WO2022218970A2 - Pyrrolobenzodiazepine conjugates - Google Patents

Pyrrolobenzodiazepine conjugates Download PDF

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Publication number
WO2022218970A2
WO2022218970A2 PCT/EP2022/059730 EP2022059730W WO2022218970A2 WO 2022218970 A2 WO2022218970 A2 WO 2022218970A2 EP 2022059730 W EP2022059730 W EP 2022059730W WO 2022218970 A2 WO2022218970 A2 WO 2022218970A2
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group
alkyl
methyl
conjugate according
methoxy
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PCT/EP2022/059730
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French (fr)
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WO2022218970A3 (en
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Philip Wilson Howard
Luke Masterton
Arnaud Charles Tiberghien
Ian Hutchinson
Stephen John Gregson
Thais CAILLEAU
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Medimmune Limited
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Publication of WO2022218970A3 publication Critical patent/WO2022218970A3/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
    • A61K47/68035—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a pyrrolobenzodiazepine
    • 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
    • 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
    • A61K47/6863—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 stomach or intestines 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • PBD pyrrolobenzodiazepine
  • PBDs pyrrolobenzodiazepines
  • Antibiotics, 41, 1366-1373 (1988)), prothracarcin 20 (Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, et al., J.
  • PBDs are of the general structure: 25 They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring.
  • All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring.
  • the PBD dimers are thought to form sequence-selective DNA lesions such as the palindromic 5’-Pu-GATC-Py-3’ interstrand cross-link (Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al., Biochemistry, 44, 4135-4147) which is thought to be mainly responsible for their biological activity.
  • the first dimers (Bose, D.S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992)) were of the general formula: where n is from 3 to 6. The compounds where n were 3 and 5 showed promising cytoxicity 20 in vitro.
  • This compound 30 008188229 3 was found to have “exquisite cytotoxicity in the picomolar region ... some 9000-fold more potent that DSB-120.” This compound (also discussed in Gregson, S., et al., J. Med.
  • WO 2007/085930 describes the preparation of dimer PBD compounds having linker groups 5 for connection to a cell binding agent, such as an antibody.
  • the linker is present in the bridge linking the monomer PBD units of the dimer.
  • Dimer PBD compounds having linker groups for connection to a cell binding agent, such as an antibody are described in WO 2011/130598.
  • the linker in these compounds is 10 attached to one of the available N10 positions, and are generally cleaved by action of an enzyme on the linker group.
  • the dimer PBD compounds have either endo or exo unsaturation in the C-ring.
  • WO2014/096368 discloses PBD dimer conjugates linked via a N10 where the PBD moieity 15 which is not linked to the antibody is non-alkylating. Disclosure The present disclosure provides conjugates comprising PBD dimers linked via the N10 of a non-alkylating PBD moiety (i.e. the released moiety has a secondary amine at the N10 20 position, rather than an imine or equivalent group), and the precursor drug linkers used to make such conjugates.
  • a first aspect of the present disclosure provides conjugates of formula I: L - (D L ) p (I) 25 wherein L is a Ligand unit (i.e., a targeting agent), p is from 1 to 20, D L is a Drug Linker unit of formula I’:
  • I' R LL is a linker for connection to the Ligand Unit, which is 008188229 5 O H LL N G Q X IIIa' , wherein Q is: , where Q X is such that Q is an amino-acid residue, a dipeptide residue, 5 a tripeptide residue, or a tetrapeptide issue;
  • G LL is a linker connected to the Ligand Unit;
  • 10 D represents either group D1 or D2: D1 D2 ; the dotted line indicates the optional presence of a double bond between C2 and C3
  • R N2 is H or C 1-4 alkyl
  • aromatic rings e.g. benzene or pyridine
  • Y and Y’ are selected from O, S, and NH
  • R 6’ , R 7’ , and R 9’ are selected from the same groups as R 6 , R 7 and R 9 respectively
  • the Ligand unit is a targeting agent that binds to a target moiety.
  • the Ligand unit can, for example, specifically bind to a cell component (a Cell Binding Agent) or to other target molecules of interest.
  • the Ligand unit can be, for example, a protein, polypeptide or peptide, such as an antibody, an antigen-binding 15 fragment of an antibody, or other binding agent, such as an Fc fusion protein.
  • At least the N10 position bearing a linking group releases a non-alkylating PBD (i.e. the released moiety has a secondary amine at the N10 position, rather than an imine or equivalent group).
  • Dimers containing at least one secondary amine moiety are unable to 20 covalently cross-link DNA, and may be less toxic than dimers with two imine moieties (which can covalently cross-link DNA).
  • avoiding the presence of a C11 hydroxy group adjacent the carbamate on the N10 nitrogen may increase the stability of the carbamate. 25
  • the proximity of the hydroxy to the carbonyl of the carbamate allows for the formation of an internal hydrogen bond which could catalyse a more facile nucleophilic attack on the carbonyl.
  • a second aspect of the present disclosure comprises a compound with the formula II: II and salts and solvates thereof, wherein D, R 2 , R 6 , R 7 , R 9 , Y, R”, Y’, D’, R 22 , R 6’ , R 7’ , R 9’ , R 20 and R 21 (including the presence 5 or absence of double bonds between C2 and C3 and C2’ and C3’ respectively) are as defined in the first aspect of the disclosure; R L is a linker for connecting to a Ligand unit, which is: , where Q and X are as defined in the first aspect and G L is a linker for connecting to a10 Ligand unit.
  • a third aspect of the present disclosure provides the use of a conjugate of the first aspect of the disclosure in the manufacture of a medicament for treating a proliferative disease.
  • the third aspect also provides a conjugate of the first aspect of the disclosure for use in the 15 treatment of a proliferative disease.
  • the third aspect also provides a method of treating a proliferative disease comprising administering a therapeutically effective amount of a conjugate of the first aspect of the disclosure to a patient in need thereof.
  • One of ordinary skill in the art is readily able to determine whether or not a candidate 20 conjugate treats a proliferative condition for any particular cell type. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described in the examples below.
  • a fourth aspect of the present disclosure provides the synthesis of a conjugate of the first aspect of the disclosure comprising conjugating a compound (drug linker) of the second aspect of the disclosure with a Ligand Unit.
  • a fifth aspect of the present disclosure provides a method of making a compound of formula IV: from a compound of formula V: 10 using the Mitsunobu reaction; where R 8 is selected from: (a) OMe, OCH2Ph, OH, OProt O and -Y’-R”-Hal; (b) ; and 15 (c) 008188229 12 20 R 9' 21 R R N Y' Y H R'' Vc N 7' R 6' D' O R where D, R 2 , R 6 , R 7 , R 9 , Y, R”, Y’, D’, R 22 , R 6’ , R 7’ , R 9’ , R 20 and R 21 are as defined in the first aspect of the disclosure; 5 Hal is a halogen
  • a sixth aspect of the present disclosure provides a compound of formula VI: D, R 2 , R 6 , R 7 , R 9 , Y, R”, Y’, D’, R 6’ , R 7’ , R 9’ and R 22 (including the presence or absence of double bonds between C2 and C3 and C2’ and C3’ respectively) are as defined in the first20 aspect of the disclosure.
  • Substituents The phrase “optionally substituted” as used herein, pertains to a parent group which may be unsubstituted or which may be substituted. 5 Unless otherwise specified, the term “substituted” as used herein, pertains to a parent group which bears one or more substituents.
  • substituted is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group.
  • substituents are well known, and 10 methods for their formation and introduction into a variety of parent groups are also well known. Examples of substituents are described in more detail below.
  • C1-12 alkyl as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated).
  • C1-4 alkyl as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a 20 carbon atom of a hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated).
  • alkyl includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below. 25 Examples of saturated alkyl groups include, but are not limited to, methyl (C 1 ), ethyl (C 2 ), propyl (C 3 ), butyl (C 4 ), pentyl (C 5 ), hexyl (C 6 ) and heptyl (C 7 ).
  • saturated linear alkyl groups include, but are not limited to, methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), n-butyl (C 4 ), n-pentyl (amyl) (C 5 ), n-hexyl (C 6 ) and n-heptyl (C 7 ).
  • saturated branched alkyl groups include iso-propyl (C 3 ), iso-butyl (C 4 ), sec-butyl (C 4 ), tert-butyl (C 4 ), iso-pentyl (C 5 ), and neo-pentyl (C 5 ).
  • C 2-12 Alkenyl refers to an alkyl group having35 one or more carbon-carbon double bonds.
  • C2-12 alkynyl refers to an alkyl group having one or more carbon-carbon triple bonds.
  • unsaturated alkynyl groups include, but are not limited to, ethynyl (-C ⁇ CH) and 2-propynyl (propargyl, -CH2-C ⁇ CH).
  • C3-12 cycloalkyl refers to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.
  • cycloalkyl groups include, but are not limited to, those derived from: saturated monocyclic hydrocarbon compounds: cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C 7 ), methylcyclopropane (C 4 ), dimethylcyclopropane (C 5 ), methylcyclobutane (C 5 ), 20 dimethylcyclobutane (C 6 ), methylcyclopentane (C 6 ), dimethylcyclopentane (C 7 ) and methylcyclohexane (C 7 ); unsaturated monocyclic hydrocarbon compounds: cyclopropene (C 3 ), cyclobutene (C 4 ), cyclopentene (C 5 ), cyclohexene (C 6 ), methylcyclopropene (C 4 ), dimethylcyclopropene (C 5 ), methylcyclobuten
  • C 3-20 heterocyclyl refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms.
  • each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
  • 35 008188229 15 the prefixes (e.g. C3-20, C3-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms.
  • C5-6heterocyclyl pertains to a heterocyclyl group having 5 or 6 ring atoms.
  • monocyclic heterocyclyl groups include, but are not limited to, those derived from: N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5),10 piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7); O1: oxirane (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxole (dihydrofuran) (C5)
  • substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C 5 ), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C 6 ), such as allopyranose, 30 altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.
  • furanoses C 5
  • arabinofuranose such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse
  • pyranoses C 6
  • allopyranose 30 altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.
  • C 5-20 aryl refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which 35 moiety has from 5furth to 20 ring atoms.
  • C 5-7 aryl refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of 008188229 16 an aromatic compound, which moiety has from 5 to 7 ring atoms
  • C5-10 aryl pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 10 ring atoms.
  • each ring has from 5 to 7 ring atoms. 5
  • the prefixes e.g.
  • C3-20, C5-7, C5-6, C5-10, etc. denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms.
  • C5-6 aryl as used herein, pertains to an aryl group having 5 or 6 ring atoms. 10
  • the ring atoms may be all carbon atoms, as in “carboaryl groups”. Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e.
  • phenyl (C6), naphthalene (C10), azulene (C10), anthracene (C14), phenanthrene (C14), naphthacene (C18), and pyrene (C16).
  • aryl groups which comprise fused rings include, but are not limited to, groups derived from indane (e.g.2,3-dihydro-1H- indene) (C9), indene (C9), isoindene (C9), tetraline (1,2,3,4-tetrahydronaphthalene (C10), acenaphthene (C 12 ), fluorene (C 13 ), phenalene (C 13 ), acephenanthrene (C 15 ), and 20 aceanthrene (C 16 ).
  • the ring atoms may include one or more heteroatoms, as in “heteroaryl groups”.
  • Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from: 25 N 1 : pyrrole (azole) (C 5 ), pyridine (azine) (C 6 ); O 1 : furan (oxole) (C 5 ); S 1 : thiophene (thiole) (C 5 ); N 1 O 1 : oxazole (C 5 ), isoxazole (C 5 ), isoxazine (C 6 ); N 2 O 1 : oxadiazole (furazan) (C 5 ); 30 N 3 O 1 : oxatriazole (C 5 ); N 1 S 1 : thiazole (C 5 ), isothiazole (C 5 ); N 2 : imidazole (1,3-diazole) (C 5 ), pyrazole (1,2-diazole) (C 5 ), pyridazine (1,2-diazine) (C 6 ), pyr
  • heteroaryl which comprise fused rings include, but are not limited to: C9 (with 2 fused rings) derived from benzofuran (O1), isobenzofuran (O1), indole (N1), isoindole (N1), indolizine (N1), indoline (N1), isoindoline (N1), purine (N4) (e.g., adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzisoxazole (N1O1), 5 benzodioxole (O2), benzofurazan (N2O1), benzotriazole (N3), benzothiofuran (S1), benzothiazole (N1S1), benzothiadiazole (N2S); C10 (with 2 fused rings) derived from chromene (O1), isochromene (O1), isochromene (O
  • R is an ether substituent, for example, a C 1-7 alkyl group (also referred to as a C 1-7 alkoxy group, discussed below), a C 3-20 heterocyclyl group (also referred to as a C 3-20 heterocyclyloxy group), or a C 5-20 aryl group (also referred to as a C 5-20 aryloxy group), preferably a C 1-7 alkyl group.
