cathepsin or plasmin, or glyosidases such as, for example, ß- glucuronidases
  • groups which can be cleaved chemically in vivo are in particular disulphide, hydrazone, acetal and aminal groups
  • groups which can be cleaved enzymatically in vivo are in particular the 2-8-oligopeptide group, especially a dipeptide group, a tripeptide group or a glycoside group.
  • Peptide cleavage sites are disclosed in Bioconjugate Chem.
  • linker which are stable in vivo are distinguished by a high stability (preferably less than 5% metabolites after 24 hours in plasma) and do not have the chemically or enzymatically in vivo cleavable groups mentioned herein.
  • the invention relates to a conjugate as described supra, wherein the linker–Z’- represents one of the following general structures (i) to (iii):
  • attachment group L2 represents a coupling group to the binder or a single bond.
  • coupling is preferably to a cysteine residue or a lysine residue of the binder.
  • coupling can be to a tyrosine residue, glutamine residue or to an unnatural amino acid of the binder.
  • the unnatural amino acids may contain, for example, aldehyde or keto groups (such as, for example, formylglycine) or azide or alkyne groups (see Lan & Chin, Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins, Chem.Rev. 2014, 114, 4764- 4806).
  • the invention relates to a conjugate as described supra, wherein the in vivo cleavable group SG represents a 2-8 oligopeptide group, preferably a
  • the invention relates to a conjugate as described supra, wherein L1 and L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -S-, -SO-, SO 2 , -NH-, -CO-, -NMe-, -NHNH-, -SO 2 NHNH-, -NHCO-, -CONH-, - CONHNH-, arylene groups, heteroarylene groups, straight C 1 -C 6 -alkylene groups, branched C 1 -C 6 -alkylene groups, C 3 -C 7 -cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 hetero
  • L2 is preferably derived from a group which reacts with the sulphhydryl group of the cysteine.
  • substituents independently selected from the group consisting of halogen, -NHCONH 2 , -COOH, -OH, -NH 2 , NH-CNNH 2 , sulphonamide, sulphone, sulphoxide and sulphonic acid.
  • L2 is preferably derived from a group which reacts with the sulphhydryl group of the cysteine. These include haloacetyls, maleimides, aziridines, acryloyls, arylating compounds, vinylsulphones, pyridyl disulphides, TNB thiols and disulphide-reducing agents.
  • the invention relates to a conjugate as described supra, wherein L2 represents:
  • # 1 represents the attachment point to the binder
  • # 2 represents the attachment point to the group L1, L1’ or SG.
  • the invention relates to a conjugate as described supra, wherein the linker–Z’- represents one of the following general structures (i) to (iii): (i) ⁇ –L1-SG-L2- ⁇ (ii) ⁇ –L1-SG-L1’-L2- ⁇ (iii) ⁇ –L1-L2- ⁇ wherein ⁇ represents the attachment point to D; ⁇ represents the attachment point to AB; SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal; L1, L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than onceby one or more groups independently selected from: -O-, -S-, -SO-, SO 2 , -NH-, -CO-
  • * and # represent the points of attachment of said group with the rest of the compound, optionally substituted with one or more substituents independently selected from the group consisting of halogen, -NHCONH 2 , -COOH, -OH, -NH 2 , NH-CNNH 2 , sulphonamide, sulphone, sulphoxide and sulphonic acid;
  • L2 represents:
  • # 1 represents the attachment point to the binder
  • # 2 represents the attachment point to the group L1, L1’ or SG.
  • the invention relates to a conjugate as described supra, wherein L2 represents one or more of the following three formulae:
  • # 1 represents the attachment point to the binder
  • # 2 represents the attachment point to the group L1, L1’ or SG
  • # 1 represents the attachment point to the binder
  • # 2 represents the attachment point to the group L1, L1’ or SG
  • the amide group at # 2 is connected to L1, L1’ or SG via the group–CH 2 -C(O)-.
  • the invention relates to a conjugate as described supra, wherein SG is a 2-8 oligopeptide.
  • the invention relates to a conjugate as described supra, wherein the 2-8 oligopeptide consists of amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine.
  • the 2-8 oligopeptide consists of amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine.
  • the invention relates to a conjugate as described supra, wherein L1 and L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -NH-, -CO-, -NHCO-, -CONH-, phenyl and
  • * and # represent the points of attachment of said group with the rest of the compound, optionally substituted with one or more substituents independently selected from the group consisting of -F, -Cl, -COOH, -OH, and -NH 2 .
  • the invention relates to a conjugate as described supra, wherein L1 and L1’ represent, independently of each other, one of the general structures (iv) or (v): (iv) –A’-(NR 10 CO)-B’- (v) –A’-(CONR 10 )-B’- wherein: A’ represents C 1 -C 6 -alkyl, (C 1 -C 2 -alkyl)-(phenylene), and (C 1 -C 3 -alkyl)-(NR 11 )-(C 2 -alkyl); optionally substituted with one or more substituents independently selected from–F and -Cl; B’ represents a straight-chain or branched hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from -O-, -NH-, -CO-, -NHCO-, and -CONH-; optionally substituted with–COOH; R
  • the invention relates to a conjugate of general formula (II)
  • AB stands for a binder
  • Z’ stands for a linker
  • n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8; wherein: A represents:
  • R 1 represents, independently of each other, halogen, hydroxy, C 1 -C 3 -alkyl, C 1 -C 3 - haloalkyl, C 1 -C 3 -alkoxy, C 1 -C 3 -haloalkoxy, -N(H)R 6 , -N(R 6 )R 7 or -NH 2 ;
  • R 2 represents H, C 1 -C 6 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 4 -haloalkyl or phenyl,
  • phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • n 0, 1, 2 or 3
  • * and # represent the points of attachment of said group with the rest of the compound, said group being optionally substituted with one or more substituents independently selected from the group consisting of:
  • -Z’- represents one of the following general structures (i) to (iii): (i) ⁇ –L1-SG-L2- ⁇ (ii) ⁇ –L1-SG-L1’-L2- ⁇ (iii) ⁇ –L1-L2- ⁇ wherein ⁇ represents the attachment point to A; ⁇ represents the attachment point to AB; SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide,
  • L1, L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -S-, -SO-, SO 2 , -NH-, -CO-, -NMe-, -NHNH-, -SO 2 NHNH-, -NHCO-, -CONH-, - CONHNH-, arylene groups, heteroarylene groups, straight C 1 -C 6 -alkylene groups, branched C 1 -C 6 -alkylene groups, C 3 -C 7 -cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the group consisting of
  • * and # represent the points of attachment of said group with the rest of the compound, optionally substituted with one or more substituents independently selected from the group consisting of halogen, -NHCONH 2 , -COOH, -OH, -NH 2 , NH-CNNH 2 , sulphonamide, sulphone, sulphoxide and sulphonic acid;
  • L2 represents:
  • # 1 represents the attachment point to the binder
  • # 2 represents the attachment point to the group L1, L1’ or SG; or the enantiomers, diastereomers, salts, solvates or salts of solvates thereof.
  • the invention relates to a conjugate as described supra, wherein
  • the invention relates to a conjugate as described supra, wherein the linker–Z’- represents one of the following general structures (i) to (iii):
  • ⁇ represents the attachment point to D
  • ⁇ represents the attachment point to AB
  • SG represents an in vivo cleavable group
  • L1 and L1’ represent, independently of each other, an in vivo non-cleavable organic group
  • L2 represents an attachment group
  • the invention relates to a conjugate as described supra, wherein the in vivo cleavable group SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal.
  • the in vivo cleavable group SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal.
  • the invention relates to a conjugate as described supra, wherein L1 and L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -S-, -SO-, SO 2 , -NH-, -CO-, -NMe-, -NHNH-, -SO 2 NHNH-, -NHCO-, -CONH-, - CONHNH-, arylene groups, heteroarylene groups, straight C 1 -C 6 -alkylene groups, branched C 1 -C 6 -alkylene groups, C 3 -C 7 -cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the group consisting of
  • the invention relates to a conjugate as described supra, wherein L2 represents:
  • the invention relates to a conjugate as described supra, wherein the linker–Z’- represents one of the following general structures (i) to (iii): (i) ⁇ –L1-SG-L2- ⁇ (ii) ⁇ –L1-SG-L1’-L2- ⁇ (iii) ⁇ –L1-L2- ⁇ wherein ⁇ represents the attachment point to D; ⁇ represents the attachment point to AB; SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal; L1, L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O
  • * and # represent the points of attachment of said group with the rest of the compound, optionally substituted with one or more substituents independently selected from the group consisting of halogen, -NHCONH 2 , -COOH, -OH, -NH 2 , NH-CNNH 2 , sulphonamide, sulphone, sulphoxide and sulphonic acid;
  • # 1 represents the attachment point to the binder
  • # 2 represents the attachment point to the group L1, L1’ or SG.
  • the invention relates to a conjugate as described supra, wherein L2 represents one or more of the following three formulae:
  • # 1 represents the attachment point to the binder
  • # 2 represents the attachment point to the group L1, L1’ or SG
  • # 1 represents the attachment point to the binder
  • # 2 represents the attachment point to the group L1, L1’ or SG
  • the amide group at # 2 is connected to L1, L1’ or SG via the group–CH 2 -C(O)-.
  • the invention relates to a conjugate as described supra, wherein SG is a 2-8 oligopeptide.
  • the invention relates to a conjugate as described supra, wherein the 2-8 oligopeptide consists of amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine.
  • the 2-8 oligopeptide consists of amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine.
  • the invention relates to a conjugate as described supra, wherein L1 and L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -NH-, -CO-, -NHCO-, -CONH-, phenyl and _ ; optionally substituted with one or more substituents independently selected from the group consisting of -F, -Cl, -COOH, -OH, and -NH 2 .
  • the invention relates to a conjugate as described supra, wherein L1 and L1’ represent, independently of each other, one of the general structures (iv) or (v): (iv) –A’-(NR 10 CO)-B’- (v) –A’-(CONR 10 )-B’- wherein: A’ represents C 1 -C 6 -alkyl, (C 1 -C 2 -alkyl)-(phenylene), and (C 1 -C 3 -alkyl)-(NR 11 )-(C 2 -alkyl); optionally substituted with one or more substituents independently selected from–F and -Cl;
  • B’ represents a straight-chain or branched hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from :-O-, -NH-, -CO-, -NHCO-, and -CONH-; optionally substituted with–COOH;
  • R 10 , R 11 represent, independently of each other hydrogen or C 1 -C 3 -alkyl; or R 10 , R 11 together with the nitrogens to which they are attached form a 6-membered nitrogen containing heterocycloalkyl group.
  • the invention relates to a conjugate of general formula (II)
  • AB stands for a binder
  • Z’ stands for a linker
  • n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8; wherein: A represents:
  • R 1 represents, independently of each other, halogen, hydroxy, C 1 -C 3 -alkyl, C 1 -C 3 - haloalkyl, C 1 -C 3 -alkoxy, C 1 -C 3 -haloalkoxy, -N(H)R 6 , -N(R 6 )R 7 or -NH 2 ;
  • R 2 represents H, C 1 -C 6 -alkyl, C 3 -C 6 -cycloalkyl, C 1 -C 4 -haloalkyl or phenyl, wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
  • n 0, 1, 2 or 3
  • * and # represent the points of attachment of said group with the rest of the compound, said group being optionally substituted with one or more substituents independently selected from the group consisting of:
  • -Z’- represents one of the following general structures (i) to (iii): (i) ⁇ –L1-SG-L2- ⁇ (ii) ⁇ –L1-SG-L1’-L2- ⁇ (iii) ⁇ –L1-L2- ⁇ wherein ⁇ represents the attachment point to A; ⁇ represents the attachment point to AB; SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulf
  • L2 represents:
  • # 1 represents the attachment point to the binder
  • # 2 represents the attachment point to the group L1, L1’ or SG; or the enantiomers, diastereomers, salts, solvates or salts of solvates thereof.
  • the invention relates to a conjugate as described supra, wherein
  • the invention relates to a conjugate as described supra, wherein L1 and L1’, independently of each other, are those below, where r in each case independently of one another represents a number from 1 to 20, preferably from 1 to 15, particularly preferably from 2 to 20, especially preferably from 2 to 10.
  • the invention relates to a conjugate as described supra,
  • SG comprises 2-6 amino acids selected from the group comprising: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine.
  • the invention relates to a conjugate as described supra, wherein SG comprises 2-3 amino acids selected from the group comprising: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine.
  • SG comprises 2-3 amino acids selected from the group comprising: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine.
  • the invention relates to a conjugate as described supra, wherein SG comprises 2-3 amino acids selected from the group comprising: alanine, glycine, histidine, isoleucine, leucine, methionine, serine, citrulline and valine.
  • the invention relates to a conjugate as described supra, wherein SG comprises 2 amino acids selected from the group comprising: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine.
  • SG comprises 2 amino acids selected from the group comprising: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine.
  • the invention relates to a conjugate as described supra, wherein SG comprises 2 amino acids selected from the group comprising: alanine, glycine, histidine, isoleucine, leucine, methionine, serine, citrulline and valine.
  • the table C below provides examples showing preferred ’ (i) ⁇ –L1-SG-L2- ⁇ (iii) ⁇ –L1-SG-L1’-L2- ⁇ (iii) ⁇ –L1-L2- ⁇ where r in each case independently of one another repr bly from 1 to 10, particularly preferably from 1 to 8.
  • the groups L1 and L1’ below are rea eft-hand symbol in the Table C below denotes the linkage site to ⁇ - or ⁇ -L1-SG-, respectively, and the righ w denotes the linkage site to -SG-L1’-L2- ⁇ , -SG-L1’- L2- ⁇ or -L2- ⁇ . It is understood that SG and L1’ are op TABLE C L1 SG L1’
  • # 1 represents the attachment point to the binder
  • # 2 represents the attachment point to the group L1, L1’ or SG.
  • r in each case independently of one another represents a number from 1 to 15, preferably from 1 to 10, particularly preferably from 1 to 8.
  • groups L1 and L1’ below are read from left to right, meaning that the left-hand symbol in the Table D below denotes the linkage site to ⁇ - or ⁇ -L1-SG-, respectively, and the right-hand symbol in the Table D below denotes the linkage site to -SG-L2- ⁇ , -SG-L1’-L2- ⁇ or -L2- ⁇ .
  • # 2 represents the attachment point to the group L1, L1’ or SG.
  • the invention relates to a conjugate as described supra, wherein SG comprises valine and alanine.
  • the invention relates to a conjugate as described supra, wherein SG comprises valine and citrulline.
  • the invention relates to a conjugate as described supra, wherein SG comprises alanine-valine.
  • the invention relates to a conjugate as described supra, wherein SG comprises citrulline-alanine.
  • the invention relates to a conjugate as described supra, wherein SG comprises (C-terminus)-Ala-Val-(N-terminus) or (C-terminus)-Cit-Val-(N- terminus).
  • the invention relates to a conjugate as described supra, wherein SG is (C-terminus)-Ala-Val-(N-terminus) or (C-terminus)-Cit-Val-(N-terminus).
  • the invention relates to a conjugate of a binder or a derivative thereof with one or more molecules of an active component, wherein the active component is a NAMPT inhibitor, which is conjugated to the binder via a linker Z’.
  • the conjugate has the formula: A B Z' D
  • n wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8, and D stands for a NAMPT inhibitor of Formula (IVa):
  • C 1 represents a phenylene, heteroarylene (preferably a 6-membered heteroarylene), 5- to 7-membered heterocycloalkylene (preferably a 6-membered heterocycloalkylene) or C 3 -C 6 - cycloalkylene (preferably a C 6 -cycloalkylene) group, in which C 3 -C 6 -cycloalkylene is optionally partially unsaturated, said groups being optionally substituted with one or more substituents independently selected from the group consisting of:
  • a 1 represents a group selected from 5- to 7-membered heterocycloalkylene, 6 to 10-membered fused heterocycloalkylene and heteroarylene,
  • phenyl and C 3 -C 6 -cycloalkyl groups are optionally substituted with one or more substituents independently selected from the group consisting of:
  • halogen hydroxy, cyano, C 1 -C 3 -alkyl, C 1 -C 3 -alkoxy, C 1 -C 3 -haloalkoxy, -NH 2 , -N(H)R 6 and -N(R 6 )R 7 ; q is 0, 1, 2 or 3 (preferably 1),
  • n 0, 1, 2 or 3 (preferably 1)
  • the conjugate has the formula: AB+ Z'- D] n wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8, and D stands for a NAMPT inhibitor of Formula (IVb):
  • H Z Y ( IVb) wherein # represents the point of attachment to linker Z’; in which: A 1 represents a group selected from 5- to 7-membered heterocycloalkylene, 6 to 10-membered fused heterocycloalkylene and heteroarylene, said group being optionally substituted with one or more substituents independently selected from the group consisting of:
  • phenyl and C 3 -C 6 -cycloalkyl groups are optionally substituted with one or more substituents independently selected from the group consisting of:
  • halogen hydroxy, cyano, C 1 -C 3 -alkyl, C 1 -C 3 -alkoxy, C 1 -C 3 -haloalkoxy, -NH 2 , -N(H)R 6 and -N(R 6 )R 7 ; q is 0, 1, 2 or 3 (preferably 1),
  • n 0, 1, 2 or 3 (preferably 1)
  • the conjugate has the formula: A B Z' D
  • n wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8, and D stands for a NAMPT inhibitor of Formula (IVc):
  • a 1 represents a group selected from 5- to 7-membered heterocycloalkylene, 6 to 10-membered fused heterocycloalkylene and heteroarylene, said group being optionally substituted with one or more substituents independently selected from the group consisting of:
  • the invention relates to compounds selected from the group consisting of: tert-butyl ⁇ 4-[3- ⁇ 4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl ⁇ -5,5- dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl ⁇ carbamate

Abstract

Conjugate of a binder having formula (AA) wherein AB stands for a binder, Z' stands for a linker, D stands for an active component which is a NAMPT inhibitor and its use as pharmaceuticals.

Description

Antibody drug conjugates (ADCs) with NAMPT inhibitors
Field of application of the invention
The present invention relates to novel conjugates of a binder or a derivative thereof with one or more molecules of an active component, wherein the active component is a NAMPT inhibitor, which is conjugated to the binder via a linker Z’ as described and defined herein, and methods for their preparation, their use for the treatment and/or prophylaxis of disorders, in particular of hyper-proliferative disorders.
Background of the invention
Nicotinamide adenine dinucleotide (NAD) is a biologically important coenzyme that plays a critical role in many cell metabolism-related transformations and in cell signaling [Lin, S-J.; Guarente L. Current Opinion Cell Biol. 2003, 15, 241–146; Ziegler M. Eur. J. Biochem. 2000, 267, 1550–1564]. In mammalian cells, the two step salvaging of NAD+ from nicotinamide (NAM)– nicotinamide pathway - is the most efficient process compared to the de novo synthesis of NAD+ from the essential amino acid L-tryptophan which takes mainly place in the liver [Schramm V. L. et al. PNAS 2009, 106, 13748–13753]. NAMPT (nicotinamide phosphoribosyltransferase also known as pre-B-cell-colony-enhancing factor (PBEF) and visfatin, NMPRT, NMPETase or NAmPRTase, international nomenclature E.C.2.4.2.12) catalyzes the first step of this process, the phosphoribosylation of NAM to NMN (nicotinamide mononucleotide) which is further converted to NAD+ by NMNAT (nicotinamide mononucleotide adenylyltransferase). NAMPT is the rate-limiting enzyme in the production of NAD+ and its inhibition leads to a rapid depletion of NAD+ [Deng Y. et al. Bioanalysis 2014, 6, 1145–1457]. In general, an altered cell metabolism is one of the basic characteristics of cancer cells as hypothesized by Otto Heinrich Warburg [Warburg, O. Über den Stoffwechsel der Carcinomzelle. Klin. Wochenschr.4, 534–536 (1925)]. As cancer cells proliferate continuously, these cells have to adapt to a stressful and dynamic microenvironment. This results in an increased need for energy, macromolecules and the maintenance of the cellular redox status by cancer cells [Cairns R. A. et al. Nature Rev. 2011, 11, 85–95]. With this regard, NAD+ is used as electron carrier in glycolysis, which is up-regulated in cancer cells due to the Warburg effect, as well as in mitochondrial oxidative phosphorylation. Further, NAD+ serves as a substrate for several enzymes, for example poly-ADP-ribose polymerases (PARPs) and sirtuins (SIRTs) which are involved in DNA repair and gene expression,   processes often aberrantly regulated in cancer cells and leading to consumption of NAD+ [Berger F et al. 2004 Trends Biochem. Sci. 29, 111–118]. Phosphorylated forms of NAD+/NADH also exist and are often employed for biosynthetic and/or cell protection purposes in addition to energy generation. They are also involved in the cellular response to oxidative stress [Massudi H. Redox Rep.2012, 17, 28–46]. For these reasons, many cancer cells have an increased need for NAD+ and its synthesis is constantly required, rendering cancer cells particularly sensitive to NAMPT inhibition. Moreover, it was demonstrated that NAMPT is implicated in the regulation of cell viability during genotoxic or oxidative stress and that NAMPT inhibitors are potentially useful for the treatment of e.g. inflammation, metabolic disorders and cancer [Tong L. et al. Expert Opin. Ther. Targets 2007, 11, 695–705; Galli, M. et al. Cancer Res.2010, 70, 8–11, J. Med. Chem 2013, 56, 6279– 6296]. Daporinad also known as APO866, FK866, WK175 or WK22 ((E)-N-[4-(I-benzoylpiperidin-4- yl)butyl]-3-(pyrldine-3-yl)-acrylamide) is a highly potent and selective inhibitor of NAMPT which interferes with NAD biosynthesis, ATP generation and induces cell death. In vivo efficacy of daporinad was shown in murine renal cell carcinoma model RENCA [Drevs J. et al. Anticancer Res 2003, 23, 4853-4858]. Clinical trials with daporinad have been completed for the treatment of chronic lymphocytic leukemia (CLL), cutaneous T cell lymphoma (CTL), and advanced melanoma [ClinicalTrials.gov Identifier: NCT00435084, NCT00431912, NCT00432107]. CHS-828 also known as GMX1778 (N-[6-(4-chlorophenoxy)hexyl]-N'-cyano-N''-4-pyridinyl- guanidine), an inhibitor of NAMPT as well as an inhibitor of NF- ^B pathway activity [Hassan S. B. et al. Anticancer Res 2006, 26, 4431-4436], showed highly cytotoxic effects in vitro and in vivo in human breast and lung cancer cell line-derived in vivo models [Hijarnaa PJ et al. Cancer Res. 1999, 59, 5751–5757]. A Phase I study for this compound in patients with solid tumors was published in the year 2002 [Hovstadius P et al. ClinCancerRes 2002, 9, 2843–2850]. Best observed responses in the clinical trials were stable disease. Therefore, it has been assumed that the lack of significant activity in clinical trials may result from the inability to dose NAMPT inhibitors to higher drug exposures due to dose-limiting toxicities [Sampath D. et al. Pharmacology and Therapeutics 2015, 151, 16–31]. Combining targeting of a cytotoxic drug to cancer cells eg by employing a binder or an antibody may improve the therapeutic window and may thus result in better tolerability and better clinical responses. The present invention relates to novel conjugates of a binder or a derivative thereof with one or more molecules of an active component, wherein the active component is a NAMPT inhibitor, which is conjugated to the binder via a linker.   A number of chemical compounds have been shown to act as NAMPT inhibitors. For example, Bioorganic & Medicinal Chemistry Letters (2013), 23, 4875–4885; WO 2014111871 and WO 2013067710 discloses 1,3-dihydro-2H-isoindoles as NAMPT inhibitors. DE10010423, WO9206087 and WO2006064189 disclose 1-alkyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl derivatives which may be useful for the treatment of anemia, cardiovascular and diglyceride acyltransferase (DGAT) mediated disorders (e.g. diabetes), respectively. WO2012067965 discloses 4-oxo-3,4-dihydrophthalazine phenyl cyclic urea derivatives which may be useful as NAMPT and ROCK inhibitors. Despite the progress made during the last decades in the treatment of uncontrolled proliferative cellular processes in humans and animals, like cancer diseases, there is still a huge unmet medical need to expand therapeutic options especially based on new drugs selectively addressing new targets.
Summary of the Invention Therefore, inhibitors of NAMPT represent valuable compounds that should complement therapeutic options either as single agents or in combination with other drugs, particularly those NAMPT inhibitors with increased selectivity over other biological targets. It is thus an object of the present invention to provide substances which may be of benefit for cancer therapy. To achieve this object, the invention provides conjugates of a binder or derivatives thereof with one or more active compound molecules, the active compound molecule being a NAMPT inhibitor attached to the binder via a linker Z’. The binder is preferably a binder protein or peptide, particularly preferably a human, humanized or chimeric monoclonal antibody or an antigen-binding fragment thereof. The conjugate according to the invention can be represented by the general formula:
Figure imgf000004_0001
wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, and D stands for an active component. According to the invention, the active component (NAMPT inhibitor) may have the Formula (I) below:
 
Figure imgf000005_0001
wherein: A represents:
Figure imgf000005_0002
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2;
t is 0, 1, 2 or 3; R2 represents H, C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, and -N(R6)R7 ; R3 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8); R4 represents C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl;
R5 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or
  R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8), q is 0, 1, 2 or 3,
m is 0, 1, 2 or 3,
with the proviso that q + m is 2, 3 or 4 ;
Figure imgf000006_0002
represents a group which is selected from :
Figure imgf000006_0001
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I),
said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, R6(H)N- and -N(R6)R7;
R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl); R8, R9 represent, independently of each other, hydrogen, C1-C3-alkyl, C3-C6-cycloalkyl or - C(=O)-(C1-C3-alkyl); or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer. The inventors have found a number of methods to attach the binder to the NAMPT inhibitor in order to achieve the object mentioned above. According to the invention, the NAMPT inhibitor may be attached to the binder via a linker Z’ at position # in formula (I). The conjugates according to the invention can have chemically labile linkers, enzymatically labile linkers or stable linkers.
The linker–Z’- may represent one of the following general structures (i) to (iii):
  (i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ wherein § represents the attachment point to D; §§ represents the attachment point to AB; SG represents an in vivo cleavable group, L1 represents an in vivo non-cleavable organic group, and L2 represents an attachment group. SG may represent a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal. L1, L1’ may represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -S-, -SO-, SO2, -NH-, -CO-, -NMe-, -NHNH-, -SO2NHNH-, -NHCO-, -CONH-, - CONHNH-, arylene groups, heteroarylene groups, straight C1-C6-alkylene groups, branched C1-C6-alkylene groups, C3-C7-cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the group consisting of
N, O,S, -SO- and–SO2- (preferably
Figure imgf000007_0002
in which * and # represent the points of attachment of said group with the rest of the compound, optionally substituted with one or more substituents selected from the group consisting of halogen, -NHCONH2, -COOH, -OH, -NH2, -NH-CNNH2, sulphonamide, sulphone, sulphoxide and sulphonic acid. L2 may represent:
Figure imgf000007_0001
 
Figure imgf000008_0001
wherein #1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG.
The invention furthermore provides processes for preparing the conjugates according to the invention, and also precursors and intermediates or salts thereof useful for their preparation. The preparation of the conjugates according to the invention regularly comprises the following steps: (i) Preparation of a linker precursor which optionally carries protective groups; (ii) Conjugation of the linker precursor to the derivative, which optionally carries protective groups, of a low-molecular weight NAMPT inhibitor (preferably a NAMPT inhibitor having Formula (I), giving a NAMPT inhibitor/linker conjugate which optionally carries protective groups; (iii) Attachment of a reactive group to the NAMPT inhibitor/linker conjugate; (iv) Removal of any protective groups present in the NAMPT inhibitor/linker conjugate and (v) Conjugation of the binder to the NAMPT inhibitor/linker conjugate, giving the binder/NAMPT inhibitor conjugate according to the invention.
 
Attachment of the reactive group may also take place during preparation of the linker precursor (e.g. during step (i) above)) rather than after the construction of an optionally protected NAMPT inhibitor/linker precursor conjugate. Depending on the linker, succinimide-linked ADCs may, after conjugation, be converted according to Scheme A into the open-chain succinamides, which have an advantageous stability profile. As illustrated above, conjugation of the linker precursor to a low-molecular weight NAMPT inhibitor may take place at position # in formula (I). In the synthesis steps prior to the conjugation, any functional groups present may also be present in protected form. Prior to the conjugation step, these protective groups are removed by known methods of peptide chemistry. Conjugation can take place chemically by various routes. In particular, it is optionally possible to modify the low-molecular weight NAMPT inhibitor for conjugation to the linker, for example by introduction of protective groups or leaving groups to facilitate substitution. In linker Z’, position #1 of group L2 preferably reacts with an amino or thiol group on binder AB to form a covalent bond, preferably with a cysteine or a lysine residue in a protein of AB. The cysteine residue in a protein may of course be present naturally in the protein, may be introduced by biochemical methods or, preferably, may be generated by prior reduction of disulphides of the binder.
Detailed Description of the Invention
Definitions Constituents that are optionally substituted as stated herein, may be substituted, unless otherwise noted, one or more times, independently from one another at any possible position. When any variable occurs more than one time in any constituent or in different constituents, each definition is independent. For example, for any component of formula (I) in which R1, R6, R7, R8, R9, R10 and/or R11 occur more than one time, each definition of R1, R6, R7, R8, R9, R10 and R11 is independent. Should a constituent be composed of more than one part, e.g. -(C1-C2 alkyl)-(phenylene)-, the position of a possible substituent can be at any of these parts at any suitable position. A hyphen at the beginning or at the end of the constituent marks the point of attachment to the rest of the molecule. Should a ring be substituted, the substitutent(s) could be at any suitable position of the ring, also on a ring nitrogen atom if suitable.  
The term“comprising” when used in the specification includes“consisting of”. If it is referred to“as mentioned above”,“mentioned above” or“supra” within the description it is referred to any of the disclosures made within the specification in any of the preceding pages. “suitable” within the sense of the invention means chemically possible to be made by methods within the knowledge of a skilled person. The term“compound” as used in, for example,“compound of the present invention”, “compound of the invention”,“compound described supra”,“compound described herein”, “compound defined supra”,“compound defined herein”, used throughout the specification refer to NAMPT inhibitors (e.g. NAMPT inhibitors (D) of formula (I)) and intermediates (e.g. the NAMPT inhibitor-linker-intermediates of general formula (III)) used to prepare the ADC conjugates of the present invention, as well as metabolites of the ADC conjugates of the present invention. The terms as mentioned in the present text have preferably the following meanings: The term“halogen atom”,“halo-” or“Hal-” is to be understood as meaning a fluorine, chlorine, bromine or iodine atom. The term“C1-C6-alkyl” is to be understood as meaning a linear or branched, saturated, monovalent hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms, e.g. a methyl, ethyl, propyl, butyl, pentyl, hexyl, iso-propyl, iso-butyl, sec-butyl, tert-butyl, iso-pentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neo-pentyl, 1,1-dimethylpropyl, 4- methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3- dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl group, or an isomer thereof. Particularly, said group has 1, 2, 3 or 4 carbon atoms (“C1-C4-alkyl”), e.g. a methyl, ethyl, propyl, butyl, iso-propyl, iso-butyl, sec-butyl, tert- butyl group, more particularly 1, 2 or 3 carbon atoms (“C1-C3-alkyl”), e.g. a methyl, ethyl, n- propyl- or iso-propyl group. The term“C1-C3-haloalkyl” is to be understood as meaning a linear or branched, saturated, monovalent hydrocarbon group in which the term“C1-C3-alkyl” is defined supra, and in which one or more hydrogen atoms is replaced by a halogen atom, identically or differently, i.e. one halogen atom being independent from another. Particularly, said halogen atom is F. Said C1-
 
C3-haloalkyl group is, for example,–CF3, -CHF2, -CH2F, -CF2CF3, -CH2CH2F, -CH2CHF2, - CH2CF3, -CH2CH2CF3, or -CH(CH2F)2. Particularly, said group has 1, 2 or 3 carbon atoms. The term“C1-C3-alkoxy” is to be understood as meaning a linear or branched, saturated, monovalent, hydrocarbon group of formula–O-(C1-C3-alkyl), in which the term“C1-C3-alkyl” is defined supra, e.g. a methyl, ethyl, n-propyl- or iso-propyl group, or an isomer thereof. Particularly, said group has 1, 2 or 3 carbon atoms. The term“C1-C3-haloalkoxy” is to be understood as meaning a linear or branched, saturated, monovalent C1-C3-alkoxy group, as defined supra, in which one or more of the hydrogen atoms is replaced, identically or differently, by a halogen atom. Particularly, said halogen atom is F. Said C1-C3-haloalkoxy group is, for example,–OCF3, -OCHF2, -OCH2F, -OCF2CF3, or - OCH2CF3. Particularly, said group has 1, 2 or 3 carbon atoms. The term“C3-C6-cycloalkyl” is to be understood as meaning a saturated, monovalent, monocyclic hydrocarbon ring which contains 3, 4, 5 or 6 carbon atoms (“C3-C6-cycloalkyl”). Said C3-C6-cycloalkyl group is for example, a monocyclic hydrocarbon ring, e.g. a cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl ring. Particularly, said group has 3 carbon atoms (“C3- cycloalkyl”), i.e. a cyclopropyl group. The term“5- to 7-membered heterocycloalkyl” is to be understood as meaning a saturated or partially unsaturated, monovalent, mono- or bicyclic hydrocarbon ring which contains 3, 4, 5 or 6 carbon atoms, and one or two heteroatom-containing group selected from O, N, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8), preferably O, N, NR8, S, S(=O), S(=O)2, more preferably O, N, NR8, in which R8 and R9 are as defined herein, said heterocycloalkyl group being attached to the rest of the molecule via a carbon atom or, if present, a nitrogen atom, of the heterocycloalkyl ring.
Particularly, without being limited thereto, said heterocycloalkyl can be a 5-membered ring, such as, but not limited to, tetrahydrofuranyl, pyrrolidinyl or pyrrolinyl, or a 6-membered ring, such as, but not limited to, tetrahydropyranyl, piperidinyl, morpholinyl or piperazinyl, or a 7- membered ring, such as, but not limited to, an azepanyl ring, for example. Optionally, said heterocycloalkyl can be benzo fused.
As mentioned supra, said 5- to 7-membered heterocycloalkyl can be partially unsaturated, i.e. it can contain one or more double bonds, such as, without being limited thereto, a 2,5-dihydro- 1H-pyrrolyl, for example, or, it may be benzofused, such as, without being limited thereto, a dihydroisoquinolinyl ring, for example.
 
The term“fused heterocycloalkylene” means a bicyclic, saturated or partially unsaturated heterocycle with 6, 7, 8, 9 or 10 ring atoms in total (“6 to 10-membered fused heterocycloalkylene), in which the two rings share two adjacent ring atoms, which“fused heterocycloalkyl” contains one or two identical or different ring heteroatoms from the series: O, N, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8), preferably O, N, NR8, S, S(=O), S(=O)2, more preferably O, N, NR8, in which R8 and R9 are as defined herein, it being possible for said fused heterocycloalkyl group to be attached to the rest of the molecule via any one of the carbon atoms or, if present, a nitrogen atom.
Said fused heterocycloalkyl group is, for example, azabicyclo[3.3.0]octylene, azabicyclo[4.3.0]nonylene, diazabicyclo[4.3.0]nonylene, oxazabicyclo[4.3.0]nonylene, thiazabicyclo[4.3.0]nonylene, 5,6,7,8-tetrahydrophthalazinylene, 1,2,5,6,7,8- hexahydrophthalazinylene, 1,2-dihydrophthalazinylene, 1,2-dihydroisoquinolinylene, 1,2- dihydropyrrolo[1,2-d][1,2,4]triazinylene,  isoindolinylene, 4,5-dihydro-3H-2,3- benzodiazepinylene or azabicyclo[4.4.0]decylene. The term“heteroatom containing group” is understood as meaning a heteroatom, such as, O and S, or a group contining a heteroatom, such as NR8, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8). The term“heteroarylene group” is understood as meaning a bivalent, monocyclic, or bicyclic aromatic ring having 5, 6, 8, 9, 10, 11, 12, 13 or 14 ring atoms (a“5- to 14-membered heteroarylene” group), particularly 5, 6, 9 or 10 ring atoms (a“5-, 6-, 9- or 10-membered heteroaryl” group), more particularly 5 or 6 ring atoms (a“5- to 6-membered heteroarylene” group), which contains at least one ring heteroatom and optionally one, two or three further ring heteroatoms from the series: N, O and/or S. It is understood that any heteroarylene group is attached to the rest of the molecule via carbon atoms of the heteroarylenic ring or, if present, a nitrogen atom.
Particularly, without being limited thereto, said heteroarylene group can be a 5-membered ring, such as, but not limited to, thienylene, furanylene, pyrrolylene, oxazolylene, thiazolylene, imidazolylene, pyrazolylene, isoxazolylene, isothiazolylene, thia-4H-pyrazolylene, furylene, triazolylene (1,2,4-triazolylene, 1,3,4-triazolylene or 1,2,3-triazolylene), thiadiazolylene (1,3,4- thiadiazolylene, 1,2,5-thiadiazolylene, 1,2,3-thiadiazolylene or 1,2,4-thiadiazolylene) and oxadiazolylene (1,3,4-oxadiazolylene, 1,2,5-oxadiazolylene, 1,2,3-oxadiazolylene or 1,2,4- oxadiazolylene), etc., and benzo derivatives thereof, such as, for example, benzofuranylene, benzothienylene, benzoxazolylene, benzisoxazolylene, benzimidazolylene, benzotriazolylene,  
indazolylene, indolylene, isoindolylene, etc.; or a 6-membered ring such as but not limited to, pyridylene, pyridazinylene, pyrimidinylene, pyrazinylene, triazinylene, etc., and benzo derivatives thereof, such as, for example, quinolinylene, quinazolinylene, isoquinolinylene, etc.. In general, and unless otherwise mentioned, the heteroarylenic radicals include all the possible isomeric forms thereof, e.g. the positional isomers thereof. Thus, for some illustrative non- restricting example, the term pyridinylene includes pyridin-2-ylene, pyridin-3-ylene and pyridin- 4-ylene; or the term thienylene includes thien-2-ylene and thien-3-ylene. Unless otherwise noted, any heteroatom of a heteroarylic ring with unsatisfied valences mentioned herein is assumed to have the hydrogen atom(s) to satisfy the valences. The term“C1-C6”, as used throughout this text, e.g. in the context of the definition of“C1-C6- alkyl” is to be understood as meaning an alkyl group having a finite number of carbon atoms of 1 to 6, i.e.1, 2, 3, 4, 5, or 6 carbon atoms. It is to be understood further that said term“C1- C6” is to be interpreted as any sub-range comprised therein, e.g. C1-C6 , C2-C5 , C3-C4 , C1-C2 , C1-C3 , C1-C4 , C1-C5 ; particularly C1-C2 , C1-C3 , C1-C4 , C1-C5, C1-C6; more particularly C1-C4. Further, as used herein, the term“C3-C6”, as used throughout this text, e.g. in the context of the definition of“C3-C6-cycloalkyl”, is to be understood as meaning a cycloalkyl group having a finite number of carbon atoms of 3 to 6, i.e.3, 4, 5 or 6 carbon atoms. It is to be understood further that said term“C3-C6” is to be interpreted as any sub-range comprised therein, e.g. C3- C6 , C4-C5 , C3-C5 , C3-C4 , C4-C6, C5-C6 ; particularly C3-C6. The term "substituted" means that one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. The term "optionally substituted" means optional substitution with the specified groups, radicals or moieties. Ring system substituent means a substituent attached to an aromatic or nonaromatic ring system which, for example, replaces an available hydrogen on the ring system.
 
As used herein, the term“one or more”, e.g. in the definition of the substituents of the compounds of the general formulae of the present invention, is understood as meaning“one, two, three, four or five, particularly one, two, three or four, more particularly one, two or three, even more particularly one or two”. The invention also includes all suitable isotopic variations of a compound of the invention. An isotopic variation of a compound of the invention is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually or predominantly found in nature. Examples of isotopes that can be incorporated into a compound of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine, chlorine, bromine and iodine, such as 2H (deuterium), 3H (tritium), 11C, 13C, 14C, 15N, 17O, 18O, 32P, 33P, 33S, 34S, 35S, 36S, 18F, 36Cl, 82Br, 123I, 124I, 125I, 129I and 131I, respectively. Certain isotopic variations of a compound of the invention, for example, those in which one or more radioactive isotopes such as 3H or 14C are incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence is preferred in some circumstances. Isotopic variations of a compound of the invention can generally be prepared by conventional procedures known by a person skilled in the art such as by the illustrative methods or by the preparations described in the examples hereafter using appropriate isotopic variations of suitable reagents. Where the plural form of the word compounds, salts, polymorphs, hydrates, solvates and the like, is used herein, this is taken to mean also a single compound, salt, polymorph, isomer, hydrate, solvate or the like. By "stable compound' is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. The compounds of this invention optionally contain one or more asymmetric centres, depending upon the location and nature of the various substituents desired. Asymmetric carbon atoms are present in the (R) or (S) configuration, resulting in racemic mixtures in the case of a single asymmetric centre, and diastereomeric mixtures in the case of multiple asymmetric centres. In certain instances, asymmetry may also be present due to restricted
 
rotation about a given bond, for example, the central bond adjoining two substituted aromatic rings of the specified compounds. The compounds of the present invention optionally contain sulphur atoms which are asymmetric, such as an asymmetric sulfoxide, of structure:
*
Figure imgf000015_0001
example, in which * indicates atoms to which the rest of the molecule can be bound.
Substituents on a ring may also be present in either cis or trans form. It is intended that all such configurations (including enantiomers and diastereomers), are included within the scope of the present invention. Preferred compounds are those which produce the more desirable biological activity. Separated, pure or partially purified isomers and stereoisomers or racemic or diastereomeric mixtures of the compounds of this invention are also included within the scope of the present invention. The purification and the separation of such materials can be accomplished by the technicques provided herein or by (other) standard techniques known in the art. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example, by the formation of diastereoisomeric salts using an optically active acid or base or formation of covalent diastereomers. Examples of appropriate acids are tartaric, diacetyltartaric, ditoluoyltartaric and camphorsulfonic acid. Mixtures of diastereoisomers can be separated into their individual diastereomers on the basis of their physical and/or chemical differences by methods known in the art, for example, by chromatography or fractional crystallisation. The optically active bases or acids are then liberated from the separated diastereomeric salts. A different process for separation of optical isomers involves the use of chiral chromatography (e.g., chiral HPLC columns), with or without conventional derivatisation, optimally chosen to maximise the separation of the enantiomers. Suitable chiral HPLC columns are manufactured by Daicel, e.g., Chiracel OD and Chiracel OJ among many others, all routinely selectable. Enzymatic separations, with or without derivatisation, are also useful. The optically active compounds of this invention can likewise be obtained by chiral syntheses utilizing optically active starting materials. In order to limit different types of isomers from each other reference is made to IUPAC Rules Section E (Pure Appl Chem 45, 11-30, 1976).  
The present invention includes all possible stereoisomers of the compounds of the present invention as single stereoisomers, or as any mixture of said stereoisomers, e.g. R- or S- isomers, or E- or Z-isomers, in any ratio. Isolation of a single stereoisomer, e.g. a single enantiomer or a single diastereomer, of a compound of the present invention is achieved by any suitable state of the art method, such as chromatography, especially chiral chromatography, for example. Further, the compounds of the present invention may exist as tautomers. The present invention includes all possible tautomers of the compounds of the present invention as single tautomers, or as any mixture of said tautomers, in any ratio. Further, the compounds of the present invention can exist as N-oxides, which are defined in that at least one nitrogen of the compounds of the present invention is oxidised. The present invention includes all such possible N-oxides. Accordingly, the present invention includes all possible salts, polymorphs, metabolites, hydrates, solvates, prodrugs (e.g.: esters) thereof, and diastereoisomeric forms of the NAMPT inhibtors or precursors (including intermediates) thereof as single salt, polymorph, metabolite, hydrate, solvate, prodrug (e.g.: esters) thereof, or diastereoisomeric form, or as mixture of more than one salt, polymorph, metabolite, hydrate, solvate, prodrug (e.g.: esters) thereof, or diastereoisomeric form in any ratio. The compounds of the present invention can exist as a hydrate, or as a solvate, wherein the compounds of the present invention contain polar solvents, in particular water, methanol or ethanol for example as structural element of the crystal lattice of the compounds. The amount of polar solvents, in particular water, may exist in a stoichiometric or non-stoichiometric ratio. In the case of stoichiometric solvates, e.g. a hydrate, hemi-, (semi-), mono-, sesqui-, di-, tri-, tetra-, penta- etc. solvates or hydrates, respectively, are possible. The present invention includes all such hydrates or solvates. Further, the compounds of the present invention can exist in free form, e.g. as a free base, or as a free acid, or as a zwitterion, or can exist in the form of a salt. Said salt may be any salt, either an organic or inorganic addition salt, particularly any pharmaceutically acceptable organic or inorganic addition salt, customarily used in pharmacy.
 
The term“pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present invention. For example, see S. M. Berge, et al.“Pharmaceutical Salts,” J. Pharm. Sci.1977, 66, 1-19. A suitable pharmaceutically acceptable salt of the compounds of the present invention may be, for example, an acid-addition salt of a compound of the present invention bearing a nitrogen atom, in a chain or in a ring, for example, which is sufficiently basic, such as an acid-addition salt with an inorganic acid, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, bisulfuric, phosphoric, or nitric acid, for example, or with an organic acid, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3-hydroxy-2-naphthoic, nicotinic, pamoic, pectinic, persulfuric, 3-phenylpropionic, picric, pivalic, 2-hydroxyethanesulfonate, itaconic, sulfamic, trifluoromethanesulfonic, dodecylsulfuric, ethansulfonic, benzenesulfonic, para-toluenesulfonic, methansulfonic, 2- naphthalenesulfonic, naphthalinedisulfonic, camphorsulfonic acid, citric, tartaric, stearic, lactic, oxalic, malonic, succinic, malic, adipic, alginic, maleic, fumaric, D-gluconic, mandelic, ascorbic, glucoheptanoic, glycerophosphoric, aspartic, sulfosalicylic, hemisulfuric, or thiocyanic acid, for example. Further, another suitably pharmaceutically acceptable salt of a compound of the present invention which is sufficiently acidic, is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a physiologically acceptable cation, for example a salt with N-methyl-glucamine, dimethyl-glucamine, ethyl-glucamine, lysine, dicyclohexylamine, 1,6-hexadiamine, ethanolamine, glucosamine, sarcosine, serinol, tris-hydroxy-methyl- aminomethane, aminopropandiol, sovak-base, 1-amino-2,3,4-butantriol. Additionally, basic nitrogen containing groups may be quaternised with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, and dibutyl sulfate; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and strearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others. Those skilled in the art will further recognise that acid addition salts of the claimed compounds may be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods. Alternatively, alkali and alkaline earth metal salts of acidic compounds of the invention are prepared by reacting the compounds of the invention with the appropriate base via a variety of known methods.  
The present invention includes all possible salts of the compounds of the present invention as single salts, or as any mixture of said salts, in any ratio. In the present text, in particular in the Experimental Section, for the synthesis of intermediates and of examples of the present invention, when a compound is mentioned as a salt form with the corresponding base or acid, the exact stoichiometric composition of said salt form, as obtained by the respective preparation and/or purification process, is, in most cases, unknown. Unless specified otherwise, suffixes to chemical names or structural formulae such as "hydrochloride", "trifluoroacetate", "sodium salt", or "x HCl", "x CF3COOH", "x Na+", for example, are to be understood as not a stoichiometric specification, but solely as a salt form. The salts include water-insoluble and, particularly, water-soluble salts. This applies analogously to cases in which synthesis intermediates or example compounds or salts thereof have been obtained, by the preparation and/or purification processes described, as solvates, such as hydrates with (if defined) unknown stoichiometric composition. Furthermore, derivatives of the conjugates described herein and the salts thereof which are converted into a conjugate as described herein or a salt thereof in a biological system (bioprecursors or pro-drugs) are covered by the invention. Said biological system is e.g. a mammalian organism, particularly a human subject. The bioprecursor is, for example, converted into a conjugate as described herein or a salt thereof by metabolic processes. Furthermore, the present invention includes all possible crystalline forms, or polymorphs, of the conjugates of the present invention, either as single polymorph, or as a mixture of more than one polymorph, in any ratio. In the context of the properties of the conjugates of the present invention the term “pharmacokinetic profile” means one single parameter or a combination thereof including permeability, bioavailability, exposure, and pharmacodynamic parameters such as duration, or magnitude of pharmacological effect, as measured in a suitable experiment. Conjugates with improved pharmacokinetic profiles can, for example, be used in lower doses to achieve the same effect, may achieve a longer duration of action, or a may achieve a combination of both effects.
 
The term“combination” in the present invention is used as known to persons skilled in the art and may be present as a fixed combination, a non-fixed combination or kit-of-parts. A“fixed combination” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein the said first active ingredient and the said second active ingredient are present together in one unit dosage or in a single entity. One example of a“fixed combination” is a pharmaceutical composition wherein the said first active ingredient and the said second active ingredient are present in admixture for simultaneous administration, such as in a formulation. Another example of a“fixed combination” is a pharmaceutical combination wherein the said first active ingredient and the said second active ingredient are present in one unit without being in admixture. A non-fixed combination or“kit-of-parts” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein the said first active ingredient and the said second active ingredient are present in more than one unit. One example of a non- fixed combination or kit-of-parts is a combination wherein the said first active ingredient and the said second active ingredient are present separately. The components of the non-fixed combination or kit-of-parts may be administered separately, sequentially, simultaneously, concurrently or chronologically staggered. Any such combination of a compound of formula (I) of the present invention with an anti-cancer agent as defined below is an embodiment of the invention. The term“(chemotherapeutic) anti-cancer agents”, includes but is not limited to:
131I-chTNT, abarelix, abiraterone, aclarubicin, adalimumab, ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alectinib, alemtuzumab, alendronic acid, alitretinoin, altretamine, amifostine, aminoglutethimide, hexyl aminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anethole dithiolethione, anetumab ravtansine, angiotensin II, antithrombin III, aprepitant, arcitumomab, arglabin, arsenic trioxide, asparaginase, atezolizumab, axitinib, azacitidine, basiliximab, belotecan, bendamustine, besilesomab, belinostat, bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin, blinatumomab, bortezomib, buserelin, bosutinib, brentuximab vedotin, busulfan, cabazitaxel, cabozantinib, calcitonine, calcium folinate, calcium levofolinate, capecitabine, capromab, carbamazepine carboplatin, carboquone, carfilzomib, carmofur, carmustine, catumaxomab, celecoxib, celmoleukin, ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin, cladribine, clodronic acid, clofarabine, cobimetinib, copanlisib , crisantaspase, crizotinib, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daratumumab, darbepoetin alfa, dabrafenib, dasatinib, daunorubicin,  
decitabine, degarelix, denileukin diftitox, denosumab, depreotide, deslorelin, dianhydrogalactitol, dexrazoxane, dibrospidium chloride, dianhydrogalactitol, diclofenac, dinutuximab, docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin + estrone, dronabinol, eculizumab, edrecolomab, elliptinium acetate, elotuzumab, eltrombopag, endostatin, enocitabine, enzalutamide, epirubicin, epitiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine, ethinylestradiol, etoposide, everolimus, exemestane, fadrozole, fentanyl, filgrastim, fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide, folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant, gadobutrol, gadoteridol, gadoteric acid meglumine, gadoversetamide, gadoxetic acid, gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, Glucarpidase, glutoxim, GM-CSF, goserelin, granisetron, granulocyte colony stimulating factor, histamine dihydrochloride, histrelin, hydroxycarbamide, I-125 seeds, lansoprazole, ibandronic acid, ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate, interferon alfa, interferon beta, interferon gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab, irinotecan, Itraconazole, ixabepilone, ixazomib, lanreotide, lansoprazole, lapatinib, Iasocholine, lenalidomide, lenvatinib, lenograstim, lentinan, letrozole, leuprorelin, levamisole, levonorgestrel, levothyroxine sodium, lisuride, lobaplatin, lomustine, lonidamine, masoprocol, medroxyprogesterone, megestrol, melarsoprol, melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate, methoxsalen, methylaminolevulinate, methylprednisolone, methyltestosterone, metirosine, mifamurtide, miltefosine, miriplatin, mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane, mitoxantrone, mogamulizumab, molgramostim, mopidamol, morphine hydrochloride, morphine sulfate, nabilone, nabiximols, nafarelin, naloxone + pentazocine, naltrexone, nartograstim, necitumumab, nedaplatin, nelarabine, neridronic acid, netupitant/palonosetron, nivolumab, pentetreotide, nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nintedanib, nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, olaparib, olaratumab, omacetaxine mepesuccinate, omeprazole, ondansetron, oprelvekin, orgotein, orilotimod, osimertinib, oxaliplatin, oxycodone, oxymetholone, ozogamicine, p53 gene therapy, paclitaxel, palbociclib, palifermin, palladium-103 seed, palonosetron, pamidronic acid, panitumumab, panobinostat, pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxy PEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b, pembrolizumab, pemetrexed, pentazocine, pentostatin, peplomycin, Perflubutane, perfosfamide, Pertuzumab, picibanil, pilocarpine, pirarubicin, pixantrone, plerixafor, plicamycin, poliglusam, polyestradiol phosphate, polyvinylpyrrolidone + sodium hyaluronate, polysaccharide-K, pomalidomide, ponatinib, porfimer sodium, pralatrexate, prednimustine, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, racotumomab, radium-223 chloride, radotinib, raloxifene, raltitrexed, ramosetron,  
ramucirumab, ranimustine, rasburicase, razoxane, refametinib , regorafenib, risedronic acid, rhenium-186 etidronate, rituximab, rolapitant, romidepsin, romiplostim, romurtide, roniciclib , samarium (153Sm) lexidronam, sargramostim, satumomab, secretin, siltuximab, sipuleucel-T, sizofiran, sobuzoxane, sodium glycididazole, sonidegib, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, talimogene laherparepvec, tamibarotene, tamoxifen, tapentadol, tasonermin, teceleukin, technetium (99mTc) nofetumomab merpentan, 99mTc-HYNIC-[Tyr3]- octreotide, tegafur, tegafur + gimeracil + oteracil, temoporfin, temozolomide, temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide, thiotepa, thymalfasin, thyrotropin alfa, tioguanine, tocilizumab, topotecan, toremifene, tositumomab, trabectedin, trametinib, tramadol, trastuzumab, trastuzumab emtansine, treosulfan, tretinoin, trifluridine + tipiracil, trilostane, triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan, ubenimex, valatinib , valrubicin, vandetanib, vapreotide, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vismodegib, vorinostat, vorozole, yttrium-90 glass microspheres, zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin.
The invention provides conjugates of a binder or derivative thereof with one or more active compound molecules, the active compound molecule being a NAMPT inhibitor attached to the binder via a linker Z’. In accordance with a first aspect, the invention relates to a conjugate of a binder or a derivative thereof with one or more molecules of an active compound that has the formula:
Figure imgf000021_0001
n wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8, and D stands for an active component having Formula (I) described herein. The binder is preferably a binder peptide or protein such as, for example, an antibody. Furthermore, if n is greater than 1, the linker is preferably attached to different amino acids of the same chemical nature of the binder peptide or protein or derivative thereof. Particular preference is given to binding to different cysteine or lysine residues of the binder, even more preferable is binding to different cysteine residues of the binder. Binders which can be used according to the invention, NAMPT inhibitors which can be used according to the invention and linkers which can be used according to the invention which can be used in combination without any limitation are described below. In particular, the binders represented in each case as preferred or particularly preferred can be employed in combination  
with the NAMPT inhibitors represented in each case as preferred or particularly preferred, optionally in combination with the linkers represented in each case as preferred or particularly preferred.
NAMPT inhibitors
Despite the fact that various inhibitors of NAMPT are known, there remains a need for selective NAMPT inhibitors to be used for the treatment of diseases such as hyper-proliferative diseases, which offer one or more advantages such as:
^ improved activity and / or efficacy, allowing e.g. a dose reduction
^ improved side effect profile, such as fewer undesired side effects, lower intensity of side effects, or reduced (cyto)toxicity
^ improved physicochemical properties, such as solubility in water, body fluids, and aqueous formulations, e.g. for intravenous administration
^ improved pharmacokinetic properties, allowing e.g. for dose reduction or an easier dosing scheme
^ improved duration of action, e.g. by improved pharmacokinetics and / or improved target residence time
^ easier drug substance manufacturing e.g. by shorter synthetic routes or easier purification. The NAMPT inhibitors used in the binder drug conjugates according to the invention preferably show anti-proliferative activity in tumor cell lines, such as THP-1, U251 MG, MV-4-11, MDA- MB-453, NCI-N87 or SK-OV-3, for example. According to the present invention, the NAMPT inhibitors (D) are described by Formula (I):
(
Figure imgf000022_0001
wherein: A represents:  
Figure imgf000023_0001
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2;
t is 0, 1, 2 or 3; R2 represents H, C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl, wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, and -N(R6)R7; R3 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8); R4 represents C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl;
R5 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8), q is 0, 1, 2 or 3,
m is 0, 1, 2 or 3,
with the proviso that q + m is 2, 3 or 4;
 
Figure imgf000024_0002
represents a group which is selected from :
Figure imgf000024_0003
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I),
said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, R6(H)N- and -N(R6)R7; R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl) ; R8, R9 represent, independently of each other, hydrogen, C1-C3-alkyl, C3-C6-cycloalkyl or - C(=O)-(C1-C3-alkyl); or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer.
In a second aspect, the invention relates to a conjugate as described supra, wherein:
A represents:
Figure imgf000024_0001
,
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2;
t is 0, 1, 2 or 3; R2 represents H, C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl,
  wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy and -N(H)R6, -N(R6)R7;
R3 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or
R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2; R4 represents C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl;
R5 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or
R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, q is 0, 1, 2 or 3,
m is 0, 1, 2 or 3,
with the proviso that q + m is 2, 3 or 4 ;
Figure imgf000025_0002
represents a group which is selected from :
Figure imgf000025_0001
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I) ,
said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, and C1-C3-haloalkoxy- ;
R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl, or -C(=O)-(C1-C3- alkyl) ; R8 represents, independently of each other hydrogen, C1-C3-alkyl, C3-C6-cycloalkyl or - C(=O)-(C1-C3-alkyl) ; or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer.  
In a third aspect, the invention relates to a conjugate as described supra,
wherein:
A represents:
Figure imgf000026_0001
,
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2;
t is 0, 1, 2 or 3; R2 represents H, C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy and -N(H)R6, -N(R6)R7 ; and R3 represents H or C1-C3-alkyl; or
R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8; R4 represents C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl;
R5 represents H or C1-C3-alkyl; or
R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8; q is 0, 1, 2 or 3,
m is 0, 1, 2 or 3,
with the proviso that q + m is 2, 3 or 4 ;
 
Figure imgf000027_0002
represents a group which is selected from :
Figure imgf000027_0003
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I);
R6, R7 represent, independently of each other C1-alkyl or C3-cycloalkyl; R8 represents, independently of each other hydrogen, C1-C3-alkyl or C3-C6-cycloalkyl; or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer. In a fourth aspect, the invention relates to a conjugate as described supra,
wherein:
A represents:
Figure imgf000027_0001
,
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy or -NH2, -N(H)R6;
t is 0, 1 or 2; R2 represents H, C1-C4-alkyl, C3-C6-cycloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy and C1-C3-haloalkoxy;
R3 represents H or C1-C3-alkyl; or
R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8;
  R4 represents C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl;
R5 represents H or C1-C3-alkyl; or
R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8;
epresents a group selected from : (
Figure imgf000028_0001
,
Figure imgf000028_0002
, in which * represents the point of attachment of said group with the rest of the compound of formula (I) ;
Figure imgf000028_0004
represents a group which is selected from :
Figure imgf000028_0003
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I),
R6 represents, independently of each other, C1-alkyl or C3-cycloalkyl;
R8 represents, independently of each other, hydrogen, or C1-C3-alkyl; or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer. In a fifth aspect, the invention relates to a conjugate as described supra,
wherein:
A represents:  
Figure imgf000029_0004
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, F, Cl, Br, hydroxy, methyl, CF3, methoxy, - NH2, or -N(H)(CH3);
t is 0, 1 or 2, preferably 0; R2 represents H, methyl, propan-2-yl or phenyl,
R3 represents H, or methyl; R4 represents methyl;
R5 represents H or methyl;
Figure imgf000029_0001
represents a group selected from :
Figure imgf000029_0002
pre era y , 
in which * represents the point of attachment of said group with the rest of the compound of formula (I);
Figure imgf000029_0003
represents:
 
Figure imgf000030_0002
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I), or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer. According to another aspect, the invention relates to a conjugate as described supra, wherein: A represents:
Figure imgf000030_0001
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2;
t is 0 or 1; R2 represents H, C1-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, and -N(R6)R7 ; R3 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8);  
R4 represents C1-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl;
R5 represents H, C1-alkyl or C1-C3-haloalkyl; or R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8), q is 1,
m is 1;
Figure imgf000031_0001
represen s
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I),
said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, R6(H)N- and -N(R6)R7;
R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl) ; R8, R9 represent, independently of each other, hydrogen, C1-C3-alkyl, C3-C6-cycloalkyl or - C(=O)-(C1-C3-alkyl) ; or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer.
According to another aspect, the invention relates to a conjugate as described supra, wherein:
A represents:
Figure imgf000031_0002
,   wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2;
t is 0; R2 represents H, C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy and -N(H)R6, -N(R6)R7;
R3 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or
R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2; R4 represents C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl;
R5 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or
R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, q is 1,
m is 1;
Figure imgf000032_0001
represents
Figure imgf000032_0002
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I) ,
said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, and C1-C3-haloalkoxy- ;
R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl, or -C(=O)-(C1-C3- alkyl) ;
 
R8 represents, independently of each other hydrogen, C1-C3-alkyl, C3-C6-cycloalkyl or - C(=O)-(C1-C3-alkyl) ; or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer. According to another aspect, the invention relates to a conjugate as described supra, wherein:
A represents:
Figure imgf000033_0001
,
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2;
t is 0; R2 represents H, C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy and -N(H)R6, -N(R6)R7 ; and R3 represents H or C1-C3-alkyl; or
R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8; R4 represents C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl;
R5 represents H or C1-C3-alkyl; or
R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8; q is 1,   m is 1;
represents
Figure imgf000034_0002
Figure imgf000034_0003
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I) ;
R6, R7 represent, independently of each other C1-alkyl or C3-cycloalkyl;
R8 represents, independently of each other hydrogen, C1-C3-alkyl or C3-C6-cycloalkyl; or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer. According to another aspect, the invention relates to a conjugate as described supra, wherein:
A represents:
Figure imgf000034_0001
,
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy or -NH2, -N(H)R6;
t is 0; R2 represents H, C1-C4-alkyl, C3-C6-cycloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy and C1-C3-haloalkoxy;
R3 represents H or C1-C3-alkyl; or
R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8; R4 represents C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl;   R5 represents H or C1-C3-alkyl; or
R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8;
represents
Figure imgf000035_0001
Figure imgf000035_0002
in which * represents the point of attachment of said group with the rest of the compound of formula (I) ;
represents
Figure imgf000035_0003
Figure imgf000035_0004
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I),
R6 represents, independently of each other, C1-alkyl or C3-cycloalkyl;
R8 represents, independently of each other, hydrogen, or C1-C3-alkyl;
or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer. According to another aspect, the invention relates to a conjugate as described supra, wherein:
A represents:
Figure imgf000035_0005
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, F, Cl, Br, hydroxy, methyl, CF3, methoxy, - NH2, or -N(H)(CH3);
t is 0;  
R2 represents H, methyl, propan-2-yl or phenyl,
R3 represents H, or methyl; R4 represents methyl;
R5 represents H or methyl;
Figure imgf000036_0001
represents a group ,
in which * represents the point of attachment of said group with the rest of the compound of formula (I);
represents:
Figure imgf000036_0002
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I),
or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: A represents:
Figure imgf000036_0003
,
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’;   R1 represents, independently of each other, halogen, C1-C3-alkoxy, or -NH2;
t is 0, or 1, preferably 0; R2 represents C1-C6-alkyl, or phenyl,
R3 represents H, R4 represents C1-C6-alkyl;
R5 represents H, or C1-C3-alkyl; q is 1,
m is 1;
Figure imgf000037_0001
represents
Figure imgf000037_0002
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I);
or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: A represents:
Figure imgf000037_0003
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; wherein R2 and R3 are as defined supra. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra,  
wherein: A represents:
d
Figure imgf000038_0001
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; wherein R4 and R5 are as defined supra. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2.
wherein R6 and R7 are as defined herein for the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy or -NH2, -N(H)R6,
wherein R6 is as defined herein for the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R1 represents, independently of each other, F, Cl, Br, hydroxy, methyl, CF3, methoxy, - NH2, or -N(H)(CH3). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R1 represents, independently of each other, halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3- haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2,
wherein R6 and R7 are as defined herein for the compound of formula (I).  
In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R1 represents halogen, C1-C3-alkoxy or -NH2. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R1 represents F, Br, methoxy or -NH2. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R1 represents F, Cl, Br, Me, or NH2. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R1 represents F, Cl, Me, or NH2, and
t is 0 or 1. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: t is 0, 1, 2 or 3. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: t is 0, 1 or 2. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: t is 0, or 1. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: t is 0.  
In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: t is 1. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: t is 2. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R2 represents H, methyl, propan-2-yl or phenyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R2 represents H, C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy and -N(H)R6, -N(R6)R7, wherein R6 and R7 are as defined herein for the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R2 represents H, C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy and -N(H)R6, -N(R6)R7 , wherein R6 and R7 are as defined herein for the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R2 represents H, C1-C4-alkyl, C3-C6-cycloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
 
halogen, C1-C3-alkyl, C1-C3-alkoxy and C1-C3-haloalkoxy. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R2 represents methyl, propan-2-yl or phenyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R3 represents H, or methyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R3 represents H, C1-C3-alkyl or C1-C3-haloalkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R3 represents H or C1-C3-alkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R3 represents H. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8); wherein R8 and R9 are as defined herein for the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:
 
R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, wherein R8 is as defined herein for the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8; wherein R8 is as defined herein for the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R4 represents C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R4 represents C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R4 represents methyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R5 represents H, C1-C3-alkyl or C1-C3-haloalkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R5 represents H or C1-C3-alkyl.
 
In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R5 represents H or methyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8), wherein R8 and R9 are as defined herein for the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, wherein R8 is as defined herein for the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8; wherein R8 is as defined herein for the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: q is 0, 1, 2 or 3,
m is 0, 1, 2 or 3,
with the proviso that q + m is 2, 3 or 4. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:
 
q is 1 or 2,
m is 1 or 2,
with the proviso that q + m is 2 or 3. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:
represents a group selected from :
Figure imgf000044_0001
,
Figure imgf000044_0002
in which * represents the point of attachment of said group with the rest of the compound of formula (I), wherein R1 and t are as defined herein for the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:
epresents a group:
Figure imgf000044_0003
, in which * represents the point of attachment of said group with the rest of the compound of formula (I), wherein R1 and t are as defined herein for the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:
  represents a group:
,
Figure imgf000045_0001
in which * represents the point of attachment of said group with the rest of the compound of formula (I), wherein R1 is as defined herein for the compound of formula (I) and t is 0 or 1. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:
Figure imgf000045_0005
represents a group .  In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:
Figure imgf000045_0004
) represents a group which is selected from :
Figure imgf000045_0003
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:
represents a group which is selected from :
Figure imgf000045_0002
 
Figure imgf000046_0002
in which * and represent the points of attachment of said group with the rest of the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:
Figure imgf000046_0001
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R6 represents, independently of each other, C1-alkyl or C3-cycloalkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R6, R7 represent, independently of each other C1-alkyl or C3-cycloalkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:
 
R6 represents, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:
R7 represents, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1- C3-alkyl). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R6 represents, independently of each other, C1-alkyl or C3-cycloalkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R7 represents, independently of each other, C1-alkyl or C3-cycloalkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R8, R9 represent, independently of each other, hydrogen, C1-C3-alkyl, C3-C6-cycloalkyl or - C(=O)-(C1-C3-alkyl). In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R8 represents, independently of each other hydrogen, C1-C3-alkyl or C3-C6-cycloalkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein: R8 represents, independently of each other, hydrogen, or C1-C3-alkyl. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein:  
R8 represents, independently of each other, hydrogen, or methyl. A further aspect of the invention are conjugates as described supra, which are present as their salts. Yet another aspect of the invention are conjugates as described supra in which the salt is a pharmaceutically acceptable salt. It is to be understood that the present invention relates to any sub-combination within any embodiment or aspect of the present invention of conjugates as described supra. More particularly still, the present invention covers conjugates that are disclosed in the Example section of this text, infra. In accordance with another aspect, the present invention covers methods of preparing conjugates of the present invention, said methods comprising the steps as described in the Experimental Section herein. Linkers
The literature discloses various options for covalently coupling (conjugating) organic molecules to binders such as, for example antibodies (see, for example, K. Lang and J. W. Chin. Chem. Rev. 2014, 114, 4764-4806, M. Rashidian et al. Bioconjugate Chem. 2013, 24, 1277-1294). Preference according to the invention is given to conjugation of the NAMPT inhibitors to an antibody via one or more sulphur atoms of cysteine residues of the antibody which are either already present as free thiols or generated by reduction of disulphide bridges, and/or via one or more NH groups of lysine residues of the antibody. However, it is also possible to attach the NAMPT inhibitor to the antibody via tyrosine residues, via glutamine residues, via residues of unnatural amino acids, via free carboxyl groups or via sugar residues of the antibody. For coupling, use is made of linkers. Linkers can be categorized into the group of the linkers which can be cleaved in vivo and the group of the linkers which are stable in vivo (see L. Ducry and B. Stump, Bioconjugate Chem.21, 5-13 (2010)). The linkers which can be cleaved in vivo have a group which can be cleaved in vivo, where, in turn, a distinction may be made between groups which are chemically cleavable in vivo and groups which are enzymatically cleavable in vivo. "Chemically cleavable in vivo" and "enzymatically cleavable in vivo" means that the linkers or groups are cleaved at or in the target cell by the chemically or enzymatically different environment therein (e.g. lower pH; elevated glutathione concentration; presence of lysosomal  
enzymes such as cathepsin or plasmin, or glyosidases such as, for example, ß- glucuronidases), thus releasing the low-molecular weight NAMPT inhibitor or a derivative thereof. Groups which can be cleaved chemically in vivo are in particular disulphide, hydrazone, acetal and aminal groups; groups which can be cleaved enzymatically in vivo are in particular the 2-8-oligopeptide group, especially a dipeptide group, a tripeptide group or a glycoside group. Peptide cleavage sites are disclosed in Bioconjugate Chem. 2002, 13, 855- 869, and Bioorganic & Medicinal Chemistry Letters 8 (1998) 3341-3346 and also Bioconjugate Chem. 1998, 9, 618-626. These include, for example, valine-alanine, valine-lysine, valine- citrulline, alanine-lysine and phenylalanine-lysine (optionally with additional amide group). Linkers which are stable in vivo are distinguished by a high stability (preferably less than 5% metabolites after 24 hours in plasma) and do not have the chemically or enzymatically in vivo cleavable groups mentioned herein. In accordance with a sixth aspect, the invention relates to a conjugate as described supra, wherein the linker–Z’- represents one of the following general structures (i) to (iii):
(i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ wherein § represents the attachment point to D; §§ represents the attachment point to AB; SG represents an in vivo cleavable group, L1 and L1’ represent, independently of each other, an in vivo non-cleavable organic group, and L2 represents an attachment group. Attachment group L2 represents a coupling group to the binder or a single bond. Here, coupling is preferably to a cysteine residue or a lysine residue of the binder. Alternatively, coupling can be to a tyrosine residue, glutamine residue or to an unnatural amino acid of the binder. The unnatural amino acids may contain, for example, aldehyde or keto groups (such as, for example, formylglycine) or azide or alkyne groups (see Lan & Chin, Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins, Chem.Rev. 2014, 114, 4764- 4806). In accordance with a seventh aspect, the invention relates to a conjugate as described supra, wherein the in vivo cleavable group SG represents a 2-8 oligopeptide group, preferably a
  dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal. In accordance with a eighth aspect, the invention relates to a conjugate as described supra, wherein L1 and L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -S-, -SO-, SO2, -NH-, -CO-, -NMe-, -NHNH-, -SO2NHNH-, -NHCO-, -CONH-, - CONHNH-, arylene groups, heteroarylene groups, straight C1-C6-alkylene groups, branched C1-C6-alkylene groups, C3-C7-cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the group consisting of
N, O, S, -SO- and–SO2- (preferably
Figure imgf000050_0002
in which * and # represent the points of attachment of said group with the rest of the compound,
optionally substituted with one or more substituents independently selected from the group consisting of halogen, -NHCONH2, -COOH, -OH, -NH2, NH-CNNH2, sulphonamide, sulphone, sulphoxide and sulphonic acid. When the linker is attached to a cysteine side chain or a cysteine residue, L2 is preferably derived from a group which reacts with the sulphhydryl group of the cysteine. These include haloacetyls, maleimides, aziridines, acryloyls, arylating compounds, vinylsulphones, pyridyl disulphides, TNB thiols and disulphide-reducing agents. These groups generally react in an electrophilic manner with the sulphhydryl bond, forming a sulphide (e.g. thioether) or disulphide bridge. In accordance with a ninth aspect, the invention relates to a conjugate as described supra, wherein L2 represents:
Figure imgf000050_0001
 
Figure imgf000051_0001
wherein #1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG.
In accordance with a tenth aspect, the invention relates to a conjugate as described supra, wherein the linker–Z’- represents one of the following general structures (i) to (iii): (i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ wherein § represents the attachment point to D; §§ represents the attachment point to AB; SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal; L1, L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than onceby one or more groups independently selected from: -O-, -S-, -SO-, SO2, -NH-, -CO-, -NMe-, -NHNH-, -SO2NHNH-, -NHCO-, - CONH-, -CONHNH-, arylene groups, heteroarylene groups, straight C1-C6-   alkylene groups, branched C1-C6-alkylene groups, C3-C7-cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the group consisting of N, O, S, -SO- and–SO2- (preferably
Figure imgf000052_0001
in which * and # represent the points of attachment of said group with the rest of the compound, optionally substituted with one or more substituents independently selected from the group consisting of halogen, -NHCONH2, -COOH, -OH, -NH2, NH-CNNH2, sulphonamide, sulphone, sulphoxide and sulphonic acid; L2 represents:
Figure imgf000052_0002
wherein #1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG.
In accordance with an eleventh aspect, the invention relates to a conjugate as described supra, wherein L2 represents one or more of the following three formulae:
 
Figure imgf000053_0001
wherein #1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG, wherein in a preferred embodiment over 60% of the attachment points to the binder, even more preferred over 80% of the attachment points to the binder, preferably over 90% of the attachment points to the binder, preferably over 95% of the attachment points to the binder in respect to the total number of attachments of the linker to the binder, are represented by one of the two structures:
Figure imgf000053_0002
wherein, in a particularly preferred embodiment, the amide group at #2 is connected to L1, L1’ or SG via the group–CH2-C(O)-.
In accordance with a twelfth aspect, the invention relates to a conjugate as described supra, wherein SG is a 2-8 oligopeptide.
In accordance with a thirteenth aspect, the invention relates to a conjugate as described supra, wherein the 2-8 oligopeptide consists of amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine.
In accordance with a fourteenth aspect, the invention relates to a conjugate as described supra, wherein L1 and L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from:   -O-, -NH-, -CO-, -NHCO-, -CONH-, phenyl and
Figure imgf000054_0001
in which * and # represent the points of attachment of said group with the rest of the compound, optionally substituted with one or more substituents independently selected from the group consisting of -F, -Cl, -COOH, -OH, and -NH2.
In accordance with a fifteenth aspect, the invention relates to a conjugate as described supra, wherein L1 and L1’ represent, independently of each other, one of the general structures (iv) or (v): (iv) –A’-(NR10CO)-B’- (v) –A’-(CONR10)-B’- wherein: A’ represents C1-C6-alkyl, (C1-C2-alkyl)-(phenylene), and (C1-C3-alkyl)-(NR11)-(C2-alkyl); optionally substituted with one or more substituents independently selected from–F and -Cl; B’ represents a straight-chain or branched hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from -O-, -NH-, -CO-, -NHCO-, and -CONH-; optionally substituted with–COOH; R10, R11 represent, independently of each other, hydrogen or C1-C3 alkyl; or R10, R11 together with the nitrogens to which they are attached form a 6-membered nitrogen containing heterocycloalkyl group.
In accordance with a sixteenth aspect, the invention relates to a conjugate of general formula (II)
 
Figure imgf000055_0001
wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8; wherein: A represents:
Figure imgf000055_0002
wherein * represents the point of attachment to the rest of the compound and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2;
t is 0, 1, 2 or 3; R2 represents H, C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, and -N(R6)R7 ; R3 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8); R4 represents C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl;   R5 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8), q is 0, 1, 2 or 3,
m is 0, 1, 2 or 3,
with the proviso that q + m is 2, 3 or 4 ;
Figure imgf000056_0001
) represents a group which is selected from :
Figure imgf000056_0002
in which * and # represent the points of attachment of said group with the rest of the compound, said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, R6(H)N- and -N(R6)R7;
R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl); R8, R9 represent, independently of each other, hydrogen, C1-C3-alkyl, C3-C6-cycloalkyl or - C(=O)-(C1-C3-alkyl); -Z’- represents one of the following general structures (i) to (iii): (i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ wherein § represents the attachment point to A; §§ represents the attachment point to AB; SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal;
  L1, L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -S-, -SO-, SO2, -NH-, -CO-, -NMe-, -NHNH-, -SO2NHNH-, -NHCO-, -CONH-, - CONHNH-, arylene groups, heteroarylene groups, straight C1-C6-alkylene groups, branched C1-C6-alkylene groups, C3-C7-cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the group consisting of
N, O and S, -SO- and–SO2- (preferably
Figure imgf000057_0002
in which * and # represent the points of attachment of said group with the rest of the compound, optionally substituted with one or more substituents independently selected from the group consisting of halogen, -NHCONH2, -COOH, -OH, -NH2, NH-CNNH2, sulphonamide, sulphone, sulphoxide and sulphonic acid; L2 represents:
Figure imgf000057_0001
wherein #1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG;   or the enantiomers, diastereomers, salts, solvates or salts of solvates thereof. According to another aspect, the invention relates to a conjugate as described supra, wherein
Figure imgf000058_0001
in which * represents the point of attachment of said group with the rest of the compound;
represents unsubstituted
Figure imgf000058_0003
Figure imgf000058_0002
in which * and # represent the points of attachment of said group with the rest of the compound, t is 0, q is 1, and m is 1. According to another aspect, the invention relates to a conjugate as described supra, wherein the linker–Z’- represents one of the following general structures (i) to (iii):
(i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ wherein § represents the attachment point to D; §§ represents the attachment point to AB; SG represents an in vivo cleavable group, L1 and L1’ represent, independently of each other, an in vivo non-cleavable organic group, and L2 represents an attachment group.
 
According to another aspect, the invention relates to a conjugate as described supra, wherein the in vivo cleavable group SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal. According to another aspect, the invention relates to a conjugate as described supra, wherein L1 and L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -S-, -SO-, SO2, -NH-, -CO-, -NMe-, -NHNH-, -SO2NHNH-, -NHCO-, -CONH-, - CONHNH-, arylene groups, heteroarylene groups, straight C1-C6-alkylene groups, branched C1-C6-alkylene groups, C3-C7-cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the group consisting of
N, O and S, -SO- or–SO2- (preferably
Figure imgf000059_0001
in which * and # represent the points of attachment of said group with the rest of the compound, optionally substituted with one or more substituents selected from the group consisting of halogen, -NHCONH2, -COOH, -OH, -NH2, NH-CNNH2, sulphonamide, sulphone, sulphoxide and sulphonic acid. According to another aspect, the invention relates to a conjugate as described supra, wherein L2 represents:
,
Figure imgf000059_0002
 
wherein #1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG. According to another aspect, the invention relates to a conjugate as described supra, wherein the linker–Z’- represents one of the following general structures (i) to (iii): (i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ wherein § represents the attachment point to D; §§ represents the attachment point to AB; SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal; L1, L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -S-, -SO-, SO2, -NH-, -CO-, -NMe-, -NHNH-, -SO2NHNH-, -NHCO-, -CONH-, - CONHNH-, arylene groups, heteroarylene groups, straight C1-C6-alkylene groups, branched C1-C6-alkylene groups, C3-C7-cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the group consisting of
N, O and S, -SO- or–SO2- (preferably
Figure imgf000060_0001
in which * and # represent the points of attachment of said group with the rest of the compound, optionally substituted with one or more substituents independently selected from the group consisting of halogen, -NHCONH2, -COOH, -OH, -NH2, NH-CNNH2, sulphonamide, sulphone, sulphoxide and sulphonic acid;  
L2 represents:
,
Figure imgf000061_0001
wherein #1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG.
According to another aspect, the invention relates to a conjugate as described supra, wherein L2 represents one or more of the following three formulae:
Figure imgf000061_0002
wherein #1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG, wherein in a preferred embodiment over 60% of the attachment points to the binder, even more preferred over 80% of the attachment points to the binder, preferably over 90% of the attachment points to the binder, preferably over 95% of the attachment points to the binder in
 
respect to the total number of attachments of the linker to the binder, are represented by one of the two structures:
Figure imgf000062_0001
, wherein, in a particularly preferred embodiment, the amide group at #2 is connected to L1, L1’ or SG via the group–CH2-C(O)-. According to another aspect, the invention relates to a conjugate as described supra, wherein SG is a 2-8 oligopeptide. According to another aspect, the invention relates to a conjugate as described supra, wherein the 2-8 oligopeptide consists of amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine. According to another aspect, the invention relates to a conjugate as described supra, wherein L1 and L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -NH-, -CO-, -NHCO-, -CONH-, phenyl and
Figure imgf000062_0002
_ ; optionally substituted with one or more substituents independently selected from the group consisting of -F, -Cl, -COOH, -OH, and -NH2. According to another aspect, the invention relates to a conjugate as described supra, wherein L1 and L1’ represent, independently of each other, one of the general structures (iv) or (v): (iv) –A’-(NR10CO)-B’- (v) –A’-(CONR10)-B’- wherein: A’ represents C1-C6-alkyl, (C1-C2-alkyl)-(phenylene), and (C1-C3-alkyl)-(NR11)-(C2-alkyl); optionally substituted with one or more substituents independently selected from–F and -Cl;
 
B’ represents a straight-chain or branched hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from :-O-, -NH-, -CO-, -NHCO-, and -CONH-; optionally substituted with–COOH; R10, R11 represent, independently of each other hydrogen or C1-C3-alkyl; or R10, R11 together with the nitrogens to which they are attached form a 6-membered nitrogen containing heterocycloalkyl group. According to another aspect, the invention relates to a conjugate of general formula (II)
Figure imgf000063_0001
wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8; wherein: A represents:
Figure imgf000063_0002
wherein * represents the point of attachment to the rest of the compound and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2;
t is 0, 1, 2 or 3; R2 represents H, C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl,   wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, and -N(R6)R7 ; R3 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8); R4 represents C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl;
R5 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8), q is 0, 1, 2 or 3,
m is 0, 1, 2 or 3,
with the proviso that q + m is 2, 3 or 4 ;
Figure imgf000064_0001
represents a group which is selected from :
Figure imgf000064_0002
in which * and # represent the points of attachment of said group with the rest of the compound, said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, R6(H)N- and -N(R6)R7;
R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl) ; R8, R9 represent, independently of each other, hydrogen, C1-C3-alkyl, C3-C6-cycloalkyl or - C(=O)-(C1-C3-alkyl); -Z’- represents one of the following general structures (i) to (iii):   (i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ wherein § represents the attachment point to A; §§ represents the attachment point to AB; SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal; L1, L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -S-, -SO-, SO2, -NH-, -CO-, -NMe-, - NHNH-, -SO2NHNH-, -NHCO-, -CONH-, -CONHNH-, arylene groups, heteroarylene groups, straight C1-C6-alkylene groups, branched C1-C6-alkylene groups, C3-C7-cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the group consisting of N, O and S, -SO- or–SO2-
(preferably
Figure imgf000065_0002
optionally substituted with one or more substituents independently selected from the group consisting of halogen, -NHCONH2, -COOH, -OH, -NH2, NH-CNNH2, sulphonamide, sulphone, sulphoxide and sulphonic acid; L2 represents:
Figure imgf000065_0001
 
Figure imgf000066_0001
wherein #1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG; or the enantiomers, diastereomers, salts, solvates or salts of solvates thereof.
According to another aspect, the invention relates to a conjugate as described supra, wherein
Figure imgf000066_0002
in which * represents the point of attachment of said group with the rest of the compound;
represents unsubstituted
Figure imgf000066_0004
Figure imgf000066_0003
in which * and # represent the points of attachment of said group with the rest of the compound, t is 0, q is 1, and m is 1.
 
In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein L1 and L1’, independently of each other, are those below, where r in each case independently of one another represents a number from 1 to 20, preferably from 1 to 15, particularly preferably from 2 to 20, especially preferably from 2 to 10. It is understood that the groups L1 and L1’ below are read from left to right, meaning that the left-hand symbol (or dashed line) in the Table A below denotes the linkage site to §- or §-L1-SG- and the right-hand symbol (or dashed line) in the Table A below denotes the linkage site to -SG-L2-§§, -SG-L1’-L2-§§, or -L2-§§. Table A
Figure imgf000067_0001
 
•
Figure imgf000068_0001
 
Figure imgf000069_0001
 
'
Figure imgf000070_0001
 
, " *
Figure imgf000071_0001
 
Figure imgf000072_0001
Further Examples of a linker moiety L1 and L1’, independently of each other, are given in the Table B below. It is understood that the groups L1 or L1’ below are read from left to right, meaning that the left-hand symbol in the Table B below denotes the linkage site to §- or §- L1-SG- and the right-hand symbol in the Table B below denotes the linkage site to -SG-L2- §§, -SG-L1’-L2-§§, or -L2-§§. Table B
 
Figure imgf000073_0001
 
*
Figure imgf000074_0001
 
Figure imgf000075_0001
 
Figure imgf000076_0001
 
Figure imgf000077_0001
 
(
) ) )
Figure imgf000078_0001
In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra,  
wherein SG comprises 2-6 amino acids selected from the group comprising: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein SG comprises 2-3 amino acids selected from the group comprising: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein SG comprises 2-3 amino acids selected from the group comprising: alanine, glycine, histidine, isoleucine, leucine, methionine, serine, citrulline and valine. In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein SG comprises 2 amino acids selected from the group comprising: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine.
In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein SG comprises 2 amino acids selected from the group comprising: alanine, glycine, histidine, isoleucine, leucine, methionine, serine, citrulline and valine.
  The table C below provides examples showing preferred ’ (i) §–L1-SG-L2-§§ (iii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ where r in each case independently of one another repr bly from 1 to 10, particularly preferably from 1 to 8. It is understood that the groups L1 and L1’ below are rea eft-hand symbol in the Table C below denotes the linkage site to §- or §-L1-SG-, respectively, and the righ w denotes the linkage site to -SG-L1’-L2-§§, -SG-L1’- L2-§§ or -L2-§§. It is understood that SG and L1’ are op TABLE C L1 SG L1’
‐S‐S‐   I o o
0 o
—It CH
. . , #1
(disulfide)  HO #
HO 1
- % , 2 N 1 ,
/t 2
. - rN .‘ il i*id
o
• o
H - - r 0 or
' 0
. 2)(.
1
#  
 
Figure imgf000080_0001
F     0 o o
NH O #
,,2 HO HO
-- - - ”- , 2 N 1 ,
- 0 # 1-1 #2
0 1 N -14
0 0 or 0
2().
# 1
#  
0
0 0
#1 H # 1 „2 HO O
- #-N , 2 N ,
# 1-1 # 1 2 N
#i-i
0 0 0 or 0
2().
# 1
#  
0
0 0
#1 # 1 „2 HO H O
0 #-N , 2 N ,
- #1-1 # 2 N
#i-i
/ 0 0 0 or
0
) 2 .(.
# 1
#  
0
0 0 „2 # HO #
H0 /1 I "-- , 2 N ,
# 1-1 # 2 N
# 1-1
0 0 0 or 0
) 2 .(.
# 1
#  
 
Figure imgf000081_0001
0 .     O O O
H •
• N 0 # HO
. N ••• .,.,2 HO #
r H r '. tr- , 2 N 1 ,
# H 2 N
i* H
0
O O or 0
2().
# 1
#  
O 1 O 0
tti „ O
,,2 # H
.— HO
, ,
ic 2 Eid # 2
#H
0 0 o or 0
2().
# 1
#  
o 0 o
#1 # 1 „2 HO HO
. ,,.— , ,
ic 2 Ei N d # 2 N
#H
O #1 or O
 
0
.
} :#:2(##1 HO
fr— HO O
, 2 N ,
tA H # 2 N
i* H
0
O O or 0
) 2 .(.
# 1
#
 
Figure imgf000082_0001
o
H 0 O     o o
N Ho #
2 # HO
_4_ [ _/ ] 0 H #— , 2 N 1 ,
# 1-1 2 N
# 1-1 o o o or o
) #2 (, 1
#  
0 o
o tti 2 #1 HO
#— HO
, 2 ,
igH # 2 N
#1-1
0 0 0 or ) 0
2().
# 1
#  
o 0 o
# #1 . HO HO
#— , 2 N ,
tA 1-1 # 2 N
#1-1 o o 0
0
2
# 1
 
0
. 0 0
2 # Ho #
HO
#- , 2 1 ,
ttl-I 2
#1-1
0 0 0
0
2
1
 
 
Figure imgf000083_0001
 
Figure imgf000084_0001
      wherein for the group L2
#1 represents the attachment point to the binder,
#2 represents the attachment point to the group L1, L1’ or SG.
 
 
The table D below provides examples showing more preferred combinations of linker Z’: (i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§
where r in each case independently of one another represents a number from 1 to 15, preferably from 1 to 10, particularly preferably from 1 to 8. It is understood that the groups L1 and L1’ below are read from left to right, meaning that the left-hand symbol in the Table D below denotes the linkage site to §- or §-L1-SG-, respectively, and the right-hand symbol in the Table D below denotes the linkage site to -SG-L2-§§, -SG-L1’-L2-§§ or -L2-§§. TABLE D
 
Ex L2 
pl
29 0 0 0
HO
42 # H)O L-41
, 2,N or 2
#•*" H #
# 1-)1 r 0 0
0    30 0
0 0
HO,•,.#1 #1
#2 HO
, 2 or
#1-1 # 2„N
#""H 0 0 0   (In .
: 0 . 0 0
HO,•,.#1 m #2 #1 HO
, 2 N or
#1-1 # 2„
#""H te   0 0 o   31 0
0 0
HO.,•,#i #
42 HO
, 2 N or
# 1-1 # 2,N
#--H 0 0 o  
 
Figure imgf000086_0001
32 0
0 0 #
42 H0
H0).#1 , 2,,N or
it'" H # 2
0 0 #-HNy 0   33 0 0
#
42 H0 °
H0 , or
#42 #1 2
#''' H 0 0 0 )   34 0 0 0
#2 #1 H0
H0 ,"" 1 , or
#2 1-i #1 2
#1-1 0 0 0   35 0
0 0
041 #
42 H0
H
, 2 or 2,.N
#1-1 #1 #- H 0 0
0    
Figure imgf000087_0001
36 0
0 0
.k......41 #
42 H0
H0 , 2 or 2
#•'...H # N
# 1-1)r 0 0 0   37 o  
38 o o o
,.,4ti #2 #1 H0
H0 , or
#2 1-i # 2 N
#"'"H o o o   39 o
o o 1
i # H0
H0)(-"*"41 , 2 or 2
#''...H #
# 1-1)r 0 0 0   40
I •
.. .  
 