  • R is an alkyl group, for example, a C 1-7 alkyl group.
  • C 1-7 alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n- propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy).
  • 35 008188229 18 Oxo (keto, -one): O.
  • R is an acyl substituent, for example, a C1-7 alkyl group (also referred to as C1-7 alkylacyl or C1-7 alkanoyl), a C3-20 heterocyclyl group (also referred to as 5 C3-20 heterocyclylacyl), or a C5-20 aryl group (also referred to as C5-20 arylacyl), preferably a C1-7 alkyl group.
  • a C1-7 alkyl group also referred to as C1-7 alkylacyl or C1-7 alkanoyl
  • C3-20 heterocyclyl group also referred to as 5 C3-20 heterocyclylacyl
  • C5-20 aryl group also referred to as C5-20 arylacyl
  • Carboxy (carboxylic acid): -C( O)OH.
  • R 1 and R 2 are independently amino substituents, for example, hydrogen, a C1-7 alkyl group (also referred to as C1-7 alkylamino or di-C1-7 alkylamino), a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably H or a C 1-7 alkyl group, or, in the 20 case of a “cyclic” amino group, R 1 and R 2 , taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms.
  • R 1 and R 2 are independently amino substituents, for example, hydrogen, a C1-7 alkyl group (also referred to as C1-7 alkylamino or di-C1-7 alkylamino), a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably H or a C 1-7 alkyl group, or, in the 20 case of a “cyclic” amino group, R 1 and R 2
  • Amino groups may be primary (-NH 2 ), secondary (-NHR 1 ), or tertiary (-NHR 1 R 2 ), and in cationic form, may be quaternary (- + NR 1 R 2 R 3 ).
  • Examples of amino groups include, but are not limited to, -NH 2 , -NHCH 3 , -NHC(CH 3 ) 2 , -N(CH 3 ) 2 , -N(CH 2 CH 3 ) 2 , and -NHPh.
  • Examples of cyclic amino 25 groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
  • Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): -C( O)NR 1 R 2 , wherein R 1 and R 2 are independently amino substituents, as defined for amino groups.
  • C3-12 alkylene refers to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 3 to 12 20 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated.
  • alkylene includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below.
  • linear saturated C 3-12 alkylene groups include, but are not limited to, -(CH 2 ) n - 25 where n is an integer from 3 to 12, for example, -CH 2 CH 2 CH 2 - (propylene), -CH 2 CH 2 CH 2 CH 2 - (butylene), -CH 2 CH 2 CH 2 CH 2 CH 2 - (pentylene) and -CH 2 CH 2 CH 2 CH- 2 CH 2 CH 2 CH 2 - (heptylene).
  • Examples of branched saturated C 3-12 alkylene groups include, but are not limited to, 30 -CH(CH 3 )CH 2 -, -CH(CH 3 )CH 2 CH 2 -, -CH(CH 3 )CH 2 CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -, -CH 2 CH(CH 3 )CH 2 CH 2 -, -CH(CH 2 CH 3 )-, -CH(CH 2 CH 3 )CH 2 -, and -CH 2 CH(CH 2 CH 3 )CH 2 -.
  • Examples of alicyclic saturated C3-12 alkylene groups include, but are not limited to, cyclopentylene (e.g. cyclopent-1,3-ylene), and cyclohexylene 10 (e.g. cyclohex-1,4-ylene).
  • C3-12 cycloalkylenes examples include, but are not limited to, cyclopentenylene (e.g.4-cyclopenten-1,3-ylene), cyclohexenylene (e.g.2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-15 1,4-ylene).
  • cyclopentenylene e.g.4-cyclopenten-1,3-ylene
  • cyclohexenylene e.g.2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-15 1,4-ylene
  • the subscript refers to the number of atoms in the chain including the heteroatoms.
  • the chain -C2H4-O- C 2 H 4 - would be a C 5 group.
  • the Ligand Unit may be of any kind, and include a protein, polypeptide, peptide and a non- 30 peptidic agent that specifically binds to a target molecule.
  • the Ligand unit may be a protein, polypeptide or peptide.
  • the Ligand unit may be a cyclic polypeptide.
  • Ligand units can include antibodies or a fragment of an antibody that contains at least one target molecule-binding site, lymphokines, 008188229 21 hormones, growth factors, or any other cell binding molecule or substance that can specifically bind to a target.
  • the terms “specifically binds” and “specific binding” refer to the binding of an antibody or 5 other protein, polypeptide or peptide to a predetermined molecule (e.g., an antigen).
  • the antibody or other molecule binds with an affinity of at least about 1x10 7 M -1 , and binds to the predetermined molecule with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or a closely-related molecule.
  • a non-specific molecule e.g., BSA, casein
  • Ligand units include those agents described for use in WO 2007/085930, which is incorporated herein.
  • the Ligand unit is a Cell Binding Agent that binds to an extracellular15 target on a cell.
  • a Cell Binding Agent can be a protein, polypeptide, peptide or a non- peptidic agent.
  • the Cell Binding Agent may be a protein, polypeptide or peptide. In some embodiments, the Cell Binding Agent may be a cyclic polypeptide. The Cell Binding Agent also may be antibody or an antigen-binding fragment of an antibody. Thus, in one embodiment, the present disclosure provides an 20 antibody-drug conjugate (ADC).
  • a cell binding agent may be of any kind, and include peptides and non-peptides. These can include antibodies or a fragment of an antibody that contains at least one binding site, 25 lymphokines, hormones, hormone mimetics, vitamins, growth factors, nutrient-transport molecules, or any other cell binding molecule or substance.
  • the cell binding agent is a linear or cyclic peptide comprising 4-30, 30 preferably 6-20, contiguous amino acid residues. In this embodiment, it is preferred that one cell binding agent is linked to one monomer or dimer pyrrolobenzodiazepine compound.
  • the cell binding agent comprises a peptide that binds integrin ⁇ ⁇ ⁇ 6 . The 35 peptide may be selective for ⁇ ⁇ ⁇ 6 over XYS. 008188229 22
  • the cell binding agent comprises the A20FMDV-Cys polypeptide.
  • the A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC.
  • a variant of the A20FMDV-Cys sequence may be used wherein one, two, three, four, five, six, seven, eight, nine or ten amino acid residues are substituted with another amino acid 5 residue.
  • the polypeptide may have the sequence NAVXXXXXXXXXXXXXXRTC.
  • antibody herein is used in the broadest sense and specifically covers 10 monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), multivalent antibodies and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854- 4861).
  • Antibodies may be murine, human, humanized, chimeric, or derived from other species.
  • An antibody is a protein generated by the immune system that is capable of 15 recognizing and binding to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York).
  • a target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody.
  • An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease.
  • the immunoglobulin can be of any 25 type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
  • the immunoglobulins can be derived from any species, including human, murine, or rabbit origin.
  • “Antibody fragments” comprise a portion of a full length antibody, generally the antigen 30 binding or variable region thereof.
  • Examples of antibody fragments include Fab, Fab', F(ab') 2 , and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody 35 molecules; and multispecific antibodies formed from antibody fragments.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being 5 directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
  • the modifier “monoclonal” 10 indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant 15 DNA methods (see, US 4816567).
  • the monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol.
  • the monoclonal antibodies herein specifically include chimeric antibodies, humanized antibodies and human antibodies.
  • Examples of cell binding agents include those agents described for use in 25 WO 2007/085930, which is incorporated herein.
  • Modified antibodies suitable for use in the present disclosure include those where cysteines have been inserted in selected sites in antibodies. These are described in Dimasi, N., et al., Molecular Pharmaceutics, 2017, 14, 1501-1516 (DOI: 30 10.1021/acs.molpharmaceut.6b00995) and WO2015/157595.
  • antibodies which have been modified by insertion of a cysteine after the S239 position are of use.
  • antibodies which have been modified by insertion of a non-natural 35 amino acid at F241 may be used.
  • a non-natural amino acid as employed herein refers to an amino acid which is other than one of the twenty-one 008188229 24 naturally occurring amino acids.
  • the non-natural amino acids are generally derived from natural amino acids.
  • Derived from a natural amino acid refers to the fact that the non- natural amino acid is based on (or incorporates) or is similar to the structure of natural amino acid, for example the alkylene chain in lysine may be shortened to provide a 3- 5 carbon chain as opposed to the natural 4 carbon chain but the structural relationship or similarity to lysine still exists.
  • derivatives of natural amino acids include modifications such as incorporating a functional group, lengthening or shortening an alkylene chain, adding one or more substituents to a nitrogen, oxygen, sulfur in a side chain or converting a nitrogen, oxygen or sulfur into a different functional group or a 10 combination of any of the same. Usually the majority of modifications will be the addition of structure in the non-natural amino acid.
  • modification may include removed or replacing an atom naturally found in an amino acid.
  • the non-natural amino has a formula (AAII): RX-XAA1-O0-1C(O)-amino-acid-residue (AAII) 15 wherein: RX represents an unsaturated group selected from a: i) C4-9 linear conjugated diene, ii) C5-14 carbocyclyl comprising a conjugated diene, and iii) a 5 to 14 membered heterocyclyl comprising 1, 2 or 3 heteroatoms selected O, N 20 and S, and a conjugated diene, wherein i), ii) and iii) may bear up to five substituents, (such as one, two or three substituents) for example, the substituents are independently selected from C1-3 alkyl, oxo, halogen, sulfo, sulfhydryl, amino, -C1-3alkyleneN3, or -
  • amino acid residue referred to in AAII is as defined for AAI above.
  • the amino acid residue refers to an amino acid comprising the -NH 2 and -COOH groups.
  • the amino acid residue in formula AAII may additionally comprise an R group of a natural amino acid.
  • the amino acid residue in 10 formula AAII may be derived from a natural amino acid but have its natural R group replaced with RX-XAA1-O 0-1 C(O).
  • the non-natural amino acid is a residue of the structure of formula 15 wherein X2 represents -C-, -C(R’)-, -CH2 or O; R’ represents H or C1-3 alkyl, Ra represents 20 i) a saturated or unsaturated branched or unbranched C1-8 alkylene chain, wherein at least one carbon (for example 1, 2 or 3 carbons) is replaced by a heteroatom selected from O, N, S(O)0-3, wherein said chain is optionally, substituted by one or more groups independently selected from oxo,25 halogen, amino; or ii) together with a carbon from the 5 membered ring represents a cyclopropane ring linked to a saturated or unsaturated (in particular saturated) branched or unbranched C1-6 alkylene chain, wherein at least one carbon (for example 1, 2 or 3 30 carbons) is replaced by a heteroatom selected from O, N, S(O)0-3, wherein said chain is optionally, substituted by one
  • Ra is -(CH 2 )mC(O)-, -CH 2 (CH 3 )C(O)-, -(CH 2 )mCH 2 OC(O)-, -CHCHCH 2 OC(O)-, or -OCH 2 CH 2 COC(O)- and m represents 0 or 1.
  • Rb is H, -OC 1-3 alkyl, -CH 3 , -CH(CH 3 ) 2 , CH 2 OH, -CH 2 N 3 , or –CCH.
  • Rc is H, -OC 1-3 alkyl, -CH 3 , -CH(CH 3 ) 2 , CH 2 OH, -CH 2 N 3 , or –CCH.
  • Rd is H, -OC 1-3 alkyl, -CH 3 , -CH(CH 3 ) 2 , CH 2 OH, -CH 2 N 3 , or –CCH.
  • Re represents H or -CH 2 OCH 2 CH 2 N 3.
  • the non-natural amino acid is a residue of the structure of formula (AAIIIa): 25 (AAIIIa) wherein Ra, Rb, Rc, Rd, Re and X2 are defined above.
  • the non-natural amino acid has the structure of formula (AAIIIb): 30 008188229 27 (AAIIIb) wherein Ra, Rb, Rc, Rd, Re and X2 are defined above.
  • the non-natural amino acid has the structure of formula (AAIIIc): (AAIIIc) 5 wherein Ra, Rb, Rc, Rd, Re are defined above and X2’ is -C- or -CR’ as defined above.
  • Generally compounds, for example formula (AAIII), (AAIIIa), (AAIIIb) and (AAIIIc) will at most contain only one azide group. 10
  • the non-natural amino acid is selected from the group comprising:
  • the non-natural amino acid is selected from:
  • Antibodies which have been modified by the insertion of CP2-NNAA are of particular use in the present invention. These are described in as described in WO2019/224340, and Roy 5 et al., MAbs 12 (1): 1684749 (doi:10.1080/19420862.2019.1684749), which are both incorporated herein by reference.
  • antibodies with this insertion at F241 may be used, e.g. modified Herceptin.
  • Tumour-associate antigens and cognate antibodies for use in embodiments of the present 10 disclosure are listed below, and are described in more detail on pages 14 to 86 of WO 2017/186894, which is incorporated herein.