Figure imgf000088_0001
41 o   42 0 0
HO.0....#1 .2 #1 H0
, 2 or
# 1-i # 2 N
#1-1 0 0
0   44 0
0 0
.,•,41 42 N # H0
HO
, 2 or
1*1-1 # 2 N
#••'.1H 0 0 0  
45 o
o o 1 HO,•,41 #
42 H0
, 2 N or
1*1-i # 2 N
4"..1H 0 0 0   46 0 0 0
°41 i #1 H0
HO
0 , or
#2 l # #2 [1 0 0 0  
 
Figure imgf000089_0001
47 0
0 0
)(,....,41 i # H0
H0 , 2 N or
W N #2
1*1-1 # N
1-1)( 0 o
o   48 0
0 0 #1
i H0 #1
H0 , 2 1 or 2 N #i-i
111-1 0 0
0   49 0
0 0 tti #
#2 H0
H0 '' , or
#il # 2
#1-1 0 0 0   50 0
0 0
....311 #1
  #2 H0
H0 , 2 or
# i-i # 2 N
# 1-1 0 0 0   51 0
0 0 #1
i N H0 H0)(•-.•"# 0 , 1 or
#2 Fl
o 0 #2-Hy
0   52 0 o 0
#1
0 #2 H0 H0 )L2t1
, 1 or
#2 Fl #42 1 "srK 0 0 ii
0  
 
Figure imgf000090_0001
53 0 o 0
)1_,,,,41 #
#2 H0
H0 , 2,N or
it H #
o 0 #24y0   54 o
i :
  55 0 0
# 1 0
#1 #2 H0 H0
, 2 N 1 or
1 * H 2
ItH 0 o 0   56 0 0 0
HO.,,41 #
#2 H0
, 2,N or
  2 N
#- H #
it H 0 0
0   57 0
0 0
)1_,,,,41 #
42 N H0
HO
, 2, or
  it H
0 0 # # 2 -H0y
  58 0
0 0
0.,41 #
#2 H0
H
, 2 N or ,N
# 1-1 #
#•- H 0 0 0  
 
Figure imgf000091_0001
59 0
0 0
#1 ) #
#2 H0
H0)(',• , 2,,N or
#0- H #
0 #2-Hy
0 0   60 0 o 0 #1
0 #2 # H0
H0 , # 2 -H or
# 2
# H 0 0 ''
0   61 0 ° o
#1 #1 #2 H0
H0)(',• , 2,,N 1 or
#- H
0 0 #2-NHy
0   62 0
0 0 #2 # H0
H0-''1 , or
#2 H # 2 N
#H 0 0 o   63 0
0 0
#1 #2 # H0
H0-•'' , or
W N # 2 H N
# 2_,N
#'' H 0 0 o   64 -- o 0 o „ - #1 s-- #2 # H0
H0 \- , #2 N or
  H # #2 N ''
'''H ° 0 0
 
 
Figure imgf000092_0001
65 0
0 0 #2 # H0
H0)('--41 , 2,N or
it•- H # 2
#H).r o o o   68 0 0 0
- 11
-- #2 #1 H0
H0 #1 \- , 2 or
  # 1-1 # 2
#iH 0 0 0   69 0
° 0 0
0#1 #1
/ N #2_ H0
H
H , 2 or
#1-1 # 2,N
#'' H 0 0 0   70
1 '
•
. .
:   71 0 0 0 1
#2 #1 H0 #
H0 , ,#2FI or
# ,2,N
# H 0 0 0  
 
Figure imgf000093_0001
72 O 0 O
#1 ) N 1 0 #
#2 H0
HOK" , 2 or
it•-"H # 2 N
#1H)r 0
O O     0 O O
#
#2 H0
H0 '" , 2 or
# 1-1 # 2 N
#
O 0 O   73 - 0 0
\-- #1 - #1 H0
- ID
\- #2 H 0
, 2 N or
  #1-i # #2 N
*"..H o o 0  
75 o
i :
 
) W 76 O O 0.O,.,#1
#1 H0
#2 H
, 2 or
#1-1 # 2..N
#*"H O 0 O  
 
Figure imgf000094_0001
77A      0
o o o H i # H0 #
H0)(',- 1 ,
N 0H 2,N or 2
it•-H #
i. #1H)r
0 0
'''/' 0 0 .   0
 
78A  0     0 O
0
H
o #1 H0
Nly/ #2
N., H0 #1 H , 2 or
O # 11 2
#1 4t 1H 0 0 O   80A      0
0 O 0 ... #2 #1 H0 H 0 #1
, 2 N or 2,N ) N # 11 #
#'' H H . 0 0 0  
 
81A  N1 H H     o 0 o
N,ir..„..-... #
42 H0
H0)(-,'#1 , 2_,N or 2
it•- H #
0 #1H)r O 0 0
O   - _
HN
0
_\,,..,
0 - 24
C H 3
82A  0     0 O
0
H
o # H
) N).r/ N /\.)‹ #2 0
H0)(',- #1 H , 2,N or 2
0 it•- H # N
#1H)r 0 0
0   wherein for the group L2 #1 represents the attachment point to the binder,
 
#2 represents the attachment point to the group L1, L1’ or SG.
 
In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein SG comprises valine and alanine.
In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein SG comprises valine and citrulline.
In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein SG comprises alanine-valine.
In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein SG comprises citrulline-alanine.
In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein SG comprises (C-terminus)-Ala-Val-(N-terminus) or (C-terminus)-Cit-Val-(N- terminus).
In a further embodiment of the above-mentioned aspects, the invention relates to a conjugate as described supra, wherein SG is (C-terminus)-Ala-Val-(N-terminus) or (C-terminus)-Cit-Val-(N-terminus).
 
According to another aspect, the invention relates to a conjugate of a binder or a derivative thereof with one or more molecules of an active component, wherein the active component is a NAMPT inhibitor, which is conjugated to the binder via a linker Z’.
According to a further aspect of the present invention, the conjugate has the formula: AB Z' D
n wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8, and D stands for a NAMPT inhibitor of Formula (IVa):
Figure imgf000098_0001
wherein # represents the point of attachment to linker Z’; in which: C1 represents a phenylene, heteroarylene (preferably a 6-membered heteroarylene), 5- to 7-membered heterocycloalkylene (preferably a 6-membered heterocycloalkylene) or C3-C6- cycloalkylene (preferably a C6-cycloalkylene) group, in which C3-C6-cycloalkylene is optionally partially unsaturated, said groups being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, R6(H)N- and -N(R6)R7; A1 represents a group selected from 5- to 7-membered heterocycloalkylene, 6 to 10-membered fused heterocycloalkylene and heteroarylene,
 
said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, oxo (C=O), C1-C6-alkyl, C1-C4-alkoxy, -NH2, R6(H)N-, - N(R6)R7, C3-C6-cycloalkyl and phenyl, in which C1-C6-alkyl and C1-C4-alkoxy groups are optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, -N(H)R6 and -N(R6)R7, in which phenyl and C3-C6-cycloalkyl groups are optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, -N(H)R6 and -N(R6)R7; q is 0, 1, 2 or 3 (preferably 1),
m is 0, 1, 2 or 3 (preferably 1),
with the proviso that q + m is 2, 3 or 4; R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl); wherein the active component is conjugated to the binder via a linker Z’ (Z’ and the binder are as defined in the aspects and/or embodiments defined herein).
According to a further aspect of the present invention, the conjugate has the formula: AB+ Z'- D] n wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8, and D stands for a NAMPT inhibitor of Formula (IVb):
 
[ ] q
V-W
[ ]m 4 7-A 1 -#
H Z=Y (IVb) wherein # represents the point of attachment to linker Z’; in which: A1 represents a group selected from 5- to 7-membered heterocycloalkylene, 6 to 10-membered fused heterocycloalkylene and heteroarylene, said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, oxo (C=O), C1-C6-alkyl, C1-C4-alkoxy, -NH2, R6(H)N-, - N(R6)R7, C3-C6-cycloalkyl and phenyl, in which C1-C6-alkyl and C1-C4-alkoxy groups are optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, -N(H)R6 and -N(R6)R7, in which phenyl and C3-C6-cycloalkyl groups are optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, -N(H)R6 and -N(R6)R7; q is 0, 1, 2 or 3 (preferably 1),
m is 0, 1, 2 or 3 (preferably 1),
with the proviso that q + m is 2, 3 or 4; V W
*
Figure imgf000100_0001
represents a group which is selected from:
Figure imgf000100_0002
in which * and # represent the points of attachment of said group with the rest of the compound,  
said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, R6(H)N- and -N(R6)R7,
(preferably unsubstituted
Figure imgf000101_0001
R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl); wherein the active component is conjugated to the binder via a linker Z’ (Z’ and the binder are as defined in the aspects and/or embodiments defined herein).
According to a further aspect of the present invention, the conjugate has the formula: AB Z' D
n wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8, and D stands for a NAMPT inhibitor of Formula (IVc):
Figure imgf000101_0002
wherein # represents the point of attachment to linker Z’; in which: A1 represents a group selected from 5- to 7-membered heterocycloalkylene, 6 to 10-membered fused heterocycloalkylene and heteroarylene, said group being optionally substituted with one or more substituents independently selected from the group consisting of:
 
halogen, hydroxy, cyano, oxo (C=O), C1-C6-alkyl, C1-C4-alkoxy, -NH2, R6(H)N-, - N(R6)R7, C3-C6-cycloalkyl and phenyl, in which C1-C6-alkyl and C1-C4-alkoxy groups are optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, -N(H)R6 and -N(R6)R7, R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl); wherein the active component is conjugated to the binder via a linker Z’ (Z’ and the binder are as defined in the aspects and/or embodiments defined herein).
In accordance with a further aspect, the invention relates to compounds selected from the group consisting of: tert-butyl {4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5- dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}carbamate
N-{4-[1-(4-aminobutyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide trifluoroacetate
tert-butyl N2-(tert-butoxycarbonyl)-N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-L- asparaginate
tert-butyl {3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5- dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}carbamate
N-{4-[1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(3-hydroxypropyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-(4-{1-[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide S-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-  
5,6-dihydropyridazin-1(4H)-yl]propyl} ethanethioate
tert-butyl N2-(tert-butoxycarbonyl)-N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-L- asparaginate
ethyl 4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]butanoate
4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]butanoic acid
tert-butyl {3-[(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}benzoyl)amino]propyl}carbamate tert-butyl {(28S)-35-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]-27,31-dioxo-2,5,8,11,14,17,20,23-octaoxa- 26,32-diazapentatriacontan-28-yl}carbamate
N5-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]propyl}-N1-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl-L- glutamamide
L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluorophenyl)-L-alaninamide trifluoroacetate
N-(tert-butoxycarbonyl)-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L-alaninamide L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L-alaninamide
N-{4-[4-methyl-1-(4-nitrobenzyl)-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(4-aminobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L- alaninamide
L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L-alaninamide
 
N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L- alaninamide
L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5- dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L-alaninamide trifluoroacetate methyl 4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}benzoate
4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]methyl}benzoic acid
N-{4-[1-{4-[(3-aminopropyl)carbamoyl]benzyl}-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(3-fluoro-4-nitrobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2- fluorophenyl)-L-alaninamide
L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluorophenyl)-L-alaninamide
N-(4-{1-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)butyl]-6-oxo-4-phenyl-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide N-{4-[5,5-dimethyl-1-(4-nitrobenzyl)-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(4-nitrobenzyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(4-aminobenzyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N,N'-(disulfanediylbis{(1-oxopropane-3,1-diyl)iminobutane-4,1-diyl[4-methyl-6-oxo-5,6- dihydropyridazine-1,3(4H)-diyl]benzene-4,1-diyl})bis(1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide)
N,N'-(disulfanediylbis{propane-3,1-diyl[4-methyl-6-oxo-5,6-dihydropyridazine-1,3(4H)- diyl]benzene-4,1-diyl})bis(1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide)
N-{4-[4-methyl-6-oxo-1-(3-sulfanylpropyl)-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide  
N-{4-[1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5-dimethyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-L-asparagine N-{4-[1-(3-aminopropyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide hydrochloride
N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]propyl}-L-asparagine hydrochloride
tert-butyl 4-{2-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}piperazine-1-carboxylate N-(4-{4-methyl-6-oxo-1-[2-(piperazin-1-yl)ethyl]-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
tert-butyl {4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}carbamate
tert-butyl {3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}carbamate
N-{4-[1-(2-hydroxyethyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide N-(4-{1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide trifluoroacetate N-{4-[1-(2-aminoethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide N-{4-[1-(2-hydroxyethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide N-(4-{1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-5,5-dimethyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
 
N-{4-[1-(2-aminoethyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide {1-[2-({2-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5-dimethyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}amino)-2-oxoethyl]-2,5-dioxopyrrolidin-3-yl}-L- cysteine trifluoroacetate {1-[2-({2-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}amino)-2-oxoethyl]-2,5-dioxopyrrolidin-3-yl}-L- cysteine trifluoroacetate S-{(3S)-1-[6-({3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}amino)-6-oxohexyl]-2,5-dioxopyrrolidin-3- yl}-L-cysteine
S-{(3S)-1-[6-(4-{2-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}piperazin-1-yl)- 6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine
N-(4-{4-methyl-6-oxo-1-[4-({3-[(3-sulfanylpropanoyl)amino]propyl}carbamoyl)benzyl]-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide S-{1-[6-({3-[(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}benzoyl)amino]propyl}amino)-6- oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine
N6-[5-({4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-5-oxopentanoyl]-L-lysine
N-{4-[4-methyl-6-oxo-1-{4-[(3-sulfanylpropanoyl)amino]butyl}-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(3-aminopropyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(4-amino-3-fluorobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Methyl N2-(tert-butoxycarbonyl)-N6-[5-({4-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-5- oxopentanoyl]-L-lysinate
N2-(tert-butoxycarbonyl)-N6-[5-({4-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-5- oxopentanoyl]-L-lysine
 
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]-L-valyl-rel- N-(4-{[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluorophenyl)-L-alaninamide
S-{1-[6-({4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L- cysteine
N-{4-[1-(4-aminobutyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
S-(1-{6-[(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluorophenyl)amino]-6-oxohexyl}-2,5- dioxopyrrolidin-3-yl)-L-cysteine
S-{1-[6-({4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-6-oxo-4- phenyl-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L- cysteine
N-{4-[1-(4-aminobutyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(4-aminobenzyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide,
N2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]-N-{4-[3-{4- [(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]butyl}-L-asparagine,S-{(3R)-1-[6-({6-[(4S)-3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]hexyl}amino)-6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine,
S-{(3R)-1-[6-({5-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carbonyl)amino]phenyl}- 4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]pentyl}amino)-6-oxohexyl]-2,5-dioxopyrrolidin- 3-yl}-L-cysteine,
N-{4-[1-(5-aminopentyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide, and
N-{4-[1-(6-aminohexyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide,
or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N- oxide, tautomer or stereoisomer.
 
In a further aspect of the present invention, one or more of these compounds may be used as an intermediate to provide a conjugate as described supra. In another aspect of the present invention, one or more of these compounds may be a metabolite obtainable by the cleavage of a conjugate as described supra. In accordance with a seventeenth aspect, the invention relates to a conjugate as described supra, which is selected from the group consisting of:
Figure imgf000108_0001
 
Figure imgf000109_0001
 
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
( s
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
N N O
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
wherein n is a number from 1 to 50, and the antibody is selected from an anti-HER2-antibody, an anti-CD123-antibody, an anti-B7H3-antibody, an anti-C4.4a-antibody, or an antigen binding fragment thereof. NAMPT inhibitor - linker-intermediates and preparation of the conjugates The conjugates according to the invention are prepared by initially providing the low-molecular weight NAMPT inhibitor with a linker. The intermediate obtained in this manner is then reacted with the binder (preferably antibody). The NAMPT inhibitor-linker-intermediates are conjugates of general formula (III):
Figure imgf000135_0001
wherein Z’ stands for a linker; wherein: A represents:
Figure imgf000135_0002
wherein * represents the point of attachment to the rest of the compound and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2;
t is 0, 1, 2 or 3;   R2 represents H, C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, and -N(R6)R7 ; R3 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S , S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8); R4 represents C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl;
R5 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8), q is 0, 1, 2 or 3,
m is 0, 1, 2 or 3,
with the proviso that q + m is 2, 3 or 4 ;
Figure imgf000136_0001
represents a group which is selected from :
Figure imgf000136_0002
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I),
said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, R6(H)N- and -N(R6)R7;
R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl) ; R8, R9 represent, independently of each other, hydrogen, C1-C3-alkyl, C3-C6-cycloalkyl or - C(=O)-(C1-C3-alkyl);   -Z’- represents one of the following general structures (i) to (iii): (i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ wherein § represents the attachment point to A; §§ represents the attachment point to an antibody; SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal; L1, L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more of -O-, -S-, -SO-, SO2, -NH-, -CO-, -NMe-, -NHNH-, -SO2NHNH-, -NHCO-, - CONH-, -CONHNH-, arylene groups, heteroarylene groups, cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the
group consisting of N, O and S, -SO- or–SO2- (preferably
Figure imgf000137_0002
optionally substituted with one or more substituents selected from the group consisting of halogen, -NHCONH2, -COOH, -OH, -NH2, NH-CNNH2, sulphonamide, sulphone, sulphoxide or sulphonic acid; L2 represents:
Figure imgf000137_0001
 
Figure imgf000138_0001
wherein #1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG; or the enantiomers, diastereomers, salts, solvates or salts of solvates thereof.
Preferably, for coupling to a cysteine residue, one of the conjugates below is reacted with the cysteine-containing binder such as an antibody, which is optionally partially reduced for this purpose:
 
Figure imgf000139_0001
where R1, V, W, Z, Y, A, t, q and m have the same meaning as in formula (I), SG, L1 and L1’ have the same meaning as described above. The conjugate may be employed, for example, in the form of its trifluoroacetic acid salt. For the reaction with the binder such as, for example, the antibody, the conjugate is preferably used in a 2- to 20-fold molar excess, preferably in a 5- to 16-fold molar excess with respect to the binder. For an intermediate coupling to a cysteine residue, the reactions can be illustrated as follows:
 
Figure imgf000140_0001
where R1, V, W, Z, Y, A, Z’, t, q and m have the same meaning as described supra. Preferably PBS buffer is employed with DMSO, wherein the DMSO does not exceed 10% of the total volume. In accordance with the invention, this gives preferably conjugates of general formula (II):
Figure imgf000140_0002
AB represents an antibody attached via a cysteine or a lysine residue and n is a number from 1 to 50. With particular preference, AB is a human, humanized or chimeric monoclonal antibody or an antigen-binding fragment thereof, in particular an anti-HER2-antibody, an anti-CD123- antibody, an anti-B7H3-antibody, an anti-C4.4a-antibody, or an antigen binding fragment thereof. Examples of conjugates of general formula (II) are:
 
Figure imgf000141_0001
 
)
Figure imgf000142_0001
 
Figure imgf000143_0001
 
Figure imgf000144_0001
 
Figure imgf000145_0001
 
Figure imgf000146_0001
 
Figure imgf000147_0001
 
Figure imgf000148_0001
 
Figure imgf000149_0001
 
Figure imgf000150_0001
 
Figure imgf000151_0001
 
Figure imgf000152_0001
 
Figure imgf000153_0001
 
( s
Figure imgf000154_0001
 
Figure imgf000155_0001
Depending on the linker, succinimide-linked ADCs may, after conjugation, be converted into the open-chain succinamides, which have an advantageous stability profile.
Figure imgf000155_0002
The open-chain succinamides represented in Scheme A will be collectively represented as follows:
Figure imgf000155_0003
This reaction (ring opening) can be carried out at pH 7.5 to 9, preferably at pH 8, at a temperature of from 20°C to 37°C, for example by stirring. The preferred stirring time is 8 to 30 hours. In the above formulae, R1, V, W, Z, Y, A, Z’, t, q and m have the same meaning as described above. AB is an antibody coupled via a cysteine residue or a lysine residue. With particular preference, AB is an anti-HER2-antibody, an anti-CD123-antibody, an anti-B7H3-antibody, an anti-C4.4a-antibody, or an antigen binding fragment thereof.
Binders
In the broadest sense, the term "binder" is understood to mean a molecule which binds to a target molecule present at a certain target cell population to be addressed by the binder/active compound conjugate. The term binder is to be understood in its broadest meaning and also comprises, for example, lectins, proteins capable of binding to certain sugar chains, and phospholipid-binding proteins. Such binders include, for example, high-molecular weight proteins (binding proteins), polypeptides or peptides (binding peptides), non-peptidic (e.g. aptamers (US5,270,163), review by Keefe AD., et al., Nat. Rev. Drug) Discov. 2010; 9:537- 550), or vitamins) and all other cell-binding molecules or substances. Binding proteins are, for example, antibodies and antibody fragments or antibody mimetics such as, for example, affibodies, adnectins, anticalins, DARPins, avimers, nanobodies (review by Gebauer M. et al., Curr. Opinion in Chem. Biol. 2009; 13:245-255; Nuttall S.D. et al., Curr. Opinion in Pharmacology 2008; 8:608-617). Binding peptides are, for example, ligands of a ligand/receptor pair such as, for example, VEGF of the ligand/receptor pair VEGF/KDR, such as transferrin of the ligand/receptor pair transferrin/transferrin receptor or cytokine/cytokine receptor, such as TNFalpha of the ligand/receptor pair TNFalpha/TNFalpha receptor. The literature also discloses various options of covalent coupling (conjugation) of organic molecules to antibodies. Preference according to the invention is given to the conjugation of the toxophores to the antibody via one or more sulphur atoms of cysteine residues of the antibody and/or via one or more NH groups of lysine residues of the antibody. However, it is also possible to bind the toxophore to the antibody via free carboxyl groups or via sugar residues of the antibody. In one embodiment of the present invention, the linker Z’ of the conjugate is bound to a cysteine side chain on the binder AB.  
The binder or a derivative thereof may be a binding peptide or–protein or a derivative of a binding peptide or–protein. In a further embodiment of the present invention, each molecule of the active component binds to different amino acids of the binding peptide or–protein or their derivatives respectively, via a linker. According to one aspect of the present invention, the conjugate averages 1.2 to 50 molecules of the active components per binder. In accordance with a further aspect of the present invention, the binding peptide or protein represents an antibody or wherein the derivative of the binding peptide or -protein comprises one of the following groups:
Figure imgf000157_0001
respectively. In one aspect of the present invention, the binder binds to a cancer target-molecule. In a further aspect of the present invention, the binder binds to an extracellular target molecule. After binding to the extracellular target molecule, the binder may be internalized in the expressing cell of the target molecule and is processed intracellularly, preferably through the lysosomal pathway. In one embodiment the binding peptide or–protein is a human, humanized or chimeric monoclonal antibody, or an antigen-binding fragment thereof. Preferably the binding peptide or -protein is an anti-HER2-antibody, an anti-CD123-antibody, an anti-B7H3-antibody, an anti-C4.4a-antibody, or an antigen binding fragment thereof. A "target molecule" in the broadest sense is understood to mean a molecule which is present in the target cell population and which may be a protein (for example a receptor of a growth factor) or a non-peptidic molecule (for example a sugar or phospholipid). It is preferably a receptor or an antigen.
The term "extracellular" target molecule describes a target molecule, attached to the cell, which is located at the outside of a cell, or the part of a target molecule which is located at the outside of a cell, i.e. a binder may bind on an intact cell to its extracellular target molecule. An extracellular target molecule may be anchored in the cell membrane or be a component of the cell membrane. The person skilled in the art is aware of methods for identifying extracellular  
target molecules. For proteins, this may be by determining the transmembrane domain(s) and the orientation of the protein in the membrane. These data are usually deposited in protein databases (e.g. SwissProt). The term "cancer target molecule" describes a target molecule which is more abundantly present on one or more cancer cell species than on non-cancer cells of the same tissue type. Preferably, the cancer target molecule is selectively present on one or more cancer cell species compared with non-cancer cells of the same tissue type, where selectively describes an at least two-fold enrichment on cancer cells compared to non-cancer cells of the same tissue type (a "selective cancer target molecule"). The use of cancer target molecules allows the selective therapy of cancer cells using the conjugates according to the invention. The binder can be attached to the linker via a bond. Attachment of the binder can be via a heteroatom of the binder. Heteroatoms according to the invention of the binder which can be used for attachment are sulphur (in one embodiment via a sulphhydryl group of the binder), oxygen (according to the invention by means of a carboxyl or hydroxyl group of the binder) and nitrogen (in one embodiment via a primary or secondary amine group or amide group of the binder). These heteroatoms may be present in the natural binder or are introduced by chemical methods or methods of molecular biology. According to the invention, the attachment of the binder to the toxophore has only a minor effect on the binding activity of the binder with respect to the target molecule. In a preferred embodiment, the attachment has no effect on the binding activity of the binder with respect to the target molecule. In accordance with the present invention, the term "antibody" is to be understood in its broadest meaning and comprises immunoglobulin molecules, for example intact or modified monoclonal antibodies, polyclonal antibodies or multispecific antibodies (e.g. bispecific antibodies). An immunoglobulin molecule preferably comprises a molecule having four polypeptide chains, two heavy chains (H chains) and two light chains (L chains) which are typically linked by disulphide bridges. Each heavy chain comprises a variable domain of the heavy chain (abbreviated VH) and a constant domain of the heavy chain. The constant domain of the heavy chain may, for example, comprise three domains CH1, CH2 and CH3. Each light chain comprises a variable domain (abbreviated VL) and a constant domain. The constant domain of the light chain comprises a domain (abbreviated CL). The VH and VL domains may be subdivided further into regions having hypervariability, also referred to as complementarity determining regions (abbreviated CDR) and regions having low sequence variability (framework region, abbreviated FR). Typically, each VH and VL region is composed of three CDRs and up to four FRs. For example from the amino terminus to the carboxy terminus in the following order: FR1,  
CDR1, FR2, CDR2, FR3, CDR3, FR4. An antibody may be obtained from any suitable species, e.g. rabbit, llama, camel, mouse or rat. In one embodiment, the antibody is of human or murine origin. An antibody may, for example, be human, humanized or chimeric. The term "monoclonal" antibody refers to antibodies obtained from a population of substantially homogeneous antibodies, i.e. individual antibodies of the population are identical except for naturally occurring mutations, of which there may be a small number. Monoclonal antibodies recognize a single antigenic binding site with high specificity. The term monoclonal antibody does not refer to a particular preparation process. The term "intact" antibody refers to antibodies comprising both an antigen-binding domain and the constant domain of the light and heavy chain. The constant domain may be a naturally occurring domain or a variant thereof having a number of modified amino acid positions. The term "modified intact" antibody refers to intact antibodies fused via their amino terminus or carboxy terminus by means of a covalent bond (e.g. a peptide bond) with a further polypeptide or protein not originating from an antibody. Furthermore, antibodies may be modified such that, at defined positions, reactive cysteines are introduced to facilitate coupling to a toxophore (see Junutula et al. Nat Biotechnol.2008 Aug;26(8):925-32). The term "human" antibody refers to antibodies which can be obtained from a human or which are synthetic human antibodies. A "synthetic" human antibody is an antibody which is partially or entirely obtainable in silico from synthetic sequences based on the analysis of human antibody sequences. A human antibody can be encoded, for example, by a nucleic acid isolated from a library of antibody sequences of human origin. An example of such an antibody can be found in Söderlind et al., Nature Biotech.2000, 18:853-856. The term "humanized" or "chimeric" antibody describes antibodies consisting of a non-human and a human portion of the sequence. In these antibodies, part of the sequences of the human immunoglobulin (recipient) are replaced by sequence portions of a non-human immunoglobulin (donor). In many cases, the donor is a murine immunoglobulin. In the case of humanized antibodies, amino acids of the CDR of the recipient are replaced by amino acids of the donor. Sometimes, amino acids of the framework, too, are replaced by corresponding amino acids of the donor. In some cases the humanized antibody contains amino acids present neither in the recepient nor in the donor, which were introduced during the optimization of the antibody. In the case of chimeric antibodies, the variable domains of the donor immunoglobulin are fused with the constant regions of a human antibody.  
The term complementarity determining region (CDR) as used herein refers to those amino acids of a variable antibody domain which are required for binding to the antigen. Typically, each variable region has three CDR regions referred to as CDR1, CDR2 and CDR3. Each CDR region may embrace amino acids according to the definition of Kabat and/or amino acids of a hypervariable loop defined according to Chotia. The definition according to Kabat comprises, for example, the region from about amino acid position 24– 34 (CDR1), 50– 56 (CDR2) and 89– 97 (CDR3) of the variable light chain and 31– 35 (CDR1), 50– 65 (CDR2) and 95– 102 (CDR3) of the variable heavy chain (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The definition according to Chotia comprises, for example, the region from about amino acid position 26– 32 (CDR1), 50– 52 (CDR2) and 91–96 (CDR3) of the variable light chain and 26– 32 (CDR1), 53– 55 (CDR2) and 96– 101 (CDR3) of the variable heavy chain (Chothia and Lesk; J Mol Biol 196: 901-917 (1987)). In some cases, a CDR may comprise amino acids from a CDR region defined according to Kabat and Chotia.
Depending on the amino acid sequence of the constant domain of the heavy chain, antibodies may be categorized into different classes. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be divided into further subclasses. (Isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The constant domains of the heavy chain, which correspond to the different classes, are referred to as [alpha/ ^], [delta/ ^], [epsilon/ ^], [gamma/ ^] and [my/ ^]. Both the three-dimensional structure and the subunit structure of antibodies are known. The term "functional fragment" or "antigen-binding antibody fragment" of an antibody/immunoglobulin is defined as a fragment of an antibody/immunoglobulin (e.g. the variable domains of an IgG) which still comprises the antigen binding domains of the antibody/immunoglobulin. The "antigen binding domain" of an antibody typically comprises one or more hypervariable regions of an antibody, for example the CDR, CDR2 and/or CDR3 region. However, the "framework" or "skeleton" region of an antibody may also play a role during binding of the antibody to the antigen. The framework region forms the skeleton of the CDRs. Preferably, the antigen binding domain comprises at least amino acids 4 to 103 of the variable light chain and amino acids 5 to 109 of the variable heavy chain, more preferably amino acids 3 to 107 of the variable light chain and 4 to 111 of the variable heavy chain, particularly preferably the complete variable light and heavy chains, i.e. amino acids 1– 109 of the VL and 1 to 113 of the VH (numbering according to WO97/08320).
 
"Functional fragments" or "antigen-binding antibody fragments" of the invention encompass, non-conclusively, Fab, Fab’, F(ab’)2 and Fv fragments, diabodies, Single Domain Antibodies (DAbs), linear antibodies, individual chains of antibodies (single-chain Fv, abbreviated to scFv); and multispecific antibodies, such as bi and tri-specific antibodies, for example, formed from antibody fragments C. A. K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, editors (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag. Antibodies other than "multispecific" or "multifunctional" antibodies are those having identical binding sites. Multispecific antibodies may be specific for different epitopes of an antigen or may be specific for epitopes of more than one antigen (see, for example WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., 1991, J. Immunol. 147:60 69; U. S. Pat. Nos. 4,474,893; 4,714,681 ; 4,925,648; 5,573,920; 5,601,819; or Kostelny et al., 1992, J. Immunol. 148: 1547 1553). An F(ab’)2 or Fab molecule may be constructed such that the number of intermolecular disulphide interactions occurring between the Ch1 and the CL domains can be reduced or else completely prevented. "Epitopes" refer to protein determinants capable of binding specifically to an immunoglobulin or T cell receptors. Epitopic determinants usually consist of chemically active surface groups of molecules such as amino acids or sugar side chains or combinations thereof, and usually have specific 3-dimensional structural properties and also specific charge properties.
"Functional fragments" or "antigen-binding antibody fragments" may be fused with another polypeptide or protein, not originating from an antibody, via the amino terminus or carboxyl terminus thereof, by means of a covalent bond (e.g. a peptide linkage). Furthermore, antibodies and antigen-binding fragments may be modified by introducing reactive cysteines at defined locations, in order to facilitate coupling to a toxophore (see Junutula et al. Nat Biotechnol. 2008 Aug; 26(8):925-32). Polyclonal antibodies can be prepared by methods known to a person of ordinary skill in the art. Monoclonal antibodies may be prepared by methods known to a person of ordinary skill in the art (Köhler and Milstein, Nature, 256, 495-497, 1975). Human and humanized monoclonal antibodies may be prepared by methods known to a person of ordinary skill in the art (Olsson et al., Meth Enzymol.92, 3-16 or Cabilly et al. US 4,816,567 or Boss et al. US 4,816,397). A person of ordinary skill in the art is aware of diverse methods for preparing human antibodies and fragments thereof, such as, for example, by means of transgenic mice (N Lonberg and D Huszar, Int Rev Immunol.1995; 13(1):65-93) or Phage Display Technologien (Clackson et al., Nature. 1991 Aug 15;352(6336):624-8). Antibodies of the invention may be obtained from  
recombinant antibody libraries consisting for example of the amino acid sequences of a multiplicity of antibodies compiled from a large number of healthy volunteers. Antibodies may also be produced by means of known recombinant DNA technologies. The nucleic acid sequence of an antibody can be obtained by routine sequencing or is available from publically accessible databases. An“isolated” antibody or binder has been purified to remove other constituents of the cell. Contaminating constituents of a cell which may interfere with a diagnostic or therapeutic use are, for example, enzymes, hormones, or other peptidic or non-peptidic constituents of a cell. A preferred antibody or binder is one which has been purified to an extent of more than 95% by weight, relative to the antibody or binder (determined for example by Lowry method, UV- Vis spectroscopy or by SDS capillary gel electrophoresis). Moreover an antibody which has been purified to such an extent that it is possible to determine at least 15 amino acids of the amino terminus or of an internal amino acid sequence, or which has been purified to homogeneity, the homogeneity being determined by SDS-PAGE under reducing or non- reducing conditions (detection may be determined by means of Coomassie Blau staining or preferably by silver coloration). However, an antibody is normally prepared by one or more purification steps. The term“specific binding” or“binds specifically” refers to an antibody or binder which binds to a predetermined antigen/target molecule. Specific binding of an antibody or binder typically describes an antibody or binder having an affinity of at least 10-7 M (as Kd value; i.e. preferably those with smaller Kd values than 10-7 M), with the antibody or binder having an at least two times higher affinity for the predetermined antigen/target molecule than for a non-specific antigen/target molecule (e.g. bovine serum albumin, or casein) which is not the predetermined antigen/target molecule or a closely related antigen/target molecule. The antibodies preferably have an affinity of at least 10-7 M (as Kd value; in other words preferably those with smaller Kd values than 10-7 M), preferably of at least 10-8 M, more preferably in the range from 10-9 M to 10-11 M. The Kd values may be determined, for example, by means of surface plasmon resonance spectroscopy. The antibody-drug conjugates of the invention likewise exhibit affinities in these ranges. The affinity is preferably not substantially affected by the conjugation of the drugs (in general, the affinity is reduced by less than one order of magnitude, in other words, for example, at most from 10-8 M to 10-7 M).
 
The antibodies used in accordance with the invention are also notable preferably for a high selectivity. A high selectivity exists when the antibody of the invention exhibits an affinity for the target protein which is better by a factor of at least 2, preferably by a factor of 5 or more preferably by a factor of 10, than for an independent other antigen, e.g. human serum albumin (the affinity may be determined, for example, by means of surface plasmon resonance spectroscopy). Furthermore, the antibodies of the invention that are used are preferably cross-reactive. In order to be able to facilitate and better interpret preclinical studies, for example toxicological or activity studies (e.g. in xenograft mice), it is advantageous if the antibody used in accordance with the invention not only binds the human target protein but also binds the species target protein in the species used for the studies. In one embodiment the antibody used in accordance with the invention, in addition to the human target protein, is cross-reactive to the target protein of at least one further species. For toxicological and activity studies it is preferred to use species of the families of rodents, dogs and non-human primates. Preferred rodent species are mouse and rat. Preferred non-human primates are rhesus monkeys, chimpanzees and long-tailed macaques. In one embodiment the antibody used in accordance with the invention, in addition to the human target protein, is cross-reactive to the target protein of at least one further species selected from the group of species consisting of mouse, rat and long-tailed macaque (Macaca fascicularis). Especially preferred are antibodies used in accordance with the invention which in addition to the human target protein are at least cross-reactive to the mouse target protein. Preference is given to cross-reactive antibodies whose affinity for the target protein of the further non-human species differs by a factor of not more than 50, more particularly by a factor of not more than ten, from the affinity for the human target protein. Antibodies directed against a cancer target molecule
The target molecule towards which the binder, for example an antibody or an antigen-binding fragment thereof, is directed is preferably a cancer target molecule. The term "cancer target molecule" describes a target molecule which is more abundantly present on one or more cancer cell species than on non-cancer cells of the same tissue type. Preferably, the cancer target molecule is selectively present on one or more cancer cell species compared with non- cancer cells of the same tissue type, where selectively describes an at least two-fold enrichment on cancer cells compared to non-cancer cells of the same tissue type (a "selective cancer target molecule"). The use of cancer target molecules allows the selective therapy of cancer cells using the conjugates according to the invention.  
Antibodies which are specific against an antigen, for example cancer cell antigen, can be prepared by a person of ordinary skill in the art by means of methods with which he or she is familiar (such as recombinant expression, for example) or may be acquired commercially (as for example from Merck KGaA, Germany). Examples of known commercially available antibodies in cancer therapy are Erbitux® (cetuximab, Merck KGaA), Avastin® (bevacizumab, Roche) and Herceptin® (trastuzumab, Genentech). Trastuzumab is a recombinant humanized monoclonal antibody of the IgG1kappa type which in a cell-based assay (Kd = 5 nM) binds the extracellular domains of the human epidermal growth receptor with high affinity. The antibody is produced recombinantly in CHO cells. In a preferred embodiment, the target molecule is a selective cancer target molecule.
In a particularly preferred embodiment, the target molecule is a protein. In one embodiment, the target molecule is an extracellular target molecule. In a preferred embodiment, the extracellular target molecule is a protein. Cancer target molecules are known to those skilled in the art. Examples of these are listed below. Examples of cancer target molecules are: (1) EGF receptor (NCBI reference sequence NP_005219.2, Gene ID: 1956) (2) mesothelin (SwissProt reference Q13421-3), where mesothelin is encoded by amino acids 296-598. Amino acids 37-286 are coding for the megakaryocyte-potentiating factor. Mesothelin is anchored in the cell membrane via a GPI anchor and is localized extracellularly. (3) carboanhydrase IX (CA9, SwissProt reference Q16790, Gene ID: 768) (4) C4.4a (NCBI reference sequence NP_055215.2; synonym LYPD3, Gene ID: 27076) (5) CD52 (NCBI reference sequence NP_001794.2) (6) Her2 (NCBI reference sequence NP_004439.2; synonym ERBB2, Gene ID: 2064) (7) CD20 (NCBI reference sequence NP_068769.2) (8) the lymphocyte activation antigen CD30 (SwissProt ID P28908) (9) the lymphocyte adhesion molecule CD22 (SwissProt ID P20273, Gene ID: 933)
 
(10) the myeloid cell surface antigen CD33 (SwissProt ID P20138, Gene ID: 945) (11) the transmembrane glycoprotein NMB (GPNMB, SwissProt ID Q14956) (12) the adhesion molecule CD56 (SwissProt ID P13591) (13) the surface molecule CD70 (SwissProt ID P32970, Gene ID: 970) (14) the surface molecule CD74 (SwissProt ID P04233, Gene ID: 972) (15) the B-lymphocyte antigen CD19 (SwissProt ID P15391, Gene ID: 930) (16) the surface protein mucin-1 (MUC1, SwissProt ID P15941, Gene ID: 4582) (17) the surface protein CD138 (SwissProt ID P18827) (18) the integrin subunit alphaV (Genbank Accession No.: NP_002201.1, Gene ID: 3685) (19) the teratocarcinoma-derived growth factor 1 protein TDGF1 (Genbank Accession No.: NP_003203.1, Gene ID: 6997) (20) the prostate-specific membrane antigen PSMA (Swiss Prot ID: Q04609) (21) the tyrosine protein kinase EPHA2 (Swiss Prot ID: P29317, Gene ID: 1969) (22) the surface protein SLC44A4 (Genbank Accession No: NP_001171515.1, Gene ID: 80736) (23) the surface protein BMPR1B (SwissProt: O00238) (24) the transport protein SLC7A5 (SwissProt: Q01650) (25) the epithelial prostate antigen STEAP1 (SwissProt: Q9UHE8, Gene ID: 26872) (26) the ovarian carcinoma antigen MUC16 (SwissProt: Q8WXI7; Gene ID: 94025) (27) the transport protein SLC34A2 (SwissProt: O95436, Gene ID: 10568) (28) the surface protein SEMA5B (SwissProt: Q9P283) (29) the surface protein LYPD1 (SwissProt: Q8N2G4) (30) the endothelin receptor type B EDNRB (SwissProt: P24530, Gene ID: 1910) (31) the ring finger protein RNF43 (SwissProt: Q68DV7) (32) the prostate carcinoma-associated protein STEAP2 (SwissProt: Q8NFT2)  
(33) the cation channel TRPM4 (SwissProt: Q8TD43) (34) the complement receptor CD21 (SwissProt: P20023) (35) the B-cell antigen receptor complex-associated protein CD79b (SwissProt: P40259, Gene ID: 974) (36) the cell adhesion antigen CEACAM6 (SwissProt: P40199) (37) the dipeptidase DPEP1 (SwissProt: P16444) (38) the interleukin receptor IL2Ralpha (SwissProt: Q9UHF4, Gene ID: 3559) (39) the proteoglycan BCAN (SwissProt: Q96GW7) (40) the ephrin receptor EPHB2 (SwissProt: P29323) (41) the prostate stem cell-associated protein PSCA (Genbank Accession No: NP_005663.2) (42) the surface protein LHFPL3 (SwissProt: Q86UP9) (43) the receptor protein TNFRSF13C (SwissProt: Q96RJ3) (44) the B-cell antigen receptor complex-associated protein CD79a (SwissProt: P11912) (45) the chemokine receptor CXCR5 (SwissProt: P32302) (46) the ion channel P2X5 (SwissProt: Q93086) (47) the lymphocyte antigen CD180 (SwissProt: Q99467) (48) the receptor protein FCRL1 (SwissProt: Q96LA6) (49) the receptor protein FCRL5 (SwissProt: Q96RD9) (50) the MHC class II molecule Ia antigen HLA-DOB (Genbank Accession No: NP_002111.1) (51) the T-cell protein VTCN1 (SwissProt: Q7Z7D3) (52) TWEAKR (FN14, Gene ID: 51330) (53) the lymphocyte antigen CD37 (Swiss Prot: P11049, Gene ID: 951) (54) the FGF receptor 2; FGFR2 (Gene ID: 2263; official symbol: FGFR2). The FGFR2 receptor occurs in different splice variants (alpha, beta, IIIb, IIIc). All splice variants may act as target molecule. (55) the transmembrane glycoprotein B7H3 (CD276; Gene ID: 80381)  
(56) the B cell receptor BAFFR (CD268; Gene ID: 115650)
(57) the receptor protein ROR 1 (Gene ID: 4919)
(58) the surface receptor IL3RA (CD123; Gene ID: 3563)
(59) the CXC chemokine receptor CXCR5 (CD185; Gene ID 643)
(60) the receptor protein syncytin (Gene ID 30816)
(61) the aspartate beta-hydroxylase (ASPH; Gene ID 444)
(62) the cell-surface glycoprotein CD44 (Gene ID: 960)
(63) CDH15 (cadherin 15, Gene ID: 1013)
(64) the cell-surface glycoprotein CEACAM5 (Gene ID: 1048)
(65) the cell adhesion molecule L1-like (CHL1, Gene ID: 10752)
(66) the receptor tyrosine kinase c-Met (Gene ID: 4233)
(67) the Notch ligand DLL3 (Gene ID: 10683)
(68) the ephrin A4 (EFNA4, Gene ID: 1945)
(69) the ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3, Gene ID: 5169) (70) the coagulation factor III (F3, Gene ID: 2152)
(71) the FGF receptor 3 (FGFR3, Gene ID: 2261)
(72) the folate hydrolase FOLH1 (Gene ID: 2346)
(73) the folate receptor 1 (FOLR1; Gene ID: 2348)
(74) the transmembrane glycoprotein nmb (GPNMB, Gene ID: 10457)
(75) the guanylate cyclase 2C (GUCY2C, Gene ID: 2984)
(76) the KIT proto-oncogene receptor tyrosine kinase (Gene ID: 3815)
(77) the lysosomal associated membrane protein 1 (LAMP1, Gene ID: 3916)
(78) the Lewis-Y carbohydrate antigen
(79) the lymphocyte antigen 6 complex, locus E (LY6E, Gene ID: 4061)
(80) the protein NOTCH3 (Gene ID: 4854)
 
(81) the protein tyrosine kinase 7 (PTK7, Gene ID: 5754) (82) the nectin cell adhesion molecule 4 (PVRL4, NECTIN4, Gene ID: 81607) (83) the transmembrane protein syndecan 1 (SDC1, Gene ID: 6382) (84) the SLAM family member 7 (SLAMF7, Gene ID: 57823) (85) the transport protein SLC39A6 (Gene ID: 25800) (86) the SLIT and NTRK like family member 6 (SLITRK6, Gene ID: 84189) (87) the cell surface receptor TACSTD2 (Gene ID: 4070) (88) the receptor protein TNFRSF8 (Gene ID: 943) (89) the receptor protein TNFSF13B (Gene ID: 10673) (90) the trophoblast glycoprotein TPBG (Gene ID: 7162) (91) the cell surface receptor TROP2 (TACSTD2, Gene ID: 4070) (93) the galanin-like G protein-coupled receptor KISS1R (GPR54, Gene ID: 84634) (94) the transmembrane protein SLAMF6 (Gene ID: 114836)
(95) the lymphocyte antigen 6 complex, locus G6D (Gene ID: 58530) Further examples of cancer target molecules according to the invention are listed below.
Figure imgf000168_0001
 
Figure imgf000169_0001
 
Figure imgf000170_0001
 
Figure imgf000171_0001
 
Figure imgf000172_0001
 
Figure imgf000173_0001
In one embodiment the binder is a binding protein. In a preferred embodiment the binder is an antibody, an antigen-binding antibody fragment, a multispecific antibody or an antibody mimetic. Preferred antibody mimetics are affibodies, adnectins, anticalins, DARPins, avimers, or nanobodies. Preferred multispecific antibodies are bispecific and trispecific antibodies. In a preferred embodiment the binder is an antibody or an antigen-binding antibody fragment, more preferably an isolated antibody or an isolated antigen-binding antibody fragment. Preferred antigen-binding antibody fragments are Fab, Fab’, F(ab’)2 and Fv fragments, diabodies, DAbs, linear antibodies and scFv. Particularly preferred are Fab, diabodies and scFv. In a particularly preferred embodiment the binder is an antibody. Particularly preferred are monoclonal antibodies or antigen-binding antibody fragments thereof. Further particularly preferred are human, humanized or chimeric antibodies or antigen-binding antibody fragments thereof.
 
Antibodies or antigen-binding antibody fragments which bind cancer target molecules may be prepared by a person of ordinary skill in the art using known processes, such as, for example, chemical synthesis or recombinant expression. Binders for cancer target molecules may be acquired commercially or may be prepared by a person of ordinary skill in the art using known processes, such as, for example, chemical synthesis or recombinant expression. Further processes for preparing antibodies or antigen-binding antibody fragments are described in WO 2007/070538 (see page 22 "Antibodies"). The person skilled in the art knows how processes such as phage display libraries (e.g. Morphosys HuCAL Gold) can be compiled and used for discovering antibodies or antigen-binding antibody fragments (see WO 2007/070538, page 24 ff and AK Example 1 on page 70, AK Example 2 on page 72). Further processes for preparing antibodies that use DNA libraries from B cells are described for example on page 26 (WO 2007/070538). Processes for humanizing antibodies are described on page 30-32 of WO2007070538 and in detail in Queen, et al., Pros. Natl. Acad. Sci. USA 86:10029-10033, 1989 or in WO 90/0786. Furthermore, processes for the recombinant expression of proteins in general and of antibodies in particular are known to the person skilled in the art (see, for example, in Berger and Kimrnel (Guide to Molecular Cloning Techniques, Methods in Enzymology, Vo1. 152, Academic Press, Inc.); Sambrook, et al., (Molecular Cloning: A Laboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3); Current Protocols in Molecular Biology, (F. M. Ausabel et al. [Eds.], Current Protocols, Green Publishing Associates, Inc. / John Wiley & Sons, Inc.); Harlow et al., (Monoclonal Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988, Paul [Ed.]); Fundamental Immunology, (Lippincott Williams & Wilkins (1998)); and Harlow, et al., (Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1998)). The person skilled in the art knows the corresponding vectors, promoters and signal peptides which are necessary for the expression of a protein/antibody. Commonplace processes are also described in WO 2007/070538 on pages 41–45. Processes for preparing an IgG1 antibody are described for example in WO 2007/070538 in Example 6 on page 74 ff. Processes which allow the determination of the internalization of an antibody after binding to its antigen are known to the skilled person and are described for example in WO 2007/070538 on page 80. The person skilled in the art is able to use the processes described in WO 2007/070538 that have been used for preparing carboanhydrase IX (Mn) antibodies in analogy for the preparation of antibodies with different target molecule specificity. anti-EGFR antibodies
Examples of antibodies which bind the cancer target molecules EGFR are cetuximab (INN number 7906), panitumumab (INN number 8499), nimotuzumab (INN number 8545), "TPP- 4030", and "TPP-5653". Cetuximab (Drug Bank Accession Number DB00002) is a chimeric  
anti-EGFR1 antibody which is produced in SP2/0 mouse myeloma cells and is sold by ImClone Systems Inc/Merck KgaA/Bristol-Myers Squibb Co. Cetuximab is indicated for the treatment of metastasizing, EGFR expressing, colorectal carcinoma with wild type K-Ras gene. It has an affinity of 10-10M. In a preferred embodiment, the anti-EGFR antibodies are selected from the group consisting of cetuximab, panitumumab, nimotuzumab, zalutumumab, necitumumab, matuzumab, RG- 716, GT-MAB 5.2-GEX, ISU-101, ABT-806, SYM-004, MR1-1, SC-100, MDX-447, DXL-1218, "TPP-4030", and "TPP-5653". In a particularly preferred embodiment the anti-EGFR antibodies are selected from the group consisting of cetuximab, panitumumab, nimotuzumab, zalutumumab, necitumumab and matuzumab. The person skilled in the art knows of processes which can be used to prepare further antibodies, from the CDR regions of the abovementioned antibodies by means of sequence variations, these further antibodies having a similar or better affinity and/or specificity for the target molecule. anti-HER2 antibodies
An example of an antibody binding to the cancer target molecule Her2 is trastuzumab (Genentech). Trastuzumab is a humanized antibody used inter alia for the treatment of breast cancer. Further examples of antibodies binding to HER2 are, in addition to trastuzumab (INN 7637, CAS No.: RN: 180288-69-1) and Pertuzumab (CAS No.: 380610-27-5), the antibodies disclosed in WO 2009/123894-A2, WO 200/8140603-A2 or in WO 2011/044368-A2. An example of an anti-HER2 conjugate is trastuzumab-emtansine (INN-No. 9295). By reference, these antibodies and antigen-binding fragments thereof are incorporated herein, and they can be used in the context of the present invention. Anti-TWEAKR antibodies
According to the invention, use may be made of anti-TWEAKR antibodies. Examples of anti-TWEAKR antibodies and antigen-binding fragments are described in WO 2014/198817 (A1) and WO 2015/189143 (A1). By reference, all antibodies of WO 2014/ 198817 (A1) and WO 2015/189143 (A1) are hereby incorporated into the description of the present invention, and they can be used in the present invention. The sequences of the  
antibodies are shown in Table 31 and Table 32 of WO 2014/198817 (A1). Preference is given to antibodies, antigen-binding fragments and variants of the antibodies derived from the antibodies referred to as TPP-2090 and TPP-2658. In a further embodiment, the anti- TWEAKR antibodies or antigen-binding antibody fragments comprise at least one, two or three CDR amino acid sequences of an antibody listed in Table 31 or Table 32 of WO 2014/198817 (A1). In addition, antibodies which bind to TWEAKR are known to the person skilled in the art, see, for example, WO2009/020933(A2) (e.g. PDL-192) or WO2009140177 (A2) (e.g. BIIB036 (P4A8)).
Further examples of anti-TWEAKR antibodies and antigen-binding fragments are ITEM-4 and ITEM-4 derived antibodies. ITEM-4 is an anti-TWEAKR antibody described by Nakayama et al. (Nakayama, et al., 2003, Biochem Biophy Res Comm, 306:819-825). Humanized variants of this antibody based on CDR grafting are described by Zhou et al. (Zhou et al., 2013, J Invest Dermatol. 133(4):1052-62) and in WO 2009/020933. ITEM-4 is a moderately agonistically or agonistically acting anti-TWEAKR antibody.
One preferred example of an anti-TWEAKR antibody is TPP-2090. See Figures 1 and 2 for sequence listings.
anti-CD123-antibody
According to the invention, use is made of an anti-CD123 antibody or an antigen-binding fragment thereof, preferably one selected from those described below or modified by suitable mutation. In addition, the person skilled in the art is familiar with antibodies binding to CD123. Sun et al. (Sun et al., 1996, Blood 87(1):83-92) describe the generation and properties of the monoclonal antibody 7G3, which binds to the N-terminal domain of IL-3Ra, CD123. US Patent Number 6,177,078 (Lopez) relates to the anti-CD123 antibody 7G3. A chimeric variant of this antibody (CSL360) is described in WO 2009/070844, and a humanized version (CSL362) in WO 2012/021934. The sequence of the 7G3 antibody is disclosed in EP2426148. Based on the publication of the sequences of the variable regions (VH and VL) of 7G3 (EP2426148), the TPP-5969 was obtained by CDR grafting in human framework regions. Antibody TPP-5969 is humanized variants of 7G3 as a subtype of human IgG1 kappa. Further optimization of the sequences resulted in humanized and germlined antibodies TPP-8987 and TPP-9476. Based on the publication of the sequences of the variable regions (VH and VL) of 12F1 (WO 2013/173820), TPP-6013 was obtained by fusion of the variable domains of the donor  
immunoglobulin (VH and VL) with the constant regions of a human antibody. TPP-6013 is a chimeric variant of 12F1 where the variable regions VH and VL are linked to the constant regions (CL, CH1, CH2, CH3) of a human IgG1 of the kappa subtype. This 12F1 sequence represents also the starting point of the humanized and germlined antibodies TPP-8988 and TPP-9342.
In the present application, reference is made to the following highly preferred anti-CD123 antibodies:“TPP-8987”,“TPP-9476”,“TPP-8988” and“TPP-9342”.
TPP-8987 and TPP-9476 are humanized and germlined variants of the antibody 7G3.
TPP-8988 and TPP-9342 are humanized and germlined variants of the antibody 12F1 Preferred anti-CD123 antibodies include TPP-8987, TPP-8988, TPP-9342, and TPP-9476. See Figures 1 and 2 for sequence listings.
anti-B7H3-antibody
The anti-B7H3 antibodies were generated, for example, by screening of a phage display library for recombinant murine B7H3 (murine CD276; Gene ID: 102657) and human B7H3 (human CD276; Gene ID: 80381) expressing cells. Particularly the antibodies TPP-6497 and TPP-8382 are important examples. The antibodies obtained in this manner were reformatted into the human IgG1 format. These two antibodies were used for the working examples described here. In addition, antibodies which bind to B7H3 are known to the person skilled in the art. Examples of anti-B7H3 antibodies include TPP-6497, TPP-6499,TPP-6501, TPP-6502, TPP- 6515, TPP-8322, TPP-8382, TPP-8564, TPP-8565, TPP-8567, and TPP-8568.
Preferred anti-B7H3 antibodies include TPP-8382 and TPP-8567.
See Figures 1 and 2 for sequence listings.
anti-C4.4a-antibody 
According to the invention, use may be made of C4.4a antibodies. Examples of anti-C4.4a antibodies and antigen-binding fragments are described in WO 2012/143499 A2. By reference, all antibodies of WO 2012/143499 A2 are hereby incorporated into the description of the present invention, and they can be used in the present invention. The sequences of the antibodies are given in Table 1 of WO 2012/143499 A2, where each row shows the respective CDR amino acid sequences of the variable light chain or the variable heavy chain of the antibody listed in column 1.  
In one embodiment, the anti-C4.4a antibodies or antigen-binding antibody fragments thereof are, after binding to a cell expressing C4.4a, internalized by the cell. In a further embodiment, the anti-C4.4a antibodies or antigen-binding antibody fragments comprise at least one, two or three CDR amino acid sequences of an antibody listed in Table 1 of WO 2012/143499 A2 or Table 2 of WO 2012/143499 A2. Preferred embodiments of such antibodies are likewise listed in WO 2012/143499 A2 and incorporated herein by reference. An example of an anti-C4.4a-antibody is TPP-509.
See Figures 1 and 2 for sequence listings. Antibody Sequences In the present application, reference is made to the following preferred antibodies shown in the Table of Figure 1.
TPP-509 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 9 and a region of the light chain corresponding to SEQ ID NO: 10.
TPP-1015 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 29 and a region of the light chain corresponding to SEQ ID NO: 30.
TPP-5969 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 39 and a region of the light chain corresponding to SEQ ID NO: 40.
TPP-6013 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 49 and a region of the light chain corresponding to SEQ ID NO: 50.
TPP-8987 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 59 and a region of the light chain corresponding to SEQ ID NO: 60.
TPP-8988 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 69 and a region of the light chain corresponding to SEQ ID NO: 70.
TPP-9342 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 79 and a region of the light chain corresponding to SEQ ID NO: 80.
TPP-9476 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 89 and a region of the light chain corresponding to SEQ ID NO: 90.
TPP-6497 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 99 and a region of the light chain corresponding to SEQ ID NO: 100.
TPP-6499 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 109 and a region of the light chain corresponding to SEQ ID NO: 110.
TPP-6501 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 119 and a region of the light chain corresponding to SEQ ID NO: 120.
 
TPP-6502 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 129 and a region of the light chain corresponding to SEQ ID NO: 130.
TPP-6515 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 139 and a region of the light chain corresponding to SEQ ID NO: 140.
TPP-8322 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 149 and a region of the light chain corresponding to SEQ ID NO: 150.
TPP-8382 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 159 and a region of the light chain corresponding to SEQ ID NO: 160.
TPP-8564 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 169 and a region of the light chain corresponding to SEQ ID NO: 170.
TPP-8565 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 179 and a region of the light chain corresponding to SEQ ID NO: 180.
TPP-8567 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 189 and a region of the light chain corresponding to SEQ ID NO: 190.
TPP-8568 is an antibody comprising a region of the heavy chain corresponding to SEQ ID NO: 199 and a region of the light chain corresponding to SEQ ID NO: 200. TPP-509 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 1 and a variable region of the light chain corresponding to SEQ ID NO: 5. TPP-1015 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 21 and a variable region of the light chain corresponding to SEQ ID NO: 25. TPP-5969 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 31 and a variable region of the light chain corresponding to SEQ ID NO: 35. TPP-6013 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 41 and a variable region of the light chain corresponding to SEQ ID NO: 45. TPP-8987 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 51 and a variable region of the light chain corresponding to SEQ ID NO: 55. TPP-8988 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 61 and a variable region of the light chain corresponding to SEQ ID NO: 65. TPP-9342 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 71 and a variable region of the light chain corresponding to SEQ ID NO: 75. TPP-9476 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 81 and a variable region of the light chain corresponding to SEQ ID NO: 85. TPP-6497 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 91 and a variable region of the light chain corresponding to SEQ ID NO: 95. TPP-6499 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 101 and a variable region of the light chain corresponding to SEQ ID NO: 105.  
TPP-6501 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 111 and a variable region of the light chain corresponding to SEQ ID NO: 115. TPP-6502 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 121 and a variable region of the light chain corresponding to SEQ ID NO: 125. TPP-6515 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 131 and a variable region of the light chain corresponding to SEQ ID NO: 135. TPP-8322 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 141 and a variable region of the light chain corresponding to SEQ ID NO: 145. TPP-8382 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 151 and a variable region of the light chain corresponding to SEQ ID NO: 155. TPP-8564 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 161 and a variable region of the light chain corresponding to SEQ ID NO: 165. TPP-8565 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 171 and a variable region of the light chain corresponding to SEQ ID NO: 175. TPP-8567 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 181 and a variable region of the light chain corresponding to SEQ ID NO: 185. TPP-8568 is an antibody which comprises a variable region of the heavy chain corresponding to SEQ ID NO: 191 and a variable region of the light chain corresponding to SEQ ID NO: 195. TPP-509 is an anti-C4.4a antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 2, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 3, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 4, and
a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 6, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 7, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 8.
TPP-1015 is an anti-HER2 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 22, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 23, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 24, and
a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 26, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 27, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 28. TPP-5969 is is an anti-CD123 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 32, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 33, and the variable CDR3  
sequence of the heavy chain, as shown in SEQ ID NO: 34, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 36, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 37, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 38.
TPP-6013 is is an anti-CD123 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 42, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 43, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 44, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 46, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 47, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 48.
TPP-8987 is is an anti-CD123 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 52, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 53, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 54, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 56, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 57, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 58.
TPP-8988 is is an anti-CD123 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 62, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 63, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 64, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 66, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 67, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 68.
TPP-9342 is is an anti-CD123 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 72, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 73, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 74, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 76, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 77, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 78.
TPP-9476 is is an anti-CD123 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 82, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 83, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 84, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 86, the variable CDR2  
sequence of the light chain, as shown in SEQ ID NO: 87, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 88.
TPP-6497 is is an anti-B7H3 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 92, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 93, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 94, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 96, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 97, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 98.
TPP-6499 is is an anti-B7H3 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 102, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 103, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 104, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 106, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 107, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 108.
TPP-6501 is is an anti-B7H3 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 112, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 113, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 114, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 116, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 117, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 118.
TPP-6502 is is an anti-B7H3 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 122, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 123, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 124, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 126, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 127, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 128.
TPP-6515 is is an anti-B7H3 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 132, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 133, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 134, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 136, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 137, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 138.  
TPP-8322 is is an anti-B7H3 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 142, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 143, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 144, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 146, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 147, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 148.
TPP-8382 is is an anti-B7H3 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 152, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 153, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 154, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 156, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 157, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 158.
TPP-8564 is is an anti-B7H3 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 162, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 163, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 164, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 166, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 167, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 168.
TPP-8565 is is an anti-B7H3 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 172, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 173, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 174, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 176, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 177, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 178.
TPP-8567 is is an anti-B7H3 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 182, the variable CDR2 sequence of the heavy chain, as shown in SEQ ID NO: 183, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 184, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 186, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 187, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 188.
TPP-8568 is is an anti-B7H3 antibody which comprises a variable heavy chain comprising the variable CDR1 sequence of the heavy chain, as shown in SEQ ID NO: 192, the variable CDR2  
sequence of the heavy chain, as shown in SEQ ID NO: 193, and the variable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 194, and a variable light chain comprising the variable CDR1 sequence of the light chain, as shown in SEQ ID NO: 196, the variable CDR2 sequence of the light chain, as shown in SEQ ID NO: 197, and the variable CDR3 sequence of the light chain, as shown in SEQ ID NO: 198. DNA molecules of the invention The present invention also relates to the DNA molecules that encode an antibody of the invention or antigen-binding fragment thereof. These sequences are optimized in certain cases for mammalian expression. DNA molecules of the invention are not limited to the sequences disclosed herein, but also include variants thereof. DNA variants within the invention may be described by reference to their physical properties in hybridization. The skilled worker will recognize that DNA can be used to identify its complement and, since DNA is double stranded, its equivalent or homolog, using nucleic acid hybridization techniques. It also will be recognized that hybridization can occur with less than 100% complementarity. However, given appropriate choice of conditions, hybridization techniques can be used to differentiate among DNA sequences based on their structural relatedness to a particular probe. For guidance regarding such conditions see, Sambrook et al., 1989 supra and Ausubel et al., 1995 (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons). Structural similarity between two polynucleotide sequences can be expressed as a function of "stringency" of the conditions under which the two sequences will hybridize with one another. As used herein, the term "stringency" refers to the extent that the conditions disfavor hybridization. Stringent conditions strongly disfavor hybridization, and only the most structurally related molecules will hybridize to one another under such conditions. Conversely, non-stringent conditions favor hybridization of molecules displaying a lesser degree of structural relatedness. Hybridization stringency, therefore, directly correlates with the structural relationships of two nucleic acid sequences. Hybridization stringency is a function of many factors, including overall DNA concentration, ionic strength, temperature, probe size and the presence of agents which disrupt hydrogen bonding. Factors promoting hybridization include high DNA concentrations, high ionic strengths, low temperatures, longer probe size and the absence of agents that disrupt hydrogen bonding. Hybridization typically is performed in two phases: the“binding” phase and the“washing” phase.
 
Functionally Equivalent DNA Variants Yet another class of DNA variants within the scope of the invention may be described with reference to the product they encode. These functionally equivalent polynucleotides are characterized by the fact that they encode the same peptide sequences due to the degeneracy of the genetic code. It is recognized that variants of DNA molecules provided herein can be constructed in several different ways. For example, they may be constructed as completely synthetic DNAs. Methods of efficiently synthesizing oligonucleotides are widely available. See Ausubel et al., section 2.11, Supplement 21 (1993). Overlapping oligonucleotides may be synthesized and assembled in a fashion first reported by Khorana et al., J. Mol. Biol. 72:209-217 (1971); see also Ausubel et al., supra, Section 8.2. Synthetic DNAs preferably are designed with convenient restriction sites engineered at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector. As indicated, a method of generating variants is to start with one of the DNAs disclosed herein and then to conduct site-directed mutagenesis. See Ausubel et al., supra, chapter 8, Supplement 37 (1997). In a typical method, a target DNA is cloned into a single-stranded DNA bacteriophage vehicle. Single-stranded DNA is isolated and hybridized with an oligonucleotide containing the desired nucleotide alteration(s). The complementary strand is synthesized and the double stranded phage is introduced into a host. Some of the resulting progeny will contain the desired mutant, which can be confirmed using DNA sequencing. In addition, various methods are available that increase the probability that the progeny phage will be the desired mutant. These methods are well known to those in the field and kits are commercially available for generating such mutants. Recombinant DNA constructs and expression The present invention further provides recombinant DNA constructs comprising one or more of the nucleotide sequences encoding the preferred antibodies of the present invention. The recombinant constructs of the present invention can be used in connection with a vector, such as a plasmid, phagemid, phage or viral vector, into which a DNA molecule encoding an antibody of the invention or antigen-binding fragment thereof or variant thereof is inserted. An antibody, antigen binding portion, or variant thereof provided herein can be prepared by recombinant expression of nucleic acid sequences encoding light and heavy chains or portions thereof in a host cell. To express an antibody, antigen binding portion, or variant thereof recombinantly a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the light and/or heavy chains or portions thereof such that  
the light and heavy chains are expressed in the host cell. Standard recombinant DNA methodologies are used to prepare and/or obtain nucleic acids encoding the heavy and light chains, incorporate these nucleic acids into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. No.4,816,397 by Boss et al.. In addition, the nucleic acid sequences encoding variable regions of the heavy and/or light chains can be converted, for example, to nucleic acid sequences encoding full-length antibody chains, Fab fragments, or to scFv. The VL- or VH-encoding DNA fragment can be operatively linked, (such that the amino acid sequences encoded by the two DNA fragments are in-frame) to another DNA fragment encoding, for example, an antibody constant region or a flexible linker. The sequences of human heavy chain and light chain constant regions are known in the art (see e.g., Kabat, E. A., el al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. To create a polynucleotide sequence that encodes a scFv, the VH- and VL-encoding nucleic acids can be operatively linked to another fragment encoding a flexible linker such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554). To express the antibodies, antigen binding fragments thereof or variants thereof standard recombinant DNA expression methods can be used (see, for example, Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For example, DNA encoding the desired polypeptide can be inserted into an expression vector which is then transfected into a suitable host cell. Suitable host cells are prokaryotic and eukaryotic cells. Examples for prokaryotic host cells are e.g. bacteria, examples for eukaryotic hosts cells are yeasts, insects and insect cells, plants and plant cells, transgenic animals, or mammalian cells. In some embodiments, the DNAs encoding the heavy and light chains are inserted into separate vectors. In other embodiments, the DNA encoding the heavy and light chains is inserted into the same vector. It is understood that the design of the expression vector, including the selection of regulatory sequences is affected by factors such as the choice of the host cell, the level of expression of protein desired and whether expression is constitutive or inducible.  
Therefore, an embodiment of the present invention are also host cells comprising the vector or a nucleic acid molecule, whereby the host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell. Another embodiment of the present invention is a method of using the host cell to produce an antibody and antigen binding fragments, comprising culturing the host cell under suitable conditions and recovering said antibody. Therefore another embodiment of the present invention is the production of the antibodies according to this invention with the host cells of the present invention and purification of these antibodies to at least 95% homogeneity by weight. Bacterial Expression Useful expression vectors for bacterial use are constructed by inserting a DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desirable, to provide amplification within the host. Suitable prokaryotic hosts for transformation include but are not limited to E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus. Bacterial vectors may be, for example, bacteriophage-, plasmid- or phagemid-based. These vectors can contain a selectable marker and a bacterial origin of replication derived from commercially available plasmids typically containing elements of the well-known cloning vector pBR322 (ATCC 37017). Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is de-repressed/induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide libraries, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Therefore, an embodiment of the present invention is an expression vector comprising a nucleic acid sequence encoding for the novel antibodies of the present invention.  
Antibodies of the present invention or antigen-binding fragments thereof or variants thereof include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic host, including, for example, E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, preferably, from E. coli cells. Mammalian Expression Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. Expression of the antibodies may be constitutive or regulated (e.g. inducible by addition or removal of small molecule inductors such as Tetracyclin in conjunction with Tet system). For further description of viral regulatory elements, and sequences thereof, see e.g., U.S.5,168,062 by Stinski, U.S.4,510,245 by Bell et al. and U.S. 4,968,615 by Schaffner et al.. The recombinant expression vectors can also include origins of replication and selectable markers (see e.g., U.S.4,399,216, 4,634,665 and U.S. 5,179,017). Suitable selectable markers include genes that confer resistance to drugs such as G418, puromycin, hygromycin, blasticidin, zeocin/bleomycin or methotrexate or selectable marker that exploit auxotrophies such as Glutamine Synthetase (Bebbington et al., Biotechnology (N Y). 1992 Feb;10(2):169-75), on a host cell into which the vector has been introduced. For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, neo gene confers resistance to G418, the bsd gene from Aspergillus terreus confers resistance to blasticidin, puromycin N-acetyl-transferase confers resistance to puromycin, the Sh ble gene product confers resitance to zeocin, and resistance to hygromycin is conferred by the E. coli hygromycin resistance gene (hyg or hph). Selectable markers like DHFR or Glutamine Synthetase are also useful for amplification techniques in conjunction with MTX and MSX. Transfection of the expression vector into a host cell can be carried out using standard techniques such as electroporation, nucleofection, calcium-phosphate precipitation, lipofection, polycation-based transfection such as polyethlylenimine (PEI)-based transfection and DEAE-dextran transfection. Suitable mammalian host cells for expressing the antibodies, antigen binding fragments thereof or variants thereof provided herein include Chinese Hamster Ovary (CHO cells) such as CHO-K1, CHO-S, CHO-K1SV [including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 and Urlaub et al., Cell. 1983 Jun;33(2):405-  
12, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621; and other knockout cells exemplified in Fan et al., Biotechnol Bioeng. 2012 Apr;109(4):1007-15], NS0 myeloma cells, COS cells, HEK293 cells, HKB11 cells, BHK21 cells, CAP cells, EB66 cells, and SP2 cells. Expression might also be transient or semi-stable in expression systems such as HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E, HEK293-Freestyle, HKB11, Expi293F, 293EBNALT75, CHO Freestyle, CHO-S, CHO-K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO-3E7 or CAP-T cells (for instance Durocher et al., Nucleic Acids Res. 2002 Jan 15;30(2):E9). In some embodiments, the expression vector is designed such that the expressed protein is secreted into the culture medium in which the host cells are grown. The antibodies, antigen binding fragments thereof or variants thereof can be recovered from the culture medium using standard protein purification methods. Purification Antibodies of the invention or antigen-binding fragments thereof or variants thereof can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to ammonium sulfate or ethanol precipitation, acid extraction, Protein A chromatography, Protein G chromatography, anion or cation exchange chromatography, phospho-cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be employed for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein by reference. Antibodies of the present invention or antigen-binding fragments thereof or variants thereof include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from an eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the antibody of the present invention can be glycosylated or can be non- glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20. In preferred embodiments, the antibody is purified (1) to greater than 95% by weight of antibody as determined e.g. by the Lowry method, UV-Vis spectroscopy or by by SDS-Capillary Gel electrophoresis (for example on a Caliper LabChip GXII, GX 90 or Biorad Bioanalyzer device),  
and in further preferred embodiments more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated naturally occurring antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. Metabolites Following introduction of the ADCs into tumour cells and subsequent dissociation of the conjugate, a cytotoxic metabolite may be formed that is released within the tumour cell and can unfold its action therein directly and selectively. For the purpose of the present invention the term metabolite is understood as the product of the (e.g., enzymatic or chemical) cleavage of the conjugate of a binder or a derivative thereof according to the present invention. The present invention thus also relates to metabolites obtainable by the cleavage of any of the conjugates described herein. Accordingly, the metabolite will comprise a molecule of an active component, wherein the active component is a NAMPT inhibitor. In one embodiment, if the conjugate according to the invention comprises a stable linker, the metabolite of the NAMPT inhibitor as such comprises an amino acid residue, preferably a cysteine or a lysine residue of the binder protein or peptide. In another embodiment, if the conjugate according to the invention comprises a cleavable linker, the metabolite of the NAMPT inhibitor as such is possibly connected with only part of the linker moiety, i.e. the metabolite of the NAMPT inhibitor as such does not comprise a cysteine and/or a lysine residue of the binder protein or peptide. General Procedures The conjugates according to the invention can be prepared according to the following schemes 1 through 44.
 
The schemes and procedures described below illustrate synthetic routes to the compounds of formula (I) of the invention and are not intended to be limiting. It is obvious to the person skilled in the art that the order of transformations as exemplified in the Schemes can be modified in various ways. The order of transformations exemplified in the Schemes is therefore not intended to be limiting. In addition, interconversion of any of the substituents, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, V, W, Y or Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. The racemic and chiral synthesis of dihydropyridazinones is described in the following representative patents and journals: WO2011138427; US4666902; US20080027041; EP185964; EP196005; EP175363; EP240026; EP400519; EP344634; DE10010423; WO2001064652; DE10010426; DE10010430; DE2304977; Chem. Pharm. Bull. 46(1), 84-96 (1998); J. Med. Chem. 39, 297-303 (1996); J. Med. Chem. 50, 3242-3255 (2007); Bioorganic & Medicinal Chemistry Letters 21, 5493-5497 (2011).
  One route for the preparation of compounds of formula (Ia) is described in Scheme 1.
Scheme 1
Figure imgf000192_0001
Scheme 1: Route for the preparation of compounds of formula (Ia), wherein R1, R2, R3, q, m, t, V, W, Y and Z have the meaning as given for general formula (I), supra. X represents a leaving group such as for example a Cl or Br atom, and X1 represents a leaving group such as for example a Cl, Br or I, or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group).
 
B1 represents a leaving group such as, for example, a haloalkyl such as, for example, trichloromethyl or a imide such as, for example, pyrrolidine-2,5-dione. PG1 represents an amine protecting group as for example an acetyl group. In addition, interconversion of any of the substituents R1, R2, R3, B1, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-1, 1-2, 1-4, 1-7, 1-9 and 1-11 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. A suitably substituted aromatic amine of general formula (1-1), such as, for example, N- phenylacetamide, can be reacted with a suitable substituted acid chloride (1-2), such as, for example, 2-chloropropanoyl chloride, in the presence of a Lewis acid, such as, for example, aluminium trichloride, in a suitable solvent system, such as, for example, dichloromethane, at temperatures ranging from - 20°C to boiling point of the respective solvent, preferably the reaction is carried out at 0°C, to furnish intermediates of general formula (1-3).
Intermediates of general formula (1-3) can be converted to intermediates of general formula (1-5) by reaction with a suitably alkyl malonate of the general formula (1-4), such as, for example, dimethyl malonate, in the presence of a suitable base, such as, for example sodium hydride, in a suitable solvent system, such as, for example, DMF, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 0°C. Intermediates of general formula (1-5) can be reacted with a suitable Broensted acid, such as, for example, hydrochloric acid or sulphuric acid, at temperatures ranging from 0°C to boiling point of the respective Broensted acid, preferably the reaction is carried out at 100°C, to furnish intermediates of general formula (1-6).
 
Intermediates of general formula (1-6) can be converted to intermediates of general formula (1-8) by reaction with a suitable hydrazine of the formula (1-7), such as, for example, hydrazine monohydrate, in a suitable solvent system, such as, for example, propan-1-ol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 80°C. Intermediates of general formula (1-8) are treated with a carbonate of general formula (1-9), such as, for example, 1,1'-[carbonylbis(oxy)]dipyrrolidine-2,5-dione, in the presence of a suitable base, such as for example, N,N-dimethylpyridin-4-amine, in a suitable solvent system, such as, for example, DMF, at a temperature between 0°C and the boiling point of the respective solvent, preferably the reaction is carried out at room temperature to form the desired intermediate of general formula(1-10). Intermediates of general formula (1-10) can be converted to compounds of formula (Ia) by reaction with a suitably substituted amine of the general formula (1-11), such as, for example, 1-(pyridin-3-yl)piperazine, in the presence of a suitable base, such as, for example triethylamine, in a suitable solvent system, such as, for example, DMF, in a temperature range from 0°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. An alternative route for the preparation of compounds of formula (Ia) is described in Scheme 2.
Scheme 2
 
Figure imgf000195_0001
Scheme 2: Route for the preparation of compounds of formula (Ia), wherein R1, R2, R3, q, m, t, V, W, Y and Z have the meaning as given for general formula (I), supra. B1 represents a leaving group such as for example a halo alkyl such for example trichloromethyl or a imid such as, for example pyrrolidine-2,5-dione. PG1 represents an amine protecting group such as, for example, an acetyl group. In addition, interconversion of any of the substituents R1, R2, R3, B1, q, m, t, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-1, 1-7, 1-9, 1-11, and 1-12 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs.  
A suitably substituted aromatic amine of general formula (1-1), such as, for example, N- phenylacetamide, can be reacted with a suitable substituted dihydrofuran-2,5-dione (1-12), such as, for example, 3-methyldihydrofuran-2,5-dione, in the presence of a Lewis acid, such as, for example, aluminium trichloride, in a suitable solvent system, such as, for example, DMF, at temperatures ranging from - 20°C to boiling point of the respective solvent, preferably the reaction is carried out at 0°C, to furnish intermediates of general formula (1-13). Intermediates of general formula (1-13) can be converted to intermediates of general formula (1-14) by reaction with a hydrazine of the formula (1-7), such as, for example, hydrazine monohydrate, in a suitable solvent system, such as, for example, propan-1-ol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 0°C. Intermediates of general formula (1-14) can be reacted with a suitable Broensted acid, such as, for example, hydrochloric acid or sulphuric acid, at temperatures ranging from 0°C to boiling point of the respective Broensted acid, preferably the reaction is carried out at 100°C, to furnish intermediates of general formula (1-8). Intermediates of general formula (1-8) are treated with a carbonate of general formula (1-9), such as, for example, 1,1'-[carbonylbis(oxy)]dipyrrolidine-2,5-dione, in the presence of a suitable base, such as for example, N,N-dimethylpyridin-4-amine, in a suitable solvent system, such as, for example, DMF, at a temperature between 0°C and the boiling point of the respective solvent, preferably the reaction is carried out at room temperature to form the desired intermediate of general formula(1-10). Intermediates of general formula (1-10) can be converted to compounds of formula (Ia) by reaction with a suitably substituted amine of the general formula (1-11), such as, for example, 1-(pyridin-3-yl)piperazine, in the presence of a suitable base, such as, for example triethylamine, in a suitable solvent system, such as, for example, DMF, in a temperature range from 0°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. A route for the preparation of compounds of formula (1-8) is described in Scheme 3. Scheme 3
 
Figure imgf000197_0001
Scheme 3: Route for the preparation of compounds of formula (1-8), wherein R2, R3, V, W, Y and Z have the meaning as given for general formula (I), supra. X2 represents a leaving group such as for example a Cl, Br or I atom. In addition, interconversion of any of the substituents R2, R3, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W.
 
Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-1, 1-2, 1-4, 1-7, and 1-20 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. A suitably substituted aromatic halide of general formula (1-15), such as, for example, N- bromobenzene, can be reacted with a suitable substituted acid chloride (1-2), such as, for example, 2-chloropropanoyl chloride, in the presence of a Lewis acid, such as, for example, aluminium trichloride, in a suitable solvent system, such as, for example, dichloromethane, at temperatures ranging from - 20°C to boiling point of the respective solvent, preferably the reaction is carried out at 0°C, to furnish intermediates of general formula (1-16). Intermediates of general formula (1-16) can be converted to intermediates of general formula (1-17) by reaction with a suitably alkyl malonate of the general formula (1-4), such as, for example, dimethyl malonate, in the presence of a suitable base, such as, for example sodium hydride, in a suitable solvent system, such as, for example, DMF, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 0°C. Intermediates of general formula (1-17) can be reacted with a suitable Broensted acid, such as, for example, hydrochloric acid or sulphuric acid, at temperatures ranging from 0°C to boiling point of the respective Broensted acid, preferably the reaction is carried out at 100°C, to furnish intermediates of general formula (1-18). Intermediates of general formula (1-18) can be converted to intermediates of general formula (1-19) by reaction with a suitable hydrazine of the formula (1-7), such as, for example, hydrazine monohydrate, in a suitable solvent system, such as, for example, propan-1-ol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 80°C. Intermediates of general formula (1-19) can be reacted with a suitable substituted carbamate, such as, for example tert-butyl carbamate (1-20), in the presence of a suitable base, such as, for example caesium carbonate, and a suitable palladium catalyst, such as for example bis(dibenzylideneacetone)-palladium(0), in the presence of a suitable ligand, such as for example 9(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine), in a suitable solvent system, such as, for example, 1,4-dioxane, in a temperature range from room temperature to  
the boiling point of the respective solvent, preferably the reaction is carried out at at 110°C to furnish compounds of formula (1-21). Alternatively the following palladium catalysts can be used: allylpalladium chloride dimmer, dichlorobis(benzonitrile)palladium (II), palladium (II) acetate, palladium (II) chloride, tetrakis(triphenylphosphine)palladium (0), tris(dibenzylideneacetone)dipalladium (0), chloro(2'-amino-1,1'-biphenyl-2-yl)palladium(II) dimer, (2'-amino-1,1'-biphenyl-2-yl)methanesulfonatopalladium(II) dimer, trans-di(m- acetato)bis[o-(di-o-tolylphosphino)benzyl]dipalladium(II) [cataCXium® C], allylchloro[1,3- bis(2,4,6-trimethylphenyl)imidazol-2-ylidene]palladium(II), allylchloro[1,3-bis(2,6- diisopropylphenyl)imidazol-2-ylidene]palladium(II), chloro[(1,3-dimesitylimidazol-[1,3- bis(2,4,6-trimethylphenyl)-1,3-dihydro-2H-imidazol-2-ylidene](chloro){2- [(dimethylamino)methyl]phenyl}palladium, chloro[(1,2,3-N)-3-phenyl-2-propenyl][1,3-bis(2,6- di-iso-propylphenyl)imidazol-2-ylidene]palladium(II), [2-(acetylamino)phenyl]{1,3-bis[2,6- di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2-ylidene}chloropalladium, {1,3-bis[2,6- di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2-ylidene}(chloro){2- [(dimethylamino)methyl]phenyl} palladium, {1,3-bis[2,6-di(propan-2-yl)phenyl]-2,3-dihydro-1H- imidazol-2-yl}(dichloro)(3-chloropyridine-kappaN)palladium, [1,3-bis(2,6-diisopropylphenyl) imidazol-2-ylidene](3-chloropyridyl)palladium(II) dichloride, [2-(acetylamino)-4- methoxyphenyl]{1,3-bis[2,6-di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2- ylidene}chloropalladium, {1,3-bis[2,6-di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2- ylidene}(chloro){2-[(dimethylamino)methyl]-3,5-dimethoxyphenyl}palladium, dichloro[1,3- bis(2,6-di-3-pentylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II), dichloro(di-µ- chloro)bis[1,3-bis(2,6-di-iso-propylphenyl)imidazol-2-ylidene]dipalladium(II), 2-(2'-di-tert- butylphosphine)biphenylpalladium(II) acetate, chloro[dicyclohexyl(2',6'-dimethoxybiphenyl-2- yl)-lambda5-phosphanyl][2-(phenyl-kappaC2)ethanaminato-kappaN]palladium, [2-(2- aminoethyl)phenyl](chloro)palladium - di-tert-butyl[2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, {dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane}{2-[2- (methylazanidyl-kappaN)ethyl]phenyl-kappaC1}palladium, chloro(2-dicyclohexylphosphino- 2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl) palladium(II), [2',6'-bis(propan-2- yloxy)biphenyl-2-yl](dicyclohexyl)phosphane - [2-(2-aminoethyl)phenyl](chloro)palladium, [2- (2-aminoethyl)phenyl](chloro){dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]-lambda5- phosphanylidene}palladium, 2'-(dicyclohexylphosphanyl)-N,N,N',N'-tetramethylbiphenyl-2,6- diamine - (2'-aminobiphenyl-2-yl)(chloro)palladium, chloro(2-dicyclohexylphosphino-2',6'-di- iso-propoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II), [2'-(azanidyl- kappaN)biphenyl-2-yl-kappaC2](chloro){dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]- lambda5-phosphanyl}palladium, (2'-aminobiphenyl-2-yl)(methanesulfonato- kappaO)palladium - di-tert-butyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane, (2'-  
aminobiphenyl-2-yl)palladium(1+) methanesulfonate - di-tert-butyl[2',4',6'-tri(propan-2- yl)biphenyl-2-yl]phosphane, dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane - [2-(2-aminoethyl)phenyl](chloro)palladium, (2'-aminobiphenyl-2- yl)palladium(1+) methanesulfonate - 2'-(dicyclohexylphosphanyl)-N,N,N',N'- tetramethylbiphenyl-2,6-diamine, sodium 2'-(dicyclohexylphosphanyl)-2,6-dimethoxybiphenyl- 3-sulfonate - (2'-aminobiphenyl-2-yl)(chloro)palladium, chloro(2-dicyclohexylphosphino- 2',4',6'-tri-iso-propyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl]palladium(II), (2'-aminobiphenyl-2- yl)(methanesulfonato-kappaO)palladium - [2',6'-bis(propan-2-yloxy)biphenyl-2- yl](dicyclohexyl)phosphane, (2'-aminobiphenyl-2-yl)(methanesulfonato-kappaO)palladium - dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane, (2'-aminobiphenyl-2- yl)palladium(1+) methanesulfonate - dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane - (2'-aminobiphenyl-2-yl)(chloro)palladium, (2'-aminobiphenyl-2-yl)(methanesulfonato- kappaO)palladium - di-tert-butyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, (2'-aminobiphenyl-2-yl)(methanesulfonato-kappaO)palladium - dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane or the following ligands: racemic-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl, rac-BINAP, 1,1'-bis(diphenyl- phosphino)ferrocene, bis(2-diphenylphosphinophenyl)ether, di-tert-butylmethylphosphonium tetrafluoroborate, 2-(di-tert-butylphosphino)biphenyl, tri-tert-butylphosphonium tetrafluoroborate, tri-2-furylphosphine, tris(2,4-di-tert-butylphenyl)phosphite, tri-o- tolylphosphine, (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine), dicyclohexyl(2',4',6'-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, di-tert-butyl (2',4',6'- triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, di-tert-butyl(2',4',6'-triiso propylbiphenyl-2- yl)phosphine, dicyclohexyl(2',4',6'-triisopropylbiphenyl-2-yl) phosphine, di-tert-butyl(2',4',6'- triisopropyl-3-methoxy-6-methylbiphenyl-2-yl)phos-phine, di-tert-butyl(2',4',6'-triisopropyl- 3,4,5,6-tetramethylbiphenyl-2-yl) phosphine, adamantan-1-yl(adamantan-2-yl)(2',4',6'- triisopropyl-3,6-dimethoxybiphenyl-2-yl) phosphine, dicyclohexyl(2',6'-dimethoxybiphenyl-2- yl)phosphine, dicyclohexyl(2',6'-diisopropoxybiphenyl-2-yl)phosphine, 2'- (dicyclohexylphosphino)-N,N-dimethyl-biphenyl-2-amine, 2'-(di-tert-butylphosphino)-N,N- dimethylbiphenyl-2-amine, 2'-(di-phenylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6- diamine, di-tert-butyl(2',4',6'-tricyclohexyl-3,6-dimethoxybiphenyl-2-yl)phosphine, bis[3,5- bis(trifluoromethyl)phe-nyl] (2',4',6'-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, biphenyl-2-yl(di-tert-butyl)phosphine, dicyclohexyl(2'-methylbiphenyl-2-yl)phosphine, biphenyl-2-yl (dicyclohexyl)phosphine, 2'-(dicyclohexylphosphino)-N,N-dimethylbiphenyl-2- amine, 2'-(dicyclohexylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6-diamine, sodium 2'- (dicyclohexylphosphino)-2,6-diisopropylbiphenyl-4-sulfonate, sodium 2'-   (dicyclohexylphosphino)-2,6-dimethoxybiphenyl-3-sulfonate, 1,1'-binaphthalen-2-yl(di-tert- butyl)phosphine. Intermediates of general formula (1-21) can be converted to intermediates of general formula (1-8) by reaction with suitable Broensted acid, such as, for example trifluoroactic acid, in a suitable solvent system, such as, for example, dichloromethane, in a temperature range from – 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. A route for the preparation of compounds of formula (1-8) is described in Scheme 4.
Figure imgf000201_0001
Scheme 4: Route for the preparation of compounds of formula (1-8), wherein R2, R3, V, W, Y and Z have the meaning as given for general formula (I), supra. X2 represents a leaving group such as for example a Cl or Br atom or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). PG1 represents an amine protecting group, such as, for example, an acetyl group. In addition, interconversion of any of the substituents R2, R3, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the  
person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-7, 1-22, and 1-23 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. A suitably substituted aromatic ketone of general formula (1-22), such as, for example, N-(4- propionylphenyl)acetamide, can be reacted with a suitable substituted intermediate of general formula (1-23), such as, for example, ethyl bromoacetate, in the presence of a suitable base, such as, for example, lithium 1,1,1,3,3,3-hexamethyldisilazan-2-ide, in a suitable solvent system, such as, for example, THF, at temperatures ranging from - 100°C to boiling point of the respective solvent, preferably the reaction is carried out at - 78°C, to furnish intermediates of general formula (1-24). Intermediates of general formula (1-24) can be converted to intermediates of general formula (1-14) by reaction with a suitable hydrazine of the formula (1-7), such as, for example, hydrazine monohydrate, in a suitable solvent system, such as, for example, propan-1-ol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 0°C. Intermediates of general formula (1-14) can be reacted with a suitable Broensted acid, such as, for example, hydrochloric acid or sulphuric acid, at temperatures ranging from 0°C to boiling point of the respective Broensted acid, preferably the reaction is carried out at 100°C, to furnish intermediates of general formula (1-8). A route for the preparation of compounds of formula (1-26) is described in Scheme 5.
Scheme 5
 
Figure imgf000203_0001
Scheme 5: Route for the preparation of compounds of formula (1-26), wherein q, m, t and R1 have the meaning as given for general formula (I), supra. B2 represents a group such as, for example, a H, Cl or Br atom or an nitro group. In addition, interconversion of any of the substituents can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-11 and 1-25 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. Intermediates of general formula (1-11) can be converted to intermediates of general formula (1-26) by reaction with a suitable substituted carbamate of the general formula (1-25), such as, for example, 4-nitrophenyl carbamate, in a suitable solvent system, such as, for example, ethanol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 79°C. An alternative route for the preparation of compounds of formula (Ia) is described in Scheme 6.
Scheme 6
 
Figure imgf000204_0001
Scheme 6: Route for the preparation of compounds of formula (Ia), wherein R1, R2, R3, A, B, C, D, n , m, V, W, Y and Z have the meaning as given for general formula (I), supra. X2 represents a leaving group such as for example a Cl, Br or I atom or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). In addition, interconversion of any of the substituents R1, R2, R3, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Intermediates of general formula (1-19) can be reacted with a suitable urea of the general formula (1-26), such as, for example 4-(pyridin-3-yl)piperazine-1-carboxamide, in the presence of a suitable base, such as, for example caesium carbonate, and a suitable palladium catalyst, such as for example bis(dibenzylideneacetone)-palladium(0), in the presence of a suitable ligand, such as for example 9(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine), in a suitable solvent system, such as, for example, 1,4-dioxane, in a temperature range from room temperature to the boiling point of the respective solvent, preferably the reaction is carried out at at 110°C to furnish compounds of formula (Ia). Alternatively the following palladium catalysts can be used: allylpalladium chloride dimmer, dichlorobis(benzonitrile)palladium (II), palladium (II) acetate, palladium (II) chloride, tetrakis(triphenylphosphine)palladium (0), tris(dibenzylideneacetone)dipalladium (0), chloro(2'-amino-1,1'-biphenyl-2-yl)palladium(II) dimer, (2'-amino-1,1'-biphenyl-2-yl)methanesulfonatopalladium(II) dimer, trans-di(m-  
acetato)bis[o-(di-o-tolylphosphino)benzyl]dipalladium(II) [cataCXium® C], allylchloro[1,3- bis(2,4,6-trimethylphenyl)imidazol-2-ylidene]palladium(II), allylchloro[1,3-bis(2,6- diisopropylphenyl)imidazol-2-ylidene]palladium(II), chloro[(1,3-dimesitylimidazol-[1,3- bis(2,4,6-trimethylphenyl)-1,3-dihydro-2H-imidazol-2-ylidene](chloro){2- [(dimethylamino)methyl]phenyl}palladium, chloro[(1,2,3-N)-3-phenyl-2-propenyl][1,3-bis(2,6- di-iso-propylphenyl)imidazol-2-ylidene]palladium(II), [2-(acetylamino)phenyl]{1,3-bis[2,6- di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2-ylidene}chloropalladium, {1,3-bis[2,6- di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2-ylidene}(chloro){2- [(dimethylamino)methyl]phenyl} palladium, {1,3-bis[2,6-di(propan-2-yl)phenyl]-2,3-dihydro-1H- imidazol-2-yl}(dichloro)(3-chloropyridine-kappaN)palladium, [1,3-bis(2,6-diisopropylphenyl) imidazol-2-ylidene](3-chloropyridyl)palladium(II) dichloride, [2-(acetylamino)-4- methoxyphenyl]{1,3-bis[2,6-di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2- ylidene}chloropalladium, {1,3-bis[2,6-di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2- ylidene}(chloro){2-[(dimethylamino)methyl]-3,5-dimethoxyphenyl}palladium, dichloro[1,3- bis(2,6-di-3-pentylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II), dichloro(di-µ- chloro)bis[1,3-bis(2,6-di-iso-propylphenyl)imidazol-2-ylidene]dipalladium(II), 2-(2'-di-tert- butylphosphine)biphenylpalladium(II) acetate, chloro[dicyclohexyl(2',6'-dimethoxybiphenyl-2- yl)-lambda5-phosphanyl][2-(phenyl-kappaC2)ethanaminato-kappaN]palladium, [2-(2- aminoethyl)phenyl](chloro)palladium - di-tert-butyl[2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, {dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane}{2-[2- (methylazanidyl-kappaN)ethyl]phenyl-kappaC1}palladium, chloro(2-dicyclohexylphosphino- 2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl) palladium(II), [2',6'-bis(propan-2- yloxy)biphenyl-2-yl](dicyclohexyl)phosphane - [2-(2-aminoethyl)phenyl](chloro)palladium, [2- (2-aminoethyl)phenyl](chloro){dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]-lambda5- phosphanylidene}palladium, 2'-(dicyclohexylphosphanyl)-N,N,N',N'-tetramethylbiphenyl-2,6- diamine - (2'-aminobiphenyl-2-yl)(chloro)palladium, chloro(2-dicyclohexylphosphino-2',6'-di- iso-propoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II), [2'-(azanidyl- kappaN)biphenyl-2-yl-kappaC2](chloro){dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]- lambda5-phosphanyl}palladium, (2'-aminobiphenyl-2-yl)(methanesulfonato- kappaO)palladium - di-tert-butyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane, (2'- aminobiphenyl-2-yl)palladium(1+) methanesulfonate - di-tert-butyl[2',4',6'-tri(propan-2- yl)biphenyl-2-yl]phosphane, dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane - [2-(2-aminoethyl)phenyl](chloro)palladium, (2'-aminobiphenyl-2- yl)palladium(1+) methanesulfonate - 2'-(dicyclohexylphosphanyl)-N,N,N',N'- tetramethylbiphenyl-2,6-diamine, sodium 2'-(dicyclohexylphosphanyl)-2,6-dimethoxybiphenyl- 3-sulfonate - (2'-aminobiphenyl-2-yl)(chloro)palladium, chloro(2-dicyclohexylphosphino- 2',4',6'-tri-iso-propyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl]palladium(II), (2'-aminobiphenyl-2-  
yl)(methanesulfonato-kappaO)palladium - [2',6'-bis(propan-2-yloxy)biphenyl-2- yl](dicyclohexyl)phosphane, (2'-aminobiphenyl-2-yl)(methanesulfonato-kappaO)palladium - dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane, (2'-aminobiphenyl-2- yl)palladium(1+) methanesulfonate - dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane - (2'-aminobiphenyl-2-yl)(chloro)palladium, (2'-aminobiphenyl-2-yl)(methanesulfonato- kappaO)palladium - di-tert-butyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, (2'-aminobiphenyl-2-yl)(methanesulfonato-kappaO)palladium - dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane or the following ligands: racemic-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl, rac-BINAP, 1,1'-bis(diphenyl- phosphino)ferrocene, bis(2-diphenylphosphinophenyl)ether, di-tert-butylmethylphosphonium tetrafluoroborate, 2-(di-tert-butylphosphino)biphenyl, tri-tert-butylphosphonium tetrafluoroborate, tri-2-furylphosphine, tris(2,4-di-tert-butylphenyl)phosphite, tri-o- tolylphosphine, (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine), dicyclohexyl(2',4',6'-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, di-tert-butyl (2',4',6'- triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, di-tert-butyl(2',4',6'-triiso propylbiphenyl-2- yl)phosphine, dicyclohexyl(2',4',6'-triisopropylbiphenyl-2-yl) phosphine, di-tert-butyl(2',4',6'- triisopropyl-3-methoxy-6-methylbiphenyl-2-yl)phos-phine, di-tert-butyl(2',4',6'-triisopropyl- 3,4,5,6-tetramethylbiphenyl-2-yl) phosphine, adamantan-1-yl(adamantan-2-yl)(2',4',6'- triisopropyl-3,6-dimethoxybiphenyl-2-yl) phosphine, dicyclohexyl(2',6'-dimethoxybiphenyl-2- yl)phosphine, dicyclohexyl(2',6'-diisopropoxybiphenyl-2-yl)phosphine, 2'- (dicyclohexylphosphino)-N,N-dimethyl-biphenyl-2-amine, 2'-(di-tert-butylphosphino)-N,N- dimethylbiphenyl-2-amine, 2'-(di-phenylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6- diamine, di-tert-butyl(2',4',6'-tricyclohexyl-3,6-dimethoxybiphenyl-2-yl)phosphine, bis[3,5- bis(trifluoromethyl)phe-nyl] (2',4',6'-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, biphenyl-2-yl(di-tert-butyl)phosphine, dicyclohexyl(2'-methylbiphenyl-2-yl)phosphine, biphenyl-2-yl (dicyclohexyl)phosphine, 2'-(dicyclohexylphosphino)-N,N-dimethylbiphenyl-2- amine, 2'-(dicyclohexylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6-diamine, sodium 2'- (dicyclohexylphosphino)-2,6-diisopropylbiphenyl-4-sulfonate, sodium 2'- (dicyclohexylphosphino)-2,6-dimethoxybiphenyl-3-sulfonate, 1,1'-binaphthalen-2-yl(di-tert- butyl)phosphine. An alternative route for the preparation of compounds of formula (Ia) is described in Scheme 7.
 