  • BMPR1B bone morphogenetic protein receptor-type IB
  • E16 LAT1, SLC7A5
  • STEAP1 six transmembrane epithelial antigen of prostate
  • 0772P CA125, MUC16
  • MPF MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin
  • Napi3b NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b)
  • Sema 5b FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain
  • CXCR5 Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays35 a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, 008188229 31 (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and 20 presents them to CD4+ T lymphocytes); 273 aa, pI: 6.56, MW: 30820.TM: 1 [P] Gene Chromosome: 6p21.3) (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by 5 extracellular ATP, may be involved
  • CD72 B-cell differentiation antigen CD72, Lyb-2
  • 359 aa pI: 8.66, MW: 40225, TM: 1 5 [P] Gene Chromosome: 9p13.3).
  • LY64 Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis); 661 aa, pI: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12).
  • FcRH1 Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation
  • Gene Chromosome: 1q21-1q22) 35)
  • IRTA2 Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; 20 deregulation of the gene by translocation occurs in some B cell malignancies); 977 aa, pI: 6.88, MW: 106468, TM: 1 [P] Gene Chromosome: 1q21) (36)
  • TENB2 TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin
  • NM175060 15 (55) GRP78 – HSPA5 (heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) (56) CD70 (CD70 molecule) L08096 (57) Stem Cell specific antigens.
  • the cell binding agent may be labelled, for example to aid detection or purification of the agent either prior to incorporation as a conjugate, or as part of the conjugate.
  • the label may be a biotin label.
  • the cell binding agent may be labelled with a radioisotope.
  • the compounds of the present disclosure may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a conjugate of formula II.
  • the term “therapeutically effective amount” is an amount sufficient to show benefit to a patient. Such 35 benefit may be at least amelioration of at least one symptom.
  • compositions according to the present disclosure, and for use in 10 accordance with the present disclosure may comprise, in addition to the active ingredient, i.e.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may comprise a solid carrier or an adjuvant.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or 20 vegetable oils, mineral oil or synthetic oil.
  • Physiological saline solution dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • a capsule may comprise a solid carrier such a gelatin.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • the Conjugates can be used to treat proliferative disease and autoimmune disease.
  • proliferative disease pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo. 35 008188229 35
  • proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g.
  • lung cancer small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian 5 carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis.
  • cancers of interest include, but are not limited to, haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin 10 lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin.
  • malignancies such as leukemias and lymphomas, such as non-Hodgkin 10 lymphoma
  • subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin.
  • autoimmune disease examples include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic 15 arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves’ disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn’s disease), anaphylaxis, allergic reaction, Sjögren’s syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener’s granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, 20 Schmidt’s syndrome, autoimmune uveitis, Addison’s disease, adrenalitis, thyroiditis, Hashimoto’s thyroiditis, autoimmune
  • the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture’s syndrome, rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren’s syndrome, Hashimoto’s thyroiditis, Graves’ disease, primary biliary cirrhosis, Wegener’s granulomatosis, tuberculosis, or graft versus host disease), or Th2-lymphocytes 15 (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn’s syndrome, systemic sclerosis, or chronic graft versus host disease).
  • B lymphocytes e.g., systemic lupus erythemato
  • disorders involving dendritic cells involve disorders of Th1- lymphocytes or Th2-lymphocytes.
  • the autoimmunie disorder is a T cell-mediated immunological disorder. 20
  • the amount of the Conjugate administered ranges from about 0.01 to about 10 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administerd ranges from about 0.05 to about 5 mg/kg per 25 dose. In some embodiments, the amount of the Conjugate administerd ranges from about 0.1 to about 5 mg/kg per dose.
  • the amount of the Conjugate administered ranges from about 0.1 to about 4 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to 30 about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 2 mg/kg per dose.
  • Drug loading The drug loading (p) is the average number of PBD drugs per cell binding agent, e.g. 35 antibody. Where the compounds of the disclosure are bound to cysteines, drug loading may range from 1 to 8 drugs (D) per cell binding agent, i.e.
  • compositions of conjgates include collections of cell binding agents, e.g. antibodies, conjugated with a range of drugs, from 1 to 8. Where the compounds of the disclosure are bound to lysines, drug loading may range from 1 to 80 drugs (D) per cell binding agent, although an upper limit of 40, 20, 5 10 or 8 may be preferred.
  • compositions of conjgates include collections of cell binding agents, e.g. antibodies, conjugated with a range of drugs, from 1 to 80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8.
  • the average number of drugs per antibody in preparations of ADC from conjugation 10 reactions may be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, and electrophoresis.
  • the quantitative distribution of ADC in terms of p may also be determined.
  • ELISA the averaged value of p in a particular preparation of ADC may be determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res.11:843-852).
  • the 15 distribution of p (drug) values is not discernible by the antibody-antigen binding and detection limitation of ELISA.
  • ELISA assay for detection of antibody-drug conjugates does not determine where the drug moieties are attached to the antibody, such as the heavy chain or light chain fragments, or the particular amino acid residues.
  • separation, purification, and characterization of homogeneous ADC where p is a 20 certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.
  • Such techniques are also applicable to other types of conjugates.
  • p may be limited by the number of attachment sites on 25 the antibody.
  • an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached.
  • Higher drug loading e.g. p >5
  • 30 Typically, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction.
  • An antibody may contain, for example, many lysine residues that do not react with the Drug Linker. Only the most reactive lysine groups may react with an amine-reactive linker reagent. Also, only the most reactive cysteine thiol groups may react with a thiol-reactive linker reagent. Generally, antibodies do not contain 35 many, if any, free and reactive cysteine thiol groups which may be linked to a drug moiety.
  • cysteine thiol residues in the antibodies of the compounds exist as disulfide bridges 008188229 38 and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP, under partial or total reducing conditions.
  • DTT dithiothreitol
  • the loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of Drug Linker relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) 5 partial or limiting reductive conditions for cysteine thiol modification.
  • Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges.
  • Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two 10 reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut’s reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by engineering one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine 15 amino acid residues).
  • a reducing agent such as DTT (dithiothreitol).
  • the engineered cysteine thiols may react with linker reagents or the drug-linker reagents of the present disclosure which have thiol-reactive, electrophilic groups such as maleimide or alpha-halo amides to form ADC with cysteine engineered antibodies and the PBD drug moieties.
  • the location of the drug moiety can thus be 25 designed, controlled, and known.
  • the drug loading can be controlled since the engineered cysteine thiol groups typically react with thiol-reactive linker reagents or drug-linker reagents in high yield.
  • Engineering an IgG antibody to introduce a cysteine amino acid by substitution at a single site on the heavy or light chain gives two new cysteines on the symmetrical antibody.
  • a drug loading near 2 can be achieved with near homogeneity of30 the conjugation product ADC.
  • the resulting product is a mixture of ADC compounds with a distribution of drug moieties attached to an 35 antibody, e.g.1, 2, 3, etc.
  • Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) may separate compounds in the mixture 008188229 39 by drug loading value.
  • the antibody-drug conjugate compositions of the disclosure include mixtures of antibody-drug conjugate compounds where the antibody has one or more PBD drug moieties and where the drug moieties may be attached to the antibody at various amino acid residues.
  • the average number of dimer pyrrolobenzodiazepine groups per cell binding agent is in the range 1 to 20. In some embodiments the range is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4, and 4 to 8.
  • the Therapeutic Index of a particular drug-linker/conjugate can be calculated by dividing 20 the maximum tolerated single dose (MTD) of a non-targeted ADC in rat, by the minimal effective single dose (MED) of a comparable targeted ADC in mouse.
  • the MED may be the single dose necessary to achieve tumour stasis in an in vivo model at 28 days.
  • R 8 should be selected from: (a) OH and -Y’-R”-Hal; (b) 15 . 008188229 41 Further derivation When R 8 is (Vc), compounds of formula II may be synthesised from compounds of formula IV by converting R Lpre into R L .
  • the precursor of R L (R Lpre ) will comprise: 5 Where Prot N is an amine protecting group, such as Alloc.
  • R 8 is -Y’-R”-Hal
  • compounds of formula IV where R 8 is (Vc) may be synthesised by coupling a compound of formula VIa: to the compound of formula IV when R 8 is -Y’-R”-Hal.
  • R 8 is –OH
  • Y’ is O
  • compounds of formula IV where R 8 is (Vc) may be synthesised by coupling a compound of formula VIb: to the compound of formula IV when R 8 is –OH.
  • Compounds of formula V may be synthesised from compounds of formula VII: 008188229 42 Lpre R O O 9 R O OProt 8 R NH H (VII) 7 R N 6 R O D by removal of the hydroxyl protecting group (Prot O ) where R 8 is selected from: (a) OMe, OCH 2 Ph, OH and –Y’-R”-Hal; 5 b) and R 6 , R 7 , R 9 , R 11a , R 6’ , R 7’ , R 9’ , Y, R”, Y’, D and D’ are as defined in the first aspect of the10 disclosure.
  • Compounds of the sixth aspect of the disclosure may be made by analogous methods to those described above.
  • the reduced antibody is then cooled to 0°C and alkylated with 1.5 equivalents of maleimide drug-linker per antibody thiol. After 1 hour, the reaction is 10 quenched by the addition of 5 equivalents of N-acetyl cysteine. Quenched drug-linker is removed by gel filtration over a PD-10 column. The ADC is then sterile-filtered through a 0.22 ⁇ m syringe filter. Protein concentration can be determined by spectral analysis at 280 nm and 329 nm, respectively, with correction for the contribution of drug absorbance at 280 nm.
  • R 6’ , R 7’ , R 9’ , and Y’ are selected from the same groups as R 6 , R 7 , R 9 , and Y respectively. In some of these embodiments, R 6’ , R 7’ , R 9’ , and Y’ are the same25 as R 6 , R 7 , R 9 , and Y respectively.
  • R 22 is the same as R 2 . N10’-C11’ 30
  • R 20 is H and R 21 is H.
  • R 20 and R 21 together form a nitrogen-carbon double bond between35 the nitrogen and carbon atoms to which they are bound.
  • the amino acids in the dipeptide may be any combination of natural amino acids.
  • the dipeptide may be the site of action for cathepsin-mediated cleavage. 5
  • Other dipeptide combinations may be used, including those described by Dubowchik et al., 5 Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference.
  • the amino acid side chain is derivatised, where appropriate.
  • an amino group or carboxy group of an amino acid side chain may be derivatised.
  • an amino group NH2 of a side chain amino acid, such as lysine is a derivatised form selected from the group consisting of NHR and NRR’.
  • a carboxy group COOH of a side chain amino acid, such as aspartic acid is a derivatised form selected from the group consisting of COOR, CONH2, CONHR 15 and CONRR’.
  • the amino acid side chain is chemically protected, where appropriate.
  • the side chain protecting group may be a group as discussed above.
  • the present inventors have established that protected amino acid sequences are cleavable by 20 enzymes. For example, it has been established that a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.
  • Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog. Additional protecting group strategies are set out25 in Protective Groups in Organic Synthesis, Greene and Wuts.
  • Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality: Arg: Z, Mtr, Tos; 30 Asn: Trt, Xan; Asp: Bzl, t-Bu; Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt; Glu: Bzl, t-Bu; Gln: Trt, Xan; 35 His: Boc, Dnp, Tos, Trt; Lys: Boc, Z-Cl, Fmoc, Z, Alloc; 008188229 47 Ser: Bzl, TBDMS, TBDPS; Thr: Bz; Trp: Boc; Tyr: Bzl, Z, Z-Br.
  • the side chain protection is selected to be orthogonal to a group provided as, or as part of, a capping group, where present.
  • the removal of the side chain protecting group does not remove the capping group, or any protecting group functionality that is part of the capping group.
  • the amino acids selected are those having no reactive side chain functionality.
  • An example of a preferred group is: .
  • R 20 groups include: 20 . Dimer link
  • Y and Y’ are both O. 008188229 48
  • R’’ is a C3-7 alkylene group with no substituents.
  • R’’ is a C3, C5 or C7 alkylene.
  • R’’ may be a C3 or C5 alkylene.
  • R is a group of formula: 5
  • R is a group of formula: 10
  • R and R’ are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups, wherein the optional substituents are 15 selected from C1-12 alkyl, C3-20 heterocyclyl, C5-20 aryl, halo, hydroxy, ether, alkoxy, oxo, acyl, carboxy, ester, amino, amido, nitro, and cyano.
  • R 9 is H.
  • R 6 is selected from H, OH, OR, SH, NH2, nitro and halo, and may be selected from H or halo. In some of these embodiments R 6 is H. In some embodiments, R 7 is selected from H, OH, OR, SH, SR, NH 2 , NHR, NRR’, and halo. In some of these embodiments R 7 is selected from H, OH and OR, where R is 25 selected from optionally substituted C 1-7 alkyl, C 3-10 heterocyclyl and C 5-10 aryl groups. R may be more preferably a C 1-4 alkyl group, which may or may not be substituted. A substituent of interest is a C 5-6 aryl group (e.g. phenyl).