Figure imgf000207_0001
Scheme 7: Route for the preparation of compounds of formula (I), wherein R1, R2, R3, q, m, t, V, W, Y and Z have the meaning as given for general formula (Ia), supra. L2 represents a group such as, for example, a H, Cl or Br atom or an nitro group. In addition, interconversion of any of the substituents R1, R2, R3, q, m, t, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs.
Compounds 1-11 and 1-27 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs.
  Intermediates of general formula (1-8) can be converted to intermediates of general formula
(1-28) by reaction with a suitably chloroformiate of the general formula (1-27), such as, for example, 4-nitrophenyl carbonochloridate, in the presence of a suitable base, such as, for example, triethylamine, in a suitable solvent system, such as, for example, toluene, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. Intermediates of general formula (1-28) can be converted to compounds of formula (Ia) by reaction with a suitably amine of the general formula (1-11), such as, for example, 1-(pyridin- 3-yl)piperazine, in a suitable solvent system, such as, for example, ethanol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 79°C. A route for the preparation of compounds of formula (1-8) is described in Scheme 8.
Figure imgf000208_0001
Scheme 8: Route for the preparation of compounds of formula (1-8), wherein R2, R3, V, W, Y and Z have the meaning as given for general formula (I), supra.
 
B5 represents a leaving group such as for example an aryl sulfonate such as for example p- toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). B3 and B4 represent a H atom or an substituted alkyl group. B3 and B4 can form a ring system. PG1 represents an amine protecting group as for example an acetyl group or a tert- butyloxycarbonyl group. In addition, interconversion of any of the substituents wherein R2, R3, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-7 and 1-32 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. A suitably substituted dihydrofuran-2,5-dione of general formula (1-12), such as, for example, 3-methyldihydrofuran-2,5-dione (1-22), can be reacted with a suitable hydrazine of formula (1-7), such as, for example, hydrazine monohydrate, in a suitable solvent system, such as, for example, acetonitrile, at temperatures ranging from 0°C to boiling point of the respective solvent, preferably the reaction is carried out at 90°C, to furnish intermediates of general formula (1-29). Intermediates of general formula (1-29) can be converted to intermediates of general formula (1-31) by reaction with a suitably acid anhydride, such as, for example, trifluoromethane sulfonic anhydride, in the presence of a suitable base, such as, for example, triethylamine, in a suitable solvent system, such as, for example, acetonitrile, in a temperature range from - 78°C to the boiling point of the respective solvent, preferably the reaction is carried out at - 20°C. Intermediates of general formula (1-31) can be reacted with a suitable boronic acid derivative of the general formula (1-32), such as, for example, {4-[(tert- butoxycarbonyl)amino]phenyl}boronic acid, in the presence of a suitable base, such as, for  
example sodium carbonate, and a suitable palladium catalyst, such as for example tetrakis(triphenylphosphine)palladium (0), in a suitable solvent system, such as, for example, 1,4-dioxane and water, in a temperature range from room temperature to the boiling point of the respective solvent, preferably the reaction is carried out at at 80°C to furnish compounds of formula (1-8). Alternatively the following palladium catalysts can be used: allylpalladium chloride dimmer, dichlorobis(benzonitrile)palladium (II), palladium (II) acetate, palladium (II) chloride, bis(dibenzylideneacetone)-palladium(0), tris(dibenzylideneacetone)dipalladium (0), chloro(2'-amino-1,1'-biphenyl-2-yl)palladium(II) dimer, (2'-amino-1,1'-biphenyl-2-yl)methanesulfonatopalladium(II) dimer, trans-di(m- acetato)bis[o-(di-o-tolylphosphino)benzyl]dipalladium(II) [cataCXium® C], allylchloro[1,3- bis(2,4,6-trimethylphenyl)imidazol-2-ylidene]palladium(II), allylchloro[1,3-bis(2,6- diisopropylphenyl)imidazol-2-ylidene]palladium(II), chloro[(1,3-dimesitylimidazol-[1,3- bis(2,4,6-trimethylphenyl)-1,3-dihydro-2H-imidazol-2-ylidene](chloro){2- [(dimethylamino)methyl]phenyl}palladium, chloro[(1,2,3-N)-3-phenyl-2-propenyl][1,3-bis(2,6- di-iso-propylphenyl)imidazol-2-ylidene]palladium(II), [2-(acetylamino)phenyl]{1,3-bis[2,6- di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2-ylidene}chloropalladium, {1,3-bis[2,6- di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2-ylidene}(chloro){2- [(dimethylamino)methyl]phenyl} palladium, {1,3-bis[2,6-di(propan-2-yl)phenyl]-2,3-dihydro-1H- imidazol-2-yl}(dichloro)(3-chloropyridine-kappaN)palladium, [1,3-bis(2,6-diisopropylphenyl) imidazol-2-ylidene](3-chloropyridyl)palladium(II) dichloride, [2-(acetylamino)-4- methoxyphenyl]{1,3-bis[2,6-di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2- ylidene}chloropalladium, {1,3-bis[2,6-di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2- ylidene}(chloro){2-[(dimethylamino)methyl]-3,5-dimethoxyphenyl}palladium, dichloro[1,3- bis(2,6-di-3-pentylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II), dichloro(di-µ- chloro)bis[1,3-bis(2,6-di-iso-propylphenyl)imidazol-2-ylidene]dipalladium(II), 2-(2'-di-tert- butylphosphine)biphenylpalladium(II) acetate, chloro[dicyclohexyl(2',6'-dimethoxybiphenyl-2- yl)-lambda5-phosphanyl][2-(phenyl-kappaC2)ethanaminato-kappaN]palladium, [2-(2- aminoethyl)phenyl](chloro)palladium - di-tert-butyl[2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, {dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane}{2-[2- (methylazanidyl-kappaN)ethyl]phenyl-kappaC1}palladium, chloro(2-dicyclohexylphosphino- 2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl) palladium(II), [2',6'-bis(propan-2- yloxy)biphenyl-2-yl](dicyclohexyl)phosphane - [2-(2-aminoethyl)phenyl](chloro)palladium, [2- (2-aminoethyl)phenyl](chloro){dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]-lambda5- phosphanylidene}palladium, 2'-(dicyclohexylphosphanyl)-N,N,N',N'-tetramethylbiphenyl-2,6- diamine - (2'-aminobiphenyl-2-yl)(chloro)palladium, chloro(2-dicyclohexylphosphino-2',6'-di- iso-propoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II), [2'-(azanidyl- kappaN)biphenyl-2-yl-kappaC2](chloro){dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]-  
lambda5-phosphanyl}palladium, (2'-aminobiphenyl-2-yl)(methanesulfonato- kappaO)palladium - di-tert-butyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane, (2'- aminobiphenyl-2-yl)palladium(1+) methanesulfonate - di-tert-butyl[2',4',6'-tri(propan-2- yl)biphenyl-2-yl]phosphane, dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane - [2-(2-aminoethyl)phenyl](chloro)palladium, (2'-aminobiphenyl-2- yl)palladium(1+) methanesulfonate - 2'-(dicyclohexylphosphanyl)-N,N,N',N'- tetramethylbiphenyl-2,6-diamine, sodium 2'-(dicyclohexylphosphanyl)-2,6-dimethoxybiphenyl- 3-sulfonate - (2'-aminobiphenyl-2-yl)(chloro)palladium, chloro(2-dicyclohexylphosphino- 2',4',6'-tri-iso-propyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl]palladium(II), (2'-aminobiphenyl-2- yl)(methanesulfonato-kappaO)palladium - [2',6'-bis(propan-2-yloxy)biphenyl-2- yl](dicyclohexyl)phosphane, (2'-aminobiphenyl-2-yl)(methanesulfonato-kappaO)palladium - dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane, (2'-aminobiphenyl-2- yl)palladium(1+) methanesulfonate - dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane - (2'-aminobiphenyl-2-yl)(chloro)palladium, (2'-aminobiphenyl-2-yl)(methanesulfonato- kappaO)palladium - di-tert-butyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, (2'-aminobiphenyl-2-yl)(methanesulfonato-kappaO)palladium - dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane or the following ligands: racemic-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl, rac-BINAP, 1,1'-bis(diphenyl- phosphino)ferrocene, bis(2-diphenylphosphinophenyl)ether, di-tert-butylmethylphosphonium tetrafluoroborate, 2-(di-tert-butylphosphino)biphenyl, tri-tert-butylphosphonium tetrafluoroborate, tri-2-furylphosphine, tris(2,4-di-tert-butylphenyl)phosphite, tri-o- tolylphosphine, (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine), dicyclohexyl(2',4',6'-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, di-tert-butyl (2',4',6'- triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, di-tert-butyl(2',4',6'-triiso propylbiphenyl-2- yl)phosphine, dicyclohexyl(2',4',6'-triisopropylbiphenyl-2-yl) phosphine, di-tert-butyl(2',4',6'- triisopropyl-3-methoxy-6-methylbiphenyl-2-yl)phos-phine, di-tert-butyl(2',4',6'-triisopropyl- 3,4,5,6-tetramethylbiphenyl-2-yl) phosphine, adamantan-1-yl(adamantan-2-yl)(2',4',6'- triisopropyl-3,6-dimethoxybiphenyl-2-yl) phosphine, dicyclohexyl(2',6'-dimethoxybiphenyl-2- yl)phosphine, dicyclohexyl(2',6'-diisopropoxybiphenyl-2-yl)phosphine, 2'- (dicyclohexylphosphino)-N,N-dimethyl-biphenyl-2-amine, 2'-(di-tert-butylphosphino)-N,N- dimethylbiphenyl-2-amine, 2'-(di-phenylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6- diamine, di-tert-butyl(2',4',6'-tricyclohexyl-3,6-dimethoxybiphenyl-2-yl)phosphine, bis[3,5- bis(trifluoromethyl)phe-nyl] (2',4',6'-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, biphenyl-2-yl(di-tert-butyl)phosphine, dicyclohexyl(2'-methylbiphenyl-2-yl)phosphine, biphenyl-2-yl (dicyclohexyl)phosphine, 2'-(dicyclohexylphosphino)-N,N-dimethylbiphenyl-2-   amine, 2'-(dicyclohexylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6-diamine, sodium 2'- (dicyclohexylphosphino)-2,6-diisopropylbiphenyl-4-sulfonate, sodium 2'- (dicyclohexylphosphino)-2,6-dimethoxybiphenyl-3-sulfonate, 1,1'-binaphthalen-2-yl(di-tert- butyl)phosphine. Intermediates of general formula (1-14) can be reacted with a suitable Broensted acid, such as, for example, trifluoroacetic acid, in a suitable solvent system, such as, for example, dichloromethane, at temperatures ranging from 0°C to boiling point of the respective solvent, preferably the reaction is carried out at room temperature, to furnish intermediates of general formula (1-8). An route for the preparation of compounds of formula (1-33) is described in Scheme 9.
Figure imgf000212_0001
Scheme 9: Route for the preparation of compounds of formula (1-8), wherein L1, R2, R3, V, W, Y and Z have the meaning as given for general formula (I), supra. X1 represents a leaving group such as for example a Cl, Br or I, or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). In addition, interconversion of any of the substituents R1, R2, L1, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of  
functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-7 and 1-35 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. Intermediates of general formula (1-6) can be converted to intermediates of general formula (1-34) by reaction with a suitable hydrazine of the general formula (1-7), such as, for example, hydrazine hydrate (1:1), in a suitable solvent system, such as, for example, propan-1-ol, in a temperature range from 0°C to the boiling point of the respective solvent, preferably the reaction is carried out at 100°C. Intermediates of general formula (1-34) are treated with an intermediate of general formula (1- 35), such as, for example, ethyl trifluoromethanesulfonate, in the presence of a suitable base, such as for example, sodium hydride, in the presence of a suitable phase transfer catalyst, such as for example, N,N,N-tributylbutan-1-aminium iodide in a suitable solvent system, such as, for example, DMF, at a temperature between 0°C and the boiling point of the respective solvent, preferably the reaction is carried out at room temperature to form the desired intermediate of general formula(1-8). One route for the preparation of compounds of formula (1-44) is described in Scheme 10.
Scheme 10
 
Figure imgf000214_0001
Scheme 10: Route for the preparation of compounds of formula (1-44), wherein V, W, Y and Z have the meaning as given for general formula (I), supra. PG1 represents an amine protecting group as for example an acetyl group. In addition, interconversion of any of the substituents V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs.  
Compounds 1-1, 1-36, 1-37, 1-38 and 1-41 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. A suitably substituted aromatic amine of general formula (1-1), such as, for example, N- phenylacetamide, can be reacted with a suitable substituted acid chloride (1-36), such as, for example, propanoyl chloride, in the presence of a Lewis acid, such as, for example, aluminium trichloride, in a suitable solvent system, such as, for example, dichloromethane, at temperatures ranging from - 20°C to boiling point of the respective solvent, preferably the reaction is carried out at 0°C, to furnish intermediates of general formula (1-37).
Intermediates of general formula (1-37) can be converted to intermediates of general formula (1-39) by reaction with a suitably glyoxylic acid derivative of the general formula (1-38), such as, for example, glyoxylic acid monohydrate, in the presence of a suitable base, such as, for example sodium hydroxide, in the presence of a suitable phase transfer catalyst, such as, for example benzyltriethylammonium chloride, in a suitable solvent system, such as, for example, methanol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. Intermediates of general formula (1-39) can be reacted with a suitable Broensted acid, such as, for example, hydrochloric acid, in a suitable solvent system, such as, for example, methanol, at temperatures ranging from - 20°C to boiling point of the respective Broensted acid, preferably the reaction is carried out at 0°C, to furnish intermediates of general formula (1-40). Intermediates of general formula (1-40) can be converted to intermediates of general formula (1-42) by reaction with a suitably sulfonyl chloride of the general formula (1-41), such as, for example, methanesulfonyl chloride, in the presence of a suitable base, such as, for example triethylamine, in a suitable solvent system, such as, for example, trichloromethane, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 0°C. Intermediates of general formula (1-42) are treated with a suitable base, such as, for example DBU, in a suitable solvent system, such as, for example, toluene, at a temperature between 0°C and the boiling point of the respective solvent, preferably the reaction is carried out at 120 °C to form the desired intermediate of general formula(1-43).   Intermediates of general formula (1-43) can be converted to intermediates of general formula
(1-44) by reaction with a suitable base, such as, for example sodium hydroxide, in a suitable solvent system, such as, for example, ethanol, in a temperature range from 0°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. One route for the preparation of compounds of formulae (1-47 and 1-48) is described in Scheme 11.
Figure imgf000216_0001
Scheme 10: Route for the preparation of compounds of formulae (1-47 and 1-48), wherein R1, V, W, Y and Z have the meaning as given for general formula (I), supra. PG1 represents an amine protecting group as for example an acetyl group. In addition, interconversion of any of the substituents V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W.  
Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-7 is either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. Intermediates of general formula (1-44) can be hydrogenated at a suitable hydrogen pressure, such as, for example, 80 atmospheres, in the presence of a chiral catalyst, such as, for example, Ru(OAc)2(S-BINAP), in a suitable solvent system, such as, for example, methanol, at temperatures ranging from - 20°C to boiling point of the respective solvent, preferably the reaction is carried out at room temperature, to furnish intermediates of general formula (1-45). Alternatively the following palladium catalysts can be used: [Ru(OAc)2(p-cymene)] or the following ligands: (R)-(+)-2,2'-Bis(diphenylphosphino)-6,6'-dimethoxy-1,1'-biphenyl ( (R)-MeO-BIPHEP); (S)-(+)- 2,2'-Bis(diphenylphosphino)-6,6'-dimethoxy-1,1'-biphenyl ( (S)-MeO-BIPHEP); (R)-(+)-2,2'- Bis(di-2-furanylphosphino)-6,6'-dimethoxy-1,1'-biphenyl; (S)-(+)-2,2'-Bis(di-2- furanylphosphino)-6,6'-dimethoxy-1,1'-biphenyl; (R)-(+)-2,2'-Bis[di(3,5-xylyl)phosphino]-6,6'- dimethoxy-1,1'-biphenyl; (S)-(+)-2,2'-Bis[di(3,5-xylyl)phosphino]-6,6'-dimethoxy-1,1'-biphenyl; (R)-(+)-2,2'-Bis[di(3,5-di-t-butyl-4-methoxyphenyl)phosphino]-6,6'-dimethoxy-1,1'-biphenyl, (S)-(+)-2,2'-Bis[di(3,5-di-t-butyl-4-methoxyphenyl)phosphino]-6,6'-dimethoxy-1,1'-biphenyl, (R)-(+)-2,2'-Bis(di-i-propylphosphino)-6,6'-dimethoxy-1,1'-biphenyl; (S)-(+)-2,2'-Bis(di-i- propylphosphino)-6,6'-dimethoxy-1,1'-biphenyl; (R)-(-)-2,2'-Bis[di(3,5-di-t- butylphenyl)phosphino]-6,6'-dimethoxy-1,1'-biphenyl; (S)-(-)-2,2'-Bis[di(3,5-di-t- butylphenyl)phosphino]-6,6'-dimethoxy-1,1'-biphenyl; (R)-(-)-5,5'-Bis(diphenylphosphino)-4,4'- bi-1,3-benzodioxole; (S)-(-)-5,5'-Bis(diphenylphosphino)-4,4'-bi-1,3-benzodioxole; (2R,3R)-(-)- Bis(diphenylphosphino)butane ((R,R)-CHIRAPHOS); (2S,3S)-(-)- Bis(diphenylphosphino)butane ((S,S)-CHIRAPHOS); (R)-(-)-5,5'-Bis(diphenylphosphino)-4,4'- bi-1,3-benzodioxole; (S)-(-)-5,5'-Bis(diphenylphosphino)-4,4'-bi-1,3-benzodioxole; (R)-(+)- 4,12-Bis(diphenylphosphino)-[2.2]-paracyclophane ( (R)-PHANEPHOS); (S)-(+)-4,12- Bis(diphenylphosphino)-[2.2]-paracyclophane ( (S)-PHANEPHOS); (R)-(+)-2,2'- Bis(diphenylphosphino)-6,6'-dimethyl-1,1'-biphenyl; (S)-(+)-2,2'-Bis(diphenylphosphino)-6,6'- dimethyl-1,1'-biphenyl; (R)-(4,4',5,5'-tetramethyl-3,3'-bithiene-2,2'- diyl)bis(diphenylphosphine); (S)-(4,4',5,5'-tetramethyl-3,3'-bithiene-2,2'- diyl)bis(diphenylphosphine).
 
Chiral hydrogenations are described in Asymmetric Catalysis on Industrial Scale, Challenges, Approaches and Solutions. Blaser, H.-U.: Federsel, H.-J.: Eds, Wiley-VCH, Weinheim, 2nd Edition, 2010.
Intermediates of general formula (1-45) can be converted to intermediates of general formula (1-46) by reaction with a suitable hydrazine of the formula (1-7), such as, for example, hydrazine monohydrate, in a suitable solvent system, such as, for example, propan-1-ol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 80°C. Intermediates of general formula (1-44) can be hydrogenated at a suitable hydrogen pressure, such as, for example, 80 atmospheres, in the presence of a chiral catalyst, such as, for example, Ru(OAc)2(S-BINAP), in a suitable solvent system, such as, for example, methanol, at temperatures ranging from - 20°C to boiling point of the respective solvent, preferably the reaction is carried out at room temperature, to furnish intermediates of general formula (1-47). Alternatively the following palladium catalysts can be used: [Ru(OAc)2(p-cymene)] or the following ligands: (R)-(+)-2,2'-Bis(diphenylphosphino)-6,6'-dimethoxy-1,1'-biphenyl ( (R)-MeO-BIPHEP); (S)-(+)- 2,2'-Bis(diphenylphosphino)-6,6'-dimethoxy-1,1'-biphenyl ( (S)-MeO-BIPHEP); (R)-(+)-2,2'- Bis(di-2-furanylphosphino)-6,6'-dimethoxy-1,1'-biphenyl; (S)-(+)-2,2'-Bis(di-2- furanylphosphino)-6,6'-dimethoxy-1,1'-biphenyl; (R)-(+)-2,2'-Bis[di(3,5-xylyl)phosphino]-6,6'- dimethoxy-1,1'-biphenyl; (S)-(+)-2,2'-Bis[di(3,5-xylyl)phosphino]-6,6'-dimethoxy-1,1'-biphenyl; (R)-(+)-2,2'-Bis[di(3,5-di-t-butyl-4-methoxyphenyl)phosphino]-6,6'-dimethoxy-1,1'-biphenyl, (S)-(+)-2,2'-Bis[di(3,5-di-t-butyl-4-methoxyphenyl)phosphino]-6,6'-dimethoxy-1,1'-biphenyl, (R)-(+)-2,2'-Bis(di-i-propylphosphino)-6,6'-dimethoxy-1,1'-biphenyl; (S)-(+)-2,2'-Bis(di-i- propylphosphino)-6,6'-dimethoxy-1,1'-biphenyl; (R)-(-)-2,2'-Bis[di(3,5-di-t- butylphenyl)phosphino]-6,6'-dimethoxy-1,1'-biphenyl; (S)-(-)-2,2'-Bis[di(3,5-di-t- butylphenyl)phosphino]-6,6'-dimethoxy-1,1'-biphenyl; (R)-(-)-5,5'-Bis(diphenylphosphino)-4,4'- bi-1,3-benzodioxole; (S)-(-)-5,5'-Bis(diphenylphosphino)-4,4'-bi-1,3-benzodioxole; (2R,3R)-(-)- Bis(diphenylphosphino)butane ((R,R)-CHIRAPHOS); (2S,3S)-(-)- Bis(diphenylphosphino)butane ((S,S)-CHIRAPHOS); (R)-(-)-5,5'-Bis(diphenylphosphino)-4,4'- bi-1,3-benzodioxole; (S)-(-)-5,5'-Bis(diphenylphosphino)-4,4'-bi-1,3-benzodioxole; (R)-(+)- 4,12-Bis(diphenylphosphino)-[2.2]-paracyclophane ( (R)-PHANEPHOS); (S)-(+)-4,12- Bis(diphenylphosphino)-[2.2]-paracyclophane ( (S)-PHANEPHOS); (R)-(+)-2,2'- Bis(diphenylphosphino)-6,6'-dimethyl-1,1'-biphenyl; (S)-(+)-2,2'-Bis(diphenylphosphino)-6,6'- dimethyl-1,1'-biphenyl; (R)-(4,4',5,5'-tetramethyl-3,3'-bithiene-2,2'-   diyl)bis(diphenylphosphine); (S)-(4,4',5,5'-tetramethyl-3,3'-bithiene-2,2'- diyl)bis(diphenylphosphine). Chiral hydrogenations are described in Asymmetric Catalysis on Industrial Scale, Challenges, Approaches and Solutions. Blaser, H.-U.: Federsel, H.-J.: Eds, Wiley-VCH, Weinheim, 2nd Edition, 2010.
Intermediates of general formula (1-47) can be converted to intermediates of general formula (1-48) by reaction with a suitable hydrazine of the general formula (1-7), such as, for example, hydrazine monohydrate, in a suitable solvent system, such as, for example, propan-1-ol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 80°C. One route for the preparation of compounds of formula (1-14) is described in Scheme 12.
Figure imgf000219_0001
Scheme 12: Route for the preparation of compounds of formula (1-14), wherein PG1, R2, R3, V, W, Y and Z have the meaning as given for general formula (I), supra.  
X3 represents a halogen atom such as for example a Cl or Br atom. PG1 represents an amine protecting group as for example a fluorenylmethyloxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl or tert-butyloxycarbonyl group. In addition, interconversion of any of the substituents PG1, R2, R3, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-7, 1-49, 1-50 and 1-54 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. Compounds of formula (1-49) are reacted with a compound of formula (1-50) as mentioned above with a peptide coupling agent, for example N-(3-Dimethylaminopropyl)-N¢- ethylcarbodiimide hydrochloride, in the presence of 1-hydroxy-7-azabenzotriazole in a suitable solvent, such as, for example, N,N-dimethylformamid, in the presence of a suitable base, such as, for example, triethylamine in a temperature range from– 10 °C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature, to furnish compounds of formula (1-51).
Appropriate peptide synthesis methods and their applications are well-known to the person skilled in the art (see for example N. Leo Benoitin in Chemistry of Peptide Synthesis, CRC Press 2005; John Jones in Amino Acids and Peptide Synthesis, Oxford University Press, 2002 and Norbert Sewald and Hans-Dieter Jakubke in Peptides: Chemistry and Biology, Wiley-VCH, 2009).
Intermediates of general formula (1-51) can be converted to intermediates of general formula (1-53) by reaction with a suitably Grignard reagent of the general formula (1-52), such as, for example, benzylmagnesium chloride, in a suitable solvent system, such as, for example, tetrahydrofurane, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 0 °C.  
Intermediates of general formula (1-53) can be converted to intermediates of general formula (1-55) by reaction with a suitably acetic acid derivative of the general formula (1-54), such as, for example, ethyl bromoacetate, in the presence of a suitable base, such as, for example, sodium hydride, in a suitable solvent system, such as, for example, N,N-dimethylformamide, in a temperature range from - 80°C to the boiling point of the respective solvent, preferably the reaction is carried out at - 40 °C. Intermediates of general formula (1-55) can be converted to intermediates of general formula (1-56) by reaction with a suitably base, such as, for example, sodium hydroxide, in a suitable solvent system, such as, for example, methanol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. Intermediates of general formula (1-56) can be converted to intermediates of general formula (1-57) by reaction with a suitable hydrazine of the formula (1-7), such as, for example, hydrazine monohydrate, in a suitable solvent system, such as, for example, ethanol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 80°C. Intermediates of general formula (1-57) can be converted to intermediates of general formula (1-8) by reaction with a suitable Broensted acid, such as, for example trifluoroacetic acid, in a suitable solvent system, such as, for example, dichloromethane, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 0 °C. One route for the preparation of compounds of formula (1-19) is described in Scheme 13. Scheme 13
 
Figure imgf000222_0001
Scheme 13: Route for the preparation of compounds of formula (1-19), wherein R2, R3, V, W, Y and Z have the meaning as given for general formula (I), supra (R9 represents hydrogen is not shown in the above chemical structures). X2 represents a leaving group such as for example a Cl, Br or I atom and X3 represents a halogen atom such as for example a Cl or Br atom. In addition, interconversion of any of the substituents R2, R3, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-7, 1-49, 1-50 and 1-54 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs.
 
Compounds of formula (1-49) are reacted with a compound of formula (1-50) as mentioned above with a peptide coupling agent, for example N-(3-Dimethylaminopropyl)-N¢- ethylcarbodiimide hydrochloride, in the presence of 1-hydroxy-7-azabenzotriazole in a suitable solvent, such as, for example, N,N-dimethylformamid, in the presence of a suitable base, such as, for example, triethylamine in a temperature range from– 10 °C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature, to furnish compounds of formula (1-51).
Appropriate peptide synthesis methods and their applications are well-known to the person skilled in the art (see for example N. Leo Benoitin in Chemistry of Peptide Synthesis, CRC Press 2005; John Jones in Amino Acids and Peptide Synthesis, Oxford University Press, 2002 and Norbert Sewald and Hans-Dieter Jakubke in Peptides: Chemistry and Biology, Wiley-VCH, 2009).
Intermediates of general formula (1-51) can be converted to intermediates of general formula (1-53) by reaction with a suitably Grignard reagent of the general formula (1-52), such as, for example, benzylmagnesium chloride, in a suitable solvent system, such as, for example, tetrahydrofurane, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 0 °C. Intermediates of general formula (1-53) can be converted to intermediates of general formula (1-55) by reaction with a suitably acetic acid derivative of the general formula (1-54), such as, for example, ethyl bromoacetate, in the presence of a suitable base, such as, for example, sodium hydride, in a suitable solvent system, such as, for example, N,N-dimethylformamide, in a temperature range from - 80°C to the boiling point of the respective solvent, preferably the reaction is carried out at - 40 °C. Intermediates of general formula (1-55) can be converted to intermediates of general formula (1-18) by reaction with a suitably base, such as, for example, sodium hydroxide, in a suitable solvent system, such as, for example, methanol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. Intermediates of general formula (1-18) can be converted to intermediates of general formula (1-19) by reaction with a suitable hydrazine of the general formula (1-7), such as, for example, hydrazine monohydrate, in a suitable solvent system, such as, for example, ethanol, in a   temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 80°C. A route for the preparation of compounds of formula (Ia) is described in Scheme 14. Scheme 14
Figure imgf000224_0001
Scheme 14: Route for the preparation of compounds of formula (Ia), wherein R1, R2, R9, n, m, A, B, C, D, Q, V, W, Y and Z have the meaning as given for general formula (I), supra.  
X2 represents a leaving group such as for example a Cl or Br atom or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). B1 represents a leaving group such as, for example, a haloalkyl such as, for example, trichloromethyl, or a imide such as, for example, pyrrolidine-2,5-dione or 4-nitrophenyl. In addition, interconversion of any of the substituents R1, R2, R3, B1, A, B, C, D, Q, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-7, 1-9, 1-11, 1-23, 1-56 and 1-59 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. A suitably substituted aromatic ketone of general formula (1-56), such as, for example, 1-(4- bromophenyl)-2-methylpropan-1-one, can be reacted with a suitable substituted intermediate of general formula (1-23), such as, for example, ethyl bromoacetate, in the presence of a suitable base, such as, for example, lithium 1,1,1,3,3,3-hexamethyldisilazan-2-ide, in a suitable solvent system, such as, for example, THF, at temperatures ranging from - 100°C to boiling point of the respective solvent, preferably the reaction is carried out at - 78°C, to furnish intermediates of general formula (1-57). Intermediates of general formula (1-57) can be converted to intermediates of general formula (1-58) by reaction with a suitably hydrazine of the general formula (1-7), such as, for example, methylhydrazine, in a suitable solvent system, such as, for example, propan-1-ol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 0°C. Intermediates of general formula (1-58) can be reacted with a suitable substituted imine, such as, for example 1,1-diphenylmethanimine (1-59), in the presence of a suitable base, such as, for example caesium carbonate, and a suitable palladium catalyst, such as for example bis(dibenzylideneacetone)-palladium(0), in the presence of a suitable ligand, such as for  
example 9(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine), in a suitable solvent system, such as, for example, 1,4-dioxane, in a temperature range from room temperature to the boiling point of the respective solvent, preferably the reaction is carried out at at 110°C and subsequent treatment with a suitable Broensted acid, such as, for example, hydrochloric acid or sulphuric acid, at temperatures ranging from 0 °C to the boiling point of the respective Broensted acid, preferably the reaction is carried out at 100 °C, to furnish compounds of formula (1-60). Alternatively the following palladium catalysts can be used: allylpalladium chloride dimmer, dichlorobis(benzonitrile)palladium (II), palladium (II) acetate, palladium (II) chloride, tetrakis(triphenylphosphine)palladium (0), tris(dibenzylideneacetone)dipalladium (0), chloro(2'-amino-1,1'-biphenyl-2-yl)palladium(II) dimer, (2'-amino-1,1'-biphenyl-2-yl)methanesulfonatopalladium(II) dimer, trans-di(m- acetato)bis[o-(di-o-tolylphosphino)benzyl]dipalladium(II) [cataCXium® C], allylchloro[1,3- bis(2,4,6-trimethylphenyl)imidazol-2-ylidene]palladium(II), allylchloro[1,3-bis(2,6- diisopropylphenyl)imidazol-2-ylidene]palladium(II), chloro[(1,3-dimesitylimidazol-[1,3- bis(2,4,6-trimethylphenyl)-1,3-dihydro-2H-imidazol-2-ylidene](chloro){2- [(dimethylamino)methyl]phenyl}palladium, chloro[(1,2,3-N)-3-phenyl-2-propenyl][1,3-bis(2,6- di-iso-propylphenyl)imidazol-2-ylidene]palladium(II), [2-(acetylamino)phenyl]{1,3-bis[2,6- di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2-ylidene}chloropalladium, {1,3-bis[2,6- di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2-ylidene}(chloro){2- [(dimethylamino)methyl]phenyl} palladium, {1,3-bis[2,6-di(propan-2-yl)phenyl]-2,3-dihydro-1H- imidazol-2-yl}(dichloro)(3-chloropyridine-kappaN)palladium, [1,3-bis(2,6-diisopropylphenyl) imidazol-2-ylidene](3-chloropyridyl)palladium(II) dichloride, [2-(acetylamino)-4- methoxyphenyl]{1,3-bis[2,6-di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2- ylidene}chloropalladium, {1,3-bis[2,6-di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2- ylidene}(chloro){2-[(dimethylamino)methyl]-3,5-dimethoxyphenyl}palladium, dichloro[1,3- bis(2,6-di-3-pentylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II), dichloro(di-µ- chloro)bis[1,3-bis(2,6-di-iso-propylphenyl)imidazol-2-ylidene]dipalladium(II), 2-(2'-di-tert- butylphosphine)biphenylpalladium(II) acetate, chloro[dicyclohexyl(2',6'-dimethoxybiphenyl-2- yl)-lambda5-phosphanyl][2-(phenyl-kappaC2)ethanaminato-kappaN]palladium, [2-(2- aminoethyl)phenyl](chloro)palladium - di-tert-butyl[2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, {dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane}{2-[2- (methylazanidyl-kappaN)ethyl]phenyl-kappaC1}palladium, chloro(2-dicyclohexylphosphino- 2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl) palladium(II), [2',6'-bis(propan-2- yloxy)biphenyl-2-yl](dicyclohexyl)phosphane - [2-(2-aminoethyl)phenyl](chloro)palladium, [2- (2-aminoethyl)phenyl](chloro){dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]-lambda5- phosphanylidene}palladium, 2'-(dicyclohexylphosphanyl)-N,N,N',N'-tetramethylbiphenyl-2,6- diamine - (2'-aminobiphenyl-2-yl)(chloro)palladium, chloro(2-dicyclohexylphosphino-2',6'-di-  
iso-propoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II), [2'-(azanidyl- kappaN)biphenyl-2-yl-kappaC2](chloro){dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]- lambda5-phosphanyl}palladium, (2'-aminobiphenyl-2-yl)(methanesulfonato- kappaO)palladium - di-tert-butyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane, (2'- aminobiphenyl-2-yl)palladium(1+) methanesulfonate - di-tert-butyl[2',4',6'-tri(propan-2- yl)biphenyl-2-yl]phosphane, dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane - [2-(2-aminoethyl)phenyl](chloro)palladium, (2'-aminobiphenyl-2- yl)palladium(1+) methanesulfonate - 2'-(dicyclohexylphosphanyl)-N,N,N',N'- tetramethylbiphenyl-2,6-diamine, sodium 2'-(dicyclohexylphosphanyl)-2,6-dimethoxybiphenyl- 3-sulfonate - (2'-aminobiphenyl-2-yl)(chloro)palladium, chloro(2-dicyclohexylphosphino- 2',4',6'-tri-iso-propyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl]palladium(II), (2'-aminobiphenyl-2- yl)(methanesulfonato-kappaO)palladium - [2',6'-bis(propan-2-yloxy)biphenyl-2- yl](dicyclohexyl)phosphane, (2'-aminobiphenyl-2-yl)(methanesulfonato-kappaO)palladium - dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane, (2'-aminobiphenyl-2- yl)palladium(1+) methanesulfonate - dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane - (2'-aminobiphenyl-2-yl)(chloro)palladium, (2'-aminobiphenyl-2-yl)(methanesulfonato- kappaO)palladium - di-tert-butyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, (2'-aminobiphenyl-2-yl)(methanesulfonato-kappaO)palladium - dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane or the following ligands: racemic-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl, rac-BINAP, 1,1'-bis(diphenyl- phosphino)ferrocene, bis(2-diphenylphosphinophenyl)ether, di-tert-butylmethylphosphonium tetrafluoroborate, 2-(di-tert-butylphosphino)biphenyl, tri-tert-butylphosphonium tetrafluoroborate, tri-2-furylphosphine, tris(2,4-di-tert-butylphenyl)phosphite, tri-o- tolylphosphine, (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine), dicyclohexyl(2',4',6'-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, di-tert-butyl (2',4',6'- triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, di-tert-butyl(2',4',6'-triiso propylbiphenyl-2- yl)phosphine, dicyclohexyl(2',4',6'-triisopropylbiphenyl-2-yl) phosphine, di-tert-butyl(2',4',6'- triisopropyl-3-methoxy-6-methylbiphenyl-2-yl)phos-phine, di-tert-butyl(2',4',6'-triisopropyl- 3,4,5,6-tetramethylbiphenyl-2-yl) phosphine, adamantan-1-yl(adamantan-2-yl)(2',4',6'- triisopropyl-3,6-dimethoxybiphenyl-2-yl) phosphine, dicyclohexyl(2',6'-dimethoxybiphenyl-2- yl)phosphine, dicyclohexyl(2',6'-diisopropoxybiphenyl-2-yl)phosphine, 2'- (dicyclohexylphosphino)-N,N-dimethyl-biphenyl-2-amine, 2'-(di-tert-butylphosphino)-N,N- dimethylbiphenyl-2-amine, 2'-(di-phenylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6- diamine, di-tert-butyl(2',4',6'-tricyclohexyl-3,6-dimethoxybiphenyl-2-yl)phosphine, bis[3,5- bis(trifluoromethyl)phe-nyl] (2',4',6'-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine,  
biphenyl-2-yl(di-tert-butyl)phosphine, dicyclohexyl(2'-methylbiphenyl-2-yl)phosphine, biphenyl-2-yl (dicyclohexyl)phosphine, 2'-(dicyclohexylphosphino)-N,N-dimethylbiphenyl-2- amine, 2'-(dicyclohexylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6-diamine, sodium 2'- (dicyclohexylphosphino)-2,6-diisopropylbiphenyl-4-sulfonate, sodium 2'- (dicyclohexylphosphino)-2,6-dimethoxybiphenyl-3-sulfonate, 1,1'-binaphthalen-2-yl(di-tert- butyl)phosphine.
Alternatively, compound of structure 1-58 can be reacted with ammonia in a copper(I)oxide mediated coupling reaction in a suitable solvent such as ethylene glycol at a temperature between r.t. and 100°C, preferably between 80°C and 100°C to give compound of structure 1- 60.
Intermediates of general formula (1-60) are treated with a carbonate of general formula (1-9), such as, for example, 1,1'-[carbonylbis(oxy)]dipyrrolidine-2,5-dione, in the presence of a suitable base, such as for example, N,N-dimethylpyridin-4-amine, in a suitable solvent system, such as, for example, DMF, at a temperature between 0°C and the boiling point of the respective solvent, preferably the reaction is carried out at room temperature to form the desired intermediate of general formula(1-61). Intermediates of general formula (1-61) can be converted to compounds of formula (Ia) by reaction with a suitably substituted amine of the general formula (1-11), such as, for example, 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine or a salt thereof, in the presence of a suitable base, such as, for example triethylamine, in a suitable solvent system, such as, for example, DMF, in a temperature range from 0°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. A route for the preparation of compounds of formula (Ib) is described in Scheme 15.
Scheme 15
 
Figure imgf000229_0001
Scheme 15: Route for the preparation of compounds of formula (Ib), wherein R1, R4, R5, q, m, t, V, W, Y and Z have the meaning as given for general formula (I), supra. X2 represents a leaving group such as for example a Cl or Br atom or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). B1 represents a leaving group such as, for example, a C1-C3-haloalkyl such as, for example, trichloromethyl, or an imid such as, for example, pyrrolidine-2,5-dione, or a 4-nitrophenyl.  
In addition, interconversion of any of the substituents R1, R4, R5, B1, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-7, 1-9, 1-11, 1-59, 1-62, and 1-63 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. A suitably substituted aromatic ketone of general formula (1-62), such as, for example, 1-(4- bromophenyl)ethanone, can be reacted with a suitable substituted intermediate of general formula (1-63), such as, for example, ethyl 2-bromo-2-methylpropanoate, in the presence of a suitable base, such as, for example, lithium 1,1,1,3,3,3-hexamethyldisilazan-2-ide, in a suitable solvent system, such as, for example, THF, at temperatures ranging from - 100°C to boiling point of the respective solvent, preferably the reaction is carried out at - 78°C, to furnish intermediates of general formula (1-64). Intermediates of general formula (1-64) can be converted to intermediates of general formula (1-65) by reaction with a suitable hydrazine of the formula (1-7), such as, for example, hydrazine monohydrate, in a suitable solvent system, such as, for example, propan-1-ol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 0°C. Intermediates of general formula (1-65) can be reacted with a suitable substituted imine, such as, for example 1,1-diphenylmethanimine (1-59), in the presence of a suitable base, such as, for example caesium carbonate, and a suitable palladium catalyst, such as for example bis(dibenzylideneacetone)-palladium(0), in the presence of a suitable ligand, such as for example 9(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine), in a suitable solvent system, such as, for example, 1,4-dioxane, in a temperature range from room temperature to the boiling point of the respective solvent, preferably the reaction is carried out at at 110°C and subsequent treatment with a suitable Broensted acid, such as, for example, hydrochloric acid or sulphuric acid, at temperatures ranging from 0 °C to the boiling point of the respective
 
Broensted acid, preferably the reaction is carried out at 100 °C, to furnish compounds of formula (1-66). Alternatively the following palladium catalysts can be used: allylpalladium chloride dimmer, dichlorobis(benzonitrile)palladium (II), palladium (II) acetate, palladium (II) chloride, tetrakis(triphenylphosphine)palladium (0), tris(dibenzylideneacetone)dipalladium (0), chloro(2'-amino-1,1'-biphenyl-2-yl)palladium(II) dimer, (2'-amino-1,1'-biphenyl-2-yl)methanesulfonatopalladium(II) dimer, trans-di(m- acetato)bis[o-(di-o-tolylphosphino)benzyl]dipalladium(II) [cataCXium® C], allylchloro[1,3- bis(2,4,6-trimethylphenyl)imidazol-2-ylidene]palladium(II), allylchloro[1,3-bis(2,6- diisopropylphenyl)imidazol-2-ylidene]palladium(II), chloro[(1,3-dimesitylimidazol-[1,3- bis(2,4,6-trimethylphenyl)-1,3-dihydro-2H-imidazol-2-ylidene](chloro){2- [(dimethylamino)methyl]phenyl}palladium, chloro[(1,2,3-N)-3-phenyl-2-propenyl][1,3-bis(2,6- di-iso-propylphenyl)imidazol-2-ylidene]palladium(II), [2-(acetylamino)phenyl]{1,3-bis[2,6- di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2-ylidene}chloropalladium, {1,3-bis[2,6- di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2-ylidene}(chloro){2- [(dimethylamino)methyl]phenyl} palladium, {1,3-bis[2,6-di(propan-2-yl)phenyl]-2,3-dihydro-1H- imidazol-2-yl}(dichloro)(3-chloropyridine-kappaN)palladium, [1,3-bis(2,6-diisopropylphenyl) imidazol-2-ylidene](3-chloropyridyl)palladium(II) dichloride, [2-(acetylamino)-4- methoxyphenyl]{1,3-bis[2,6-di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2- ylidene}chloropalladium, {1,3-bis[2,6-di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2- ylidene}(chloro){2-[(dimethylamino)methyl]-3,5-dimethoxyphenyl}palladium, dichloro[1,3- bis(2,6-di-3-pentylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II), dichloro(di-µ- chloro)bis[1,3-bis(2,6-di-iso-propylphenyl)imidazol-2-ylidene]dipalladium(II), 2-(2'-di-tert- butylphosphine)biphenylpalladium(II) acetate, chloro[dicyclohexyl(2',6'-dimethoxybiphenyl-2- yl)-lambda5-phosphanyl][2-(phenyl-kappaC2)ethanaminato-kappaN]palladium, [2-(2- aminoethyl)phenyl](chloro)palladium - di-tert-butyl[2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, {dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane}{2-[2- (methylazanidyl-kappaN)ethyl]phenyl-kappaC1}palladium, chloro(2-dicyclohexylphosphino- 2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl) palladium(II), [2',6'-bis(propan-2- yloxy)biphenyl-2-yl](dicyclohexyl)phosphane - [2-(2-aminoethyl)phenyl](chloro)palladium, [2- (2-aminoethyl)phenyl](chloro){dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]-lambda5- phosphanylidene}palladium, 2'-(dicyclohexylphosphanyl)-N,N,N',N'-tetramethylbiphenyl-2,6- diamine - (2'-aminobiphenyl-2-yl)(chloro)palladium, chloro(2-dicyclohexylphosphino-2',6'-di- iso-propoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II), [2'-(azanidyl- kappaN)biphenyl-2-yl-kappaC2](chloro){dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]- lambda5-phosphanyl}palladium, (2'-aminobiphenyl-2-yl)(methanesulfonato- kappaO)palladium - di-tert-butyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane, (2'- aminobiphenyl-2-yl)palladium(1+) methanesulfonate - di-tert-butyl[2',4',6'-tri(propan-2-  
yl)biphenyl-2-yl]phosphane, dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane - [2-(2-aminoethyl)phenyl](chloro)palladium, (2'-aminobiphenyl-2- yl)palladium(1+) methanesulfonate - 2'-(dicyclohexylphosphanyl)-N,N,N',N'- tetramethylbiphenyl-2,6-diamine, sodium 2'-(dicyclohexylphosphanyl)-2,6-dimethoxybiphenyl- 3-sulfonate - (2'-aminobiphenyl-2-yl)(chloro)palladium, chloro(2-dicyclohexylphosphino- 2',4',6'-tri-iso-propyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl]palladium(II), (2'-aminobiphenyl-2- yl)(methanesulfonato-kappaO)palladium - [2',6'-bis(propan-2-yloxy)biphenyl-2- yl](dicyclohexyl)phosphane, (2'-aminobiphenyl-2-yl)(methanesulfonato-kappaO)palladium - dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane, (2'-aminobiphenyl-2- yl)palladium(1+) methanesulfonate - dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane - (2'-aminobiphenyl-2-yl)(chloro)palladium, (2'-aminobiphenyl-2-yl)(methanesulfonato- kappaO)palladium - di-tert-butyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, (2'-aminobiphenyl-2-yl)(methanesulfonato-kappaO)palladium - dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane or the following ligands: racemic-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl, rac-BINAP, 1,1'-bis(diphenyl- phosphino)ferrocene, bis(2-diphenylphosphinophenyl)ether, di-tert-butylmethylphosphonium tetrafluoroborate, 2-(di-tert-butylphosphino)biphenyl, tri-tert-butylphosphonium tetrafluoroborate, tri-2-furylphosphine, tris(2,4-di-tert-butylphenyl)phosphite, tri-o- tolylphosphine, (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine), dicyclohexyl(2',4',6'-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, di-tert-butyl (2',4',6'- triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, di-tert-butyl(2',4',6'-triiso propylbiphenyl-2- yl)phosphine, dicyclohexyl(2',4',6'-triisopropylbiphenyl-2-yl) phosphine, di-tert-butyl(2',4',6'- triisopropyl-3-methoxy-6-methylbiphenyl-2-yl)phosphine, di-tert-butyl(2',4',6'-triisopropyl- 3,4,5,6-tetramethylbiphenyl-2-yl) phosphine, adamantan-1-yl(adamantan-2-yl)(2',4',6'- triisopropyl-3,6-dimethoxybiphenyl-2-yl) phosphine, dicyclohexyl(2',6'-dimethoxybiphenyl-2- yl)phosphine, dicyclohexyl(2',6'-diisopropoxybiphenyl-2-yl)phosphine, 2'- (dicyclohexylphosphino)-N,N-dimethyl-biphenyl-2-amine, 2'-(di-tert-butylphosphino)-N,N- dimethylbiphenyl-2-amine, 2'-(di-phenylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6- diamine, di-tert-butyl(2',4',6'-tricyclohexyl-3,6-dimethoxybiphenyl-2-yl)phosphine, bis[3,5- bis(trifluoromethyl)phe-nyl] (2',4',6'-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, biphenyl-2-yl(di-tert-butyl)phosphine, dicyclohexyl(2'-methylbiphenyl-2-yl)phosphine, biphenyl-2-yl (dicyclohexyl)phosphine, 2'-(dicyclohexylphosphino)-N,N-dimethylbiphenyl-2- amine, 2'-(dicyclohexylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6-diamine, sodium 2'- (dicyclohexylphosphino)-2,6-diisopropylbiphenyl-4-sulfonate, sodium 2'-  
(dicyclohexylphosphino)-2,6-dimethoxybiphenyl-3-sulfonate, 1,1'-binaphthalen-2-yl(di-tert- butyl)phosphine.
Alternatively, compound of structure 1-65 can be reacted with ammonia in a copper(I)oxide mediated coupling reaction in a suitable solvent such as ethylene glycol at a temperature between r.t. and 100°C, preferably between 80°C and 100°C to give compound of structure 1- 66. Intermediates of general formula (1-66) are treated with a carbonate of general formula (1-9), such as, for example, 1,1'-[carbonylbis(oxy)]dipyrrolidine-2,5-dione, in the presence of a suitable base, such as for example, N,N-dimethylpyridin-4-amine, in a suitable solvent system, such as, for example, DMF, at a temperature between 0°C and the boiling point of the respective solvent, preferably the reaction is carried out at room temperature to form the desired intermediate of general formula(1-67). Intermediates of general formula (1-67) can be converted to compounds of formula (Ib) by reaction with a suitably substituted amine of the general formula (1-11), such as, for example, 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine or a salt thereof, in the presence of a suitable base, such as, for example triethylamine, in a suitable solvent system, such as, for example, DMF, in a temperature range from 0°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. A route for the preparation of compounds of formula (1-65) is described in Scheme 16.
Scheme 16
 
Figure imgf000234_0001
Scheme 16: Route for the preparation of compounds of formula (1-65), wherein R4, R5, V, W, Y and Z have the meaning as given for general formula (I), supra. X2 represents a leaving group such as for example a Cl or Br atom or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group).
In addition, interconversion of any of the substituents R4, R5, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-7 and 1-68 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs.
  A suitably substituted aromatic halide of general formula (1-15), such as, for example, N- bromobenzene, can be reacted with a suitable substituted anhydride (1-68), such as, for example, 3,3-dimethyldihydrofuran-2,5-dione, in the presence of a Lewis acid, such as, for example, aluminium trichloride, in a suitable solvent system, such as, for example, dichloromethane, at temperatures ranging from - 20°C to boiling point of the respective solvent, preferably the reaction is carried out at 0°C, to furnish intermediates of general formula (1-69). Intermediates of general formula (1-69) can be converted to intermediates of general formula (1-65) by reaction with a suitably hydrazine of the general formula (1-7), such as, for example, methylhydrazine, in a suitable solvent system, such as, for example, propan-1-ol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 80°C.
A route for the preparation of compounds of formula (1-33a) is described in Scheme 17.
Figure imgf000235_0001
Scheme 17: Route for the preparation of compounds of formula (1-70), wherein L1, R4, R5, V, W, Y and Z have the meaning as given for general formula (I), supra.
 
X1 represents a leaving group such as for example a Cl, Br or I, or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). X2 represents a leaving group such as for example a Cl, Br or I atom. In addition, interconversion of any of the substituents R1, R10, R11, L1, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-33 and 1-35 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. Intermediates of general formula (1-69) can be converted to intermediates of general formula (1-74) by reaction with a suitably substituted hydrazine of the general formula (1-33), such as, for example, hydrazine hydrate (1:1), in a suitable solvent system, such as, for example, propan-1-ol, in a temperature range from 0°C to the boiling point of the respective solvent, preferably the reaction is carried out at 100°C. Intermediates of general formula (1-74) are treated with an intermediate of general formula (1- 35), such as, for example, ethyl trifluoromethanesulfonate, in the presence of a suitable base, such as for example, sodium hydride, in the presence of a suitable phase transfere catalyst, such as for example, N,N,N-tributylbutan-1-aminium iodide in a suitable solvent system, such as, for example, DMF, at a temperature between 0°C and the boiling point of the respective solvent, preferably the reaction is carried out at room temperature to form the desired intermediate of general formula (1-70). A route for the preparation of compounds of formula (1-64) is described in Scheme 18.
Scheme 18
 
Figure imgf000237_0001
Scheme 17: Route for the preparation of compounds of formula (1-64), wherein R4, R5, V, W, Y and Z have the meaning as given for general formula (I), supra. X2 represents a leaving group such as for example a Cl or Br atom or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). R13 represents an alkyl or aryl group such as, for example, methyl, ethyl or phenyl. In addition, interconversion of any of the substituents R10, R11, R13, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compound 1-75 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. A suitably substituted aromatic ketone of general formula (1-62), such as, for example, 1-(4- bromophenyl)ethanone, can be reacted with a suitable substituted intermediate of general formula (1-75), such as, for example, [(1-ethoxyvinyl)oxy](trimethyl)silane - propane (1:1), in the presence of a suitable base, such as, for example, lithium 1,1,1,3,3,3-hexamethyldisilazan- 2-ide, in a suitable solvent system, such as, for example, THF, at temperatures ranging from - 100°C to boiling point of the respective solvent, preferably the reaction is carried out at - 78°C, to furnish intermediates of general formula (1-64).   A route for the preparation of compounds of formula (1-64) is described in Scheme 19.
Figure imgf000238_0001
Scheme 18: Route for the preparation of compounds of formula (1-64), wherein R4, R5, V, W, Y and Z have the meaning as given for general formula (I), supra. X2 represents a leaving group such as for example a Cl or Br atom or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group).
In addition, interconversion of any of the substituents R4, R5, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compound 1-75 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. A suitably substituted aromatic ketone of general formula (1-76), such as, for example, 2- bromo-1-(4-bromophenyl)ethanone, can be reacted with a suitable substituted intermediate of general formula (1-77), such as, for example, ethyl 2-methylpropanoate, in the presence of a suitable base, such as, for example, lithium 1,1,1,3,3,3-hexamethyldisilazan-2-ide, in a suitable  
solvent system, such as, for example, THF, at temperatures ranging from - 100°C to boiling point of the respective solvent, preferably the reaction is carried out at - 78°C, to furnish intermediates of general formula (1-64). A route for the preparation of compounds of formula (1-64) is described in Scheme 20. Scheme 20
(
Figure imgf000239_0001
Scheme 20: Route for the preparation of compounds of formula (1-8), wherein R4, R5, V, W, Y and Z have the meaning as given for general formula (I), supra. X represents a leaving group such as for example a Cl or Br atom, and X2 represents a leaving group such as for example a Cl, Br or I atom. In addition, interconversion of any of the substituents R4, R5, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compound 1-78 is either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs. A suitably substituted aromatic halide of general formula (1-15), such as, for example, N- bromobenzene, can be reacted with a suitable substituted acid chloride (1-78), such as, for example, ethyl 4-chloro-2,2-dimethyl-4-oxobutanoate, in the presence of a Lewis acid, such   as, for example, aluminium trichloride, in a suitable solvent system, such as, for example, dichloromethane, at temperatures ranging from - 20°C to boiling point of the respective solvent, preferably the reaction is carried out at 0°C, to furnish intermediates of general formula (1-64).
One route for the preparation of compounds of formula (I) is described in Scheme 21.
Figure imgf000240_0001
Scheme 21: Route for the preparation of compounds of formula (Id), wherein R1, R4, q, m, t, V, W, Y and Z have the meaning as given for general formula (I), supra and R5 is hydrogen (not shown in the above structures). X2 represents a leaving group such as for example a Cl, Br or I, or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). B1 represents a leaving group such as, for example, a C1-C3-haloalkyl such as, for example, trichloromethyl, an imid such as, for example pyrrolidine-2,5-dione or 4-nitrophenyl. In addition, interconversion of any of the substituents R1, R4, V, W, Y and Z can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art (see for example T.W. Greene and P.G.M. Wuts in Protective Groups in Organic Synthesis, 3rd edition, Wiley 1999). Specific examples are described in the subsequent paragraphs. Compounds 1-7, 1-9, 1-11, 1-76 and 1-79 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs.
Intermediates of general formula (1-76) can be converted to intermediates of general formula (1-80) by reaction with a suitably alkyl malonate of the general formula (1-79), such as, for example, diethyl methylmalonate, in the presence of a suitable base, such as, for example sodium hydride, in a suitable solvent system, such as, for example, DMF, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 0°C. Intermediates of general formula (1-80) can be reacted with a suitable Broensted acid, such as, for example, hydrochloric acid or sulphuric acid, at temperatures ranging from 0°C to boiling point of the respective Broensted acid, preferably the reaction is carried out at 100°C, to furnish intermediates of general formula (1-81).
 
Intermediates of general formula (1-81) can be converted to intermediates of general formula (1-82) by reaction with a suitably hydrazine of the general formula (1-7), such as, for example, methylhydrazine, in a suitable solvent system, such as, for example, propan-1-ol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 80°C. Intermediates of general formula (1-82) can be reacted with a suitable substituted imine, such as, for example 1,1-diphenylmethanimine (1-59), in the presence of a suitable base, such as, for example caesium carbonate, and a suitable palladium catalyst, such as for example bis(dibenzylideneacetone)-palladium(0), in the presence of a suitable ligand, such as for example 9(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine), in a suitable solvent system, such as, for example, 1,4-dioxane, in a temperature range from room temperature to the boiling point of the respective solvent, preferably the reaction is carried out at at 110°C and subsequent treatment with a suitable Broensted acid, such as, for example, hydrochloric acid or sulphuric acid, at temperatures ranging from 0 °C to the boiling point of the respective Broensted acid, preferably the reaction is carried out at 100 °C, to furnish compounds of formula (1-83d). Alternatively the following palladium catalysts can be used: allylpalladium chloride dimmer, dichlorobis(benzonitrile)palladium (II), palladium (II) acetate, palladium (II) chloride, tetrakis(triphenylphosphine)palladium (0), tris(dibenzylideneacetone)dipalladium (0), chloro(2'-amino-1,1'-biphenyl-2-yl)palladium(II) dimer, (2'-amino-1,1'-biphenyl-2-yl)methanesulfonatopalladium(II) dimer, trans-di(m- acetato)bis[o-(di-o-tolylphosphino)benzyl]dipalladium(II) [cataCXium® C], allylchloro[1,3- bis(2,4,6-trimethylphenyl)imidazol-2-ylidene]palladium(II), allylchloro[1,3-bis(2,6- diisopropylphenyl)imidazol-2-ylidene]palladium(II), chloro[(1,3-dimesitylimidazol-[1,3- bis(2,4,6-trimethylphenyl)-1,3-dihydro-2H-imidazol-2-ylidene](chloro){2- [(dimethylamino)methyl]phenyl}palladium, chloro[(1,2,3-N)-3-phenyl-2-propenyl][1,3-bis(2,6- di-iso-propylphenyl)imidazol-2-ylidene]palladium(II), [2-(acetylamino)phenyl]{1,3-bis[2,6- di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2-ylidene}chloropalladium, {1,3-bis[2,6- di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2-ylidene}(chloro){2- [(dimethylamino)methyl]phenyl} palladium, {1,3-bis[2,6-di(propan-2-yl)phenyl]-2,3-dihydro-1H- imidazol-2-yl}(dichloro)(3-chloropyridine-kappaN)palladium, [1,3-bis(2,6-diisopropylphenyl) imidazol-2-ylidene](3-chloropyridyl)palladium(II) dichloride, [2-(acetylamino)-4- methoxyphenyl]{1,3-bis[2,6-di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2- ylidene}chloropalladium, {1,3-bis[2,6-di(propan-2-yl)phenyl]-1,3-dihydro-2H-imidazol-2- ylidene}(chloro){2-[(dimethylamino)methyl]-3,5-dimethoxyphenyl}palladium, dichloro[1,3- bis(2,6-di-3-pentylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II), dichloro(di-µ- chloro)bis[1,3-bis(2,6-di-iso-propylphenyl)imidazol-2-ylidene]dipalladium(II), 2-(2'-di-tert-  
butylphosphine)biphenylpalladium(II) acetate, chloro[dicyclohexyl(2',6'-dimethoxybiphenyl-2- yl)-lambda5-phosphanyl][2-(phenyl-kappaC2)ethanaminato-kappaN]palladium, [2-(2- aminoethyl)phenyl](chloro)palladium - di-tert-butyl[2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, {dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane}{2-[2- (methylazanidyl-kappaN)ethyl]phenyl-kappaC1}palladium, chloro(2-dicyclohexylphosphino- 2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl) palladium(II), [2',6'-bis(propan-2- yloxy)biphenyl-2-yl](dicyclohexyl)phosphane - [2-(2-aminoethyl)phenyl](chloro)palladium, [2- (2-aminoethyl)phenyl](chloro){dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]-lambda5- phosphanylidene}palladium, 2'-(dicyclohexylphosphanyl)-N,N,N',N'-tetramethylbiphenyl-2,6- diamine - (2'-aminobiphenyl-2-yl)(chloro)palladium, chloro(2-dicyclohexylphosphino-2',6'-di- iso-propoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II), [2'-(azanidyl- kappaN)biphenyl-2-yl-kappaC2](chloro){dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]- lambda5-phosphanyl}palladium, (2'-aminobiphenyl-2-yl)(methanesulfonato- kappaO)palladium - di-tert-butyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane, (2'- aminobiphenyl-2-yl)palladium(1+) methanesulfonate - di-tert-butyl[2',4',6'-tri(propan-2- yl)biphenyl-2-yl]phosphane, dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane - [2-(2-aminoethyl)phenyl](chloro)palladium, (2'-aminobiphenyl-2- yl)palladium(1+) methanesulfonate - 2'-(dicyclohexylphosphanyl)-N,N,N',N'- tetramethylbiphenyl-2,6-diamine, sodium 2'-(dicyclohexylphosphanyl)-2,6-dimethoxybiphenyl- 3-sulfonate - (2'-aminobiphenyl-2-yl)(chloro)palladium, chloro(2-dicyclohexylphosphino- 2',4',6'-tri-iso-propyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl]palladium(II), (2'-aminobiphenyl-2- yl)(methanesulfonato-kappaO)palladium - [2',6'-bis(propan-2-yloxy)biphenyl-2- yl](dicyclohexyl)phosphane, (2'-aminobiphenyl-2-yl)(methanesulfonato-kappaO)palladium - dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane, (2'-aminobiphenyl-2- yl)palladium(1+) methanesulfonate - dicyclohexyl[2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane - (2'-aminobiphenyl-2-yl)(chloro)palladium, (2'-aminobiphenyl-2-yl)(methanesulfonato- kappaO)palladium - di-tert-butyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2- yl]phosphane, (2'-aminobiphenyl-2-yl)(methanesulfonato-kappaO)palladium - dicyclohexyl[3,6-dimethoxy-2',4',6'-tri(propan-2-yl)biphenyl-2-yl]phosphane or the following ligands: racemic-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl, rac-BINAP, 1,1'-bis(diphenyl- phosphino)ferrocene, bis(2-diphenylphosphinophenyl)ether, di-tert-butylmethylphosphonium tetrafluoroborate, 2-(di-tert-butylphosphino)biphenyl, tri-tert-butylphosphonium tetrafluoroborate, tri-2-furylphosphine, tris(2,4-di-tert-butylphenyl)phosphite, tri-o- tolylphosphine, (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine), dicyclohexyl(2',4',6'-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, di-tert-butyl (2',4',6'-  
triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, di-tert-butyl(2',4',6'-triiso propylbiphenyl-2- yl)phosphine, dicyclohexyl(2',4',6'-triisopropylbiphenyl-2-yl) phosphine, di-tert-butyl(2',4',6'- triisopropyl-3-methoxy-6-methylbiphenyl-2-yl)phos-phine, di-tert-butyl(2',4',6'-triisopropyl- 3,4,5,6-tetramethylbiphenyl-2-yl) phosphine, adamantan-1-yl(adamantan-2-yl)(2',4',6'- triisopropyl-3,6-dimethoxybiphenyl-2-yl) phosphine, dicyclohexyl(2',6'-dimethoxybiphenyl-2- yl)phosphine, dicyclohexyl(2',6'-diisopropoxybiphenyl-2-yl)phosphine, 2'- (dicyclohexylphosphino)-N,N-dimethyl-biphenyl-2-amine, 2'-(di-tert-butylphosphino)-N,N- dimethylbiphenyl-2-amine, 2'-(di-phenylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6- diamine, di-tert-butyl(2',4',6'-tricyclohexyl-3,6-dimethoxybiphenyl-2-yl)phosphine, bis[3,5- bis(trifluoromethyl)phe-nyl] (2',4',6'-triisopropyl-3,6-dimethoxybiphenyl-2-yl)phosphine, biphenyl-2-yl(di-tert-butyl)phosphine, dicyclohexyl(2'-methylbiphenyl-2-yl)phosphine, biphenyl-2-yl (dicyclohexyl)phosphine, 2'-(dicyclohexylphosphino)-N,N-dimethylbiphenyl-2- amine, 2'-(dicyclohexylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6-diamine, sodium 2'- (dicyclohexylphosphino)-2,6-diisopropylbiphenyl-4-sulfonate, sodium 2'- (dicyclohexylphosphino)-2,6-dimethoxybiphenyl-3-sulfonate, 1,1'-binaphthalen-2-yl(di-tert- butyl)phosphine.
Alternatively, compound of structure 1-82 can be reacted with ammonia in a copper(I)oxide mediated coupling reaction in a suitable solvent such as ethylene glycol at a temperature between r.t. and 100°C, preferably between 80°C and 100°C to give compound of structure 1- 83d. Intermediates of general formula (1-83d) are treated with a carbonate of general formula (1- 9), such as, for example, 1,1'-[carbonylbis(oxy)]dipyrrolidine-2,5-dione, in the presence of a suitable base, such as for example, N,N-dimethylpyridin-4-amine, in a suitable solvent system, such as, for example, DMF, at a temperature between 0°C and the boiling point of the respective solvent, preferably the reaction is carried out at room temperature to form the desired intermediate of general formula (1-84). Intermediates of general formula (1-84) can be converted to compounds of formula (Id) by reaction with a suitably substituted amine of the general formula (1-11), such as, for example, 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine or a salt thereof, in the presence of a suitable base, such as, for example triethylamine, in a suitable solvent system, such as, for example, DMF, in a temperature range from 0°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. It is known to the person skilled in the art that, if there are a number of reactive centers on a starting or intermediate compound, it may be necessary to block one or more reactive centers  
temporarily by protective groups in order to allow a reaction to proceed specifically at the desired reaction center. A detailed description for the use of a large number of proven protective groups is found, for example, in T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, 1999, 3rd Ed., or in P. Kocienski, Protecting Groups, Thieme Medical Publishers, 2000. The compounds according to the invention are isolated and purified in a manner known per se, e.g. by distilling off the solvent in vacuo and recrystallizing the residue obtained from a suitable solvent or subjecting it to one of the customary purification methods, such as chromatography on a suitable support material. Furthermore, reverse phase preparative HPLC of compounds of the present invention which possess a sufficiently basic or acidic functionality, may result in the formation of a salt, such as, in the case of a compound of the present invention which is sufficiently basic, a trifluoroacetate or formate salt for example, or, in the case of a compound of the present invention which is sufficiently acidic, an ammonium salt for example. Salts of this type can either be transformed into its free base or free acid form, respectively, by various methods known to the persion skilled in the art, or be used as salts in subsequent biological assays. Additionally, the drying process during the isolation of compounds of the present invention may not fully remove traces of cosolvents, especially such as formic acid or trifluoroacetic acid, to give solvates or inclusion complexes. The person skilled in the art will recognise which solvates or inclusion complexes are acceptable to be used in subsequent biological assays. It is to be understood that the specific form (e.g. salt, free base, solvate, inclusion complex) of a compound of the present invention as isolated as described herein is not necessarily the only form in which said compound can be applied to a biological assay in order to quantify the specific biological activity. Salts of the compounds of formula (Ia), (Ib), (Ic), or (Id) according to the invention can be obtained by dissolving the free compound in a suitable solvent (for example a ketone such as acetone, methylethylketone or methylisobutylketone, an ether such as diethyl ether, tetrahydrofuran or dioxane, a chlorinated hydrocarbon such as methylene chloride or chloroform, or a low molecular weight aliphatic alcohol such as methanol, ethanol or isopropanol) which contains the desired acid or base, or to which the desired acid or base is then added. The acid or base can be employed in salt preparation, depending on whether a mono- or polybasic acid or base is concerned and depending on which salt is desired, in an equimolar quantitative ratio or one differing therefrom. The salts are obtained by filtering, reprecipitating, precipitating with a non-solvent for the salt or by evaporating the solvent. Salts obtained can be converted into the free compounds which, in turn, can be converted into salts. In this manner, pharmaceutically unacceptable salts, which can be obtained, for example, as  
process products in the manufacturing on an industrial scale, can be converted into pharmaceutically acceptable salts by processes known to the person skilled in the art. Especially preferred are hydrochlorides and the process used in the examples section.
Pure diastereomers and pure enantiomers of the compounds and salts according to the invention can be obtained e.g. by asymmetric synthesis, by using chiral starting compounds in synthesis and by splitting up enantiomeric and diasteriomeric mixtures obtained in synthesis.
Enantiomeric and diastereomeric mixtures can be split up into the pure enantiomers and pure diastereomers by methods known to a person skilled in the art. Preferably, diastereomeric mixtures are separated by crystallization, in particular fractional crystallization, or chromatography. Enantiomeric mixtures can be separated e.g. by forming diastereomers with a chiral auxiliary agent, resolving the diastereomers obtained and removing the chiral auxiliary agent. As chiral auxiliary agents, for example, chiral acids can be used to separate enantiomeric bases such as e.g. mandelic acid and chiral bases can be used to separate enantiomeric acids via formation of diastereomeric salts. Furthermore, diastereomeric derivatives such as diastereomeric esters can be formed from enantiomeric mixtures of alcohols or enantiomeric mixtures of acids, respectively, using chiral acids or chiral alcohols, respectively, as chiral auxiliary agents. Additionally, diastereomeric complexes or diastereomeric clathrates may be used for separating enantiomeric mixtures. Alternatively, enantiomeric mixtures can be split up using chiral separating columns in chromatography. Another suitable method for the isolation of enantiomers is the enzymatic separation. Optionally, compounds of the formula (Ia), (Ib), (Ic), or (Id) can be converted into their salts, or, optionally, salts of the compounds of the formula (I) or (II) can be converted into the free compounds. Corresponding processes are customary for the skilled person. Optionally, compounds of the formula (Ia), (Ib), (Ic), or (Id) can be converted into their N-oxides. The N-oxide may also be introduced by way of an intermediate. N-oxides may be prepared by treating an appropriate precursor with an oxidizing agent, such as meta-chloroperbenzoic acid, in an appropriate solvent, such as DCM, at suitable temperatures, such as from 0 °C to 40 °C, whereby room temperature is generally preferred. Further corresponding processes for forming N-oxides are customary for the skilled person. One preferred aspect of the invention is the process for the preparation of the conjugates and compounds according to the examples, as well as the intermediates used for their preparation.  
Optionally, compounds of the formula (Ia), (Ib), (Ic), or (Id) can be converted into their salts, or, optionally, salts of the compounds of the formula (I) or (II) can be converted into the free compounds. Corresponding processes are customary for the skilled person. The NAMPT inhibitors of formula (IVa), (IVb) and (IVc):
4
Figure imgf000247_0001
wherein # represents the point of attachment to linker Z’ and q, m, C1, A1, V, W, Z, Y are as defined herein, can be made according to any of the methods described in the present application, or alternatively, according to any of the methods described in WO2012067965 (each of the methods and compounds of WO2012067965 being incorporated herein by reference, in its entirety). A method to synthesize compounds according to structure (II) is alkylation of compounds 1- 68, meaning compounds of structure (Ia), (Ib), or (Id), at the pyridazinone NH with a compound of type 1-35 as depicted in scheme 22.
 
Figure imgf000248_0001
Scheme 22: Scheme for the preparation of compounds of formula (I) via alkylation of compounds of formula 1-68, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A. X1 represents a leaving group such as for example a Cl, Br or I, or an aryl sulfonate such as for example p- toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). Conditions for this alkylation can be typical conditions known to those skilled in the art like treatment of 1-68 with a suitable base, such as, for example sodium hydride, in a suitable solvent system, such as, for example, DMF, followed by addition of an alkylating compound of structure 1-35 in a temperature range from -20°C to the boiling point of the respective solvent, preferably the reaction is carried out between 0°C and r.t.. An alternative method of synthesizing compounds of structure (II) is displayed in scheme 23.
Figure imgf000248_0002
Scheme 22: Scheme for the preparation of compounds of formula (I) via alkylation of compounds of formula (II), wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A. B2 represents a group such as, for example, a H, Cl or Br atom or an nitro group.  
An aniline of structure 1-83 is reacted with a chloroformate of structure 1-27 in a manner as described above to give carbamate 1-89 which is then reacted with amine of general structure 1-11 in manner as described above to give compound of general structure (II).
Compounds of type of type 1-88 which are compounds of general structure (II) wherein L1 bears an amino substituent can be prepared by a route as described in scheme 23. Aniline 1- 83 is reacted with an alkylating agent of structure 1-84 wherein R13 is a protected amine group like NH-PG1, phthalimide, nitro or azide under conditions known to those skilled in the art to give N-alkylated compound of structure 1-85. These methods include treatment of 1-83 with a suitable base, such as, for example sodium hydride, in a suitable solvent system, such as, for example, DMF, followed by addition of an alkylating compound of structure 1-84 in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out between 0°C and r.t.. Aniline of structure 1-85 is then reacted with a chloroformate of structure 1-27 such as, for example, 4-nitrophenyl carbonochloridate, in the presence of a suitable base, such as, for example, triethylamine, in a suitable solvent system, such as, for example, toluene, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. The resulting compound of structure 1-86 is then reacted with an amine of structure 1-11 such as, for example, 1-(pyridin-3-yl)piperazine, in a suitable solvent system, such as, for example, ethanol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 79°C. The resulting compound of structure 1-87 is then converted into amine of structure 1-88 by transforming N-protected moiety R13 to NH2 by methods known to those skilled in the art, like acidic cleavage of the Boc group with TFA or hydrochloric acid, cleavage of the phthalimide group with hydrazine or methyl amine, reduction of the nitro group with iron/acetic acid or hydrogenation with palladium on charcoal under a hydrogen atmosphere or reduction of the azide group by hydrogenation with palladium on charcoal under a hydrogen atmosphere or by Staudinger-type reduction with triphenylphosphine to give compound of structure 1-88.
 
Figure imgf000250_0001
Scheme 23: Route for the preparation of compounds of formula 1-88, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A X1 represents a leaving group such as for example a Cl, Br or I, or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). B2 represents a group such as, for example, a H, Cl or Br atom or an nitro group. PG1 represents an amine protecting group as for example an acetyl group or a tert-butyloxycarbonyl group. An alternative route for the synthesis of structure1-85 is depicted in scheme 24.
 
Figure imgf000251_0001
Scheme 24: Alternative route for the preparation of compound of structure 1-85, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A. X1 represents a leaving group such as for example a Cl, Br or I, or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). X2 represents a leaving group such as for example a Cl, Br or I atom. B2 represents a group such as, for example, a H, Cl or Br atom or an nitro group. PG1 represents an amine protecting group as for example an acetyl group or a tert-butyloxycarbonyl group.
Compound 1-89 is reacted with an alkylating agent of structure 1-84 wherein R13 is a protected amine group like NH-PG1, phthalimide, nitro or azide under conditions known to those skilled in the art to give N-alkylated compound of structure 1-90. These methods include treatment of 1-89 with a suitable base, such as, for example sodium hydride, in a suitable solvent system, such as, for example, DMF, followed by addition of an alkylating compound of structure 1-84 in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out between 0°C and r.t.. 1-90 can then be converted into aniline 1-85 by reaction with a suitable substituted imine, such as, for example 1,1-diphenylmethanimine (1- 59) as described in scheme 21. Alternatively, compounds of structure 1-90 can be reacted with ammonia in a copper(I)oxide mediated coupling reaction in a suitable solvent such as ethylene glycol at a temperature between r.t. and 100°C, preferably between 80°C and 100°C to give compound of structure 1-85. An alternative method for the synthesis of compound of general structure 1-87 is described in scheme 24a.  
Figure imgf000252_0001
Scheme 24a: Alternative route for the preparation of compound of structure 1-87, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A. X1 represents a leaving group such as for example a Cl, Br or I, or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). X2 represents a leaving group such as for example a Cl, Br or I atom. B2 represents a group such as, for example, a H, Cl or Br atom or an nitro group. PG1 represents an amine protecting group as for example an acetyl group or a tert-butyloxycarbonyl group.
Pyridazinone of general structure 1-87 is reacted with an alkylating agent of structure 1-84 wherein R13 is a protected amine group like NH-PG1, phthalimide, nitro or azide under conditions known to those skilled in the art to give N-alkylated compound of structure 1-87. These methods include treatment of 1-87 with a suitable base, such as, for example sodium hydride, in a suitable solvent system, such as, for example, DMF, followed by addition of an alkylating compound of structure 1-84 in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out between 0°C and r.t.. Compounds of structure (1-95) which are compounds of general structure (II) wherein L1 bears an hydroxyl substituent can be prepared by a route as described in scheme 25. Aniline 1-83 is reacted with an alkylating agent of structure 1-91 wherein PG2 as a hydroxyl protecting group like trimethylsilyl or tert-butyldimethylsilyl under conditions known to those skilled in the art to give N-alkylated compound of structure 1-92. These methods include treatment of 1-83 with a suitable base, such as, for example sodium hydride, in a suitable solvent system, such as, for example, DMF, followed by addition of an alkylating compound of structure 1-91 in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the   reaction is carried out between 0°C and r.t.. Aniline of structure 1-92 is then reacted with a chloroformate of structure 1-27 such as, for example, 4-nitrophenyl carbonochloridate, in the presence of a suitable base, such as, for example, triethylamine, in a suitable solvent system, such as, for example, toluene, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. The resulting compound of structure 1-93 is then reacted with an amine of structure 1-11 such as, for example, 1-(pyridin-3-yl)piperazine, in a suitable solvent system, such as, for example, ethanol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 79°C. The resulting compound of structure 1-94 is then converted into amine of structure 1-94 by deprotecting the hydroxyl group by methods known to those skilled in the art, like tetra-n-butylammonium fluoride or Brønsted acids like hydrochloric acid for the cleavage of O-silyl groups to give compound of structure 1-95.
Figure imgf000253_0001
Scheme 25: Route for the preparation of compounds of formula 1-95, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A.   An alternative route for the synthesis of structure1-92 and the corresponding free hydroxyl compound of structure 1-97 is depicted in scheme 26.
Figure imgf000254_0001
Scheme 26: Alternative route for the preparation of compound of structure 1-92 and hydroxyl compound 1-97, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A.
Compound 1-89 is reacted with an alkylating agent of structure 1-90 wherein PG2 is a hydroxyl protecting group like trimethylsilyl or tert-butyldimethylsilyl under conditions known to those skilled in the art to give N-alkylated compound of structure 1-96. These methods include treatment of 1-89 with a suitable base, such as, for example sodium hydride, in a suitable solvent system, such as, for example, DMF, followed by addition of an alkylating compound of structure 1-90 in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out between 0°C and r.t.. 1-90 can then be converted into aniline 1-92 by reaction with a suitable substituted imine, such as, for example 1,1- diphenylmethanimine (1-59) as described in scheme 21. In the course of this reaction the O- protecting group PG2 can be cleaved yielding the free hydroxyl compound of structure 1-97. Besides 1-97 can be synthesized by deprotecting the hydroxyl group in 1-92 by methods known to those skilled in the art, like tetra-n-butylammonium fluoride or Brønsted acids like hydrochloric acid for the cleavage of O-silyl groups.
Alternatively, compound of structure 1-96 can be reacted with ammonia in a copper(I)oxide mediated coupling reaction in a suitable solvent such as ethylene glycol at a temperature between r.t. and 100°C, preferably between 80°C and 100°C to give compound of structure 1- 97 or 1-92.
  An alternative route for the synthesis of compounds of structure 1-95 is depicted in scheme
27.
Figure imgf000255_0001
Scheme 27: Alternative route for the synthesis of compound of structure 1-95, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A. Aniline of structure 1-97 is reacted with a chloroformate of structure 1-27 such as, for example, 4-nitrophenyl carbonochloridate, in the presence of a suitable base, such as, for example, triethylamine, in a suitable solvent system, such as, for example, toluene, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. The resulting compound of structure 1-98 is then reacted with an amine of structure 1-11 such as, for example, 1-(pyridin-3-yl)piperazine under conditions described above to give compound of structure 1-95. A further method to synthesize amino compounds of general structure 1-88 is shown in scheme 27a.
 
Figure imgf000256_0001
Scheme 27: Alternative route for the synthesis of compound of structure 1-95, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A. Hydroxy compound of general structure 1-95 is converted by a Mitsunobu reaction under conditions known to those skilled in the like e.g. treatment of 1-95 with diisopropylazodicarboxylate or diethylazodicarboylate or the like and triphenylphosphine in THF or other suitable solvents and addition of an amine source like phthalimide to give phthaloyl protected amine of general structure 1-95a. The reaction is carried out at temperatures between 0°C and 25°C. 1-95a is then reacted with hydrazine or methylamine to yield the free amine 1-88. Other suitable routes include transformation of the hydroxyl group in structure 1-95 to a leaving group like methanesulphonate, p-toluenesulphonate, bromide, iodide or chloride by methods known to those skilled in the art and reacting those activated compounds with a suitable nitrogen-nucleophile like phthalimide ot azide followed by deprotection or of the phthaloyl protected amine with suitable reagents like hydrazine or methylamine or reduction of the azide by hydrogenation with palladium on charcoal under a hydrogen atmosphere or by Staudinger- type reduction with triphenylphosphine.
 
Compounds of structure (1-100) which are compounds of general structure (II) wherein L1 bears a thiol substituent can be prepared by a route as described in scheme 28. Hydroxy compound 1-95 can be converted into the corresponding thioacetyl compound of structure 1- 99 by means of a Mitsunobu reaction under conditions known to those skilled in the like e.g. treatment of 1-95 with diisopropylazodicarboxylate or diethylazodicarboylate or the like and triphenylphosphine in THF or other suitable solvents and addition of thioacetic acid or comparable thiocarbonic acids or salts thereof. The reaction is carried out at temperatures between 0°C and 25°C. Other suitable routes include transformation of the hydroxyl group in structure 1-95 to a leaving group like methanesulphonate, p-toluenesulphonate, bromide, iodide or chloride by methods known to those skilled in the art and reacting those activated compounds with a suitable thiol-nucleophile like sodium sulfide or sodium thioacetate. S- Protected thiol of structure 1-99 can then be transformed into free thiol of structure 1-100 by cleavage of the thioacyl group under basic conditions known to those skilled in the art like treatment with sodium hydroxide, sodium methoxide, or sodium ethoxide in a suitable solvent like methanol, ethanol, water, or THF. The reaction is carried out at temperatures between 0°C and 100°C, preferably between 0°C and 25°C. (
Figure imgf000257_0001
Scheme 28: Route for the synthesis of compound of structure 1-100, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A. An further method for the synthesis of thiols of general structure 1-100 is the reduction of corresponding disulfides by methods known to those skilled in the art like e.g. reaction with triscarboxyethylphosphine or reduction with sodiumborohydride.
Carboxylic acids of structure 1-105 which are compounds of general structure (II) can be synthesized according to scheme 29. Aniline 1-83 is reacted with an alkylating agent of  
structure 1-101 wherein PG3 as a carboxylic acid protecting group like methyl, ethyl, benzyl or tert-butyl under conditions known to those skilled in the art to give N-alkylated compound of structure 1-102. These methods include treatment of 1-83 with a suitable base, such as, for example sodium hydride, in a suitable solvent system, such as, for example, DMF, followed by addition of an alkylating compound of structure 1-101 in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out between 0°C and r.t.. Aniline of structure 1-102 is then reacted with a chloroformate of structure 1-27 such as, for example, 4-nitrophenyl carbonochloridate, in the presence of a suitable base, such as, for example, triethylamine, in a suitable solvent system, such as, for example, toluene, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at room temperature. The resulting compound of structure 1-103 is then reacted with an amine of structure 1-11 such as, for example, 1-(pyridin-3-yl)piperazine, in a suitable solvent system, such as, for example, ethanol, in a temperature range from - 20°C to the boiling point of the respective solvent, preferably the reaction is carried out at 79°C. The resulting compound of structure 1-104 is then converted into carboxylic acid of structure 1-105 by deprotecting the carboxyl group by methods known to those skilled in the art, like esterification under basic conditions like e.g. sodium hydroxide, sodium methoxide or sodium ethoxide in a suitable solvent like methanol, ethanol, water or THF to give compound of structure 1-95. Alternative methods for the deprotection step include acidic deprotection of tert- butyl esters with Lewis or Brønstedt acids like BF3, TFA or hydrochloric acid or hydrogenolytic deprotection of benzyl esters with palladium on charcoal under hydrogen atmosphere in a suitable solvent like methanol, ethanol, THF or acetic acid.
 
Figure imgf000259_0001
Scheme 29: Route for the synthesis of compound of structure 1-105, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A. Amines of general structure 1-105c which is a subclass of structure 1-88 can be synthesized as shown in scheme 30.
 
Figure imgf000260_0001
Scheme 30: Route for the synthesis of compound of structure 1-105c, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A. Carboxylic acid 1-105 is coupled to an amine of structure 1-105a wherein s is 2 to 10, preferably 3 to 6 and R13 is a protected amine, phthalimide or azide as described above by peptide coupling methods known to those skilled in the art, like HATU- or T3P mediated couplings. For further peptide coupling methods see above. Protected amine 1-105b is then deprotected by methods known to those skilled in the art, like like acidic cleavage of the Boc group with TFA or hydrochloric acid, cleavage of the phthalimide group with hydrazine or methyl amine, reduction of the nitro group with iron/acetic acid or hydrogenation with palladium on charcoal under a hydrogen atmosphere or reduction of the azide group by hydrogenation with palladium on charcoal under a hydrogen atmosphere or by Staudinger-type reduction with triphenylphosphine to give compound of structure 1-105c.
Compounds with carboxylic acid bearing linkers of structure 1-114 which are a subclass of general structure 1-88 can be synthesized by reacting an amino compound of structure 1-88 with a a-amino and a-carboxyl protected amino acid of type 1-110 by amide coupling methods   known to those skilled in the art using e.g. HATU as shown in scheme 32. PG1 and PG3 are protecting groups appropriate to protect the corresponding amino or carboxyl group known to those skilled in the art (see above). Further peptide forming reactions are well described in N. Leo Benoitin in Chemistry of Peptide Synthesis, CRC Press 2005; John Jones in Amino Acids and Peptide Synthesis, Oxford University Press, 2002 and Norbert Sewald and Hans-Dieter Jakubke in Peptides: Chemistry and Biology, Wiley-VCH, 2009). The formed bis-protected amino acid of type 1-111 can then be deprotected successively at the amino and carboxyl group by methods known to those skilled in the art as already described above via compounds of structures 1-112 and 1-113 to yield compound of structure 1-114.
Figure imgf000261_0001
Scheme 32: Route for the synthesis of compound of structure 1-114, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A.
  Compounds of structure 1-127 with polyethyleneglycol (PEG) side chains in the linker which are a subclass of general structure 1-88 can be synthesized as described in scheme 34.
Figure imgf000262_0001
Scheme 34: Route for the synthesis of compound of structure 1-127, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A, and o is 1 to 5 and p is 1 to 10. An a-amine,w-carboxyl bisprotected amino acid of structure 1-122 is coupled to a polyethyleneglycolic amine of structure 1-123 by amide formation methods known to those skilled in the art (see above). The so formed amide of structure 1-124 is then deprotected at the w-carboxyl group by deprotection methods known to those skilled in the art (see above) to give free acid 1-125 which can then be coupled to amine 1-88 to give the corresponding PEGylated amide of structure 1-126. Deprotection of the amine group (methods see above) then give the corresponding amine of structure 1-127.   Peptidic linker moieties of structure 1-131 can be synthesized is described in scheme 35.
Figure imgf000263_0001
Scheme 34: Route for the synthesis of compound of structure 1-131. An a-carboxyl protected amino acid of structure 1-128 is coupled with an a-amino protected amino acid of structure 1-129 by peptide coupling methods known to those skilled in the art, like e.g. HATU. For further peptide coupling methods see above. The so formed peptide of structure 1-130 can then be deprotected at the carboxyl group my methods known to those skilled in the art (see above) to give peptide 1-131.
Maleimides of general structure 1-134 can be synthesized as described in scheme 35.
Figure imgf000263_0002
Scheme 35: Route for the synthesis of compound of structure 1-134 wherein RA and RB are amino acid side chains like e.g. methyl for alanine or isopropyl for valine and R12 is C1-C10 alkylene, preferably C1-C5 alkylene. A peptide of general structure 1-132 is coupled with an activated ester of type 1-133 to give peptidic maleimide of general structure 1-134. This reaction takes place in a suitable solvent   like DMF at a temperature between 0°C and 50°C, preferable at r.t.. Alternatively, compounds of structure 1-134 can be synthesized by coupling peptide of structure 1-132 with the corresponding free acid of structure 1-133 by typical peptide coupling methods (see above), preferably with HATU or T3P. Maleimides of general structure 1-137 can be synthesized as described in scheme 36.
Figure imgf000264_0001
Scheme 36: Route for the synthesis of compound of structure 1-135 wherein R12 is C1-C10 alkyl, preferably C1-C5 and p is 1 to 10, preferably 1 to 6. An amine of structure 1-135 is coupled with an activated ester of type 1-133 to give maleimide of general structure 1-136. This reaction takes place in a suitable solvent like DMF at a temperature between 0°C and 50°C, preferable at r.t.. Alternatively, compounds of structure 1-134 can be synthesized by coupling peptide of structure 1-132 with the corresponding free acid of structure 1-133 by typical peptide coupling methods (see above), preferably with HATU or T3P. Maleimide of structure 1-136 is then deprotected at the carboxyl moiety by means known to those skilled in the art (see above), preferably with TFA in a suitable solvent like dichloromethane for the deprotection of tert-butylesters.
Antibody-drug conjugates of general structure
Figure imgf000264_0002
can be synthesized by reacting an activated moiety Z’’-D with an antibody AB wherein linker Z’’ represents one of the following general structures (i) to (iii): (i) §–L1-SG-L2’-§§ (ii) §–L1-SG-L1’-L2’-§§   (iii) §–L1-L2’-§§ wherein § represents the attachment point to D; §§ represents the attachment point to AB; SG represents an in vivo cleavable group, L1 and L1’ represent, independently of each other, an in vivo non-cleavable organic group, and L2’ represents an activated attachment group. For the synthesis of cysteine-linked antibody-drug conjugates L2’ is a thiol-reactive group from the group of
Figure imgf000265_0001
wherein #2 represents the attachment point to the group L1, L1’ or SG and X1 represents a leaving group such as for example a Cl, Br or I, or an aryl sulfonate such as for example p- toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group). More preferably, activated moiety Z’’-D for the synthesis of cysteine-linked antibody-drug conjugates bears a maleimide as in general structure 1-153
‘
Figure imgf000265_0002
, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and
R5 are connected at the positions shown for group A and Q represents one of the following general structures (i) to (iii): (i) §–L1-SG-§§ (ii) §–L1-SG-L1’-§§ (iii) §–L1-§§ wherein § represents the attachment point to D; §§ represents the attachment point to L2’; SG represents an in vivo cleavable group, L1 and L1’ represent, independently of each other, an in vivo non-cleavable organic group, and L2’ represents an activated attachment group.
Maleimides of general structure 1-138 which are a subclass of general structure 1-153 can be synthesized by reacting amine of general structure 1-88 with activated ester of structure 1-133 as shown in scheme 37.
‘
‘
Figure imgf000266_0001
  Scheme 37: Route for the synthesis of compound of structure 1-138, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A and R12 is C1-C10 alkyl, preferably C1-C5. Amine of general structure 1-88 is reacted with an N-hydroxysuccinimidyl ester of structure 1- 133 by methods known to those skilled in the art to give maleimide of general structure 1-138. These methods include addition of a base like e.g. triethyl amine or diisoproypethylamine and a suitable solvent like DMF or THF. Alternative methods include coupling of amine 1-88 with the corresponding free acid of structure 1-133 under peptide coupling conditions known to those skilled in the art as described above. Peptidic maleimides of general structure 1-139 which are a subclass of general structure 1- 153 can be synthesized by coupling amine of general structure 1-88 with peptide of structure 1-134 as shown in scheme 38.
‘
Figure imgf000267_0001
Scheme 38: Route for the synthesis of compound of structure 1-139, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A and wherein RA and RB are amino acid side chains e.g. methyl for alanine or isopropyl for valine. This coupling reaction can be achieved by means known to those skilled in the art, e.g. peptide coupling with HATU or T3P. Alternative methods include the conversion of peptidic carboxylic acid 1-134 into the corresponding N-hydroxysuccinimidyl ester by methods known to those   skilled in the art and reaction of the N-hydroxysuccinimidyl ester with amine 1-88. For further peptide coupling methods see above. An alternative method for the preparation of maleimides of general structure 1-139 is described in scheme 39.
Figure imgf000268_0001
Scheme 39: Alternative route for the synthesis of compound of structure 1-139, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A and wherein RA and RB are amino acid side chains e.g. methyl for alanine or isopropyl for valine.  Amine of general structure 1-88 is coupled to peptide of general structure 1-131 by peptide coupling methods known to those skilled in the art to give peptide 1-140. Deprotection of the amine moiety by means known to those skilled in the art, e.g. TFA in dichloromethane for Boc-   deprotection, gives amine 1-141 which is then reacted with activated ester of general structure
1-133 as described above to give maleimide of general structure 1-139. A method to synthesize PEGylated maleimides of structure 1-142 which are a substructure of structure 1-153 is shown in scheme 40.
Figure imgf000269_0001
Scheme 40: Route for the synthesis of compound of structure 1-142, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A and wherein R12 is C1-C10 alkyl, preferably C1-C5 and p is 1 to 10, preferably 1 to 6.  Amine 1-88 is coupled to carboxylic acid 1-137 by peptide coupling methods known to those skilled in the art, e.g. peptide coupling with HATU or T3P. Alternative methods include the conversion of carboxylic acid 1-137 into the corresponding N-hydroxysuccinimidyl ester by methods known to those skilled in the art and reaction of the N-hydroxysuccinimidyl ester with amine 1-88. For further peptide coupling methods see above. A method to synthesize maleimides of structure 1-143 which are a substructure of general structure 1-153 is shown in scheme 41.
  ‘
Figure imgf000270_0002
Scheme 41: Route for the synthesis of compound of structure 1-143, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A and wherein R12 is C1-C10 alkyl, preferably C1-C5.  Carboxylic acid 1-105 is coupled to an amine of structure 1-142 by peptide coupling methods known to those skilled in the art, e.g. peptide coupling with HATU or T3P. Alternative methods include the conversion of carboxylic acid 1-105 into the corresponding N-hydroxysuccinimidyl ester by methods known to those skilled in the art and reaction of the N-hydroxysuccinimidyl ester with amine 1-142. For further peptide coupling methods see above.
For the synthesis of lysine-linked antibody-drug conjugates L2’ is an amine-reactive group from the group of
Figure imgf000270_0001
wherein #2 represents the attachment point to the group L1, L1’ or SG X1 represents a leaving group such as for example a Cl, Br or I, or an aryl sulfonate such as for example p-toluene sulfonate, or a alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate (triflate group).  
More preferably, activated moiety Z’’-D for the synthesis of lysine-linked antibody-drug conjugates bears a N-hydroxysuccinimidyl ester as in general structure 1-154
‘
Figure imgf000271_0001
 
wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A, and Q represents one of the following general structures (i) to (iii): (i) §–L1-SG-§§ (ii) §–L1-SG-L1’-§§ (iii) §–L1-§§ wherein § represents the attachment point to D; §§ represents the attachment point to L2’; SG represents an in vivo cleavable group, L1 and L1’ represent, independently of each other, an in vivo non-cleavable organic group, and L2’ represents an activated attachment group.
A method to synthesize N-hydroxysuccinimidyl esters of general structure 1-145 which are a substructure of general structure 1-154 is shown in scheme 41.
 
Figure imgf000272_0001
Scheme 41: Route for the synthesis of compound of structure 1-145, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A and wherein R12 is C1-C10 alkyl, preferably C1-C5.  Amine of general structure 1-88 is reacted with a bis-N-hydroxysuccinimidyl ester of structure 1-144 by methods known to those skilled in the art to give maleimide of general structure 1- 145. These methods include addition of a base like e.g. triethyl amine or diisoproypethylamine and a suitable solvent like DMF or THF. Alternative methods include coupling of amine 1-88 with the corresponding free acid of structure 1-133 under peptide coupling conditions known to those skilled in the art as described above.
The metabolites of the exemplified antibody-drug conjugates bearing non-cleavable linkers can be described by the general structure 1-146 for cysteine-linked ADCs and 1-147 for lysine- linked conjugates.
 
Figure imgf000273_0001
wherein Z’, and D are as defined herein. More specifically, cysteine-linked antibody-drug conjugates with non-cleavable linkers connected via a succinimide deliver metabolites of general structure 1-149. The succinimide ring can also be present in an open form described by general structures 1-149a and 1-149b. The metabolites of general structure 1-149 can be synthesized by reacting maleimide of general structure 1-138 with cysteine (1-148) in a suitable solvent like DMF at a temperature range of 0-40°C, preferably at 25°C to give cysteine-metabolite 1-149 as described in scheme 42.
 
Figure imgf000274_0001
Scheme 41: Route for the synthesis of cysteine metabolites of structure 1-149, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A.
Lysine-linked antibody-drug conjugates with non-cleavable linkers connected via an amide bond deliver metabolites of general structure 1-152. The metabolites of general structure 1- 152 can be synthesized by reacting N-hydroxysuccinimidyl esters of general structure 1-154 with a-amino,a-carboxyl-bisprotected lysine (1-150) in a suitable solvent like DMF or THF and a suitable base like triethylamine or diisopropylethylamine at a temperature range of 0-40°C, preferably at 25°C to give a-amino,a-carboxyl-bisprotected lysine-metabolite of general structure 1-151 as described in scheme 42. Deprotection at the a-amino and a-carboxyl group   of lysine conjugate 1-151 by methods known to those skilled in the art as already described above delivers the lysine metabolite of general structure 1-152. An alternative method for the synthesis of 1-151 include coupling of a-amino,a-carboxyl-bisprotected lysine (1-150) with the corresponding free acid of structure 1-145 under peptide coupling conditions known to those skilled in the art as described above.
Figure imgf000275_0001
Scheme 42: Route for the synthesis of cysteine metabolites of structure 1-149, wherein R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A. 
Antibody-drug conjugates of general structure
Figure imgf000275_0002
can be synthesized by reacting activated moiety of general structure Z’’-D with an antibody.   More specifically cysteine-linked antibody-drug conjugates can be synthesized according to scheme 43.
Figure imgf000276_0001
Scheme 43: Route for the synthesis of cysteine-linked antibody-drug conjugates, wherein AB, n, R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A. Antibody AB is reduced with triscarboxyethylphosphine (TCEP) to reduce the intrachain disulfides of the antibody in a suitable solvent like PBS buffer at pH 7.2 at a concentration between range between 1 mg/ml and 20 mg/ml, preferably in the range of about 10 mg/ml to 15 mg/ml, at a temperature between 4°C and 30°C, preferably at 20°C. Then maleimide 1-153 is added dissolved in a suitable solvent like DMSO, DMF, isopropanol or PBS buffer, preferably in DMSO whereas the amount of solvent should not exceed 10% of the total volume. For the synthesis of cysteine-linked antibody-drug conjugates with a corresponding ring- opened succinimide the reaction mixture can be basified to pH 8 by addition of a suitable base or buffer like PBS buffer pH 8. In case Q bears a reducible moiety like a disulfide the TCEP is removed after the reduction step prior to the addition of the maleimide 1-153 by a suitable method like size-exclusion chromatography via a Sephadex® column. Lysine-linked antibody-drug conjugates can be synthesized according to scheme 44.
 
Figure imgf000277_0001
Scheme 44: Route for the synthesis of cysteine-linked antibody-drug conjugates, wherein AB, n, R1, R2, R3, R4, R5, L1, t, q, m, V, W, Z and Y are as defined herein and R2, R3, R4 and R5 are connected at the positions shown for group A. Antibody AB is reacted with N-hydroxysuccinimidyl ester of general structure 1-154, dissolved in a suitable solvent like DMSO, DMF, isopropanol or PBS buffer, preferably in DMSO whereas the amount of solvent should not exceed 10% of the total volume, in a suitable solvent like PBS buffer at pH 7.2 at a concentration between range between 1 mg/ml and 20 mg/ml, preferably in the range of about 10 mg/ml to 15 mg/ml, at a temperature between 4°C and 30°C, preferably at 20°C.
Site specific conjugation Site specific conjugation, in which a known number of linker-drugs are consistently conjugated to defined sites might be used. There are various methods described in literature for site specific conjugation (Agarwal et al., Bioconjug. Chem.26, 176-192 (2015); Cal et al., Angew. Chem. Int. Ed. Engl.53, 10585-10587 (2014); Behrens et al., MAbs 6, 46-53 (2014); Panowski et al., MAbs 6, 34-45 (2014)). Methods for site specific conjugation include, in particular,enzymatic methods, e.g using transglutaminases (TGases), glycyltransferases or formylglycine generating enzyme (Sochaj et al., Biotechnology Advances, 33, 775– 784 2015).
One way for this attachment using transglutaminases (TGases), are literature-described approaches dealing with a site specific conjugation of binders using transglutaminase. Transglutaminases (TGase) including bacterial transglutaminase (BTG) (EC 2.3.2.13) are a family of enzymes that catalyze the formation of a covalent bond between the g-carbonyl amide group of glutamines and the primary amine of lysines. Since some TGases also accept substrates other than lysine as the amine donor, they have been used to modify proteins including antibodies at suitable acceptor glutamine residues (Jeger et al., Angewandte Chemie  
Int. Ed. Engl 49, 9995-9997 (2010); Josten et al., J. Immunol. Methods 240, 47-54 (2000); Mindt et al., Bioconjugate Chem. 19, 271-278 (2008); Dennler et al., in Antibody Drug Conjuagtes (Ducry, L., Ed.), pp 205-215, Humana Press. (2013)). On the one hand transglutaminases were used for coupling of drugs to antibodies bearing genetically artificial glutamine tags being transglutaminse acceptor glutamines introduced by genetically engineering (Strop et al., Chem. Biol.20, 161-167 (2013)). On the other hand it was reported that the conserved glutamine Q295 (Kabat numbering system of IgGs) located in the constant domain of the heavy chain is the sole g-carbonyl amide donor for bacterial transglutaminase (EC 2.3.2.13) within the backbone of a aglycosylated IgG1, whereas no acceptor glutamine is present in the backbone in IgG1 being glycosylated at position N297 (kabat numbering) of the heavy chain (Jeger et al., Angewandte Chemie Int. Ed. Engl 49, 9995-9997 (2010)). In summary, the bacterial transglutaminase can be used for the conjugation of an amine group of the linker/drug to an acceptor glutamine residue of the antibody. Such acceptor glutamines can be introduced by engineering of the antibody by mutations or by generation of aglycosylated antibodies. Such aglycosylated antibodies can be generated by deglycosylation using N-glycosidase F (PNGaseF) or by mutation of the N297 (Kabat numbering) of the glycosylation site of the heavy chain to any other amino acid. Enzymatic conjugation of such aglycosylated antibodies was described for aglycosylated antibody variants bearing the mutations N297D, N297Q (Jeger et al., Angewandte Chemie Int. Ed. Engl 49, 9995-9997 (2010)), or N297S (see patent applications WO2013092998A1 and WO2013092983A2). Enzymatic conjugation using transglutaminase of such aglycosylated antibodies provides ADCs with DAR of 2 in general, in which both heavy chains are functionalized site specifically at position Q295 (Kabat numbering). The mutation N297Q of the antibody provides 1 additional site for conjugation at each heavy chain leading for example to ADCS with DAR of 4, in which both heavy chains are functionalized site-specifically at position Q295 and Q297 (Kabat numbering). Antibody variants bearing the mutations Q295N and N297Q provide one acceptor glutamine residue at position Q297 (Simone Jeger, Site specific conjugation of tumour targeting antibodies using transglutaminase, Dissertation at ETH Zurich (2009)). There are several examples in literature describing site specific conjugation of aglycosylated antibodies via transglutaminase (e.g. Dennler et al., Bioconjugate Chemistry 19, 569-578 (2014); Lhospice et al., Molecular Pharmaceutics 12, 1863-1871 (2015)).
Methods of use The conjugates and compounds according to the invention show a valuable pharmacological and pharmacokinetic spectrum of action which could not have been predicted.  
They are therefore suitable for use as medicaments for the treatment and/or prophylaxis of disorders in humans and animals. Within the scope of the present invention, the term“treatment” includes prophylaxis. Commercial utility The conjugates and compounds of the present invention have surprisingly been found to effectively inhibit NAMPT finally resulting in cell death e.g. apoptosis and may therefore be used for the treatment or prophylaxis of diseases of uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune responses, or inappropriate cellular inflammatory responses, or diseases which are accompanied with uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune responses, or inappropriate cellular inflammatory responses, particularly in which the uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune responses, or inappropriate cellular inflammatory responses is mediated by NAMPT, such as, for example, benign and malignant neoplasia, more specifically haematological tumours, solid tumors, and/or metastases thereof, e.g. leukaemias and myelodysplastic syndrome, malignant lymphomas, head and neck tumors including brain tumors and brain metastases, tumors of the thorax including non-small cell and small cell lung tumors, gastrointestinal tumors, endocrine tumors, mammary and other gynaecological tumors, urological tumours including renal, bladder and prostate tumours, skin tumors, and sarcomas, and/or metastases thereof, especially haematological tumors, solid tumours, and/or metastases of breast, bladder, bone, brain, central and peripheral nervous system, cervix, colon, endocrine glands (e.g. thyroid and adrenal cortex), endocrine tumours, endometrium, esophagus, gastrointestinal tumors, germ cells, kidney, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, renal, small intestine, soft tissue, stomach, skin, testis, ureter, vagina and vulva as well as malignant neoplasias including primary tumors in said organs and corresponding secondary tumors in distant organs (“tumor metastases”). Haematological tumors can e.g. be exemplified by aggressive and indolent forms of leukemia and lymphoma, namely non-Hodgkins disease, chronic and acute myeloid leukemia (CML/ AML), acute lymphoblastic leukemia (ALL), Hodgkins disease, multiple myeloma and T-cell lymphoma. Also included are myelodysplastic syndrome, plasma cell neoplasia, paraneoplastic syndromes, and cancers of unknown primary site as well as AIDS related malignancies.
One aspect of the invention is the use of a conjugate or a compound described supra for the treatment of acute myeloid leukemias (AML) and/or myelodysplastic syndrome (MDS), as well  
as a method of treatment of AML and/or MDS, comprising administering an effective amount of a conjugate or a compound of the present invention. Well known and often used cancer cell lines to study AML are eg. MOLM-13 (human AML cell line), THP-1 (human monocytic leukemia cell line), KG-1 (bone marrow derived human acute myeloid leukemia cell line), MV- 4-11 (human biphenotypic B myelomonocytic leukemia, NOMO-1 (human acute myeloid leukemia cell line), NB4 (human promyelocytic leukemia cell line).
 
One aspect of the invention is the use of a conjugate or a compound described supra for the treatment of breast cancer, ovarian cancer, and/or gastric cancer, including HER2-positive breast, HER2-positive ovarian cancer, and/or HER2-positive gastric cancer, as well as a method of treatment of breast cancer, ovarian cancer, and gastric cancer, including HER2- positive breast, HER2-positive ovarian cancer, and/or HER2-positive gastric cancer, comprising administering an effective amount of a conjugate or a compound of the present invention. A well known and often used cancer cell lines to study breast cancer, including HER2-positive breast cancer is the MDA-MB-453 breast cancer cell line [Cailleau R. et al., In vitro 14 (11):911-915, 1978]. A well known and often used cancer cell lines to study ovarian cancer, including HER2-positive ovarian cancer is the HER2-positive SK-OV-3 ovarian cancer cell line. A well known and often used cancer cell lines to study gastric cancer, including HER2- positive gastric cancer is the HER2-positive NCI-N87 gastric cancer cell line. One aspect of the invention is the use of a conjugate or a compound described supra for the treatment of brain tumors, including glioblastomas, as well as a method of treatment of brain tumors, including glioblastomas, comprising administering an effective amount of a conjugate or a compound of the present invention. Well known and often used cancer cell lines to study glioblastoma are eg. U251 MG (formerly known as U-373 MG) glioblastoma astrocytoma cells (Pontén, J., Macintyre, E. H. Acta Pathol Microbiol Scand A.74, 465-486, 1968). One aspect of the invention is the use of a conjugate or a compound described supra for the treatment of lung cancer, as well as a method of treatment of lung cancer, comprising administering an effective amount of a conjugate or a compound of the present invention. Well known and often used cancer cell lines to study lung cancer are eg. A549. One aspect of the invention is the use of a conjugate or a compound described supra for the treatment of squamous cell carcinoma of head and neck, including pharynx squamous cell carcinoma, as well as a method of treatment of squamous cell carcinoma of head and neck, including pharynx squamous cell carcinoma, comprising administering an effective amount of
 
a conjugate or a compound of the present invention. Well known and often used cancer cell lines to study pharynx squamous cell carcinoma are eg. FaDu. One aspect of the invention is the use of a conjugate or a compound described supra for the treatment of epidermoid carcinoma of the vulva, as well as a method of treatment of epidermoid carcinoma of the vulva, comprising administering an effective amount of a conjugate or a compound of the present invention. Well known and often used cancer cell lines to study epidermoid carcinoma of the vulva are eg. A431.
In accordance with an aspect of the present invention therefore the invention relates to a conjugate or a compound described supra, or an N-oxide, a salt, a tautomer or a stereoisomer of said conjugate or compound, or a salt of said N-oxide, tautomer or stereoisomer particularly a pharmaceutically acceptable salt thereof, or a mixture of same, as described and defined herein, for use in the treatment or prophylaxis of a disease, especially for use in the treatment of a disease. Another particular aspect of the present invention is therefore the use of a conjugate or a compound described supra, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, for the prophylaxis or treatment of hyperproliferative diseases and/or disorders responsive to induction of cell death i.e apoptosis. The term“inappropriate” within the context of the present invention, in particular in the context of “inappropriate cellular immune responses, or inappropriate cellular inflammatory responses”, as used herein, is to be understood as preferably meaning a response which is less than, or greater than normal, and which is associated with, responsible for, or results in, the pathology of said diseases. Preferably, the use is in the treatment of hyperproliferative diseases and/or disorders responsive to induction of cell death, wherein the diseases are cancer diseases, haematological tumours, solid tumors and/or metastases thereof, e.g. leukaemias and myelodysplastic syndrome, malignant lymphomas, head and neck tumors including brain tumors and brain metastases, tumors of the thorax including non-small cell and small cell lung tumors, gastrointestinal tumors, endocrine tumors, mammary and other gynaecological tumors, urological tumors including renal, bladder and prostate tumors, skin tumors, and sarcomas, and/or metastases thereof.  
A preferred aspect is the use of a conjugate or a compound described supra for the prophylaxis and/or treatment of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), breast cancer (particularly HER2-positive breast), ovarian cancer (particularly HER2-positive ovarian cancer), gastric cancer (particularly HER2-positive gastric cancer), brain tumors (particularly glioblastoma) lung cancer, squamous cell carcinoma of head and neck (particularly pharynx squamous cell carcinoma) or epidermoid carcinoma of the vulva and/or metastases thereof. Another aspect of the present invention is the use of a conjugate or a compound described supra or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, as described herein, in the manufacture of a medicament for the treatment or prophylaxis of a disease, wherein such disease is a hyperproliferative disorder or a disorder responsive to induction of cell death e.g.apoptosis. In an embodiment the disease is a haematological tumour, a solid tumour and/or metastases thereof, e.g. leukaemias and myelodysplastic syndrome, malignant lymphomas, head and neck tumours including brain tumours and brain metastases, tumours of the thorax including non-small cell and small cell lung tumours, gastrointestinal tumours, endocrine tumours, mammary and other gynaecological tumours, urological tumours including renal, bladder and prostate tumours, skin tumours, and sarcomas, and/or metastases thereof. In a preferred embodiment the disease is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), breast cancer (particularly HER2-positive breast), ovarian cancer (particularly HER2-positive ovarian cancer), gastric cancer (particularly HER2-positive gastric cancer), a brain tumor (particularly glioblastoma) lung cancer, squamous cell carcinoma of head and neck (particularly pharynx squamous cell carcinoma) or epidermoid carcinoma of the vulva and/or metastases thereof.
Method of treating hyper-proliferative disorders
The present invention relates to a method for using a conjugate or a compound described supra and compositions thereof, to treat mammalian hyper-proliferative disorders. Conjugates and compounds can be utilized to inhibit, block, reduce, decrease, etc., cell proliferation and/or cell division, and/or produce cell death e.g. apoptosis. This method comprises administering to a mammal in need thereof, including a human, an amount of a conjugate or a compound described supra, or a pharmaceutically acceptable salt, isomer, polymorph, metabolite, hydrate, solvate or ester thereof ; etc. which is effective to treat the disorder. Hyper-proliferative disorders include but are not limited, e.g., psoriasis, keloids, and other hyperplasias affecting the skin, benign prostate hyperplasia (BPH), solid tumours, such as cancers of the breast,  
respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases. Those disorders also include lymphomas, sarcomas, and leukaemias. Examples of breast cancer include, but are not limited to invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Examples of cancers of the respiratory tract include, but are not limited to small-cell and non- small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma. Examples of brain cancers include, but are not limited to brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumour. Tumours of the male reproductive organs include, but are not limited to prostate and testicular cancer. Tumours of the female reproductive organs include, but are not limited to endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus.
Tumours of the digestive tract include, but are not limited to anal, colon, colorectal, oesophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers. Tumours of the urinary tract include, but are not limited to bladder, penile, kidney, renal pelvis, ureter, urethral and human papillary renal cancers. Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma. Examples of liver cancers include, but are not limited to hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to squamous cell carcinoma, Kaposi’s sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head-and-neck cancers include, but are not limited to laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, lip and oral cavity cancer and squamous cell. Lymphomas include, but are not limited to AIDS-related lymphoma, non-Hodgkin’s lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma, Hodgkin’s disease, and lymphoma of the central nervous system.  
Sarcomas include, but are not limited to sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia. These disorders have been well characterized in humans, but also exist with a similar etiology in other mammals, and can be treated by administering pharmaceutical compositions of the present invention. The term“treating” or“treatment” as stated throughout this document is used conventionally, e.g., the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving the condition of, etc., of a disease or disorder, such as a carcinoma. Methods of treating NAMPT disorders
The present invention also provides methods for the treatment of disorders associated with aberrant NAMPT activity. Such disorders include, but are not limited to, disorders associated with activation of NF-KB, inflammatory and tissue repair disorders; particularly rheumatoid arthritis, inflammatory bowel disease, asthma and COPD (chronic obstructive pulmonary disease), osteoarthritis, osteoporosis and fibrotic diseases; dermatosis, including psoriasis, atopic dermatitis and ultra- violet induced skin damage; autoimmune diseases including systemic lupus erythematosis, multiple sclerosis, psoriatic arthritis, ankylosing spondylitis, tissue and organ rejection, Alzheimer's disease, stroke, athersclerosis, restenosis, diabetes, glomerulonephritis, cancer, including, but not limited to, breast, prostate, lung, colon, cervix, ovary, skin, CNS, bladder, pancreas, leukaemia, lymphoma or Hodgkin's disease, cachexia, inflammation associated with infection and certain viral infections, including Acquired Immune Deficiency Syndrome (AIDS), adult respiratory distress syndrome, and ataxia telengiectasia, heart failure, hepatomegaly, cardiomegaly, diabetes, cystic fibrosis, symptoms of xenograft rejections, septic shock or asthma. Involvement of NAMPT in the treatment of cancer is described in WO 97/48696. Involvement of NAMPT in immuno-supression is described in WO 97/48397. Involvement of NAMPT for the treatment of diseases involving angiogenesis is described in WO2003/80054. Involvement of  
NAMPT for the treatment of rheumatoid arthritis and septic shock is described in WO 2008/025857. Involvement of NAMPT for the prophlaxis and treatment of ischaemia is described in WO 2009/109610. Effective amounts of conjugates or compounds of the present invention can be used to treat such disorders, including those diseases (e.g., cancer) mentioned in the Background section above. Nonetheless, such cancers and other diseases can be treated with conjugates or compounds of the present invention, regardless of the mechanism of action and/or the relationship between NAMPT and the disorder. The phrase“aberrant NAMPT activity” includes any abnormal expression or activity of the gene encoding the enzyme or of the polypeptide it encodes. Examples of such aberrant activity, include, but are not limited to, over-expression of the gene or polypeptide; gene amplification; mutations which produce constitutively-active or hyperactive enzyme activity; gene mutations, deletions, substitutions, additions, etc. The present invention also provides for methods of inhibiting a NAMPT activity, comprising administering an effective amount of a conjugate or a compound of the present invention, including salts, polymorphs, metabolites, hydrates, solvates, prodrugs (e.g.: esters) thereof, and diastereoisomeric forms thereof. NAMPT activity can be inhibited in cells (e.g., in vitro), or in the cells of a mammalian subject, especially a human patient in need of treatment. Methods of treating angiogenic disorders
The present invention also provides methods of treating disorders and diseases associated with excessive and/or abnormal angiogenesis. Inappropriate and ectopic expression of angiogenesis can be deleterious to an organism. A number of pathological conditions are associated with the growth of extraneous blood vessels. These include, e.g., diabetic retinopathy, ischemic retinal-vein occlusion, and retinopathy of prematurity [Aiello et al. New Engl. J. Med.1994, 331, 1480 ; Peer et al. Lab. Invest.1995, 72, 638], age-related macular degeneration [AMD ; see, Lopez et al. Invest. Opththalmol. Vis. Sci. 1996, 37, 855], neovascular glaucoma, psoriasis, retrolental fibroplasias, angiofibroma, inflammation, rheumatoid arthritis (RA), restenosis, in-stent restenosis, vascular graft restenosis, etc. In addition, the increased blood supply associated with cancerous and neoplastic tissue, encourages growth, leading to rapid tumour enlargement and metastasis. Moreover, the growth of new blood and lymph vessels in a tumour provides an escape route for renegade cells, encouraging metastasis and the consequence spread of the cancer. Thus,  
conjugates and compounds of the present invention can be utilized to treat and/or prevent any of the aforementioned angiogenesis disorders, e.g., by inhibiting and/or reducing blood vessel formation; by inhibiting, blocking, reducing, decreasing, etc. endothelial cell proliferation or other types involved in angiogenesis, as well as causing cell death e.g. apoptosis of such cell types. Preferably, the diseases of said method are haematological tumours, solid tumour and/or metastases thereof. The conjugates and compounds of the present invention can be used in particular in therapy and prevention i.e. prophylaxis, especially in therapy of tumour growth and metastases, especially in solid tumours of all indications and stages with or without pre-treatment of the tumour growth. Methods for Treating a Patient Diagnosed with or Suspected to have a Cancer Deficient in Nicotinic Acid Pathway
The present invention also provides methods for treating a patient diagnosed with or suspected to have a cancer deficient in nicotinic acid pathway. Said method comprises the steps of administering to the patient:
(a) an effective amount of a conjugate or a compound of the present invention; and
(b) an effective amount of nicotinic acid. Optionally, the method further comprises a step of administering to the patient:
(c) a DNA damaging agent. Methods to determine whether a cancer is deficient in nicotinic acid pathway are known to the skilled person (for example in WO2009/072004 which is incorporated herein in its entirety by reference). Suitable DNA damaging agents include, but are not limited to those described herein. The specific DNA damaging agent for each patient will vary according to the nature and severity of the condition as determined by the attending diagnostician, the activity of the specific conjugate or compound employed, the age and general condition of the patient, time of administration, route of administration, rate of excretion of the drug, drug combinations, and the like.
 
The present invention also includes a conjugate or a compound of the present invention in combination with nicotinic acid for use in the treatment of a patient diagnosed with or suspected to have a cancer deficient in nicotinic acid pathway. The present invention also includes the use of a conjugate or a compound of the present invention in combination with nicotinic acid, in the manufacture of a medicament for the treatment of a patient diagnosed with or suspected to have a cancer deficient in nicotinic acid pathway. In an embodiment the effective amount of nicotinic acid is administered intravenously. In another embodiment the effective amount of nicotinic acid is administered orally. In an embodiment the nicotinic acid is administered prior or subsequent to the administration of the conjugate or compound of the present invention. In an embodiment the nicotinic acid is administered simultaneously with the administration of the conjugate or compound of the present invention. Method for predicting the utility of administering nicotinic acid to reduce the severity of side- effects of cancer treatment with NAMPT inhibitors
The present invention also provides a method for stratifying patients according to the utility of administering nicotinic acid to reduce the severity of side-effects of cancer treatment with NAMPT inhibitors according to the present invention, the method comprising the steps of: a) determining the level of Nicotinic acid phosphoribosyltransferase (NAPRT) in a cancer of said subject; and
b) 1) in the event of a level of NAPRT, as determined in step a) above, which is lower than a predetermined threshold value, selecting said subject for sequential or simultaneous treatment with i) an effective amount of a conjugate or a compound described supra, and ii) an effective amount of nicotinic acid, a nicotinic acid precursor or a prodrug of nicotinic acid; and
2) in the event of a level of NAPRT, as determined in step a) above, which is higher than or equal to a predetermined threshold value, selecting said subject for treatment with i) an effective amount of a conjugate or a compound described supra in the absence of sequential or simultaneous treatment with ii) an effective amount of nicotinic acid, a nicotinic acid precursor or a prodrug of nicotinic acid.
 
The invention further comprises a method for the treatment or for alleviating the symptoms of a cancer in a subject, the method comprising the steps of:
a) determining the level of Nicotinic acid phosphoribosyltransferase (NAPRT) in said subject; and,
b) 1) in the event of a level of NAPRT, as determined in step a) above, which is lower than a predetermined threshold value, treating said subject sequentially or simultaneous with i) a conjugate or a compound described supra, and ii) an effective amount of nicotinic acid, a nicotinic acid precursor or a prodrug of nicotinic acid; and
2) in the event of a level of NAPRT, as determined in step a) above, which is higher than or equal to a predetermined threshold value, treating said subject with i) a conjugate or a compound described supra in the absence of sequential or simultaneous treatment with ii) an effective amount of a nicotinic acid, a nicotinic acid precursor or a prodrug of nicotinic acid. The above methods optionally comprise a further step c): administering said subject an effective amount of a DNA damaging agent. In an embodiment of the above methods the level of NAPRT is determined in the tumour tissue of said subject. In an embodiment of the above methods the level of nicotinic acid phosphoribosyltransferase (NAPRT) is determined on the level of nucleic acids encoding NAPRT, such as by RT-PCR or quantification of DNA methylation (suitable methods are provided, for example, in Shames et al., Clin Cancer Res; 19(24); 6912–23, which is incorporated herein in its entirety by reference). In an embodiment of the above methods the level of Nicotinic acid phosphoribosyltransferase (NAPRT) is determined on the level of proteins, such as in assays based on specific antibodies or other specific binding partners to NAPRT. The present invention also relates to a conjugate or a compound according to the present invention for use in a method for the treatment or for alleviating the symptoms of a cancer in a subject, said method comprising the steps a) and b) supra. The present invention also relates to NAPRT for use as a biomarker useful in a method for predicting the utility of administering a nicotinic acid, a nicotinic acid precursor or a prodrug of nicotinic acid to reduce the severity of side-effects of cancer treatment with a conjugate or a compound according to the present invention.
 
The present invention further relates to the use of NAPRT as a biomarker in selecting responsive patients to the sequential or simultaneous treatment with i) an effective amount of a conjugate or a compound described supra, and ii) an effective amount of a nicotinic acid, a nicotinic acid precursor or a prodrug of nicotinic acid. The present invention further relates to the use of Nicotinic acid phosphoribosyltransferase (NAPRT) as a biomarker in selecting patients that benefit from being treated with an effective amount of a conjugate or a compound described supra in the absence of sequential or simultaneous treatment with an effective amount of a nicotinic acid, a nicotinic acid precursor or a prodrug of nicotinic acid. The stratification of the subjects is based on a predetermined threshold value. Suitable methods to establish the predetermined threshold value are know to the skilled person (for example in WO2011006988 or in US8,912,184 which are incorporated herein in their entirety by reference). The predetermined threshold value may be, for example, set by the medical practitioner based data from a plurality of patients, e.g. at least 5 patient, or at least 20 patient, or even at least 50 patients. Hence, in order to create basis for setting the threshold value, it will be necessary to first establish or obtain data from a cohort of existing patients to determine the level of NAPRT in their tumour tissue. The level of NAPRT in tumour tissue may be determined by one of a number of methods which either directly measure NAPRT, or which in a more indirect manner correlates (or is expected to correlate) with the level of NAPRT in the tissue in question. The cohort to which reference is made is desirably matched to one or more of tumour type, age, sex, or severity of disease, in particular the tumour type. In one variant, however, the threshold value may set based on the level of NAPRT of a different tissue type than the tumour tissue in a population of human beings. This may be similar or identical patients, or may alternatively be healthy subjects. However, preferably, the threshold value is set based on the level of NAPRT in the same tissue, such as tumour tissue, as the tumour tissue in question, and obtained from plurality of patients with the same cancer indication. Methods of Sensitizing Cells to Radiation
In a distinct embodiment of the present invention, a conjugate or a compound of the present invention may be used to sensitize a cell to radiation. That is, treatment of a cell with a conjugate or a compound of the present invention prior to radiation treatment of the cell renders the cell more susceptible to DNA damage and cell death than the cell would be in the absence of any treatment with a conjugate or a compound of the invention. In one aspect, the cell is treated with at least one conjugate or compound of the invention.
 
Thus, the present invention also provides a method of killing a cell, wherein a cell is administered one or more conjugates or compounds of the invention in combination with conventional radiation therapy. The present invention also provides a method of rendering a cell more susceptible to cell death, wherein the cell is treated one or more conjugates or compounds of the invention prior to the treatment of the cell to cause or induce cell death. In one aspect, after the cell is treated with one or more conjugates or compounds of the invention, the cell is treated with at least one conjugate or compound, or at least one method, or a combination thereof, in order to cause DNA damage for the purpose of inhibiting the function of the normal cell or killing the cell. In one embodiment, a cell is killed by treating the cell with at least one DNA damaging agent. That is, after treating a cell with one or more conjugates or compounds of the invention to sensitize the cell to cell death, the cell is treated with at least one DNA damaging agent to kill the cell. DNA damaging agents useful in the present invention include, but are not limited to, chemotherapeutic agents (e.g., cisplatinum), ionizing radiation (X-rays, ultraviolet radiation), carcinogenic agents, and mutagenic agents. In another embodiment, a cell is killed by treating the cell with at least one method to cause or induce DNA damage. Such methods include, but are not limited to, activation of a cell signalling pathway that results in DNA damage when the pathway is activated, inhibiting of a cell signalling pathway that results in DNA damage when the pathway is inhibited, and inducing a biochemical change in a cell, wherein the change results in DNA damage. By way of a non- limiting example, a DNA repair pathway in a cell can be inhibited, thereby preventing the repair of DNA damage and resulting in an abnormal accumulation of DNA damage in a cell. In one aspect of the invention, a conjugate or a compound of the invention is administered to a cell prior to the radiation or other induction of DNA damage in the cell. In another aspect of the invention, a conjugate or a compound of the invention is administered to a cell concomitantly with the radiation or other induction of DNA damage in the cell. In yet another aspect of the invention, a conjugate or a compound of the invention is administered to a cell immediately after radiation or other induction of DNA damage in the cell has begun. In another aspect, the cell is in vitro. In another embodiment, the cell is in vivo. Pharmaceutical compositions of the compounds of the invention
 
This invention also relates to pharmaceutical compositions containing one or more conjugates or compounds of the present invention. These compositions can be utilised to achieve the desired pharmacological effect by administration to a patient in need thereof. A patient, for the purpose of this invention, is a mammal, including a human, in need of treatment for the particular condition or disease. Therefore, the present invention includes pharmaceutical compositions comprising a conjugate or a compound as defined supra, together with at least one pharmaceutically acceptable carrier or auxiliary. Another aspect of the invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier or auxiliary and a pharmaceutically effective amount of a conjugate or a compound, or salt thereof, of the present invention. Another aspect of the invention is a pharmaceutical composition comprising a pharmaceutically effective amount of a conjugate or a compound as defined supra and a pharmaceutically acceptable auxiliary for the treatment of a disease mentioned supra, especially for the treatment of haematological tumours, solid tumours and/or metastases thereof. Another aspect of the invention is a pharmaceutical composition comprising a conjugate or a compound as defined supra and a pharmaceutically acceptable auxiliary for the treatment of a haematological tumour, a solid tumour and/or metastases thereof. A pharmaceutically acceptable carrier or auxiliary is preferably a carrier that is non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. Carriers and auxiliaries are all kinds of additives assisting the composition to be suitable for administration. A pharmaceutically effective amount of a conjugate or a compound is preferably that amount which produces a result or exerts the intended influence on the particular condition being treated. The conjugates and compounds of the present invention can be administered with pharmaceutically-acceptable carriers or auxiliaries well known in the art using any effective conventional dosage unit forms, including immediate, slow and timed release preparations. The conjugates and compounds of this invention may be administered parenterally, that is, subcutaneously, intravenously, intraocularly, intrasynovially, intramuscularly, or  
interperitoneally, as injectable dosages of the conjugate or compound in preferably a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid or mixture of liquids such as water, saline, aqueous dextrose and related sugar solutions, an alcohol such as ethanol, isopropanol, or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, glycerol ketals such as 2,2-dimethyl-1,1-dioxolane-4-methanol, ethers such as poly(ethylene glycol) 400, an oil, a fatty acid, a fatty acid ester or, a fatty acid glyceride, or an acetylated fatty acid glyceride, with or without the addition of a pharmaceutically acceptable surfactant such as a soap or a detergent, suspending agent such as pectin, carbomers, methycellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agent and other pharmaceutical adjuvants. Illustrative of oils which can be used in the parenteral formulations of this invention are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum and mineral oil. Suitable fatty acids include oleic acid, stearic acid, isostearic acid and myristic acid. Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate. Suitable soaps include fatty acid alkali metal, ammonium, and triethanolamine salts and suitable detergents include cationic detergents, for example dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamine acetates ; anionic detergents, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates; non-ionic detergents, for example, fatty amine oxides, fatty acid alkanolamides, and poly(oxyethylene-oxypropylene)s or ethylene oxide or propylene oxide copolymers ; and amphoteric detergents, for example, alkyl-beta- aminopropionates, and 2-alkylimidazoline quarternary ammonium salts, as well as mixtures. The parenteral compositions of this invention will typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers may also be used advantageously. In order to minimise or eliminate irritation at the site of injection, such compositions may contain a non-ionic surfactant having a hydrophile-lipophile balance (HLB) preferably of from about 12 to about 17. The quantity of surfactant in such formulation preferably ranges from about 5% to about 15% by weight. The surfactant can be a single component having the above HLB or can be a mixture of two or more components having the desired HLB. Illustrative of surfactants used in parenteral formulations are the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
 
The pharmaceutical compositions may be in the form of sterile injectable aqueous suspensions. Such suspensions may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents such as, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents which may be a naturally occurring phosphatide such as lecithin, a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate, a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadeca-ethyleneoxycetanol, a condensation product of ethylene oxide with a partial ester derived form a fatty acid and a hexitol such as polyoxyethylene sorbitol monooleate, or a condensation product of an ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, for example polyoxyethylene sorbitan monooleate. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Diluents and solvents that may be employed are, for example, water, Ringer’s solution, isotonic sodium chloride solutions and isotonic glucose solutions. In addition, sterile fixed oils are conventionally employed as solvents or suspending media. For this purpose, any bland, fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. Controlled release formulations for parenteral administration include liposomal, polymeric microsphere and polymeric gel formulations that are known in the art. The compositions of the invention can also contain other conventional pharmaceutically acceptable compounding ingredients, generally referred to as carriers or diluents, as necessary or desired. Conventional procedures for preparing such compositions in appropriate dosage forms can be utilized. Such ingredients and procedures include those described in the following references, each of which is incorporated herein by reference: Powell, M.F. et al., "Compendium of Excipients for Parenteral Formulations" PDA Journal of Pharmaceutical Science & Technology 1998, 52(5), 238-311 ; Strickley, R.G "Parenteral Formulations of Small Molecule Therapeutics Marketed in the United States (1999)-Part-1" PDA Journal of Pharmaceutical Science & Technology 1999, 53(6), 324-349 ; and Nema, S. et al., "Excipients and Their Use in Injectable Products" PDA Journal of Pharmaceutical Science & Technology 1997, 51(4), 166-171. Pharmaceutical compositions according to the present invention can be illustrated as follows: i.v. solution:
 
The conjugate according to the invention is dissolved in a concentration below the saturation solubility in a physiologically acceptable solvent (e.g. isotonic saline solution, D-PBS, or a formulation with glycine and sodium chloride in citrate buffer with addition of polysorbate 80). The solution is subjected to sterile filtration and dispensed into sterile and pyrogen-free injection vessels. i.v. solution: The conjugates according to the invention can be converted to the administration forms mentioned. This can be accomplished in a manner known per se by "mixing with" or "dissolving in" inert, non-toxic, pharmaceutically suitable excipients (e.g. buffer substances, stabilizers, solubilizers, preservatives). The following, for example, may be present: amino acids (glycine, histidine, methionine, arginine, lysine, leucine, isoleucine, threonine, glutamic acid, phenylalanine and others), sugars and related compounds (glucose, saccharose, mannitol, trehalose, sucrose, mannose, lactose, sorbitol), glycerol, sodium salts, potassium, ammonium salts and calcium salts (e.g. sodium chloride, potassium chloride or disodiumhydrogenphosphate and many others), acetate/acetic acid buffer systems, phosphate buffer systems, citric acid and citrate buffer systems, trometamol (TRIS and TRIS salts), Polysorbates (e.g. Polysorbate 80 and Polysorbate 20), Poloxamers (e.g. Poloxamer 188 and Poloxamer 171), Macrogols (PEG derivatives, e.g. 3350), Triton X-100, EDTA salts, glutathione, albumins (e.g. human), urea, benzyl alcohol, phenol, chlorocresol, metacresol, benzalkonium chloride and many others.
Lyophilizate for subsequent conversion into an i.v., s.c. or i.m. solution: Alternatively the compounds of the invention may be converted into a stable lyophilizate (possibly with the aid of abovementioned excipients) and, before being administered, reconstituted with a suitable solvent (e.g. injection-grade water, isotonic saline solution) and administered.
Dose and administration Based upon standard laboratory techniques known to evaluate compounds useful for the treatment of hyper-proliferative disorders and angiogenic disorders, by standard toxicity tests and by standard pharmacological assays for the determination of treatment of the conditions identified above in mammals, and by comparison of these results with the results of known  
medicaments that are used to treat these conditions, the effective dosage of the conjugates and compounds of this invention can readily be determined for treatment of each desired indication. The amount of the active ingredient to be administered in the treatment of one of these conditions can vary widely according to such considerations as the particular conjugate or compound and dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient treated, and the nature and extent of the condition treated. The total amount of the active ingredient to be administered will generally range from about 0.001 mg/kg to about 200 mg/kg body weight per day, and preferably from about 0.01 mg/kg to about 20 mg/kg body weight per day. Clinically useful dosing schedules will range from one to three times a day dosing to once every four weeks dosing. In addition, "drug holidays" in which a patient is not dosed with a drug for a certain period of time, may be beneficial to the overall balance between pharmacological effect and tolerability. A unit dosage may contain from about 0.5 mg to about 1500 mg of active ingredient, and can be administered one or more times per day or less than once a day. The average daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous and parenteral injections, and use of infusion techniques will preferably be from 0.01 to 200 mg/kg of total body weight. Of course the specific initial and continuing dosage regimen for each patient will vary according to the nature and severity of the condition as determined by the attending diagnostician, the activity of the specific compound employed, the age and general condition of the patient, time of administration, route of administration, rate of excretion of the drug, drug combinations, and the like. The desired mode of treatment and number of doses of a compound of the present invention or a pharmaceutically acceptable salt or ester or composition thereof can be ascertained by those skilled in the art using conventional treatment tests. Combination Therapies The conjugates and compounds of this invention can be administered as the sole pharmaceutical agent or in combination with one or more other pharmaceutical agents where the combination causes no unacceptable adverse effects. Those combined pharmaceutical agents can be other agents having antiproliferative effects such as for example for the treatment of haematological tumours, solid tumours and/or metastases thereof and/or agents for the treatment of undesired side effects.The present invention relates also to such combinations. Other anti-hyper-proliferative agents suitable for use with the composition of the invention include but are not limited to those compounds acknowledged to be used in the treatment of neoplastic diseases in Goodman and Gilman's The Pharmacological Basis of Therapeutics  
(Ninth Edition), editor Molinoff et al., publ. by McGraw-Hill, pages 1225-1287, (1996), which is hereby incorporated by reference, especially (chemotherapeutic) anti-cancer agents as defined supra. The combination can be a non-fixed combination or a fixed-dose combination as the case may be. Methods of testing for a particular pharmacological or pharmaceutical property are well known to persons skilled in the art. Another embodiment of the invention are conjugates and compounds according to the claims as disclosed in the Claims section wherein the definitions are limited according to the preferred or more preferred definitions as disclosed below or specifically disclosed residues of the exemplified conjugates and compounds and subcombinations thereof. The following examples illustrate the invention in greater detail, without restricting it. Further conjugates and compounds according to the invention, of which the preparation is not explicitly described, can be prepared in an analogous way. The term“according to” within the experimental section is used in the sense that the procedure referred to is to be used“analogously to”.
Experimental Part The following table lists the abbreviations used in this paragraph and in the Intermediates and Examples section as far as they are not explained within the text body.
Figure imgf000296_0001
 
Figure imgf000297_0001
Amino acid Abbreviations Ala = Alanine
Arg = Arginine
 
Asn = Asparagine Asp = Aspartic acid Cys = Cysteine Glu = Glutamic acid Gln = Glutamine Gly = Glycine His = Histidine Ile = Isoleucine Leu = Leucine Lys = Lysine Met = Methionine Phe = Phenylalanine Pro = Proline Ser = Serine Thr = Threonine Trp = Tryptophan Tyr = Tyrosine Cit = citrulline Val = Valine The compounds of the present invention optionally contain a cleavable group SG. As used herein, when SG is a peptide the amino acid residues which constitute said peptide are herein referred to indistinctly by its three letter code, amino acid or amino acid residue (e.g. -Ala-Val- , -alanine-valine- or -alanyl-valyl-). It is understood that a hyphen at the beginning and/or at the end of the amino acid residues mark the points of attachment to the rest of the molecule. Unless stated otherwise, said hyphen may be connected to the N-terminus or the C-terminus   of the amino acid residue to which it connects. The present invention includes all such possible connecting modes (e.g.–Ala-Val- -alanine-valine- or -alanyl-valyl- includes both (C-terminus)- Ala-Val-(N-terminus) and (N-terminus)-Ala-Val-(C-terminus)). Similarly, whenever said hyphens are not present, it is understood that the end amino acid residues of the peptide chain may connect to the rest of the molecule via the N-terminus or the C-terminus of the end amino acid residues (e.g. when SG is Ala-Val-, alanine-valine- or -alanyl-valyl- it includes both (C- terminus)-Ala-Val-(N-terminus) and (N-terminus)-Ala-Val-(C-terminus)). Unless indicated otherwise, the present invention includes all such possible connecting modes.
Other abbreviations not specified herein have their meanings customary to the skilled person. The various aspects of the invention described in this application are illustrated by the following examples which are not meant to limit in any way the full scope of the invention as described herein.
Specific Experimental Descriptions NMR peak forms in the following specific experimental descriptions are stated as they appear in the spectra, possible higher order effects have not been considered. Reactions employing microwave irradiation may be run with a Biotage Initator ® microwave oven optionally equipped with a robotic unit. The reported reaction times employing microwave heating are intended to be understood as fixed reaction times after reaching the indicated reaction temperature. The compounds and intermediates produced according to the methods of the invention may require purification. Purification of organic compounds is well known to the person skilled in the art and there may be several ways of purifying the same compound. In some cases, no purification may be necessary. In some cases, the compounds may be purified by crystallization. In some cases, impurities may be stirred out using a suitable solvent. In some cases, the compounds may be purified by chromatography, particularly flash column chromatography, using for example prepacked silica gel cartridges, e.g. from Separtis such as Isolute® Flash silica gel (“SiO2”) or Isolute® Flash NH2 silica gel (“amine-coated SiO2”) in combination with a Isolera autopurifier (Biotage) and eluents such as gradients of e.g. hexane/ ethyl acetate or dichloromethane/methanol. In some cases, the compounds may be purified by preparative HPLC using for example a Waters autopurifier equipped with a diode array detector and/or on- line electrospray ionization mass spectrometer in combination with a suitable prepacked reverse phase column and eluents such as gradients of water and acetonitrile which may contain additives such as trifluoroacetic acid, formic acid or aqueous ammonia. In some cases,  
purification methods as described above can provide those compounds of the present invention which possess a sufficiently basic or acidic functionality in the form of a salt, such as, in the case of a compound of the present invention which is sufficiently basic, a trifluoroacetate or formate salt for example, or, in the case of a compound of the present invention which is sufficiently acidic, an ammonium salt for example. A salt of this type can either be transformed into its free base or free acid form, respectively, by various methods known to the person skilled in the art, or be used as salts in subsequent biological assays. It is to be understood that the specific form (e.g. salt, free base etc) of a compound of the present invention as isolated as described herein is not necessarily the only form in which said compound can be applied to a biological assay in order to quantify the specific biological activity. The purities of the intermediates and examples are ³95% as judged by LC/MS or HNMR spectra if not stated otherwise. The percentage yields reported in the following examples are based on the starting component that was used in the lowest molar amount. Air and moisture sensitive liquids and solutions were transferred via syringe or cannula, and introduced into reaction vessels through rubber septa. Commercial grade reagents and solvents were used without further purification. The term“concentrated under reduced pressure” refers to use of a Buchi rotary evaporator at a minimum pressure of approximately 15 mm of Hg. All temperatures are reported uncorrected in degrees Celsius (°C). In order that this invention may be better understood, the following examples are set forth. These examples are for the purpose of illustration only, and are not to be construed as limiting the scope of the invention in any manner. All publications mentioned herein are incorporated by reference in their entirety.
Analytical LC-MS conditions LC-MS-data given in the subsequent specific experimental descriptions refer (unless otherwise noted) to the following conditions:
Methods: Method 1: Instrument: Waters Acquity UPLC-MS SQD; column: Acquity UPLC BEH C18 1.7 50x2.1mm; Eluent A: water + 0.1% vol. formic acid (99%), Eluent B: acetonitrile; gradient: 0-  
1.6 min 1-99% B, 1.6-2.0 min 99% B; rate 0.8 ml/min; temperature: 60 °C; injection: 1 µl; DAD scan: 210-400 nm; ELSD. Method 2: Instrument: Waters Acquity UPLC-MS SQD; column: Acquity UPLC BEH C18 1.7 50x2.1mm; Eluent A: water + 0.2% vol. NH3 (32%), Eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; rate 0.8 ml/min; temperature: 60 °C; injection: 1 µl; DAD scan: 210-400 nm; ELSD. Method 3: Instrument: Waters Acquity UPLC-MS SQD; column: Acquity UPLC BEH C18 1.7 50x2.1mm; Eluent A: water + 0.2% vol. NH3 (32%), Eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; rate 0.8 ml/min; temperature: 60 °C; injection: 2 µl; DAD scan: 210-400 nm; ELSD. Method 4: Instrument: Waters Acquity UPLC-MS SQD; column: Acquity UPLC BEH C18 1.7 50x2.1mm; eluent A: water + 0.1% Vol. HCOOC (99%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow 0.8 ml/min; temperature: 60 °C; injection: 2 µl; DAD scan: 210-400 nm. Method 5: Instrument: SHIMADZU LCMS; UFLC 20-AD and LCMS 2020 MS detector; column: Shim-pack XR-ODS, 2.2 µm, 3.0 × 50 mm. eluents: acetonitrile / water + 0.05% acetic acid. Method 6: Instrument MS: Thermo Scientific FT-MS; instrument UHPLC: Thermo Scientific UltiMate 3000; column: Waters, HSST3, 2.1 x 75 mm, C18 1.8 µm; eluent A: water + 0.01% HCOOH; eluent B: acetonitrile + 0.01% HCOOH; gradient: 0.0 min 10% B ® 2.5 min 95% B ® 3.5 min 95% B; temperature: 50°C; flow: 0.90 ml/min; UV-detection: 210 nm / optimum Integration path 210-300 nm. Method 7: Instrument: Agilent MS Quad 6150; HPLC: Agilent 1290; column: Waters Acquity UPLC HSS T31.8 µ 50 x 2.1 mm; eluent A: water + 0.025 % HCOOH, eluent B: acetonitrile + 0.025 % HCOOH; gradient: 0.0 min 90% A ® 0.3 min 90% A ® 1.7 min 5% A ® 3.0 min 5% A, temperature: 50°C; flow: 1.20 ml/min; UV-detection: 205– 305 nm. Method 8: Instrument: Waters ACQUITY SQD UPLC System; column: Waters Acquity UPLC HSS T31.8 µ 50 x 1 mm; eluent A: water + 0.025% HCOOH, eluent B: 1 l Acetonitril + 0.025% HCOOH; gradient: 0.0 min 90% A ® 1.2 min 5% A ® 2.0 min 5% A, temperature: 50°C; flow: 0.40 ml/min; UV-detection: 208– 400 nm. Flash column chromatography conditions
 
“Purification by (flash) column chromatography” as stated in the subsequent specific experimental descriptions refers to the use of a Biotage Isolera purification system. For technical specifications see“Biotage product catalogue” on www.biotage.com.
Preparation of antibody/active compound conjugates (ADC) General process for generating of different anti-target antibodies The anti-CD123 antibodies were obtained by CDR grafting. The sequence of the 7G3 antibody (EP2426148) represents the starting point of the humanized antibodies such as TPP-5969. Bispecific scFv immunofusion proteins based on 12F1 are disclosed in WO 2013/173820. Based on the publication of the sequences of the variable regions (VH and VL) of 12F1 (WO 2013/173820), the following antibody sequences were obtained by fusion of the variable domains of the donor immunoglobulin (VH and VL) with the constant regions of a human antibody. A chimeric variant of 12F1 such as TPP-6013 was generated. Humanization The murine antibody sequence of the 7G3 antibody was humanized by transferring the CDRs into a human antibody skeleton. For the definition of the CDRs according to Kabat, see Andre C.R. Martin,“Protein sequence and structure analysis of antibody variable domains” in Antibody Engineering (Springer Lab Manuals), Eds.: Duebel, S. and Kontermann, R., Springer- Verlag, Heidelberg. After comparison of the murine frame sequences (without CDRs) with human germline sequences, a similar frequently occurring human frame sequence was selected. In this case, it was the heavy chain IGHV1-46-01 with the J sequence IGHJ4-03 and the light chain IGKV4-1-01 with the J segment IGKJ2. The germline sequences originated from the VBASE2 database (Retter I, Althaus HH, Münch R, Müller W: VBASE2, an integrative V gene database. Nucleic Acids Res.2005 Jan 1; 33(Database issue):D671-4). The sequences were named using the IMGT system (Lefranc, M.-P., Giudicelli, V., Ginestoux, C., Jabado- Michaloud, J., Folch, G., Bellahcene, F., Wu, Y., Gemrot, E., Brochet, X., Lane, J., Regnier, L., Ehrenmann, F., Lefranc, G. and Duroux, P. IMGT®, the international ImMunoGeneTics information system®. Nucl. Acids Res, 37, D1006-D1012 (2009); doi:10.1093/nar/gkn838). The antibody variant TPP-5969 described herein carries various point mutations differing from the human germline sequence which may influence its properties. The anti-B7H3 antibodies were generated, for example, by screening of a phage display library for recombinant murine B7H3 (murine CD276; Gene ID: 102657) and human B7H3 (human CD276; Gene ID: 80381) expressing cells. Particularly the antibodies TPP-6497 and TPP-8382  
are important examples. The antibodies obtained in this manner were reformatted into the human IgG1 format. These two antibodies were used for the working examples described here. In addition, antibodies which bind to B7H3 are known to the person skilled in the art.
General process for expressing anti-target antibodies in mammalian cells The antibodies, for example TPP-5969, TPP-6013, TPP-6497, TPP-8382 and TPP-1015 were produced in transient mammalian cell cultures as described by Tom et al., Chapter 12 in Methods Express: Expression Systems edited by Michael R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007 General process for purifying antibodies from cell supernatants The antibodies, for example TPP-5969, TPP-6013, TPP-6497, TPP-8382 and TPP-1015 were obtained from the cell culture supernatants. The cell supernatants were clarified by centrifugation of cells. The cell supernatant was then purified by affinity chromatography on a MabSelect Sure (GE Healthcare) chromatography column. To this end, the column was equilibrated in DPBS pH 7.4 (Sigma/Aldrich), the cell supernatant was applied and the column was washed with about 10 column volumes of DPBS pH 7.4 + 500 mM sodium chloride. The antibodies were eluted in 50 mM sodium acetate pH 3.5 + 500 mM sodium chloride and then purified further by gel filtration chromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4.
Checking the antigen-binding of the ADCs The capability of the binder of binding to the target molecule was checked after coupling had taken place. The person skilled in the art is familiar with multifarious methods which can be used for this purpose; for example, the affinity of the conjugate can be checked using ELISA technology or surface plasmon resonance analysis (BIAcore™ measurement). The conjugate concentration can be measured by the person skilled in the art using customary methods, for example for antibody conjugates by protein determination. (see also Doronina et al.; Nature Biotechnol. 2003; 21:778-784 and Polson et al., Blood 2007; 1102:616-623).
The following antibodies were used for the coupling reactions:
 
Figure imgf000304_0001
The letter in the ADC example number describes the respective antibody part. The letter“M” denotes the respective active metabolite derived from the ADC after intracellular degradation of the antibody and/or linker part of the ADC. In case several preparations have been made of a certain antibody-drug conjugate the different preparations are distinguished by an additional small case letter (a, b, c, etc.). For illustration, example 36Ea refers to preparation“a” of an anti-B7H3 TPP-8382-conjugate whereas example 36Eb refers to preparation“b” of the same anti-B7H3 TPP-8382-conjugate.
Procedure 1: General process for coupling to cysteine side chains Between 2 and 8 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), dissolved in PBS buffer, were added to a solution of the appropriate antibody in PBS buffer in the concentration range between 1 mg/ml and 20 mg/ml, preferably in the range of about 10 mg/ml to 15 mg/ml, and the mixture was stirred at RT for 1h. For this purpose, the solution of the respective antibody used can be employed at the concentrations stated in the working examples, or it may optionally also be diluted with PBS buffer to about half of the stated starting concentrations in order to get into the preferred concentration range. Subsequently, depending on the intended loading from 2 to 20 equivalents, preferably about 5-16 equivalents of the maleinimide precursor compound or halide precursor compound to be coupled can be added as a solution in DMSO. Here, the amount of DMSO should not exceed 10% of the total volume. The reaction was stirred in the case of maleinimide precursors for 60-240 min at RT and then applied to PBS-equilibrated PD 10 columns (Sephadex ® G-25, GE Healthcare) and eluted with PBS buffer. Generally, unless indicated otherwise, 5 mg of the antibody in question in PBS buffer were used for the reduction and the subsequent coupling. Purification on the PD10 column thus in each case afforded solutions of the respective ADCs in 3.5 ml PBS buffer. The   sample was then concentrated by ultracentrifugation and optionally rediluted with PBS buffer. If required, for better removal of low-molecular weight components, concentration by ultrafiltration was repeated after redilution with PBS buffer. For biological tests, if required, the concentrations of the final ADC samples were optionally adjusted to the range of 0.5-15 mg/ml by redilution. The respective protein concentrations, stated in the working examples, of the ADC solutions were determined. Furthermore, antibody loading (drug/mAb ratio) was determined using the methods described under procedure 5. Typically 3.0 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 8.0 equivalents of the corresponding maleimide were employed in the conjugation reaction. For the generation of species with a higher drug/antibody ratio (>4) typically 8.0 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 16.0 equivalents of the corresponding maleimide were employed in the conjugation reaction. Typically the following general procedure was used for the conjugation of 5 mg of antibody: Under argon, a solution of 0.029 mg of TCEP in 50 µl of PBS buffer (pH 7.2) was added to 5 mg of the antibody in question in 0.5 ml of PBS buffer (pH 7.2) (c~10 mg/ml). The reaction was stirred at RT for 30 min, and 0.27 µmol of the maleinimide precursor compound dissolved in 50 µl of DMSO were then added. After a further 1.5 h– 2 h of stirring at RT, the reaction was diluted with 1900 µl of PBS buffer (pH 7.2). This solution was then applied to PD 10 columns (Sephadex® G-25, GE Healthcare) which had been equilibrated with PBS buffer (pH 7.2) and was eluted with PBS buffer (pH 7.2). The eluate was then centrifuged at 10°C/4G for 5 min, and the supernatant was then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2) to a volume of 2.5 mL. Typically the following general procedure was used for the conjugation of 35 mg of antibody: Under argon, a solution of 0.201 mg of TCEP in 200 µl of PBS buffer (pH 7.2) was added to 35 mg of the antibody in question in 2.5 of PBS buffer (pH 7.2) (c~15 mg/ml). The reaction was stirred at RT for 30 min, and 1.17 µmol of the maleinimide precursor compound dissolved in 200 µl of DMSO were then added. After a further 1.5 h– 2 h of stirring at RT, the reaction was diluted with PBS buffer (pH 7.2) to a total volume of 5.0 mL. This solution was then applied to PD 10 columns (Sephadex ® G-25, GE Healthcare) which had been equilibrated with PBS buffer (pH 7.2) and was eluted with PBS buffer (pH 7.2). The eluate was then centrifuged at 10°C/4G for 5 min, and the supernatant was then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2) to a volume of 3.5 mL. Unless indicated otherwise, the immunoconjugates shown in the examples were prepared by this process. Depending on the linker, the ADCs shown in the examples may also be present  
to a lesser or higher degree in the form of the hydrolysed open-chain succinamides attached to the antibodies. Both isomeric froms could be present. In particular the ADCs containing the linker substructure
 
Figure imgf000306_0001
as connection to thiol groups of the antibodies may optionally also be prepared in a targeted manner by rebuffering after the coupling and stirring at pH 8 for about 20 h according to Scheme A via the ADCs attached via ring-closed succinamides. #1 represents the sulphur bridge to the antibody, and #2 the point of attachment to the modified payload. Such ADCs in which the linker is attached to the antibodies through hydrolysed open-chain succinamides may optionally also be prepared in a targeted manner by an exemplary procedure as follows: Procedure 2: General process for coupling to cysteine side chains with ring-opened maleimide Typically the following general procedure was used: Under argon, a solution of 0.029 mg of TCEP in 50 µl of PBS buffer (pH 7.2) was added to 5 mg of the antibody in question in 0.5 ml of PBS buffer (pH 8) (c~10 mg/ml). The reaction was stirred at RT for 30 min, and 0.27 µmol of the maleinimide precursor compound dissolved in 50 µl of DMSO were then added. After a further 1.5 h– 2 h of stirring at RT, the reaction was diluted with 1900 µl of PBS buffer (pH 8). This solution was then applied to PD 10 columns (Sephadex ® G-25, GE Healthcare) which had been equilibrated with PBS buffer pH 8 and was eluted with PBS buffer pH 8. The eluate was then stirred at RT under argon overnight. After that the eluate was then centrifuged at 10°C/4G for 5 min, and the supernatant was then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2) to a volume of 2.5 mL. The respective protein concentrations, stated in the working examples, of the ADC solutions were determined. Furthermore, antibody loading (drug/mAb ratio) was determined using the methods described under procedure 5.
 
Other potentially hydrolysis-sensitive thianylsuccinimide bridges to the antibody in the working examples contain the following linker substructures, where #1 represents the thioether linkage to the antibody and #2 the point of attachment to the modified payload:
Figure imgf000307_0001
These linker substructures represent the linking unit to the antibody and have (in addition to the linker composition) a significant effect on the structure and the profile of the metabolites formed in the tumour cells. Procedure 3: General process for coupling to lysine side chains To a solution of the appropriate antibody in PBS buffer in the concentration range between 1 mg/ml and 20 mg/ml, preferably in the range of about 10 mg/ml to 15 mg/ml, was added a solution of the corresponding final intermediate (e.g. N-hydroxysuccinimidyl ester), typically 2- 10 equivalents, in DMSO. After stirring for 30 min to 6 h at r.t. again a solution of the corresponding final intermediate (e.g. N-hydroxysuccinimidyl ester), typically 2-10 equivalents, in DMSO was added and the mixture was stirred for further 30 min to 6 h at r.t.. Preferentially, the amount of added DMSO should not exceed 10% of the total volume. After that the mixture was applied to PBS-equilibrated PD 10 columns (Sephadex ® G-25, GE Healthcare) and eluted with PBS buffer. Generally, unless indicated otherwise, 5 mg of the antibody in question in PBS buffer were used for the reduction and the subsequent coupling. Purification on the PD10  
column thus in each case afforded solutions of the respective ADCs in 3.5 ml PBS buffer. The sample was then concentrated by ultracentrifugation and optionally rediluted with PBS buffer. If required, for better removal of low-molecular weight components, concentration by ultrafiltration was repeated after redilution with PBS buffer. For biological tests, if required, the concentrations of the final ADC samples were optionally adjusted to the range of 0.5-15 mg/ml by redilution. The respective protein concentrations, stated in the working examples, of the ADC solutions were determined. Furthermore, antibody loading (drug/mAb ratio) was determined using the methods described under procedure 5. Typically the following general procedure was used for the conjugation of 5 mg of antibody: Under argon, a solution of 0.175 µmol of the final intermediate (e.g. N-hydroxysuccinimidyl ester) in 25 µl of DMSO was added to to 5 mg of the antibody in question in 0.5 ml of PBS buffer (pH 7.2) (c~10 mg/ml). The reaction was stirred at RT for 1 h and again a solution of 0.175 µmol of the final intermediate in 25 µl of DMSO was added. After stirring for further 1 h the reaction was diluted with PBS buffer (pH 7.2) to a total volume of 2.5 mL. This solution was then applied to PD 10 columns (Sephadex ® G-25, GE Healthcare) which had been equilibrated with PBS buffer (pH 7.2) and was eluted with PBS buffer (pH 7.2). The eluate was then centrifuged at 10°C/4G for 5 min, and the supernatant was then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2) to a volume of 2.5 mL. Typically the following general procedure was used for the conjugation of 35 mg of antibody: Under argon, a solution of 1.17 µmol of the final intermediate (e.g. N-hydroxysuccinimidyl ester) in 300 µl of DMSO was added to to 35 mg of the antibody in question in 5 ml of PBS buffer (pH 7.2) (c~7 mg/ml). The reaction was stirred at RT for 1 h and again a solution of 1.17 µmol of the final intermediate in 300 µl of DMSO was added. After stirring for further 1 h the reaction was diluted with PBS buffer (pH 7.2) to a total volume of 7.5 mL. This solution was then applied to PD 10 columns (Sephadex ® G-25, GE Healthcare) which had been equilibrated with PBS buffer (pH 7.2) and was eluted with PBS buffer (pH 7.2). The eluate was then centrifuged at 10°C/4G for 5 min, and the supernatant was then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2) to a volume of 3.5 mL.
Procedure 4: General process for coupling to cysteine side chains in case the final intermediate bears a reducible moiety, e.g. disulfide In case the final intermediate bears a reducible moiety like e.g. disulfide, remaining TCEP from the antibody reduction step may also cleave the reducible moiety in the final intermediate and  
should thus been removed. The general methods for the conjugation to cysteine side chains should thus be adjusted. Typically the following general procedure was used: Under argon, a solution of 0.029 mg of TCEP in 50 µl of PBS buffer (pH 7.2) was added to 5 mg of the antibody in question in 0.5 ml of PBS buffer (pH 8) (c~10 mg/ml). The reaction was stirred at RT for 30 min and was then applied to PD 10 columns (Sephadex ® G-25, GE Healthcare) which had been equilibrated with PBS buffer (pH 7.2) and was eluted with PBS buffer (pH 7.2). To the eluate 0.27 µmol of the maleinimide precursor compound dissolved in 50 µl of DMSO were then added. After a further 1.5 h– 2 h of stirring at RT, the reaction was diluted with 1900 µl of PBS buffer (pH 8). This solution was then applied to PD 10 columns (Sephadex ® G-25, GE Healthcare) which had been equilibrated with PBS buffer (pH 7.2) and was eluted with PBS buffer (pH 7.2). The eluate was then centrifuged at 10°C/4G for 5 min, and the supernatant was then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2) to a volume of 2.5 mL. This solution was then applied to PD 10 columns (Sephadex ® G-25, GE Healthcare) which had been equilibrated with PBS buffer pH 8 and was eluted with PBS buffer pH 8. The eluate was then stirred at RT under argon overnight. After that the eluate was then centrifuged at 10°C/4G for 5 min, and the supernatant was then concentrated by ultracentrifugation and rediluted with PBS buffer (pH 7.2) to a volume of 2.5 mL. The respective protein concentrations, stated in the working examples, of the ADC solutions were determined. Furthermore, antibody loading (drug/mAb ratio) was determined using the methods described under procedure 5.
Procedure 5: Determination of the antibody, the toxophore loading and the conjugation site For protein identification in addition to molecular weight determination after deglycosylation and/or denaturing, a tryptic digestion was carried out, which, after denaturing, reduction and derivatization, confirms the identity of the protein via the tryptic peptides found. The toxophor loading of the conjugates described in the working example was determined as follows: Determination of toxophor loading of lysine coupled ADCs were carried out by mass spectromic determination of the molecular weights of the individual conjugate species. Here, the conjugates were first deglycosylated using PNGaseF, acidified and after HPLC separation/desalting over a short C4 column (GromSil 300 Butyl-1 ST, 5µm, 5mmx500µm), analysed by mass spectrometry using an ESI-QTof System (Bruker Daltonik). All spectra over  
the signal in the TIC (Total Ion Chromatogram) were added and the molecular weight of the different conjugate species was calculated based on MaxEnt deconvolution. The DAR (= drug/antibody ratio) was then calculated out of the sum of toxophor number weighted coupled species divided by the sum of the singly weighted integration results of each species. Determination of cysteine coupled ADCs were carried out by mass spectrometric determination of the molecular weights of the individual conjugate species after reduction. Guanidinium hydrochloride (GuHCl) (28.6 mg) and a solution of DL-Dithiothreitol (DTT) (500 mM, 3 µL) were added to the ADC solution (1mg/mL, 50µL). The mixture was incubated at 55°C for 1 hour and then analyzed by mass spectrometry after online desalting using an ESI- QT of Bruker Daltonik. For DAR determination, all spectra over the signal in the TIC (Total Ion Chromatogram) were added and the molecular weight of the different conjugate species was calculated based on MaxEnt deconvolution of light and heavy Chain. Average loading of the antibody with toxophors was calculated from the peak areas determined by integration as double the sum of the HC-Load and the LC-load, whereas the HC-load is the sum of toxophor number weighted integration results of all heavy chain (HC) -peaks divided by the sum of the singly weighted integration results of the HC- peaks and whereas the LC-load is the sum of toxophor number weighted integration results of the light chain (LC)-peaks divided by the sum of the singly weighted integration results of all LC peaks. For determination of the ring opening percentage of the cysteine adduct, the areas of the molecular weight peaks of the ring closed and the ring opened cysteine adduct (delta in molecular weight 18 Da) was determined for the 1-fold conjugated light chain (also possible for light and heavy chain). The mean value over all variants gives the percentage of ring opened cysteine adduct. Alternatively, the toxophor loading of cysteine-linked conjugates was determined by reversed- phase chromatography of the reduced and denatured ADCs. Guanidinium hydrochloride (GuHCl) (28.6 mg) and a solution of DL-Dithiothreitol (DTT) (500 mM, 3 µl) were added to the ADC solution (1 mg/ml, 50 µl). The mixture was incubated at 55°C for one hour and analyzed by HPLC. HPLC analysis was carried out on an Agilent 1260 HPLC system with detection at 220 nm. A Polymer Laboratories PLRP-S polymeric reversed-phase column (catalogue number PL1912- 3802) (2.1 x150 mm, 8 µm particle size, 1000 Å) was used at a flow rate of 1 ml/min with the following gradient: 0 min, 25%B; 3 min, 25%B; 28 min, 50%B. Mobile phase A consisted of 0.05% trifluoroacetic acid (TFA) in water, mobile phase B of 0.05% trifluoroacetic acid in acetonitrile.  
The detected peaks were assigned by retention time comparison with the light chain (L0) and the heavy chain (H0) of the non-conjugated antibody. Peaks detected exclusively in the conjugated sample were assigned to the light chains with one toxophore (L1) or the heavy chains with one, two or three toxophors (H1, H2, H3). Average loading of the antibody with toxophors was calculated from the peak areas determined by integration as double the sum of the HC-Load and the LC-load, whereas the HC-load is the sum of toxophor number weighted integration results of all heavy chain (HC) -peaks divided by the sum of the singly weighted integration results of the HC- peaks and whereas the LC- load is the sum of toxophor number weighted integration results of the light chain (LC)-peaks divided by the sum of the singly weighted integration results of all LC peaks. Alternatively, the Drug load was determined by UV-absorption during size exclusion chromatography (SEC). Here, 50 µL of the ADC solution were analyzed by size exclusion chromatography. The analysis was carried out on an Agilent 1260 HPLC system with detection at 280 nm and detection at a toxophore related wavelength. A Superdex 20010/300 GL from GE Healthcare (Lot No: 10194037) (10 x310 mm, 13 µm particle size) was used at a flow rate of 1 ml/min with using isocratic condition. Mobile phase consisted of PBS buffer (pH 7.2). For determination of the drug load out of the SEC profile, the ratio R of the peak area of the monomer peak at the drug specific wavelength (^drug) and at 280 nm was determined. Out of this ratio R, the DAR can be calculated as follows:
^
Figure imgf000311_0001
Whereas e stands for the molar extinction coefficients of the antibody (Ab) and the drug (D). The extinction coefficients of the antibody at 280 nm and at the drug wavelength were determined experimentally. Mean values of different antibodies were used for calculation. For the Nampt toxophores (compounds without additional linker) the extinction coefficients were determined experimentally. The following wavelengths and extinction coefficients were determined and used for the DAR calculations:
Figure imgf000311_0002
 
Figure imgf000312_0002
“(UV)” in the experimental part.
-----Synthetic intermediates----- Intermediate 1
4-(4-aminophenyl)-2,2-dimethyl-4-oxobutanoic acid
Figure imgf000312_0001
To a solution of ethyl 4-(4-bromophenyl)-2,2-dimethyl-4-oxobutanoate (7.75 g, 24.7 mmol) in ethylene glycol (75 mL) was added at r.t. copper(I)oxide (70.8 mg, 495 µmol). After degassing of the mixture ammonia (12.8 g, 33 % in water, 247 mmol) was added and the mixture was heated to 85°C for 14 h. More ammonia was added sequentially every 24 h over the course of 3 days. Then the mixture was cooled to r.t. and combined with a parallel reaction mixture of the same size, acidified with 1N hydrochloric acid, and extracted with ethyl acetate (3 times). The combined organic phases were washed with aqueous sodium bicarbonate solution and dried over magnesium sulphate, to give the crude title compound (3.50 g, 47% yield, 47% purity). The crude compound was used in the next step without further purification.
LC-MS (method 1): Rt = 0.76 min; MS (ESIneg): m/z = 220 [M-H]–
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.874 (0.65), 0.890 (0.99), 0.906 (0.65), 1.011 (0.66), 1.017 (0.99), 1.035 (0.93), 1.089 (1.17), 1.090 (1.17), 1.100 (1.01), 1.105 (1.38), 1.118 (0.68), 1.123 (0.66), 1.144 (2.20), 1.154 (3.34), 1.167 (16.00), 1.172 (7.79), 1.190 (3.28), 1.987 (11.41), 2.084 (2.65), 3.105 (4.48), 3.386 (0.41), 3.999 (0.91), 4.017 (2.65), 4.035 (2.59), 4.053 (0.84), 6.017 (2.33), 6.530 (2.82), 6.534 (0.99), 6.547 (0.91), 6.551 (2.90), 7.178 (0.44), 7.199  
(0.89), 7.220 (0.50), 7.562 (0.77), 7.566 (0.78), 7.583 (1.10), 7.587 (0.73), 7.597 (0.59), 7.640 (2.80), 7.644 (0.90), 7.657 (0.89), 7.661 (2.56), 7.766 (0.44).
Intermediate 2
6-(4-aminophenyl)-4,4-dimethyl-4,5-dihydropyridazin-3(2H)-one
Figure imgf000313_0001
To a mixture of 4-(4-aminophenyl)-2,2-dimethyl-4-oxobutanoic acid (3.30 g, 50 % purity, 7.46 mmol) in n-propanol (83 mL) was added at r.t. hydrazinium hydrate (1.4 ml, 80 % purity, 22 mmol) and the mixture was heated to 100°C for 3 h. After that the mixture was cooled to r.t. and poured on ice water, extracted with ethyl acetate, washed with brine and filtered through a silicone filter. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography (SiO2, hexane/ethyl acetate gradient) to give the title compound (1.12 g, 66% yield).
LC-MS (method 1): Rt = 0.67 min; MS (ESIpos): m/z = 218 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.040 (16.00), 2.084 (1.20), 2.518 (0.43), 2.690 (4.78), 5.485 (2.73), 6.544 (2.61), 6.566 (2.70), 7.440 (2.65), 7.462 (2.47), 10.564 (1.98). Intermediate 3
4-nitrophenyl [4-(5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl]carbamate
Figure imgf000313_0002
To a mixture of 6-(4-aminophenyl)-4,4-dimethyl-4,5-dihydropyridazin-3(2H)-one (1.19 g, 90 % purity, 4.93 mmol) in THF (40 mL) was added 4-nitrophenyl carbonochloridate (CAS-No 7693- 46-1, 1.09 g, 5.42 mmol) and the mixture was heated to 60°C for 5 h. Then the mixture was concentrated under reduced pressure and directly used in the next step.
LC-MS (method 1): Rt = 1.09 min; MS (ESIpos): m/z = 383 [M+H]+
 
¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.074 (16.00), 1.739 (1.84), 1.747 (1.67), 1.751 (1.10), 1.756 (5.62), 1.760 (1.07), 1.765 (1.69), 1.772 (1.91), 2.518 (0.47), 2.812 (4.34), 3.577 (0.40), 3.581 (1.94), 3.584 (1.12), 3.587 (1.06), 3.592 (1.51), 3.596 (1.81), 3.597 (4.15), 3.604 (1.40), 3.608 (1.06), 3.612 (1.14), 3.615 (1.76), 7.552 (3.57), 7.557 (1.33), 7.563 (1.60), 7.569 (1.77), 7.575 (4.21), 7.585 (1.86), 7.741 (0.40), 7.747 (2.92), 7.751 (0.85), 7.764 (0.78), 7.768 (2.19), 8.307 (3.69), 8.313 (1.08), 8.325 (1.11), 8.331 (3.68), 10.658 (0.61), 10.837 (2.67). Intermediate 4
N-[4-(5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl]-1,3-dihydro-2H-pyrrolo[3,4- c]pyridine-2-carboxamide
Figure imgf000314_0001
To a mixture of 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine dihydrochloride (CAS-No. 6000-50-6 , 1.17 g, 6.03 mmol) and dichloromethane (20 mL) was added at r.t. N,N-diisopropylethyl amine (2.9 ml, 16 mmol) and the mixture stirred for 30 min. Then this mixture was added to a mixture of 4-nitrophenyl [4-(5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl]carbamate (2.28 g, 92 % purity, 5.49 mmol) in dichloromethane (22 mL) and the reaction mixture stirred at r.t. for 14 h. After that the precipitate was filtered off, washed with water (200 mL) and dried under reduced pressure to give the title compound (1.72 g, 85 % yield).
LC-MS (Method 1): Rt = 0.63 min; MS (ESIpos): m/z = 364 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.074 (16.00), 2.518 (0.89), 2.523 (0.70), 2.800 (4.37), 4.811 (1.83), 4.829 (1.84), 7.431 (1.00), 7.445 (1.03), 7.632 (1.25), 7.637 (0.49), 7.649 (0.80), 7.655 (4.04), 7.674 (4.03), 7.679 (0.74), 7.690 (0.48), 7.696 (1.12), 8.496 (1.52), 8.509 (1.45), 8.613 (3.81), 10.779 (2.72). Intermediate 5
tert-butyl {3-[3-(4-aminophenyl)-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]propyl}carbamate
 
Figure imgf000315_0001
6-(4-aminophenyl)-4,4-dimethyl-4,5-dihydropyridazin-3(2H)-one (1.00 g, 4.60 mmol) was dissolved in DMF (13 ml, 170 mmol) and sodium hydride (387 mg, 60% purity, 9.67 mmol) was added. The mixture was stirred until gas evolution has almost stopped and tetra-n- butylammonium iodide (170 mg, 460 µmol) was added. The mixture was cooled with an ice bath and tert-butyl (3-bromopropyl)carbamate (1.32 g, 5.52 mmol) was added slowly. The ice bath was removed and the mixture allowed to warm to room temperature and stirred overnight. Water was added and the mixture was extracted with DCM (2x) and dried. The solvent was removed under reduced pressure to give the title compound (1.2 g, 70% yield). LC-MS (METHOD 7): Rt = 0.67 min; MS (ESIpos): m/z = 275 [M-C5H7O2]+
Intermediate 6
4-nitrophenyl [4-(1-{3-[(tert-butoxycarbonyl)amino]propyl}-5,5-dimethyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl)phenyl]carbamate
)
Figure imgf000315_0002
tert-butyl {3-[3-(4-aminophenyl)-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]propyl}carbamate (1.30 g, 3.47 mmol) was dissolved in THF (28 ml, 350 mmol). 4- nitrophenyl carbonochloridate (700 mg, 3.47 mmol) was added and the mixture was stirred at room temperature overnight. The solvent was removed to give the crude title compound (1.8 g, 24% yield), which was used in the next step without further purification.
LC-MS (METHOD 6): Rt = 2.13 min; MS (ESIpos): m/z = 540 [M+H]+
Intermediate 7
tert-butyl {4-[3-(4-aminophenyl)-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}- carbamate
Figure imgf000316_0001
6-(4-aminophenyl)-4,4-dimethyl-4,5-dihydropyridazin-3(2H)-one (718 mg, 3.30 mmol) in DMF (9.4 ml, 120 mmol) was dissolved in DMF (9.4 ml, 120 mmol) and sodium hydride (278 mg, 60% purity, 6.94 mmol) was added. The mixture was stirred until gas evolution has almost stopped and tetra-n-butylammonium iodide (122 mg, 330 µmol) was added. The mixture was cooled with an ice bath and tert-butyl (4-bromobutyl)carbamate (1.00 g, 3.97 mmol) was added slowly. The ice bath was removed and the mixture was allowed to warm to room temperature and stirred overnight. Water was added and the precipitate was collected by filtration. Finally the residue was dried under reduced pressure to give the title compound The solvent was removed under reduced pressure to give the title compound (500 mg, 39% yield), which was used in the next step without further purification.
LC-MS (METHOD 7): Rt = 1.20 min; MS (ESIneg): m/z = 388 [M-H]-
 
Intermediate 8
4-nitrophenyl [4-(1-{4-[(tert-butoxycarbonyl)amino]butyl}-5,5-dimethyl-6-oxo-1,4,5,6-tetra- hydropyridazin-3-yl)phenyl]carbamate
0
Figure imgf000317_0001
To a mixture of tert-butyl {4-[3-(4-aminophenyl)-5,5-dimethyl-6-oxo-5,6-dihydropyridazin- 1(4H)-yl]butyl}carbamate (500 mg, 1.29 mmol) in THF (10 mL) was added 4-nitrophenyl carbonochloridate (CAS-No 7693-46-1, 259 mg, 1.29 mmol) and the mixture was stirred overnight at room temperature. Then the mixture was concentrated under reduced pressure and directly used in the next step. LC-MS (METHOD 6): Rt = 2.14 min; MS (ESIpos): m/z = 554 [M+H]+
Intermediate 9
Tert-butyl {4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5- dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}carbamate
 
Figure imgf000318_0001
To a solution of 4-nitrophenyl [4-(1-{4-[(tert-butoxycarbonyl)amino]butyl}-5,5-dimethyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl)phenyl]carbamate (430 mg, 777 µmol) in dichloromethane (9.3 mL) was added at r.t. 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine hydrochloride (122 mg, 777 µmol) and N,N-diisopropylethylamine (420 µl, 2.4 mmol) and the mixture stirred for 14 h at that temperature. After that the mixture was concentrated under reduced pressure and purified by column chromatography (SiO2, hexane/ethyl acetate gradient, then dichloromethane/methanol) to give the title compound (330 mg, 83% yield).
LC-MS (method 1): Rt = 0.98 min; MS (ESIpos): m/z = 535 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.035 (0.95), 1.053 (2.47), 1.071 (1.70), 1.078 (6.69), 1.357 (16.00), 1.988 (0.62), 2.518 (1.34), 2.523 (0.94), 2.830 (1.70), 2.918 (0.57), 2.933 (0.56), 3.422 (0.50), 3.435 (0.50), 3.440 (0.53), 3.452 (0.56), 3.701 (0.61), 4.355 (0.71), 4.817 (0.86), 4.834 (0.87), 7.434 (0.48), 7.447 (0.50), 7.652 (0.69), 7.675 (1.69), 7.703 (1.70), 7.725 (0.64), 8.499 (0.72), 8.511 (0.66), 8.614 (0.95), 8.638 (0.92).
Intermediate 10
N-{4-[1-(4-aminobutyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide trifluoroacetate
 
Figure imgf000319_0001
To a solution of tert-butyl {4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]- phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}carbamate (330 mg, 617 µmol) in dichloromethane (4 mL) was added at r.t. trifluoroacetic acid (240 µl, 3.1 mmol) and the mixture stirred for 14 h at that temperature. Then further trifluoroacetic acid (240 µl, 3.1 mmol) was added and the mixture was heated to 45°C for 2 h. After cooling to r.t. the mixture was concentrated under reduced pressure and the residue was dried under vacuo to give the title compound (356 mg).
LC-MS (method 1): Rt = 0.56 min; MS (ESIpos): m/z = 435 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.095 (16.00), 1.240 (0.63), 1.255 (0.58), 1.515 (0.54), 1.536 (0.96), 1.553 (0.78), 1.641 (0.41), 1.657 (0.90), 1.678 (0.90), 1.695 (0.63), 2.327 (0.47), 2.522 (1.84), 2.669 (0.50), 2.799 (0.56), 2.817 (0.98), 2.835 (1.09), 2.848 (4.68), 3.728 (0.94), 3.745 (1.90), 3.761 (0.95), 4.892 (2.82), 4.901 (2.79), 7.657 (2.86), 7.679 (4.44), 7.693 (1.45), 7.707 (1.29), 7.721 (3.92), 7.743 (1.90), 8.644 (1.51), 8.658 (1.47), 8.719 (2.24), 8.768 (2.33).
Intermediate 11
tert-butyl N2-(tert-butoxycarbonyl)-N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-L- asparaginate
 
Figure imgf000320_0001
Under an atmosphere of argon (3S)-4-tert-butoxy-3-[(tert-butoxycarbonyl)amino]-4- oxobutanoic acid (120 mg, 416 µmol) and and HATU (190 mg, 499 µmol) were dissolved in N,N-dimethylformamid (7.4 ml, 96 mmol). After 10 min. N-{4-[1-(3-aminopropyl)-5,5-dimethyl- 6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide hydrochloride (1:1) (190 mg, 416 µmol) and N,N-diisopropylethylamine (290 µl, 1.7 mmol) were added. The mixture was stirred at room temperature overnight. Subsequently, the solvent was removed under reduced pressure and the residue was purified by column chromatography on silica gel (DCM/MeOH = 10:1) to give the title compound (203 mg, 92% purity, 65% yield).
LC-MS (METHOD 6): Rt = 1.60 min; MS (ESIneg): m/z = 690 [M-H]- ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.085 (3.46), 1.365 (10.96), 1.380 (1.65), 2.837 (1.21), 3.162 (15.54), 3.175 (16.00), 4.061 (1.28), 4.075 (3.71), 4.088 (3.63), 4.101 (1.20), 7.675 (1.25), 7.707 (1.18).
Intermediate 12
tert-butyl {3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5- dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}carbamate
 
Figure imgf000321_0001
4-nitrophenyl [4-(1-{3-[(tert-butoxycarbonyl)amino]propyl}-5,5-dimethyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl)phenyl]carbamate (1.80 g, 30 % purity, 1.00 mmol) was dissolve in dichloromethane (12 ml, 190 mmol). N,N-diisopropylethylamine (540 µl, 3.1 mmol) and 2,3- dihydro-1H-pyrrolo[3,4-c]pyridine hydrochloride (1:1), (157 mg, 1.00 mmol) were added. The mixture was stirred at room temperature overnight. Subsequently, the solvent was removed under reduced pressure and the residue was purified by column chromatography on silica gel (cyclohexane/ethyl acetate, gradient) to give the title compound (120 mg, 29% purity, 20% yield).
LC-MS (method 1): Rt = 0.97 min; MS (ESIpos): m/z = 521 [M+H]+
1H-NMR (500 MHz, DMSO-d6) d [ppm]: 0.918 (0.64), 0.933 (1.53), 0.947 (0.76), 1.065 (3.05), 1.079 (7.19), 1.103 (0.73), 1.155 (0.74), 1.181 (0.42), 1.199 (0.44), 1.213 (0.88), 1.231 (0.94), 1.240 (2.35), 1.248 (5.21), 1.255 (5.05), 1.261 (5.71), 1.270 (5.77), 1.284 (4.14), 1.313 (0.52), 1.317 (0.60), 1.328 (0.43), 1.346 (1.26), 1.352 (1.68), 1.365 (4.81), 1.372 (7.60), 1.703 (0.51), 1.718 (0.57), 1.732 (0.45), 2.361 (0.41), 2.482 (0.87), 2.514 (1.99), 2.518 (1.29), 2.522 (1.02), 2.806 (0.68), 2.837 (1.83), 2.932 (1.01), 2.944 (0.71), 2.958 (0.52), 3.117 (0.96), 3.132 (1.10), 3.144 (1.06), 3.149 (0.87), 3.156 (0.56), 3.497 (0.43), 3.502 (0.41), 3.590 (0.50), 3.602 (0.65), 3.671 (1.18), 3.695 (0.66), 3.710 (0.79), 3.724 (0.47), 4.140 (0.48), 4.672 (0.66), 4.817 (0.74), 4.835 (0.82), 5.758 (16.00), 6.929 (0.48), 6.947 (0.47), 7.434 (0.65), 7.444 (0.83), 7.466 (0.59), 7.654 (0.54), 7.660 (0.93), 7.678 (1.66), 7.708 (1.49), 7.726 (0.80), 8.109 (0.49), 8.127 (0.43), 8.499 (0.78), 8.509 (0.73), 8.614 (1.02), 8.655 (0.86).
 