  • substituents at the 7- positions are OMe and OCH 2 Ph.
  • Other substituents of particular interest are dimethylamino (i.e. –NMe 2 ); -(OC 2 H 4 ) q OMe, where q is from 0 to 2; nitrogen-containing C 6 30 heterocyclyls, including morpholino, piperidinyl and N-methyl-piperazinyl. 008188229 49
  • D and D’ In some embodiments, D and D’ are D1 and D’1 respectively. 5 In some embodiments, D and D’ are D2 and D’2 respectively.
  • R 2 When there is a double bond present between C2 and C3, R 2 is selected from: 10 (a) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene; (b) C1-5 saturated aliphatic alkyl; (c) C3-6 saturated cycloalkyl; 15 , wherein each of R 11 , R 12 and R 13 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R 2 group is no more than 5; (e) , wherein one of R 15a and R 15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo methyl,20 methoxy; pyridy
  • R 2 When R 2 is a C5-10 aryl group, it may be a C5-7 aryl group.
  • a C5-7 aryl group may be a phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl.
  • R 2 is preferably phenyl.
  • R 2 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
  • 008188229 50 When R 2 is a C5-10 aryl group, it may be a C8-10 aryl, for example a quinolinyl or isoquinolinyl group.
  • the quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position.
  • the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3- 5 yl and quinolin-6-yl may be preferred.
  • the isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred.
  • R 2 is a C5-10 aryl group, it may bear any number of substituent groups. It preferably 10 bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be at any position.
  • R 2 is C5-7 aryl group
  • a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably ⁇ or ⁇ to the bond 15 to the remainder of the compound. Therefore, where the C5-7 aryl group is phenyl, the substituent is preferably in the meta- or para- positions, and more preferably is in the para- position.
  • R 2 is a C 8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any 20 number of substituents at any position of the quinoline or isoquinoline rings.
  • R 2 substituents when R 2 is a C 5-10 aryl group 25
  • R 2 substituents when R 2 is a C 5-10 aryl group is halo, it is preferably F or Cl, more preferably Cl.
  • a substituent on R 2 when R 2 is a C 5-10 aryl group is ether it may in some embodiments be an alkoxy group, for example, a C 1-7 alkoxy group (e.g.
  • methoxy, ethoxy or it may in some 30 embodiments be a C 5-7 aryloxy group (e.g phenoxy, pyridyloxy, furanyloxy).
  • the alkoxy group may itself be further substituted, for example by an amino group (e.g. dimethylamino).
  • a substituent on R 2 when R 2 is a C 5-10 aryl group is C 1-7 alkyl it may preferably be a C 1-4 35 alkyl group (e.g. methyl, ethyl, propryl, butyl).
  • a substituent on R 2 when R 2 is a C5-10 aryl group is C3-7 heterocyclyl, it may in some embodiments be C6 nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by 5 C1-4 alkyl groups. If the C6 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom.
  • a substituent on R 2 when R 2 is a C5-10 aryl group is bis-oxy-C1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene. 10
  • a substituent on R 2 when R 2 is a C5-10 aryl group is ester this is preferably methyl ester or ethyl ester.
  • Particularly preferred substituents when R 2 is a C5-10 aryl group include methoxy, ethoxy,15 fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl- thiophenyl.
  • Other particularly preferred substituents for R 2 are dimethylaminopropyloxy and carboxy.
  • Particularly preferred substituted R 2 groups when R 2 is a C 5-10 aryl group include, but are20 not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4- fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4- cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl.
  • R 2 group is 4-nitrophenyl.
  • R 2 groups of particular interest include 4-(4-25 methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl.
  • Other R 2 groups When R 2 is C 1-5 saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of 30 these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched.
  • R 2 When R 2 is C 3-6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl. 35 008188229 52 12 R 13 R When R 2 11 is R , each of R 11 , R 12 and R 13 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R 2 group is no more than 5. In some embodiments, the total number of carbon atoms in the R 2 group is no more than 4 or no more than 3.
  • one of R 11 , R 12 and R 13 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
  • two of R 11 , R 12 and R 13 are H, with the other group being selected10 from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
  • the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that are not H are methyl. 15
  • R 11 is H.
  • R 12 is H.
  • R 13 is H.
  • R 11 and R 12 are H. In some embodiments, R 11 and R 13 are H. 25 In some embodiments, R 12 and R 13 are H.
  • An R 2 group of particular interest is: .
  • R 2 is , one of R 15a and R 15b is H and the other is selected from: 30 phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, 008188229 53 methoxy; pyridyl; and thiophenyl.
  • the group which is not H is optionally substituted phenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
  • R 14 is selected from: H; C 1-3 saturated alkyl; C 2-3 alkenyl; C 2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted. In some embodiments, R 14 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some10 of these embodiments, R 14 is selected from H and methyl.
  • R 16a and R 16b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a 15 group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R 16a and R 16b is H, the other is selected from nitrile and a C1-4 alkyl ester.
  • R 2 is H. 20 In some embodiments, .
  • it is preferred that R 16a and R 16b are both H. In other embodiments, it is preferred that R 16a and R 16b are both methyl.
  • R 16a and R 16b are H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
  • the group which is not H is selected from methyl and ethyl.
  • R 22 is selected from: (a) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 5 alkylene; (b) C1-5 saturated aliphatic alkyl; (c) C3-6 saturated cycloalkyl; (d) , wherein each of R 31 , R 32 and R 33 are independently selected from H, C 1-3 saturated alkyl, C 2-3 alkenyl, C 2-3 alkynyl and cyclopropyl, where the total number of10 carbon atoms in the R 22 group is no more than 5; R25b (e) , wherein one of R 25a and R 25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from
  • R 22 When R 22 is a C5-10 aryl group, it may be a C5-7 aryl group.
  • a C5-7 aryl group may be a phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl.
  • R 22 is preferably phenyl.
  • R 22 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
  • R 22 When R 22 is a C5-10 aryl group, it may be a C8-10 aryl, for example a quinolinyl or isoquinolinyl group.
  • the quinolinyl or isoquinolinyl group may be bound to the PBD core 25 through any available ring position.
  • the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may be preferred.
  • the isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be30 preferred. 008188229 55
  • R 22 is a C5-10 aryl group, it may bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be any position.
  • R 22 is C5-7 aryl group
  • a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably ⁇ or ⁇ to the bond to the remainder of the compound. Therefore, where the C5-7 aryl group is phenyl, the substituent is preferably in the meta- or para- positions, and more preferably is in the para-10 position.
  • R 22 is a C8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings.
  • R 22 substituents when R 22 is a C5-10 aryl group If a substituent on R 22 when R 22 is a C5-10 aryl group is halo, it is preferably F or Cl, more preferably Cl. 20 If a substituent on R 22 when R 22 is a C 5-10 aryl group is ether, it may in some embodiments be an alkoxy group, for example, a C 1-7 alkoxy group (e.g.
  • methoxy, ethoxy or it may in some embodiments be a C 5-7 aryloxy group (e.g phenoxy, pyridyloxy, furanyloxy).
  • the alkoxy group may itself be further substituted, for example by an amino group (e.g. 25 dimethylamino).
  • a substituent on R 22 when R 22 is a C 5-10 aryl group is C 1-7 alkyl, it may preferably be a C 1-4 alkyl group (e.g. methyl, ethyl, propryl, butyl).
  • a substituent on R 22 when R 22 is a C 5-10 aryl group is C 3-7 heterocyclyl, it may in some embodiments be C 6 nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by C 1-4 alkyl groups. If the C 6 nitrogen containing heterocyclyl group is piperazinyl, the said35 further substituent may be on the second nitrogen ring atom.
  • R 22 when R 22 is a C5-10 aryl group is bis-oxy-C1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene. If a substituent on R 22 when R 22 is a C5-10 aryl group is ester, this is preferably methyl ester 5 or ethyl ester. Particularly preferred substituents when R 22 is a C5-10 aryl group include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl- thiophenyl.
  • R 22 are dimethylaminopropyloxy10 and carboxy.
  • Particularly preferred substituted R 22 groups when R 22 is a C5-10 aryl group include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4- fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4- 15 cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl.
  • R 22 group is 4-nitrophenyl.
  • R 22 groups of particular interest include 4-(4- methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl.
  • 20 Other R 22 groups When R 22 is C 1-5 saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched.
  • R 22 When R 22 is C 3-6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.
  • each of R 31 , R 32 and R 33 are independently selected from H, 30 C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R 22 group is no more than 5. In some embodiments, the total number of carbon atoms in the R 22 group is no more than 4 or no more than 3.
  • one of R 31 , R 32 and R 33 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
  • two of R 31 , R 32 and R 33 are H, with the other group being selected 5 from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
  • the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that are not H are methyl. 10
  • R 31 is H.
  • R 32 is H.
  • R 33 is H. 15 In some embodiments, R 31 and R 32 are H. In some embodiments, R 31 and R 33 are H. 20 In some embodiments, R 32 and R 33 are H. 2
  • An R 2 group of particular interest is: . R25b When R 22 is , one of R 25a and R 25b is H and the other is selected from: phenyl, 25 which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the group which is not H is optionally substituted phenyl. If the phenyl optional substituent is halo, it is preferably fluoro.
  • the phenyl group is unsubstituted.
  • R 24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from 008188229 58 halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted.
  • R 24 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R 24 is selected from H and methyl. 5 When there is a single bond present between C2’ and C3’, R 22 is H or , where R 26a and R 26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R 26a and R 26b is10 H, the other is selected from nitrile and a C1-4 alkyl ester. In some embodiments, R 22 is H.
  • R 26a and R 26b are both H. In other embodiments, it is preferred that R 26a and R 26b are both methyl. 20 In further embodiments, it is preferred that one of R 26a and R 26b is H, and the other is selected from C 1-4 saturated alkyl, C 2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted. In these further embodiment, it may be further preferred that the group which is not H is selected from methyl and ethyl.
  • D L is of formula I’-a, I’-b, I’-c or I’-d: 008188229 59 I'-a I'-b I'-c I'-d where the dotted line represents the possible presence of a double bond between C2 and C3 and C2’ and C3’; where there is no double bond between C2 and C3 and C2’ and C3’, R 2a and R 22a are the same and are selected from: 008188229 60 (a) H; and ; where there is a double bond between C2 and C3 and C2’ and C3’ R 2a and R 22a are the 5 same and are selected from: 10 ; R 1a is selected from methyl and benzyl; 15 R 20 , R 21 and R LL are as defined above.
  • 20 G L G L may be selected from: 008188229 61 00
  • G LL is selected from G LL1-1 and G LL1-2 . In some of these 5 embodiments, G LL is G LL1-1 . In other embodiments, G LL is G LL1-1A .
  • G LL1-1A may be formed by a Diels-Alder reaction between G L1-1 and a spirocyclopropyl- cyclopentadiene of formula: Such a group can be incorporated into the antibody via the addition of a 10 linker or by incorporating a non-natural amino acid into the polypeptide sequence, as described in WO2019/224340, which is incorporated herein by reference).
  • 5 a may be 0, 1, 2, 3, 4 or 5.
  • a is 0 to 3. In some of these embodiments, a is 0 or 1.
  • a is 0.
  • b may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.
  • b is10 0 to 12.
  • b is 0 to 8, and may be 0, 2, 4 or 8.
  • c may be 0 or 1.
  • d may be 0, 1, 2, 3, 4 or 5.
  • d is 0 to 3.
  • d is 1 or 2.
  • d is 2.
  • a is 0, c is 1 and d is 2, and b may be from 0 to 8.
  • b is 0, 4 or 8. 20
  • Q is an amino acid residue.
  • the amino acid may a natural amino acids or a non-natural amino acid.
  • Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp,25 where Cit is citrulline.
  • Q comprises a dipeptide residue.
  • the amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids.
  • the dipeptide comprises natural amino acids.
  • the linker is a 30 cathepsin labile linker
  • the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.
  • Q is selected from: C O -Phe-Lys- NH , 008188229 65 C O -Val-Ala- NH , C O -Val-Lys- NH , C O -Ala-Lys- NH , C O -Val-Cit- NH , 5 CO -Phe-Cit- NH , C O -Leu-Cit- NH , C O -Ile-Cit- NH , C O -Phe-Arg- NH , and C O -Trp-Cit- NH ; 10 where Cit is citrulline.
  • Q is selected from: C O -Phe-Lys- NH , C O -Val-Ala- NH , 15 CO -Val-Lys- NH , C O -Ala-Lys- NH , C O -Val-Cit- NH .
  • Q is selected from CO -Phe-Lys- NH , CO -Val-Cit- NH and CO -Val-Ala- NH .
  • Other dipeptide combinations of interest include: C O -Gly-Gly- NH , C O -Pro-Pro- NH , and C O -Val-Glu- NH .
  • Q is a tripeptide residue.
  • the amino acids in the tripeptide may be 30 any combination of natural amino acids and non-natural amino acids.
  • the tripeptide comprises natural amino acids.
  • the linker is a cathepsin labile linker
  • the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide then is a recognition site for cathepsin.
  • Q is a tetrapeptide residue.
  • the amino acids in the tetrapeptide 5 may be any combination of natural amino acids and non-natural amino acids.
  • the tetrapeptide comprises natural amino acids.
  • the linker is a cathepsin labile linker
  • the tetrapeptide is the site of action for cathepsin-mediated cleavage.
  • NH - represents the N-terminus
  • Glu represents the residue of glutamic acid, i.e.: 20 ⁇ Glu represents the residue of glutamic acid when bound via the ⁇ -chain, i.e.: .
  • the amino acid side chain is chemically protected, where appropriate.
  • the side chain protecting group may be a group as discussed below.
  • Protected amino 25 acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin. 008188229 67 Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog, and as described above.
  • the C11 substituent may be in the 5 following stereochemical arrangement relative to neighbouring groups: 10 In other embodiments, the C11 substituent may be in the following stereochemical arrangement relative to neighbouring groups: .
  • Examples 20 General Information Reaction progress was monitored by LCMS using the short run conditions outlined below. Extraction and chromatography solvents (HPLC grade) were bought from VWR, U.K. All other chemicals were purchased from Aldrich (standard reagent grades). Visualization of TLC was achieved with UV light or iodine vapor unless otherwise stated. Extraction and 25 chromatography solvents were bought and used without further purification from VWR UK. All fine chemicals were purchased from Sigma-Aldrich or TCI Europe unless otherwise stated.
  • Pegylated reagents were obtained from Quanta Biodesign US via Stratech UK, or Purepeg. Column chromatography was performed on an Isolera (Biotage) automated system using normal phase SNAP Ultra cartridges purchased from Biotage. 30 The LC/MS conditions were as follow: The analytical LC/MS conditions (for reaction monitoring and purity determination) were as follows: Positive mode electrospray mass spectrometry was performed using a Shimadzu Nexera®/Prominence® LCMS-2020. Mobile phases used were solvent A (H 2 O with 0.1% 35 formic acid) and solvent B (CH 3 CN with 0.1% formic acid).
  • Flow rate was 0.8 10 mL/minute (for 3-minute run) and 0.6 mL/minute (for 15-minute run). Detection was at 254 nm.
  • the column was a Waters Acquity UPLC® BEH Shield RP181.7 ⁇ m 2.1 mm x 50 mm fitted with a Waters Acquity UPLC® BEH Shield RP18 VanGuard pre-column, 130 ⁇ , 1.7 ⁇ m, 2.1 mm x 5 mm at 50 °C.
  • Method 1 Gradient started with initial composition 5% B held over 25 seconds, then 10 increased from 5% B to 100% B over a 1 min 35 seconds period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 5 seconds and held there for 5 seconds. The total duration of the gradient run was 3.0 min with a sample injection volume of 2 ⁇ L and detection at 223 nm and 254 nm.
  • Method 2 Gradient started with initial composition 25% B held over 25 seconds, then 15 increased from 25% B to 100% B over a 1 min 35 seconds period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 5 seconds and held there for 5 seconds. The total duration of the gradient run was 3.0 min with a sample injection volume of 2 ⁇ L and detection at 223 nm and 254 nm.
  • Method 3 Gradient started with initial composition 5% B held over 1 min 25 seconds, then 20 increased from 5% B to 100% B over a 9 min 35 second period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 10 seconds and held there for 2 min.
  • the total duration of the gradient run was 15.0 min with a sample injection volume of 2 ⁇ L and detection at 223 nm and 254 nm.
  • the preparative HPLC conditions were as follows: reverse-phase UPLC was carried out on a Shimazdzu Prominence ® machine using a Phenomenex ® Gemini NX 5 ⁇ m C18 column 150 mm x 21.2 mm fitted with a Phenomenex ® Gemini SecurityGuard PREP Cartridge NX C18 15 mm x 21.2 mm at 50 °C. Eluents used were solvent A (H 2 O with 0.1% formic acid) and solvent B (CH 3 CN with 0.1% formic acid).
  • the reaction mixture was stirred at 40 °C for 40 min.
  • the 10 volatiles were removed under vacuum.
  • the residue was dissolved in DCM and purified by chromatography (Biotage Ultra 100 g EtOAc/EtOH 4:1 / Hex 15/85 up to 65/35 in 10 CV; elution from 45% to 53%).
  • the cleanest fractions were pooled and the solvent was removed by rotoevaporation under vacuum, followed by hard vacuum to give 6 (543 mg, 0.353 mmol, 74% Yield) as a white solid.
  • reaction mixture was stirred under argon at room temperature for 30 minutes and was found complete by LCMS.
  • Aqueous ammonium chloride (30 mL, 6 mass%) was added and the mixture was stirred vigorously.
  • the mixture was then decanted in a biotage phase separation cartridge.
  • the DCM layer was 30 evaporated to dryness under vacuum.
  • the residue was dissolved in chloroform (20 mL) and the solvent removed by rotoevaporation under vacuum at 35°C. This cycle was repeated a second time, followed by drying under hard vacuum (3 mbar, on rotoevaporator).
  • the reaction mixture was stirred at 40 °C for 3 hours at which point analysis by LC-MS revealed 50% conversion to desired product.
  • the heat was switched off and the mixture 20 stirred for 4 days where LC-MS revealed satisfactory conversion to desired product.
  • the mixture was partitioned between water (50 mL) and EtOAc (50 mL) and the layers separated.
  • the aqueous phase was extracted with EtOAc (3 x 25 mL) and the combined organic layers washed with 10% citric acid (30 mL), brine (40 mL), dried (MgSO 4 ), filtered and evaporated in vacuo to provide the crude product.
  • reaction mixture was allowed to stir for 3 hours at which point analysis by LC-MS revealed presence of starting material.
  • An additional equivalent of LiOAc was added and the mixture was heated at 40 °C for 16 hours, at this point analysis by LC-MS revealed reaction 35 completion.
  • the mixture was partitioned between water (100 mL) and EtOAc (76 mL) and the layers separated.
  • reaction mixture was allowed to stir for 3 hours heated at 60 °C, at which point analysis by LC-MS revealed 008188229 79 completion of reaction.
  • the mixture was partitioned between water (100 mL) and EtOAc (76 mL) and the layers separated. The aqueous phase was extracted with EtOAc (3 x 50 mL) and the combined organic layers washed with brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product which was purified by Isolera to give 5 16 as a white foam (2.14 g, 74% yield, LC-MS (method 1) 2.10 min, ES + m/z 1189 [M+H]).
  • the reaction mixture was stirred under argon for 10 minutes at which point analysis by LC-MS (sampled in MeOH) showed complete conversion to methyl carbamate (LC-MS 2.11 min, ES + m/z 1218 [M+H]).
  • the 35 mixture was treated with pyridine (1.50 equiv., 0.208 g, 0.213 mL, 2.63 mmol) followed by 17a (1 equiv., 1.12 g, 1.75 mmol) and dibutyltin dilaurate (0.2 equiv., 0.221 g, 0.210 mL, 008188229 80 0.350 mmol) and allowed to stir for 18 hours, analysis by LC-MS showed good conversion to desired product.
  • reaction mixture was washed with water (10 mL), dried (biotage SPE) and evaporated to dryness.
  • the resulting solid was purified by column 30 chromatography (gradient methanol / dichloromethane, 5/95 to 10/90 v/v) to leave the product as a white solid, 240 mg (78%).
  • TIPS protected phenol 30 (4.8 g, 9.64 mmol) was dissolved in DMF (40.0 mL) at 25°C. A solution of lithium acetate (1.02 g, 10.0 mmol) in water (2.0 mL) was added. The reaction was allowed to proceed at 25°C for 2h at which point completion was observed by LCMS.
  • Example 5 Compound I4 is Compound 19-4 of WO 2012/65964 (see page 253).
  • Diisopropyl azodicarboxylate (2.2 equiv., 2.22 mL, 11.3 mmol) was added to a stirred solution of I4 (1 equiv., 3.14 g, 5.14 mmol) and triphenylphosphine (3.00 equiv., 4.04 g, 10 15.42 mmol) in THF (50 mL).
  • the mixture 20 was allowed to stir at room temperature for 20 min, at which point the reaction had gone to completion (as monitored by LC/MS).
  • the reaction mixture was diluted with CH 2 Cl 2 (50 mL) and the organic phase was washed with H 2 O (2 x 50 mL) until complete pyrrolidine removal.
  • the organic phase was dried over MgSO 4 , filtered and excess solvent removed by rotary evaporation under reduced pressure.
  • the crude material was purified by silica gel25 column chromatography (CHCl 3 /MeOH ; 100% to 95:3) to afford pure product 39 (8.16 mg).
  • Analytical data: ES + 1.21 min, m/z 868.35 [M+H] + .
  • Example 7 008188229 96
  • Compound 40 is described in Tiberghien et al., Org. Process Res. Dev.2018, 22, 9, 1241- 5 1256
  • Trifluoromethanesulfonic anhydride (8.87 mL, 51.7 mmol) was added to a solution of 40 10 (20.0 g, 34.4 mmol) and 2,6-dimethylpyridine (8.1 mL, 69 mmol) in dry toluene (120 mL) at -40°C under argon.
  • the solution was stirred at -35°C for 30 min at which point completion was indicated by LCMS and TLC (EtOAc / hexane 1/3).
  • the solution was diluted with ethyl acetate (100 mL) and water (100 mL).
  • the organic layer was washed further with 0.02 N HCl (100 mL), followed by saturated bicarbonate (100 mL), and brine (50 mL).
  • the 15 organics were dried with magnesium sulfate and concentrated down to around 50 mL under vacuum.
  • the solution was diluted with toluene (100 mL).
  • the solution underwent two vacuum/argon cycles, followed by addition of potassium phosphate dibasic (36.7 g, 206 mmol, 98.0 mass%), 4-methoxyphenylboronic acid (7.01 g, 44.7 mmol), tetrakis(triphenylphosphine)palladium(0) (800 mg, 0.689 mmol), and water (30 mL).
  • the 20 reaction was stirred under argon at room temperature for 1h, at which point TLC and LCMS indicated reaction completion.
  • the mixture was diluted with ethyl acetate (100 mL) and washed with water (100 mL) and brine (50 mL). The organics were dried over magnesium sulfate and concentrated under vacuum.
  • the reaction was found to be progressing rapidly and complete after 1h by LCMS.
  • the reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (100 15 mL), followed by saturated aqueous hydrogen carbonate (50 mL), and brine (50 mL).
  • the organics were dried over magnesium sulphate.
  • the volatiles were removed under vacuum.
  • the residue was purified with a first chromatography (340 g ultra, EtOAc/Hexanes 35/65 up to 100/0 in 6 CV.
  • the mixture was heated at 50°C for 1.5h, at which point LCMS showed conversion of the 30 phenol to the mono-alkylated and bis-alkylated products.
  • the mixture was decanted, and the supernatant was evaporated to dryness.
  • the residue was loaded on a 10g silica samplet, dried under vacuum, and loaded on top of a 50g Ultra Biotage column.
  • the mixture was purified by chromatography (gradient EtOAc / Hexane 25/75 up to 100% EtOAc, followed by EtOAc / EtOH 90/10).
  • Zinc powder was added in portion (2.00 g, 30.5 mmol) and the ice bath was 5 removed. After 1h30, about 20% of hydroxylamine side product was observed. A further 1g of zinc was added, together with another 0.25 mL of acid and water. The reaction was allowed to carry on to completion over another 1.5h. The solids were removed by filtration over a bed of celite. The solution was partitioned between water (100 mL) and ethyl acetate (150 mL), separated, and washed with aqueous 10 sodium bicarbonate (150 mL). The organic phase was dried over magnesium sulfate and the volatiles were removed under vacuum.
  • the reaction was found to be progressing rapidly and complete after 3h by TLC (EtOAc). 5
  • the reaction mixture was diluted with 2-MeTHF (30 mL) and washed with water (30 mL), followed by saturated aqueous hydrogen carbonate (30 mL). The volatiles were removed under vacuum.
  • the residue was purified with a first chromatography (25 g ultra, DCM versus DCM/MeOH 4/1; gradient from 10/90 up to 35/65 up to 50/50 in 6 CV. Elution from 25/75); The pure fractions were pooled and the volatiles removed under vacuum to give 5210 (360 mg, 0.274 mmol, 75.1% Yield over 3 steps) as a white solid.