Intermediate 13
tert-butyl {3-[3-(4-aminophenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]propyl}carbamate
)
Figure imgf000322_0001
6-(4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (2.00 g, 9.84 mmol) was dissolved in DMF (19 ml, 250 mmol) and sodium hydride (827 mg, 60% purity, 20.7 mmol) was added. The mixture was stirred until gas evolution has almost stopped and tetra-n- butylammonium iodide (363 mg, 984 µmol) was added. The mixture was cooled with an ice bath and tert-butyl (3-bromopropyl)carbamate (2.81 g, 11.8 mmol) was added slowly. The ice bath was removed and the mixture allowed to warm to room temperature and stirred overnight. Water was added and the mixture was extracted with DCM (2x) and dried. The solvent was removed under reduced pressure to give 3.547 g (quant.) of the title compound.
LC-MS (METHOD 6): Rt = 1.49 min; MS (ESIpos): m/z = 361 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: -0.149 (0.51), -0.008 (4.64), 0.008 (4.00), 0.146 (0.57), 0.918 (0.69), 0.936 (1.86), 0.955 (0.92), 1.016 (2.29), 1.034 (2.29), 1.236 (0.53), 1.301 (0.50), 1.368 (16.00), 1.382 (1.97), 1.687 (0.71), 2.087 (1.11), 2.215 (0.53), 2.260 (0.57), 2.322 (0.67), 2.327 (0.88), 2.332 (0.65), 2.523 (2.36), 2.615 (0.49), 2.669 (0.93), 2.730 (11.81), 2.890 (14.25), 2.949 (0.65), 3.595 (0.43), 5.535 (1.83), 6.563 (1.75), 6.585 (1.74), 6.753 (0.42), 7.496 (1.64), 7.518 (1.58), 7.952 (1.78).
Intermediate 14
4-nitrophenyl {4-[1-{3-[(tert-butoxycarbonyl)amino]propyl}-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}carbamate
 
Figure imgf000323_0001
tert-butyl {3-[3-(4-aminophenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]propyl}carbamate (3.55 g, 9.84 mmol) was dissolved in THF (79 ml, 980 mmol). 4- nitrophenyl carbonochloridate (1.98 g, 9.84 mmol)was added and the mixture was stirred at room temperature overnight. The solvent was removed to give 5.17 g (quant.) of the crude title compound, which was used in the next step without further purification.
LC-MS (METHOD 6): Rt = 1.99 min; MS (ESIpos): m/z = 526 [M+H]+
Intermediate 15
6-(4-bromophenyl)-2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-4,4-dimethyl-4,5- dihydropyridazin-3(2H)-one
Figure imgf000323_0002
To a solution of 6-(4-bromophenyl)-4,4-dimethyl-4,5-dihydropyridazin-3(2H)-one (5.95 g, 88 % purity, 18.6 mmol) in DMF (36 mL) was added at r.t. sodium hydride (1.56 g, 60 % purity, 39.1  
mmol). After stirring for 20 min tetra-n-butylammoniumiodide (688 mg, 1.86 mmol) was added and the mixture was cooled to 0°C. Then a solution of (2-bromoethoxy)(tert- butyl)dimethylsilane (CAS-No.86864-60-0, 5.35 g, 22.3 mmol) in 2 mL DMF was added slowly and the mixture was stirred at r.t. for 4 h. After that the mixture was diluted with water and extracted with dichloromethane (3x). The combined organic extracts were washed with brine and filtered through a silicone filter. After removal of the solvent under reduced pressure the crude product was purified by column chromatography (SiO2, hexane(ethyl acetate gradient) to give the title compound (2.81 g, 63 % yield).
LC-MS (OA01a02): Rt = 1.77 min; MS (ESIpos): m/z = 439/441 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: -0.051 (0.70), -0.019 (0.65), 0.007 (0.82), 0.794 (0.88), 0.813 (0.82), 0.820 (16.00), 0.828 (1.44), 1.094 (6.57), 2.859 (1.90), 3.341 (2.30), 3.812 (0.75), 3.851 (0.69), 7.644 (0.99), 7.666 (1.53), 7.738 (1.56), 7.760 (0.95).
Intermediate 16
6-(4-aminophenyl)-2-(2-hydroxyethyl)-4,4-dimethyl-4,5-dihydropyridazin-3(2H)-one
Figure imgf000324_0001
A mixture of 6-(4-bromophenyl)-2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-4,4-dimethyl-4,5- dihydropyridazin-3(2H)-one (3.59 g, 8.17 mmol), benzophenone imine (1.2 mL, 9.8 mmol) and cesium carbonate (3.73 g, 11.4 mmol) in dioxane (32 mL) was degassed three times. Then tris(dibenzylidenacetone)dipalladium (150 mg, 163 µmol) and (rac)-2,2-bis- (diphenylphosphino)1,1-binaphthyl (203 mg, 327 µmol) was added and the mixture was again degassed and then heated to 80°C for 14 h. After cooling to r.t. the mixture was acidified with 1N hydrochloric acid to pH 1 and then heated to 60°C for 3 h. After cooling to r.t. the mixture was basified by addition of aqueous sodium bicarbonate solution to pH 9 and then extracted with ethyl acetate (3x). The combined organic extracts were washed with brine and filtered through a silicone filter. After removal of the solvent under reduced pressure the crude product was puirified by column chromatography (SiO2, hexane/ethyl acetate gradient) to give the title compound (0.54 g, 70% purity, 18% yield).
LC-MS (Method 1): Rt = 0.65 min; MS (ESIpos): m/z = 262 [M+H]+  
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.047 (16.00), 2.075 (0.41), 2.322 (0.75), 2.327 (1.10), 2.332 (0.80), 2.518 (3.51), 2.523 (2.46), 2.539 (0.75), 2.665 (0.80), 2.669 (1.16), 2.673 (0.82), 2.722 (4.39), 3.551 (1.36), 3.566 (1.55), 3.581 (0.62), 3.724 (1.14), 3.740 (1.94), 3.757 (0.80), 4.631 (0.86), 4.645 (2.18), 4.660 (0.86), 5.539 (2.50), 6.553 (2.71), 6.558 (0.80), 6.570 (0.80), 6.575 (2.76), 7.470 (2.65), 7.475 (0.77), 7.487 (0.77), 7.492 (2.45).
Intermediate 17
4-nitrophenyl {4-[1-(2-hydroxyethyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}carbamate
Figure imgf000325_0001
To a solution of 6-(4-aminophenyl)-2-(2-hydroxyethyl)-4,4-dimethyl-4,5-dihydropyridazin- 3(2H)-one (500 mg, 70 % purity, 1.34 mmol) in THF (5 mL) was added at r.t. 4-nitrophenyl carbonochloridate (CAS-No.7693-46-1, 270 mg, 1.34 mmol) and the mixture stirred for further 14 h at that temperature. Then the mixture was concentrated under reduced pressure to give the crude product (756 mg) which was directly used in the next step.
LC-MS (Method 1): Rt = 1.09 min; MS (ESIpos): m/z = 427 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.081 (16.00), 2.322 (0.67), 2.327 (0.95), 2.332 (0.68), 2.518 (3.04), 2.523 (2.02), 2.664 (0.68), 2.669 (0.99), 2.673 (0.72), 2.845 (4.14), 3.396 (2.49), 3.572 (0.87), 3.589 (2.15), 3.605 (1.19), 3.774 (1.12), 3.789 (1.89), 3.807 (0.78), 7.554 (3.71), 7.560 (1.17), 7.572 (1.54), 7.578 (5.56), 7.586 (0.72), 7.599 (1.92), 7.776 (2.99), 7.781 (0.92), 7.793 (0.78), 7.798 (2.29), 8.309 (3.82), 8.315 (1.14), 8.327 (1.14), 8.333 (3.79), 10.673 (0.65).
Intermediate 18
ethyl 4-(4-bromophenyl)-2,2-dimethyl-4-oxobutanoate
 
Figure imgf000326_0001
To a mixture of potassium tert-butanolate (22.5 g, 201 mmol) in THF (130 mL) was added at –70°C a solution of 1-(4-bromophenyl)ethanone (CAS-No. 99-90-1, 20.0 g, 100 mmol) in THF (40 mL). After stirring for 1 h at that temperature a solution of ethyl 2-bromo-2- methylpropanoate (CAS-No.600-00-0, 29 ml, 200 mmol) in THF (30 mL) was added slowly at –70°C after which the reaction mixture was stirred for 14 h at r.t. Then the mixture was poured on 1N hydrochloric acid (100 mL), the layers were separated and the aqueous phase was extracted with dichloromethane (2x). The combined organic phases were washed with brine, dried over sodium sulphate and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, hexane/ethyl acetate gradient) to give the title compound (15.5 g, 70 % purity, 35 % yield).
LC-MS (method 1): Rt = 1.40 min; MS (ESIpos): m/z = 313/315 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.005 (0.71), 1.019 (0.60), 1.029 (0.75), 1.032 (0.92), 1.036 (1.37), 1.047 (0.77), 1.050 (1.00), 1.054 (0.81), 1.057 (0.58), 1.062 (0.62), 1.073 (3.63), 1.075 (1.57), 1.090 (7.40), 1.093 (1.23), 1.108 (3.38), 1.111 (1.01), 1.114 (0.70), 1.121 (0.47), 1.138 (1.09), 1.143 (0.56), 1.156 (0.49), 1.216 (16.00), 3.330 (11.54), 3.351 (4.90), 3.929 (0.43), 3.947 (0.45), 3.977 (0.98), 3.995 (3.15), 4.012 (3.16), 4.030 (0.98), 7.727 (2.23), 7.732 (0.69), 7.744 (0.81), 7.749 (3.09), 7.798 (1.09), 7.848 (0.43), 7.879 (3.05), 7.884 (0.85), 7.896 (0.70), 7.901 (2.17).
Intermediate 19
4-(4-bromophenyl)-2,2-dimethyl-4-oxobutanoic acid
Figure imgf000326_0002
A mixture of ethyl 4-(4-bromophenyl)-2,2-dimethyl-4-oxobutanoate (6.70 g, 21.4 mmol), aqueous lithium hydroxide solution (64 ml, 1.0 M, 64 mmol), and THF (40 mL) was stirred at 55°C for 14 h. After cooling to r.t. the mixture was acidified to pH 2 by addition of concentrated hydrochloric acid. The mixture was extracted with dichloromethane and the organic phase
 
washed with brine, filtered through a silicone filter and concentrated under reduced pressure to give the title compound which was directly used in the next step.
LC-MS (method 1): Rt = 1.09 min; MS (ESIpos): m/z = 285/287 [M+H]+
Intermediate 20
6-(4-bromophenyl)-4,4-dimethyl-4,5-dihydropyridazin-3(2H)-one
Figure imgf000327_0001
To a mixture of 4-(4-bromophenyl)-2,2-dimethyl-4-oxobutanoic acid (18.4 g, 64.5 mmol) in n- propanol (110 ml, 1.4 mol) was added at r.t. hydrazine hydrate (6.3 ml, 99 % purity, 130 mmol) and the mixture was heated to 100°C for 2 h. After cooling to r.t. water was added and the mixture was extracted with ethyl acetate (3x). The combined organic extracts were washed with brine, filtered through a silicone filter and concentrated under reduced pressure to give the title compound (11.9 g, 88% purity, 58 % yield).
LC-MS (Method 2): Rt = 0.64 min; MS (ESIpos): m/z = 281 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.070 (16.00), 2.829 (4.86), 7.614 (2.02), 7.619 (0.73), 7.631 (0.91), 7.636 (3.61), 7.642 (0.53), 7.686 (0.54), 7.693 (3.57), 7.698 (0.96), 7.709 (0.72), 7.714 (1.96), 10.957 (1.63).
Intermediate 21
2-{2-[3-(4-bromophenyl)-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}-1H- isoindole-1,3(2H)-dione
 
Figure imgf000328_0001
To a solution of 6-(4-bromophenyl)-4,4-dimethyl-4,5-dihydropyridazin-3(2H)-one (1.49 g, 5.30 mmol) in DMF (20 mL) was added at 0°C sodium hydride (233 mg, 60 % on mineral oil, 5.83 mmol). After stirring at r.t. for 30 min the mixture was cooled again to 0°C and a solution of 2- (2-chloroethyl)-1H-isoindole-1,3(2H)-dione (CAS-No. 6270-06-0, 1.17 g, 5.56 mmol) in DMF (5 mL) was added dropwise. After stirring at r.t. for 14 h the reaction was quenched by addition of water. The mixture was extracted with dichloromethane (3x) and the combined organic extracts were washed with brine, filtered through a silicone filter and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, hexane/ethyl acetate gradient) and preparative HPLC to give the title compound (377 mg, 16% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 122x50 mm. Eluent A: water + 0.1 Vol- % HCOOH; Eluent B: acetonitrile; gradient: 0-20 min 30-70% B. rate 250 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 1.40 min; MS (ESIpos): m/z = 454/456 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.973 (16.00), 1.154 (0.61), 1.172 (1.23), 1.190 (0.64), 1.987 (2.42), 2.749 (4.77), 3.864 (0.86), 3.876 (1.38), 3.882 (1.14), 3.890 (1.24), 4.003 (1.22), 4.011 (1.17), 4.017 (1.90), 4.029 (0.85), 4.035 (0.76), 7.400 (1.38), 7.405 (0.56), 7.416 (0.93), 7.422 (4.45), 7.426 (1.01), 7.437 (0.93), 7.442 (4.59), 7.448 (0.88), 7.459 (0.57), 7.464 (1.37), 7.747 (1.11), 7.754 (0.92), 7.755 (1.12), 7.759 (1.10), 7.762 (1.28), 7.769 (3.04), 7.777 (1.53), 7.785 (3.19), 7.791 (1.45), 7.795 (1.09), 7.798 (1.14), 7.800 (1.00), 7.807 (1.11).
 
Intermediate 22
2-{2-[3-(4-aminophenyl)-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}-1H- isoindole-1,3(2H)-dione
Figure imgf000329_0001
A mixture of 2-{2-[3-(4-bromophenyl)-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}- 1H-isoindole-1,3(2H)-dione (377 mg, 830 µmol), benzophenone imine (1.2 ml, 1000 µmol) and cesium carbonate (379 mg, 1.16 mmol) in dioxane (3.s mL) was degassed three times. Then tris(dibenzylidenacetone)dipalladium (15.2 mg, 16.6 µmol) and (rac)-2,2-bis- (diphenylphosphino)1,1-binaphthyl (20.7 mg, 33.2 µmol) was added and te mixture was again degassed. After that the reaction mixture was heated to 80°C for 14 h after which the mixture was cooled to r.t., acidified to pH 1 by addition of hydrochloric acid and stirred at 60°C for 3 h. After cooling to r.t. the mixture was basified to pH 9 by addition of aqueous sodium bicarbonate solution and extracted with ethyl acetate (3x). The combined organic extracts were washed with brine, filtered through a silicone filter and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, hexane/ethyl acetate gradient) to give the title compound (200 mg, 70 % purity, 43 % yield).
LC-MS (Method 1): Rt = 0.99 min; MS (ESIpos): m/z = 391 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.939 (16.00), 1.154 (1.82), 1.172 (3.71), 1.189 (1.91), 1.987 (6.97), 2.613 (4.62), 3.838 (1.01), 3.850 (1.75), 3.864 (1.58), 3.946 (1.44), 3.961 (1.72), 3.972 (1.04), 3.999 (0.68), 4.016 (1.76), 4.034 (1.72), 4.052 (0.59), 5.488 (3.31), 6.370 (2.69), 6.391 (2.81), 7.158 (2.83), 7.180 (2.65), 7.773 (1.34), 7.781 (1.60), 7.788 (1.71), 7.795 (3.10), 7.805 (1.45), 7.817 (3.23), 7.823 (1.80), 7.830 (1.64), 7.838 (1.29).
Intermediate 23
4-nitrophenyl (4-{1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-5,5-dimethyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl}phenyl)carbamate  
Figure imgf000330_0002
To a solution of 2-{2-[3-(4-aminophenyl)-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]ethyl}-1H-isoindole-1,3(2H)-dione (200 mg, 512 µmol) in THF (3 mL) was added 4- nitrophenyl carbonochloridate (CAS-No. 7693-46-1, 103 mg, 512 µmol) and the mixture was heated to 60°C for 3 h. After cooling to r.t. the mixture was concentrated under reduced pressure to give the title compound (310 mg) which was directly used in the next step.
LC-MS (Method 2): Rt = 1.34 min; MS (ESIpos): m/z = 555 [M+H]+
Intermediate 24
6-(4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one
Figure imgf000330_0001
A mixture of (3S)-4-(4-aminophenyl)-3-methyl-4-oxobutanoic acid (CAS-Nr.42075-29-6, 20.0 g, 96.5 mmol) and hydrazine hydrate (14 mL, 290 mmol) in n-propanol (160 mL) was heated to 100°C for 4 h. After cooling to r.t. the precipitate was filtered off, washed with water and dried under reduced pressure to give the title compound (10.2 g, 49 % yield).
LC-MS (Method 1): Rt = 0.57 min; MS (ESIpos): m/z = 204 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.998 (15.87), 1.017 (16.00), 2.115 (2.96), 2.117 (2.98), 2.156 (3.52), 2.159 (3.40), 2.496 (0.82), 2.537 (2.50), 2.554 (2.89), 2.579 (2.32), 2.596 (2.22), 3.230 (1.49), 3.233 (1.59), 3.247 (2.19), 3.251 (2.19), 3.265 (1.51), 3.269 (1.44), 5.471 (10.64), 6.521 (1.36), 6.528 (11.87), 6.533 (3.44), 6.545 (3.61), 6.550 (12.71), 6.557 (1.24),
  7.431 (1.40), 7.437 (11.26), 7.442 (3.40), 7.454 (3.36), 7.459 (10.81), 7.466 (1.12), 10.634 (7.21).
Intermediate 25
6-(4-aminophenyl)-2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-methyl-4,5-dihydropyridazin- 3(2H)-one
Figure imgf000331_0001
To a solution of 6-(4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (4.01 g, 19.7 mmol) in DMF (38 mL) was added at r.t. sodium hydride (1.66 g, 60 % on mineral oil, 41.4 mmol). After stirring for 20 min at this temperature tetra-n-butylammoniumiodide (729 mg, 1.97 mmol) was added. Then the mixture was cooled to 0°C and (2-bromoethoxy)(tert- butyl)dimethylsilane (CAS-No.86864-60-0, 5.66 g, 23.7 mmol) was added. After stirring at r.t. for 14 h water was added and the mixture was extracted with dichloromethane (3x). The combined organic extracts were washed with brine, filtered through a silicone filter and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, hexane/ethyl acetate gradient) to give the title compound (4.07 g, 93 % purity, 53 % yield).
LC-MS (Method 1): Rt = 1.34 min; MS (ESIpos): m/z = 363 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: -0.085 (0.69), -0.001 (6.54), 0.002 (6.45), 0.760 (0.94), 0.810 (1.21), 0.818 (16.00), 0.825 (1.03), 1.020 (1.72), 1.038 (1.71), 1.986 (0.50), 2.082 (3.40), 3.780 (0.96), 5.539 (1.40), 6.554 (1.27), 6.576 (1.32), 7.495 (1.25), 7.516 (1.16).
Intermediate 26
4-nitrophenyl {4-[1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}carbamate  
Figure imgf000332_0001
To a solution of 6-(4-aminophenyl)-2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-methyl-4,5- dihydropyridazin-3(2H)-one (4.07 g, 93 % purity, 10.5 mmol) in THF (45 mL) was added 4- nitrophenyl carbonochloridate (CAS-No. 7693-46-1, 2.11 g, 10.5 mmol) and the mixture was heated to 60°C for 3 h. After cooling to r.t. the mixture was concentrated under reduced pressure to give the title compound (5.60 g) which was directly used in the next step.
LC-MS (Method 2): Rt = 1.01 min; MS (ESIneg): m/z = 5 [M-H]-
Intermediate 27
N-{4-[1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
)
Figure imgf000332_0002
To a mixture of 4-nitrophenyl {4-[1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}carbamate (4.64 g, 90 % purity, 7.93 mmol) and N,N- diisopropylethylamine (4.3 ml, 25 mmol) in dichloromethane (30 mL) was added at r.t. 2,3- dihydro-1H-pyrrolo[3,4-c]pyridine dihydrochloride, Cas No 6000-50-6 (1.53 g, 7.93 mmol).   Then further N,N-diisopropylethylamine (1.5 ml) and THF (25 mL) was added and the mixture was heated to 60°C. After stirring for 4 h the mixture was cooled to r.t. and poured on water. To this mixture was added 1N aqueous sodium hydroxide solution and the mixture was extracted with dichloromethane (2x). The combined organic extracts were washed with brine, filtered through a silicone filter and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, ethyl acetate/ethanol gradient) to give the title compound (1.72 g, 75 % purity, 32% yield).
LC-MS (Method 2): Rt = 1.31 min; MS (ESIpos): m/z = 508 [M+H]+
Intermediate 28
6-(4-aminophenyl)-2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-5-methyl-4,5-dihydropyridazin- 3(2H)-one
Figure imgf000333_0001
To a solution of 6-(4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (5.00 g, 24.6 mmol) in DMF (47 mL) was added at r.t. sodium hydride (2.07 g, 60 % purity, 51.7 mmol). After stirring for 20 min at this temperature tetra-n-butylammoniumiodide (909 mg, 2.46 mmol) was added. Then the mixture was cooled to 0°C (3-bromopropoxy)(tert-butyl)dimethylsilane (CAS- No.89031-84-5, 6.8 mL, 30 mmol) was added. After stirring at r.t. for 4 h water was added and the mixture was extracted with dichloromethane (3x). The combined organic extracts were washed with brine, filtered through a silicone filter and concentrated under reduced pressure. The crude product was purified by column chromatography (amine-coated SiO2, dichloromethane/ethyl acetate gradient) to give the title compound (6.00 g, 83 % purity, 54 % yield).
LC-MS (Method 2): Rt = 1.49 min; MS (ESIpos): m/z = 376 [M+H]+
  Intermediate 29
4-nitrophenyl {4-[1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}carbamate
Figure imgf000334_0001
To a solution of 6-(4-aminophenyl)-2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-5-methyl-4,5- dihydropyridazin-3(2H)-one (7.00 g, 85 % purity, 15.8 mmol) in THF (130 mL) was added 4- nitrophenyl carbonochloridate (CAS-No. 7693-46-1, 3.19 g, 15.8 mmol) and the mixture was heated to 60°C for 5 h. After cooling to r.t. the mixture was concentrated under reduced pressure to give the title compound (310 mg) which was directly used in the next step.
Intermediate 30
N-{4-[1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000334_0002
 
To a solution of 4-nitrophenyl{4-[1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}carbamate (7.50 g, 90 % purity, 12.5 mmol) in dichloromethane (150 mL) was added at r.t. N,N-diisopropylethylamine (8.9 ml, 51 mmol) and 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine dihydrochloride (CAS-No. 6000-50-6, 2.41 g, 12.5 mmol). After stirring for further 14 h at this temperature the mixture was concentrated under reduced pressure and the crude product was purified by column chromatography (dichloromethane/ethanol gradient) to give the title compound (2.10 g, ~2:1 mixture with the free hydroxyl compound).
LC-MS (Method 2): Rt = 1.36 min; MS (ESIpos): m/z = 522 [M+H]+
Intermediate 31
N-{4-[1-(3-hydroxypropyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000335_0001
Alternative A A mixture of N-{4-[1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-4-methyl-6-oxo-1,4,5,6-tetra- hydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide from the previous step (2.10 g) and tetra-n-butylammonium fluoride trihydrate (3.81 g, 12.1 mmol) and THF (33 mL) was stirred at r.t. for 14 h. After that the mixture was diluted with water and extracted with ethyl acetate. The organic phase was filtered through a silicone filter and concentrated under reduced pressure. The crude product was purified by column chromatography (amine-coated SiO2, dichloromethane/ethanol gradient and SiO2, dichloromethane/ethanol gradient) to give the title compound (1.30 g, 83 % purity, 66 % yield). LC-MS (Method 2): Rt = 0.71 min; MS (ESIpos): m/z = 408 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.915 (6.17), 0.933 (16.00), 0.951 (7.39), 1.034 (3.40), 1.051 (7.93), 1.069 (4.70), 1.277 (1.73), 1.296 (3.15), 1.314 (3.13), 1.333 (1.71), 1.524 (0.68),  
1.544 (1.50), 1.553 (1.47), 1.564 (1.90), 1.572 (1.45), 1.584 (1.29), 3.141 (2.29), 3.162 (1.98), 3.169 (1.69), 3.183 (2.16), 3.403 (0.65), 3.416 (0.67), 3.421 (1.80), 3.433 (2.19), 3.438 (1.64), 3.447 (0.68), 3.451 (1.82), 3.455 (0.63), 3.468 (0.60), 4.355 (1.10), 4.367 (2.15), 4.380 (1.04), 4.481 (0.70), 5.760 (4.97), 7.669 (0.55), 7.691 (1.08), 7.729 (1.06), 7.752 (0.47), 8.497 (0.53), 8.510 (0.49), 8.611 (0.67), 8.683 (0.59).
Alternative B To a solution of 4-nitrophenyl{4-[1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}carbamate (7.50 g, 90 % purity, 12.5 mmol) in dichloromethane (150 mL) was added at r.t. N,N-diisopropylethylamine (8.9 ml, 51 mmol) and 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine dihydrochloride (CAS-No. 6000-50-6, 2.41 g, 12.5 mmol). After stirring for further 14 h at this temperature the mixture was concentrated under reduced pressure and the crude product was purified by column chromatography (dichloromethane/ethanol gradient) and subsequent trituration with hot methanol to give the title compound (1.26 g, 24% yield).
LC-MS (Method 1): Rt = 0.61 min; MS (ESIpos): m/z = 408 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.053 (16.00), 1.071 (15.88), 1.223 (0.84), 1.240 (3.86), 1.256 (4.62), 1.272 (2.41), 1.723 (1.17), 1.742 (4.02), 1.759 (6.35), 1.775 (4.42), 1.793 (1.33), 2.273 (2.97), 2.277 (3.26), 2.314 (3.94), 2.318 (4.26), 2.323 (2.45), 2.327 (2.61), 2.332 (1.77), 2.337 (0.80), 2.518 (8.04), 2.523 (5.31), 2.660 (0.76), 2.665 (1.85), 2.669 (2.61), 2.674 (2.13), 2.679 (2.93), 2.696 (2.89), 2.720 (2.37), 2.737 (2.09), 3.159 (3.58), 3.172 (3.62), 3.372 (1.65), 3.385 (2.33), 3.403 (1.65), 3.420 (3.82), 3.433 (4.94), 3.436 (8.12), 3.449 (8.24), 3.452 (4.78), 3.465 (3.26), 3.639 (1.33), 3.657 (2.53), 3.674 (2.37), 3.691 (2.93), 3.708 (1.33), 3.883 (1.33), 3.901 (2.85), 3.919 (1.97), 3.934 (2.33), 3.952 (1.09), 4.095 (0.80), 4.109 (0.76), 4.457 (5.15), 4.470 (11.74), 4.483 (4.74), 4.818 (8.24), 4.835 (8.36), 7.435 (4.58), 7.447 (4.70), 7.655 (0.96), 7.661 (8.44), 7.667 (2.97), 7.679 (3.86), 7.684 (14.95), 7.690 (2.21), 7.726 (2.41), 7.732 (15.12), 7.737 (4.02), 7.749 (2.81), 7.754 (7.76), 8.499 (7.44), 8.511 (6.95), 8.614 (9.33), 8.645 (8.60).
Intermediate 32
N-(4-{1-[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
 
Figure imgf000337_0002
To a solution of triphenylphosphine (1.61 g, 6.14 mmol) and phthalimide (CAS-No. 85-41-6, 451 mg, 3.07 mmol) in THF (37 mL) was added 0°C N-{4-[1-(3-hydroxypropyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (1.25 g, 100 % purity, 3.07 mmol) at and diisopropyl azodicarboxylate (1.2 ml, 6.1 mmol). After stirring at r.t. for 14 h the mixture was filtrated and the filter washed with THF. The filtrate was concentrated under reduced pressure and the crude product was purified by column chromatography (SiO2, dichloromethane/ethanol gradient) to give the title compound (1.03 g, 70 % purity, 44 % yield).
LC-MS (Method 2): Rt = 0.96 min; MS (ESIpos): m/z = 537 [M+H]+
Intermediate 33
S-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]propyl} ethanethioate
Figure imgf000337_0001
To a solution of diisopropyl azodicarboxylate (280 µl, 1.4 mmol) and triphenylphosphine (373 mg, 1.42 mmol) in THF (6 mL) was added 0°C thioacetic acid (100 µl, 1.4 mmol) and N-{4-[1-   (3-hydroxypropyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carboxamide (290 mg, 712 µmol) After stirring at r.t. for 14 h more thioacetic aced was added and the mixture stirred for further 4 h at r.t. after which the mixture was diluted with saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate. The organic phase was washed with brine, filtered through a silicone filter and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, dichloromethane/ethanol gradient) to give the title compound (160 mg, 96 % purity, 46 % yield).
LC-MS (Method 1): Rt = 0.80 min; MS (ESIpos): m/z = 466 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.053 (0.55), 1.075 (4.08), 1.093 (4.01), 1.836 (1.00), 1.854 (1.47), 1.872 (1.03), 2.287 (0.88), 2.290 (0.93), 2.320 (16.00), 2.327 (2.11), 2.331 (1.59), 2.447 (0.76), 2.518 (3.15), 2.523 (2.16), 2.665 (0.56), 2.669 (0.77), 2.674 (0.56), 2.701 (0.63), 2.717 (0.80), 2.742 (0.67), 2.834 (1.12), 2.851 (1.79), 2.855 (1.92), 2.870 (0.97), 2.872 (0.98), 3.159 (10.35), 3.172 (11.95), 3.676 (0.62), 3.693 (0.65), 3.710 (0.79), 3.884 (0.79), 3.901 (0.67), 3.917 (0.65), 4.082 (1.00), 4.096 (2.97), 4.109 (2.78), 4.122 (0.86), 4.818 (2.41), 4.835 (2.37), 5.759 (1.43), 7.434 (1.32), 7.447 (1.33), 7.660 (2.10), 7.666 (0.84), 7.678 (1.13), 7.683 (3.97), 7.689 (0.76), 7.730 (3.91), 7.735 (1.10), 7.747 (0.83), 7.752 (2.01), 8.499 (2.03), 8.511 (1.87), 8.614 (2.63), 8.616 (2.60), 8.646 (2.34).
Intermediate 34
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanine
N ) N
Figure imgf000338_0001
To a solution of L-valyl-L-alanine (CAS-No. 27493-61-4, 244 mg, 1.30 mmol) in DMF (7.5 mL) was added at r.t. 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (CAS- No. 55750-63-5, 400 mg, 1.30 mmol) and triethylamine (360 µl, 2.6 mmol). After stirring for 14 h at this temperature the mixture was concentrated under reduced pressure, taken up in ethyl acetate and washed with sodium bicarbonate solution. After washing with brine the organic phase was filtered through a silicone filter and concentrated under reduced pressure to give the title compound (133 mg, 95% purity, 25% yield).
LC-MS (Method 1): Rt = 0.75 min; MS (ESIpos): m/z = 381 [M+H]+
 
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.800 (4.00), 0.816 (4.11), 0.845 (3.83), 0.862 (3.88), 1.146 (0.77), 1.165 (1.21), 1.184 (1.03), 1.244 (4.46), 1.263 (4.43), 1.451 (1.22), 1.458 (1.41), 1.475 (1.63), 1.491 (1.14), 1.917 (0.64), 1.934 (0.63), 2.078 (0.64), 2.097 (0.78), 2.144 (0.82), 2.727 (12.31), 2.729 (12.50), 2.888 (16.00), 3.335 (2.44), 3.346 (3.21), 3.363 (3.58), 3.380 (1.79), 4.128 (0.68), 4.145 (1.02), 4.160 (1.01), 4.163 (0.94), 4.177 (0.91), 4.182 (0.99), 4.200 (0.74), 5.758 (0.91), 7.003 (12.71), 7.008 (2.14), 7.763 (1.04), 7.786 (1.01), 7.950 (2.08), 8.189 (0.93), 8.207 (0.90).
Intermediate 35
4-nitrophenyl {4-[4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}carbamate
Figure imgf000339_0001
To a solution of 6-(4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (13.2 g, 64.8 mmol) in THF (530 mL) was added 4-nitrophenyl carbonochloridate (CAS-No.7693-46-1, 14.4 g, 71.3 mmol) and the mixture was heated to 60°C for 5 h. After cooling to r.t. the mixture was concentrated under reduced pressure to give the title compound which was directly used in the next step.
Intermediate 36
N-{4-[4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4- c]pyridine-2-carboxamide
Figure imgf000339_0002
To a suspension of 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine dihydrochloride (CAS-No. 6000-50- 6, 13.8 g, 71.3 mmol) and dichloromethane (490 mL) was added N,N-diisopropylethylamine  
(34 ml, 190 mmol) at r.t. and the mixture stirred for 30 min. Then this mixture was added to a solution of 4-nitrophenyl {4-[4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}carbamate (23.9 g, 64.8 mmol) from the previous step in dichloromethane and the mixture was stirred at r.t. for 14 h. After that the precipitate was filtered off, washed with water (500 mL) and dried under redcued pressure to give the title compound (21.9 g, 95 % purity, 92 % yield).
LC-MS (Method 1): Rt = 0.55 min; MS (ESIpos): m/z = 351 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.063 (15.83), 1.081 (16.00), 1.240 (1.02), 1.255 (1.01), 2.198 (3.24), 2.238 (3.73), 2.518 (1.60), 2.522 (1.08), 2.641 (2.45), 2.658 (3.09), 2.682 (2.48), 2.699 (2.20), 3.351 (1.68), 3.355 (1.72), 3.369 (2.34), 3.373 (2.34), 3.387 (1.63), 3.391 (1.54), 4.811 (8.63), 4.829 (8.67), 5.758 (15.50), 7.428 (4.51), 7.430 (4.59), 7.441 (4.70), 7.443 (4.72), 7.639 (6.96), 7.645 (2.60), 7.656 (3.73), 7.662 (15.74), 7.667 (2.90), 7.689 (2.88), 7.695 (15.56), 7.701 (3.57), 7.712 (2.59), 7.717 (6.42), 8.495 (7.13), 8.508 (6.68), 8.613 (14.35), 10.866 (10.49).
Intermediate 37
tert-butyl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan- 18-oate
)
Figure imgf000340_0001
To a solution of tert-butyl 15-amino-4,7,10,13-tetraoxapentadecanoate triethylamine (1.6 g, 12.4 mmol) dissolved in 30 ml of N,N-Dimethylformamide was added dropwise. The reaction mixture was stirred at room temperature for 3 hours. Then the reaction was quenched by water and the mixture was extracted with ethyl acetate. The organic layer was dried over sodium sulfate, filtered and concentrated to dryness. The residue was purified by preparative HPLC to yield 2.2 g (38.5%) of the product.
HPLC: X-Bridge C18 30x150 mm, 5 µm, phase A: water/formic acid (0.1%), phase B: acetonitrile (20% up to 45% in 8 min), Rt = 5.5 min.
MS (method 5, ESIpos): m/z = 459 [M+H]+  
1H-NMR (300 MHz, DMSO-d6): d [ppm] 1.40 (s, 9H), 2.35-2.41 (t, 2H), 3.12-3.23 (t, 2H), 3.35- 3.45 (t, 2H), 3.45-3.53 (m, 12H), 3.53-3.61 (t, 2H), 4.02 (s, 2H), 7.10 (s, 2H), 8.16-8.23 (t, 1H).
Intermediate 38
1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid
Figure imgf000341_0001
To a solution of tert-butyl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15- tetraoxa-3- azaoctadecan-18-oate (2.1g, 4.58mmol) in 30 mL of dichlormethane, 2 mL of trifluoroacetic acid was added. The mixture was stirred for 5 h at room temperature. Then the mixture was concentrated to dryness. The residue was purified by prep-HPLC to yield 1.1 g (60%) of the product.
MS (ESIpos): m/z = 403 (M+H)+
1H-NMR (300 MHz, DMSO-d6): d [ppm] 2.35-2.41 (t, 2H), 3.10-3.22 (t, 2H), 3.40-3.53 (m, 14H), 3.53-3.72 (t, 2H), 4.02 (s, 2H), 7.09 (s, 2H), 8.20-8.24 (t, 1H), 12.17 (br, 1H).
Intermediate 39
tert-butyl N2-(tert-butoxycarbonyl)-N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-L- asparaginate
 
Figure imgf000342_0001
Under an atmosphere of argon (3S)-4-tert-butoxy-3-[(tert-butoxycarbonyl)amino]-4- oxobutanoic acid (727 mg, 2.51 mmol) and and HATU (1.15 g, 3.02 mmol) were dissolved in N,N-dimethylformamid (44 ml, 580 mmol). After 10 min. N-{4-[1-(3-aminopropyl)-4-methyl-6- oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide hydrochloride (1:1) (1.11 g, 2.51 mmol) and N,N-diisopropylethylamine (1.8 ml, 10 mmol) were added. The mixture was stirred at room temperature overnight. Subsequently, water (10 ml) was added, the formed precipitate was filtered and washed with small amounts of water (2x1ml). Finally, the residue was dried under reduced pressure and purified by column chromatography on silica gel (DCM/MeOH = 10:1) to give the title compound (50 mg, 3% yield). LC-MS (METHOD 6): Rt = 1.43 min; MS (ESIneg): m/z = 676 [M-H]- ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: -0.008 (1.23), 0.008 (0.93), 1.062 (2.38), 1.080 (2.40), 1.360 (15.27), 1.365 (16.00), 1.742 (0.74), 2.329 (0.68), 4.910 (1.59), 4.936 (1.52), 7.653 (1.40), 7.671 (0.70), 7.676 (2.20), 7.739 (2.22), 7.744 (0.69), 7.761 (1.52), 7.879 (0.63), 8.678 (0.74), 8.691 (0.71), 8.714 (1.38), 8.806 (1.11).
Intermediate 40
ethyl 4-[3-(4-aminophenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butanoate
 
Figure imgf000343_0001
6-(4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (2.20 g, 10.8 mmol) was dissolved in DMF (20 ml, 260 mmol) and sodium hydride (909 mg, 60% purity, 22.7 mmol) was added. The mixture was stirred until gas evolution has almost stopped and tetra-n- butylammonium iodide (400 mg, 1.08 mmol) was added. The mixture was cooled with an ice bath and 4-bromobutanoate (1.9 ml, 13 mmol) was added slowly. The ice bath was removed and the mixture allowed to warm to room temperature and stirred overnight. Aqueous saturated ammonium chloride solution was added and the mixture was extracted with DCM (2x). The organic layer was washed with water and dried. Then the mixture was concentrated under reduced pressure and the crude product was purified by preparative HPLC (ACN/water, gradient) to give the title compound (1.36 g, 37% yield).
Instrument: Gilson Abimed 305 pump, KNAUER UV detector K-2501, Varian fraction collector model 701A, Prepcon 5 software. Column: Chromatorex C18, 10µm, 125x40 mm. Eluent A: water + 0.1 Vol-% TFA; Eluent B: acetonitrile; gradient: 0-22 min 10-50% B; rate 50 ml/min; temperature 25°C. LC-MS (METHOD 7): Rt = 1.01 min; MS (ESIpos): m/z = 318 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: -0.008 (0.86), 1.025 (9.01), 1.043 (9.11), 1.143 (7.58), 1.161 (16.00), 1.179 (7.82), 1.829 (2.36), 1.847 (3.83), 1.865 (2.56), 2.244 (1.86), 2.282 (2.20), 2.285 (2.30), 2.295 (3.56), 2.313 (6.62), 2.331 (3.13), 2.367 (1.23), 2.637 (1.40), 2.654 (1.56), 2.679 (1.40), 2.695 (1.20), 2.711 (1.23), 3.303 (1.10), 3.318 (1.53), 3.335 (1.06), 3.624 (1.53), 3.641 (2.36), 3.658 (2.69), 3.674 (3.26), 3.691 (2.49), 3.787 (5.46), 3.804 (7.22), 3.821 (7.22), 3.837 (7.45), 3.855 (6.75), 4.010 (5.22), 4.028 (9.95), 4.045 (9.58), 4.063 (4.36), 6.729 (1.80), 6.749 (2.26), 7.576 (3.09), 7.597 (3.13).
Intermediate 41
ethyl 4-[4-methyl-3-(4-{[(4-nitrophenoxy)carbonyl]amino}phenyl)-6-oxo-5,6-dihydropyridazin- 1(4H)-yl]butanoate  
Figure imgf000344_0002
Ethyl 4-[3-(4-aminophenyl)-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butanoate (1.36 g, 4.28 mmol) was dissolved in THF (30 ml, 370 mmol).4-nitrophenyl carbonochloridate (864 mg, 4.28 mmol) was added and the mixture was stirred at room temperature overnight. The solvent was removed to give the crude title compound (2.10 g, 82% yield, 81% purity), which was used in the next step without further purification.
Intermediate 42
ethyl 4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]butanoate
Figure imgf000344_0001
Under an atmosphere of argon ethyl 4-[4-methyl-3-(4-{[(4- nitrophenoxy)carbonyl]amino}phenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butanoate (2.07 g, 4.28 mmol) was dissolve in dichloromethane (50 ml, 780 mmol). N,N-diisopropylethylamine (3.4 ml, 19 mmol) and 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine dihydrochloride (909 mg, 4.71  
mmol) were added. The mixture was stirred at room temperature overnight. Subsequently, the solvent was removed under reduced pressure and the residue was purified by preparative HPLC (ACN/water, gradient) to give the title compound (1.07 g, 52% yield).
Instrument: Gilson Abimed 305 pump, KNAUER UV detector K-2501, Varian fraction collector model 701A, Prepcon 5 software. Column: Reprosil C18, 10 µm, 290x100 mm. Eluent A: water + 0.1 Vol-% TFA; Eluent B: acetonitrile; gradient: 0-22 min 30-80% B; rate 250 ml/min; temperature 25°C. LC-MS (METHOD 8): Rt = 0.63 min; MS (ESIpos): m/z = 464 [M+H]+
¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.008 (1.58), 1.059 (9.88), 1.077 (9.96), 1.150 (7.76), 1.168 (16.00), 1.185 (7.89), 1.836 (0.79), 1.854 (2.80), 1.871 (4.35), 1.888 (2.99), 1.906 (0.84), 2.282 (2.31), 2.314 (4.30), 2.324 (3.95), 2.332 (8.05), 2.350 (3.18), 2.366 (0.41), 2.523 (1.88), 2.670 (0.63), 2.695 (1.63), 2.712 (2.12), 2.737 (1.52), 2.753 (1.41), 3.360 (0.54), 3.381 (1.28), 3.396 (1.77), 3.415 (1.22), 3.667 (0.68), 3.684 (1.44), 3.700 (1.69), 3.717 (2.07), 3.734 (0.93), 3.814 (0.98), 3.831 (2.10), 3.847 (1.66), 3.864 (1.44), 3.881 (0.63), 4.017 (2.56), 4.035 (7.67), 4.053 (7.56), 4.071 (2.45), 4.819 (6.48), 4.837 (6.53), 7.433 (2.99), 7.445 (3.13), 7.662 (4.57), 7.684 (8.79), 7.728 (8.79), 7.750 (4.46), 8.499 (3.81), 8.511 (3.70), 8.615 (5.44), 8.632 (5.61), 11.679 (0.54).
Intermediate 43
4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]butanoic acid
Figure imgf000345_0001
4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]butanoate (54.0 mg, 116 µmol) was dissolved in THF (840 µl). Water  
(840 µl) and lithium hydroxide monohydrate (9.78 mg, 233 µmol) were added and the mixture was stirred at room temperature for 3.5 h. The mixture was acidified by the addition of 1N aqueous hydrochloric acid (pH = 3) and concentrated under reduced Pressure to give 72 mg of the crude title compound, which was used in the next step without further purification. LC-MS (METHOD 6): Rt = 0.83 min; MS (ESIpos): m/z = 436 [M+H]+
Intermediate 44
tert-butyl {3-[(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}benzoyl)amino]propyl}carbamate
Figure imgf000346_0001
To a solution of 4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}benzoic acid (655 mg, 1.35 mmol) in DMF (8 mL) was added at r.t.1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3- oxid hexafluorophosphate (HATU, 618 mg, 1.63 mmol), tert-butyl (3-aminopropyl)carbamate (CAS-No.75178-96-0, 260 mg, 1.49 mmol) and N,N-diisopropylethylamine (470 µl, 2.7 mmol) and the mixture stirred for 14 h at that temperature. Then the mixture was diluted with water, extracted with dichloromethane, and the organic extract was washed with brine and filtered through a silicone filter. Removal of the solvent under redcued pressure gave the crude product (1.20 g) which was directly used in the next step.
LC-MS (Method 1): Rt = 0.87 min; MS (ESIpos): m/z = 640 [M+H]+
 
¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.042 (0.49), 1.060 (0.50), 1.223 (0.53), 1.240 (2.43), 1.256 (3.26), 1.272 (1.82), 1.357 (3.98), 1.375 (2.65), 2.686 (5.26), 2.727 (12.43), 2.729 (12.38), 2.888 (16.00), 7.664 (0.59), 7.700 (0.59), 7.775 (0.49), 7.796 (0.42), 7.950 (1.91).
Intermediate 45
benzyl N2-(tert-butoxycarbonyl)-N-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl-L-alpha- glutaminate
Figure imgf000347_0001
(2S)-5-(benzyloxy)-2-[(tert-butoxycarbonyl)amino]-5-oxopentanoic acid (691 mg, 2.05 mmol) was dissolved in DMF (25 ml, 330 mmol). HATU (934 mg, 2.46 mmol), N,N- diisopropylethylamine (1.1 ml, 6.1 mmol) and 2,5,8,11,14,17,20,23-octaoxapentacosan-25- amine (890 µl, 2.5 mmol) were added and the mixture was stirred at room temperature overnight. Water was added and the mixture was extracted with DCM (3x). The combined organic layers were dried and evaporated under reduced pressure. Purification by preparative HPLC (ACN/water + 0.1% TFA, gradient) provided the title compound (645 mg, 45% yield). Instrument: Gilson Abimed 305 pump, KNAUER UV detector K-2501, Varian fraction collector model 701A, Prepcon 5 software. Column: Chromatorex C18, 10µm, 125x40 mm. Eluent A: water + 0.1 Vol-% TFA; Eluent B: acetonitrile; gradient: 0-22 min 10-70% B; rate 50 ml/min; temperature 25°C.
Preparative HPLC: Column Chromatorex C18, 10 µm, 125x40 mm. Eluent A: water + 0.1 Vol-% TFA; Eluent B: acetonitrile; gradient: 0-22 min 10-70% B. Rate 50 ml/min; temperature 25°C.  
LC-MS (METHOD 6): Rt = 1.73 min; MS (ESIpos): m/z = 703 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: -0.008 (0.67), 1.370 (4.36), 3.235 (5.50), 3.395 (0.76), 3.409 (0.75), 3.411 (0.76), 3.420 (0.88), 3.427 (0.67), 3.434 (1.07), 3.484 (5.31), 3.491 (5.17), 3.501 (15.77), 3.503 (16.00), 3.507 (3.51), 3.515 (1.03), 3.517 (1.01), 5.078 (1.94), 7.357 (2.65).
Intermediate 46
N2-(tert-butoxycarbonyl)-N-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl-L-alpha-glutamine
)
I
Figure imgf000348_0001
Benzyl N2-(tert-butoxycarbonyl)-N-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl-L-alpha- glutaminate (645 mg, 918 µmol) were dissolved in ethanol (50 ml). Palladium on charcoal (10% Pd/C, 140 mg) was added and the mixture was hydrogenated under normal pressure at room temperature for 2 h. The mixture was filtered and the filtrate was evaporated under reduced pressure to give the title compound (505 mg, 90% yield).
LC-MS (METHOD 7): Rt = 0.93 min; MS (ESIneg): m/z = 611 [M-H]- ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: -0.008 (1.99), 0.008 (1.59), 1.375 (2.89), 3.238 (3.88), 3.437 (0.68), 3.496 (1.38), 3.504 (8.70), 3.507 (16.00).
Intermediate 47
tert-butyl {(28S)-35-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]-27,31-dioxo-2,5,8,11,14,17,20,23-octaoxa-  
26,32-diazapentatriacontan-28-yl}carbamate
Figure imgf000349_0001
Under an atmosphere of argon N-{4-[1-(3-aminopropyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide hydrochloride (1:1), (36.1 mg, 81.6 µmol) and N,N-diisopropylethylamine (71 µl, 410 µmol) were dissolved in DMF (420 µl). N2-(tert-butoxycarbonyl)-N-2,5,8,11,14,17,20,23- octaoxapentacosan-25-yl-L-alpha-glutamine (50.0 mg, 81.6 µmol) dissolved in DMF (1 ml) and HATU (37.2 mg, 97.9 µmol) were added and the mixture was stirred at room temperature overnight. Then the mixture was concentrated under reduced pressure and the crude product was purified by preparative HPLC (ACN/water, gradient) to give the title compound (28 mg, 34% yield). The product was used in the next step without further purification.
Instrument: Gilson Abimed 305 pump, KNAUER UV detector K-2501, Varian fraction collector model 701A, Prepcon 5 software. Column: Chromatorex C18, 10µm, 125x30 mm. Eluent A: water + 0.1 Vol-% TFA; Eluent B: acetonitrile; gradient: 0-22 min 10-50% B; rate 50 ml/min; temperature 25°C.
LC-MS (METHOD 6): Rt = 1.16 min; MS (ESIpos): m/z = 1001 [M+H]+
Intermediate 48
N5-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]propyl}-N1-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl-L- glutamamide  
Figure imgf000350_0002
tert-butyl {(28S)-35-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]-27,31-dioxo-2,5,8,11,14,17,20,23-octaoxa- 26,32-diazapentatriacontan-28-yl}carbamate (28.0 mg, 28.0 µmol) was dissolved in Hydrogen chloride solution (4N in dioxane, 70 µl, 280 µmol) and DCM (1 ml). The mixture was stirred for 3 h at room temperature and subsequently concentrated under reduced pressure to give the title compound (26 mg, quant.)
LC-MS (MCW_SQ-HSST3): Rt = 0.53 min; MS (ESIneg): m/z = 900 [M-H]-
Intermediate 49
N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-L-alanine
Figure imgf000350_0001
H-Val-Ala-OH, 3.5 g (18.6 mmol) was dissolved in 100 ml of N,N-Dimethylformamide. Triethylamine, 3.8 g (37.2 mmol) and N-succinimidyl maleimidoacetate, 4.7 g (18.6 mmol) in 10 ml of N,N-Dimethylformamide was added. The reaxtion mixture was stirred for 1h at room temperature. water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over sodium sulfate, filtered and evaporated to dryness. The crude was purified by preparative HPLC to yield 2.10 g (35 %) of the product.
HPLC: Welch Ultimate XB-C18 50*250 mm, 10 µm, phase A: water/TFA (0.05%), phase B: ACN (10-45%, 35 min), Rt = 25-27 min
MS (method 5, ESIpos): m/z = 459 (M+H)+  
1H-NMR (300 MHz, DMSO-d6): d [ppm] 0.72-0.89 (m, 6H), 1.29-1.30 (d, 3H), 1.93-1.99 (t, 1H), 4.12 (s, 2H), 4.13-4.25 (m, 2H), 7.10 (s, 2H), 8.26-8.29 (d, 1H), 8.35-8.37 (d, 1H), 12.42 (s, 1H).
Intermediate 50
methyl N-(tert-butoxycarbonyl)-L-valyl-L-alaninate
Figure imgf000351_0001
A mixture of N-(tert-butoxycarbonyl)-L-valine (CAS-No. 13734-41-3, 7.00 g, 32.2 mmol), methyl L-alaninate hydrochloride (CAS-No. 2491-20-5, 4.50 g, 32.2 mmol), N-methyl- morpholine (11 ml, 97 mmol) and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5- b]pyridinium 3-oxid hexafluorophosphate) (HATU, 12.3 g, 32.2 mmol) in DMF (250 mL) was stirred at r.t. for 14 h. Then the solvent was removed under reduced pressure and the residue was purified by column chromatography (SiO2, hexane/ethyl acetate gradient) to give the title compound (10.4 g, 90% purity, 96% yield).
LC-MS (Method 2): Rt = 0.99 min; MS (ESIpos): m/z = 303 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.803 (1.64), 0.821 (1.75), 0.855 (1.85), 0.872 (1.89), 1.265 (2.98), 1.283 (2.99), 1.373 (12.16), 3.158 (12.96), 3.171 (16.00), 3.330 (9.29), 4.080 (1.19), 4.093 (3.92), 4.107 (3.61), 4.120 (1.00).
Intermediate 51
N-(tert-butoxycarbonyl)-L-valyl-L-alanine
Figure imgf000351_0002
A mixture of methyl N-(tert-butoxycarbonyl)-L-valyl-L-alaninate (9.70 g, 32.1 mmol), lithium hydroxide monohydrate (2.69 g, 64.2 mmol), THF (170 mL) and water (76 mL) was stirred at r.t. for 14 h. After that the mixture was acidified with trifluoroacetic acid and extracted with ethyl acetate. The organic extract was washed with brine, filtered through a silicone filter and concentrated under reduced pressure. The crude product was purified by column   chromatography (SiO2, dichloromethane/ethanol gradient) to give the title compound (7.14 g, 90 % purity, 69 % yield).
LC-MS (Method 2): Rt = 0.53 min; MS (ESIpos): m/z = 289 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.797 (2.15), 0.813 (2.32), 0.851 (2.38), 0.868 (2.52), 0.945 (0.58), 0.955 (0.62), 0.962 (0.64), 0.972 (0.60), 1.251 (3.61), 1.270 (3.70), 1.306 (0.73), 1.324 (0.76), 1.350 (0.94), 1.373 (16.00), 1.528 (2.71), 1.903 (0.41), 3.822 (0.47), 4.171 (0.45), 4.189 (0.71), 4.207 (0.48), 6.590 (0.60), 6.613 (0.58), 8.101 (0.89), 8.119 (0.80).
Intermediate 52
N-(tert-butoxycarbonyl)-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L-alaninamide
Figure imgf000352_0001
A mixture of N-{4-[1-(4-aminobutyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}- 1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (277 mg, 90 % purity, 593 µmol), N-(tert- butoxycarbonyl)-L-valyl-L-alanine (285 mg, 90 % purity, 890 µmol), (1- [bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) (HATU, 338 mg, 890 µmol) and N-methylmorpholine (200 µl, 1.8 mmol) in DMF (5 mL) was stirred at r.t. for 14 h. After that the mixture was concentrated under reduced pressure and the residue was purified by column chromatography (amine-coated SiO2,   dichloromethane/methanol gradient) to give the title compound (440 mg, 86 % purity, 92 % yield).
LC-MS (method 1): Rt = 0.89 min; MS (ESIneg): m/z = 689 [M-H]- ¹H-NMR (500 MHz, DMSO-d6) d [ppm]: 0.773 (0.62), 0.786 (0.66), 0.809 (0.77), 0.816 (1.07), 0.823 (1.04), 0.828 (1.15), 1.051 (0.87), 1.065 (0.91), 1.148 (0.75), 1.155 (0.90), 1.157 (0.72), 1.162 (1.19), 1.169 (1.19), 1.172 (0.92), 1.176 (0.47), 1.253 (0.42), 1.263 (0.43), 1.268 (0.51), 1.353 (0.53), 1.372 (6.28), 1.379 (2.79), 1.955 (2.71), 1.987 (0.94), 2.084 (1.07), 2.687 (16.00), 2.728 (10.46), 2.781 (2.51), 2.888 (13.12), 2.941 (3.71), 4.815 (0.57), 4.836 (0.58), 7.660 (0.69), 7.678 (0.98), 7.728 (0.96), 7.745 (0.55), 7.951 (1.68), 8.499 (0.52), 8.509 (0.50), 8.614 (0.75), 8.640 (0.60).
Intermediate 53
L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L-alaninamide
Figure imgf000353_0001
A mixture of N-(tert-butoxycarbonyl)-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin- 2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L- alaninamide (435 mg, 86 % purity, 542 µmol), trifluoroacetic acid (420 µl, 5.4 mmol) and dichloromethane (7 mL) was stirred at r.t. for 14 h. After that the mixture was concentrated under reduced pressure and coevaporated twice with toluene. The residue was triturated with ethyl acetate at 0°C to give the title compound (300 mg, 94 % yield).
LC-MS (method 1): Rt = 0.61 min; MS (ESIneg): m/z = 589 [M-H]-   ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.899 (10.76), 0.913 (11.66), 0.951 (0.71), 0.963 (0.67), 0.982 (0.51), 1.051 (5.91), 1.069 (5.97), 1.154 (4.18), 1.172 (8.55), 1.189 (5.13), 1.199 (4.94), 1.206 (5.27), 1.217 (4.92), 1.224 (4.81), 1.319 (0.41), 1.400 (1.81), 1.418 (1.64), 1.567 (0.65), 1.584 (1.59), 1.602 (1.90), 1.619 (1.32), 1.955 (0.42), 1.986 (16.00), 2.015 (1.19), 2.031 (1.17), 2.047 (0.75), 2.163 (0.40), 2.281 (1.66), 2.322 (2.31), 2.674 (1.37), 2.686 (2.45), 2.717 (1.00), 2.728 (4.50), 2.888 (4.60), 2.941 (0.43), 3.057 (0.72), 3.075 (1.27), 3.093 (1.58), 3.110 (1.35), 3.127 (0.76), 3.377 (0.91), 3.395 (1.30), 3.412 (0.86), 3.593 (2.03), 3.627 (0.92), 3.852 (0.78), 3.865 (0.93), 3.885 (0.81), 3.898 (0.63), 3.999 (1.26), 4.016 (3.74), 4.034 (3.67), 4.052 (1.21), 4.308 (1.21), 4.325 (1.87), 4.344 (1.33), 4.362 (0.46), 4.895 (5.98), 4.906 (5.86), 7.655 (4.25), 7.678 (6.75), 7.698 (2.49), 7.712 (2.45), 7.733 (6.33), 7.755 (3.57), 7.950 (0.75), 8.039 (5.29), 8.052 (4.69), 8.529 (1.82), 8.548 (1.76), 8.649 (3.27), 8.662 (3.22), 8.723 (4.40), 8.772 (4.99).
Intermediate 54
N-{4-[4-methyl-1-(4-nitrobenzyl)-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000354_0001
To a solution of N-{4-[4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carboxamide (5.00 g, 14.3 mmol) was added at r.t. sodium hydride (749 mg, 55 % on mineral oil, 17.2 mmol) and the mixture was stirred for 30 min. After that tetra-n-butylammonium iodide (529 mg, 1.43 mmol) and 1-(bromomethyl)-4-nitrobenzene (CAS-No.100-11-8, 3.71 g, 17.2 mmol) was added and the mixture stirred at r.t. for 14 h. Then the mixture was concentrated under reduced pressure and taken up in dichloromethane/methanol. The solid was filitered off and washed with dichloromethane. The filtrate was concentrated under reduced pressure and the crude product was purified by column chromatography (1.15 g, 16 % yield).  
LC-MS (method 1): Rt = 0.88 min; MS (ESIpos): m/z = 485 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.851 (0.53), 0.915 (0.66), 0.934 (1.58), 0.951 (0.75), 1.035 (1.76), 1.052 (3.60), 1.071 (15.91), 1.090 (15.82), 1.144 (0.53), 1.232 (1.85), 2.377 (3.56), 2.416 (4.09), 2.518 (14.37), 2.522 (9.89), 2.827 (2.37), 2.843 (2.99), 2.868 (2.46), 2.885 (2.20), 3.422 (1.10), 3.435 (1.14), 3.440 (1.93), 3.452 (1.36), 3.461 (2.51), 3.478 (1.71), 4.342 (0.48), 4.355 (0.97), 4.367 (0.48), 4.807 (9.41), 4.825 (9.67), 5.004 (3.47), 5.043 (6.55), 5.112 (6.20), 5.151 (3.43), 5.758 (5.49), 7.427 (5.19), 7.439 (5.49), 7.562 (11.34), 7.585 (11.96), 7.645 (8.04), 7.667 (16.00), 7.705 (16.00), 7.728 (7.38), 8.211 (14.99), 8.216 (4.57), 8.228 (4.70), 8.233 (13.41), 8.494 (7.60), 8.507 (7.34), 8.608 (10.55), 8.643 (9.36).
Intermediate 55
N-{4-[1-(4-aminobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000355_0001
A mixture of N-{4-[4-methyl-1-(4-nitrobenzyl)-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}- 1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (1.65 g, 3.41 mmol), iron (2.85 g, 51.1 mmol) and acetic acid (19 ml, 340 mmol) in ethanol (20 ml) was heated to 90°C for 3 h. The hot mixture was then filtered through Celite® and the filter cake was washed with ethanol. The filtrate was concentrated under reduced pressure and the residue was taken up in dichloromethane/methanol (4:1), washed with 10% aqueous sodium carbonate solution and brine. After filtration of the organic phase through a silicone filter the solvent was remoed under reduced pressure and the crude product was purified by column chromatography (SiO2, dichloromethane/methanol gradient) to give the title compound (300 mg, 70% purity, 14% yield).
LC-MS (method 2): Rt = 0.85 min; MS (ESIpos): m/z = 455 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.852 (0.76), 1.072 (8.09), 1.090 (8.16), 1.233 (1.96), 1.986 (0.51), 2.006 (0.51), 2.318 (1.39), 2.323 (2.85), 2.327 (3.92), 2.332 (2.85), 2.337 (1.33),  
2.378 (2.15), 2.416 (3.16), 2.420 (3.04), 2.518 (16.00), 2.523 (11.26), 2.660 (1.26), 2.665 (2.85), 2.669 (3.86), 2.673 (2.66), 2.679 (1.14), 2.827 (1.20), 2.844 (1.52), 2.869 (1.26), 2.886 (1.08), 3.442 (0.89), 3.461 (1.26), 3.478 (0.82), 4.808 (4.74), 4.827 (4.68), 5.005 (1.77), 5.044 (3.23), 5.112 (3.16), 5.152 (1.71), 7.427 (2.53), 7.441 (2.53), 7.556 (0.89), 7.563 (5.82), 7.568 (2.02), 7.580 (2.15), 7.585 (6.07), 7.591 (0.89), 7.645 (4.24), 7.650 (1.64), 7.662 (2.28), 7.668 (8.28), 7.673 (1.52), 7.701 (1.71), 7.706 (8.16), 7.712 (2.15), 7.724 (1.52), 7.729 (3.79), 8.205 (1.14), 8.212 (7.78), 8.217 (2.40), 8.228 (2.34), 8.234 (6.89), 8.240 (0.82), 8.495 (3.48), 8.507 (3.23), 8.608 (4.68), 8.643 (4.62).
Intermediate 56
N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L- alaninamide
Figure imgf000356_0001
A mixture of N-{4-[1-(4-aminobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}- 1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (300 mg, 70 % purity, 462 µmol), N-(tert- butoxycarbonyl)-L-valyl-L-alanine (200 mg, 100 % purity, 693 µmol), (1- [bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) (HATU, 264 mg, 693 µmol) and N-methylmorpholine (150 µl, 1.4 mmol) in DMF (3.5 mL) was stirred at r.t. for 14 h. After that the mixture was concentrated under  
reduced pressure and the residue was purified by preparative HPLC to give the title compound (90.0 mg, 89 % purity, 24 % yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x30 mm. Eluent A: water + 0.1 Vol- % HCOOH; Eluent B: acetonitrile; gradient: 0-8 min 15-55% B. rate 150 ml/min, temperature 25°C.
LC-MS (Method 2): Rt = 1.03 min; MS (ESIpos): m/z = 725 [M+H]+
Intermediate 57
L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L-alaninamide
Figure imgf000357_0001
A mixture of N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin- 2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L- alaninamide (70.0 mg, 96.6 µmol), trifluoroacetic acid (74 µl, 970 µmol) and dichloromethane (1.2 mL) was stirred at r.t. for 14 h. After that the mixture was concentrated under reduced pressure and coevaporated twice with toluene. The residue was purified by preparative HPLC to give the title compound (4.00 mg, 6% yield).  
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: XBridge C18 5µM 100x30 mm. Eluent A: water + 0.1 Vol-% ammonia; Eluent B: acetonitrile; gradient: 0-20 min 15-55% B. rate 60 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.74 min; MS (ESIneg): m/z = 623 [M-H]- ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.755 (3.47), 0.760 (2.73), 0.772 (3.61), 0.777 (2.65), 0.854 (3.54), 0.862 (2.58), 0.870 (3.47), 0.879 (2.36), 1.012 (3.47), 1.030 (3.54), 1.234 (1.99), 1.268 (2.58), 1.275 (3.24), 1.285 (2.58), 1.292 (3.10), 1.879 (0.52), 1.893 (0.52), 1.909 (0.44), 1.986 (0.59), 2.006 (0.59), 2.332 (3.32), 2.337 (1.40), 2.366 (0.88), 2.518 (16.00), 2.523 (11.58), 2.673 (3.10), 2.679 (1.33), 2.753 (0.52), 2.769 (0.66), 2.794 (0.52), 2.810 (0.44), 2.990 (0.81), 3.390 (0.44), 3.408 (0.59), 4.429 (0.44), 4.737 (0.96), 4.775 (1.25), 4.811 (2.29), 4.828 (2.29), 4.979 (1.18), 5.016 (0.96), 7.241 (2.58), 7.263 (2.95), 7.429 (1.33), 7.442 (1.33), 7.530 (2.36), 7.547 (1.70), 7.551 (1.77), 7.642 (2.06), 7.665 (4.13), 7.702 (3.91), 7.724 (1.77), 8.496 (1.92), 8.508 (1.77), 8.610 (2.51), 8.636 (2.36), 10.004 (1.25), 10.020 (0.96).
Intermediate 58
tert-butyl {4-[methoxy(methyl)carbamoyl]phenyl}carbamate
Figure imgf000358_0001
To a solution of 4-[(tert-butoxycarbonyl)amino]benzoic acid (43.5 g, 183 mmol) in DMF (260 mL) was added triethylamine (103 mL, 734 mmol), N,O-dimethylhydroxylamine hydrochloride (26.8 g, 275 mmol), 1-hydroxy-7-azabenzotriazol (HOAt, 37.4 g, 275 mmol) and 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 70.3 g, 367 mmol). The reaction was stirred at RT overnight. Aqueous saturated sodium bicarbonate solution (200 mL) was added and the reaction was extracted with ethyl acetate. The organic extracts were washed with aqueous saturated sodium bicarbonate solution, brine (2 x 100 mL), dried over solid sodium sulfate and concentrated under redcued pressure. The crude product was purified by column chromatography (SiO2, hexane/ethyl acetate gradient) to give the title compound (53.6 g, 77% purity, 80%).  
LC-MS (Method 1): Rt = 1.06 min; MS (ESIneg): m/z = 279 [M–H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.154 (0.49), 1.172 (1.03), 1.190 (0.51), 1.482 (16.00), 1.988 (1.68), 2.728 (1.31), 2.888 (1.63), 3.226 (6.06), 3.331 (6.55), 7.494 (0.59), 7.516 (1.73), 7.539 (2.24), 7.561 (0.70), 9.624 (0.67).
Intermediate 59
tert-butyl [4-(phenylacetyl)phenyl]carbamate
Figure imgf000359_0001
Tert-butyl {4-[methoxy(methyl)carbamoyl]phenyl}carbamate (26.8 g, 95.7 mmol) was dissolved in tetrahydrofuran (894 mL). The mixture was cooled to 0°C and benzyl magnesium chloride (2M in THF, 191 mL, 383 mmol) was added. The reaction stirred at room temperature overnight. The mixture was combined with a second reaction mixture of the same size and was quenched by addition of 1N hydrochloric acid and extracted with ethyl acetate. The combined organic layers were filtered through a silicone filter and concentrated under reduced pressure. The crude product was purified by column chromatography (hexane/ethyl acetate gradient) to give the title compound (46.1 g, 83% purity, 64% yield).
LC-MS (Method 1): Rt = 1.36 min; MS (ESIpos): m/z = 312 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.484 (16.00), 4.291 (2.89), 7.213 (0.63), 7.243 (0.71), 7.260 (1.72), 7.264 (1.22), 7.280 (1.40), 7.295 (0.80), 7.298 (1.13), 7.300 (0.76), 7.571 (1.28), 7.593 (1.40), 7.959 (1.60), 7.981 (1.42), 9.793 (0.89).
Intermediate 60
ethyl 4-{4-[(tert-butoxycarbonyl)amino]phenyl}-4-oxo-3-phenylbutanoate
 
Figure imgf000360_0001
To a solution of sodium hydride (3.83 g, 160 mmol) in DMF (480 mL) was added at–40°C portionwise tert-butyl [4-(phenylacetyl)phenyl]carbamate (23.0 g, 83% purity, 61.4 mmol). After warming to r.t. the mixture stirred at that temperature for 1 h. Then the mixture was cooled again to–40°C and ethyl bromoacetate (320 µl, 2.9 mmol) was added. The mixture was warmed to r.t. and stirred at that temperature for 14 h. After that the mixture was combined with a reaction mixture of the same size, diluted with saturated aqueous ammonium chloride solution and extracted with ethyl acetate (3x). The combined organic extracts were washed with brine and filtered through a silicone filter. After removal of the solvent under reduced pressure the crude product was purified by column chromatography (SiO2, hexane/ethyl acetate gradient) to give the title compound (43.4 g, 90% purity, 80% yield).
LC-MS (Method 1): Rt = 1.43 min; MS (ESIneg): m/z = 396 [M–H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.084 (1.84), 1.103 (3.96), 1.120 (1.88), 1.153 (0.49), 1.172 (0.98), 1.189 (0.50), 1.463 (16.00), 1.484 (2.77), 1.986 (1.93), 2.687 (0.47), 3.159 (0.43), 3.971 (0.55), 3.989 (1.71), 4.008 (1.68), 4.017 (0.48), 4.025 (0.52), 4.035 (0.44), 7.188 (0.66), 7.206 (0.51), 7.256 (0.71), 7.260 (0.61), 7.272 (0.60), 7.275 (1.47), 7.294 (1.00), 7.318 (1.38), 7.321 (1.48), 7.339 (0.79), 7.343 (0.53), 7.498 (1.35), 7.520 (1.43), 7.931 (1.60), 7.953 (1.44), 9.751 (1.05).
Intermediate 61
4-{4-[(tert-butoxycarbonyl)amino]phenyl}-4-oxo-3-phenylbutanoic acid
I
Figure imgf000360_0002
 
To a solution of ethyl 4-{4-[(tert-butoxycarbonyl)amino]phenyl}-4-oxo-3-phenylbutanoate (21.7 g, 90% purity, 49.1 mmol) in methanol (430 mL) was added at r.t. aqueous 2M sodium hydroxide solution (174 mL, 348 mmol) and the mixture stirred at that temperature for 14 h. After that the mixture was combined with a reaction mixture of the same size, acidified to pH 5 by addition of 1N hydrochloric acid and extracted with ethyl acetate (3x). The combined organic extracts were washed with brine, filtered through a silicone filter and concentrated under reduced pressure. To give the title compound (42.0 g, 100%).
LC-MS (Method 1): Rt = 1.22 min; MS (ESIneg): m/z = 368 [M–H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.153 (0.90), 1.171 (1.83), 1.189 (0.90), 1.462 (16.00), 1.484 (2.89), 1.907 (2.52), 1.986 (3.39), 2.558 (0.41), 2.590 (0.48), 3.098 (0.41), 3.125 (0.43), 3.336 (0.75), 4.016 (0.75), 4.034 (0.73), 4.291 (0.43), 7.182 (0.65), 7.200 (0.52), 7.253 (0.72), 7.273 (1.44), 7.291 (0.96), 7.317 (1.57), 7.334 (0.79), 7.338 (0.54), 7.497 (1.37), 7.518 (1.43), 7.931 (1.63), 7.953 (1.47), 7.959 (0.43), 9.743 (1.11).
Intermediate 62
tert-butyl [4-(6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl)phenyl]carbamate
Figure imgf000361_0001
To a solution of 4-{4-[(tert-butoxycarbonyl)amino]phenyl}-4-oxo-3-phenylbutanoic acid (41.0 g, 111 mmol) in ethanol (1.3 L) was added at r.t. hydrazine hydrate (27.8 g, 64% purity, 555 mmol) and the mixture was heated to 80°C for 80 h. After cooling to r.t. the mixture was concentrated under reduced pressure to approximately 1/3 of the original volume. The precipitate was filtered off, washed with water and dried under vacuum at 50°C for 14 h to give the title compound (22.8 g, 53%).
LC-MS (Method 2): Rt = 1.17 min; MS (ESIpos): m/z = 366 [M+H]+
1H-NMR (500 MHz, DMSO-d6) d [ppm]: 1.457 (16.00), 2.450 (0.49), 2.484 (0.64), 3.027 (0.46), 3.045 (0.42), 4.636 (0.47), 4.651 (0.48), 7.171 (1.05), 7.185 (1.31), 7.188 (1.02), 7.231 (0.73),  
7.246 (0.49), 7.295 (1.04), 7.311 (1.39), 7.325 (0.53), 7.436 (0.96), 7.454 (1.09), 7.635 (1.79), 7.639 (0.52), 7.649 (0.49), 7.653 (1.42), 9.509 (0.69), 11.043 (1.37).
Intermediate 63
6-(4-aminophenyl)-5-phenyl-4,5-dihydropyridazin-3(2H)-one
Figure imgf000362_0001
To a solution of tert-butyl {4-[6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}carbamate (15.2 g, 80 % purity, 33.2 mmol) in dichloromethane (560 mL) was added at r.t. trifluoroacetic acid (26 mL, 330 mmol) and the mixture stirred for 2 h at that temperature. After cooling to 0°C the mixture was neutralized with sodium hydroxide and extracted with dichloromethane. The extracts were combined with extracts from a parallel reaction mixture (tert-butyl {4-[6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}carbamate (22.2 g, 59.5 mmol), trifluoroacetic acid (45.8 mL, 595 mmol) and dichloromethane (1000 mL)) and concentrated under reduces pressure. The residue was triturated with dichloromethane/methanol to give the title compound (18.8 mmol, 75% yield).
LC-MS (method 1): Rt = 0.79 min; MS (ESIpos): m/z = 266 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.456 (0.49), 2.396 (4.00), 2.437 (4.44), 2.518 (1.88), 2.523 (1.34), 2.925 (3.26), 2.945 (4.02), 2.967 (3.48), 2.986 (3.00), 4.548 (4.06), 4.566 (4.11), 5.496 (14.62), 5.760 (0.44), 6.484 (1.61), 6.491 (16.00), 6.495 (4.34), 6.508 (4.53), 6.513 (15.89), 6.519 (1.70), 7.166 (8.81), 7.179 (2.71), 7.184 (11.86), 7.188 (9.18), 7.200 (1.23), 7.204 (2.14), 7.207 (1.28), 7.216 (1.72), 7.222 (6.56), 7.228 (1.77), 7.237 (3.37), 7.240 (4.85), 7.244 (2.19), 7.286 (9.74), 7.290 (3.85), 7.302 (6.77), 7.305 (12.78), 7.319 (1.93), 7.322 (4.67), 7.325 (2.70), 7.418 (1.70), 7.425 (14.89), 7.430 (4.42), 7.442 (4.31), 7.447 (14.31), 7.453 (1.56), 10.838 (10.76).
Intermediate 64
4-nitrophenyl {4-[6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}carbamate
 
Figure imgf000363_0002
To a solution of 6-(4-aminophenyl)-5-phenyl-4,5-dihydropyridazin-3(2H)-one (6.00 g, 22.6 mmol) in THF (180 mL) was added at r.t.4-nitrophenyl carbonochloridate (CAS-No. 7693-46- 1, 5.01 g, 24.9 mmol) and the mixture was heated to 60°C for 7 h. After cooling to r.t. the precipitate was filtered off and the filtrate was concentrated under reduced pressure to give the title compound (10.6 g, 76% purity, 83% yield) which was directly used in the next step. LC-MS (method 1): Rt = 1.15 min; MS (ESIpos): m/z = 431 [M+H]+
Intermediate 65
N-{4-[6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4- c]pyridine-2-carboxamide
Figure imgf000363_0001
To a suspension of 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine dihydrochloride (CAS-No. 6000-50- 6, 1.88 g, 9.71 mmol) in dichloromethane (35 mL) was added at r.t. N,N-diisopropylethylamine (4.6 ml, 26 mmol). After stirring at that temperature for 30 min the mixture was added to a solution of 4-nitrophenyl {4-[6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}carbamate (5.00 g, 76 % purity, 8.83 mmol) in dichloromethane (35 mL) and the   mixture stirred for 14 h. Then the precipitate was filtered off, washed with water and dried under reduced pressure to give the title compound (3.10 g, 84% yield).
LC-MS (method 1): Rt = 0.70 min; MS (ESIneg): m/z = 410 [M-H]- ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 2.456 (4.84), 2.518 (3.77), 2.522 (2.46), 3.012 (2.86), 3.031 (3.64), 3.053 (3.22), 3.074 (2.54), 4.657 (4.13), 4.675 (4.24), 4.786 (11.10), 4.805 (10.97), 7.189 (8.93), 7.208 (11.47), 7.211 (9.30), 7.222 (2.30), 7.240 (6.15), 7.259 (4.29), 7.305 (9.19), 7.324 (11.41), 7.342 (3.98), 7.415 (5.79), 7.428 (5.92), 7.574 (9.67), 7.597 (15.72), 7.654 (16.00), 7.676 (8.88), 8.486 (7.78), 8.498 (7.28), 8.586 (10.61), 8.598 (11.66), 11.049 (12.07).
Intermediate 66
Methyl N2-(tert-butoxycarbonyl)-N6-[5-({4-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-5- oxopentanoyl]-L-lysinate
Figure imgf000364_0001
A mixture of N-(4-{(4S)-1-[4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-4- methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine- 2-carboxamide (142 mg, 225 µmol), methyl N2-(tert-butoxycarbonyl)-L-lysinate (70.2 mg, 270 µmol), N,N-diisopropylethylamine (98 µl, 560 µmol) and DMF (2.6 mL) was stirred at r.t. for 4 h. After that the mixture was purified by preparative HPLC to give the crude product (55.0 mg, 31% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: XBridge C18 10µM 100x30 mm. eluent A: water + 0.1% ammonia; eluent B: acetonitrile; gradient: 0-20, min 15-55% B. rate 60 ml/min, temperature 25°C.
LC-MS (method 2): Rt = 0.98 min; MS (ESIpos): m/z = 777 [M+H]-
Intermediate 67
N2-(tert-butoxycarbonyl)-N6-[5-({4-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-5- oxopentanoyl]-L-lysine
 
Figure imgf000366_0001
A mixture of methyl N2-(tert-butoxycarbonyl)-N6-[5-({4-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}- amino)-5-oxopentanoyl]-L-lysinate (55.0 mg, 70.8 µmol), lithium hydroxide monohydrate (8.9 mg, 212 µmol), water (3 mL) and THF (6 mL) was stirred at r.t. for 14 h. After that the mixture was neutrlized by addition of TFA and concentrated under reduced pressure to give the crude title compound which was directly used in the next step.
Intermediate 68
N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L- alaninamide
 
Figure imgf000367_0001
A mixture of N-{4-[1-(4-aminobenzyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (300 mg, 640 µmol), N-(tert- butoxycarbonyl)-L-valyl-L-alanine (222 mg, 768 µmol), [Bis(dimethylamino)methylene]-1H- 1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) (HATU, 365 mg, 960 µmol), N- methylmorpholine (210 µl, 1.9 mmol) and DMF (5 mL) was stirred at r.t. for 14 h. After that the mixture was concentrated under reduced pressure and the residue was purified by column chromatography (dichloromethane/ethanol gradient) to give the title compound (210 mg, 94% purity, 44% yield).
LC-MS (method 1): Rt = 1.02 min; MS (ESIpos): m/z = 739 [M+H]- 1H-NMR (400 MHz, DMSO-d6) delta [ppm]: 0.793 (0.25), 0.810 (0.29), 0.834 (0.46), 0.846 (0.62), 0.862 (0.37), 0.904 (0.04), 1.098 (2.15), 1.139 (0.05), 1.231 (0.06), 1.272 (0.50), 1.289 (0.50), 1.332 (1.52), 1.368 (1.80), 1.888 (0.04), 1.904 (0.05), 1.923 (0.09), 1.936 (0.05), 1.954 (0.04), 2.083 (16.00), 2.240 (0.05), 2.327 (0.06), 2.523 (0.23), 2.680 (0.92), 2.877 (0.55), 3.025 (0.16), 3.340 (0.20), 3.742 (0.18), 3.801 (0.06), 3.823 (0.07), 3.839 (0.05), 4.395 (0.07), 4.413 (0.11), 4.431 (0.08), 4.809 (0.42), 4.826 (0.43), 4.855 (0.49), 5.758 (0.04), 6.714 (0.08), 6.735 (0.07), 6.832 (0.06), 6.853 (0.06), 7.226 (0.35), 7.247 (0.39), 7.427 (0.23), 7.440 (0.24), 7.490 (0.16), 7.501 (0.16), 7.511 (0.19), 7.520 (0.34), 7.541 (0.26), 7.561 (0.14), 7.583 (0.12), 7.638 (0.24), 7.660 (0.76), 7.678 (0.61), 7.700 (0.19), 8.047 (0.09), 8.064 (0.09), 8.253 (0.05), 8.271 (0.05), 8.494 (0.32), 8.506 (0.31), 8.512 (0.18), 8.515 (0.17), 8.533 (0.15), 8.536 (0.14), 8.609 (0.45), 8.636 (0.40), 8.743 (0.14), 8.746 (0.14), 8.753 (0.14), 8.757 (0.13), 9.761 (0.10), 9.975 (0.12).   Intermediate 69
L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5- dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L-alaninamide trifluoroacetate
Figure imgf000368_0001
A mixture of N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin- 2-ylcarbonyl)amino]phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)- L-alaninamide (210 mg, 284 µmol), trifluoroacetic acid (220 µl, 2.8 mmol), 1 drop DMF and dichloromethane (1.8 mL) was stirred at r.t. for 72 h. After that the mixture was concentrated under reduced pressure and the residue taken up in dichloromethane. After addition of trifluoroacetic acid (650 µl, 8.5 mmol) the mixture was stirred at r,.t. for 14 h. After that the mixture was concentrated under reduced pressure and coevaporated twice with toluene to give the title compound (300 mg, 80% purity). LC-MS (method 1): Rt = 0.74 min; MS (ESIpos): m/z = 639 [M+H]- 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.920 (2.57), 0.927 (3.62), 0.935 (6.87), 0.944 (3.96), 0.952 (4.98), 1.074 (0.48), 1.101 (15.81), 1.230 (0.41), 1.308 (1.96), 1.328 (3.54), 1.346 (2.61), 1.462 (4.51), 1.736 (11.89), 2.058 (0.48), 2.072 (0.61), 2.327 (0.44), 2.518 (1.65), 2.523 (1.14), 2.548 (0.67), 2.669 (0.45), 2.728 (10.91), 2.806 (5.75), 2.888 (16.00), 3.056 (0.47), 3.346 (0.73), 3.378 (0.55), 3.596 (0.99), 3.610 (0.58), 3.624 (0.67), 3.955 (0.58), 3.985 (0.46), 4.481 (0.45), 4.498 (0.71), 4.516 (0.45), 4.563 (0.48), 4.859 (2.64), 4.889 (3.01), 4.901 (2.85), 7.237 (1.84), 7.243 (1.67), 7.259 (2.17), 7.264 (1.56), 7.501 (0.61), 7.512 (0.65), 7.524 (2.22), 7.533 (0.89), 7.545 (3.05), 7.566 (1.23), 7.636 (1.98), 7.659 (4.46), 7.687 (4.77), 7.693 (1.85), 7.698 (1.56), 7.709 (2.76), 7.950 (1.75), 8.070 (1.82), 8.524 (0.52), 8.527 (0.57), 8.545 (0.52), 8.548
 