  • Herceptin and R347 antibodies engineered to have cysteine inserted between the 239 and 240 positions were produced following the methods described in Dimasi, N., et al., Molecular Pharmaceutics, 2017, 14, 1501-1516 (DOI: 5 10.1021/acs.molpharmaceut.6b00995).
  • HerC239i-9 ADC (ConjA) HerC239i (30 mg, 200 nmol) was loaded onto solid support and reduced, reoxidised, conjugated to Compound 9, purified, released from the resin and formulated into 25 mM 10 Histidine, 200 mM Sucrose pH 6.0 buffer containing 0.02% Tween-80 according to patent # WO2012140433.
  • R347C239i-9 ADC (ConjB) R347C239i (300 mg, 2 ⁇ mol) was loaded onto solid support and reduced, reoxidised, 30 conjugated to Compound 9, purified, released from the resin and formulated into 25 mM Histidine, 200 mM Sucrose pH 6.0 buffer containing 0.02% Tween-80 according to patent # WO2012140433.
  • HerC239i-22 ADC (ConjC) A 10 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline pH 15 7.4 (PBS) was added (80 molar equivalent/antibody, 14.9 micromoles, 1.49 mL) to a 10 mL solution of HerC239i (28 mg, 187 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 3.0 mg/mL. The reduction mixture was allowed to react at room temperature for 3 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking.
  • TCEP tris(2-carboxyethyl)phosphine
  • PBS phosphate-buffered saline pH 15 7.4
  • EDTA ethylenediaminetetraacetic acid
  • the 20 reduced antibody was buffer exchanged, via spin filter centrifugation using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent.
  • Compound 22 was added as a DMSO solution (10 30 molar equivalent/antibody, 1.87 micromoles, in 0.93 mL DMSO) to ⁇ 8.4 mL of this reoxidised antibody solution (28 mg, 187 nanomoles) for a 10% (v/v) final DMSO concentration.
  • the solution left to react at room temperature for 1 hour at room temperature with gentle shaking, then the conjugation was quenched by addition of N- acetyl cysteine (9.33 micromoles, 93 ⁇ L at 100 mM) for 30 min at room temperature, then 35 purified by spin filtration into PBS using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile-filtered and analysed.
  • HerC239i-28 ADC ConjD
  • DTT DL -dithiothreitol
  • PBS phosphate-buffered saline pH 7.4
  • EDTA ethylenediaminetetraacetic acid
  • the reduced antibody was buffer exchanged, via spin filter centrifugation, into a reoxidation 25 buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent.
  • a 50 mM solution of dehydroascorbic acid (DHAA, 20 molar equivalent/antibody, 6.67 micromoles, 133.3 ⁇ L) in DMSO was added and the reoxidation mixture was allowed to react for 16 hours at room temperature with gentle (60 rpm) shaking at an antibody concentration of 4.0 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the 30 cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC).
  • the reoxidation mixture was then sterile-filtered and diluted to 2.2 mg/mL in a conjugation buffer containing PBS and 1 mM EDTA.
  • Compound 28 was added as a DMSO solution (10 molar equivalent/antibody, 1.33 micromoles, in 1.0 mL DMSO) to 9.0 mL of this reoxidised antibody solution (20 mg, 133 nanomoles) for a 10% (v/v) final DMSO concentration and a 35 final antibody concentration of 2.0 mg/mL.
  • HerC239i-55 ADC (ConjF) A 10 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (80 molar equivalent/antibody, 16 micromoles, 1.6 mL) to a 10 mL solution of HerC239i (30 mg, 200 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 3.0 mg/mL. 25 The reduction mixture was allowed to react at room temperature for 3.5 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking.
  • TCEP tris(2-carboxyethyl)phosphine
  • PBS phosphate-buffered saline pH 7.4
  • EDTA ethylenediaminetetraacetic acid
  • the reduced antibody was buffer exchanged, via spin filter centrifugation using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent.
  • the reoxidation mixture was then sterile-filtered and 35 diluted in a conjugation buffer containing PBS and 1 mM EDTA and 33% (v/v) propylene glycol for a final antibody concentration of ⁇ 2 mg/mL.
  • Compound 55 was added as a 008188229 109 DMSO solution (15 molar equivalent/antibody, 3 micromoles, in 1.5 mL DMSO) to ⁇ 13.5 mL of this reoxidised antibody solution (30 mg, 200 nanomoles) for a 10% (v/v) final DMSO concentration.
  • High monomeric purity fractions corresponding to ConjF peak were pooled, diluted 3-5x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A 10 and loaded onto a 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP column, eluting with 0 ⁇ 100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 100 mL (20 CV) at 5 mL/min.
  • HIC Hydrophobic Interaction Chromatography
  • Drug-to-antibody ratio 2 (DAR 2) fractions were pooled and diluted 3-5x with 10 mM sodium phosphate pH 6.0 CHT Buffer A and loaded onto a 5 mL Bio-Scale Mini CHT ceramic hydroxyapatite 40 ⁇ m Type II cartridge, eluting with 0 ⁇ 100%15 10 mM sodium phosphate, 1 M sodium chloride pH 6.0 CHT Buffer B over 125 mL (25 CV) at 5 mL/min. High monomeric purity fractions were pooled and buffer exchanged into PBS via spin filtration using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile-filtered and analysed.
  • Example 9 - PBD Cytotoxicity Assay 35 The potency of the PBD molecules were measured via in vitro cytotox assays in the carcinoma cell line NCI-N87. 008188229 110 Solid PBD material was dissolved in DMSO to a 2 mM stock solution, from which eight serial dilutions were made at a 1:10 ratio in DMSO and stored at -20°C until use. Adherent NCI-N87 cells were washed with D-PBS and detached with Trypsin-EDTA, cell 5 density and viability were then determined in duplicate by Trypan blue exclusion assay using an automated cell counter (LUNA-IITM).
  • LUNA-IITM automated cell counter
  • Cell suspension was diluted to 1 x 10 5 cells/ml in growth media (RPMI 1640 with Glutamax + 10% (v/v) HyCloneTM Fetal Bovine Serum) and vortexed before dispensing 2mL per well into sterile 3 mL polypropylene plates. Warhead dilutions were then dispensed into the appropriate wells at 10 ⁇ l/well and 10 mixed by repeat pipetting. For control wells 10 ⁇ l of DMSO was dispensed onto 2 mL cell suspension, and thoroughly mixed.100 ⁇ l of each sample was then aliquoted into 2 replicate wells of a sterile flat 96-well microplate and incubated in a 37°C CO2-gassed (5%) incubator.
  • growth media RPMI 1640 with Glutamax + 10% (v/v) HyCloneTM Fetal Bovine Serum
  • cell viability was measured by CellTiter 96 TM Aqueous One (MTS) assay, which was dispensed at 20 ⁇ l/well and incubated 15 for 4 hours at 37°C, 5%CO2. Plates were then read on an EnVision TM Multi-label Plate Reader (Perkin Elmer) using absorbance at 490 nm. Cell survival percentage was calculated from the mean absorbance of the 2 replicate wells for each sample, compared to the mean absorbance in the two control wells treated with20 DMSO only (100%).
  • MTS CellTiter 96 TM Aqueous One
  • Example 10 ADC cytotoxicity method MTS The concentration and viability of cells from a sub-confluent (80-90% confluency) T75 flask 30 are measured by trypan blue staining, and counted using the LUNA-IITM Automated Cell Counter. Cells were diluted to 2x10 5 /ml, dispensed (50 ⁇ l per well) into 96-well flat-bottom plates.
  • a stock solution (1 ml) of antibody drug conjugate (ADC) (20 ⁇ g/ml) was made by dilution of filter-sterilised ADC into cell culture medium.
  • a set of 8x 10-fold dilutions of stock ADC were made in a 24-well plate by serial transfer of 100 ⁇ l into 900 ⁇ l of cell culture medium.
  • 5 ADC dilution was dispensed (50 ⁇ l per well) into 4 replicate wells of the 96-well plate, containing 50 ⁇ l cell suspension seeded the day previously. Control wells received 50 ⁇ l cell culture medium.
  • the 96-well plate containing cells and ADCs was incubated at 37C in a CO2-gassed incubator for the exposure time.
  • MTS MTS (Promega) was dispensed (20 ⁇ l per well) into each well and incubated for 4 hours at 37C in the CO2-gassed incubator. Well absorbance was measured at 490 nm. Percentage cell survival was calculated from the mean absorbance in the 4 ADC-treated wells compared to the mean absorbance in the 4 control untreated wells (100%). IC50 was determined from 15 the dose-response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose-response curve with variable slope. ADC incubation times were 4 days with MDA-MB-468 and 7 days for NCI-N87.
  • mice Female severe combined immune-deficient mice (Fox Chase SCID®, C.B-17/Icr-Prkdcscid, Charles River) were: A. ten weeks old with a body weight (BW) range of 16.9 to 21.9 grams on Day 1 of the study; B. eight weeks old with a body weight (BW) range of 16.5 to 21.6 grams on Day 1 of the 30 study.
  • BW body weight
  • mice were fed ad libitum water (reverse osmosis, 1 ppm Cl), and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fibre.
  • the mice were housed on irradiated Enricho’cobs TM Laboratory Animal 008188229 112 Bedding in static micro-isolators on a 12-hour light cycle at 20–22°C (68–72°F) and 40– 60% humidity.
  • CR Discovery Services specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care.
  • Tumour Cell Culture 10 Human NCI-N87 gastric carcinoma lymphoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin G sodium, 100 ⁇ g/mL streptomycin sulfate and 25 ⁇ g/mL gentamicin. The cells were grown in tissue culture flasks in a humidified incubator at 37 °C, in an atmosphere of 5% CO2 and 95% air.
  • NCI-N87 cells used for implantation were harvested during log phase growth and Re- suspended in phosphate buffered saline (PBS) containing 50% MatrigelTM (BD Biosciences).
  • PBS phosphate buffered saline
  • MatrigelTM MatrigelTM
  • mice Fourteen days later, designated as Day 1 of the study, mice were sorted according to calculated tumour size into groups each consisting of ten animals with individual tumour volumes ranging from: 25 A.108 to 172 mm 3 and group mean tumour volumes of 131 mm 3 ; B.108 to 144 mm 3 and group mean tumour volumes of 113-115 mm 3 .
  • A.ConjA (1 mg/kg) was administered intravenously once on Day 1 (qd x 1).
  • a vehicle- treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 85.
  • B. ConjD (4 mg/kg) was administered intravenously once on Day 1 (qd x 1).
  • a vehicle- 5 treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 79.
  • Each mouse was euthanized when its tumour reached the endpoint volume of 800 mm 3 or on the final day, whichever came first. The time to endpoint (TTE) was calculated for each mouse.
  • TTD Tumor Growth Delay
  • the data set consisted of the first observation that exceeded the endpoint volume used in analysis and the three consecutive observations that immediately preceded the attainment of this endpoint volume.
  • the calculated TTE is usually less than the TP date, the day on which the animal was euthanized for tumor size. Animals with tumors that 25 did not reach the endpoint volume were assigned a TTE value equal to the last day of the study. In instances in which the log-transformed calculated TTE preceded the day prior to reaching endpoint or exceeded the day of reaching tumor volume endpoint, a linear interpolation was performed to approximate the TTE.
  • NTRa non-treatment-related causes due to accident
  • NTRu unknown 30 etiology
  • TGD tumor growth delay
  • TGI tumor growth inhibition Tumor growth inhibition
  • Percent tumor growth inhibition (%TGI) was defined as the difference between the MTV of the designated control group and the MTV of the drug- treated group, expressed as a percentage of the MTV of the control group:
  • the data set for TGI analysis included all animals in a group, except those that died due to 20 treatment-related (TR) or non-treatment-related (NTR) causes prior to the day of TGI analysis.
  • TR treatment-related
  • NTR non-treatment-related
  • Treatment efficacy may be determined from the tumor volumes of animals remaining in the 25 study on the last day.
  • the MTV (n) was defined as the median tumor volume on the last day of the study in the number of animals remaining (n) whose tumors had not attained the endpoint volume.
  • Treatment efficacy may also be determined from the incidence and magnitude of regression responses observed during the study. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal.
  • PR partial regression
  • CR complete regression
  • the tumor volume was 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm 3 for one or more of these three measurements.
  • the tumor volume was less than 13.5 mm 3 for three consecutive measurements during the course of the study.
  • Acceptable toxicity was defined as a group mean body weight (BW) loss of less than 20% during the study and no more than 10% TR deaths. Dosing was 15 suspended in any group where mean weight loss exceeded acceptable limits. If group mean body weight recovered to acceptable levels, then dosing was modified to lower levels and/or reduced frequency then resumed. Deaths were classified as TR if it was attributable to treatment side effects as evidenced by clinical signs and/or necropsy. A TR classification was also assigned to deaths by unknown causes during the dosing period or within 14 20 days of the last dose. A death was classified as non-treatment-related (NTR) if there was no evidence that death was related to treatment side effects.
  • NTR non-treatment-related
  • NTR deaths are further categorized as follows: NTRa describes deaths due to accidents or human error; NTRm is assigned to deaths thought to result from tumor dissemination by invasion and/or metastasis based on necropsy results; NTRu describes deaths of unknown causes that 25 lack available evidence of death related to metastasis, tumor progression, accident or human error. It should be noted that treatment side effects cannot be excluded from deaths classified as NTRu.
  • Statistical and Graphical Analyses 30 GraphPad Prism 8.0 for Windows was used for all statistical analysis and graphical presentations. Study groups experiencing toxicity beyond acceptable limits (>20% group mean body weight loss or greater than 10% treatment-related deaths) or having fewer than five evaluable observations, were not included in the statistical analysis.
  • the logrank test was employed to assess the significance of the difference between the overall survival 35 experiences of two groups.
  • the logrank test analyzes the individual TTEs for all animals in a group, except those lost to the study due to NTR death.
  • Statistical analyses of the 008188229 116 differences between Day 19 median tumor volumes (MTVs) of control and treated groups were accomplished using the Mann-Whitney U-test.
  • significance level P 0.05.
  • Prism summarizes test results as not significant (ns) at P > 0.05, significant (symbolized by “*”) at 0.01 ⁇ P ⁇ 0.05, very 5 significant (“**”) at 0.001 ⁇ P ⁇ 0.01, and extremely significant (“***”) at P ⁇ 0.001.
  • R N2 is H or C1-4 alkyl
  • aromatic rings e.g. benzene or pyridine
  • Y and Y’ are selected from O, S, and NH
  • a conjugate according to statement 1 wherein both Y and Y’ are O. 3.
  • R is either a group of formula: (a) 008188229 123 where r is 1 or 2; or (b) 5 5.
  • a conjugate according to any one of statements 1 to 5, wherein R 6 is H. 10 7.
  • a conjugate according to statement 8 wherein R 7 is a methoxy group. 10.
  • 20. A conjugate according to statement 19, wherein the total number of carbon atoms in the R 2 group is no more than 4. 15 21.
  • R 2 is the group: 008188229 125 * .
  • D’ is D’1
  • R 22 is a C1-5 saturated aliphatic alkyl group.
  • R 20 is H and R 21 is10 H.
  • a 20 and R 20 is selected from: 008188229 129 008188229 130 60.
  • D L is of formula I’-a, I’-b, I’-c or I’-d: 008188229 131 I'-c I'-d where the dotted line represents the possible presence of a double bond between C2 and C3 and C2’ and C3’; where there is no double bond between C2 and C3 and C2’ and C3’, R 2a and R 22a are the same and are selected from: 5 (a) H; and ; where there is a double bond between C2 and C3 and C2’ and C3’ R 2a and R 22a are the same and are selected from: 10 008188229 132 * (f) ; O O (g) ; and N N (h) ; R 1a is selected from methyl and benzyl; 5 R 20 , R 21 and R LL are as defined above.
  • R L and R 11b are as defined in statement 1.
  • Q is an amino acid residue selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp. 10 67.
  • 71. A conjugate according to any one of statements 1 to 70, wherein a is 0 to 3.
  • 72. A conjugate according to statement 71, wherein a is 0. 10 73.
  • 74. A conjugate according to statement 73, wherein b is 0 to 8. 15 75.
  • Ar represents a C5-6 arylene group, e.g. phenylene and X 1 represents C 1-4 alkyl.
  • Ar represents a C5-6 arylene group, e.g. phenylene and X 1 represents C 1-4 alkyl.
  • G LL is selected from G LL1-1 and G LL1-2 .
  • 84 A conjugate according to statement 83, wherein G LL is G LL1-1 . 10 85.
  • G LL is G LL1- 1A .
  • the Ligand Unit is 15 an antibody or an active fragment thereof.
  • the antibody or antibody fragment is an antibody or antibody fragment for a tumour-associated antigen. 20 88.
  • the antibody or antibody fragment is an antibody which binds to one or more tumor-associated antigens or cell- surface receptors selected from (1)-(89): (1) BMPR1B; (2) E16; 25 (3) STEAP1; (4) 0772P; (5) MPF; (6) Napi3b; (7) Sema 5b; 30 (8) PSCA hlg; (9) ETBR; (10) MSG783; 008188229 136 (11) STEAP2; (12) TrpM4; (13) CRIPTO; (14) CD21; 5 (15) CD79b; (16) FcRH2; (17) HER2; (18) NCA; (19) MDP; 10 (20) IL20R-alpha; (21) Brevican; (22) EphB2R; (23) ASLG659; (24) PSCA; 15 (25) GEDA; (26) BAFF-R; (27) CD22; (28) CD79a; (29) CXCR5; 20 (30) HLA-DOB; (3
  • R L is a linker for connecting to a Ligand unit, which is: 008188229 139 O H L N G Q X IIIa , where Q and X are as defined in any one of statements 1 and 66 to 80 and G L is a linker for connecting to a Ligand unit. 5 93.
  • 96. A compound according to statement 95, wherein G L is G L1-1 . 10 97.
  • a composition comprising a mixture of conjugates according to any one of statements 1 to 91, wherein the average p in the mixture of conjugate compounds is about 1 to about 8. 98.
  • a pharmaceutical composition comprising the conjugate of any one of statements 1 to 91, and a pharmaceutically acceptable diluent, carrier or excipient. 008188229 141 100.
  • a method of medical treatment comprising administering to a patient the pharmaceutical composition of statement 99.
  • a method of synthesis of a conjugate according to any one of statements 1 to 91 comprising conjugating a compound according to any one of statements 92 to 96 with a30 Ligand Unit. 109.
  • a compound of formula VI: VI 5 D, R 2 , R 6 , R 7 , R 9 , Y, R”, Y’, D’, R 6’ , R 7’ , R 9’ and R 22 are as defined in any one of statements 1 to 65. 10 119.

Abstract

A conjugate of formula (I): L - (DL)p, wherein L is a Ligand unit (i.e., a targeting agent), p is from 1 to 20, DL is a Drug Linker unit of formula (I'); RLL is a linker for connection to the Ligand Unit, D represents D1; D' represents D'1.

Description

008188229 1 PYRROLOBENZODIAZEPINE CONJUGATES The present disclosure relates to conjugates comprising pyrrolobenzodiazepine (PBD) or related moity dimers linked via the N10 of a non-alkylating PBD-related moiety, and the 5 precursor drug linkers used to make such conjugates. Background to the disclosure Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu. The first PBD antitumour antibiotic,10 anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc., 87, 5793- 5795 (1965); Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogues (Thurston, et al., Chem. Rev.1994, 433-465 (1994)). Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145- 15 148 (1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180487; Thurston, et al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41, 1366-1373 (1988)), prothracarcin 20 (Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of the general structure: 25
Figure imgf000003_0001
They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N=C), a carbinolamine(NH-CH(OH)), or a carbinolamine methyl ether (NH- CH(OMe)) at the N10-C11 position which is the electrophilic centre responsible for 30 alkylating DNA. All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity 008188229 2 with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp.3-11 (1975); Hurley and Needham- VanDevanter, Acc. Chem. Res., 19, 230-237 (1986)). Their ability to form an adduct in the minor groove, enables them to interfere with DNA processing, hence their use as 5 antitumour agents. It has been previously disclosed that the biological activity of these molecules can be potentiated by joining two PBD units together through their C8/C’-hydroxyl functionalities via a flexible alkylene linker (Bose, D.S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992); 10 Thurston, D.E., et al., J. Org. Chem., 61, 8141-8147 (1996)). The PBD dimers are thought to form sequence-selective DNA lesions such as the palindromic 5’-Pu-GATC-Py-3’ interstrand cross-link (Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al., Biochemistry, 44, 4135-4147) which is thought to be mainly responsible for their biological activity. 15 The first dimers (Bose, D.S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992)) were of the general formula:
Figure imgf000004_0001
where n is from 3 to 6. The compounds where n were 3 and 5 showed promising cytoxicity 20 in vitro. However when antitumor activity of the the n=3 compound (DSB-120) was studied (Walton, M., et al., Cancer Chemother Pharmacol (1996) 38: 431. doi:10.1007/s002800050507), this was found not to be as promising. This lack of promise was thought to be “a consequence of low tumour selectivity and drug uptake as a result of high protein binding and/or extensive drug metabolism in vivo”. 25 In order to improve on these compounds, compounds were investigated (Gregson, S.J., et al., Chem. Commun., 1999, 797-798. doi: 10.1039/A809791G) with the “inclusion of C2/C2’ substituents that should follow the contour of the host minor groove”. This compound 30
Figure imgf000004_0002
008188229 3 was found to have “exquisite cytotoxicity in the picomolar region … some 9000-fold more potent that DSB-120.” This compound (also discussed in Gregson, S., et al., J. Med. Chem., 44, 737-748 (2001); 5 Alley, M.C., et al., Cancer Research, 64, 6700-6706 (2004); and Hartley, J.A., et al., Cancer Research, 64, 6693-6699 (2004)) has been involved in clinical trials as a standalone agent, for example, NCT02034227 investigating its use in treating Acute Myeloid Leukemia and Chronic Lymphocytic Leukemia (see: https://www.clinicaltrials.gov/ct2/show/NCT02034227). 10 Dimeric PBD compounds bearing C2 aryl substituents alongside endo-unsaturation, such as SG2202 (ZC-207), are disclosed in WO 2005/085251:
Figure imgf000005_0001
, and in WO2006/111759, bisulphites of such PBD compounds, for example SG2285 (ZC-15 423):
Figure imgf000005_0002
These compounds have been shown to be highly useful cytotoxic agents (Howard, P.W., et al., Bioorg. Med. Chem. (2009), doi: 10.1016/j.bmcl.2009.09.012). 20 In a review of PBD containing ADCs (Mantaj, J., et al., Angew. Chem. Int. Ed. (2016), 55, 2-29; DOI: 10.1002/anie.201510610), the SAR of PBD dimers is discussed. The summary of the SAR is presented in Figure 3 - B “C2-exo and C1-C2/C2-C3 unsaturation enhances activity”. A more detailed discussion is found at section 2.4 which says: 25 “DSB-120 has poor activity in vivo, attributed partly to its high reactivity with cellular thiol- containing molecules such as glutathione. However, introduction of C2/C2’-exo unsaturation as in SJG-136 led to an overall increase in DNA-binding affinity and 008188229 4 cytotoxicity, and a lower reactivity toward cellular nucleophiles with more of the agent potentially reaching its target DNA.” WO 2007/085930 describes the preparation of dimer PBD compounds having linker groups 5 for connection to a cell binding agent, such as an antibody. The linker is present in the bridge linking the monomer PBD units of the dimer. Dimer PBD compounds having linker groups for connection to a cell binding agent, such as an antibody, are described in WO 2011/130598. The linker in these compounds is 10 attached to one of the available N10 positions, and are generally cleaved by action of an enzyme on the linker group. The dimer PBD compounds have either endo or exo unsaturation in the C-ring. WO2014/096368 discloses PBD dimer conjugates linked via a N10 where the PBD moieity 15 which is not linked to the antibody is non-alkylating. Disclosure The present disclosure provides conjugates comprising PBD dimers linked via the N10 of a non-alkylating PBD moiety (i.e. the released moiety has a secondary amine at the N10 20 position, rather than an imine or equivalent group), and the precursor drug linkers used to make such conjugates. A first aspect of the present disclosure provides conjugates of formula I: L - (DL)p (I) 25 wherein L is a Ligand unit (i.e., a targeting agent), p is from 1 to 20, DL is a Drug Linker unit of formula I’: I'
Figure imgf000006_0001
RLL is a linker for connection to the Ligand Unit, which is 008188229 5 O H LL N G Q X IIIa' , wherein Q is:
Figure imgf000007_0001
, where QX is such that Q is an amino-acid residue, a dipeptide residue, 5 a tripeptide residue, or a tetrapeptide issue; X is:
Figure imgf000007_0002
, where a = 0 to 5, b = 0 to 16, c = 0 or 1, d = 0 to 5; GLL is a linker connected to the Ligand Unit; 10 D represents either group D1 or D2:
Figure imgf000007_0003
D1 D2 ; the dotted line indicates the optional presence of a double bond between C2 and C3; when there is a double bond present between C2 and C3, R2 is selected from the group consisting of: 15 (ia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene; (ib) C1-5 saturated aliphatic alkyl; (ic) C3-6 saturated cycloalkyl; 008188229 6 12 R 13 R 11 (id) R , wherein each of R11, R12 and R13 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R2 group is no more than 5;
Figure imgf000008_0001
(ie) , wherein one of R15a and R15b is H and the other is selected from: 5 phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (if)
Figure imgf000008_0002
, where R14 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; 10 when there is a single bond present between C2 and C3, R2 is selected from H, OH, F, diF
Figure imgf000008_0003
, where R16a and R16b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R16a and R16b is H, the other is selected from nitrile and a C1-4 alkyl 15 ester; D’ represents either group D’1 or D’2:
Figure imgf000008_0004
D'1 D'2 wherein the dotted line indicates the optional presence of a double bond between C2’ and C3’; 20 when there is a double bond present between C2’ and C3’, R22 is selected from the group consisting of: 008188229 7 (iia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene; (iib) C1-5 saturated aliphatic alkyl; 5 (iic) C3-6 saturated cycloalkyl; (iid)