(0.52), 8.638 (0.99), 8.645 (1.82), 8.651 (0.82), 8.659 (1.90), 8.700 (0.86), 8.718 (3.09), 8.758 (0.69), 8.762 (0.91), 8.769 (3.09), 8.796 (0.69), 8.815 (0.56), 10.129 (1.37), 10.165 (1.03).
-----Examples------ Example 1
methyl 4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}benzoate
Figure imgf000369_0001
To a solution of N-{4-[4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carboxamide (1.50 g, 4.16 mmol) in DMF (10 mL) was added at r.t. sodium hydride (200 mg, 60 % on mineral oil, 5.00 mmol). After stirring for 20 min tetra-n- butylammoniumiodide (154 mg, 369 µmol) was addedand the mixture was cooled to 0°C. Then methyl 4-(chloromethyl)benzoate (CAS-No. 34040-64-7, 0.92 g, 5.00 mmol) was added the mixture was stirred at r.t. for 14 h. After addition of water the mixture was extracted with dichloromethane (3x). The combined organic phases were filtered through a silicone filter and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, dichloromethane/ethanol gradient) to give the title compound (1.24 mg, 59 % yield).
LC-MS (Method 1): Rt = 0.85 min; MS (ESIpos): m/z = 498 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.035 (1.51), 1.053 (6.85), 1.070 (5.57), 2.084 (0.57), 2.360 (0.95), 2.363 (1.00), 2.401 (1.15), 2.405 (1.02), 2.518 (1.25), 2.523 (0.84), 2.808 (0.66), 2.825 (0.84), 2.850 (0.68), 2.867 (0.59), 3.423 (0.97), 3.435 (0.88), 3.440 (1.16), 3.453 (0.89),  
3.832 (16.00), 4.345 (0.42), 4.358 (0.82), 4.807 (2.71), 4.824 (2.72), 4.919 (1.13), 4.958 (1.66), 5.080 (1.60), 5.118 (1.09), 7.426 (2.18), 7.431 (3.61), 7.436 (2.27), 7.447 (1.45), 7.452 (3.42), 7.642 (2.17), 7.647 (0.88), 7.659 (1.33), 7.665 (4.58), 7.670 (0.94), 7.692 (1.01), 7.698 (4.59), 7.703 (1.25), 7.715 (0.86), 7.720 (1.97), 7.922 (0.84), 7.927 (4.19), 7.931 (1.43), 7.943 (1.39), 7.947 (3.69), 8.493 (2.21), 8.506 (2.05), 8.606 (2.89), 8.608 (2.86), 8.640 (2.63).
Example 2
4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]methyl}benzoic acid
Figure imgf000370_0001
A mixture of methyl 4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}- 4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}benzoate (890 mg, 1.79 mmol), aqueous lithium hydroxide solution (5.4 ml, 1.0 M, 5.4 mmol) and THF (7.3 ml, 89 mmol) was heated to 50°C for 2 h. After cooling to 0°C the mixture was acidified to pH 2 by addition of 4N hydrochloric acid. The precipitate was filtered off, washed with water and dried under reduced pressure to give the title compound (360 mg, 42 % yield). A second crop (231 mg, 95 % purity, 25 % yield) was received as more product precipitated from the filtrate which was also filtred off, washed with water and dried under reduced pressure.
LC-MS (Method 1): Rt = 0.74 min; MS (ESIpos): m/z = 484 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.055 (14.19), 1.073 (14.41), 1.231 (0.54), 2.318 (0.82), 2.323 (1.90), 2.327 (2.72), 2.331 (1.95), 2.337 (0.91), 2.363 (3.31), 2.401 (3.67), 2.518 (10.06), 2.523 (6.98), 2.660 (0.86), 2.665 (1.90), 2.669 (2.58), 2.674 (1.77), 2.678 (0.86), 2.808 (2.13), 2.824 (2.76), 2.849 (2.27), 2.867 (1.95), 3.426 (2.27), 3.445 (2.76), 3.461 (1.90), 4.821 (9.52), 4.833 (9.56), 4.911 (3.81), 4.949 (5.39), 5.072 (5.21), 5.111 (3.72), 7.402 (10.65), 7.407 (3.76), 7.424 (11.42), 7.457 (4.31), 7.471 (4.35), 7.637 (1.04), 7.643 (7.80), 7.648 (2.86), 7.660 (3.94), 7.666 (16.00), 7.671 (2.86), 7.697 (3.08), 7.702 (16.00), 7.707 (3.85), 7.719 (2.76),  
7.724 (7.03), 7.897 (2.40), 7.901 (14.46), 7.906 (4.35), 7.918 (4.31), 7.922 (12.74), 7.927 (2.08), 8.510 (6.71), 8.522 (6.35), 8.625 (9.52), 8.650 (8.93), 12.904 (2.13).
Example 3
N-{4-[1-{4-[(3-aminopropyl)carbamoyl]benzyl}-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000371_0001
A mixture of tert-butyl {3-[(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)- amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}benzoyl)amino]propyl}- carbamate (120.0 mg, 188 µmol), trifluoroacetic acid (144 µl, 2.0 mmol) and dichloromethane (1.5 mL) was stirred at r.t. for 14 h. Then the mixture was concentrated under reduced pressure and the crude product was purified by preparative HPLC to give the title compound (80.0 mg, 90% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 120x30 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-8 min 15-55% B. rate 150 ml/min, temperature 25°C.
LC-MS (Method 2): Rt = 0.83 min; MS (ESIneg): m/z = 538 [M-H]- 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.043 (11.89), 1.061 (11.94), 1.738 (0.96), 1.755 (3.04), 1.773 (3.83), 1.793 (3.04), 1.809 (1.07), 2.318 (1.07), 2.323 (2.48), 2.327 (3.55), 2.331 (2.54), 2.337 (1.18), 2.360 (2.76), 2.398 (3.15), 2.518 (14.25), 2.523 (10.14), 2.659 (1.07),  
2.665 (2.54), 2.669 (3.49), 2.674 (2.37), 2.678 (1.07), 2.796 (3.49), 2.813 (5.07), 2.831 (3.15), 2.838 (3.27), 2.847 (2.08), 2.855 (1.92), 3.286 (2.76), 3.302 (6.42), 3.317 (7.10), 3.334 (5.69), 3.424 (2.54), 3.442 (2.65), 3.463 (1.80), 4.811 (7.49), 4.827 (7.55), 4.861 (3.55), 4.899 (4.28), 5.090 (4.11), 5.128 (3.27), 7.382 (8.79), 7.403 (9.63), 7.440 (4.00), 7.452 (4.17), 7.636 (3.04), 7.642 (9.46), 7.647 (5.46), 7.659 (6.42), 7.664 (16.00), 7.670 (4.56), 7.697 (2.99), 7.703 (13.92), 7.708 (3.89), 7.720 (2.59), 7.725 (6.37), 7.731 (1.13), 7.788 (1.92), 7.794 (11.61), 7.798 (3.94), 7.810 (3.61), 7.814 (10.03), 7.819 (1.80), 8.504 (5.30), 8.517 (5.01), 8.559 (1.86), 8.574 (3.94), 8.588 (1.80), 8.617 (7.61), 8.648 (7.83).
Example 4
N-{4-[1-(3-fluoro-4-nitrobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000372_0001
To a solution of N-{4-[4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carboxamide (1.50 g, 4.29 mmol) in DMF (10 mL) was added at r.t. sodium hydride (206 mg, 60 % on mineral oil, 5.15 mmol). After stirring for 20 min tetra-n- butylammoniumiodide (159 mg, 429 µmol) was added and the mixture was cooled to 0°C. Then 4-(bromomethyl)-2-fluoro-1-nitrobenzene (CAS-No. 131858-37-2, 1.21 g, 5.15 mmol) was added and the mixture was stirred at r.t. for 14 h. After addition of water the mixture was extracted with dichloromethane (3x). The combined organic phases were filtered through a silicone filter and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, dichloromethane/ethanol gradient) to give the title compound (950 mg, 44 % yield).
LC-MS (Method 1): Rt = 0.90 min; MS (ESIpos): m/z = 504 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.000 (4.82), 0.924 (0.77), 0.943 (2.02), 0.961 (0.91), 1.043 (7.24), 1.060 (16.00), 1.078 (7.77), 1.094 (11.85), 1.113 (11.85), 2.384 (2.45), 2.387  
(2.67), 2.425 (3.01), 2.429 (2.70), 2.525 (1.05), 2.529 (0.71), 2.864 (1.73), 2.881 (2.28), 2.906 (1.85), 2.923 (1.61), 3.414 (1.07), 3.426 (1.17), 3.431 (3.28), 3.443 (3.55), 3.448 (3.43), 3.454 (1.34), 3.461 (3.71), 3.465 (1.60), 3.472 (1.82), 3.475 (1.82), 3.478 (2.03), 3.490 (1.22), 4.340 (1.99), 4.353 (3.90), 4.366 (1.87), 4.813 (6.66), 4.832 (6.71), 5.064 (11.06), 7.342 (2.82), 7.345 (2.82), 7.364 (2.92), 7.366 (2.99), 7.424 (3.72), 7.437 (3.87), 7.460 (3.04), 7.464 (2.90), 7.490 (3.00), 7.495 (2.86), 7.656 (5.84), 7.660 (2.15), 7.678 (12.59), 7.713 (12.58), 7.718 (3.08), 7.731 (2.15), 7.736 (5.32), 8.137 (3.59), 8.158 (5.51), 8.178 (3.26), 8.310 (1.14), 8.494 (5.86), 8.507 (5.46), 8.608 (7.55), 8.647 (6.81).
Example 5
N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2- fluorophenyl)-L-alaninamide
Figure imgf000373_0001
A mixture of N-{4-[1-(4-amino-3-fluorobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (379 mg, 90 % purity, 723 µmol), N-(tert-butoxycarbonyl)-L-valyl-L-alanine (250 mg, 867 µmol), N-methyl-morpholine (240 µl, 2.2 mmol) and (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3- oxid hexafluorophosphate) (HATU, 412 mg, 1.08 mmol) in DMF (6 mL) was stirred at r.t. for 14 h. After that the mixture was concentrated under redcued pressure and the residue was  
purified by column chromatography (SiO2, dichloromethane/ethanol gradient) and preparative HPLC to give the title compound (150 mg, 27 % yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 122x50 mm. Eluent A: water + 0.1 Vol- % ammonia; Eluent B: acetonitrile; gradient: 0-20 min 15-55% B. rate 250 ml/min, temperature 25°C.
LC-MS (method 2): Rt = 1.11 min; MS (ESIpos): m/z = 743 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.787 (1.21), 0.804 (1.42), 0.827 (4.20), 0.833 (3.06), 0.844 (4.48), 0.852 (2.56), 1.026 (1.92), 1.033 (2.28), 1.040 (1.99), 1.045 (1.99), 1.052 (1.99), 1.234 (2.42), 1.256 (7.54), 1.266 (8.11), 1.274 (4.55), 1.281 (3.20), 1.292 (3.63), 1.298 (2.56), 1.358 (9.39), 1.897 (0.43), 1.987 (0.64), 2.006 (0.57), 2.318 (1.42), 2.323 (3.13), 2.327 (4.48), 2.331 (3.27), 2.337 (1.85), 2.382 (1.07), 2.518 (16.00), 2.523 (11.09), 2.660 (1.42), 2.665 (3.20), 2.669 (4.41), 2.674 (3.06), 2.678 (1.35), 2.805 (0.50), 2.834 (0.43), 3.306 (0.78), 3.410 (0.50), 3.429 (0.71), 3.449 (0.43), 3.751 (0.43), 4.811 (3.06), 4.829 (3.20), 4.855 (0.78), 4.954 (0.64), 4.992 (0.71), 7.091 (1.00), 7.112 (1.07), 7.135 (0.57), 7.144 (0.64), 7.163 (0.57), 7.173 (0.57), 7.429 (1.56), 7.443 (1.64), 7.648 (2.28), 7.671 (4.48), 7.710 (3.77), 7.732 (2.06), 7.755 (0.57), 7.773 (0.50), 7.794 (0.50), 8.100 (0.43), 8.496 (2.42), 8.508 (2.20), 8.610 (3.27), 8.643 (2.70), 9.567 (0.57), 9.712 (0.57).
Example 6
L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluorophenyl)-L-alaninamide
 
Figure imgf000375_0001
A mixture of N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin- 2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluoro- phenyl)-L-alaninamide (150 mg, 202 µmol), trifluoroacetic acid (160 µl, 2.0 mmol) and dichloromethane (1.3 mL) was stirred at r.t. for 14 h. After that the mixture was concentrated under reduced pressure and coevaporated two times with toluene. The residue was purified by preparative HPLC to give the title compound (84.0 mg, 65 % yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: XBridge C18 5µM 100x30 mm. Eluent A: water + 0.1 Vol-% ammonia; Eluent B: acetonitrile; gradient: 0-20 min 15-55% B. rate 60 ml/min, temperature 25°C.
LC-MS (Method 2): Rt = 0.93 min; MS (ESIpos): m/z = 643 [M+H]+
Example 7
N-(4-{1-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)butyl]-6-oxo-4-phenyl-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
 
Figure imgf000376_0001
To a solution of N-{4-[6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide (600 mg, 1.46 mmol) in DMF (3 mL) was added at r.t. sodium hydride (87.5 mg, 60 % on mineral oil, 2.19 mmol) and the mixture stirred for 20 min at that temperature. Then tetra-n-butylammonium iodide (53.9 mg, 146 µmol) was added and the mixture was cooled to 0°C. After that 2-(4-bromobutyl)-1H-isoindole-1,3(2H)-dione (CAS- No.5394-18-3, 494 mg, 1.75 mmol) was added the the mixture was warmed to r.t.. After strring for 1 h at that temperature the mixture was diluted with water, extracted with dichloromethane, and the organic extracts were washed with brine and filtered through silicone filter. The filtrate was concentrated under reduced pressure and the crude product was purified by column chromatography (SiO2, dichloromethane/ethanol gradient) to give the title compound (525 mg, 57 % yield) which was directly used in the next step.
LC-MS (method 2): Rt = 1.13 min; MS (ESIpos): m/z = 613 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.915 (0.91), 0.934 (2.39), 0.952 (1.14), 1.138 (0.63), 1.297 (0.46), 1.316 (0.51), 1.572 (1.50), 1.589 (2.44), 1.603 (2.62), 1.626 (1.78), 1.642 (1.90), 1.658 (1.40), 2.085 (16.00), 2.518 (6.78), 2.523 (4.67), 2.536 (2.64), 2.574 (2.23), 2.674 (1.09), 2.729 (2.39), 2.888 (2.92), 3.043 (1.30), 3.062 (1.73), 3.084 (1.52), 3.103 (1.19), 3.616 (3.45), 3.633 (1.88), 3.673 (0.58), 3.691 (0.94), 3.706 (1.07), 3.723 (1.14), 3.736 (0.86), 3.838 (0.66), 3.855 (1.30), 3.871 (1.04), 3.888 (0.84), 4.677 (2.01), 4.695 (2.08), 4.797 (5.56), 4.814 (5.43), 5.759 (0.61), 7.137 (4.67), 7.155 (5.69), 7.189 (1.14), 7.207 (3.17), 7.226 (2.23), 7.276 (4.60), 7.295 (6.02), 7.314 (2.26), 7.421 (3.02), 7.434 (3.25), 7.578 (5.26), 7.601 (8.33), 7.661 (8.41), 7.684 (4.88), 7.821 (3.99), 7.829 (4.11), 7.834 (3.63), 7.836 (3.96), 7.842 (8.23), 7.852 (2.06), 7.858 (2.18), 7.868 (8.84), 7.874 (4.57), 7.877 (3.48), 7.881 (4.19), 7.889 (3.94), 8.490 (4.62), 8.503 (4.27), 8.602 (6.40), 8.614 (5.41).
Example 8
N-{4-[5,5-dimethyl-1-(4-nitrobenzyl)-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
 
Figure imgf000377_0001
To a solution of N-[4-(5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl]-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (1.72 g, 4.73 mmol) in DMF (11 mL) was added at r.t. sodium hydride (227 mg, 60 % on mineral oil, 5.68 mmol) and the mixture stirred for 30 min at that temperature. Then tetra-n-butylammonium iodide (44.9 mg, 122 µmol) was added and the mixture was cooled to 0°C. After that 1-(bromomethyl)-4-nitrobenzene (CAS- No.100-11-8, 1.23 g, 5.68 mmol) was added the the mixture was warmed to r.t.. After strring for 14 h at that temperature the mixture was diluted with water and dichloromethane. The precipitate was filtered off and dried under vacuum to give the title compound (1.35 g, 90% purity, 51% yield). 50 mg of this product were further purified by preparative HPLC to give the pure title compound (15.0 mg, 1% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x30 mm. Eluent A: water + 0.1 Vol- % HCOOH; Eluent B: acetonitrile; gradient: 0-8 min 15-55% B. rate 150 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 1.04 min; MS (ESIpos): m/z = 499 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.133 (16.00), 1.232 (0.53), 2.518 (4.25), 2.523 (2.87), 2.673 (0.70), 2.949 (4.23), 4.806 (2.24), 4.824 (2.27), 5.057 (3.72), 7.427 (1.23), 7.440 (1.26), 7.550 (2.72), 7.573 (2.75), 7.641 (1.50), 7.664 (4.10), 7.686 (4.17), 7.709 (1.37), 8.202 (3.55), 8.224 (3.11), 8.494 (1.71), 8.507 (1.62), 8.608 (2.46), 8.644 (2.19).
Example 9
N-{4-[1-(4-nitrobenzyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
 
Figure imgf000378_0001
To a solution of N-{4-[6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide (500 mg, 1.22 mmol) in DMF (4 mL) was added at r.t. sodium hydride (63.6 mg, 60 % on mineral oil, 1.46 mmol) and the mixture stirred for 20 min at that temperature. Then tetra-n-butylammonium iodide (44.9 mg, 122 µmol) was added and the mixture was cooled to 0°C. After that 1-(bromomethyl)-4-nitrobenzene (CAS-No. 100-11- 8, 315 mg, 1.46 mmol) was added the the mixture was warmed to r.t.. After strring for 14 h at that temperature the mixture was concentrated under reduced pressure and the residue was triturated with dichloromethane/methanol. The solids were filtered off and purified by preparative HPLC to give the title compound (14.0 mg, 2 % yield). The filtrate was concentrated under reduced pressure and the residue was purified by column chromatography (SiO2, dichloromethane/methanol gradient) to give a second crop of the title compound (305 mg, 90% purity, 41%).
HPLC: Instrument: Waters Autopurification system SQD; column: Waters XBrigde C18 5µ 100x30mm; eluent A: water + 0.2% Vol. ammonia (32%), eluent B: acetonitrile; gradient: 0.00 – 0.50 min 5% B, 25mL/min, 0.51– 5.50 min 10-100% B, 70mL/min, 5.51– 6.50 min 100% B, 70mL/min. Temperature: 25°C; injection: 2500 µl; DAD scan: 210-400 nm.
LC-MS (method 3): Rt = 1.10 min; MS (ESIneg): m/z = 545 [M-H]- 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.116 (0.53), 1.134 (1.23), 1.151 (0.69), 1.900 (0.43), 2.318 (1.01), 2.323 (2.29), 2.327 (3.15), 2.331 (2.35), 2.337 (1.07), 2.518 (16.00), 2.523 (11.20), 2.540 (2.77), 2.661 (2.45), 2.665 (4.00), 2.669 (3.63), 2.673 (2.45), 2.678 (1.12), 2.703 (1.97), 2.895 (1.01), 2.903 (0.80), 2.994 (0.59), 3.190 (1.23), 3.209 (1.65), 3.231 (1.39), 3.251 (1.17), 3.920 (2.03), 4.755 (2.35), 4.774 (6.24), 4.788 (7.47), 4.803 (6.24), 5.026 (1.92), 5.064 (3.09), 5.138 (3.09), 5.177 (1.76), 5.496 (4.69), 6.787 (0.43), 6.933 (0.53), 6.989 (0.59), 7.012 (7.04), 7.023 (0.69), 7.030 (3.95), 7.035 (1.49), 7.047 (1.81), 7.052 (4.85), 7.062 (2.99), 7.068 (3.63), 7.078 (3.73), 7.087 (3.63), 7.173 (0.85), 7.196 (2.56), 7.202 (2.51), 7.207 (1.71), 7.217 (3.57), 7.221 (3.73), 7.231 (9.33), 7.237 (8.00), 7.245 (4.69), 7.248 (4.80), 7.257 (1.49), 7.320 (1.23), 7.328 (1.33), 7.343 (3.31), 7.356 (2.77), 7.361 (5.01), 7.365 (2.13), 7.374 (1.12), 7.414   (4.43), 7.427 (4.80), 7.445 (1.23), 7.452 (4.59), 7.457 (2.03), 7.462 (1.44), 7.468 (1.71), 7.474 (3.95), 7.479 (1.28), 7.484 (1.07), 7.528 (0.91), 7.535 (6.03), 7.540 (2.45), 7.552 (2.40), 7.557 (6.51), 7.563 (1.07), 7.590 (4.91), 7.595 (1.81), 7.607 (2.88), 7.612 (7.68), 7.617 (1.60), 7.629 (1.01), 7.642 (3.68), 7.664 (4.05), 7.672 (1.87), 7.678 (7.73), 7.683 (2.40), 7.695 (2.08), 7.700 (4.69), 8.115 (0.64), 8.137 (0.69), 8.176 (0.96), 8.182 (1.17), 8.189 (7.79), 8.194 (2.77), 8.198 (1.28), 8.206 (2.40), 8.211 (7.15), 8.217 (1.17), 8.230 (0.91), 8.237 (4.80), 8.242 (1.55), 8.254 (1.60), 8.259 (4.37), 8.265 (0.69), 8.300 (0.43), 8.486 (5.44), 8.498 (5.71), 8.505 (3.63), 8.596 (7.95), 8.623 (4.75).
Example 10
N-{4-[1-(4-aminobenzyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000379_0001
A mixture of N-{4-[1-(4-nitrobenzyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}- 1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (305 mg, 558 µmol), iron (467 mg, 8.37 mmol), acetic acid (3.2 ml, 56 mmol), and ethanol (3.3 ml) was heated to 90°C for 2.5 h. After that the hot mixture was filtered through Celite®, the filter cake washed with ethanol, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, dichloromethane/methanol gradient) and preparative HPLC to give the title compound (45.8 mg, 15 % yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x20 mm. Eluent A: water + 0.1 Vol- % TFA; Eluent B: acetonitrile; gradient: 0-22 min 15-55% B. rate 50 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.79 min; MS (ESIpos): m/z = 517 [M+H]+
 
¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.137 (0.50), 1.233 (0.43), 2.024 (0.57), 2.084 (0.43), 2.336 (1.28), 2.518 (16.00), 2.522 (11.09), 2.539 (0.64), 2.549 (1.21), 2.673 (2.99), 2.678 (1.28), 3.067 (0.71), 3.086 (1.00), 3.109 (0.85), 3.128 (0.64), 4.561 (1.42), 4.596 (1.71), 4.651 (1.07), 4.669 (1.14), 4.780 (5.76), 4.801 (5.55), 4.952 (1.71), 4.987 (1.56), 5.028 (4.55), 5.090 (5.76), 5.139 (5.97), 6.500 (4.48), 6.509 (5.97), 6.514 (2.28), 6.521 (5.05), 6.525 (2.20), 6.530 (5.97), 6.570 (0.43), 6.908 (11.66), 6.971 (1.99), 6.976 (2.35), 6.984 (4.62), 6.991 (3.34), 6.995 (2.77), 7.006 (4.55), 7.008 (6.40), 7.013 (2.06), 7.026 (1.85), 7.031 (5.76), 7.099 (4.91), 7.121 (4.62), 7.134 (0.85), 7.141 (1.00), 7.155 (3.20), 7.164 (3.63), 7.170 (4.20), 7.175 (4.91), 7.180 (3.34), 7.183 (4.84), 7.187 (3.98), 7.300 (0.92), 7.308 (1.28), 7.322 (3.84), 7.330 (1.28), 7.336 (3.34), 7.341 (6.12), 7.355 (1.21), 7.358 (1.07), 7.416 (3.34), 7.429 (3.48), 7.447 (5.90), 7.452 (1.78), 7.469 (5.05), 7.574 (3.13), 7.596 (4.84), 7.660 (4.98), 7.682 (2.77), 8.487 (4.27), 8.499 (7.47), 8.598 (7.96).
Example 11
N,N'-(disulfanediylbis{(1-oxopropane-3,1-diyl)iminobutane-4,1-diyl[4-methyl-6-oxo-5,6- dihydropyridazine-1,3(4H)-diyl]benzene-4,1-diyl})bis(1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide)
 
Figure imgf000381_0001
A mixture of N-{4-[1-(4-aminobutyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}- 1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (20.0 mg, 47.6 µmol), 1,1'-{disulfanediyl- bis[(1-oxopropane-3,1-diyl)oxy]}dipyrrolidine-2,5-dione (CAS-No. 57757-57-0, 48.1 mg, 119 µmol) and N,N-diisopropylethylamine (29 µl, 170 µmol) in DMF (1 mL) was stirred at r.t. for 14 h. After that the mixture was filtrated and the filtrate was purified by preparative HPLC to give the title compound (9.00 mg, 18% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x30 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-8 min 15-55% B. rate 150 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.73 min; MS (ESIpos): m/z = 1015 [M]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.047 (2.56), 1.065 (2.60), 1.222 (2.61), 1.240 (15.02), 1.256 (16.00), 1.272 (9.33), 1.373 (0.47), 1.391 (0.69), 1.411 (0.63), 1.584 (0.56), 1.603 (0.67), 1.622 (0.47), 2.274 (0.61), 2.313 (0.72), 2.414 (0.81), 2.432 (1.80), 2.449 (0.99), 2.518 (0.98),   2.523 (0.65), 2.590 (2.00), 2.669 (0.57), 2.687 (0.52), 2.712 (0.43), 2.727 (2.63), 2.839 (1.06), 2.857 (2.02), 2.875 (0.92), 2.888 (3.00), 3.043 (0.40), 3.060 (0.97), 3.075 (0.96), 3.106 (0.43), 3.117 (0.46), 3.124 (1.28), 3.135 (1.29), 3.143 (1.27), 3.154 (1.25), 3.161 (0.41), 3.378 (0.44), 3.580 (0.41), 3.597 (0.97), 3.607 (0.98), 3.613 (1.26), 3.623 (1.21), 3.630 (1.05), 3.639 (0.83), 3.646 (0.46), 3.838 (0.46), 4.911 (1.91), 4.942 (1.85), 7.647 (1.59), 7.669 (2.50), 7.729 (2.48), 7.751 (1.43), 7.808 (0.76), 7.822 (0.81), 7.938 (0.40), 7.951 (1.11), 8.705 (0.63), 8.718 (0.64), 8.739 (1.61), 8.834 (0.98).
Example 12
N,N'-(disulfanediylbis{propane-3,1-diyl[4-methyl-6-oxo-5,6-dihydropyridazine-1,3(4H)- diyl]benzene-4,1-diyl})bis(1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide)
Figure imgf000382_0001
A mixture of {3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl} ethanethioate (157 mg, 337 µmol), aqueous sodium hydroxide solution (340 µl, 2.0 M, 670 µmol) and ethanol (2 mL) was stirred at r.t. for 14 h. After that the mixture was diluted with water, acidified to pH 5 by addition of 1N hydrochloric acid and extracted three times with dichloromethane/methanol (4:1). The combined organic extracts were filtered through a silicone filter, concentrated under reduced pressure and the crude product was purified by two concsecutive preparative HPLCs to give the title compound (12.0 mg, 4 % yield).   1. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-8 min 15-55% B. rate 150 ml/min, temperature 25°C.
2. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-8 min 10-50% B. rate 150 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.88 min; MS (ESIpos): m/z = 846 [M+H]+
Example 13
N-{4-[4-methyl-6-oxo-1-(3-sulfanylpropyl)-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000383_0001
In the same reaction as for the synthesis of N,N'-(disulfanediylbis{propane-3,1-diyl[4-methyl- 6-oxo-5,6-dihydropyridazine-1,3(4H)-diyl]benzene-4,1-diyl})bis(1,3-dihydro-2H-pyrrolo[3,4-c]- pyridine-2-carboxamide) was isolated after two preparative HPLCs to give the title compound (10.0 mg, 7% yield).
1. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-8 min 15-55% B. rate 150 ml/min, temperature 25°C.
2. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-  
1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-8 min 10-50% B. rate 150 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.79 min; MS (ESIpos): m/z = 424 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.055 (14.61), 1.074 (14.82), 1.232 (0.83), 1.855 (1.11), 1.872 (4.31), 1.889 (6.68), 1.906 (4.80), 1.922 (1.25), 2.074 (0.97), 2.289 (2.78), 2.292 (3.06), 2.318 (1.39), 2.322 (3.06), 2.327 (4.80), 2.332 (6.12), 2.335 (3.97), 2.404 (2.64), 2.420 (2.30), 2.427 (5.29), 2.443 (8.35), 2.461 (4.38), 2.463 (4.52), 2.478 (10.43), 2.480 (11.76), 2.518 (16.00), 2.523 (10.16), 2.539 (5.77), 2.660 (1.25), 2.664 (2.78), 2.669 (4.03), 2.673 (2.92), 2.678 (1.25), 2.708 (2.16), 2.725 (2.78), 2.749 (2.23), 2.767 (1.95), 3.380 (1.39), 3.384 (1.46), 3.397 (2.02), 3.415 (1.39), 3.715 (1.11), 3.732 (2.43), 3.748 (2.57), 3.765 (3.06), 3.782 (1.32), 3.905 (1.46), 3.922 (3.13), 3.939 (2.64), 3.956 (2.37), 3.972 (1.04), 4.913 (7.86), 4.943 (7.44), 7.649 (0.90), 7.655 (8.63), 7.660 (2.85), 7.672 (3.41), 7.678 (13.08), 7.684 (1.81), 7.738 (1.95), 7.743 (13.15), 7.749 (3.48), 7.761 (2.85), 7.766 (7.86), 7.772 (1.04), 7.791 (2.71), 7.804 (2.71), 8.690 (4.52), 8.704 (4.38), 8.735 (7.58), 8.819 (6.75).
Example 14
N-{4-[1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000384_0001
To a solution of N-{4-[4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carboxamide (1.50 g, 4.29 mmol) in DMF (8 mL) was added at r.t.  
sodium hydride (361 mg, 60 % on mineral oil, 9.02 mmol) and the mixture stirred for 20 min at that temperature. After cooling to 0°C (3-bromopropoxy)(tert-butyl)dimethylsilane (CAS-No. 89031-84-5, 1.30 g, 5.15 mmol) was added and the mixture was warmed to r.t. and stirred for further 3 h. Then the mixture was diluted with water, extracted with dichloromethane (3x), and the combined organic extracts were washed with brine, filtered through a silicone filter, and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, dichloromethane/ethanol gradient) to give the title compound (1.25 g, 54% yield).
LC-MS (Method 1): Rt = 1.20 min; MS (ESIpos): m/z = 522 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.000 (4.85), 0.004 (4.77), 0.807 (0.45), 0.820 (0.64), 0.841 (0.83), 0.848 (16.00), 0.855 (0.74), 1.027 (1.20), 1.031 (0.96), 1.045 (1.20), 1.048 (0.81), 3.613 (0.89), 5.737 (0.50), 7.634 (0.67), 7.657 (1.16), 7.708 (1.16), 7.731 (0.60), 8.477 (0.58), 8.592 (0.73), 8.594 (0.72), 8.616 (0.66).
Example 15
N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5-dimethyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-L-asparagine
Figure imgf000385_0001
tert-butyl N2-(tert-butoxycarbonyl)-N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-L- asparaginate (203 mg, 92 % purity, 270 µmol) was dissolved in hydrogen chloride solution (4N in dioxane, 670 µl, 2.7 mmol) and DCM (10 ml, 160 mmol). The mixture was stirred for 1 h at  
room temperature and concentrated. Again hydrogen chloride solution (4N in dioxane, 670 µl, 2.7 mmol) and DCM (10 ml, 160 mmol) were added and the solution was stirred at room temperature overnight. Subsequently, the mixture was concentrated under reduced pressure to give the crude title compound (150 mg, 96% yield, 93% purity).
LC-MS (METHOD 6): Rt = 0.71 min; MS (ESIneg): m/z = 534 [M-H]- ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: -0.008 (2.01), 0.008 (1.99), 1.075 (2.12), 1.089 (16.00), 1.104 (1.75), 1.235 (1.07), 1.255 (1.37), 1.271 (1.57), 1.283 (1.17), 1.299 (1.11), 1.413 (0.99), 1.440 (0.64), 1.596 (0.89), 1.758 (1.15), 1.776 (1.61), 1.793 (1.11), 2.524 (1.78), 2.725 (1.24), 2.739 (2.16), 2.751 (1.27), 2.848 (4.46), 3.105 (1.27), 3.122 (1.29), 3.129 (1.26), 3.462 (1.16), 3.474 (1.45), 3.492 (1.46), 3.502 (1.21), 3.666 (1.18), 3.671 (0.98), 3.680 (1.51), 3.700 (1.54), 3.708 (1.07), 3.712 (1.38), 3.717 (0.84), 3.724 (1.00), 3.729 (1.45), 3.748 (2.15), 3.765 (1.22), 4.144 (0.98), 4.156 (0.97), 4.933 (2.70), 4.972 (2.47), 7.663 (1.95), 7.686 (3.84), 7.719 (3.98), 7.741 (1.76), 7.858 (0.77), 8.224 (2.46), 8.236 (2.70), 8.724 (0.93), 8.738 (0.94), 8.804 (1.34), 8.861 (1.51).
Example 16
N-{4-[1-(3-aminopropyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide hydrochloride
Figure imgf000386_0001
tert-butyl {3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5- dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}carbamate (263 mg, 505 µmol) was dissolved in hydrogen chloride solution (4N in dioxane, 1.3 ml, 5.1 mmol) and DCM (5 ml, 78 mmol). The mixture was stirred at room temperature and concentrated under reduced pressure to give the crude title compound (229 mg, 91% yield, 92% purity).
LC-MS (METHOD 6): Rt = 0.71 min; MS (ESIneg): m/z = 419 [M-H]- gib16082-1-2.76969065.1h-nmr.2.1
 
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.088 (2.21), 1.102 (16.00), 1.235 (0.73), 1.258 (10.07), 1.274 (13.79), 1.287 (8.78), 1.292 (3.69), 1.304 (8.49), 1.909 (0.97), 1.926 (1.04), 1.946 (0.82), 2.711 (0.41), 2.792 (0.67), 2.806 (0.99), 2.830 (0.99), 2.846 (0.88), 2.879 (4.05), 3.091 (0.47), 3.101 (0.55), 3.109 (1.30), 3.120 (1.35), 3.128 (1.34), 3.138 (1.32), 3.146 (0.58), 3.157 (0.51), 3.446 (0.69), 3.451 (0.73), 3.457 (0.77), 3.462 (1.01), 3.468 (0.83), 3.472 (1.07), 3.475 (1.11), 3.490 (1.21), 3.492 (1.20), 3.497 (1.00), 3.503 (1.24), 3.508 (1.09), 3.514 (1.09), 3.519 (1.09), 3.565 (1.70), 3.568 (5.64), 3.576 (1.53), 3.583 (1.86), 3.593 (1.88), 3.599 (2.08), 3.609 (2.06), 3.616 (1.81), 3.626 (1.75), 3.632 (1.34), 3.642 (1.25), 3.651 (1.06), 3.656 (1.05), 3.662 (1.01), 3.667 (1.12), 3.671 (1.06), 3.678 (1.10), 3.681 (1.11), 3.699 (1.04), 3.701 (1.01), 3.709 (0.87), 3.713 (0.91), 3.718 (0.73), 3.724 (0.69), 3.729 (0.66), 3.738 (0.67), 3.790 (1.49), 3.807 (2.26), 3.823 (1.19), 4.949 (2.08), 4.997 (1.94), 5.755 (0.68), 7.676 (1.69), 7.681 (0.78), 7.693 (1.16), 7.698 (3.44), 7.726 (0.86), 7.731 (3.66), 7.736 (1.01), 7.748 (0.69), 7.754 (1.60), 7.882 (1.05), 7.914 (0.87), 8.760 (0.87), 8.773 (0.81), 8.849 (1.05), 8.900 (1.31).
Example 17
N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]propyl}-L-asparagine hydrochloride
Figure imgf000387_0001
tert-butyl N2-(tert-butoxycarbonyl)-N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-L- asparaginate (50.0 mg, 100 % purity, 73.8 µmol) was dissolved in hydrogen chloride solution (4N in dioxane, 180 µl, 740 µmol) and DCM (380 µl). The mixture was stirred at room temperature for 4 h and concentrated under reduced pressure to give the crude title compound (41 mg, 99% yield, 99% purity).  
LC-MS (Method 8): Rt = 0.43 min; MS (ESIneg): m/z = 520 [M-H]- ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: -0.008 (4.90), 0.008 (4.27), 1.069 (7.65), 1.087 (7.81), 1.235 (1.58), 1.596 (15.78), 1.753 (1.99), 1.771 (2.81), 1.789 (1.99), 2.294 (1.87), 2.333 (2.43), 2.367 (1.30), 2.691 (1.55), 2.710 (2.53), 2.717 (2.56), 2.732 (5.31), 2.743 (2.75), 3.112 (2.02), 3.387 (1.87), 3.407 (2.28), 3.421 (1.99), 3.446 (1.77), 3.451 (1.83), 3.462 (2.50), 3.475 (2.91), 3.490 (3.13), 3.503 (3.19), 3.509 (2.72), 3.560 (3.67), 3.568 (16.00), 3.636 (4.65), 3.655 (4.68), 3.667 (4.87), 3.671 (4.90), 3.681 (4.46), 3.699 (3.76), 3.708 (3.10), 3.713 (3.29), 3.717 (2.59), 3.724 (2.43), 3.859 (1.26), 3.878 (1.71), 3.894 (1.52), 3.913 (1.33), 4.144 (1.42), 4.158 (1.42), 4.926 (5.03), 4.960 (4.81), 5.754 (0.79), 7.667 (4.27), 7.689 (7.15), 7.743 (7.18), 7.766 (3.98), 7.820 (1.45), 7.831 (1.52), 8.208 (3.32), 8.227 (2.02), 8.241 (2.66), 8.255 (1.33), 8.709 (2.47), 8.722 (2.40), 8.786 (4.21), 8.841 (3.92).
Example 18
tert-butyl 4-{2-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}piperazine-1-carboxylate
Figure imgf000388_0001
To a solution of N-{4-[4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carboxamide (1.10 g, 90 % purity, 2.84 mmol) in DMF (7 mL) was added at r.t. sodium hydride (136 mg, 60 % on mineral oil, 3.41 mmol) and the mixture stirred for 20 min at that temperature. After cooling to 0°C tert-butyl 4-(2-bromoethyl)piperazine-1- carboxylate (CAS-No. 655225-01-7, 1.00 g, 3.41 mmol) was added and the mixture was warmed to r.t. and stirred for further 14 h. Then the mixture was diluted with water, extracted with dichloromethane (3x), and the combined organic extracts were washed with brine, filtered through a silicone filter, and concentrated under reduced pressure to give the title compound  
(1.02 g, 87 % purity, 56 % yield).50.0 mg of the product were purified by preparative HPLC to the pure title compound (24.0 mg, 2% yield).
HPLC: Instrument: Waters Autopurification system SQD; column: Waters XBrigde C18 5µ 100x30mm; Eluent A: water + 0.2% vol. ammonia (32%), Eluent B: acetonitrile; gradient: 0.00 – 0.50 min 5% B, 25mL/min, 0.51– 5.50 min 10-100% B, 70mL/min, 5.51– 6.50 min 100% B, 70mL/min. Temperature: 25°C; injection: 2500 µl; DAD scan: 210-400 nm.
LC-MS (method 3): Rt = 1.06 min; MS (ESIpos): m/z = 562 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.087 (1.57), 1.106 (1.59), 1.375 (16.00), 2.318 (0.67), 2.323 (0.71), 2.327 (0.90), 2.331 (0.66), 2.373 (0.79), 2.518 (2.85), 2.523 (2.04), 2.556 (0.83), 2.665 (0.60), 2.669 (0.94), 2.673 (0.59), 3.226 (1.03), 4.817 (1.02), 4.835 (1.00), 7.435 (0.55), 7.448 (0.57), 7.656 (0.92), 7.679 (1.58), 7.733 (1.59), 7.755 (0.88), 8.499 (0.84), 8.512 (0.78), 8.615 (1.14), 8.642 (1.02).
Example 19
N-(4-{4-methyl-6-oxo-1-[2-(piperazin-1-yl)ethyl]-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000389_0001
A mixture of tert-butyl 4-{2-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]- phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}piperazine-1-carboxylate (1.00 g, 1.78 mmol), trifluoroacetic acid (690 µl, 8.9 mmol) and dichloromethane (9.2 ml, 140 mmol) was stirred at r.t. for 14 h. After that the mixture was concentrated under reduced pressure and coevaporated twice with toluene. The crude product was purified by column chromatography (amine-coated SiO2, dichloromethane/ethanol gradient) to give the title compound (620 mg,  
90% purity, 68% yield). 50.0 mg of the product were further purified by preparative HPLC to give 28.0 mg (3% yield) of the pure title compound.
HPLC: Instrument: Waters Autopurification system SQD; column: Waters XBrigde C18 5µ 100x30mm; Eluent A: water + 0.2% vol. ammonia (32%), Eluent B: acetonitrile; gradient: 0.00 – 0.50 min 5% B, 25mL/min, 0.51– 5.50 min 10-100% B, 70mL/min, 5.51– 6.50 min 100% B, 70mL/min. Temperature: 25°C; injection: 2500 µl; DAD scan: 210-400 nm.
LC-MS (Method 1): Rt = 0.54 min; MS (ESIpos): m/z = 462 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.064 (0.69), 1.082 (1.26), 1.096 (15.60), 1.114 (15.80), 1.231 (0.49), 2.271 (4.77), 2.310 (7.29), 2.323 (4.83), 2.327 (5.17), 2.331 (4.51), 2.452 (1.46), 2.468 (3.63), 2.518 (6.37), 2.523 (4.63), 2.540 (1.17), 2.591 (7.57), 2.602 (11.97), 2.613 (6.63), 2.660 (3.03), 2.665 (2.49), 2.669 (2.51), 2.677 (3.43), 2.702 (2.40), 2.718 (2.09), 3.376 (3.46), 3.393 (2.17), 3.570 (0.89), 3.586 (1.83), 3.603 (1.83), 3.620 (2.03), 3.635 (1.00), 4.078 (1.20), 4.094 (2.74), 4.111 (2.43), 4.128 (2.40), 4.145 (0.97), 4.816 (9.40), 4.834 (9.43), 7.433 (5.23), 7.447 (5.40), 7.653 (9.31), 7.675 (16.00), 7.697 (0.71), 7.728 (15.11), 7.750 (7.89), 8.498 (8.63), 8.511 (8.06), 8.534 (0.49), 8.614 (11.06), 8.640 (9.51).
Example 20
tert-butyl {4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}carbamate
Figure imgf000390_0001
To a solution of N-{4-[4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carboxamide (2.00 mg, 5.72 mmol) in DMF (11 mL) was added at r.t. sodium hydride (481 mg, 60% on mineral oil, 12.0 mmol) and the mixture stirred for 20 min at  
that temperature. After that tetra-n-butylammoniumiodide (211 mg, 572 µmol) was added and the mixture was cooled to 0°C. Then tert-butyl (4-bromobutyl)carbamate (CAS-No. 164365- 88-2, 2.02 g, 8.01 mmol) was added and the mixture was warmed to r.t. and stirred for 1.5 h. After that more tert-butyl (4-bromobutyl)carbamate (300 mg) was added and the mixture stirred for further 14 h. Then the mixture was diluted with water, extracted with dichloromethane (4x), and the combined organic extracts were washed with brine, filtered through a silicone filter, and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, dichloromethane/ethanol gradient) to give the title compound (1.17 g, 37 % yield).
LC-MS (Method 1): Rt = 0.88 min; MS (ESIpos): m/z = 521 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.934 (0.90), 0.952 (0.40), 1.051 (1.42), 1.069 (1.43), 1.354 (16.00), 2.084 (5.17), 2.518 (0.46), 2.922 (0.58), 2.937 (0.58), 4.817 (0.88), 4.834 (0.88), 7.433 (0.50), 7.446 (0.51), 7.660 (0.85), 7.682 (1.49), 7.727 (1.48), 7.750 (0.74), 8.498 (0.81), 8.511 (0.75), 8.613 (1.06), 8.641 (0.87).
Example 21
tert-butyl {3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}carbamate
Figure imgf000391_0001
To a solution of N-{4-[4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carboxamide (600 mg, 1.72 mmol) in DMF (3 mL) was added at r.t. sodium hydride (86.5 mg, 3.61 mmol) and the mixture stirred for 20 min at that temperature. After that tetra-n-butylammoniumiodide (63.4 mg, 172 µmol) was added and the mixture was cooled to 0°C. Then tert-butyl tert-butyl (3-bromopropyl)carbamate (CAS-No.83948-53-2 , 491 mg, 2.06 mmol) was added and the mixture was warmed to r.t. and stirred for 14 h at that  
temperature. Then the mixture was diluted with water, extracted with dichloromethane (4x), and the combined organic extracts were washed with brine, filtered through a silicone filter, and concentrated under reduced pressure to give the title compound (930 mg, 92% purity, 98% yield). 62.0 mg of the product were further purified by preparative HPLC to give the pure title compound (16.5 mg, 2% yield).
HPLC: Instrument: Waters Autopurification system SQD; column: Waters XBrigde C18 5µ 100x30mm; Eluent A: water + 0.2% vol. ammonia (32%), Eluent B: acetonitrile; gradient: 0.00 – 0.50 min 5% B, 25mL/min, 0.51– 5.50 min 10-100% B, 70mL/min, 5.51– 6.50 min 100% B, 70mL/min. Temperature: 25°C; injection: 2500 µl; DAD scan: 210-400 nm.
LC-MS (Method 1): Rt = 0.89 min; MS (ESIpos): m/z = 507 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.057 (2.53), 1.075 (2.54), 1.363 (1.52), 1.372 (16.00), 1.715 (0.69), 2.278 (0.53), 2.317 (0.66), 2.699 (0.49), 2.947 (0.70), 2.964 (0.66), 4.818 (1.45), 4.835 (1.46), 7.431 (0.79), 7.433 (0.81), 7.444 (0.83), 7.446 (0.84), 7.661 (1.44), 7.666 (0.49), 7.678 (0.66), 7.683 (2.64), 7.731 (2.57), 7.737 (0.68), 7.754 (1.35), 8.498 (1.33), 8.511 (1.24), 8.613 (1.63), 8.614 (1.67), 8.647 (1.37).
Example 22
N-{4-[1-(2-hydroxyethyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000392_0001
To a solution of 4-nitrophenyl {4-[1-(2-hydroxyethyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydro- pyridazin-3-yl]phenyl}carbamate (757 mg, 80 % purity, 1.42 mmol) in dichloromethane (40 mL) was added at r.t. N,N-diisopropylethylamine (770 µl, 4.4 mmol) and 2,3-dihydro-1H-pyrrolo[3,4- c]pyridine dihydrochloride (CAS-No. 6000-50-6, 274 mg, 1.42 mmol) and the mixture was heated to 60°C for 4 h. After cooling to r.t. the mixture was diluted with 1N aqueous sodium hydroxide solution and extracted twice with dichloromethane. The combined organic extracts  
were washed with brine and filtered through a silicone filter. The residue residue on the filter was dried at 50°C under vacuum to give the title compound (446 mg, 94 % purity, 72 % yield). The filtrate was concentrated under reduced pressure and the residue was taken triturated with dichloromethane/methanol at 0°C and filtered. The filtratewas concentrated under reduced pressure and purified by preparative HPLC to give a second crop of the title compound (20.0 mg, 97% purity, 3% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-8 min 10-50% B. rate 150 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.66 min; MS (ESIpos): m/z = 408 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.050 (0.92), 1.083 (16.00), 2.322 (0.86), 2.327 (1.20), 2.331 (0.86), 2.518 (5.97), 2.523 (4.13), 2.540 (0.80), 2.665 (0.84), 2.669 (1.20), 2.674 (0.84), 2.835 (4.09), 3.575 (1.10), 3.592 (2.42), 3.607 (1.40), 3.773 (1.34), 3.788 (2.12), 3.806 (1.10), 4.893 (2.14), 4.905 (2.04), 7.648 (1.72), 7.670 (3.50), 7.710 (4.03), 7.732 (1.68), 8.640 (0.98), 8.653 (0.92), 8.703 (1.80), 8.766 (1.48).
Example 23
N-(4-{1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide trifluoroacetate
Figure imgf000393_0001
 
To a solution of triphenylphosphine (1.14 g, 4.35 mmol) and phthalimide (CAS-No. 85-41-6, 320 mg, 2.17 mmol) in THF (20 mL) was added at 0°C N-{4-[1-(2-hydroxyethyl)-4-methyl-6- oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carbox- amide (950 mg, 91 % purity, 2.17 mmol) and thereupon diisopropyl azodicarboxylate (860 µl, 4.3 mmol). After warming to r.t. the reaction mixture was stirred for 14 h and then concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, hexane/ethyl acetate gradient) to give the title compound (1.07 g, 81% purity, 63% yield). 100 mg of the product were further purified by preparative HPLC to give the pure title compound (59 mg, 4% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x20 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-8 min 10-50% B. rate 60 ml/min, temperature 25°C. LC-MS (Method 1): Rt = 0.78 min; MS (ESIpos): m/z = 523 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.978 (15.90), 0.996 (16.00), 2.083 (2.58), 2.221 (3.54), 2.260 (4.34), 2.322 (1.14), 2.326 (1.50), 2.331 (1.14), 2.518 (5.37), 2.523 (5.26), 2.541 (3.43), 2.565 (2.50), 2.582 (2.09), 2.664 (1.11), 2.668 (1.52), 2.673 (1.08), 3.105 (1.50), 3.117 (1.08), 3.299 (1.86), 3.314 (2.61), 3.331 (1.78), 3.350 (0.54), 3.741 (2.04), 3.753 (2.27), 3.766 (1.86), 3.838 (1.16), 3.853 (1.03), 3.861 (2.14), 3.873 (4.23), 3.889 (4.34), 3.896 (3.17), 3.906 (2.81), 3.931 (1.06), 3.941 (0.77), 3.986 (0.93), 4.011 (3.10), 4.022 (5.24), 4.035 (4.65), 4.048 (2.04), 4.072 (0.70), 4.888 (10.37), 4.910 (10.22), 7.395 (8.21), 7.400 (3.15), 7.417 (14.45), 7.466 (16.00), 7.471 (4.26), 7.488 (8.18), 7.740 (4.26), 7.753 (4.36), 7.773 (0.85), 7.782 (7.23), 7.790 (7.10), 7.795 (6.30), 7.797 (6.43), 7.804 (12.85), 7.813 (3.05), 7.821 (3.02), 7.831 (14.09), 7.838 (7.35), 7.840 (5.63), 7.845 (7.33), 7.852 (6.25), 7.861 (0.88), 8.648 (9.57), 8.665 (5.24), 8.679 (4.88), 8.790 (7.90).
Example 24
N-{4-[1-(2-aminoethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
 
Figure imgf000395_0001
A mixture of N-(4-{1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (970 mg, 81 % purity, 1.50 mmol), hydrazine monohydrate (290 µl, 6.0 mmol) and THF (5 mL) was stirred at r.t. for 14 h. After that the mixture was diluted with water and washed with ethyl acetate. The aqueous phase was concentrated under reduced pressure to give the title compound (630 mg, 89% purity, 95% yield).
LC-MS (Method 2): Rt = 0.72 min; MS (ESIpos): m/z = 393 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.064 (1.49), 1.082 (1.41), 1.739 (16.00), 1.858 (2.96), 1.909 (0.56), 2.518 (0.95), 2.523 (0.69), 2.767 (0.50), 2.784 (0.99), 2.801 (0.50), 3.688 (0.41), 4.817 (0.84), 4.834 (0.85), 7.434 (0.45), 7.445 (0.46), 7.660 (0.75), 7.683 (1.42), 7.728 (1.42), 7.750 (0.70), 7.841 (1.24), 7.849 (1.12), 7.856 (1.11), 7.864 (1.47), 8.052 (1.35), 8.060 (1.01), 8.066 (1.11), 8.074 (1.12), 8.498 (0.72), 8.511 (0.67), 8.613 (0.91), 8.652 (0.76).
For further purification the product was taken up in water and diluted with saturated aqueous sodium bicarbonate solution and washed again with ethyl acetate. Then the aqueous phase was extracted six times with dichloromethane/isopropanol (4:1), and the combined organic extracts were washed with brine and filtered through a silicone filter. After concentration of the filtrate under reduced pressure the crude product was purified preparative HPLC. The product containing fractions were taken up in water, extracted five times with dichloromethane/isopropanol (4:1), and the combined organic extracts were filtered through silicone filter and concentrated under redcued pressure. The crude product was again purified by preparative HPLC and the product containing fractions were dried under vacuum via a Kugelrohr apparatus and the residue was purified by preparative HPLC to give the title compound (59.0 mg, 10% yield).
1./2. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: XBridge C185µM 100x30 mm. Eluent A: water + 0.1% ammonia; Eluent B: acetonitrile; gradient: 0-20 min 10-50% B. rate 60 ml/min, temperature 25°C.  
3. HPLC: Instrument: Waters Autopurification system; column: Waters XBrigde C18 5µ 100x30mm; eluent A: water + 0.2 Vol-% ammonia (32%), eluent B: methanol; gradient: 0.00– 0.50 min 32% B (25->70mL/min), 0.51–5.50 min 32-51% B (70mL/min), DAD scan: 210-400 nm. LC-MS (method 3): Rt = 1.02 min; MS (ESIpos): m/z = 393 [M+H]+
Example 25
N-{4-[1-(2-hydroxyethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000396_0001
To a solution of N-{4-[1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (1.72 g, 75 % purity, 2.54 mmol) in THF (50 mL) was added at r.t. tetra-n-butylammonium fluoride (1.0 M in THF, 2.5 ml, 2.5 mmol). After stirring for 1 h at that temperature more tetra-n- butylammonium fluoride (1.0 M in THF, 1.25 ml, 1.25 mmol) was added the mixture stirred for further 4 h. After that the mixture was diluted with water, extracted with ethyl acetate (3x) and the combined organic extracts were washed with saturated aqueous sodium bicarbonate solution and filtered through a silicone filter. After removal of the solvent under reduced pressure the crude product was purified by column chromatography (SiO2, ethyl acetate/ethanol gradient) to give the title compound (950 mg, 91% purity, 86% yield).
LC-MS (Method 1): Rt = 0.57 min; MS (ESIpos): m/z = 394 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.064 (14.99), 1.081 (14.81), 1.154 (4.34), 1.171 (8.83), 1.189 (4.30), 1.987 (16.00), 2.113 (1.70), 2.273 (3.02), 2.276 (3.29), 2.314 (3.93), 2.318 (3.74), 2.327 (1.04), 2.332 (0.73), 2.518 (2.81), 2.523 (1.77), 2.669 (0.98), 2.673 (1.05), 2.679 (2.53), 2.696 (2.91), 2.720 (2.34), 2.737 (2.11), 3.361 (1.71), 3.378 (2.34), 3.392 (1.61), 3.573 (1.46), 3.579 (1.71), 3.590 (4.01), 3.594 (3.99), 3.607 (5.14), 3.622 (2.98), 3.663 (1.75), 3.676 (1.77), 3.680 (1.31), 3.694 (2.71), 3.709 (2.59), 3.726 (1.43), 3.892 (1.57), 3.908 (3.20), 3.924  
(2.71), 3.940 (2.56), 3.957 (0.99), 3.998 (1.17), 4.016 (3.39), 4.034 (3.33), 4.052 (1.10), 4.671 (4.16), 4.685 (9.46), 4.699 (3.88), 4.816 (8.71), 4.834 (8.68), 7.431 (4.62), 7.433 (4.69), 7.446 (4.87), 7.654 (1.19), 7.660 (8.10), 7.665 (2.99), 7.677 (4.12), 7.683 (14.47), 7.688 (2.71), 7.722 (2.50), 7.728 (14.42), 7.734 (3.90), 7.746 (2.98), 7.751 (7.23), 7.756 (1.22), 8.497 (7.22), 8.510 (6.83), 8.613 (9.19), 8.614 (9.40), 8.643 (8.23).
Example 26
N-(4-{1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-5,5-dimethyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000397_0001
To a solution of 4-nitrophenyl (4-{1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-5,5- dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)carbamate (310 mg, 90 % purity, 502 µmol) in dichloromethane (5 mL) was added at r.t. N,N-diisopropylethylamine (270 µl, 1.6 mmol) and 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine dihydrochloride (CAS-No. 6000-50-6 , 97.0 mg, 502 µmol). After stirring for 20 h at that temperature more N,N-diisopropylethylamine (1.5 mL) was added an the mixture was diluted with THF (25 mL) and heated to 60°C for 1 h. After cooling to r.t. the mixture was dilutd with water, basified by addition of 1N aqueous sodium hydroxide solution and extracte with dichloromethane (2x). The combined organic extracts were washed with brine and filtered through a silicone filter. After removel of the solvent the crude product was purified by column chromatography (SiO2, hexane/ethyl acetate gradient) to give tihe title compound (183 mg, 66% yield).
LC-MS (Method 1): Rt = 0.90 min; MS (ESIpos): m/z = 537 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.974 (16.00), 1.137 (0.67), 1.154 (1.76), 1.172 (3.66), 1.190 (1.78), 1.988 (6.18), 2.323 (0.61), 2.327 (0.87), 2.332 (0.62), 2.518 (2.92), 2.523 (1.97), 2.665 (0.64), 2.669 (0.90), 2.673 (0.64), 2.724 (4.21), 3.868 (0.81), 3.879 (1.30), 3.886 (1.13),  
3.894 (1.23), 3.992 (1.11), 3.999 (1.46), 4.008 (1.24), 4.017 (2.04), 4.034 (1.33), 4.053 (0.42), 4.805 (2.11), 4.823 (2.11), 7.377 (2.21), 7.382 (0.84), 7.395 (1.01), 7.400 (3.37), 7.432 (1.11), 7.447 (1.20), 7.477 (3.67), 7.482 (1.01), 7.494 (0.80), 7.499 (2.26), 7.777 (1.68), 7.785 (1.62), 7.790 (1.40), 7.792 (1.50), 7.799 (3.14), 7.808 (0.81), 7.814 (0.71), 7.825 (3.46), 7.832 (1.85), 7.834 (1.39), 7.839 (1.75), 7.846 (1.69), 8.499 (1.69), 8.512 (1.62), 8.580 (2.16), 8.613 (2.24), 8.615 (2.27).
Example 27
N-{4-[1-(2-aminoethyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000398_0001
A mixture of N-(4-{1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-5,5-dimethyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (143 mg, 266 µmol), hydrazine monohydrate (14 µL, 290 µmol) and THF (2 mL) was stirred at r.t. for 14 h. After that the mixture was diluted with water and extracted with ethyl acetate (3x). The combined organic extracts were washed with brine and filtered through a silicone filter. After removal of the solvent under reduced pressure the crude product was purified by preparative HPLC to give the title compound (64.0 mg, 58% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: XBridge C185µM 100x30 mm. Eluent A: water + 0.1% ammonia; Eluent B: acetonitrile; gradient: 0-20 min 15-55% B. rate 60 ml/min, temperature 25°C.
LC-MS (Method 2): Rt = 0.78 min; MS (ESIpos): m/z = 407 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.082 (16.00), 2.322 (0.56), 2.327 (0.80), 2.332 (0.58), 2.518 (2.51), 2.523 (1.71), 2.664 (0.54), 2.669 (0.81), 2.673 (0.58), 2.743 (0.85), 2.760 (1.57), 2.777 (0.91), 2.847 (4.09), 3.691 (0.93), 3.708 (1.54), 3.725 (0.87), 4.815 (1.91), 4.832 (1.91),  
7.433 (1.04), 7.435 (1.06), 7.447 (1.08), 7.651 (1.49), 7.657 (0.58), 7.668 (0.84), 7.674 (3.77), 7.679 (0.76), 7.696 (0.77), 7.701 (3.69), 7.707 (0.83), 7.719 (0.53), 7.724 (1.34), 8.498 (1.65), 8.511 (1.54), 8.614 (2.15), 8.641 (1.95).
-----Final Intermediates--- Intermediate 70
N-{4-[1-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)-5,5-dimethyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000399_0001
To a solution of N-{4-[1-(2-aminoethyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (50.0 mg, 123 µmol) in DMF (5.3 mL) was added at r.t.1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione (CAS-No. 55750-61-3, 31.0 mg, 123 µmol) and N,N-diisopropylethylamine (43 µl, 250 µmol). After stirring at that temperature for 14 h the mixture was purified by preparative HPLC to give the title compound (66.2 mg, 96%).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-8 min 10-50% B. rate 150 ml/min, temperature 25°C. LC-MS (Method 1): Rt = 0.68 min; MS (ESIpos): m/z = 544 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.064 (16.00), 2.296 (1.14), 2.300 (2.51), 2.304 (3.49), 2.309 (2.46), 2.314 (1.09), 2.568 (1.31), 2.638 (1.14), 2.642 (2.57), 2.646 (3.49), 2.651 (2.46), 2.656 (1.09), 2.789 (4.00), 3.304 (1.54), 3.319 (1.60), 3.334 (0.86), 3.567 (2.46), 3.737 (1.54), 3.752 (2.34), 3.768 (1.31), 3.924 (5.94), 4.115 (0.51), 4.857 (4.23), 7.062 (13.49), 7.113 (0.63),  
7.632 (2.40), 7.637 (0.91), 7.649 (1.09), 7.654 (3.83), 7.701 (3.83), 7.707 (0.97), 7.719 (0.74), 7.724 (1.94), 8.179 (1.03), 8.193 (0.51), 8.583 (0.97), 8.596 (0.97), 8.668 (2.00), 8.706 (1.54).
Intermediate 71
N-{4-[1-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000400_0001
To a solution of N-{4-[1-(2-aminoethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (46.7 mg, 119 µmol) in DMF (5.1 mL) was added at r.t.1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione (CAS-No. 55750-61-3, 30.0 mg, 119 µmol) and N,N-diisopropylethylamine (41 µl, 240 µmol). After stirring at that temperature for 14 h the mixture was purified by preparative HPLC to give the title compound (42.0 mg, 65%).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-8 min 10-50% B. rate 150 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.58 min; MS (ESIpos): m/z = 530 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.852 (0.57), 1.044 (4.54), 1.062 (4.60), 1.234 (1.39), 2.280 (1.01), 2.318 (2.33), 2.323 (3.84), 2.327 (4.47), 2.331 (3.02), 2.336 (1.39), 2.518 (15.87), 2.523 (11.15), 2.660 (1.39), 2.665 (3.15), 2.669 (4.60), 2.673 (3.09), 2.678 (1.57), 2.685 (1.01), 2.710 (0.76), 2.728 (0.63), 3.315 (0.63), 3.331 (1.20), 3.347 (1.45), 3.357 (1.51), 3.385 (1.20), 3.399 (1.01), 3.713 (1.89), 3.729 (2.14), 3.745 (2.27), 3.762 (2.39), 3.778 (1.83), 3.806 (1.64), 3.821 (1.95), 3.838 (1.70), 3.855 (1.45), 3.954 (7.24), 4.111 (0.69), 4.862 (1.13), 4.890 (3.15),  
7.077 (0.50), 7.085 (16.00), 7.660 (3.15), 7.683 (4.60), 7.748 (4.35), 7.771 (2.65), 8.208 (0.57), 8.223 (1.13), 8.237 (0.57), 8.623 (1.01), 8.637 (0.94), 8.701 (2.39), 8.748 (1.57).
Intermediate 72
N-{4-[1-(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
)
Figure imgf000401_0001
A mixture of N-{4-[1-(3-aminopropyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}- 1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (25.0 mg, 90 % purity, 55.4 µmol), 1-{6- [(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (CAS-No. 55750-63-5, 17.1 mg, 55.4 µmol), N,N-diisopropylethylamine (14 µl, 83 µmol) and DMF (850 µL) was stirred at r.t. for 14 h. After that the mixture was purified by column chromatography (SiO2, dichloromethane/ethanol gradient) to give the title compound (18.0 mg, 53% yield).
LC-MS (Method 1): Rt = 0.74 min; MS (ESIpos): m/z = 601 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.061 (11.04), 1.078 (10.79), 1.173 (4.84), 1.232 (3.84), 1.467 (8.00), 1.703 (3.66), 1.719 (4.96), 1.738 (3.60), 2.002 (4.40), 2.020 (6.95), 2.038 (3.66), 2.279 (3.22), 2.323 (6.70), 2.590 (3.22), 2.670 (4.78), 2.701 (2.73), 2.725 (2.17), 2.742 (1.86), 3.045 (4.78), 3.059 (4.71), 3.362 (11.60), 3.380 (6.95), 3.606 (1.86), 3.623 (1.98), 3.638 (2.17), 3.847 (2.17), 3.865 (1.98), 3.880 (1.80), 4.815 (9.86), 4.834 (9.74), 6.989 (16.00), 7.434 (3.97), 7.447 (4.03), 7.659 (5.71), 7.681 (8.99), 7.731 (8.99), 7.754 (6.51), 7.771 (3.97), 8.499 (4.40), 8.511 (4.22), 8.615 (7.19), 8.641 (6.33).
Intermediate 73
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{3-[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-  
yl]propyl}-L-alaninamide
Figure imgf000402_0001
To a solution of N-{4-[1-(3-aminopropyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (60.0 mg, 143 µmol) in acetonitrile (5 mL) was added at r.t. N,N-diisopropylethylamine (200 µl, 1.1 mmol) and the mixture stirred for 10 min at that temperature. Then propylphosphonic anhydride (T3P, 170 µl, 570 µmol) was added and the mixture stirred for further 2.5 h. After that the mixture was concentrated under reduced pressure and the residue was purified by column chromatography (SiO2, dichloromethane/isopropanol) to give the title compound (6.00 mg, 5% yield).
LC-MS (Method 1): Rt = 0.81 min; MS (ESIpos): m/z = 770 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.786 (3.12), 0.802 (4.12), 0.814 (4.07), 0.831 (3.76), 0.915 (1.64), 0.933 (3.87), 0.951 (1.95), 1.026 (15.69), 1.041 (16.00), 1.055 (4.50), 1.074 (4.49), 1.137 (3.21), 1.161 (1.38), 1.184 (3.56), 1.187 (3.58), 1.202 (3.36), 1.205 (3.29), 1.230 (1.44), 1.255 (0.71), 1.295 (1.27), 1.315 (1.14), 1.333 (0.78), 1.423 (0.98), 1.442 (1.71), 1.460 (2.02), 1.478 (1.57), 1.492 (0.92), 1.729 (1.86), 1.898 (0.87), 1.942 (0.73), 1.959 (0.70), 2.083 (1.56), 2.090 (0.86), 2.109 (1.02), 2.115 (1.75), 2.125 (0.98), 2.143 (0.98), 2.216 (0.86), 2.277 (1.15), 2.318 (1.41), 2.477 (1.37), 2.683 (0.86), 2.699 (1.17), 2.724 (0.87), 3.074 (0.84), 3.137 (0.89), 3.152 (0.77), 3.180 (0.75), 3.372 (2.73), 3.390 (1.51), 3.410 (1.03), 3.754 (0.74), 3.769 (0.92), 3.784 (0.71), 4.105 (0.64), 4.122 (0.88), 4.126 (0.91), 4.143 (0.72), 4.187 (0.66), 4.205 (0.96), 4.223 (0.64), 4.815 (3.92), 4.833 (3.90), 6.992 (7.57), 7.432 (1.95), 7.445 (1.89), 7.659 (2.57), 7.681 (4.42), 7.728 (3.94), 7.741 (1.27), 7.751 (2.21), 7.789 (0.99), 7.817 (1.41), 7.932 (0.81), 7.950 (0.85), 8.497 (2.34), 8.509 (2.16), 8.613 (3.63), 8.645 (2.74).
 
Intermediate 74
N-{4-[(4S)-1-(2-{4-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]piperazin-1-yl}ethyl)-4- methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine- 2-carboxamide
Figure imgf000403_0001
A mixture of N-(4-{(4S)-4-methyl-6-oxo-1-[2-(piperazin-1-yl)ethyl]-1,4,5,6-tetrahydropyridazin- 3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (93.0 mg, 90 % purity, 181 µmol), 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (55.9 mg, 181 µmol), N,N-diisopropylethylamine (47 µl, 270 µmol) and DMF (2.8 mL) was stirred at r.t. for 14 h. After that the mixture was concentrated under reduced pressure and the crude product was purified by preparative HPLC to give the title compound (48.0 mg, 96% purity, 39% yield). HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x20 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-22 min 10-50% B. rate 50 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.67 min; MS (ESIpos): m/z = 655 [M+H]+
1H-NMR (600 MHz, DMSO-d6) d [ppm]: -0.005 (0.82), 0.006 (1.07), 1.096 (1.97), 1.102 (5.58), 1.114 (4.92), 1.169 (0.57), 1.182 (1.07), 1.195 (1.64), 1.208 (1.15), 1.221 (0.49), 1.416 (0.41), 1.428 (0.90), 1.440 (1.48), 1.452 (1.89), 1.462 (1.89), 1.474 (1.81), 1.487 (1.23), 1.499 (0.41), 1.735 (0.66), 2.224 (1.81), 2.236 (2.63), 2.249 (1.48), 2.289 (1.23), 2.315 (1.48), 2.353 (2.05), 2.382 (2.46), 2.384 (4.02), 2.387 (5.42), 2.390 (4.02), 2.432 (0.98), 2.518 (16.00), 2.521 (14.85), 2.524 (11.73), 2.555 (1.89), 2.566 (2.22), 2.612 (3.53), 2.615 (5.09), 2.619 (3.69), 2.682 (0.98), 2.693 (1.23), 2.709 (1.15), 2.720 (0.82), 3.014 (0.49), 3.021 (0.49), 3.287 (0.41),  
3.307 (1.56), 3.357 (3.36), 3.369 (4.84), 3.381 (2.87), 3.388 (1.48), 3.399 (0.90), 3.636 (0.49), 3.646 (0.82), 3.658 (0.82), 3.669 (0.82), 4.095 (0.49), 4.106 (0.90), 4.117 (0.82), 4.129 (0.74), 4.817 (2.87), 4.837 (2.79), 6.997 (12.39), 7.435 (1.97), 7.443 (1.56), 7.662 (3.36), 7.677 (4.43), 7.713 (0.57), 7.720 (0.66), 7.737 (4.51), 7.752 (2.71), 8.314 (9.27), 8.496 (0.90), 8.501 (2.63), 8.510 (2.05), 8.616 (3.20), 8.630 (1.07), 8.639 (2.87).
Intermediate 75
N-(4-{1-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,19-dioxo-7,10,13,16-tetraoxa-4,20- diazatricosan-23-yl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000404_0001
To a solution of N-{4-[1-(3-aminopropyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (60.0 mg, 143 µmol) and 19- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oic acid (CAS-No. 1263045-16-4, 131 mg, 315 µmol) in acetonitrile (5 mL) was added at r.t. N,N- diisopropylethylamine (200 µl, 1.1 mmol). After stirring at r.t. for 10 min propylphosphonic anhydride (T3P, 170 µl, 570 µmol) was added and the mixture stirred for further 14 h at that temperature. After that the mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC to give the title compound (19.0 mg, 95% purity, 16% yield). HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x20 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-22 min 10-50% B. rate 50 ml/min, temperature 25°C.  
LC-MS (Method 1): Rt = 0.70 min; MS (ESIpos): m/z = 805 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.061 (2.29), 1.080 (2.79), 1.098 (0.78), 1.109 (0.43), 1.232 (1.09), 1.715 (0.57), 1.732 (0.84), 1.751 (0.57), 2.277 (1.23), 2.293 (2.03), 2.299 (1.52), 2.309 (1.45), 2.318 (2.21), 2.322 (2.01), 2.327 (1.84), 2.332 (1.43), 2.336 (1.39), 2.518 (4.92), 2.523 (3.26), 2.665 (0.90), 2.669 (1.27), 2.673 (0.96), 2.679 (0.55), 2.701 (0.43), 2.724 (0.43), 3.067 (0.78), 3.085 (0.74), 3.126 (1.23), 3.141 (1.52), 3.155 (0.86), 3.361 (2.72), 3.411 (1.11), 3.463 (4.36), 3.473 (10.71), 3.478 (16.00), 3.490 (4.69), 3.563 (1.84), 3.581 (3.67), 3.599 (2.05), 3.617 (0.51), 3.635 (0.53), 3.651 (0.59), 3.853 (0.45), 4.817 (1.93), 4.834 (1.95), 5.758 (0.82), 6.994 (1.11), 6.998 (6.08), 7.434 (1.02), 7.447 (0.98), 7.660 (1.29), 7.667 (0.88), 7.683 (2.31), 7.689 (1.11), 7.732 (2.09), 7.754 (1.31), 7.850 (0.59), 8.020 (0.55), 8.498 (1.39), 8.511 (1.27), 8.614 (1.93), 8.644 (1.31).
Intermediate 76
N-(4-{1-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19- diazadocosan-22-yl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carboxamide
)
Figure imgf000405_0001
To a solution of N-{4-[1-(3-aminopropyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (60.0 mg, 97 % purity, 143 µmol) and 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan- 18-oic acid (127 mg, 315 µmol) in acetonitrile (5 mL) was added at r.t. N,N- diisopropylethylamine (200 µl, 1.1 mmol). After stirring at r.t. for 10 min propylphosphonic anhydride (T3P, 170 µl, 570 µmol) was added and the mixture stirred for further 14 h at that  
temperature. After that the mixture was concentrated under reduced pressure and the residue was purified by preparative to give the title compound (7.1 mg, 95% purity, 6% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x20 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-22 min 10-50% B. rate 50 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.66 min; MS (ESIpos): m/z = 791 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.916 (0.73), 1.061 (3.73), 1.079 (3.53), 1.231 (0.81), 1.482 (0.58), 1.715 (1.13), 1.733 (1.54), 1.750 (1.09), 2.279 (2.19), 2.295 (2.72), 2.311 (1.65), 2.322 (1.56), 2.331 (0.69), 2.523 (2.35), 2.665 (0.56), 2.669 (0.73), 2.674 (0.65), 2.683 (0.70), 2.700 (0.76), 2.725 (0.66), 2.742 (0.58), 3.051 (0.75), 3.067 (1.43), 3.085 (1.36), 3.102 (0.67), 3.166 (0.56), 3.172 (0.73), 3.186 (1.64), 3.200 (1.84), 3.214 (1.00), 3.375 (2.76), 3.389 (3.96), 3.404 (2.64), 3.477 (10.77), 3.485 (16.00), 3.493 (12.31), 3.568 (2.09), 3.583 (3.29), 3.600 (1.91), 3.618 (0.93), 3.634 (0.92), 3.651 (0.98), 3.835 (0.50), 3.853 (0.83), 3.870 (0.74), 3.886 (0.70), 4.009 (4.36), 4.816 (2.95), 4.833 (2.89), 7.085 (6.16), 7.433 (1.29), 7.445 (1.18), 7.662 (1.91), 7.684 (3.19), 7.732 (3.04), 7.754 (1.72), 7.840 (0.67), 7.853 (1.03), 7.867 (0.56), 8.234 (0.65), 8.498 (1.09), 8.510 (0.99), 8.613 (1.57), 8.648 (1.89).
Intermediate 77
N-{4-[1-(2-{4-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]piperazin-1-yl}ethyl)-4-methyl-6- oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide
 
I
z
0
0
0
A mixture of N-(4-{4-methyl-6-oxo-1-[2-(piperazin-1-yl)ethyl]-1,4,5,6-tetrahydropyridazin-3- yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (40 mg, 82 µmol), 1-{2-[(2,5- dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione (CAS-No. 55750-61-3, 23 mg, 91 µmol) and N,N-diisopropylethylamine (29 µl, 165 µmol) in DMF (1.3 mL) was stirred at r.t. for 14 h. After that the mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC to give the title compound (12.0 mg, 24% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x20 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-22 min 10-50% B. rate 50 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.55 min; MS (ESIpos): m/z = 599 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.852 (0.39), 1.090 (0.65), 1.105 (4.34), 1.123 (4.02), 1.233 (1.00), 2.287 (0.97), 2.318 (1.00), 2.323 (2.19), 2.327 (3.19), 2.332 (2.01), 2.336 (0.83), 2.390 (1.69), 2.403 (1.22), 2.419 (0.68), 2.434 (0.79), 2.446 (0.97), 2.463 (1.18), 2.518 (7.89), 2.523 (5.74), 2.573 (0.90), 2.590 (4.84), 2.606 (0.93), 2.660 (0.79), 2.665 (1.72), 2.669 (2.33), 2.673 (1.69), 2.679 (1.04), 2.699 (0.90), 2.724 (0.72), 2.741 (0.54), 3.378 (0.90), 3.393 (1.04), 3.412 (1.33), 3.425 (1.43), 3.655 (0.61), 3.671 (0.54), 3.688 (0.65), 4.105 (0.72), 4.123 (0.61), 4.140 (0.57), 4.324 (5.56), 4.817 (2.80), 4.836 (2.83), 7.106 (16.00), 7.114 (0.50), 7.433 (1.54), 7.446 (1.54), 7.660 (2.76), 7.678 (1.69), 7.683 (4.34), 7.726 (0.57), 7.742 (4.20), 7.764 (2.37), 8.139 (2.22), 8.499 (2.04), 8.511 (1.90), 8.614 (2.91), 8.641 (2.65).
 
Intermediate 78
N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]propyl}-N2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L- asparagine
Figure imgf000408_0001
Under an atmosphere of argon N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-L-asparagine hydrochloride (1:1) (415 mg, 67 % purity, 498 µmol) was dissolved in DMF (25 ml, 330 mmol). 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione, Cas No 55750-63-5 (276 mg, 897 µmol) and N,N-diisopropylethylamine (430 µl, 2.5 mmol) were added and the mixture was stirred at romm temperature overnight. The mixture was concentrated under reduced pressure and purified with preparative HPLC to give the titel compound (24 mg, 6% yield, 90% purity).
Instrument: Gilson Abimed 305 pump, KNAUER UV detector K-2501, Varian fraction collector model 701A, Prepcon 5 software. Column: Chromatorex C18, 10µm, 125x30 mm. Eluent A: water + 0.1 Vol-% TFA; Eluent B: acetonitrile; gradient: 0-22 min 10-80% B; rate 50 ml/min; temperature 25°C.
LC-MS (METHOD 6): Rt = 0.91 min; MS (ESIneg): m/z = 713 [M-H]-  
1H-NMR (400 MHz, DMSO-d6) d [ppm]: -0.157 (0.66), 0.138 (0.57), 0.758 (0.60), 0.994 (1.17), 1.010 (1.31), 1.055 (9.04), 1.073 (9.10), 1.139 (2.68), 1.157 (3.76), 1.175 (3.85), 1.198 (2.25), 1.220 (1.83), 1.238 (4.42), 1.253 (5.33), 1.268 (3.02), 1.299 (0.77), 1.319 (0.74), 1.347 (0.83), 1.438 (5.56), 1.455 (4.91), 1.616 (0.71), 1.709 (2.48), 1.726 (3.48), 1.744 (2.51), 1.761 (0.91), 2.033 (2.85), 2.052 (4.96), 2.070 (2.65), 2.148 (0.74), 2.166 (1.23), 2.185 (0.66), 2.277 (2.37), 2.318 (2.99), 2.359 (1.37), 2.406 (1.31), 2.424 (1.45), 2.443 (2.51), 2.561 (1.28), 2.585 (1.54), 2.603 (3.08), 2.623 (0.80), 2.642 (3.17), 2.650 (3.79), 2.672 (1.63), 2.690 (1.80), 2.702 (1.54), 2.714 (1.51), 2.731 (1.45), 2.763 (2.48), 2.780 (1.45), 2.800 (4.53), 2.924 (1.17), 3.044 (1.68), 3.060 (3.31), 3.077 (3.17), 3.094 (1.57), 3.119 (0.66), 3.129 (0.71), 3.137 (0.54), 3.148 (0.54), 3.157 (0.54), 3.317 (4.16), 3.335 (6.87), 3.353 (4.76), 3.369 (3.22), 3.386 (2.57), 3.402 (1.37), 3.608 (1.34), 3.617 (1.34), 3.625 (1.40), 3.633 (1.40), 3.642 (1.40), 3.650 (1.40), 3.841 (1.37), 3.850 (1.25), 3.867 (1.14), 3.875 (1.11), 4.440 (1.43), 4.459 (2.45), 4.473 (2.48), 4.492 (1.40), 4.898 (7.50), 4.917 (6.84), 5.066 (0.60), 5.078 (0.51), 5.746 (6.05), 6.971 (16.00), 6.995 (3.11), 7.644 (4.85), 7.666 (7.87), 7.728 (8.30), 7.750 (6.67), 7.844 (1.37), 7.858 (2.62), 7.871 (1.34), 7.945 (2.60), 7.965 (2.51), 8.663 (1.60), 8.701 (5.36), 8.788 (1.97).
Intermediate 79
N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]amino}-4-oxobutyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000409_0001
Under an atmosphere of argon 4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butanoic acid (25.3 mg, 58.0 µmol), (36.1 mg, 81.6 µmol) and N,N-diisopropylethylamine (40 µl, 230 µmol) were dissolved in DMF (1.8 ml).1-(6-aminohexyl)-1H-pyrrole-2,5-dione hydrochloride (1:1), Cas No 75238-09-4 (14.8 mg, 63.8 µmol) and HATU (33.1 mg, 87.0 µmol) were added and the mixture
 
was stirred at room temperature for 2.5 h. Then the mixture was purified by preparative HPLC (ACN/water + 0.1% TFA, gradient) to give the title compound (15.5 mg, 44% yield).
Instrument: Gilson Abimed 305 pump, KNAUER UV detector K-2501, Varian fraction collector model 701A, Prepcon 5 software. Column: Chromatorex C18, 10µm, 125x30 mm. Eluent A: water + 0.1 Vol-% TFA; Eluent B: acetonitrile; gradient: 0-22 min 5-35% B; rate 50 ml/min; temperature 25°C.
LC-MS (METHOD 6): Rt = 1.12 min; MS (ESIneg): m/z = 612 [M-H]- ¹H-NMR (500 MHz, DMSO-d6) d [ppm]: -0.007 (1.25), 1.063 (8.65), 1.077 (8.64), 1.153 (1.07), 1.168 (2.38), 1.182 (2.09), 1.213 (2.47), 1.236 (3.00), 1.317 (1.15), 1.331 (2.31), 1.345 (2.69), 1.360 (1.67), 1.445 (2.28), 1.459 (3.04), 1.474 (2.27), 1.488 (1.11), 1.803 (1.93), 1.817 (2.79), 1.833 (2.14), 1.847 (0.84), 2.066 (2.74), 2.082 (3.84), 2.096 (2.14), 2.283 (2.06), 2.315 (2.46), 2.332 (0.81), 2.362 (0.79), 2.636 (0.63), 2.688 (1.26), 2.701 (1.65), 2.721 (1.33), 2.734 (1.25), 2.888 (1.64), 2.972 (1.49), 2.986 (3.31), 2.997 (3.30), 3.011 (1.52), 3.169 (5.51), 3.352 (3.41), 3.366 (5.89), 3.380 (3.81), 3.387 (2.26), 3.402 (1.52), 3.610 (0.96), 3.624 (1.60), 3.637 (1.72), 3.651 (1.91), 3.665 (1.17), 3.793 (1.52), 3.807 (2.32), 3.820 (2.20), 3.834 (2.26), 3.848 (1.63), 3.965 (1.54), 4.038 (1.89), 4.052 (1.81), 4.896 (7.01), 6.985 (16.00), 7.011 (0.68), 7.658 (4.73), 7.676 (6.98), 7.691 (1.86), 7.732 (7.74), 7.750 (5.32), 8.696 (4.70), 8.773 (0.81).
Intermediate 80
N-{4-[1-{4-[(3-{[3-({3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3- oxopropyl}disulfanyl)propanoyl]amino}propyl)carbamoyl]benzyl}-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
 
Figure imgf000411_0001
A mixture of N-{4-[1-{4-[(3-aminopropyl)carbamoyl]benzyl}-4-methyl-6-oxo-1,4,5,6-tetrahydro- pyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (35.0 mg, 64.9 µmol), 1,1'-{disulfanediylbis[(1-oxopropane-3,1-diyl)oxy]}dipyrrolidine-2,5-dione (CAS-No. 57757-57-0, 31.5 mg, 77.8 µmol) and N,N-diisopropylethylamine (23 µl, 130 µmol) in DMF (1 mL) was stirred at r.t. for 2 h. After that the mixture was purified by preparative HPLC to give the title compound (20.0 mg, 93% purity, 35% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x20 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-22 min 15-55% B. rate 50 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.76 min; MS (ESIpos): m/z = 829 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.037 (4.55), 1.044 (5.49), 1.056 (4.95), 1.062 (5.22), 1.231 (0.67), 1.599 (1.27), 1.616 (2.01), 1.627 (2.21), 1.645 (1.41), 1.662 (0.40), 2.318 (1.27), 2.323 (3.01), 2.327 (4.22), 2.331 (3.01), 2.337 (1.41), 2.349 (1.34), 2.394 (1.67), 2.435 (1.67), 2.453 (5.29), 2.470 (7.90), 2.518 (16.00), 2.523 (11.31), 2.591 (2.88), 2.660 (1.41), 2.665 (3.01), 2.669 (4.15), 2.674 (2.88), 2.678 (1.27), 2.800 (6.56), 2.815 (3.01), 2.832 (1.00), 2.840 (1.14), 2.857 (2.41), 2.874 (3.28), 2.893 (1.67), 2.899 (2.14), 2.916 (3.82), 2.934 (1.61), 2.968   (1.00), 2.971 (1.00), 2.987 (3.35), 3.002 (2.08), 3.033 (0.87), 3.050 (0.74), 3.058 (0.87), 3.074 (4.02), 3.089 (6.09), 3.106 (2.74), 3.114 (1.41), 3.119 (1.07), 3.236 (2.28), 3.250 (2.08), 3.307 (0.74), 3.436 (1.00), 4.804 (4.82), 4.824 (4.82), 4.859 (1.34), 4.866 (1.41), 4.897 (1.67), 4.904 (1.87), 5.070 (1.87), 5.102 (1.27), 5.108 (1.34), 7.357 (3.41), 7.363 (4.02), 7.378 (4.08), 7.384 (3.95), 7.419 (1.54), 7.429 (2.14), 7.441 (1.74), 7.638 (2.74), 7.641 (3.41), 7.660 (6.23), 7.664 (6.16), 7.690 (1.21), 7.695 (5.42), 7.701 (6.09), 7.706 (1.61), 7.718 (2.81), 7.724 (2.48), 7.773 (4.28), 7.778 (4.95), 7.783 (1.67), 7.794 (4.22), 7.799 (3.95), 7.955 (1.00), 7.969 (1.74), 7.980 (0.94), 8.384 (1.21), 8.398 (2.48), 8.411 (1.14), 8.489 (2.41), 8.494 (2.74), 8.501 (2.34), 8.507 (2.48), 8.604 (3.55), 8.607 (3.82), 8.633 (3.21), 8.639 (3.28).
Intermediate 81
N-{4-[1-{4-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)hexanoyl]amino}propyl)carbamoyl]benzyl}-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000412_0001
A mixture of N-{4-[1-{4-[(3-aminopropyl)carbamoyl]benzyl}-4-methyl-6-oxo-1,4,5,6-tetrahydro- pyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (35.0 mg, 64.9 µmol), 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (CAS-No. 55750-  
63-5, 24.0 mg, 77.8 µmol) and N,N-diisopropylethylamine (23 µl, 130 µmol) in DMF (1 mL) was stirred at r.t. for 2 h. After the the mixture was purified by preparative to give the title compound (25.0 mg, 94% purity, 49% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x20 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-22 min 15-55% B. rate 50 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.80 min; MS (ESIpos): m/z = 733 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.043 (4.09), 1.061 (4.12), 1.161 (1.30), 1.180 (0.92), 1.439 (1.36), 1.447 (1.48), 1.458 (1.84), 1.467 (1.81), 1.476 (1.36), 1.485 (1.16), 1.581 (1.07), 1.598 (1.60), 1.616 (1.16), 2.001 (1.51), 2.019 (2.61), 2.038 (1.36), 2.323 (1.30), 2.327 (1.84), 2.331 (1.33), 2.356 (0.95), 2.394 (1.10), 2.518 (7.70), 2.523 (5.39), 2.665 (1.33), 2.669 (1.84), 2.673 (1.27), 2.815 (0.77), 3.051 (1.54), 3.067 (1.57), 3.216 (1.48), 3.231 (1.45), 3.341 (2.46), 3.359 (3.35), 3.377 (1.72), 4.808 (2.61), 4.825 (2.61), 4.865 (1.16), 4.903 (1.45), 5.071 (1.36), 5.109 (1.04), 6.985 (16.00), 7.363 (2.84), 7.380 (1.36), 7.384 (3.08), 7.426 (1.39), 7.441 (1.48), 7.640 (2.22), 7.645 (0.95), 7.657 (1.19), 7.662 (4.39), 7.699 (4.41), 7.705 (1.21), 7.717 (0.83), 7.722 (2.04), 7.774 (1.01), 7.778 (4.09), 7.783 (2.28), 7.795 (1.69), 7.799 (3.64), 8.401 (1.24), 8.494 (2.19), 8.507 (2.04), 8.608 (2.87), 8.637 (2.55).
Intermediate 82
N-{4-[1-{4-[(3-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}propyl)carbamoyl]benzyl}- 4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4- c]pyridine-2-carboxamide
 
Figure imgf000414_0001
A mixture of N-{4-[1-{4-[(3-aminopropyl)carbamoyl]benzyl}-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (200 mg, 371 µmol), 1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H-pyrrole-2,5-dione (CAS-No. 55750-61-3, 140 mg, 556 µmol) and N,N-diisopropylethylamine (130 µl, 740 µmol) in DMF (5.7 mL) was stirred ar r.t for 14 h. After that the mixture was concentrated under reduced pressure and purified by column chromatography (SiO2, dichloromethane/methanol gradient) and preparative HPLC to give the title compound (10.0 mg, 80% purity, 3% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x30 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-8 min 10-50% B. rate 150 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.71 min; MS (ESIpos): m/z = 677 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.852 (0.69), 1.042 (0.76), 1.061 (0.69), 1.232 (2.77), 2.318 (1.26), 2.323 (2.77), 2.327 (3.84), 2.332 (2.65), 2.337 (1.26), 2.518 (16.00), 2.523 (11.02), 2.540 (1.07), 2.660 (1.26), 2.665 (2.83), 2.669 (3.84), 2.674 (2.71), 2.678 (1.20), 4.000 (1.20), 4.809 (0.44), 7.089 (2.39), 7.361 (0.57), 7.382 (0.50), 7.663 (0.76), 7.700 (0.69), 7.773 (0.63), 7.794 (0.57).
 
Intermediate 83
N-(4-{1-[4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000415_0001
A mixture of N-{4-[1-(4-aminobutyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}- 1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (535 mg, 90% purity, 1.14 mmol), 1,1'- [(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione (CAS-No. 79642-50-5, 448 mg, 1.37 mmol) and N,N-diisopropylethylamine (399 µl, 2.29 mol) in DMF (18 mL) was stirred at r.t. for 14 h. After that the mixture was concentrated under reduced pressure and the residue was purified by column chromatography (SiO2, dichloromethane/isopropanol gradient) and prepararative HPLC to give the title compound (48.0 mg, 96% purity, 6% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x20 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-22 min 10-50% B. rate 50 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.71 min; MS (ESIpos): m/z = 630 [M+H]- 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.053 (9.41), 1.071 (9.51), 1.376 (1.56), 1.394 (2.37), 1.413 (1.90), 1.431 (0.76), 1.567 (0.78), 1.584 (1.98), 1.603 (2.35), 1.621 (1.62), 1.638 (0.70), 1.656 (0.81), 1.675 (1.20), 1.693 (0.91), 1.769 (0.63), 1.788 (2.35), 1.807 (3.62), 1.825 (2.61),   1.844 (0.78), 2.037 (1.02), 2.056 (1.56), 2.074 (3.99), 2.124 (0.94), 2.142 (4.20), 2.160 (5.60), 2.178 (2.45), 2.278 (2.37), 2.318 (3.13), 2.327 (1.95), 2.331 (1.30), 2.336 (0.63), 2.518 (8.23), 2.523 (5.68), 2.540 (0.78), 2.584 (16.00), 2.639 (3.34), 2.658 (6.31), 2.665 (1.95), 2.669 (2.24), 2.677 (4.59), 2.695 (2.08), 2.720 (1.64), 2.737 (1.43), 2.804 (11.99), 3.028 (0.47), 3.043 (1.95), 3.060 (3.75), 3.074 (3.07), 3.091 (1.12), 3.368 (1.49), 3.386 (1.82), 3.404 (1.25), 3.564 (0.57), 3.584 (0.63), 3.601 (1.43), 3.618 (1.54), 3.634 (1.82), 3.651 (0.76), 3.831 (0.73), 3.848 (1.62), 3.865 (1.36), 3.882 (1.30), 3.899 (0.57), 4.818 (5.99), 4.836 (6.12), 7.433 (3.41), 7.445 (3.57), 7.657 (4.64), 7.663 (2.87), 7.680 (8.65), 7.686 (3.88), 7.730 (8.94), 7.752 (4.59), 7.821 (0.68), 7.839 (1.17), 7.853 (2.11), 7.867 (1.02), 8.498 (4.72), 8.511 (4.48), 8.614 (6.78), 8.640 (4.79), 8.691 (0.89).
Intermediate 84
N-{4-[1-(4-{[3-({3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3- oxopropyl}disulfanyl)propanoyl]amino}butyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000416_0001
A mixture of N-{4-[1-(4-aminobutyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}- 1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (85.0 mg, 90% purity, 182 µmol), 1,1'- {disulfanediylbis[(1-oxopropane-3,1-diyl)oxy]}dipyrrolidine-2,5-dione (CAS-No. 57757-57-0, 110 mg, 273 µmol) and N,N-diisopropylethylamine (48 µl, 273 µmol) in DMF (2.8 mL) was  
stirred at r.t. for 14 h. After that the mixture was filtered and the filtrate was purified by preparative HPLC to give the title compound (18.0 mg, 95% purity, 13% yield).
1. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-8 min 15-55% B. rate 150 ml/min, temperature 25°C.
2. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x20 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-22 min 10-50% B. rate 50 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.82 min; MS (ESIpos): m/z = 710 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.852 (0.43), 1.054 (7.94), 1.073 (8.00), 1.233 (1.53), 1.381 (1.34), 1.399 (1.95), 1.418 (1.65), 1.437 (0.79), 1.575 (0.73), 1.593 (1.65), 1.612 (1.89), 1.629 (1.34), 2.280 (1.83), 2.318 (3.36), 2.323 (4.52), 2.327 (4.52), 2.331 (2.99), 2.337 (1.40), 2.421 (0.85), 2.432 (2.44), 2.439 (2.20), 2.449 (5.31), 2.456 (2.38), 2.467 (4.46), 2.518 (16.00), 2.523 (11.24), 2.584 (0.98), 2.591 (12.95), 2.601 (1.95), 2.619 (0.98), 2.660 (1.40), 2.665 (3.18), 2.669 (4.40), 2.674 (3.42), 2.678 (2.50), 2.695 (1.65), 2.720 (1.22), 2.737 (1.04), 2.803 (7.27), 2.840 (1.16), 2.857 (2.20), 2.871 (2.02), 2.886 (2.75), 2.903 (4.95), 2.921 (2.08), 2.959 (1.28), 2.978 (4.40), 2.993 (2.75), 3.053 (1.28), 3.067 (5.07), 3.082 (6.47), 3.102 (2.26), 3.387 (3.36), 3.405 (1.95), 3.585 (0.55), 3.602 (1.22), 3.619 (1.22), 3.635 (1.40), 3.652 (0.61), 3.829 (0.67), 3.846 (1.40), 3.863 (1.10), 3.880 (1.16), 3.898 (0.49), 4.830 (5.31), 4.840 (5.31), 7.466 (2.20), 7.479 (2.20), 7.659 (4.89), 7.681 (8.12), 7.732 (8.24), 7.754 (4.15), 7.935 (1.04), 7.950 (2.02), 7.963 (0.98), 8.517 (3.48), 8.529 (3.24), 8.633 (4.89), 8.648 (4.89), 10.542 (0.92).
Intermediate 85
N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-{3-[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]propyl}-L-alaninamide
 
Figure imgf000418_0001
To a solution of N-{4-[1-(3-aminopropyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (60.0 mg, 143 µmol) and N- [(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-L-alanine (102 mg, 315 µmol) in acetonitrile (5 mL) was added at r.t. N,N-diisopropylethylamine (200 µl, 1.1 mmol). After stirring for 5 min min propylphosphonic anhydride (T3P, 170 µl, 570 µmol) was added and the mixture stirred for further 1 h at that temperature. After that the mixture was concentrated under reduced pressure and purified by preparative HPLC and column chromatography (SiO2, dichloromethane/methanol gradient) to give the title compound (4.0 mg, 75% purity, 3% yield). HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x30 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-8 min 10-50% B. rate 150 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.71 min; MS (ESIpos): m/z = 714 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.793 (6.01), 0.807 (6.20), 0.810 (6.57), 0.821 (6.46), 0.829 (4.88), 0.836 (7.17), 0.847 (6.61), 0.852 (7.32), 0.864 (3.15), 0.868 (3.31), 0.878 (0.86), 0.918 (1.73), 0.921 (2.89), 0.936 (7.77), 0.939 (7.62), 0.950 (6.69), 0.955 (10.22), 0.967 (5.78), 0.971 (6.95), 0.986 (4.51), 1.058 (6.72), 1.077 (7.81), 1.095 (1.99), 1.190 (5.93), 1.194 (6.05), 1.208 (6.12), 1.211 (5.90), 1.233 (1.84), 1.270 (1.20), 1.276 (1.16), 1.288 (1.09), 1.297 (1.01), 1.314 (0.83), 1.335 (0.98), 1.352 (0.94), 1.358 (0.79), 1.378 (0.86), 1.430 (0.68), 1.444 (0.90), 1.447 (0.90), 1.452 (1.39), 1.456 (1.54), 1.463 (2.22), 1.466 (1.73), 1.473 (1.50), 1.483 (3.23), 1.493 (3.00), 1.499 (1.43), 1.502 (1.39), 1.505 (1.46), 1.532 (1.80), 1.544 (1.62), 1.557 (1.58), 1.683 (1.20), 1.726 (2.63), 1.743 (2.29), 1.927 (0.83), 1.943 (1.31), 1.961 (1.39), 1.977 (1.13), 2.102 (1.05), 2.285 (1.39), 2.322 (3.42), 2.327 (3.46), 2.332 (1.99), 2.336 (0.90), 2.518 (7.47), 2.523 (4.96), 2.660 (0.86), 2.665 (1.77), 2.669 (2.40), 2.673 (1.77), 2.679 (1.01), 2.687 (1.16), 2.705 (1.43), 2.729 (1.35), 2.746 (1.31), 3.074 (1.80), 3.088 (1.73), 3.376 (1.99), 3.391 (2.52),  
3.410 (2.37), 3.607 (3.57), 3.622 (3.94), 3.639 (4.17), 3.656 (3.98), 3.835 (3.72), 3.850 (3.87), 3.869 (3.64), 3.882 (3.31), 4.109 (7.36), 4.164 (2.67), 4.181 (2.40), 4.187 (2.33), 4.204 (2.40), 4.217 (2.55), 4.235 (2.07), 4.875 (5.07), 4.893 (4.88), 6.958 (0.86), 7.079 (16.00), 7.086 (1.31), 7.213 (0.90), 7.623 (0.86), 7.655 (4.39), 7.678 (7.25), 7.684 (2.67), 7.692 (2.67), 7.736 (5.97), 7.754 (3.57), 7.759 (3.83), 7.776 (1.01), 7.793 (0.86), 7.807 (1.58), 7.822 (0.90), 8.116 (1.20), 8.134 (1.31), 8.234 (1.24), 8.256 (1.24), 8.633 (1.01), 8.707 (3.68), 8.759 (1.39).
Intermediate 86
N5-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]propyl}-N2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- N1-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl-L-glutamamide
Figure imgf000419_0001
Under an atmosphere of argon N5-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-N1- 2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl-L-glutamamide (26.0 mg, 28.9 µmol) was dissolved in DMF (980 µl, 13 mmol). 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H- pyrrole-2,5-dione (16.0 mg, 51.9 µmol) and N,N-diisopropylethylamine (25 µl, 140 µmol) were added and the mixture was stirred at room temperature overnight. Then the mixture was purified by preparative HPLC to give the title compound (5.0 mg, 14% yield, 87% purity).
 
Instrument: Gilson Abimed 305 pump, KNAUER UV detector K-2501, Varian fraction collector model 701A, Prepcon 5 software. Column: Chromasil C18, 5µm, 125x20 mm. Eluent A: water + 0.1 Vol-% TFA; Eluent B: acetonitrile; gradient: 0-22 min 10-80% B; rate 50 ml/min; temperature 25°C.
LC-MS (method 8): Rt = 0.65 min; MS (ESIneg): m/z = 1093 [M-H]-
Intermediate 87
N-(4-{1-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,8,15-trioxo-11,12-dithia-4,7,16- triazaicosan-20-yl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000420_0001
A mixture of N-{4-[1-(4-{[3-({3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl}disulfanyl)- propanoyl]amino}butyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide (19.5 mg, 27.5 µmol), N-(2-aminoethyl)-3-(2,5-dioxo- 2,5-dihydro-1H-pyrrol-1-yl)propanamide trifluoroacetate (CAS-No. 1301739-85-4, 13.4 mg, 41.2 µmol) N,N-diisopropylethylamine (9.6 µl, 55 µmol) and DMF (210 µL) was stirred at r.t. for 14 h. After that the mixture was purified by preparative HPLC to give the title compound (11.2 mg, 90% purity, 46% yield).  
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + TFA; Eluent B: acetonitrile; gradient: 0-8 min 10-50% B. rate 150 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.70 min; MS (ESIpos): m/z = 806 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.852 (0.86), 1.054 (4.98), 1.072 (4.91), 1.233 (2.39), 1.379 (0.93), 1.396 (1.33), 1.416 (1.13), 1.434 (0.53), 1.590 (1.06), 1.610 (1.26), 1.627 (0.86), 1.907 (0.93), 1.986 (0.60), 2.006 (0.53), 2.074 (2.79), 2.279 (1.33), 2.288 (1.66), 2.306 (2.32), 2.318 (2.85), 2.323 (4.65), 2.327 (4.91), 2.331 (3.12), 2.337 (1.46), 2.421 (2.99), 2.439 (6.64), 2.457 (4.38), 2.518 (15.87), 2.523 (10.95), 2.574 (0.53), 2.591 (1.00), 2.608 (0.53), 2.660 (1.39), 2.665 (2.99), 2.669 (4.18), 2.674 (3.05), 2.678 (1.99), 2.696 (1.00), 2.720 (0.80), 2.737 (0.66), 2.848 (3.45), 2.857 (2.26), 2.866 (6.37), 2.884 (2.85), 3.037 (4.38), 3.065 (2.26), 3.080 (2.06), 3.097 (0.80), 3.370 (0.86), 3.388 (0.93), 3.405 (0.66), 3.575 (1.86), 3.594 (2.39), 3.612 (1.86), 3.635 (0.86), 3.844 (0.86), 3.860 (0.73), 3.877 (0.73), 4.818 (3.05), 4.835 (3.05), 7.002 (16.00), 7.434 (1.66), 7.447 (1.66), 7.658 (2.52), 7.680 (4.32), 7.730 (5.05), 7.752 (2.59), 7.952 (1.93), 7.967 (1.20), 7.978 (1.06), 8.498 (2.52), 8.511 (2.32), 8.614 (3.25), 8.642 (2.46).
Intermediate 88
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]methyl}-2-fluorophenyl)-L-alaninamide
 
Figure imgf000422_0001
A mixture of L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]- phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluorophenyl)-L-alaninamide (50.0 mg, 77.8 µmol), 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (CAS-No 55750-63-5, 26.4 mg, 85.6 µmol), N,N-diisopropylethylamine (27 µl, 160 µmol) and DMF (1.2 mL) was stirred at r.t. for 14 h. After that the mixure was purified by preparative HPLC to give the title compound (54.0 mg, 83% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% ammonia; Eluent B: acetonitrile; gradient: 0-20, min 15-55% B. rate 150 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.91 min; MS (ESIneg): m/z = 834 [M-H]- ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.786 (3.27), 0.803 (3.65), 0.819 (6.85), 0.836 (7.29), 0.852 (1.12), 1.033 (6.10), 1.052 (6.18), 1.115 (0.82), 1.135 (1.34), 1.154 (1.34), 1.179 (0.89), 1.199 (0.45), 1.233 (1.86), 1.268 (4.47), 1.277 (4.02), 1.287 (4.61), 1.295 (3.65), 1.440 (2.16), 1.881 (0.45), 1.898 (0.74), 1.915 (0.74), 1.938 (0.67), 1.955 (0.45), 1.987 (0.52), 2.006 (0.45), 2.053 (0.45), 2.070 (0.74), 2.088 (1.04), 2.106 (1.04), 2.123 (1.19), 2.140 (1.12), 2.159 (0.74), 2.332 (3.42), 2.337 (2.53), 2.379 (1.56), 2.518 (16.00), 2.523 (11.61), 2.673 (3.13), 2.679 (1.34), 2.792 (0.89), 2.808 (1.19), 2.833 (0.97), 2.850 (0.82), 3.312 (2.16), 3.347 (2.08), 3.353 (2.75), 3.371 (1.19), 3.411 (0.82), 3.429 (1.04), 3.447 (0.74), 4.113 (0.74), 4.134 (1.12), 4.153 (1.04), 4.173 (0.74), 4.178 (0.74), 4.195 (0.60), 4.480 (0.67), 4.498 (1.49), 4.516 (1.56), 4.534   (0.74), 4.813 (4.54), 4.829 (4.24), 4.855 (2.23), 4.948 (1.93), 4.987 (1.27), 6.981 (14.07), 6.996 (12.95), 7.085 (1.64), 7.106 (1.71), 7.140 (1.64), 7.169 (1.56), 7.429 (2.31), 7.442 (2.38), 7.648 (2.68), 7.668 (5.28), 7.708 (5.73), 7.731 (3.05), 7.747 (0.82), 7.799 (1.12), 7.816 (1.41), 7.837 (0.74), 7.845 (1.19), 7.866 (1.12), 8.209 (0.89), 8.226 (0.89), 8.340 (1.12), 8.359 (1.04), 8.496 (3.57), 8.508 (3.27), 8.610 (4.69), 8.641 (3.57), 9.629 (1.93), 9.660 (1.41).
Intermediate 89
N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000423_0001
A mixture of N-{4-[1-(4-aminobutyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}- 1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (100 mg, 228 µmol), 1-{6-[(2,5- dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (CAS-No. 55750-63-5, 84.5 mg, 274 µmol), N,N-diisopropylethylamine (60 µl, 340 µmol) and DMF (3.5 mL) was stirred at r.t. for 14 h. After that the mixture was filtered and the the filtrate was purified by preparative HPLC to give the title compound (65.0 mg, 46% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% HCOOH; Eluent B: acetonitrile; gradient: 0-20, min 15-55% B. rate 150 ml/min, temperature 25°C.
  LC-MS (method 1): Rt = 0.77 min; MS (ESIneg): m/z = 612 [M-H]- ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.051 (3.73), 1.069 (3.71), 1.124 (0.59), 1.145 (1.15), 1.163 (0.81), 1.233 (0.45), 1.357 (0.66), 1.375 (0.93), 1.395 (0.89), 1.403 (0.70), 1.413 (0.87), 1.422 (1.17), 1.434 (1.44), 1.441 (1.57), 1.451 (1.61), 1.470 (1.00), 1.572 (0.79), 1.591 (0.89), 1.610 (0.66), 1.980 (1.36), 1.998 (2.29), 2.017 (1.15), 2.074 (1.72), 2.278 (0.85), 2.317 (1.32), 2.322 (1.40), 2.326 (1.44), 2.331 (0.95), 2.336 (0.45), 2.518 (5.22), 2.522 (3.63), 2.659 (0.49), 2.664 (1.04), 2.668 (1.42), 2.673 (1.23), 2.677 (0.93), 2.693 (0.74), 2.718 (0.59), 2.735 (0.49), 3.017 (0.57), 3.033 (1.38), 3.048 (1.36), 3.065 (0.53), 3.340 (3.54), 3.357 (1.68), 3.367 (0.53), 3.386 (0.64), 3.404 (0.42), 3.595 (0.55), 3.611 (0.55), 3.628 (0.66), 3.845 (0.66), 3.861 (0.53), 3.878 (0.53), 4.816 (2.23), 4.833 (2.21), 6.992 (16.00), 7.433 (1.23), 7.446 (1.23), 7.654 (2.23), 7.676 (3.82), 7.725 (3.86), 7.748 (2.42), 7.761 (1.02), 7.774 (0.49), 8.498 (1.97), 8.511 (1.80), 8.614 (2.48), 8.635 (2.25).
Intermediate 90
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]butyl}-L-alaninamide
Figure imgf000424_0001
 
A mixture of L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]- phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L-alaninamide (300 mg, 90 % purity, 457 µmol), 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (CAS- No. 55750-63-5, 141 mg, 457 µmol), N,N-diisopropylethylamine (240 µl, 1.4 mmol) and DMF (7 mL) was stirred at r.t. for 14 h. After that the mixture was concentrated under reduced pressure and the residue was taken up in 0°C cold methanol/ethyl acetate and filtered. The filtrate was concentrated under reduced pressure and the crude product was purified by column chromatography (SiO2, dichloromethane/isopropanol gradient) to give the title compound (146 mg, 40% yield).
LC-MS (method 1): Rt = 0.83 min; MS (ESIpos): m/z = 784 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.777 (3.43), 0.794 (3.74), 0.805 (3.72), 0.822 (3.72), 0.833 (0.68), 0.852 (0.53), 1.048 (3.06), 1.067 (3.09), 1.151 (2.62), 1.157 (2.89), 1.169 (3.00), 1.174 (2.81), 1.202 (0.64), 1.233 (1.06), 1.390 (0.79), 1.408 (0.77), 1.427 (0.81), 1.446 (1.38), 1.463 (1.66), 1.482 (1.15), 1.575 (0.72), 1.595 (0.83), 1.613 (0.60), 1.907 (0.74), 1.924 (0.60), 1.941 (0.60), 2.089 (0.55), 2.107 (0.79), 2.125 (0.70), 2.142 (0.74), 2.160 (0.53), 2.275 (0.74), 2.327 (1.34), 2.332 (0.94), 2.337 (0.43), 2.518 (4.45), 2.523 (3.28), 2.590 (3.74), 2.689 (0.66), 2.714 (0.53), 2.731 (0.43), 3.038 (0.49), 3.054 (0.45), 3.086 (0.47), 3.101 (0.57), 3.119 (0.43), 3.347 (0.83), 3.358 (2.64), 3.375 (1.49), 3.387 (0.60), 3.608 (0.40), 3.619 (0.43), 4.093 (0.70), 4.111 (0.77), 4.115 (0.79), 4.132 (0.66), 4.180 (0.66), 4.198 (0.98), 4.216 (0.62), 4.816 (2.13), 4.835 (2.15), 6.980 (1.60), 6.993 (16.00), 7.432 (1.19), 7.446 (1.23), 7.656 (2.00), 7.679 (3.55), 7.725 (3.32), 7.747 (1.74), 7.777 (1.09), 7.798 (1.77), 7.894 (0.83), 7.913 (0.91), 8.498 (1.94), 8.511 (1.83), 8.613 (2.49), 8.640 (2.13).
Intermediate 91
N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]methyl}-2-fluorophenyl)-L-alaninamide
 
Figure imgf000426_0001
A mixture of L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]- phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluorophenyl)-L-alaninamide trifluoroacetate (34.8 mg, 46.0 µmol), 1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H- pyrrole-2,5-dione (CAS-No. 55750-61-3, 17.4 mg, 69.0 µmol), N,N-diisopropylethylamine (32 µl, 180 µmol) and DMF (710 µL) was stirred at r.t. for 14 h. After that the mixture was purified by preparative HPLC to give the title compound (3.00 mg, 8% yield).
1. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% HCOOH; Eluent B: acetonitrile; gradient: 0-20, min 15-55% B. rate 150 ml/min, temperature 25°C.
2. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x20 mm. Eluent A: water + 0.1% HCOOH; Eluent B: acetonitrile; gradient: 0-20, min 15-55% B. rate 50 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.82 min; MS (ESIneg): m/z = 778 [M-H]- 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.795 (1.88), 0.812 (2.20), 0.827 (4.47), 0.831 (5.65), 0.844 (4.78), 0.848 (5.65), 1.036 (2.51), 1.041 (3.29), 1.055 (2.75), 1.059 (3.22), 1.233 (1.80), 1.273 (3.37), 1.284 (2.59), 1.290 (3.61), 1.301 (2.12), 1.909 (0.39), 1.926 (0.55), 1.943 (0.63), 1.986 (0.47), 2.005 (0.47), 2.318 (1.41), 2.322 (3.22), 2.327 (4.63), 2.332 (3.37), 2.336 (1.65),  
2.345 (0.78), 2.385 (0.86), 2.518 (16.00), 2.523 (11.29), 2.660 (1.49), 2.665 (3.29), 2.669 (4.71), 2.673 (3.29), 2.679 (1.33), 2.820 (0.47), 2.845 (0.47), 3.306 (0.71), 3.415 (0.55), 3.432 (0.71), 3.450 (0.47), 4.102 (2.20), 4.107 (5.65), 4.166 (0.63), 4.187 (0.71), 4.194 (0.47), 4.204 (0.63), 4.216 (0.47), 4.514 (0.47), 4.523 (0.55), 4.541 (0.78), 4.559 (0.55), 4.811 (2.98), 4.830 (3.53), 4.856 (0.78), 4.869 (1.10), 4.959 (1.10), 4.997 (0.71), 7.027 (5.96), 7.029 (5.49), 7.079 (6.43), 7.090 (1.10), 7.110 (1.18), 7.133 (0.78), 7.143 (0.63), 7.162 (0.78), 7.167 (0.71), 7.430 (1.65), 7.442 (1.65), 7.651 (2.51), 7.673 (4.78), 7.699 (0.71), 7.713 (2.75), 7.718 (4.24), 7.736 (1.65), 7.740 (2.12), 7.815 (0.71), 8.267 (0.55), 8.281 (0.94), 8.289 (0.71), 8.302 (0.86), 8.354 (0.55), 8.370 (0.55), 8.406 (0.86), 8.425 (0.86), 8.496 (2.35), 8.508 (2.20), 8.610 (3.22), 8.644 (2.98), 9.658 (1.33), 9.685 (0.78).
Intermediate 92
N-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]methyl}phenyl)-L-alaninamide
Figure imgf000427_0001
A mixture of L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]- phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L-alaninamide trifluoroacetate (35.5 mg, 48.0 µmol), 1-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-1H- pyrrole-2,5-dione (CAS-No. 55750-61-3, 12.1 mg, 48.0 µmol), N,N-diisopropylethylamine (25 µl, 140 µmol) and DMF (740 µL) was stirred at r.t. for 14 h. After that the mixture was purified by preparative HPLC to give the title compound (8.00 mg, 22% yield).  
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x20 mm. Eluent A: water + 0.1% HCOOH; Eluent B: acetonitrile; gradient: 0-20, min 15-55% B. rate 50 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.81 min; MS (ESIpos): m/z = 762 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.805 (1.79), 0.822 (2.02), 0.836 (2.25), 0.848 (2.49), 0.852 (3.18), 0.859 (2.72), 0.865 (2.49), 0.876 (2.10), 1.015 (2.95), 1.033 (2.95), 1.233 (1.79), 1.266 (2.33), 1.278 (2.49), 1.284 (2.49), 1.295 (2.10), 1.919 (0.47), 1.938 (0.54), 1.955 (0.54), 1.972 (0.47), 1.987 (0.54), 2.005 (0.54), 2.318 (1.55), 2.322 (3.42), 2.327 (4.97), 2.332 (3.65), 2.336 (1.63), 2.371 (0.54), 2.518 (16.00), 2.523 (11.34), 2.660 (1.40), 2.665 (3.18), 2.669 (4.50), 2.673 (3.18), 2.678 (1.40), 3.306 (0.70), 3.413 (0.54), 4.109 (2.25), 4.124 (2.72), 4.370 (0.47), 4.412 (0.47), 4.743 (0.54), 4.780 (0.70), 4.809 (2.10), 4.827 (2.10), 4.981 (0.78), 5.017 (0.62), 7.039 (3.18), 7.045 (3.03), 7.077 (4.43), 7.214 (1.17), 7.231 (1.55), 7.236 (1.71), 7.253 (1.24), 7.429 (1.17), 7.441 (1.17), 7.520 (1.40), 7.532 (1.55), 7.541 (1.24), 7.553 (1.24), 7.647 (1.32), 7.667 (2.41), 7.670 (2.25), 7.704 (1.86), 7.712 (1.94), 7.727 (1.01), 7.734 (0.93), 8.255 (0.47), 8.277 (0.54), 8.291 (0.54), 8.308 (0.47), 8.349 (0.54), 8.369 (0.47), 8.447 (0.54), 8.466 (0.47), 8.495 (1.48), 8.508 (1.40), 8.609 (2.10), 8.637 (2.02), 9.746 (1.01), 9.917 (0.85).
Intermediate 93
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]methyl}phenyl)-L-alaninamide
 
Figure imgf000429_0001
A mixture of L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]- phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L-alaninamide trifluoroacetate (35.5 mg, 48.0 µmol), 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H- pyrrole-2,5-dione (CAS-No. 55750-63-5, 14.8 mg, 48.0 µmol), N,N-diisopropylethylamine (25 µl, 140 µmol) and DMF (740 µL) was stirred at r.t. for 14 h. After that the mixture was filtered and the filtrate was purified by preparative HPLC and column chromatography (SiO2, dichloromethane/isopropanol gradient)´to give the title compound (10.0 mg, 25% yield). HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x20 mm. Eluent A: water + 0.1% HCOOH; Eluent B: acetonitrile; gradient: 0-20, min 15-55% B. rate 50 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.88 min; MS (ESIneg): m/z = 816 [M-H]- ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.797 (0.79), 0.813 (1.00), 0.825 (1.29), 0.841 (1.72), 0.851 (2.30), 0.867 (1.43), 1.009 (1.36), 1.027 (1.65), 1.167 (0.57), 1.234 (2.87), 1.267 (1.94), 1.286 (1.79), 1.353 (0.65), 1.388 (3.66), 1.459 (0.86), 1.986 (0.65), 2.005 (0.57), 2.323 (3.73), 2.327 (4.59), 2.364 (0.65), 2.522 (16.00), 2.665 (3.23), 2.669 (4.38), 2.729 (0.65), 2.888 (0.72), 3.369 (0.57), 4.811 (1.22), 4.828 (1.15), 6.978 (2.15), 6.993 (1.94), 7.229 (0.93), 7.250 (1.08), 7.429 (0.57), 7.441 (0.65), 7.610 (0.65), 7.631 (0.65), 7.639 (0.72), 7.660 (1.29), 7.700 (1.15), 7.719 (0.65), 8.496 (0.79), 8.508 (0.79), 8.610 (1.22), 8.633 (0.86), 9.743 (0.57).   Intermediate 94
N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-fluorobenzyl)-4- methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine- 2-carboxamide
Figure imgf000430_0001
A mixture of N-{4-[1-(4-amino-3-fluorobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (30.0 mg, 63.5 µmol), 6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (CAS-No.55750-53-3, 16.1 mg, 76.2 µmol), (1- [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoro- phosphate) (HATU, 36.2 mg, 95.2 µmol), N-methylmorpholine (21 µl, 190 µmol) and DMF (1 mL) was stirred at r.t. for 14 h. After that the mixture was filtered and the filtrate was purified by preparative HPLC to give the title compound (18.0 mg, 40% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% HCOOH; Eluent B: acetonitrile; gradient: 0-8, min 15-55% B. rate 150 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.90 min; MS (ESIpos): m/z = 666 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.036 (3.71), 1.055 (3.74), 1.230 (1.66), 1.244 (1.26), 1.478 (0.96), 1.497 (1.19), 1.516 (1.06), 1.535 (1.06), 1.556 (1.19), 1.574 (0.83), 2.302 (0.96), 2.318 (2.09), 2.322 (2.88), 2.327 (2.68), 2.332 (1.99), 2.337 (1.69), 2.381 (0.93), 2.518 (7.19), 2.523 (5.57), 2.660 (0.66), 2.665 (1.52), 2.669 (2.15), 2.673 (1.49), 2.812 (0.70), 3.364 (1.95), 3.382 (3.15), 3.399 (1.66), 4.815 (2.78), 4.829 (2.39), 4.854 (1.49), 4.949 (1.36), 4.986 (0.93), 6.994 (16.00), 7.070 (0.86), 7.074 (0.93), 7.091 (0.89), 7.095 (0.99), 7.121 (1.09), 7.125 (0.89), 7.150 (1.03), 7.154 (0.89), 7.428 (1.26), 7.440 (1.29), 7.650 (2.15), 7.656 (0.80), 7.668 (1.23),  
7.673 (4.21), 7.713 (4.21), 7.719 (1.06), 7.731 (0.83), 7.736 (1.99), 7.765 (1.13), 8.409 (0.66), 8.495 (2.02), 8.508 (1.92), 8.609 (2.52), 8.647 (2.39), 9.615 (1.56).
Intermediate 95
N-{4-[1-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-3-fluorobenzyl)-4-methyl-6- oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide
Figure imgf000431_0001
A mixture of N-{4-[1-(4-amino-3-fluorobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (40.0 mg, 84.7 µmol), (2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid (CAS-No. 25021-08-3, 15.8 mg, 102 µmol), (1- [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoro- phosphate) (HATU, 48.3 mg, 127 µmol), N-methylmorpholine (28 µl, 250 µmol) and DMF (1 mL) was stirred at r.t. for 14 h. After that the mixture was filtered and the filtrate was purified by preparative HPLC to give the title compound (11.0 mg, 20% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% HCOOH; Eluent B: acetonitrile; gradient: 0-8, min 15-55% B. rate 150 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.79 min; MS (ESIpos): m/z = 610 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.033 (3.66), 1.051 (3.69), 2.323 (1.40), 2.327 (1.98), 2.332 (1.40), 2.337 (0.73), 2.346 (0.85), 2.385 (0.92), 2.518 (7.24), 2.523 (5.19), 2.660 (0.64), 2.665 (1.44), 2.669 (2.02), 2.674 (1.37), 2.679 (0.64), 2.815 (0.73), 3.414 (0.95), 3.432 (1.19),   3.449 (1.13), 3.553 (1.16), 4.316 (4.49), 4.826 (0.89), 4.863 (1.40), 4.905 (2.50), 4.933 (2.32), 4.955 (1.50), 4.993 (0.95), 7.089 (0.89), 7.105 (0.89), 7.110 (0.95), 7.130 (16.00), 7.163 (1.01), 7.167 (0.92), 7.181 (0.70), 7.192 (1.01), 7.197 (0.95), 7.639 (2.47), 7.644 (0.95), 7.656 (1.16), 7.662 (3.97), 7.716 (4.06), 7.722 (1.16), 7.734 (0.92), 7.739 (2.23), 7.759 (0.82), 7.780 (1.95), 7.801 (0.95), 8.682 (1.16), 8.696 (1.13), 8.727 (2.26), 8.810 (1.74), 10.115 (1.59).
Intermediate 96
N-(4-{1-[4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-6-oxo-4-phenyl- 1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000432_0001
A mixture of N-{4-[1-(4-aminobutyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}- 1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (123 mg, 90 % purity, 229 µmol), 1,1'- [(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione (CAS-No. 79642-50-5, 89.8 mg, 275 µmol), N,N-diisopropylethylamine (80 µl, 460 µmol) and DMF (3.5 mL) was stirred at r.t. for 14 h. After that the mixture was concentrated under reduced pressure and the residue was purified by column chromatography (SiO2, dichloromethane/isopropanol gradient) to give the title compound (23.0 mg, 14% yield).
LC-MS (method 1): Rt = 0.83 min; MS (ESIneg): m/z = 692 [M-H]- ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.363 (2.08), 1.381 (3.42), 1.399 (2.69), 1.417 (0.90), 1.595 (0.73), 1.612 (1.74), 1.629 (2.64), 1.647 (2.41), 1.665 (1.46), 1.688 (0.79), 1.707 (0.51), 1.786 (0.84), 1.805 (3.03), 1.824 (4.66), 1.843 (3.37), 1.862 (1.18), 2.050 (0.45), 2.069 (0.73), 2.074 (0.79), 2.087 (0.45), 2.157 (3.87), 2.176 (6.46), 2.195 (3.14), 2.518 (14.15), 2.522 (9.54), 2.539 (1.40), 2.548 (2.64), 2.584 (8.42), 2.589 (3.20), 2.669 (4.44), 2.675 (9.15), 2.694 (3.99), 2.805 (16.00), 3.050 (3.20), 3.069 (5.22), 3.082 (2.47), 3.090 (2.92), 3.110 (1.80), 3.652 (0.62), 3.669 (1.24), 3.686 (1.57), 3.702 (1.74), 3.719 (0.84), 3.812 (0.84), 3.830 (1.85), 3.846 (1.52), 3.863 (1.40), 3.880 (0.62), 4.686 (2.64), 4.705 (2.69), 4.793 (6.62), 4.810 (6.85), 7.157 (6.01), 7.174 (7.41), 7.217 (1.29), 7.234 (4.10), 7.253 (2.92), 7.297 (6.06), 7.316 (7.86), 7.334 (2.98),   7.418 (3.76), 7.431 (3.82), 7.596 (6.40), 7.619 (9.38), 7.632 (1.18), 7.692 (9.94), 7.714 (5.84), 7.834 (1.46), 7.848 (2.92), 7.862 (1.52), 8.488 (5.50), 8.500 (5.16), 8.599 (7.75), 8.615 (6.18).
Intermediate 97
N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-6-oxo-4-phenyl- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000433_0001
A mixture of N-{4-[1-(4-aminobutyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}- 1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (77.3 mg, 90 % purity, 144 µmol), 1-{6- [(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (CAS-No. 55750-63-5, 53.3 mg, 173 µmol), N,N-diisopropylethylamine (38 µl, 220 µmol) and DMF (2.2 mL) was stirred at r.t. for 14 h. After that the mixture was filtered and the filtrate was purified by preparative HPLC to give the title compound (27.0 mg, 26% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% HCOOH; Eluent B: acetonitrile; gradient: 0-8, min 15-55% B. rate 150 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.89 min; MS (ESIpos): m/z = 676 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.139 (0.86), 1.160 (1.49), 1.178 (1.10), 1.199 (0.50), 1.350 (1.08), 1.369 (1.66), 1.386 (1.27), 1.412 (0.66), 1.431 (1.66), 1.450 (2.70), 1.469 (2.65), 1.488 (1.41), 1.508 (0.44), 1.602 (0.80), 1.620 (1.19), 1.638 (1.08), 1.655 (0.66), 1.999 (1.74), 2.017 (2.92), 2.036 (1.49), 2.468 (1.43), 2.472 (1.46), 2.518 (6.95), 2.523 (4.44), 2.540 (2.21), 2.548 (1.32), 2.587 (1.24), 3.018 (0.52), 3.035 (1.19), 3.048 (1.88), 3.058 (1.27), 3.067 (1.52), 3.089 (1.02), 3.108 (0.72), 3.298 (0.63), 3.346 (4.58), 3.364 (1.79), 3.666 (0.58), 3.682 (0.72),   3.699 (0.74), 3.826 (0.83), 3.842 (0.66), 3.859 (0.58), 4.685 (1.13), 4.704 (1.16), 4.792 (3.06), 4.809 (3.01), 6.994 (16.00), 7.011 (0.52), 7.156 (2.62), 7.174 (3.23), 7.215 (0.63), 7.233 (1.85), 7.252 (1.27), 7.296 (2.62), 7.315 (3.34), 7.334 (1.24), 7.419 (1.71), 7.431 (1.71), 7.594 (3.06), 7.617 (4.44), 7.688 (4.58), 7.710 (2.76), 7.745 (0.69), 7.759 (1.32), 7.773 (0.66), 8.488 (2.48), 8.501 (2.32), 8.600 (3.53), 8.615 (2.90).
Intermediate 98
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[(4R)-3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-6-oxo-4-phenyl-5,6-dihydropyridazin-1(4H)- yl]butyl}-L-alaninamide
Figure imgf000434_0001
A mixture of N-{4-[(4R)-1-(4-aminobutyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (180 mg, 60 % purity, 224 µmol), N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-L-alanine (85.4 mg, 224 µmol), (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoro- phosphate) (HATU, 85.1 mg, 224 µmol), N-methylmorpholine (62 µl, 560 µmol) and DMF (4.5 mL) was stirred at r.t. for 14 h. After that the mixture was filtered and the filtrate was purified by preparative HPLC to give the title compound (34.0 mg, 17% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% HCOOH; Eluent B: acetonitrile; gradient: 0-8, min 15-55% B. rate 60 ml/min, temperature 25°C.
 
LC-MS (method 1): Rt = 1.02 min; MS (ESIpos): m/z = 846 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.785 (4.88), 0.802 (5.35), 0.812 (4.79), 0.829 (4.88), 0.857 (0.68), 1.147 (1.37), 1.168 (4.28), 1.174 (4.06), 1.186 (4.32), 1.192 (3.76), 1.232 (0.60), 1.356 (0.90), 1.375 (1.45), 1.394 (1.33), 1.413 (0.77), 1.429 (1.07), 1.448 (2.10), 1.467 (2.65), 1.484 (2.05), 1.604 (0.77), 1.622 (1.11), 1.640 (1.03), 1.656 (0.68), 1.723 (0.60), 1.919 (0.51), 1.935 (0.77), 1.952 (0.77), 1.969 (0.47), 2.075 (0.51), 2.093 (0.81), 2.111 (1.07), 2.128 (1.03), 2.146 (1.07), 2.164 (0.68), 2.181 (0.47), 2.518 (16.00), 2.523 (10.95), 2.540 (3.34), 2.549 (1.63), 2.590 (1.37), 3.006 (0.43), 3.025 (0.60), 3.043 (1.28), 3.062 (1.58), 3.083 (1.41), 3.103 (1.24), 3.217 (0.56), 3.341 (2.48), 3.359 (4.15), 3.377 (2.87), 3.449 (2.18), 3.647 (0.68), 3.664 (0.81), 3.680 (0.81), 3.693 (0.77), 3.819 (0.64), 3.831 (0.68), 3.852 (0.64), 3.863 (0.51), 4.103 (0.90), 4.120 (1.11), 4.124 (1.11), 4.141 (0.98), 4.198 (0.94), 4.216 (1.33), 4.234 (0.90), 4.686 (1.20), 4.705 (1.20), 4.863 (4.92), 6.977 (0.81), 6.992 (13.90), 7.153 (2.57), 7.171 (3.34), 7.215 (0.77), 7.233 (1.80), 7.252 (1.24), 7.296 (2.57), 7.316 (3.25), 7.334 (1.28), 7.429 (0.81), 7.590 (3.42), 7.612 (4.62), 7.644 (0.98), 7.657 (0.98), 7.693 (4.32), 7.716 (2.87), 7.783 (1.41), 7.809 (1.45), 7.908 (0.94), 7.926 (0.94), 8.608 (1.63), 8.621 (1.45), 8.669 (2.70), 8.728 (2.31).
Intermediate 99
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin- 1(4H)-yl]methyl}phenyl)-L-alaninamide
 
Figure imgf000436_0001
A mixture of L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]- phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L-alaninamide trifluoroacetate (300 mg, 80 % purity, 319 µmol), 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6- oxohexyl}-1H-pyrrole-2,5-dione (147 mg, 478 µmol), N,N-diisopropylethylamine (220 µl, 1.3 mmol) and DMF (5 mL) was stirred at r.t. for 14 h. After that the mixture was filtered and the filtrate was purified by two consecutive preparative HPLCs, column chromatography (SiO2, dichloromethane/ethanol gradient) and again preparative HPLC to give the title compound (2.30 mg, 1% yield). 1. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% HCOOH; Eluent B: acetonitrile; gradient: 0-8, min 15-55% B. rate 150 ml/min, temperature 25°C.
2. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A:  
water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-8, min 15-55% B. rate 150 ml/min, temperature 25°C.
3. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x20 mm. Eluent A: water + 0.1% HCOOH; Eluent B: acetonitrile; gradient: 0-8, min 15-55% B. rate 150 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.99 min; MS (ESIpos): m/z = 832 [M+H]+
¹H-NMR (400 MHz, CHLOROFORM-d) d [ppm]: 0.000 (0.91), 0.816 (3.95), 0.833 (4.09), 0.883 (2.11), 0.892 (2.38), 0.900 (2.37), 0.909 (2.06), 1.114 (16.00), 1.146 (0.57), 1.166 (0.75), 1.184 (1.06), 1.213 (1.15), 1.232 (0.92), 1.252 (0.47), 1.330 (2.64), 1.348 (2.67), 1.367 (1.91), 1.384 (1.82), 1.429 (0.62), 1.448 (0.82), 1.456 (0.71), 1.475 (1.08), 1.493 (1.30), 1.512 (1.06), 1.524 (0.93), 1.543 (1.07), 1.562 (1.29), 1.584 (1.48), 1.604 (1.71), 1.636 (2.53), 1.970 (0.53), 1.987 (0.62), 2.004 (0.55), 2.108 (0.56), 2.127 (0.95), 2.149 (1.19), 2.167 (1.39), 2.186 (0.70), 2.652 (4.09), 3.353 (0.71), 3.371 (1.29), 3.385 (1.47), 3.402 (2.03), 3.421 (1.08), 4.007 (0.53), 4.222 (0.51), 4.241 (0.76), 4.261 (0.48), 4.529 (0.43), 4.548 (0.67), 4.565 (0.54), 4.577 (0.48), 4.802 (3.38), 4.831 (3.46), 4.869 (3.82), 6.246 (0.86), 6.267 (0.73), 6.579 (4.55), 6.588 (7.05), 6.600 (1.33), 6.685 (0.68), 6.912 (0.50), 6.930 (0.63), 7.211 (1.27), 7.224 (1.39), 7.230 (1.57), 7.243 (1.91), 7.252 (1.63), 7.264 (2.06), 7.392 (2.14), 7.414 (2.98), 7.418 (2.63), 7.436 (2.26), 7.441 (2.50), 7.470 (1.25), 7.491 (1.03), 7.555 (1.38), 7.573 (2.76), 7.595 (1.64), 8.516 (1.47), 8.539 (1.26), 8.613 (0.80).
Intermediate 100
N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-5,5-dimethyl-6- oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide
 
Figure imgf000438_0001
A mixture of N-{4-[1-(4-aminobutyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide trifluoroacetate (1:1) (99.0 mg, 180 µmol), 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (41.9 mg, 199 µmol), HATU (75.5 mg, 199 µmol) and 4-methylmorpholine (60 µl, 540 µmol) in DMF (2.8 ml, 36 mmol) was stirred at r.t. for 14 h. After that the mixture was filtered and the filtrate was purified by preparative HPLC to give the title compound (9.00 mg, 8% yield).
1. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-8, min 15-55% B. rate 150 ml/min, temperature 25°C.
2. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x20 mm. Eluent A: water + 0.1% HCOOH; Eluent B: acetonitrile; gradient: 0-22, min 10-50% B. rate 50 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.85 min; MS (ESIpos): m/z = 628 [M+H]+ 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.078 (16.00), 1.128 (0.68), 1.148 (1.39), 1.166 (0.87), 1.187 (0.39), 1.233 (1.06), 1.354 (0.77), 1.373 (1.03), 1.393 (0.90), 1.408 (0.77), 1.427 (1.26), 1.437 (1.45), 1.446 (1.74), 1.454 (1.74), 1.465 (1.29), 1.472 (1.06), 1.575 (0.77), 1.594 (0.94), 1.613 (0.68), 1.982 (1.42), 2.000 (2.35), 2.019 (1.16), 2.322 (1.39), 2.326 (1.90), 2.332 (1.39), 2.518 (8.90), 2.522 (5.77), 2.539 (2.68), 2.665 (1.42), 2.669 (1.97), 2.673 (1.42), 2.831 (4.03), 3.013 (0.58), 3.030 (1.39), 3.045 (1.39), 3.062 (0.55), 3.344 (3.32), 3.362 (1.52), 3.692 (0.94),  
3.710 (1.77), 3.727 (0.87), 4.814 (2.35), 4.832 (2.32), 6.993 (11.10), 7.433 (1.23), 7.447 (1.26), 7.648 (1.74), 7.670 (4.00), 7.701 (4.10), 7.724 (1.68), 7.739 (0.55), 7.752 (1.03), 7.766 (0.52), 8.499 (1.61), 8.511 (1.52), 8.615 (2.39), 8.633 (2.26).
Figure imgf000439_0001
Fehler! Verweisquelle konnte nicht gefunden werden.Example 28M
{1-[2-({2-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5-dimethyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}amino)-2-oxoethyl]-2,5-dioxopyrrolidin-3-yl}-L- cysteine trifluoroacetate
Figure imgf000439_0002
To a solution of Intermediate 70
(N-{4-[1-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)-5,5-dimethyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide, 5.00 mg, 9.20 µmol) in DMF/water (10:1, 2.2 mL) was added at r.t. L-cysteine (3.34 mg, 27.6 µmol). After stirring for 20 min the mixture was purified by preparative HPLC to give the title compound (5.00 mg, 66% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x20 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-22, min 10-50% B. rate 50 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.61 min; MS (ESIpos): m/z = 665 [M+H]+  
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.089 (5.17), 1.145 (0.57), 1.232 (0.82), 2.318 (1.20), 2.323 (2.71), 2.327 (3.78), 2.331 (2.65), 2.337 (1.20), 2.518 (12.85), 2.523 (8.38), 2.547 (2.77), 2.561 (1.26), 2.660 (1.51), 2.665 (2.96), 2.669 (3.97), 2.673 (2.83), 2.678 (1.32), 2.728 (13.23), 2.824 (1.26), 2.889 (16.00), 2.970 (1.32), 3.225 (0.82), 3.780 (0.63), 3.951 (1.01), 4.249 (0.57), 4.841 (0.88), 7.659 (0.69), 7.681 (1.26), 7.723 (1.26), 7.746 (0.63), 7.951 (2.08), 8.298 (0.63), 8.526 (0.50), 8.539 (0.50), 8.641 (0.82), 8.658 (0.76).
Figure imgf000440_0001
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 71
(N-{4-[1-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide, 130 µg, 97 % purity, 0.23 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 0.91 mg/mL
Drug/mAb ratio: 5.1 (UV)
Example 29D
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 71
(N-{4-[1-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-  
carboxamide, 130 µg, 97 % purity, 0.23 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.59 mg/mL
Drug/mAb ratio: 0.2 (UV) Example 29A
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 71
(N-{4-[1-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide, 130 µg, 97 % purity, 0.23 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.02 mg/mL
Drug/mAb ratio: 4.7 (UV)
Example 29M
{1-[2-({2-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}amino)-2-oxoethyl]-2,5-dioxopyrrolidin-3-yl}-L- cysteine trifluoroacetate
Figure imgf000441_0001
To a solution of Intermediate 71
 
(N-{4-[1-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide, 5.00 mg, 9.44 µmol) in DMF/water (10:1, 2.3 mL) was added at r.t. L-cysteine (3.43 mg, 28.3 µmol). After stirring for 20 min the mixture was purified by preparative HPLC to give the title compound (3.00 mg, 40% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x20 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-22, min 10-50% B. rate 50 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.55 min; MS (ESIpos): m/z = 651 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.049 (2.19), 1.067 (2.09), 1.240 (1.23), 1.256 (0.99), 1.271 (0.58), 1.713 (0.48), 1.720 (0.51), 1.729 (1.26), 1.746 (0.48), 2.323 (2.12), 2.327 (2.50), 2.331 (1.71), 2.337 (0.75), 2.518 (7.73), 2.523 (5.30), 2.547 (15.38), 2.665 (1.71), 2.669 (2.32), 2.673 (1.81), 2.678 (1.06), 2.727 (13.06), 2.743 (0.62), 2.888 (16.00), 2.957 (1.09), 2.970 (1.44), 3.006 (1.30), 3.016 (1.33), 3.022 (0.99), 3.242 (2.39), 3.957 (1.78), 4.054 (0.44), 4.103 (0.55), 4.207 (1.03), 4.823 (1.47), 4.837 (1.47), 6.999 (8.55), 7.127 (9.13), 7.255 (8.58), 7.441 (0.82), 7.453 (0.82), 7.669 (1.03), 7.690 (1.64), 7.747 (2.05), 7.769 (1.20), 7.950 (1.98), 8.261 (1.20), 8.505 (1.09), 8.518 (1.06), 8.619 (1.54), 8.668 (0.82).
Example 30A
Figure imgf000442_0001
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 72 (N-{4-[1-(3- {[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 160  
µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.28 mg/mL
Drug/mAb ratio: 2.2
Example 30M
S-{(3S)-1-[6-({3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}amino)-6-oxohexyl]-2,5-dioxopyrrolidin-3- yl}-L-cysteine
Figure imgf000443_0001
To a solution of N-{4-[1-(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propyl)-4- methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine- 2-carboxamide (174 mg, 60 % purity, 174 µmol) in DMF/water (10:1, 11 mL) was added at r.t. L-cysteine (21.1 mg, 174 µmol) and the mixture stirred for 14 h at that temperature. After that the mixture concentrated under reduced pressure and the residue was purified by preparative HPLC to give the title compound (5.7 mg).
Instrument: Waters Autopurification system; Säule: Waters XBrigde C185µ 100x30mm; eluent A: water + 0.1 Vol-% HCOOH (99%), eluent B: acetonitrile; gradient: 0.00–0.50 min 5% B (25 ^70mL/min), 0.51–5.50 min 11-31% B (70mL/min), DAD scan: 210-400 nm.
LC-MS (Method 1): Rt = 0.65 min; MS (ESIpos): m/z = 721 [M+H]+ 1H-NMR (400 MHz, DMSO-d6) delta [ppm]: 0.834 (0.04), 0.852 (0.09), 1.060 (0.45), 1.079 (0.45), 1.162 (0.06), 1.182 (0.10), 1.187 (0.11), 1.196 (0.11), 1.203 (0.10), 1.232 (0.31), 1.271 (0.05), 1.276 (0.04), 1.278 (0.04), 1.285 (0.03), 1.422 (0.06), 1.439 (0.14), 1.456 (0.22), 1.474 (0.20), 1.488 (0.11), 1.493 (0.11), 1.505 (0.04), 1.713 (0.11), 1.731 (0.15), 1.749 (0.11), 1.987  
(0.06), 2.006 (0.16), 2.025 (0.23), 2.044 (0.11), 2.280 (0.11), 2.318 (0.28), 2.322 (0.45), 2.327 (0.52), 2.331 (0.36), 2.336 (0.17), 2.366 (0.05), 2.438 (0.06), 2.518 (2.56), 2.523 (1.78), 2.540 (16.00), 2.568 (0.06), 2.574 (0.06), 2.580 (0.09), 2.589 (0.08), 2.660 (0.17), 2.664 (0.37), 2.669 (0.50), 2.674 (0.37), 2.678 (0.19), 2.699 (0.09), 2.709 (0.06), 2.724 (0.06), 2.741 (0.06), 2.770 (0.04), 2.792 (0.05), 2.805 (0.04), 2.827 (0.04), 3.033 (0.08), 3.051 (0.17), 3.066 (0.17), 3.085 (0.11), 3.120 (0.09), 3.130 (0.06), 3.142 (0.14), 3.167 (0.11), 3.175 (0.06), 3.189 (0.10), 3.210 (0.04), 3.219 (0.04), 3.228 (0.05), 3.231 (0.05), 3.382 (0.28), 3.391 (0.24), 3.402 (0.16), 3.404 (0.16), 3.429 (0.06), 3.613 (0.06), 3.629 (0.06), 3.646 (0.07), 3.847 (0.06), 3.863 (0.06), 3.879 (0.06), 4.020 (0.05), 4.030 (0.06), 4.042 (0.06), 4.053 (0.05), 4.075 (0.04), 4.086 (0.04), 4.097 (0.04), 4.829 (0.27), 4.845 (0.27), 7.430 (0.16), 7.443 (0.16), 7.669 (0.23), 7.692 (0.45), 7.728 (0.44), 7.750 (0.20), 7.869 (0.06), 7.982 (0.07), 8.278 (0.06), 8.497 (0.24), 8.509 (0.21), 8.612 (0.31), 8.754 (0.15).
Example 31B
Figure imgf000444_0001
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 74 (N-{4-[1- (2-{4-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]piperazin-1-yl}ethyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide, 170 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.96 mg/mL
Drug/mAb ratio: 3.0 (UV)  
Example 31D
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 74 (N-{4-[1-(2- {4-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]piperazin-1-yl}ethyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide, 170 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.50 mg/mL
Drug/mAb ratio: 4.6 (UV)
Example 31A
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 74 (N-{4-[1-(2- {4-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]piperazin-1-yl}ethyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide, 170 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.48 mg/mL
Drug/mAb ratio: 3.2 (UV)
Example 31M
S-{(3S)-1-[6-(4-{2-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}piperazin-1-yl)- 6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine
 
Figure imgf000446_0001
A mixture of Intermediate 74 (9.35 mg, 14.3 µmol) and L-cysteine (1.73 mg, 14.3 µmol) in DMF/water (10:1, 1.9 mL) was stirred at r.t. for 14 h. After that the mixture was purified by preparative HPLC to give the title compound (2.1 mg, 91% purity, 17% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x20 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-22, min 10-50% B. rate 50 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.56 min; MS (ESIpos): m/z = 775 (M+H)+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.851 (0.51), 1.077 (3.75), 1.092 (9.06), 1.111 (9.06), 1.144 (0.51), 1.189 (1.47), 1.209 (2.58), 1.231 (3.39), 1.362 (4.00), 1.443 (3.44), 1.453 (4.15), 2.221 (2.53), 2.240 (4.00), 2.259 (2.33), 2.280 (2.28), 2.318 (3.65), 2.323 (4.30), 2.327 (4.00), 2.331 (3.04), 2.337 (2.48), 2.354 (3.49), 2.394 (1.72), 2.427 (1.72), 2.441 (1.57), 2.518 (11.44), 2.523 (7.85), 2.540 (4.10), 2.553 (3.04), 2.565 (4.76), 2.581 (2.28), 2.660 (1.16), 2.665 (2.48), 2.669 (3.59), 2.673 (3.44), 2.689 (1.87), 2.714 (1.67), 2.728 (13.72), 2.793 (0.91), 2.813 (1.01), 2.827 (1.01), 2.848 (1.01), 2.872 (1.11), 2.888 (16.00), 3.044 (0.76), 3.063 (0.81), 3.079 (1.42), 3.098 (1.47), 3.118 (1.82), 3.131 (1.72), 3.141 (3.29), 3.165 (2.28), 3.176 (1.27), 3.186 (1.97), 3.406 (5.01), 3.425 (2.68), 3.628 (0.76), 3.643 (1.37), 3.660 (1.32), 3.677 (1.47), 3.693 (0.71), 3.718 (0.46), 4.029 (1.22), 4.039 (1.32), 4.051 (1.37), 4.061 (1.22), 4.085 (1.52), 4.094 (2.23), 4.108 (2.28), 4.117 (1.42), 4.126 (1.27), 4.143 (0.56), 4.823 (5.82), 4.840 (5.87), 6.913 (0.71), 6.936 (0.76), 7.433 (3.44), 7.445 (3.54), 7.630 (0.76), 7.652 (1.16), 7.664 (5.57), 7.686 (9.52), 7.733 (10.03), 7.750 (2.73), 7.755 (5.06), 7.950 (1.97), 8.104 (0.96), 8.127 (0.96), 8.498 (5.22), 8.510 (4.86), 8.613 (6.78), 8.707 (5.22).
 
Fehler! Verweisquelle konnte nicht gefunden werden.Example 32A
Figure imgf000447_0001
 
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 75 (N-(4-{1-[1- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,19-dioxo-7,10,13,16-tetraoxa-4,20-diazatricosan-23- yl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4- c]pyridine-2-carboxamide, 210 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.50 mg/mL
Drug/mAb ratio: 2.9 (UV)
 
Fehler! Verweisquelle konnte nicht gefunden werden.Example 33A
Figure imgf000448_0001
  5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 76 (N-(4-{1-[1- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazadocosan-22- yl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4- c]pyridine-2-carboxamide, 190 µg, 95 % purity, 0.23 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.62 mg/mL
Drug/mAb ratio: 1.4 (UV)
 
Example 34B
Figure imgf000449_0001
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 77 (N-{4-[1- (2-{4-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]piperazin-1-yl}ethyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide, 160 µg, 0.27 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.28 mg/mL
Drug/mAb ratio: 2.8 (UV)
Example 34D
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 77 (N-{4-[1-(2- {4-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]piperazin-1-yl}ethyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 160 µg, 0.27 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.68 mg/mL  
Drug/mAb ratio: 4.2 (UV)
Example 34A
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 77 (N-{4-[1-(2- {4-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]piperazin-1-yl}ethyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 320 µg, 50 % purity, 0.27 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.40 mg/mL
Drug/mAb ratio: 3.4 (UV)
Example 35A
Figure imgf000450_0001
5 mg of anti HER2 TPP-1015 (c=11.7 mg/mL) were coupled with Intermediate 78 N-{3-[3-{4- [(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]propyl}-N2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L- asparagine (170 µg, 0.23 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.42 mg/mL
Drug/mAb ratio: 3.2 (UV)
 
Example 36A
Figure imgf000451_0001
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 79 N-{4-[1-(4- {[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]amino}-4-oxobutyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (140 µg, 0.23 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.56 mg/mL
Drug/mAb ratio: 2.2 (UV)
 
Example 37A
Figure imgf000452_0001
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 80 (N-{4-[1-{4- [(3-{[3-({3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl}disulfanyl)propanoyl]amino}propyl)- carbamoyl]benzyl}-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carboxamide, 280 µg, 0.33 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 2.80 mg/mL
Drug/mAb ratio: 1.1
Example 37M
N-(4-{4-methyl-6-oxo-1-[4-({3-[(3-sulfanylpropanoyl)amino]propyl}carbamoyl)benzyl]-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide  
N
H N N C H 3
O N N O O H N H N O
~ S H A mixture of N-{4-[1-{4-[(3-{[3-({3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl}disulfanyl)- propanoyl]amino}propyl)carbamoyl]benzyl}-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (19.0 mg, 22.9 µmol), 3,3',3''-phosphanetriyltripropanoic acid hydrochloride (CAS-No. 51805-45-9, 19.7 mg, 68.8 µmol) and DMF (180 µL) was stirred at r.t. for 14 h. After that the mixture was filtrated and purified bhy preparative HPLC to give the title compound (10.0 mg, 68% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x20 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-22 min 15-55% B. rate 50 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.77 min; MS (ESIpos): m/z = 628 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.044 (7.24), 1.062 (7.37), 1.231 (0.83), 1.593 (0.70), 1.611 (2.22), 1.628 (3.11), 1.646 (2.22), 1.663 (0.63), 2.257 (2.86), 2.277 (5.65), 2.297 (3.75), 2.318 (1.33), 2.323 (2.98), 2.327 (4.19), 2.331 (2.92), 2.337 (1.40), 2.347 (3.68), 2.356 (2.10), 2.364 (8.06), 2.382 (4.44), 2.394 (2.10), 2.518 (16.00), 2.523 (11.24), 2.540 (1.27), 2.591 (0.63), 2.612 (2.29), 2.629 (5.33), 2.632 (3.49), 2.646 (3.05), 2.649 (4.95), 2.660 (1.52), 2.665 (3.94), 2.669 (4.44), 2.674 (2.86), 2.678 (1.27), 2.728 (1.27), 2.730 (1.08), 2.798 (1.02), 2.815 (1.40), 2.840 (1.14), 2.857 (0.95), 2.889 (1.52), 3.071 (1.33), 3.088 (3.05), 3.103 (2.98), 3.121 (1.27), 3.221 (1.33), 3.238 (2.86), 3.254 (2.86), 3.270 (1.40), 3.418 (0.95), 3.437 (1.27), 3.454  
(0.89), 4.814 (4.51), 4.828 (4.57), 4.865 (2.03), 4.903 (2.60), 5.072 (2.48), 5.110 (1.97), 7.365 (5.33), 7.386 (5.84), 7.440 (2.22), 7.452 (2.29), 7.634 (0.51), 7.640 (4.19), 7.646 (1.46), 7.658 (2.16), 7.663 (8.44), 7.669 (1.33), 7.695 (1.46), 7.701 (8.38), 7.707 (1.97), 7.718 (1.40), 7.724 (3.75), 7.774 (1.21), 7.779 (7.17), 7.783 (2.35), 7.795 (2.16), 7.800 (6.16), 7.804 (1.08), 7.901 (0.89), 7.915 (1.71), 7.930 (0.83), 8.389 (1.14), 8.403 (2.35), 8.418 (1.08), 8.501 (3.62), 8.513 (3.37), 8.615 (4.95), 8.642 (4.63).
Example 38A
Figure imgf000454_0001
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 81 (N-{4-[1-{4- [(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propyl)carbamoyl]benzyl}-4- methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine- 2-carboxamide, 170 µg, 0.24 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.60 mg/mL
Drug/mAb ratio: 4.1 (UV)
 
Example 38M
S-{1-[6-({3-[(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}benzoyl)amino]propyl}amino)-6- oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine
Figure imgf000455_0001
To a solution of N-{4-[1-{4-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}- propyl)carbamoyl]benzyl}-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (9.35 mg, 12.8 µmol) in DMF/water (10:1, 2.0 mL) was added at r.t. L-cysteine (1.55 mg, 12.8 µmol) and the mixture stirred for 14 h at that temperature. After that the mixture purified by preparative HPLC to give the title compound (4.5 mg, 40% yield).
HPLC: Instrument: Waters Autopurification system SQD; column: Waters XBrigde C18 5µ 100x30mm; eluent A: water + 0.1% Vol. HCOOH (99%), eluent B: acetonitrile; gradient: 0,00 – 0,50 min 5% B, 25mL/min, 0,51– 5,50 min 10-100% B, 70mL/min, 5,51– 6,50 min 100% B, 70mL/min. Temperature: 25°C; Injection: 2500 µl; DAD scan: 210-400 nm.
LC-MS (method 4): Rt = 0.65 min; MS (ESIpos): m/z = 854 [M+H]+
 
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.045 (7.53), 1.062 (7.69), 1.147 (1.41), 1.171 (1.65), 1.187 (1.10), 1.232 (0.94), 1.443 (2.90), 1.461 (3.22), 1.594 (1.80), 1.612 (2.67), 1.627 (1.88), 2.004 (1.80), 2.019 (3.29), 2.037 (1.65), 2.318 (1.41), 2.322 (3.14), 2.327 (4.39), 2.332 (3.14), 2.336 (1.41), 2.356 (1.73), 2.394 (1.96), 2.518 (16.00), 2.523 (11.37), 2.539 (2.04), 2.549 (1.18), 2.556 (1.02), 2.566 (0.86), 2.602 (0.86), 2.611 (0.86), 2.660 (1.41), 2.664 (3.14), 2.669 (4.39), 2.673 (3.22), 2.678 (1.33), 2.779 (0.63), 2.799 (1.49), 2.813 (2.04), 2.839 (1.41), 2.855 (1.02), 3.040 (1.25), 3.055 (2.90), 3.071 (2.90), 3.086 (1.88), 3.124 (1.25), 3.136 (1.02), 3.147 (2.27), 3.171 (1.65), 3.181 (0.71), 3.193 (1.18), 3.233 (2.43), 3.249 (2.51), 3.273 (1.65), 3.282 (2.04), 3.373 (2.43), 3.418 (2.04), 3.436 (2.04), 3.453 (1.25), 4.016 (0.94), 4.026 (1.02), 4.038 (1.02), 4.048 (0.86), 4.064 (0.78), 4.074 (0.86), 4.087 (0.86), 4.097 (0.71), 4.814 (4.78), 4.830 (4.86), 4.869 (1.88), 4.907 (2.43), 5.066 (2.35), 5.103 (1.73), 7.359 (4.86), 7.380 (5.25), 7.427 (2.75), 7.440 (2.90), 7.649 (3.84), 7.672 (8.55), 7.698 (9.10), 7.703 (2.75), 7.720 (3.76), 7.801 (3.69), 7.808 (4.47), 7.813 (1.80), 7.818 (1.65), 7.823 (3.45), 7.829 (3.61), 7.922 (0.63), 7.937 (1.10), 7.951 (0.55), 8.098 (0.55), 8.113 (1.10), 8.126 (0.55), 8.494 (4.47), 8.506 (4.31), 8.526 (1.10), 8.539 (0.55), 8.586 (0.55), 8.606 (6.04), 8.699 (2.75), 8.710 (2.35).
 
Fehler! Verweisquelle konnte nicht gefunden werden.Example 39A
Figure imgf000457_0001
 
5 mg of tratuzumab (c=12.2 mg/mL) were coupled with Intermediate 82 (N-{4-[1-{4-[(3-{[(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}propyl)carbamoyl]benzyl}-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide, 200 µg, 80 % purity, 0.23 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.55 mg/mL
Drug/mAb ratio: 4.4 (UV)
 
Example 40B
Figure imgf000458_0001
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 83 (N-(4-{1- [4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 220 µg, 97 % purity, 0.33 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.23 mg/mL
Drug/mAb ratio: 4.1 (UV)
Example 40Ea
35 mg of anti-B7H3 TPP-8382 (c=15.69 mg/mL) were coupled with Intermediate 83 (N-(4-{1- [4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 1.84 mg, 80 % purity, 2.33 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 3.68 mg/mL
Drug/mAb ratio: 5.7
 
Example 40Eb
28.9 mg of anti-B7H3 TPP-8382 (c=15.69 mg/mL) were coupled with Intermediate 83 (N-(4- {1-[4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 1.22 mg, 80 % purity, 1.54 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 7.46 mg/mL
Drug/mAb ratio: 4.2
Example 40Da
5.25 mg of anti-B7H3 TPP-6497 (c=10.58 mg/mL) were coupled with Intermediate 83 (N-(4- {1-[4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 280 µg, 80 % purity, 0.35 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.63 mg/mL
Drug/mAb ratio: 2.3 (UV) Example 40Db
5.25 mg of anti-B7H3 TPP-6497 (c=10.58 mg/mL) were coupled with Intermediate 83 N-(4-{1- [4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 280 µg, 80 % purity, 0.35 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.13 mg/mL
Drug/mAb ratio: 4.3 (UV)
Example 40Aa
5.25 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 83 (N-(4-{1- [4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 280 µg, 80 % purity, 0.35 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.09 mg/mL  
Drug/mAb ratio: 2.9 Example 40Ab
5.25 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 83 (N-(4-{1- [4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 280 µg, 80 % purity, 0.35 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.52 mg/mL
Drug/mAb ratio: 3.7
Example 40F
5.00 mg of anti B7H3 TPP-8567 (c=17.9 mg/mL) were coupled with Intermediate 83 (N-(4-{1- [4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 217 µg, 97 % purity, 0.35 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.44 mg/mL
Drug/mAb ratio: 10.7
Example 40G
5.00 mg of anti C4.4a TPP-509 (c=14.3 mg/mL) were coupled with Intermediate 83 (N-(4-{1- [4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 217 µg, 97 % purity, 0.35 µmol) according procedure 3. After that the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 029 mg/mL
Drug/mAb ratio: 2.4
Example 40M
N6-[5-({4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-5-oxopentanoyl]-L-lysine
 
Figure imgf000461_0001
A mixture of (N2-(tert-butoxycarbonyl)-N6-[5-({4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-5- oxopentanoyl]-L-lysine, 54.2 mg, 71.0 µmol), trifluoroacetic acid (110 µl, 1.4 mmol) and dichloromethane (910 µL) was stirred at r.t. for 14 h. After that the mixture was concentrated under reduced pressure, coevaporated twice with toluene and the crude product was purified by preparative HPLC to give the title compound (27.0 mg, 57% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. eluent A: water + 0.1% TFA; eluent B: acetonitrile; gradient: 0-8, min 0-25% B. rate 150 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.59 min; MS (ESIpos): m/z = 663 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.852 (0.72), 1.050 (13.56), 1.069 (13.84), 1.144 (0.61), 1.233 (1.88), 1.273 (1.44), 1.291 (1.94), 1.316 (3.27), 1.332 (4.54), 1.345 (4.37), 1.361 (2.82), 1.378 (2.93), 1.397 (3.43), 1.417 (2.93), 1.434 (1.27), 1.561 (2.05), 1.579 (3.99), 1.598 (4.21), 1.616 (2.88), 1.636 (2.05), 1.662 (3.99), 1.680 (5.43), 1.698 (4.10), 1.716 (1.33), 1.991 (5.92), 1.996 (6.20), 2.009 (11.02), 2.027 (4.65), 2.033 (3.88), 2.275 (3.04), 2.318 (4.04), 2.322  
(3.16), 2.327 (3.65), 2.332 (2.60), 2.336 (1.11), 2.518 (13.40), 2.523 (9.47), 2.660 (1.11), 2.665 (2.49), 2.669 (3.71), 2.673 (2.99), 2.679 (2.93), 2.696 (2.60), 2.720 (2.10), 2.737 (1.72), 3.000 (3.71), 3.015 (3.60), 3.027 (3.21), 3.045 (4.93), 3.060 (4.76), 3.076 (1.94), 3.094 (2.16), 3.109 (3.54), 3.124 (1.99), 3.367 (3.21), 3.385 (2.93), 3.403 (1.94), 3.585 (0.83), 3.603 (1.83), 3.619 (1.88), 3.635 (2.21), 3.653 (0.94), 3.813 (1.00), 3.831 (2.16), 3.847 (1.77), 3.864 (1.77), 3.882 (0.78), 4.829 (7.70), 4.845 (7.81), 7.431 (4.93), 7.443 (5.20), 7.490 (0.94), 7.668 (7.14), 7.691 (14.95), 7.724 (16.00), 7.733 (5.04), 7.747 (8.03), 8.152 (1.77), 8.166 (3.54), 8.180 (1.72), 8.212 (0.72), 8.497 (7.42), 8.509 (6.81), 8.611 (9.30), 8.762 (7.36).
Example 41D
Figure imgf000462_0001
4.61 mg of anti-B7H3 TPP-6497 (c=10.58 mg/mL) were coupled with Intermediate 84 (N-{4- [1-(4-{[3-({3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl}disulfanyl)propanoyl]amino}butyl)-4- methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine- 2-carboxamide, 220 µg, 0.31 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.58 mg/mL
Drug/mAb ratio: 0.2 (UV)
 
Example 41A
4.61 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 84 (N-{4-[1- (4-{[3-({3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl}disulfanyl)propanoyl]amino}butyl)-4- methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine- 2-carboxamide, 220 µg, 0.31 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.73 mg/mL
Drug/mAb ratio: 2.7
Example 41M
N-{4-[4-methyl-6-oxo-1-{4-[(3-sulfanylpropanoyl)amino]butyl}-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000463_0001
A mixture of N-{4-[1-(4-{[3-({3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl}disulfanyl)- propanoyl]amino}butyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide (115 mg, 162 µmol) and tris(2- carboxyethyl)phosphine hydrochloride (139 mg, 486 µmol) in DMF (1.2 mL) was stirred at r.t. for 14 h. After that the mixture was purified by preparative HPLC (neutrale Methode C, Säule 125mmX30mm, Flow 150ml/Min) to give the title compound (31.0 mg, 38% yield).
LC-MS (Method 1): Rt = 0.76 min; MS (ESIpos): m/z = 509 [M+H]+
 
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.045 (11.60), 1.063 (11.67), 1.231 (1.26), 1.372 (2.10), 1.389 (2.93), 1.409 (2.52), 1.427 (1.05), 1.565 (0.98), 1.583 (2.45), 1.602 (2.86), 1.621 (2.03), 1.638 (0.70), 2.074 (5.45), 2.270 (2.79), 2.309 (3.21), 2.317 (2.03), 2.322 (3.35), 2.326 (4.40), 2.331 (3.00), 2.336 (1.40), 2.413 (3.70), 2.430 (8.31), 2.449 (5.17), 2.518 (16.00), 2.522 (10.90), 2.659 (1.68), 2.664 (3.70), 2.668 (5.94), 2.673 (3.91), 2.678 (1.89), 2.685 (2.38), 2.710 (1.82), 2.727 (1.47), 2.839 (4.54), 2.857 (8.66), 2.874 (3.77), 3.043 (1.89), 3.059 (4.33), 3.074 (4.19), 3.090 (1.61), 3.360 (1.47), 3.376 (1.96), 3.395 (1.33), 3.577 (0.77), 3.594 (1.68), 3.611 (1.75), 3.627 (2.03), 3.643 (0.84), 3.820 (0.91), 3.836 (1.96), 3.853 (1.61), 3.869 (1.61), 3.887 (0.63), 4.809 (7.41), 4.827 (7.34), 7.425 (4.05), 7.438 (4.19), 7.655 (6.85), 7.678 (12.16), 7.723 (11.88), 7.745 (6.01), 7.932 (1.68), 7.945 (3.28), 7.959 (1.61), 8.491 (6.29), 8.504 (5.80), 8.608 (8.38), 8.637 (7.41).
Fehler! Verweisquelle konnte nicht gefunden werden.Example 42A
Figure imgf000464_0001
  5.00 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 85 (N-[(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-L- alaninamide, 220 µg, 75 % purity, 0.23 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.40 mg/mL
Drug/mAb ratio: 1.8 (UV)
 
Fehler! Verweisquelle konnte nicht gefunden werden.Example 43M
N-{4-[1-(3-aminopropyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000465_0001
A mixture of N-(4-{1-[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (1.03 g, 1.92 mmol), hydrazine monohydrate (370 µl, 7.7 mmol), and THF (23 mL) was stirred at r.t. for 14 h. After that the mixture was diluted with water and washed with ethyl acetate. The aqueous phase was treated with aqueous hydrogen peroxide solution (1.4 ml, 30 % purity, 13 mmol) and after stirring for 30 min at r.t. treated with sodium thiosulphate. The solution was concentrated under reduced pressure, the residue was purified by column chromatography (amine-coated SiO2, dichloromethane/ethanol gradient) to give the title compound (0.34 g, 88% purity, 38% yield). 21.5 mg of the product were further purified by preparative HPLC to give the pure title compound (11.6 mg, 1.4% yield).
HPLC: Instrument: Waters Autopurification system; column: Waters XBrigde C18 5µ 100x30mm; eluent A: water + 0.2 Vol-% ammonia (32%), eluent B: acetonitrile; gradient: 0.00– 0.50 min 15% B (25->70mL/min), 0.51–5.50 min 15-35% B (70mL/min), DAD scan: 210-400 nm. LC-MS (Method 3): Rt = 0.75 min; MS (ESIpos): m/z = 407 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.989 (1.02), 1.007 (2.03), 1.026 (1.40), 1.035 (2.79), 1.053 (16.00), 1.072 (13.71), 1.144 (1.02), 1.232 (1.52), 1.615 (0.89), 1.632 (1.27), 1.649 (3.81), 1.665 (5.71), 1.682 (3.94), 1.700 (1.27), 1.717 (1.27), 1.739 (5.33), 1.805 (1.90), 1.819 (1.27), 1.902 (1.02), 2.071 (1.90), 2.183 (0.51), 2.279 (2.92), 2.318 (5.71), 2.322 (7.62), 2.327 (7.87), 2.331 (5.59), 2.523 (15.24), 2.532 (10.54), 2.549 (4.57), 2.665 (5.71), 2.669 (7.75), 2.673 (5.84), 2.686 (2.41), 2.703 (2.54), 2.727 (2.16), 2.744 (1.78), 3.375 (1.90), 3.393 (2.41), 3.411 (1.78), 3.422 (1.52), 3.435 (1.40), 3.440 (1.14), 3.452 (1.14), 3.634 (1.02), 3.650 (2.03), 3.666 (2.03), 3.683 (2.41), 3.700 (1.02), 3.891 (1.14), 3.908 (2.41), 3.924 (2.03), 3.941 (1.78),   3.958 (0.89), 4.118 (0.63), 4.344 (0.76), 4.357 (1.40), 4.370 (0.76), 4.818 (8.13), 4.835 (8.00), 7.434 (4.44), 7.446 (4.32), 7.660 (6.60), 7.683 (11.56), 7.731 (11.05), 7.754 (5.71), 8.499 (6.10), 8.512 (5.59), 8.553 (0.89), 8.615 (8.76), 8.645 (7.24).
Example 44A
Figure imgf000466_0001
5.00 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 86 N5-{3-[3- {4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]propyl}-N2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-N1- 2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl-L-glutamamide (260 µg, 0.23 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.65 mg/mL
Drug/mAb ratio: 1.9 (UV) Example 44C
5 mg of anti-CD123 TPP-5969 (c=13.36 mg/mL) were coupled with Intermediate 86 N5-{3-[3- {4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]propyl}-N2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-N1-   2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl-L-glutamamide (260 µg, 0.23 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.81 mg/mL
Drug/mAb ratio: 3.3 (UV) Example 44D
5.25 mg of anti-B7H3 TPP-6497 (c=10.58 mg/mL) were coupled with Intermediate 86 N5-{3- [3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]propyl}-N2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-N1- 2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl-L-glutamamide (260 µg, 0.23 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.07 mg/mL
Drug/mAb ratio: 2.9 (UV) Example 45D
Figure imgf000467_0001
5 mg of anti-B7H3 TPP-6497 (c=10.58 mg/mL) were coupled with Intermediate 87 (N-(4-{1-[1- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,8,15-trioxo-11,12-dithia-4,7,16-triazaicosan-20-yl]-4- methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-  
2-carboxamide, 210 µg, 0.27 µmol) according procedure 4 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.36 mg/mL
Drug/mAb ratio: 3.7 (UV)
Example 45A
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 87 (N-(4-{1-[1- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,8,15-trioxo-11,12-dithia-4,7,16-triazaicosan-20-yl]-4- methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine- 2-carboxamide, 210 µg, 0.27 µmol) according procedure 4 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.78 mg/mL
Drug/mAb ratio: 3.5
  Example 46E
Figure imgf000468_0001
 
35 mg of anti-B7H3 TPP-8382 (c=15.69 mg/mL) were coupled with Intermediate 88 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2- fluorophenyl)-L-alaninamide, 980 µg, 1.20 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Protein concentration: 8.56 mg/mL
Drug/mAb ratio: 4.4 (UV)
Example 46B
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 88 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2- fluorophenyl)-L-alaninamide, 220 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Protein concentration: 1.17 mg/mL
Drug/mAb ratio: 3.5 (UV)
Example 46D
5 mg of anti-B7H3 TPP-6497 (c=10.58 mg/mL) were coupled with Intermediate 88 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2- fluorophenyl)-L-alaninamide, 220 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Protein concentration: 1.61 mg/mL
Drug/mAb ratio: 4.1
Example 46A
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 88 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2- fluorophenyl)-L-alaninamide, 220 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Protein concentration: 1.05 mg/mL
 
Drug/mAb ratio: 4.1
Example 46M
N-{4-[1-(4-amino-3-fluorobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000470_0001
Method A:
Under an argon athmosphere palladium (19 mg, 10% on charcoal, 179 µmol) was added to a solution of N-{4-[1-(3-fluoro-4-nitrobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (900 mg, 1.79 mmol) in THF (12 mL) and methanol (1.5 mL). The mixture was then stirred under an hydrogen athmosphere (1 bar) for 2 h at r.t.. After that the mixture was filtered through Celite®, the filter cake was washed with methanol and the filtrated was concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, dichloromethane/ethanol gradient, then amine-coated SiO2, dichloromethane/ethanol gradient) and preparative HPLC to give the title compound (55.0 mg, 6% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C1810µM 125x30 mm. Eluent A: water; Eluent B: acetonitrile; gradient: 0-8 min 15-55% B. rate 150 ml/min, temperature 25°C.
Method B:
A mixture of N-{4-[1-(3-fluoro-4-nitrobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (930 mg, 1.85 mmol), iron  
(1.55 g, 27.8 mmol) and acetic acid (11 mL) was heated to 90°C for 3 h. After that the mixture was filtered through Celite®, the filter cake was washed with acetic acid and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (amine-coated SiO2, dichloromethane/ethanol gradient, then SiO2, dichloromethane/ethanol gradient) to give the title compound (165 mg, 80% purity, 15% yield). LC-MS (Method 2): Rt = 0.91 min; MS (ESIpos): m/z = 473 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.993 (6.45), 1.012 (6.52), 1.053 (0.53), 1.232 (0.76), 2.053 (1.06), 2.061 (7.58), 2.069 (7.43), 2.307 (1.44), 2.318 (1.44), 2.323 (3.34), 2.327 (4.78), 2.332 (3.26), 2.337 (1.59), 2.345 (1.74), 2.349 (1.52), 2.518 (16.00), 2.523 (11.68), 2.540 (1.67), 2.660 (1.36), 2.665 (3.34), 2.669 (4.70), 2.674 (3.18), 2.679 (1.36), 2.726 (0.99), 2.743 (1.21), 2.768 (0.99), 2.784 (0.83), 3.375 (0.68), 3.394 (1.06), 3.415 (0.68), 4.625 (2.27), 4.632 (1.82), 4.662 (2.58), 4.815 (3.87), 4.832 (4.02), 4.857 (3.34), 4.877 (1.74), 4.893 (1.90), 5.057 (6.45), 6.670 (1.82), 6.691 (2.35), 6.694 (2.05), 6.714 (2.20), 6.830 (1.82), 6.835 (1.90), 6.850 (1.36), 6.855 (1.44), 6.899 (1.82), 6.903 (1.52), 6.929 (1.74), 6.934 (1.52), 7.395 (0.53), 7.408 (1.14), 7.420 (0.68), 7.432 (2.12), 7.442 (2.12), 7.649 (3.64), 7.655 (1.36), 7.667 (1.82), 7.672 (7.20), 7.677 (1.21), 7.704 (1.14), 7.710 (7.13), 7.715 (1.74), 7.727 (1.44), 7.732 (3.34), 8.478 (1.52), 8.490 (1.59), 8.497 (3.56), 8.509 (3.18), 8.580 (1.21), 8.589 (1.14), 8.592 (1.06), 8.611 (4.47), 8.640 (3.87).
Example 46M1
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]-L-valyl-rel- N-(4-{[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluorophenyl)-L-alaninamide
 
Figure imgf000472_0001
A mixture of Intermediate 88 (15.0 mg, 0.02 mmol) and L-cysteine (2.39 mg, 0.02 mmol) in DMF/water (10:1, 1.6 mL) was stirred at r.t. for 14 h. After that the mixture was purified by preparative HPLC to give the title compound (7.00 mg, 40% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x20 mm. Eluent A: water + 0.1 % TFA; Eluent B: acetonitrile; gradient: 0-22, min 10-50% B. rate 50 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.80 min; MS (ESIpos): m/z = 957 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.806 (1.03), 0.821 (2.91), 0.837 (3.49), 0.842 (3.26), 0.850 (3.66), 0.859 (1.94), 0.867 (1.66), 1.037 (3.31), 1.054 (3.37), 1.145 (1.20), 1.234 (2.00), 1.281 (3.09), 1.298 (3.09), 1.415 (1.37), 1.432 (1.66), 1.944 (0.46), 1.986 (0.57), 2.005 (0.51), 2.076 (0.69), 2.096 (0.97), 2.113 (0.74), 2.323 (2.63), 2.327 (3.54), 2.331 (2.63), 2.337 (1.60), 2.382 (1.03), 2.518 (16.00), 2.523 (10.97), 2.660 (1.49), 2.665 (2.69), 2.669 (3.54), 2.673 (2.51), 2.794 (0.74), 2.810 (0.74), 2.835 (0.57), 2.853 (0.46), 3.119 (0.40), 3.136 (0.86), 3.153 (0.69), 3.183 (0.46), 3.412 (0.69), 3.431 (0.86), 3.447 (0.69), 4.049 (0.46), 4.072 (0.57), 4.082 (0.57), 4.090 (0.57), 4.105 (0.69), 4.127 (0.40), 4.483 (0.40), 4.500 (0.46), 4.819 (2.80), 4.836   (2.46), 4.859 (1.20), 4.951 (1.03), 4.988 (0.69), 7.086 (0.86), 7.108 (0.91), 7.130 (0.63), 7.159 (0.69), 7.428 (1.37), 7.441 (1.43), 7.657 (1.26), 7.680 (2.46), 7.710 (4.11), 7.733 (1.94), 7.762 (0.69), 7.777 (0.63), 7.797 (0.80), 7.817 (0.46), 8.495 (2.17), 8.508 (2.00), 8.610 (2.80), 8.678 (0.74), 8.686 (0.69), 8.719 (0.69), 8.741 (0.51), 9.602 (0.57), 9.727 (0.86).
,
Figure imgf000473_0001
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 89 (N-{4-[1- (4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 160 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.97 mg/mL
Drug/mAb ratio: 2.8
Example 47Bb
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 89 (N-{4-[1- (4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 330  
µg, 0.53 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.27 mg/mL
Drug/mAb ratio: 7.0
Example 47Bc
35 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 89 (N-{4-[1- (4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 2.29 mg, 3.73 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 6.86 mg/mL
Drug/mAb ratio: 7.4 (UV)
Example 47Da
5 mg of anti-B7H3 TPP-6497 (c=10.58 mg/mL) were coupled with Intermediate 89 (N-{4-[1-(4- {[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 160 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.30 mg/mL
Drug/mAb ratio: 4.2 (UV)
Example 47Db
5 mg of anti-B7H3 TPP-6497 (c=16.6 mg/mL) were coupled with Intermediate 89 (N-{4-[1-(4- {[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 330 µg, 0.53 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.41 mg/mL
Drug/mAb ratio: 8.6 (UV)
 
Example 47Aa
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 89 (N-{4-[1-(4- {[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 160 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.43 mg/mL
Drug/mAb ratio: 2.8 (UV)
Example 47Ab
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 89 (N-{4-[1-(4- {[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 330 µg, 0.53 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.45 mg/mL
Drug/mAb ratio: 8.2 (UV)
Example 47M
S-{1-[6-({4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L- cysteine
 
Figure imgf000476_0001
To a solution of Intermediate 89 (N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)- hexanoyl]amino}butyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 10.0 mg, 16.3 µmol) in DMF/water (10:1, 2.6 mL) was added at r.t. L-cysteine (1.97 mg, 16.3 µmol) and the mixture stirred for 14 h at that temperature. After that the mixture purified by preparative HPLC to give the title compound (7.0 mg, 58% yield).
HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. eluent A: water + 0.1% HCOOH; eluent B: acetonitrile; gradient: 0-8, min 14-55% B. rate 150 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.64 min; MS (ESIpos): m/z = 735 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.852 (0.50), 1.052 (5.91), 1.071 (6.05), 1.149 (1.15), 1.168 (1.08), 1.233 (1.15), 1.368 (1.01), 1.392 (1.59), 1.409 (2.16), 1.431 (2.59), 1.450 (2.45), 1.469 (1.37), 1.579 (1.15), 1.597 (1.37), 1.615 (0.94), 1.986 (1.80), 2.005 (2.88), 2.024 (1.44), 2.278 (1.30), 2.318 (2.81), 2.323 (3.82), 2.327 (4.90), 2.332 (3.32), 2.337 (1.44), 2.518 (16.00), 2.523 (11.75), 2.531 (1.66), 2.541 (1.37), 2.577 (0.65), 2.587 (0.65), 2.660 (1.51), 2.665 (3.39), 2.669 (4.83), 2.673 (3.68), 2.678 (2.09), 2.693 (1.15), 2.718 (0.86), 2.728 (1.15), 2.734 (0.72), 2.775 (0.58), 2.797 (0.65), 2.810 (0.65), 2.831 (0.65), 2.889 (1.23), 3.024 (0.79), 3.040 (2.31),   3.055 (2.09), 3.073 (1.15), 3.094 (0.72), 3.118 (1.01), 3.131 (0.79), 3.141 (1.80), 3.165 (1.23), 3.178 (0.43), 3.187 (0.94), 3.299 (3.03), 3.366 (2.02), 3.377 (1.59), 3.387 (2.16), 3.399 (1.23), 3.407 (1.23), 3.419 (0.72), 3.598 (0.72), 3.614 (0.79), 3.631 (0.86), 3.826 (0.43), 3.843 (0.86), 3.860 (0.72), 3.875 (0.65), 4.022 (0.79), 4.032 (0.86), 4.045 (0.86), 4.054 (0.79), 4.078 (0.65), 4.088 (0.72), 4.100 (0.72), 4.110 (0.65), 4.829 (3.32), 4.845 (3.32), 7.431 (2.02), 7.444 (2.09), 7.667 (3.24), 7.690 (6.41), 7.723 (6.70), 7.746 (2.81), 7.844 (0.79), 7.933 (0.43), 7.948 (0.94), 7.962 (0.43), 8.497 (3.32), 8.509 (3.03), 8.612 (4.04), 8.748 (3.32).
Figure imgf000477_0001
35 mg of anti-B7H3 TPP-8382 (c=15.69 mg/mL) were coupled with Intermediate 90 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L- alaninamide, 1.02 mg, 90 % purity, 1.17 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 6.56 mg/mL
Drug/mAb ratio: 4.3
Example 48Eb
 
35 mg of anti-B7H3 TPP-8382 (c=15.1 mg/mL) were coupled with Intermediate 90 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L- alaninamide, 3.25 mg, 90 % purity, 4.15 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 7.61 mg/mL
Drug/mAb ratio: 7.8
Example 48Ba
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 90 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L- alaninamide, 230 µg, 90 % purity, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.25 mg/mL
Drug/mAb ratio: 3.4 (UV)
Example 48Bb
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 90 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L- alaninamide, 460 µg, 90 % purity, 0.53 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.25 mg/mL
Drug/mAb ratio: 6.9 (UV)
Example 48Bc
35 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 90 (N-[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]butyl}-L-alaninamide, 1.02 mg, 90 % purity, 1.17 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.  
Protein concentration: 9.25 mg/mL
Drug/mAb ratio: 3.4
Example 48Bd
35 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 90 (N-[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]butyl}-L-alaninamide, 3.25 mg, 90 % purity, 2.34 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 10.72 mg/mL
Drug/mAb ratio: 7.2 (UV)
Example 48Da
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 90 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L- alaninamide, 230 µg, 90 % purity, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.64 mg/mL
Drug/mAb ratio: 4.1 (UV)
Example 48Db
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 90 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L- alaninamide, 460 µg, 90 % purity, 0.53 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.54 mg/mL
Drug/mAb ratio: 8.1 (UV)
 
Example 48Aa
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 90 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L- alaninamide, 460 µg, 90 % purity, 0.53 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.33 mg/mL
Drug/mAb ratio: 8.1 (UV)
Example 48Ab
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 90 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L- alaninamide, 230 µg, 90 % purity, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.55 mg/mL
Drug/mAb ratio: 3.9 (UV)
Example 48G
5 mg of anti anti C4.4a TPP-509 (c=9.87 mg/mL in 0.1 sodium phosphate buffer) were coupled with Intermediate 90 (N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4- [(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]butyl}-L-alaninamide, 418 µg) according procedure 1 with the deviation that 0.1 sodium phosphate buffer was used instead of PBS, and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with 0.1 sodium phosphate buffer.
Protein concentration: 0.9 mg/mL
Drug/mAb ratio: 5.0 (UV)
Example 48M
N-{4-[1-(4-aminobutyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide  
Figure imgf000481_0001
To a solution of tert-butyl {4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)- amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}carbamate (1.10 g, 77 % purity, 1.63 mmol) in dichloromethane (70 mL) was added at r.t. trifluoroacetic acid (500 µl, 6.5 mmol) and the mixture stirred for 14 h at that temperature. After that again trifluoroacetic acid (500 µl, 6.5 mmol) was added and the mixture was heated to 45°C for 1 h. After cooling to r.t. the mixture was concentrated under reduced pressure, coevaporated twice with toluene and the crude product was purified by column chromatography (amine-coeated SiO2, dichloromethane/ethanol gradient) to give the title compound (340 mg, 48% yield).
LC-MS (Method 1): Rt = 0.52 min; MS (ESIpos): m/z = 421 [M+H]+
1H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.073 (9.18), 1.082 (2.13), 1.091 (9.09), 1.523 (1.24), 1.545 (2.23), 1.562 (1.91), 1.582 (0.96), 1.637 (0.89), 1.654 (2.15), 1.671 (2.13), 1.693 (1.40), 2.084 (10.98), 2.173 (0.52), 2.301 (2.04), 2.323 (0.67), 2.327 (0.93), 2.332 (0.86), 2.339 (2.42), 2.373 (0.66), 2.518 (2.96), 2.523 (2.04), 2.548 (0.51), 2.665 (0.64), 2.669 (0.84), 2.674 (0.65), 2.689 (1.40), 2.705 (1.80), 2.730 (1.41), 2.747 (1.24), 2.806 (1.35), 2.825 (2.24), 2.840 (2.19), 2.857 (1.22), 3.166 (3.97), 3.395 (1.09), 3.409 (1.50), 3.429 (1.05), 3.616 (0.64), 3.633 (1.36), 3.649 (1.49), 3.666 (1.71), 3.683 (0.79), 3.855 (0.79), 3.872 (1.69), 3.889 (1.45), 3.906 (1.37), 3.922 (0.62), 4.872 (16.00), 5.758 (0.97), 7.599 (2.89), 7.611 (2.94), 7.636 (0.71), 7.659 (1.93), 7.664 (6.31), 7.669 (3.51), 7.682 (4.77), 7.687 (10.88), 7.699 (2.80), 7.738 (1.98), 7.743 (8.79), 7.749 (2.75), 7.761 (2.00), 7.766 (4.86), 8.594 (4.15), 8.606 (3.99), 8.671 (0.49), 8.703 (5.73), 8.713 (6.08).
Example 48M2
N-{6-[(3S)-3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1- yl]hexanoyl}-L-valyl-N-{4-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L- alaninamide 
 
Figure imgf000482_0001
LC-MS (Method 1): Rt = 0.70 min; MS (ESIpos): m/z = 905.5 [M+H]+  ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.790 (1.05), 0.807 (1.18), 0.815 (1.12), 0.832 (1.06), 1.049 (0.90), 1.067 (0.91), 1.152 (0.66), 1.158 (0.71), 1.170 (0.71), 1.175 (0.71), 1.349 (0.47), 1.367 (0.60), 1.391 (0.44), 2.088 (0.45), 2.518 (3.55), 2.522 (2.42), 2.539 (16.00), 2.673 (0.73), 2.678 (0.52), 2.690 (0.43), 3.001 (0.40), 3.017 (0.42), 4.817 (0.68), 4.835 (0.68), 7.657 (0.58), 7.680 (1.01), 7.726 (1.01), 7.749 (0.53), 8.497 (0.47), 8.510 (0.45), 8.613 (0.70), 8.649 (0.58). 
 
Figure imgf000483_0001
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 91 (N-[(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2- fluorophenyl)-L-alaninamide, 180 µg, 0.23 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.40 mg/mL
Drug/mAb ratio: 4.3 (UV)
Example 49A
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 91 (N-[(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2- fluorophenyl)-L-alaninamide, 180 µg, 0.23 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.34 mg/mL
Drug/mAb ratio: 3.6 (UV)  
Figure imgf000484_0001
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 92 (N-[(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]- pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}- phenyl)-L-alaninamide, 180 µg, 0.23 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.77 mg/mL
Drug/mAb ratio: 3.0 (UV)
Example 50D
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 92 (N-[(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]- pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}- phenyl)-L-alaninamide, 180 µg, 0.23 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.56 mg/mL  
Drug/mAb ratio: 3.9 (UV)
Example 50Aa
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 92 (N-[(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]- pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}- phenyl)-L-alaninamide, 180 µg, 0.23 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.12 mg/mL
Drug/mAb ratio: 3.7 (UV)
Example 50Ab
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 92 (N-[(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]- pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}- phenyl)-L-alaninamide, 180 µg, 0.23 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.65 mg/mL
Drug/mAb ratio: 3.6 (UV)
 
Figure imgf000486_0001
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 93 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}- phenyl)-L-alaninamide, 220 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.83 mg/mL
Drug/mAb ratio: 2.7 (UV)
Example 51D
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 93 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}- phenyl)-L-alaninamide, 220 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.57 mg/mL
Drug/mAb ratio: 4.3 (UV)
Example 51A
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 93 (N-[6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-  
c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}- phenyl)-L-alaninamide, 220 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.53 mg/mL
Drug/mAb ratio: 3.8 (UV)
Figure imgf000487_0001
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 94 (N-{4-[1- (4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-fluorobenzyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide, 180 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.83 mg/mL
Drug/mAb ratio: 2.6 (UV)
Example 52D
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 94 (N-{4-[1-(4- {[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-fluorobenzyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide, 180 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.75 mg/mL
Drug/mAb ratio: 4.0 (UV)
 
Example 52A
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 94 (N-{4-[1-(4- {[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-fluorobenzyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide, 180 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.76 mg/mL
Drug/mAb ratio: 3.2 (UV)
Example 52M
S-(1-{6-[(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluorophenyl)amino]-6-oxohexyl}-2,5- dioxopyrrolidin-3-yl)-L-cysteine
Figure imgf000488_0001
To a solution of Intermediate 94 (N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)- hexanoyl]amino}-3-fluorobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 17.0 mg, 25.5 µmol) in DMF/water (10:1, 3.8 mL) was added at r.t. L-cysteine (3.09 mg, 25.5 µmol) and the mixture stirred for 14 h at that temperature. After that the mixture purified by two consecutive preparative HPLCs to give the title compound (4.0 mg, 20% yield).
1. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x30 mm. Eluent A:  
water + 0.1% HCOOH; Eluent B: acetonitrile; gradient: 0-8, min 15-55% B. rate 150 ml/min, temperature 25°C.
2. HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB- 1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: Chromatorex C18 10µM 125x20 mm. Eluent A: water + 0.1% TFA; Eluent B: acetonitrile; gradient: 0-22, min 15-55% B. rate 50 ml/min, temperature 25°C.
LC-MS (method 1): Rt = 0.74 min; MS (ESIneg): m/z = 785 [M-H]- 1H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.852 (0.67), 1.036 (5.69), 1.054 (5.82), 1.144 (0.74), 1.240 (2.34), 1.469 (1.41), 1.488 (1.67), 1.506 (1.34), 1.534 (1.21), 1.551 (1.34), 1.570 (0.94), 2.307 (1.27), 2.318 (2.34), 2.322 (4.69), 2.326 (5.89), 2.332 (4.02), 2.336 (2.74), 2.380 (1.54), 2.518 (16.00), 2.522 (10.98), 2.536 (0.80), 2.551 (0.87), 2.561 (0.74), 2.597 (0.67), 2.607 (0.67), 2.660 (1.34), 2.664 (3.08), 2.668 (4.22), 2.673 (3.01), 2.678 (1.34), 2.772 (0.47), 2.784 (0.60), 2.794 (0.87), 2.810 (1.27), 2.835 (0.94), 2.853 (0.74), 3.018 (0.40), 3.037 (0.47), 3.110 (0.60), 3.121 (1.07), 3.126 (1.07), 3.148 (1.27), 3.172 (1.07), 3.194 (0.94), 3.251 (0.74), 3.371 (3.35), 3.389 (1.67), 3.409 (1.07), 3.429 (1.21), 3.447 (0.80), 4.010 (0.60), 4.019 (0.67), 4.033 (0.60), 4.043 (0.54), 4.066 (0.47), 4.077 (0.47), 4.089 (0.47), 4.098 (0.40), 4.816 (5.02), 4.832 (3.88), 4.854 (2.28), 4.945 (1.94), 4.982 (1.27), 6.994 (0.60), 7.069 (1.54), 7.090 (1.61), 7.110 (1.07), 7.116 (1.34), 7.144 (1.27), 7.427 (2.28), 7.440 (2.34), 7.654 (2.81), 7.676 (6.09), 7.708 (7.23), 7.731 (3.15), 7.759 (1.54), 7.779 (0.74), 8.412 (1.41), 8.494 (3.55), 8.506 (3.28), 8.607 (4.49), 8.687 (1.87), 8.699 (1.27), 9.779 (0.87), 9.958 (0.60).
Example 53D
Figure imgf000489_0001
 
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 95 (N-{4-[1-(4- {[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-3-fluorobenzyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 160 µg, 0.27 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.83 mg/mL
Drug/mAb ratio: 4.4 (UV)
Example 53A
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 95 (N-{4-[1-(4- {[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-3-fluorobenzyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 160 µg, 0.27 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.55 mg/mL
Drug/mAb ratio: 4.3 (UV)
Example 53B
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 95 (N-{4-[1- (4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-3-fluorobenzyl)-4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide, 160 µg, 0.27 µmol) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Under these conditions a part of the ADC may also be present in the ring-cyclized form.
Protein concentration: 1.84 mg/mL
Drug/mAb ratio: 3.5 (UV)
 
Example 54B
Figure imgf000491_0001
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 96 (N-(4-{1- [4-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-6-oxo-4-phenyl-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 240 µg, 95 % purity, 0.33 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.02 mg/mL
Drug/mAb ratio: 2.7
Example 54D
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 96 (N-(4-{1-[4- ({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-6-oxo-4-phenyl-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 240 µg, 95 % purity, 0.33 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.73 mg/mL
Drug/mAb ratio: 3.1
Example 54A
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 96 (N-(4-{1-[4- ({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)butyl]-6-oxo-4-phenyl-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 240
 
µg, 95 % purity, 0.33 µmol) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.47 mg/mL
Drug/mAb ratio: 3.8
Example 55D
Figure imgf000492_0001
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 97 (N-{4-[1-(4- {[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-6-oxo-4-phenyl-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 190 µg, 95 % purity, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.47 mg/mL
Drug/mAb ratio: 4.4
Example 55A
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 97 (N-{4-[1-(4- {[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-6-oxo-4-phenyl-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 190 µg, 95 % purity, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.35 mg/mL
Drug/mAb ratio: 4.4  
Example 55B
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 97 (N-{4-[1- (4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-6-oxo-4-phenyl-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 190 µg, 95 % purity, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.51 mg/mL
Drug/mAb ratio: 4.6
Example 55M
S-{1-[6-({4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-6-oxo-4- phenyl-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L- cysteine
0 i
Figure imgf000493_0001
To a solution of Intermediate 97 (N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)- hexanoyl]amino}butyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 11.9 mg, 16.7 µmol) in DMF/water (10:1, 1.9 mL) was added at r.t. L-cysteine (2.03 mg, 16.7 µmol) and the mixture stirred for 14 h at that temperature. After that the mixture was concentrated under reduced pressure and the residue was lyophilized to give the title compound (7.6 mg, 54% yield).
LC-MS (method 1): Rt = 0.74 min; MS (ESIneg): m/z = 795 [M-H]- ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.380 (0.47), 1.398 (0.41), 1.423 (0.41), 1.446 (0.55), 1.462 (0.53), 1.907 (4.63), 2.006 (0.42), 2.024 (0.67), 2.518 (2.73), 2.523 (1.86), 2.533 (0.49), 2.543 (0.68), 2.589 (0.56), 2.728 (13.21), 2.888 (16.00), 3.048 (0.61), 3.057 (0.51), 3.066  
(0.66), 3.146 (0.44), 4.803 (0.87), 4.818 (0.83), 7.157 (0.80), 7.174 (0.96), 7.236 (0.54), 7.299 (0.77), 7.318 (0.97), 7.417 (0.52), 7.430 (0.52), 7.605 (0.82), 7.628 (1.24), 7.686 (1.35), 7.709 (0.80), 7.951 (2.08), 8.486 (0.75), 8.499 (0.70), 8.598 (1.00), 8.711 (0.72).
Example 56D
Figure imgf000494_0001
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 98
(N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-6-oxo-4-phenyl-5,6-dihydropyridazin-1(4H)- yl]butyl}-L-alaninamide, 240 µg, 95 % purity, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.74 mg/mL
Drug/mAb ratio: 4.1
Example 56B
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 98
(N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-6-oxo-4-phenyl-5,6-dihydropyridazin-1(4H)- yl]butyl}-L-alaninamide, 240 µg, 95 % purity, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 2.27 mg/mL  
Drug/mAb ratio: 3.1 (UV)
Example 56A
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 98
(N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-6-oxo-4-phenyl-5,6-dihydropyridazin-1(4H)- yl]butyl}-L-alaninamide, 240 µg, 95 % purity, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.42 mg/mL
Drug/mAb ratio: 3.6 (UV)
Example 56M
N-{4-[1-(4-aminobutyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000495_0001
To a solution of N-(4-{1-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)butyl]-6-oxo-4-phenyl- 1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (520 mg, 806 µmol) in ethanol (15 mL) was added at r.t. hydrazine monohydrate (200 µl, 4.0 mmol) and the mixture was heated to reflux for 1.5 h. After cooling to r.t. the mixture was filtrated through Celite®, the filter cake washed with ethanol and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (SiO2, dichloromethane/ethanol gradient) to give the title compound (251 mg, 90 % purity, 58 % yield). LC-MS (method 1): Rt = 0.70 min; MS (ESIneg): m/z = 481 [M-H]-  
¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.137 (3.82), 1.233 (0.89), 1.294 (0.57), 1.353 (1.71), 1.528 (2.27), 1.547 (3.49), 1.567 (3.01), 1.584 (1.38), 1.617 (1.14), 1.635 (1.14), 1.665 (1.14), 1.682 (1.79), 1.701 (2.19), 1.717 (2.36), 1.736 (2.19), 1.754 (1.95), 1.769 (8.12), 1.830 (1.79), 1.851 (0.81), 1.907 (1.30), 1.930 (6.74), 1.959 (1.46), 2.024 (0.57), 2.084 (14.21), 2.115 (1.71), 2.371 (6.74), 2.421 (5.77), 2.518 (16.00), 2.523 (10.72), 2.548 (1.62), 2.567 (3.25), 2.606 (3.17), 2.631 (1.14), 2.673 (3.33), 2.816 (1.95), 2.832 (3.09), 2.850 (3.01), 2.909 (0.89), 3.053 (2.03), 3.072 (2.92), 3.093 (2.52), 3.114 (2.19), 3.492 (3.98), 3.731 (2.03), 3.748 (2.60), 3.765 (2.92), 3.798 (1.87), 3.815 (2.76), 3.832 (2.27), 3.849 (1.79), 3.866 (1.46), 4.709 (3.17), 4.726 (3.17), 4.807 (10.40), 4.818 (10.23), 7.170 (7.47), 7.188 (8.77), 7.191 (7.96), 7.232 (1.62), 7.250 (5.20), 7.269 (3.98), 7.308 (6.90), 7.327 (8.53), 7.345 (3.09), 7.464 (4.63), 7.476 (4.71), 7.603 (8.28), 7.607 (4.87), 7.625 (12.18), 7.699 (13.16), 7.704 (6.98), 7.722 (7.88), 8.516 (6.90), 8.529 (6.34), 8.627 (10.07), 8.639 (7.96).
Example 57B
Figure imgf000496_0001
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 99
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin- 1(4H)-yl]methyl}phenyl)-L-alaninamide (220 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.71 mg/mL
Drug/mAb ratio: 3.0 (UV)
 
Example 57D
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 99
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin- 1(4H)-yl]methyl}phenyl)-L-alaninamide (220 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.48 mg/mL
Drug/mAb ratio: 4.1 (UV)
Example 57A
5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 99
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin- 1(4H)-yl]methyl}phenyl)-L-alaninamide (220 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.07 mg/mL
Drug/mAb ratio: 3.8 (UV)
Example 57M
N-{4-[1-(4-aminobenzyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Figure imgf000497_0001
A mixture of N-{4-[5,5-dimethyl-1-(4-nitrobenzyl)-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (1.20 g, 2.41 mmol), iron (2.02 g, 36.1 mmol), acetic acid (14 ml, 240 mmol), and ethanol (14 ml) was heated to 75°C for 3 h.  
After that the hot mixture was filtered through Celite®, the filter cake washed with ethanol, and the filtrate was concentrated under reduced pressure. The residue was taken up in dichloromethane, washed with 10% aqueous sodium carbonate solution, the aqueous phase extracted with dichloromethane, and the combined organic phases were washed with brine and filtered through a silicone filter. After removal of the solvent under reduced pressure the crude product was purified by column chromatography (SiO2, dichloromethane/ethanol gradient) to give the title compound (375 mg, 76% purity, 25%). 30 mg of this product were again purified by preparative HPLC to give the pure title compound (12.0 mg, 1% yield). HPLC: Instrument: Labomatic HD-3000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 4000, Knauer UV detector Azura UVD 2.15, Prepcon 5 software. Column: XBridge C18 5µM 100x30 mm. Eluent A: water + 0.1 Vol-% ammonia; Eluent B: acetonitrile; gradient: 0-20 min 10-50% B. rate 60 ml/min, temperature 25°C.
LC-MS (Method 1): Rt = 0.70 min; MS (ESIpos): m/z = 469 [M+H]+ ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.079 (16.00), 1.103 (0.64), 2.518 (1.40), 2.523 (0.99), 2.834 (4.14), 4.715 (3.73), 4.812 (2.28), 4.829 (2.30), 4.970 (2.92), 6.478 (3.15), 6.483 (0.97), 6.499 (3.34), 6.960 (2.92), 6.981 (2.64), 7.429 (1.26), 7.442 (1.30), 7.638 (1.37), 7.660 (4.50), 7.678 (4.64), 7.701 (1.27), 8.496 (1.80), 8.508 (1.73), 8.611 (2.55), 8.634 (2.29).
 
Figure imgf000499_0001
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 100
N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-5,5-dimethyl-6- oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide (170 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.66 mg/mL
Drug/mAb ratio: 4.8 (UV)
Example 58A 5 mg of anti HER2 TPP-1015 (c=12.2 mg/mL) were coupled with Intermediate 100
N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-5,5-dimethyl-6- oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide (170 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.03 mg/mL
Drug/mAb ratio: 4.6 (UV)   Example 58B 5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with Intermediate 100 N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}butyl)-5,5-dimethyl-6- oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide (170 µg, 0.27 µmol) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.80 mg/mL
Drug/mAb ratio: 3.8 (UV)
Figure imgf000500_0001
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with Intermediate 100  
 N-(4-{1-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,19-dioxo-7,10,13,16-tetraoxa-4,20- diazatetracosan-24-yl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide (243 µg, 90% purity) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.65 mg/mL
Drug/mAb ratio: 3.2 (UV)
Example 58B 5 mg of anti-CD123 TPP-6013 (c=15.1 mg/mL) were coupled with Intermediate 100
 N-(4-{1-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,19-dioxo-7,10,13,16-tetraoxa-4,20- diazatetracosan-24-yl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide (243 µg, 90% purity) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.86 mg/mL
Drug/mAb ratio: 2.8 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=14.3 mg/mL) were coupled with Intermediate 100
 N-(4-{1-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,19-dioxo-7,10,13,16-tetraoxa-4,20- diazatetracosan-24-yl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide (243 µg, 90% purity) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.55 mg/mL
Drug/mAb ratio: 2.0 (UV)
 
Figure imgf000502_0001
5 mg of anti-B7H3 TPP-6497 (c=15.16 mg/mL) were coupled with Intermediate 100
 N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}benzyl)-6-oxo-4-phenyl- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (189 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 2.05 mg/mL
Drug/mAb ratio: 1.4 (UV)
Example 58B 5 mg of anti-CD123 TPP-6013 (c=15.1 mg/mL) were coupled with Intermediate 100
 N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}benzyl)-6-oxo-4-phenyl- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (189 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.97 mg/mL
Drug/mAb ratio: 1.6 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=14.3 mg/mL) were coupled with Intermediate 100
   N-{4-[1-(4-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}benzyl)-6-oxo-4-phenyl- 1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (189 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.28 mg/mL
Drug/mAb ratio: 1.2 (UV)
Example 61D
Figure imgf000503_0001
5 mg of anti-B7H3 TPP-6497 (c=16.16 mg/mL) were coupled with Intermediate 100
 rel-N-{4-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-N²[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)hexanoyl]-D-asparagine (194 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.63 mg/mL
Drug/mAb ratio: 1.4 (UV)
Example 58B 5 mg of anti-CD123 TPP-6013 (c=15.1 mg/mL) were coupled with Intermediate 100
 rel-N-{4-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-N²-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-  
yl)hexanoyl]-D-asparagine (194 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.75 mg/mL
Drug/mAb ratio: 0.9 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=14.3 mg/mL) were coupled with Intermediate 100
 rel-N-{4-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-N²-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)hexanoyl]-D-asparagine (194 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.15 mg/mL
Drug/mAb ratio: 1.0 (UV)
Example 58M
 
Figure imgf000504_0001
Example 61M was prepared similar to example 55M by reacting the corresponding maleimide with L-cysteine.
   
LC-MS (Method 1): Rt = 0.62 min; MS (ESIpos): m/z = 849 [M+H]+  ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.053 (12.37), 1.071 (12.87), 1.144 (1.85), 1.231 (1.00), 1.387 (3.98), 1.403 (3.77), 1.447 (3.77), 1.568 (1.85), 1.584 (2.70), 1.602 (2.56), 1.990 (1.92), 2.007 (3.27), 2.022 (2.70), 2.073 (2.06), 2.277 (3.06), 2.326 (4.55), 2.331 (3.06), 2.380 (0.71), 2.403 (0.85), 2.418 (1.64), 2.440 (1.71), 2.453 (2.06), 2.465 (4.69), 2.518 (16.00), 2.522 (10.45), 2.539 (1.00), 2.586 (0.85), 2.594 (0.85), 2.632 (1.07), 2.641 (1.07), 2.678 (2.92), 2.694 (2.06), 2.718 (1.64), 2.735 (1.42), 2.912 (0.92), 2.929 (1.14), 2.945 (1.28), 2.964 (1.21), 3.015 (1.07), 3.033 (2.20), 3.047 (4.05), 3.062 (3.70), 3.082 (2.99), 3.093 (2.42), 3.109 (2.20), 3.119 (1.64), 3.131 (1.92), 3.142 (1.85), 3.155 (1.92), 3.164 (2.84), 3.172 (2.28), 3.187 (1.99), 3.198 (2.28), 3.210 (2.84), 3.226 (2.92), 3.248 (3.41), 3.405 (6.40), 3.423 (3.77), 3.493 (1.99), 3.503 (2.06), 3.512 (1.92), 3.521 (1.64), 3.578 (1.85), 3.590 (2.63), 3.603 (2.49), 3.615 (1.71), 3.632 (1.64), 3.811 (1.07), 3.828 (1.35), 3.845 (1.28), 4.203 (1.21), 4.212 (1.28), 4.225 (1.28), 4.235 (1.14), 4.285 (1.21), 4.297 (1.21), 4.308 (1.28), 4.319 (1.14), 4.392 (1.00), 4.401 (1.99), 4.406 (1.71), 4.420 (2.06), 4.440 (0.64), 4.823 (7.96), 4.841 (8.04), 7.431 (4.62), 7.443 (4.69), 7.666 (6.97), 7.688 (13.01), 7.728 (13.23), 7.750 (6.19), 7.928 (2.28), 7.945 (2.77), 8.151 (5.69), 8.164 (1.64), 8.178 (0.85), 8.225 (1.56), 8.245 (1.49), 8.496 (7.11), 8.509 (6.54), 8.612 (9.60), 8.712 (4.84). 
Example 62E
Figure imgf000505_0001
5 mg of anti-B7H3 TPP-8382 (c=16.16 mg/mL) were coupled with Intermediate 100  
(342 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.67 mg/mL
Drug/mAb ratio: 5.1 (UV)
Example 58B 5 mg of anti-CD123 TPP-6013 (c=15.1 mg/mL) were coupled with Intermediate 100
(171 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.81 mg/mL
Drug/mAb ratio: 0.8 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with Intermediate 100
(342 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.62 mg/mL
Drug/mAb ratio: 2.2 (UV)
Example 58M
S-{(3R)-1-[6-({6-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]hexyl}amino)-6- oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine 
 
Figure imgf000507_0001
Figure imgf000507_0002
  Example 62M was prepared similar to example 55M by reacting the corresponding maleimide with L-cysteine.
  LC-MS (Method 1): Rt = 0.70 min; MS (ESIneg): m/z = 761 [M-H]-  ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.050 (2.23), 1.068 (2.24), 1.156 (0.44), 1.169 (0.45), 1.176 (0.44), 1.273 (1.29), 1.349 (0.47), 1.363 (0.54), 1.445 (0.91), 1.456 (0.85), 1.580 (0.42), 1.597 (0.57), 1.986 (0.57), 2.004 (0.97), 2.022 (0.48), 2.274 (0.50), 2.313 (0.57), 2.518 (1.08), 2.523 (0.98), 2.534 (0.48), 2.677 (0.41), 2.694 (0.44), 2.718 (0.46), 2.727 (13.33), 2.888 (16.00), 2.986 (0.77), 3.000 (0.78), 3.138 (0.63), 3.184 (0.46), 3.294 (0.55), 3.304 (0.75), 3.311 (0.86), 3.329 (1.52), 3.337 (1.59), 3.346 (1.16), 3.365 (0.75), 3.387 (0.81), 3.400 (0.66), 3.425 (0.42), 4.826 (1.31), 4.843 (1.27), 7.430 (0.80), 7.443 (0.81), 7.668 (1.06), 7.691 (2.33), 7.719 (2.42), 7.742 (0.97), 7.950 (2.02), 8.496 (1.19), 8.508 (1.10), 8.611 (1.53), 8.752 (0.75), 8.758 (0.75). 
  Example 63E
Figure imgf000508_0001
5 mg of anti-B7H3 TPP-8382 (c=15.1 mg/mL) were coupled with N-{4-[1-(5-{[6-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)hexanoyl]amino}pentyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (171 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.58 mg/mL
Drug/mAb ratio: 4.3 (UV)
Example 58B 5 mg of anti-CD123 TPP-6013 (c=15.1 mg/mL) were coupled with N-{4-[1-(5-{[6-(2,5-dioxo- 2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}pentyl)-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (171 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.79 mg/mL
Drug/mAb ratio: 3.1 (UV)  
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-{4-[1-(5-{[6-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)hexanoyl]amino}pentyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (171 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.12 mg/mL
Drug/mAb ratio: 1.1 (UV)
Example 58M
S-{(3R)-1-[6-({5-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]pentyl}amino)-6- oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine 
Figure imgf000509_0001
  Example 61M was prepared similar to example 55M by reacting the corresponding maleimide with L-cysteine.
LC-MS (Method 1): Rt = 0.67 min; MS (ESIneg): m/z = 747 [M-H]-  ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 1.052 (2.25), 1.070 (2.09), 1.168 (0.59), 1.258 (0.77), 1.275 (0.62), 1.424 (1.56), 1.441 (1.49), 1.585 (0.66), 1.602 (0.77), 1.620 (0.50), 1.980 (0.72), 1.998 (1.11), 2.017 (0.57), 2.274 (0.52), 2.316 (0.63), 2.539 (0.58), 2.669  
(0.45), 2.674 (0.48), 2.694 (0.46), 2.728 (13.69), 2.888 (16.00), 2.978 (0.49), 2.994 (0.96), 3.009 (0.91), 3.123 (0.44), 3.145 (0.64), 3.169 (0.46), 3.191 (0.47), 3.336 (1.81), 3.344 (1.82), 3.384 (0.94), 3.400 (0.73), 3.421 (0.48), 3.436 (0.40), 3.623 (0.41), 3.836 (0.40), 4.827 (1.54), 4.843 (1.33), 7.430 (0.82), 7.442 (0.77), 7.670 (1.12), 7.693 (2.03), 7.722 (2.19), 7.744 (0.96), 7.950 (2.44), 8.496 (1.04), 8.508 (0.93), 8.611 (1.43), 8.754 (0.84). 
Example 64E
Figure imgf000510_0001
5 mg of anti-B7H3 TPP-8382 (c=15.1 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-rel-N-{5-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]pentyl}-D- alaninamide (213 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.63 mg/mL
Drug/mAb ratio: 2.6 (UV)
 
Example 58B 5 mg of anti-CD123 TPP-6013 (c=15.1 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-rel-N-{5-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]pentyl}-D- alaninamide (213 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.83 mg/mL
Drug/mAb ratio: 1.4 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-rel-N-{5-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]pentyl}-D- alaninamide (213 µg) according procedure 1 with the deviation that 0.1 M sodium phosphate buffer was used instead of PBS and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with 0.1 M sodium phosphate buffer.
Protein concentration: 0.58 mg/mL
Drug/mAb ratio: 3.1 (UV)
 
Example 58M
Figure imgf000512_0001
Example 64M was prepared similar to Example 48M.
  LC-MS (Method 2): Rt = 0.85 min; MS (ESIneg): m/z = 433 [M-H]-  ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.920 (0.84), 0.934 (0.46), 0.939 (1.39), 0.953 (0.46), 0.957 (0.63), 1.023 (0.49), 1.026 (0.52), 1.035 (2.48), 1.041 (1.44), 1.052 (16.00), 1.070 (12.90), 1.238 (0.65), 1.256 (1.58), 1.264 (1.39), 1.277 (2.78), 1.286 (1.80), 1.295 (2.61), 1.313 (1.66), 1.332 (1.71), 1.348 (3.02), 1.365 (3.05), 1.383 (1.77), 1.387 (1.66), 1.400 (0.82), 1.405 (0.82), 1.462 (0.65), 1.514 (0.65), 1.527 (0.65), 1.562 (1.20), 1.579 (2.67), 1.598 (3.29), 1.616 (2.45), 1.633 (1.01), 1.717 (2.53), 1.726 (2.61), 1.729 (1.20), 1.755 (1.03), 1.767 (1.12), 1.856 (1.71), 1.927 (1.52), 2.084 (0.41), 2.275 (2.67), 2.314 (3.24), 2.317 (3.27), 2.322 (1.66), 2.327 (1.74), 2.331 (1.25), 2.336 (0.57), 2.482 (10.45), 2.523 (4.14), 2.665 (1.36), 2.669 (1.90), 2.674 (1.82), 2.678 (2.23), 2.694 (2.12), 2.719 (1.71), 2.736 (1.50), 3.169 (0.49), 3.368 (1.85), 3.386 (2.10), 3.404 (1.47), 3.423 (0.68), 3.569 (0.82), 3.587 (1.69), 3.602 (1.63), 3.620 (1.96), 3.636 (0.84), 3.832 (0.93), 3.849 (1.93), 3.866 (1.39), 3.882 (1.61), 3.900 (0.71), 4.817 (7.05), 4.835 (6.83), 7.433 (3.92), 7.445 (3.89), 7.658 (6.56), 7.681 (11.81), 7.728 (11.02), 7.750 (5.44), 8.498 (6.07), 8.511 (5.52), 8.614 (7.81), 8.643 (6.15). 
 
Example 65A
Figure imgf000513_0001
35 mg of anti-HER2 TPP-1015 (c=12.2 mg/mL) were coupled with N-{4-[1-(5-{[(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)acetyl]amino}pentyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (2.20 mg) according procedure 2 with the deviation that 0.1 M sodium phosphate buffer was used instead of PBS, and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with 0.1 M sodium phosphate buffer.
Protein concentration: 8.82 mg/mL
Drug/mAb ratio: 7.8 (UV)
Example 65A1 35 mg of anti-HER2 TPP-1015 (c=12.2 mg/mL) were coupled with N-{4-[1-(5-{[(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)acetyl]amino}pentyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (2.20 mg) according procedure 2 with the deviation that 0.1 M sodium phosphate buffer was used instead of PBS, and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with 0.1 M sodium phosphate buffer.
Protein concentration: 8.7 mg/mL
Drug/mAb ratio: 5.6 (UV)
 
Example 58B 5 mg of anti-CD123 TPP-6013 (c=15.1 mg/mL) were coupled with N-{4-[1-(5-{[(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)acetyl]amino}pentyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (138 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.40 mg/mL
Drug/mAb ratio: 3.4 (UV) Example 58D 5 mg of anti-B7H3 TPP-6497(c=16.61 mg/mL) were coupled with N-{4-[1-(5-{[(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)acetyl]amino}pentyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (314 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.24 mg/mL
Drug/mAb ratio: 8.7 (UV)
Example 58G 35 mg of anti-C4.4a TPP-509 (c=14.34 mg/mL) were coupled with N-{4-[1-(5-{[(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)acetyl]amino}pentyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (2.20 mg) according procedure 2, with the deviation that the 0.1 M sodium phosphate buffer was used instead of PBS, and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 2.54 mg/mL
Drug/mAb ratio: 7.1 (UV)
 
Example 58G1 35 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-{4-[1-(5-{[(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)acetyl]amino}pentyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (2.20 mg) according procedure 2, with the deviation that the 0.1 M sodium phosphate buffer was used instead of PBS, and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 5.69 mg/mL
Drug/mAb ratio: 7.1 (UV)
Example 58G2 35 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-{4-[1-(5-{[(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)acetyl]amino}pentyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (2.20 mg) according procedure 2, with the deviation that the 0.1 M sodium phosphate buffer was used instead of PBS, and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 3.52 mg/mL
Drug/mAb ratio: 6.3 (UV)
 
Example 66B
Figure imgf000516_0001
5 mg of anti-CD123 TPP-6013 (c=15.1 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-D-alanyl-rel-N-{4-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-D- asparagine (240 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.88 mg/mL
Drug/mAb ratio: 2.6 (UV)
Example 58E 5 mg of anti-B7H3 TPP-8382 (c=15.1 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-D-alanyl-rel-N-{4-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-D- asparagine (240 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.75 mg/mL
Drug/mAb ratio: 3.2 (UV)  
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-D-alanyl-rel-N-{4-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-D- asparagine (240 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.19 mg/mL
Drug/mAb ratio: 2.1 (UV)
Example 67B
Figure imgf000517_0002
E
Figure imgf000517_0001
5 mg of anti-CD123 TPP-6013 (c=15.1 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-D-alanyl-rel-N-{4-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-D- asparagine (240 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.59 mg/mL  
Drug/mAb ratio: 3.4 (UV)
Example 58E 5 mg of anti-B7H3 TPP-8382 (c=15.1 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-D-alanyl-rel-N-{4-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-D- asparagine (240 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.21 mg/mL
Drug/mAb ratio: 4.6 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-D-alanyl-rel-N-{4-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-D- asparagine (240 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.94 mg/mL
Drug/mAb ratio: 3.1 (UV)
 
Example 58M
Figure imgf000519_0001
Example 67M was prepared similar to Example 48M.
  LC-MS (Method 2): Rt = 0.94 min; MS (ESIneg): m/z = 447 [M-H]-  ¹H-NMR (400 MHz, DMSO-d6) d [ppm]: 0.932 (0.69), 0.951 (1.29), 0.969 (0.59), 1.035 (2.44), 1.052 (16.00), 1.070 (14.18), 1.295 (8.63), 1.300 (8.53), 1.308 (9.17), 1.453 (0.44), 1.586 (2.65), 1.603 (3.60), 1.619 (2.55), 1.716 (1.82), 1.724 (0.42), 2.277 (2.85), 2.318 (3.50), 2.327 (1.52), 2.522 (5.01), 2.669 (1.54), 2.677 (2.36), 2.693 (2.38), 2.718 (1.92), 2.735 (1.68), 3.138 (0.44), 3.369 (2.18), 3.388 (2.42), 3.405 (1.70), 3.426 (0.83), 3.563 (0.85), 3.580 (1.82), 3.596 (1.90), 3.612 (2.18), 3.629 (0.99), 3.844 (1.07), 3.862 (2.22), 3.879 (1.70), 3.895 (1.82), 3.913 (0.77), 4.816 (8.30), 4.834 (8.00), 7.433 (4.40), 7.446 (4.18), 7.658 (6.48), 7.681 (11.52), 7.727 (11.49), 7.749 (5.80), 8.498 (5.88), 8.510 (5.45), 8.614 (8.08), 8.644 (6.02). 
 
Example 68B
Figure imgf000520_0001
5 mg of anti-CD123 TPP-6013 (c=15.1 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-rel-N-{6-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]hexyl}-D-alaninamide (456 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.74 mg/mL
Drug/mAb ratio: 5.9 (UV)
Example 58E 5 mg of anti-B7H3 TPP-8382 (c=15.1 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-rel-N-{6-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]hexyl}-D-alaninamide (228 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.01 mg/mL
Drug/mAb ratio: 4.1 (UV)
Example 58G
 
5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-rel-N-{6-[(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]hexyl}-D-alaninamide (456 µg) according procedure 1, with the deviation that 0.1 M sodium phosphate buffer was used instead of PBS, and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with 0.1 M sodium phosphate buffer.
Protein concentration: 1.64 mg/mL
Drug/mAb ratio: 4.6 (UV)
Example 69B
Figure imgf000521_0001
Antibody 5 mg of anti-CD123 TPP-6013 (c=15.1 mg/mL) were coupled with N-[19-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-D-valyl-rel-N-{4- [(4R)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]butyl}-D-alaninamide (264 µg) according procedure 1 and the
 
reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.24 mg/mL
Drug/mAb ratio: 3.9 (UV)
Example 58D 5 mg of anti-B7H3 TPP-6497 (c=15.1 mg/mL) were coupled with N-[19-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-D-valyl-rel-N-{4-[(4R)-3- {4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]butyl}-D-alaninamide (264 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.33 mg/mL
Drug/mAb ratio: 4.6 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-[19-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-D-valyl-rel-N-{4-[(4R)-3- {4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]butyl}-D-alaninamide (264 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.38 mg/mL
Drug/mAb ratio: 2.8 (UV)
 
Example 70B
Figure imgf000523_0001
5 mg of anti-CD123 TPP-6013 (c=15.16 mg/mL) were coupled with N-(4-{1-[6-({5-[(2,5- dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)hexyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (227 µg) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.69 mg/mL
Drug/mAb ratio: 4.2 (UV)
Example 58D 5 mg of anti-B7H3 TPP-6497 (c=15.1 mg/mL) were coupled with N-(4-{1-[6-({5-[(2,5- dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)hexyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (227 µg) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.09 mg/mL
Drug/mAb ratio: 4.7 (UV)
Example 58G
 
5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-(4-{1-[6-({5-[(2,5- dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)hexyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (227 µg) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.33 mg/mL
Drug/mAb ratio: 2.8 (UV)
Example 71A
Figure imgf000524_0001
5 mg of anti-HER2 TPP-1015 (c=12.2 mg/mL) were coupled with rel-N-(4-{(4R)-1-[6-({N²-[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-N²-(14-oxo-2,5,8,11-tetraoxatetradecan-14- yl)-D-lysyl}amino)hexyl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide (527 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Protein concentration: 1.18 mg/mL
Drug/mAb ratio: 9.1 (UV)
 
Example 58E 5 mg of anti-B7H3 TPP-8382 (c=15.1 mg/mL) were coupled with rel-N-(4-{(4R)-1-[6-({N²-[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-N²-(14-oxo-2,5,8,11-tetraoxatetradecan-14- yl)-D-lysyl}amino)hexyl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide (527 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Protein concentration: 0.99 mg/mL
Drug/mAb ratio: 9.4 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with rel-N-(4-{(4R)-1-[6-({N²-[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-N²-(14-oxo-2,5,8,11-tetraoxatetradecan-14- yl)-D-lysyl}amino)hexyl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide (527 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Protein concentration: 0.30 mg/mL
Drug/mAb ratio: 7.1 (UV)
 
Example 72A
Figure imgf000526_0001
Antibody 5 mg of anti-HER2 TPP-1015 (c=12.2 mg/mL) were coupled with N-(4-{1-[1-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-3,19-dioxo-7,10,13,16-tetraoxa-4,20-diazahexacosan-26-yl]-4-methyl- 6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide (452 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.33 mg/mL
Drug/mAb ratio: 2.7 (UV)
Example 58E 5 mg of anti-B7H3 TPP-8382 (c=14.08 mg/mL) were coupled with N-(4-{1-[1-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-3,19-dioxo-7,10,13,16-tetraoxa-4,20-diazahexacosan-26-yl]-4-methyl- 6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide (452 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.33 mg/mL
Drug/mAb ratio: 2.5 (UV)
 
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with rel-N-(4-{(4R)-1-[6-({N²-[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-N²-(14-oxo-2,5,8,11-tetraoxatetradecan-14- yl)-D-lysyl}amino)hexyl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide (527 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS. Protein concentration: 1.23 mg/mL
Drug/mAb ratio: 1.1 (UV)
Example 73A
Figure imgf000527_0001
Epimer 1
5 mg of anti-HER2 TPP-1015 (c=12.2 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-L-alanyl-N-{6-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]hexyl}-D- asparaginyl-rel-L-glutamic acid (563 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.81 mg/mL
Drug/mAb ratio: 8.2 (UV)
 
Example 58E 5 mg of anti-B7H3 TPP-8382 (c=14.08 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-L-alanyl-N-{6-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]hexyl}-D- asparaginyl-rel-L-glutamic acid (563 µg) according procedure 1, with the deviation that 0.1M sodium phosphate buffer was used instead of PBS, and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with 0.1M sodium phosphate buffer.
Protein concentration: 1.38 mg/mL
Drug/mAb ratio: 8.1 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-L-alanyl-N-{6-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]hexyl}-D- asparaginyl-rel-L-glutamic acid (282 µg) according procedure 1, with the deviation that 0.1M sodium phosphate buffer was used instead of PBS, and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with 0.1M sodium phosphate buffer.
Protein concentration: 1.25 mg/mL
Drug/mAb ratio: 6.3 (UV)
 
Example 74A
Figure imgf000529_0001
5 mg of anti-HER2 TPP-1015 (c=12.2 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-L-alanyl-N-{6-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]hexyl}-D- asparaginyl-rel-L-glutamic acid (563 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.45 mg/mL
Drug/mAb ratio: 3.9 (UV)
Example 58E 5 mg of anti-B7H3 TPP-8382 (c=14.08 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-L-alanyl-N-{6-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]hexyl}-D- asparaginyl-rel-L-glutamic acid (563 µg) according procedure 1, with the deviation that 0.1M sodium phosphate buffer was used instead of PBS, and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with 0.1M sodium phosphate buffer.  
Protein concentration: 1.31 mg/mL
Drug/mAb ratio: 8.0 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-D-valyl-L-alanyl-N-{6-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4- c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]hexyl}-D- asparaginyl-rel-L-glutamic acid (282 µg) according procedure 1, with the deviation that 0.1M sodium phosphate buffer was used instead of PBS, and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with 0.1M sodium phosphate buffer.
Protein concentration: 1.29 mg/mL
Drug/mAb ratio: 5.9 (UV)
Example 75D
Figure imgf000530_0001
5 mg of anti-B7H3 TPP-6497 (c=16.61 mg/mL) were coupled with N-(4-{1-[5-({5-[(2,5- dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)pentyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (222 µg) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.60 mg/mL
Drug/mAb ratio: 2.7 (UV)
 
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-(4-{1-[5-({5-[(2,5- dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)pentyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (222 µg) according procedure 3 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.34 mg/mL
Drug/mAb ratio: 1.3 (UV)
Example 76A
)
Figure imgf000531_0001
 
5 mg of anti-HER2 TPP-1015 (c=12.2 mg/mL) were coupled with N-(4-{1-[1-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazatetracosan-24-yl]-4-methyl- 6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide (437 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.48 mg/mL
Drug/mAb ratio: 5.0 (UV)
Example 58E 5 mg of anti-B7H3 TPP-8382 (c=14.08 mg/mL) were coupled with N-(4-{1-[1-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazatetracosan-24-yl]-4-methyl- 6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide (437 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.07 mg/mL
Drug/mAb ratio: 8.9 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=11.88 mg/mL) were coupled with N-(4-{1-[1-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazatetracosan-24-yl]-4-methyl- 6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide (437 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.26 mg/mL
Drug/mAb ratio: 6.8 (UV)
 
Example 77A
Figure imgf000533_0001
5 mg of anti-HER2 TPP-1015 (c=12.2 mg/mL) were coupled with rel-N-{5-[(4R)-3-{4-[(1,3- dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]pentyl}-N²-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L- asparagine (366 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.46 mg/mL
Drug/mAb ratio: 8.3 (UV)
Example 58E 5 mg of anti-B7H3 TPP-8382 (c=14.08 mg/mL) were coupled with rel-N-{5-[(4R)-3-{4-[(1,3- dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]pentyl}-N²-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L- asparagine (366 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.50 mg/mL
Drug/mAb ratio: 9.0 (UV)  
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with rel-N-{5-[(4R)-3-{4-[(1,3- dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]pentyl}-N²-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]-L- asparagine (366 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.62 mg/mL
Drug/mAb ratio: 6.7 (UV)
Example 78A
)
Figure imgf000534_0001
 
5 mg of anti-HER2 TPP-1015 (c=12.2 mg/mL) were coupled with N-(4-{1-[1-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-3,19-dioxo-7,10,13,16-tetraoxa-4,20-diazapentacosan-25-yl]-4- methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine- 2-carboxamide (444 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.46 mg/mL
Drug/mAb ratio: 9.4 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-(4-{1-[1-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-3,19-dioxo-7,10,13,16-tetraoxa-4,20-diazapentacosan-25-yl]-4- methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine- 2-carboxamide (444 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.49 mg/mL
Drug/mAb ratio: 8.2 (UV)
Example 80A
Figure imgf000535_0001
 
5 mg of anti-HER2 TPP-1015 (c=12.2 mg/mL) were coupled with N-{4-[1-(6-{[(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)acetyl]amino}hexyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (312 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration:1.59 mg/mL
Drug/mAb ratio: 8.0 (UV)
Example 58E 5 mg of anti-B7H3 TPP-8382 (c=14.08 mg/mL) were coupled with N-{4-[1-(6-{[(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)acetyl]amino}hexyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (312 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.58 mg/mL
Drug/mAb ratio: 8.7 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with N-{4-[1-(6-{[(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)acetyl]amino}hexyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (312 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.15 mg/mL
Drug/mAb ratio: 5.1 (UV)
 
Example 81A
)
Figure imgf000537_0001
5 mg of anti-HER2 TPP-1015 (c=12.2 mg/mL) were coupled with rel-N-(4-{(4R)-1-[6-({N2-[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-N6-(74-oxo- 2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71- tetracosaoxatetraheptacontan-74-yl)-D-lysyl}amino)hexyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (997 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration:1.77 mg/mL
Drug/mAb ratio: 8.4 (UV)
Example 58E 5 mg of anti-B7H3 TPP-8382 (c=15.1 mg/mL) were coupled with rel-N-(4-{(4R)-1-[6-({N2-[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-N6-(74-oxo-  
2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71- tetracosaoxatetraheptacontan-74-yl)-D-lysyl}amino)hexyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (997 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 2.07 mg/mL
Drug/mAb ratio: 8.5 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=9.87 mg/mL) were coupled with rel-N-(4-{(4R)-1-[6-({N2-[6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-N6-(74-oxo- 2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71- tetracosaoxatetraheptacontan-74-yl)-D-lysyl}amino)hexyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (997 µg) according procedure 1 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.73 mg/mL
Drug/mAb ratio: 7.1 (UV)
Figure imgf000538_0001
 
5 mg of anti-HER2 TPP-1015 (c=12.2 mg/mL) were coupled with N-(4-{1-[1-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazapentacosan-25-yl]-4-methyl- 6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide (444 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.02 mg/mL
Drug/mAb ratio: 6.6 (UV)
Example 58E 5 mg of anti-B7H3 TPP-8382 (c=15.1 mg/mL) were coupled with N-(4-{1-[1-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazapentacosan-25-yl]-4-methyl- 6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide (444 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 1.19 mg/mL
Drug/mAb ratio: 7.9 (UV)
Example 58G 5 mg of anti-C4.4a TPP-509 (c=11.9 mg/mL) were coupled with N-(4-{1-[1-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)-2,18-dioxo-6,9,12,15-tetraoxa-3,19-diazapentacosan-25-yl]-4-methyl- 6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide (444 µg) according procedure 2 and the reaction was, after Sephadex purification, concentrated by ultracentrifugation and rediluted with PBS.
Protein concentration: 0.33 mg/mL
Drug/mAb ratio: 3.2 (UV)
CELL PROLIFERATION ASSAY for NAMPT-ADCs and NAMPT-SMOLs
Cell Titer Glo (CTG) METHOD  
Cells were plated in 75 µL growth medium per well in a 96-well plate (white/clear bottom, (#10775584, Perkin Elmer) at the indicated cell number (Table 1). The plates were incubated overnight at 37°C.24h after seeding of cells, test compounds were serially diluted in growth medium, and 25µL of four fold concentrated dilutions/well were added to the test plates. For antibody-drug-conjugates, typically semi-logarithmic dilutions from 300 nM to 0.03 nM in triplicates were used. The compound treated plates were incubated for 72 or 96 hrs at 37°C as indicated. In parallel to the addition of test compounds, time zero Cell Titer Glo Luminescent Cell Viability Assay (CTG) levels were measured in sister plates. To this end, 75 µL per well CTG solution (Promega, catalog # G755B and G756B) was added to cells in sister plates, incubated for 10 min, and luminescence was measured on a VICTOR V instrument (Perkin Elmer). After incubation for either 72 or 96 hrs (Table 1) in the presence of test compounds, 100 µl per well CTG solution was added to all test wells, incubated for 10 min and luminescence was measured on a VICTOR V instrument. Dose response curves and calculation of IC50 values (50% inhibition of proliferation) were generated using BELLA-Dose Response Curve (DRC) spreadsheets. The DRC software is a Biobook Spreadsheet that was developed by Bayer AG and Bayer Business Services on the IDBS E-Workbook Suite platform (IDBS: ID Business Solutions Ltd., Guildford, UK). Table 1: Assay conditions for cell lines in CTG cell proliferation assay
Figure imgf000540_0001
 
Figure imgf000541_0001
 
Figure imgf000542_0001
The analysis of the cytotoxic effects of the anti-C4.4a-ADCs was carried out with a panel of cell lines: MDA-MB-453: C4.4a positive A549-C4.4a B4: C4.4a positive A549-mock: C4.4a negative FaDu: C4.4a positive A431: C4.4a positive The analysis of the cytotoxic effects of the anti-HER2-ADCs was carried out with a panel of cell lines: MDA-MB-453: HER2 positive SK-OV-3: HER2 positive NCI-N87. HER2 positive
 
Cytotoxicity assay The biological activity of the compounds according to the invention can be shown in the assays described below: C-1a Determination of the cytotoxic effects of the ADCs The analysis of the cytotoxic effects of the anti-CD123-ADCs was carried out with a panel of cell lines: MOLM-13: humane akute monozytäre Leukämiezellen (AML-M5a), DSMZ, No. ACC 554, standard medium: RPMI 1640 (Fa. Gibco; #21875- 059, stab. L-Glutamin) + 20% heat inactivated FCS (Fa. Gibco, No.10500-064); CD123-positive. THP-1: human monocytic leukemia cell line , ATCC, NO. TIB-202, standard medium: RPMI 1640 (Fa. Gibco; #21875-059, stab. L-Glutamin) + 10% heat inactivated FCS (Fa. Gibco, No. 10500-064) + 2,5g Glucose (20 % Glucose Solution, Fa. Gibco, No.19002); CD123-positive. KG-1: bone marrow derived human acute myeloid leukemia cell line, DSMZ , No. ACC 14, standard medium: RPMI 1640 (Gibco; #21875-059, stab. L-Glutamin) + 10% heat inactivated FCS (Fa. Gibco, No. 10500-064) + 2,5g Glucose (20 % Glucose Solution, Fa. Gibco, No.19002); CD123-positive. MV-4-11: human peripheral blood derived macrophage of biphenotypic B myelomonocytic leukemia, ATCC No. CRL-9591, standard medium: IMDM (ATCC:30-2005) + 10% heat inactivated FCS (Fa. Gibco, No.10500-064); CD123-positive. NOMO-1: bone marrow derived human acute myeloid leukemia cell line (APL = AML FAB M5a), DSMZ , No. ACC 542, standard medium: RPMI 1640 (Gibco; #21875-059, stab. L- Glutamin) + 10% heat inactivated FCS (Fa. Gibco, No. 10500-064) + 2,5g Glucose (20 % Glucose Solution, Fa. Gibco, No.19002); CD123-positive. NB-4: bone marrow derived human acute promyelocytic leukemia cell line (APL = AML FAB M3), DSMZ, No. ACC 207, standard medium: RPMI 1640 (Gibco; #21875-059, stab. L- Glutamin) + 10% heat inactivated FCS (Fa. Gibco, No. 10500-064) + 2,5g Glucose (20 % Glucose Solution, Fa. Gibco, No.19002); CD123-negative. Cultivation of cells was performed after standard procedure recommended by American Tissue Culture Collection (ATCC) or by Leibniz-Institut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) for the respective cell line.
 
MTT-assay The test was carried out by collecting the cells after centrifugation, resuspending them in culture medium, counting and seeding the cells into a 96-well culture plate with white bottom (Costar #3610; MV-4-11: 5000 cells//well; MOLM-13: 5000 cells//well; THP-1: 8000 cells//well; KG-1: 5000 cells//well, NOMO-17000 cells//well, NB45000 cells//well in 100µl total volume).). The cells were then incubated in an incubator at 37°C and 5% carbon dioxide. After 5 h, the antibody drug conjugates were added in 10 µL of culture medium in concentrations of from 10- 5M to 10-13M to the cells (triplicates) and incubated in an incubator at 37°C and 5% carbon dioxide. After 96h, the proliferation was measured using the MTT assay (ATCC, Manassas, Virginia, USA, catalogue No.30-1010K). At the end of the selected incubation time, the MTT reagent was added and incubated with the cells for 4h, followed by lysis of the cells overnight by addition of the detergent. The dye formed was detected at 570nm (Infinite M1000 pro, Fa. Tecan). Based on the measured data the IC50 value was determined from the DRC (dose response curve). The proliferation of cells which were not treated with test substance but were otherwise treated identically was defined as the 100% value. The presented data refer to the examples described in the experimental part with the respective drug/antibody ratio. The data might change due to different drug/antibody ratios. The reported IC50-values represent mean values derived from several independent experiments or a single value. The biological effects of the anti-CD123 antibody-drug– conjugates were CD123 dependent. This could be confirmed by parallel treatment of the CD123-negative cell line NB4 with CD123-ADC where no cytotoxic activity could be observed. (Table 2) Table 2 below lists the IC50 values of representative working examples for different anti-CD123- ADCs from this assay:
Figure imgf000544_0001
 
Figure imgf000545_0001
 
Figure imgf000546_0001
NAMPT biochemical assay (hNAMPT IC50) Nicotinamide phosphoribosyltransferase (NAMPT) inhibitory activity of compounds of the present invention was quantified employing a cascade assay as described in the following paragraphs. The assay couples the conversion of nicotinamide (NAM) to nicotinamide mononucleotide (NMN) by NAMPT with the conversion of NMN to nicotine adenine dinucleotide (NAD+) by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) and the subsequent quantification of the generated NAD+ by a commercial detection kit (NAD/NADH- Glo™ Assay from Promega, # G9072). N-terminally His6-tagged recombinant full length human NAMPT and N-terminally His6-tagged recombinant full length human NMNAT1, both expressed in E. coli and purified via Ni-NTA- affinity chromatography and consecutive size exclusion chromatography, were used as enzymes. For the assay 50 nl of a 100fold concentrated solution of the test compound in DMSO was pipetted into a white low volume 384well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2.5 µl of a solution of NAMPT in aqueous NMNAT1-containing assay buffer [50 mM Tris/HCl pH 7.5, 12 mM MgCl2, 0.6 mM adenosine-tri-phosphate (ATP), 1 nM NMNAT1, 0.02 % (w/v) bovine serum albumin (Sigma-Aldrich # P7906), 0.001% (v/v) Tween-20 (Sigma- Aldrich # P7949)] were added and the mixture was incubated for 15 min at 22°C to allow pre- binding of the test compounds to the enzyme before the start of the enzyme reaction. Then the reaction was started by the addition of 2.5 µl of a solution of NAM (300 nM => final conc. in the 5 µl assay volume is 150 nM, Sigma-Aldrich #47865) and 5-phosphorylribose-1- pyrophosphate pentasodium salt (PRPP, 1.2 µM => final conc. in the 5 µl assay volume is 0.6 µM, Sigma-Aldrich P8296) in assay buffer and the resulting mixture was incubated for a reaction time of 20 min at 22°C. The concentration of NAMPT was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical final concentration in the 5 µl assay volume was 0.16 nM. The NAM conversion was stopped and the detection of the generated NAD+ started by the addition of 2.5 µl of a solution of 600 nM FK866, a NAMPT inhibitor commercially available (e.g. from Selleckchem), in  
detection reagent solution (1:4.5 fold dilution of NAD/NADH-Glo™ Detection Reagent [Promega] in water). The resulting mixture was incubated 2 h at 22°C to allow a steady-state of the detection system. Subsequently the generated luminescence was measured in a suitable luminescence reader, e.g. a Viewlux™ (Perkin-Elmer), and taken as a measure for the generated NAD+. The data were normalised (enzyme reaction without inhibitor = 0 % inhibition, all other assay components but no NAMPT = 100 % inhibition). Usually the test compounds were tested on the same microtiter plate in 11 different concentrations in the range of 20 µM to 0.1 nM (20 µM, 5.9 µM, 1.7 µM, 0.51 µM, 0.15 µM, 44 nM, 13 nM, 3.8 nM, 1.1 nM, 0.33 nM and 0.1 nM, the dilution series prepared separately before the assay on the level of the 100fold concentrated solutions in DMSO by serial 1:3.4 dilutions) in duplicate values for each concentration and IC50 values were calculated by a 4-parameter-fit.
  ty assay
C50 [mol/l]
Figure imgf000548_0001
ty assay
C50 [mol/l]
Figure imgf000549_0001
Figure imgf000550_0001
Figure imgf000551_0001
Figure imgf000552_0001
Figure imgf000553_0001
Figure imgf000554_0001
Figure imgf000555_0001
eration y NCI- 96h_ [mol/l] 2 E-10 4 E-10 9 E-8 2 E-8 0 E-8
6 E-11
0 E-11
Figure imgf000556_0001
eration y NCI- 96h_ [mol/l] 5 E-11
9 E-10
9 E-11
Figure imgf000557_0001
eration y NCI- 96h_ [mol/l] ADC
Figure imgf000558_0001
ADC
invention: tion MG 72H -11 E-12 -9
Figure imgf000559_0001
tion
MG 72H E-12 -9
Figure imgf000560_0001
tion
MG 72H
-11 -7 -9
-8 -10 -10
Figure imgf000561_0001
tion
MG 72H
-9
-8
ADU EO-PROLIF-A431 H CTG72HADC
IC50 [mol/l]
2.54 E-7 > 3.00 E-7
Figure imgf000562_0001
ADU EO-PROLIF-A431
H CTG72HADC
IC50 [mol/l]
> 3.00 E-7 > 3.00 E-7 3.38 E-8 10 3.23 E-9
8
Figure imgf000563_0001
ADU EO-PROLIF-A431
H CTG72HADC
IC50 [mol/l] -7
7
8
8
8
7
Figure imgf000564_0001
C-6 Determination of in vivo efficacy The in vivo anti-tumor efficacy of the invented NAMPT conjugates was tested in xenograft models, a method known as state of the art (e.g. WO 2005/081711; Polson et al., Cancer Res. 2009 Mar 15;69(6):2358-64). Rodents were implanted with tumor cells such as THP-1 and MV4-11 (human AML) or U251-MG (human glioblastoma) or MDA-MB-453 (human breast carcinoma) expressing the antigen of interest such as B7H3 or CD123 or C4.4a. After establishment of the tumor animals were treated by NAMPT conjugates containing binding antibodies and compared to the group treated with vehicle only. Treatment was either a single- dose or a repeated dose. Tumor areas were compared and showed reduced tumor growth of animals treated with binding NAMPT conjugates. C6a. Anti-tumor efficacy in tumor-bearing mice Tumor cells were subcutaneously inoculated in mice, 5 million THP-1 or MV4-11 cells in 100% matrigel in female C.B-17 Scid mice or 2 million U251-MG cells in matrigel/medium (1:1) in female NMRI nude mice or 5 million MDA-MB-453 cells in 100% matrigel in female NOD Scid mice or,. Established tumors of a size of approx.40 mm2 were measureable after 5 days (THP- 1 and MV4-11) or 17 days (U251 MG) or 35 days (MDA-MB-453) and indicated the date of treatment start. The invented conjugates were applied intravenously via the tail vein in a volume of 5 ml/kg. In the THP-1, MV4-11 and U251-MG models the treatment schedule was two times a week (every third or fourth day) for two weeks but was reduced to three treatments in total once per week. In the MDA-MB-453 model the treatment schedule was once per week. Per group 8-10 animals were treated and subcutaneous tumor growth measured by a caliper (tumor length x tumor width = tumor area). As a parameter of anti-tumor efficacy the ratio of the tumor area of the vehicle treated control group versus the tumor area of the conjugate-treated animals is taken. In all tested tumor models NAMPT-conjugates showed anti-tumor efficacy due to target mediated internalization of the conjugates. THP-1 cells express B7H3 as well as CD123, MV4- 11 and U251-MG cells express B7H3 only, MDA-MB-453 cells express C4.4a and HER2. Isotype-NAMPT conjugates showed also target-unspecific effects which were less pronounced and can be attributed to enhanced endocytotic activity in particular in THP-1 and MV4-11 cells. Table 7 shows the T/C values based on tumor area on defined day (D) after start of treatment.Table 7
 
Figure imgf000566_0001
 

Claims

Claims 1. A conjugate of a binder or a derivative thereof with one or more molecules of an active compound that has the formula:
Figure imgf000567_0001
wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8, and D stands for an active component of Formula (I):
Figure imgf000567_0002
wherein: A represents:
Figure imgf000567_0003
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2;
t is 0, 1, 2 or 3; R2 represents H, C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl, wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, and -N(R6)R7;   R3 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8); R4 represents C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl;
R5 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8), q is 0, 1, 2 or 3,
m is 0, 1, 2 or 3,
with the proviso that q + m is 2, 3 or 4;
Figure imgf000568_0001
represents a group which is selected from:
Figure imgf000568_0002
in which * and # represent the points of attachment of said group with, the rest of the compound of formula (I),
said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, R6(H)N- and -N(R6)R7;
R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl); R8, R9 represent, independently of each other, hydrogen, C1-C3-alkyl, C3-C6-cycloalkyl or - C(=O)-(C1-C3-alkyl); or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer.  
2. The conjugate according to claim 1, wherein:
A represents:
Figure imgf000569_0001
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2; t is 0, 1, 2 or 3; R2 represents H, C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy and -N(H)R6, -N(R6)R7 ;
R3 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or
R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S , S(=O), S(=O)2; R4 represents C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl;
R5 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or
R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S , S(=O), S(=O)2, q is 0, 1, 2 or 3,
m is 0, 1, 2 or 3,
with the proviso that q + m is 2, 3 or 4 ;
 
Figure imgf000570_0001
represents a group which is selected from :
Figure imgf000570_0002
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I) ,
said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, and C1-C3-haloalkoxy- ;
R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl, or -C(=O)-(C1-C3- alkyl) ; R8 represents, independently of each other hydrogen, C1-C3-alkyl, C3-C6-cycloalkyl or - C(=O)-(C1-C3-alkyl) ; or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer. 3. The conjugate according to claim 1 or 2, wherein:
A represents:
Figure imgf000570_0003
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2; t is 0, 1, 2 or 3; R2 represents H, C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy and -N(H)R6, -N(R6)R7 ; and   R3 represents H or C1-C3-alkyl; or
R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8; R4 represents C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl;
R5 represents H or C1-C3-alkyl; or
R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8; q is 0, 1, 2 or 3,
m is 0, 1, 2 or 3,
with the proviso that q + m is 2,
3 or 4 ;
Figure imgf000571_0002
) represents a group which is selected from :
Figure imgf000571_0003
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I) ;
R6, R7 represent, independently of each other C1-alkyl or C3-cycloalkyl; R8 represents, independently of each other hydrogen, C1-C3-alkyl or C3-C6-cycloalkyl ; or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer.
4. The conjugate according to any one of claims 1 to 3, wherein:
A represents:
Figure imgf000571_0001
  wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy or -NH2, -N(H)R6;
t is 0, 1 or 2; R2 represents H, C1-C4-alkyl, C3-C6-cycloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy and C1-C3-haloalkoxy;
R3 represents H or C1-C3-alkyl; or
R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8; R4 represents C1-C4-alkyl, C3-C6-cycloalkyl, C1-C3-haloalkyl or phenyl;
R5 represents H or C1-C3-alkyl; or
R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O and NR8;
Figure imgf000572_0003
represents a group selected from :
Figure imgf000572_0002
in which * represents the point of attachment of said group with the rest of the compound of formula (I) ;
represents a group which is selected from :
Figure imgf000572_0001
Figure imgf000573_0001
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I),
R6 represents, independently of each other, C1-alkyl or C3-cycloalkyl;
R8 represents, independently of each other, hydrogen, or C1-C3-alkyl; or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer.
5. The conjugate according to any one of claims 1 to 4, wherein:
A represents:
Figure imgf000573_0002
wherein * represents the point of attachment to the rest of the compound of formula (I) and # represents the point of attachment to linker Z’; R1 represents, independently of each other, F, Cl, Br, hydroxy, methyl, CF3, methoxy, - NH2, or -N(H)(CH3);
t is 0, 1 or 2, preferably 0; R2 represents H, methyl, propan-2-yl or phenyl,
R3 represents H, or methyl; R4 represents methyl;
R5 represents H or methyl;
Figure imgf000573_0003
represents a group selected from :
 
Figure imgf000574_0001
preferably
Figure imgf000574_0002
in which * represents the point of attachment of said group with the rest of the compound of formula (I);
represents:
Figure imgf000574_0003
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I), or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer.
6. The conjugate according to any one of claims 1-5, wherein the linker–Z’- represents one of the following general structures (i) to (iii): (i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ wherein § represents the attachment point to D; §§ represents the attachment point to AB; SG represents an in vivo cleavable group, L1 and L1’ represent, independently of each other, an in vivo non-cleavable organic group, and L2 represents an attachment group.  
7. The conjugate according to claim 6, wherein the in vivo cleavable group SG represents a 2- 8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal.
8. The conjugate according to claim 6 or 7, wherein L1 and L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -S-, -SO-, SO2, -NH-, -CO-, -NMe-, -NHNH-, -SO2NHNH-, -NHCO-, -CONH-, -CONHNH- , arylene groups, heteroarylene groups, , straight C1-C6-alkylene groups, branched C1-C6- alkylene groups, C3-C7-cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the group consisting of N, O and S, -SO- or–SO2-
(preferably
Figure imgf000575_0002
optionally substituted with one or more substituents selected from the group consisting of halogen, -NHCONH2, -COOH, -OH, -NH2, NH-CNNH2, sulphonamide, sulphone, sulphoxide or sulphonic acid.
9. The conjugate according to any one of claims 6 to 8, wherein L2 represents:
Figure imgf000575_0001
 
Figure imgf000576_0001
#1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG.
10. The conjugate according to any one of claims 1 to 9, wherein the linker–Z’- represents one of the following general structures (i) to (iii): (i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ wherein § represents the attachment point to D; §§ represents the attachment point to AB; SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal; L1, L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -S-, -SO-, SO2, -NH-, -CO-, -NMe-, -NHNH-, -SO2NHNH-, -NHCO-, -CONH-, - CONHNH-, arylene groups, heteroarylene groups, straight C1-C6-alkylene groups,   branched C1-C6-alkylene groups, C3-C7-cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the group consisting of
N, O and S, -SO- or–SO2- (preferably
Figure imgf000577_0003
optionally substituted with one or more substituents selected from the group consisting of halogen, -NHCONH2, -COOH, -OH, -NH2, NH-CNNH2, sulphonamide, sulphone, sulphoxide or sulphonic acid; L2 represents:
Figure imgf000577_0002
wherein #1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG.
11. The conjugate according to any one of claims 6 to 10, wherein L2 represents one or more of the following three formulae:
Figure imgf000577_0001
wherein  
#1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG, wherein in a preferred embodiment over 60% of the attachment points to the binder, even more preferred over 80% of the attachment points to the binder, preferably over 90% of the attachment points to the binder, preferably over 95% of the attachment points to the binder in respect to the total number of attachments of the linker to the binder, are represented by one of the two structures:
Figure imgf000578_0001
wherein, in a particularly preferred embodiment, the amide group at #2 is connected to L1, L1’ or SG via the group–CH2-C(O)-.
12. The conjugate according to any one of claims 6 to 11, wherein SG is a 2-8 oligopeptide.
13. The conjugate according to claim 12, wherein the 2-8 oligopeptide consists of amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, citrulline and valine.
14. The conjugate according to any one of claims 6 to 13, wherein L1 and L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from:
-O-, -NH-, -CO-, -NHCO-, -CONH-, phenyl and
Figure imgf000578_0002
__ ; in which * and # represent the points of attachment of said group with the rest of the compound,
 
optionally substituted with one or more substituents independently selected from the group consisting of -F, -Cl, -COOH, -OH, and -NH2.
15. The conjugate according to any one of claims 6 to 14, wherein L1 and L1’ represent, independently of each other, one of the general structures (iv) or (v): (iv) –A’-(NR10CO)-B’- (v) –A’-(CONR10)-B’- wherein: A’ represents C1-C6 alkyl, (C1-C2 alkyl)-(phenylene), and (C1-C3 alkyl)-(NR11)-(C2 alkyl); optionally substituted with one or more substituents independently selected from–F and -Cl; B’ represents a straight-chain or branched hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted once or more than once by one or more groups independently selected from: -O-, -NH-, -CO-, -NHCO-, and -CONH-; optionally substituted with–COOH; R10, R11 represent, independently of each other hydrogen or C1-C3 alkyl; or R10, R11 together with the nitrogens to which they are attached form a 6-membered nitrogen containing heterocycloalkyl group.
16. A conjugate of general formula (II)
, /
Figure imgf000579_0001
wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8; wherein:   A represents:
Figure imgf000580_0001
wherein * represents the point of attachment to the rest of the compound and # represents the point of attachment to linker Z’; R1 represents, independently of each other, halogen, hydroxy, C1-C3-alkyl, C1-C3- haloalkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, -N(R6)R7 or -NH2;
t is 0, 1, 2 or 3; R2 represents H, C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl,
wherein phenyl is optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -N(H)R6, and -N(R6)R7 ; R3 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R2 and R3 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S , S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8); R4 represents C1-C6-alkyl, C3-C6-cycloalkyl, C1-C4-haloalkyl or phenyl;
R5 represents H, C1-C3-alkyl or C1-C3-haloalkyl; or R4 and R5 together with the carbon to which they are attached form a C3-C6-cycloalkyl group or a 5- to 7-membered heterocycloalkyl group containing one heteroatom containing group selected from O, NR8, S, S(=O), S(=O)2, S(=NR8)(=NR9) and S(=O)(=NR8), q is 0, 1, 2 or 3,
m is 0, 1, 2 or 3,
with the proviso that q + m is 2, 3 or 4 ;
 
Figure imgf000581_0002
represents a group which is selected from :
Figure imgf000581_0003
in which * and # represent the points of attachment of said group with the rest of the compound of formula (I),
said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, R6(H)N- and -N(R6)R7;
R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl) ; R8, R9 represent, independently of each other, hydrogen, C1-C3-alkyl, C3-C6-cycloalkyl or - C(=O)-(C1-C3-alkyl); -Z’- represents one of the following general structures (i) to (iii): (i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ wherein § represents the attachment point to A; §§ represents the attachment point to AB; SG represents a 2-8 oligopeptide group, preferably a dipeptide group or a tripeptide group, or a disulfide, a hydrazone, a glycoside, an acetal or an aminal; L1, L1’ represent, independently of each other, a straight-chain or branched hydrocarbon chain having 1 to 40 carbon atoms which may be interrupted once or more than once by one or more of -O-, -S-, -SO-, SO2, -NH-, -CO-, -NMe-, -NHNH-, -SO2NHNH-, -NHCO-, - CONH-, -CONHNH-, arylene groups, heteroarylene groups, cyclic alkylene groups and 5- to 10-membered heterocyclic groups having up to 4 heteroatoms selected from the
group consisting of N, O and S, -SO- or–SO2- (preferably
Figure imgf000581_0001
  in which * and # represent the points of attachment of said group with the rest of the compound, optionally substituted with one or more substituents selected from the group consisting of halogen, -NHCONH2, -COOH, -OH, -NH2, NH-CNNH2, sulphonamide, sulphone, sulphoxide or sulphonic acid; L2 represents:
Figure imgf000582_0001
wherein #1 represents the attachment point to the binder, #2 represents the attachment point to the group L1, L1’ or SG; or the enantiomers, diastereomers, salts, solvates or salts of solvates thereof.
17. The conjugate according to any one of the above claims, wherein:
Figure imgf000582_0002
in which * represents the point of attachment of said group with the rest of the compound;
  represents unsubstituted
Figure imgf000583_0002
Figure imgf000583_0003
in which * and # represent the points of attachment of said group with the rest of the compound, t is 0, q is 1, and m is 1, or the enantiomers, diastereomers, salts, solvates or salts of solvates thereof.
18. The conjugate according to any one or all of the above claims, which is selected from the group consisting of:
Figure imgf000583_0001
 
Figure imgf000584_0001
Figure imgf000585_0001
 
Figure imgf000586_0001
 
Figure imgf000587_0001
 
Figure imgf000588_0001
Figure imgf000589_0001
 
Figure imgf000590_0001
 
Figure imgf000591_0001
Figure imgf000592_0001
 
Figure imgf000593_0001
 
Figure imgf000594_0001
 
Figure imgf000595_0001
 
( s
Figure imgf000596_0001
 
Figure imgf000597_0001
Figure imgf000598_0001
Figure imgf000599_0001
 
Figure imgf000600_0001
 
Figure imgf000601_0001
Figure imgf000602_0001
 
Figure imgf000603_0001
Figure imgf000604_0001
 
Figure imgf000605_0001
Figure imgf000606_0001
Figure imgf000607_0001
Figure imgf000608_0001
wherein n is a number from 1 to 50, and the antibody is preferably selected from an anti-HER2- antibody, an an anti-CD123-antibody, an anti-B7H3-antibody, an anti-C4.4a-antibody, or an antigen binding fragment thereof.
19. A compound selected from the group consisting of:
tert-butyl {4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5- dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}carbamate
N-{4-[1-(4-aminobutyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide trifluoroacetate
tert-butyl N2-(tert-butoxycarbonyl)-N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-L- asparaginate
tert-butyl {3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5- dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}carbamate
N-{4-[1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(3-hydroxypropyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-(4-{1-[3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)propyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide S-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]propyl} ethanethioate
tert-butyl N2-(tert-butoxycarbonyl)-N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-L- asparaginate
ethyl 4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]butanoate
4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]butanoic acid
tert-butyl {3-[(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}benzoyl)amino]propyl}carbamate  
tert-butyl {(28S)-35-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]-27,31-dioxo-2,5,8,11,14,17,20,23-octaoxa- 26,32-diazapentatriacontan-28-yl}carbamate
N5-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]propyl}-N1-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl-L- glutamamide
L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluorophenyl)-L-alaninamide trifluoroacetate
N-(tert-butoxycarbonyl)-L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L-alaninamide L-valyl-N-{4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}-L-alaninamide
N-{4-[4-methyl-1-(4-nitrobenzyl)-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(4-aminobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L- alaninamide
L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L-alaninamide
N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-5,5-dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L- alaninamide
L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5- dimethyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}phenyl)-L-alaninamide trifluoroacetate methyl 4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}benzoate
4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]methyl}benzoic acid
N-{4-[1-{4-[(3-aminopropyl)carbamoyl]benzyl}-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide  
N-{4-[1-(3-fluoro-4-nitrobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-(tert-butoxycarbonyl)-L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2- fluorophenyl)-L-alaninamide
L-valyl-N-(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluorophenyl)-L-alaninamide
N-(4-{1-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)butyl]-6-oxo-4-phenyl-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide N-{4-[5,5-dimethyl-1-(4-nitrobenzyl)-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(4-nitrobenzyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(4-aminobenzyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N,N'-(disulfanediylbis{(1-oxopropane-3,1-diyl)iminobutane-4,1-diyl[4-methyl-6-oxo-5,6- dihydropyridazine-1,3(4H)-diyl]benzene-4,1-diyl})bis(1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2- carboxamide)
N,N'-(disulfanediylbis{propane-3,1-diyl[4-methyl-6-oxo-5,6-dihydropyridazine-1,3(4H)- diyl]benzene-4,1-diyl})bis(1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide)
N-{4-[4-methyl-6-oxo-1-(3-sulfanylpropyl)-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5-dimethyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}-L-asparagine N-{4-[1-(3-aminopropyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide hydrochloride
N-{3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo- 5,6-dihydropyridazin-1(4H)-yl]propyl}-L-asparagine hydrochloride
tert-butyl 4-{2-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}piperazine-1-carboxylate  
N-(4-{4-methyl-6-oxo-1-[2-(piperazin-1-yl)ethyl]-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
tert-butyl {4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}carbamate
tert-butyl {3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}carbamate
N-{4-[1-(2-hydroxyethyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide N-(4-{1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-4-methyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide trifluoroacetate N-{4-[1-(2-aminoethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide N-{4-[1-(2-hydroxyethyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide N-(4-{1-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl]-5,5-dimethyl-6-oxo-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide N-{4-[1-(2-aminoethyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide {1-[2-({2-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-5,5-dimethyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}amino)-2-oxoethyl]-2,5-dioxopyrrolidin-3-yl}-L- cysteine trifluoroacetate {1-[2-({2-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}amino)-2-oxoethyl]-2,5-dioxopyrrolidin-3-yl}-L- cysteine trifluoroacetate S-{(3S)-1-[6-({3-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]propyl}amino)-6-oxohexyl]-2,5-dioxopyrrolidin-3- yl}-L-cysteine  
S-{(3S)-1-[6-(4-{2-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]ethyl}piperazin-1-yl)- 6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine
N-(4-{4-methyl-6-oxo-1-[4-({3-[(3-sulfanylpropanoyl)amino]propyl}carbamoyl)benzyl]-1,4,5,6- tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide S-{1-[6-({3-[(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4- methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}benzoyl)amino]propyl}amino)-6- oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine
N6-[5-({4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6- oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-5-oxopentanoyl]-L-lysine
N-{4-[4-methyl-6-oxo-1-{4-[(3-sulfanylpropanoyl)amino]butyl}-1,4,5,6-tetrahydropyridazin-3- yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(3-aminopropyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
N-{4-[1-(4-amino-3-fluorobenzyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide
Methyl N2-(tert-butoxycarbonyl)-N6-[5-({4-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-5- oxopentanoyl]-L-lysinate
N2-(tert-butoxycarbonyl)-N6-[5-({4-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2- ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-5- oxopentanoyl]-L-lysine
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]-L-valyl-rel- N-(4-{[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluorophenyl)-L-alaninamide
S-{1-[6-({4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L- cysteine
N-{4-[1-(4-aminobutyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide
S-(1-{6-[(4-{[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl- 6-oxo-5,6-dihydropyridazin-1(4H)-yl]methyl}-2-fluorophenyl)amino]-6-oxohexyl}-2,5- dioxopyrrolidin-3-yl)-L-cysteine  
S-{1-[6-({4-[3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-6-oxo-4- phenyl-5,6-dihydropyridazin-1(4H)-yl]butyl}amino)-6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L- cysteine
N-{4-[1-(4-aminobutyl)-6-oxo-4-phenyl-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide,
N-{4-[1-(4-aminobenzyl)-5,5-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide, 
N2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)hexanoyl]-N-{4-[3-{4- [(1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-ylcarbonyl)amino]phenyl}-4-methyl-6-oxo-5,6- dihydropyridazin-1(4H)-yl]butyl}-L-asparagine,S-{(3R)-1-[6-({6-[(4S)-3-{4-[(1,3-dihydro-2H- pyrrolo[3,4-c]pyridine-2-carbonyl)amino]phenyl}-4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)- yl]hexyl}amino)-6-oxohexyl]-2,5-dioxopyrrolidin-3-yl}-L-cysteine,
S-{(3R)-1-[6-({5-[(4S)-3-{4-[(1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carbonyl)amino]phenyl}- 4-methyl-6-oxo-5,6-dihydropyridazin-1(4H)-yl]pentyl}amino)-6-oxohexyl]-2,5-dioxopyrrolidin- 3-yl}-L-cysteine,
N-{4-[1-(5-aminopentyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide, and
N-{4-[1-(6-aminohexyl)-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridine-2-carboxamide,
or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N- oxide, tautomer or stereoisomer.
20. The conjugate according to any one or all of the above claims, wherein the linker Z’ is bound to a cysteine side chain on the binder AB.
21. The conjugate according to any one or all of the above claims, wherein the binder or a derivative thereof is a binding peptide or–protein or a derivative of a binding peptide or– protein.
22. The conjugate according to any one or all of the above claims, wherein each molecule of the active component is binding to different amino acids of the binding peptide or–protein or their derivatives respectively, via a linker.
 
23. The conjugate according to any one or all of the above claims, wherein the conjugate averages 1.2 to 50 molecules of the active components per binder.
24. The conjugate according to any one of claims 20 to 23, wherein the binding peptide or protein represents an antibody or wherein the derivative of the binding peptide or -protein comprises one of the following groups:
espectively.
Figure imgf000615_0001
25. The conjugate according to any one of the above claims, wherein the binder binds to a cancer target-molecule.
26. The conjugate according to claim 25, wherein the binder is binding to an extracellular target molecule.
27. The conjugate according to claim 26, wherein the binder, after binding to the extracellular target molecule, is internalized in the expressing cell of the target molecule and is processed intracellularly, preferably through the lysosomal pathway.
28. The conjugate according to any one of claims 21 to 27, wherein the binding peptide or– protein is a human, humanized or chimeric monoclonal antibody, or an antigen-binding fragment thereof.
29. The conjugate according to claim 28, wherein the binding peptide or -protein is, an anti- HER2-antibody; anti-CD123-antibody; anti-B7H3-antibody; an anti C4.4a-antibody; or an antigen binding fragment thereof.
 
30. A metabolite obtainable by the cleavage of any of the conjugates as defined in claims 1 to 18.
31. The metabolite according to claim 30, wherein the metabolite does not comprise a cysteine and/or a lysine residue of the binder protein or peptide.
32. A method of preparing the conjugate of any one of claims 1 to 18, according to either of the following reaction schemes:
Figure imgf000616_0001
wherein AB, n, R1, R2, R3, R4, R5, L1, L1’, t, q, m, V, W, Z and Y are defined as for any one of the preceeding claims, Q represents one of the following general structures (i) to (iii): (i) §–L1-SG-§§ (ii) §–L1-SG-L1’-§§ (iii) §–L1-§§  
wherein § represents the attachment point to D; §§ represents the attachment point to L2’; SG represents an in vivo cleavable group, L1 and L1’ represent, independently of each other, an in vivo non-cleavable organic group, and L2’ represents an activated attachment group; and R2, R3, R4 and R5 are connected at the positions shown for group A.
33. Use of a conjugate or a compound according to any one or all of the above claims for the treatment or prophylaxis of a disease.
34. The use of a conjugate or a compound according to claim 33, wherein the disease is a hyperproliferative disease and/or a disorder responsive to induction of cell death.
35. The use of a conjugate or a compound according to claim 34, wherein the hyperproliferative disease and/or disorder responsive to induction of cell death is a haematological tumour, solid tumour and/or metastases thereof.
36. The use of a conjugate or a compound according to claim 35, wherein the hyperproliferative disease and/or disorder is a cancer disease.
37. The use of a conjugate or a compound according to claim 36, wherein said cancer is selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), breast cancer (particularly HER2-positive breast), ovarian cancer (particularly HER2-positive ovarian cancer), gastric cancer (particularly HER2-positive gastric cancer), brain tumors (particularly glioblastoma), lung cancer, squamous cell carcinoma of head and neck (particularly pharynx squamous cell carcinoma) and epidermoid carcinoma of the vulva and/or metastases thereof.
38. The use of a conjugate or a compound according to claim 36 or 37, wherein said cancer disease is a cancer deficient in nicotinic acid pathway.  
39. A pharmaceutical composition comprising a conjugate or a compound according to any one or all of the above claims, together with at least one pharmaceutically acceptable carrier or auxiliary.
40. The composition according to claim 39 for the treatment of a haematological tumour, a solid tumour and/or metastases thereof.
41. A pharmaceutical combination comprising one or more first active ingredients selected from a conjugate or a compound according to any one or all of the above claims, and:
a) one or more second active ingredients selected from chemotherapeutic anti-cancer agents and target-specific anti-cancer agents;
b) radiation therapy ; and/or
c) a method or an agent which causes or induces DNA damage.
42. A conjugate of a binder or a derivative thereof with one or more molecules of an active component, wherein the active component is a NAMPT inhibitor, which is conjugated to the binder via a linker Z’.
43. The conjugate according to claim 42, wherein said conjugate has the formula:
Figure imgf000618_0001
wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8, and D stands for a NAMPT inhibitor of Formula (IVa):
Figure imgf000618_0002
wherein # represents the point of attachment to linker Z’;
 
in which: C1 represents a phenylene, heteroarylene (preferably a 6-membered heteroarylene), 5- to 7-membered heterocycloalkylene (preferably a 6-membered heterocycloalkylene) or C3-C6- cycloalkylene (preferably a C6-cycloalkylene) group, in which C3-C6-cycloalkylene is optionally partially unsaturated, said groups being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, R6(H)N- and -N(R6)R7; A1 represents a group selected from 5- to 7-membered heterocycloalkylene, 6 to 10-membered fused heterocycloalkylene and heteroarylene, said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, oxo (C=O), C1-C6-alkyl, C1-C4-alkoxy, -NH2, R6(H)N-, - N(R6)R7, C3-C6-cycloalkyl and phenyl, in which C1-C6-alkyl and C1-C4-alkoxy groups are optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, -N(H)R6 and -N(R6)R7, in which phenyl and C3-C6-cycloalkyl groups are optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, -N(H)R6 and -N(R6)R7; q is 0, 1, 2 or 3 (preferably 1),
m is 0, 1, 2 or 3 (preferably 1),
with the proviso that q + m is 2, 3 or 4; R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl); wherein the active component is conjugated to the binder via a linker Z’.  
44. The conjugate according to claim 42 or 43, wherein said conjugate has the formula:
Figure imgf000620_0001
wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8, and D stands for a NAMPT inhibitor of Formula (IVb):
Figure imgf000620_0002
wherein # represents the point of attachment to linker Z’; in which: A1 represents a group selected from 5- to 7-membered heterocycloalkylene, 6 to 10-membered fused heterocycloalkylene and heteroarylene, said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, oxo (C=O), C1-C6-alkyl, C1-C4-alkoxy, -NH2, R6(H)N-, - N(R6)R7, C3-C6-cycloalkyl and phenyl, in which C1-C6-alkyl and C1-C4-alkoxy groups are optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, -N(H)R6 and -N(R6)R7, in which phenyl and C3-C6-cycloalkyl groups are optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, -N(H)R6 and -N(R6)R7; q is 0, 1, 2 or 3 (preferably 1),
m is 0, 1, 2 or 3 (preferably 1),
  with the proviso that q + m is 2, 3 or 4;
Figure imgf000621_0001
represents a group which is selected from:
Figure imgf000621_0002
in which * and # represent the points of attachment of said group with the rest of the compound, said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, C1-C3-alkyl, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, R6(H)N- and -N(R6)R7,
(preferably represents unsubstituted
Figure imgf000621_0003
R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl); wherein the active component is conjugated to the binder via a linker Z’ (Z’ is as defined in the aspects and/or embodiments defined herein).
45. The conjugate according to any one of claims 42 to 44, wherein said conjugate has the formula:
Figure imgf000621_0004
wherein AB stands for a binder, Z’ stands for a linker, n stands for a number between 1 and 50, preferably 1.2 to 20 and especially preferred 2 to 8, and D stands for a NAMPT inhibitor of Formula (IVc):
Figure imgf000621_0005
wherein # represents the point of attachment to linker Z’; in which: A1 represents a group selected from 5- to 7-membered heterocycloalkylene, 6 to 10-membered fused heterocycloalkylene and heteroarylene, said group being optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, oxo (C=O), C1-C6-alkyl, C1-C4-alkoxy, -NH2, R6(H)N-, - N(R6)R7, C3-C6-cycloalkyl and phenyl, in which C1-C6-alkyl and C1-C4-alkoxy groups are optionally substituted with one or more substituents independently selected from the group consisting of:
halogen, hydroxy, cyano, C1-C3-alkoxy, C1-C3-haloalkoxy, -NH2, -N(H)R6 and -N(R6)R7, R6, R7 represent, independently of each other, C1-C3-alkyl, C3-C6-cycloalkyl or -C(=O)-(C1-C3- alkyl); wherein the active component is conjugated to the binder via a linker Z’.
46. The conjugate according to any one of claims 42 to 45, wherein: linker Z’ is as defined in any one of claims 6 to 16 or 18.
47. The conjugate according to any one of claims 42 to 46, wherein: binder AB is as defined in any one of claims 18, or 20 to 29.
48. The conjugate according to any one of the above claims, wherein linker Z’ represents one of the following general structures (i) to (iii): (i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ wherein
 
§ represents the attachment point to A (formula (I) and formula (II)), or to A1 (formula (IVa), formula (IVb) and formula (IVc)); §§ represents the attachment point to binder AB; L1 and L1’, independently of one another, are as defined in any one of the rows of Table A or Table B; r independently of one another represents a number from 1 to 20, preferably from 1 to 15, particularly preferably from 2 to 20, especially preferably from 2 to 10; and SG and L2 are as defined in any one of the above claims.
49. The conjugate according to any one of the above claims, wherein linker Z’ represents one of the following general structures (i) to (iii): (i) §–L1-SG-L2-§§ (ii) §–L1-SG-L1’-L2-§§ (iii) §–L1-L2-§§ wherein § represents the attachment point to A (formula (I) and formula (II)), or to A1 (formula (IVa), formula (IVb) and formula (IVc)); §§ represents the attachment point to binder AB; and L1, SG, L1’ and L2 are as defined in any one of the rows of Table C or Table D.
50. The conjugate according to any one of the above claims, wherein SG comprises (C- terminus)-Ala-Val-(N-terminus) or (C-terminus)-Cit-Val-(N-terminus) or -S-S-, particularly SG is (C-terminus)-Ala-Val-(N-terminus) or -S-S-.
51. The conjugate according to any one of the above claims, wherein the binding peptide or - protein is, an anti-HER2-antibody; anti-CD123-antibody; anti-B7H3-antibody; an anti C4.4a- antibody; or an antigen binding fragment thereof.
 
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