Figure imgf000009_0001
, wherein each of R31, R32 and R33 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R22 group is no more than 5; R25b (iie)
Figure imgf000009_0002
, wherein one of R25a and R25b is H and the other is selected from: 10 phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (iif)
Figure imgf000009_0003
, where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; 15 when there is a single bond present between C2’ and C3’, 26a * R 26b R22 is selected from H, OH, F, diF and R , where R26a and R26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester; 20 R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR’, nitro, Me3Sn and halo; where R and R’ are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups; R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR’, nitro, Me3Sn and halo; 25 R″ is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine; Y and Y’ are selected from O, S, and NH; R6’, R7’, and R9’ are selected from the same groups as R6, R7 and R9 respectively; 008188229 8 and either: (a) R20 is H and R21 is H; (b) R20 is H and R21 is =O; 5 (c) R20 and R21 together form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (d) R21 is OH or ORA, where RA is C1-4 alkyl and R20 is selected from:
Figure imgf000010_0001
(i) ;
Figure imgf000010_0002
(ii) ;
Figure imgf000010_0003
10 (iii) , where RZ is selected from:
Figure imgf000010_0004
(z-i) ; (z-ii) OC(=O)CH3; (z-iii) NO2; (z-iv) OMe; 008188229 9 (z-v) glucuronide (i.e. ); and (z-vi) NH-C(=O)-X1-NHC(=O)X2-NH-C(=O)-RZC, where -C(=O)-X1- NH- and -C(=O)-X2-NH- represent natural amino acid residues and RZC is selected from Me, OMe, CH2CH2OMe, and (CH2CH2O)2Me. 5 The square brackets around NO2 above indicate that NO2 group may or may not be present. For the avoidance of doubt, the carbon labelled C3 in D1 is adjacent the ternary N at the ring junction. The carbon labelled C3’ in D’1 is adjacent the ternary N at the ring junction. 10 The Ligand unit, described more fully below, is a targeting agent that binds to a target moiety. The Ligand unit can, for example, specifically bind to a cell component (a Cell Binding Agent) or to other target molecules of interest. The Ligand unit can be, for example, a protein, polypeptide or peptide, such as an antibody, an antigen-binding 15 fragment of an antibody, or other binding agent, such as an Fc fusion protein. At least the N10 position bearing a linking group releases a non-alkylating PBD (i.e. the released moiety has a secondary amine at the N10 position, rather than an imine or equivalent group). Dimers containing at least one secondary amine moiety are unable to 20 covalently cross-link DNA, and may be less toxic than dimers with two imine moieties (which can covalently cross-link DNA). Without wishing to be bound by theory, avoiding the presence of a C11 hydroxy group adjacent the carbamate on the N10 nitrogen may increase the stability of the carbamate. 25 The proximity of the hydroxy to the carbonyl of the carbamate allows for the formation of an internal hydrogen bond which could catalyse a more facile nucleophilic attack on the carbonyl. 008188229 10 A second aspect of the present disclosure comprises a compound with the formula II: II
Figure imgf000012_0001
and salts and solvates thereof, wherein D, R2, R6, R7, R9, Y, R”, Y’, D’, R22, R6’, R7’, R9’, R20 and R21 (including the presence 5 or absence of double bonds between C2 and C3 and C2’ and C3’ respectively) are as defined in the first aspect of the disclosure; RL is a linker for connecting to a Ligand unit, which is:
Figure imgf000012_0002
, where Q and X are as defined in the first aspect and GL is a linker for connecting to a10 Ligand unit. A third aspect of the present disclosure provides the use of a conjugate of the first aspect of the disclosure in the manufacture of a medicament for treating a proliferative disease. The third aspect also provides a conjugate of the first aspect of the disclosure for use in the 15 treatment of a proliferative disease. The third aspect also provides a method of treating a proliferative disease comprising administering a therapeutically effective amount of a conjugate of the first aspect of the disclosure to a patient in need thereof. One of ordinary skill in the art is readily able to determine whether or not a candidate 20 conjugate treats a proliferative condition for any particular cell type. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described in the examples below. 008188229 11 A fourth aspect of the present disclosure provides the synthesis of a conjugate of the first aspect of the disclosure comprising conjugating a compound (drug linker) of the second aspect of the disclosure with a Ligand Unit. 5 A fifth aspect of the present disclosure provides a method of making a compound of formula IV:
Figure imgf000013_0001
from a compound of formula V:
Figure imgf000013_0002
10 using the Mitsunobu reaction; where R8 is selected from: (a) OMe, OCH2Ph, OH, OProtO and -Y’-R”-Hal; (b)
Figure imgf000013_0003
; and 15 (c) 008188229 12 20 R 9' 21 R R N Y' Y H R'' Vc N 7' R 6' D' O R where D, R2, R6, R7, R9, Y, R”, Y’, D’, R22, R6’, R7’, R9’, R20 and R21 are as defined in the first aspect of the disclosure; 5 Hal is a halogen, such as Br; RLpre is a precursor to RL; and ProtO is a hydroxyl protecting group. In this aspect, where R8 in the compound of formula V is of formula Vb, the compound of10 formula IV would have both R20 and R21 as H due to the Mitsunobu reaction. The presence of the carbamate on the pre-N10 prevents the Mitsunobu reaction from proceeding beyond the formation of the secondary amine. 15 A sixth aspect of the present disclosure provides a compound of formula VI:
Figure imgf000014_0001
D, R2, R6, R7, R9, Y, R”, Y’, D’, R6’, R7’, R9’ and R22 (including the presence or absence of double bonds between C2 and C3 and C2’ and C3’ respectively) are as defined in the first20 aspect of the disclosure. 008188229 13 Definitions Substituents The phrase “optionally substituted” as used herein, pertains to a parent group which may be unsubstituted or which may be substituted. 5 Unless otherwise specified, the term “substituted” as used herein, pertains to a parent group which bears one or more substituents. The term “substituent” is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and 10 methods for their formation and introduction into a variety of parent groups are also well known. Examples of substituents are described in more detail below. 15 C1-12 alkyl: The term “C1-12 alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). The term “C1-4 alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a 20 carbon atom of a hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below. 25 Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), butyl (C4), pentyl (C5), hexyl (C6) and heptyl (C7). Examples of saturated linear alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), n-butyl (C4), n-pentyl (amyl) (C5), n-hexyl (C6) and n-heptyl (C7). 30 Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec-butyl (C4), tert-butyl (C4), iso-pentyl (C5), and neo-pentyl (C5). C2-12 Alkenyl: The term “C2-12 alkenyl” as used herein, pertains to an alkyl group having35 one or more carbon-carbon double bonds. 008188229 14 Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, - CH=CH2), 1-propenyl (-CH=CH-CH3), 2-propenyl (allyl, -CH-CH=CH2), isopropenyl (1- methylvinyl, -C(CH3)=CH2), butenyl (C4), pentenyl (C5), and hexenyl (C6). 5 C2-12 alkynyl: The term “C2-12 alkynyl” as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds. Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (-C≡CH) and 2-propynyl (propargyl, -CH2-C≡CH). 10 C3-12 cycloalkyl: The term “C3-12 cycloalkyl” as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms. 15 Examples of cycloalkyl groups include, but are not limited to, those derived from: saturated monocyclic hydrocarbon compounds: cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C7), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), 20 dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7) and methylcyclohexane (C7); unsaturated monocyclic hydrocarbon compounds: cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), 25 dimethylcyclobutene (C6), methylcyclopentene (C6), dimethylcyclopentene (C7) and methylcyclohexene (C7); and saturated polycyclic hydrocarbon compounds: norcarane (C7), norpinane (C7), norbornane (C7). 30 C3-20 heterocyclyl: The term “C3-20 heterocyclyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms. 35 008188229 15 In this context, the prefixes (e.g. C3-20, C3-7, C5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C5-6heterocyclyl”, as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms. 5 Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from: N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5),10 piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7); O1: oxirane (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxole (dihydrofuran) (C5), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7); S1: thiirane (C3), thietane (C4), thiolane (tetrahydrothiophene) (C5), thiane (tetrahydrothiopyran) (C6), thiepane (C7); 15 O2: dioxolane (C5), dioxane (C6), and dioxepane (C7); O3: trioxane (C6); N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C6); N1O1: tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), 20 dihydroisoxazole (C5), morpholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (C6); N1S1: thiazoline (C5), thiazolidine (C5), thiomorpholine (C6); N2O1: oxadiazine (C6); O1S1: oxathiole (C5) and oxathiane (thioxane) (C6); and, 25 N1O1S1: oxathiazine (C6). Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C5), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C6), such as allopyranose, 30 altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose. C5-20 aryl: The term “C5-20 aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which 35 moiety has from 5furth to 20 ring atoms. The term “C5-7 aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of 008188229 16 an aromatic compound, which moiety has from 5 to 7 ring atoms and the term “C5-10 aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 10 ring atoms. Preferably, each ring has from 5 to 7 ring atoms. 5 In this context, the prefixes (e.g. C3-20, C5-7, C5-6, C5-10, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C5-6 aryl” as used herein, pertains to an aryl group having 5 or 6 ring atoms. 10 The ring atoms may be all carbon atoms, as in “carboaryl groups”. Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C6), naphthalene (C10), azulene (C10), anthracene (C14), phenanthrene (C14), naphthacene (C18), and pyrene (C16). 15 Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g.2,3-dihydro-1H- indene) (C9), indene (C9), isoindene (C9), tetraline (1,2,3,4-tetrahydronaphthalene (C10), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (C15), and 20 aceanthrene (C16). Alternatively, the ring atoms may include one or more heteroatoms, as in “heteroaryl groups”. Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from: 25 N1: pyrrole (azole) (C5), pyridine (azine) (C6); O1: furan (oxole) (C5); S1: thiophene (thiole) (C5); N1O1: oxazole (C5), isoxazole (C5), isoxazine (C6); N2O1: oxadiazole (furazan) (C5); 30 N3O1: oxatriazole (C5); N1S1: thiazole (C5), isothiazole (C5); N2: imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C6), pyrimidine (1,3-diazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6); N3: triazole (C5), triazine (C6); and, 35 N4: tetrazole (C5). 008188229 17 Examples of heteroaryl which comprise fused rings, include, but are not limited to: C9 (with 2 fused rings) derived from benzofuran (O1), isobenzofuran (O1), indole (N1), isoindole (N1), indolizine (N1), indoline (N1), isoindoline (N1), purine (N4) (e.g., adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzisoxazole (N1O1), 5 benzodioxole (O2), benzofurazan (N2O1), benzotriazole (N3), benzothiofuran (S1), benzothiazole (N1S1), benzothiadiazole (N2S); C10 (with 2 fused rings) derived from chromene (O1), isochromene (O1), chroman (O1), isochroman (O1), benzodioxan (O2), quinoline (N1), isoquinoline (N1), quinolizine (N1), benzoxazine (N1O1), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinazoline 10 (N2), cinnoline (N2), phthalazine (N2), naphthyridine (N2), pteridine (N4); C11 (with 2 fused rings) derived from benzodiazepine (N2); C13 (with 3 fused rings) derived from carbazole (N1), dibenzofuran (O1), dibenzothiophene (S1), carboline (N2), perimidine (N2), pyridoindole (N2); and, C14 (with 3 fused rings) derived from acridine (N1), xanthene (O1), thioxanthene (S1), 15 oxanthrene (O2), phenoxathiin (O1S1), phenazine (N2), phenoxazine (N1O1), phenothiazine (N1S1), thianthrene (S2), phenanthridine (N1), phenanthroline (N2), phenazine (N2). The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional 20 substituents listed below. Halo: -F, -Cl, -Br, and -I. Hydroxy: -OH. 25 Ether: -OR, wherein R is an ether substituent, for example, a C1-7 alkyl group (also referred to as a C1-7 alkoxy group, discussed below), a C3-20 heterocyclyl group (also referred to as a C3-20 heterocyclyloxy group), or a C5-20 aryl group (also referred to as a C5-20 aryloxy group), preferably a C1-7alkyl group. 30 Alkoxy: -OR, wherein R is an alkyl group, for example, a C1-7 alkyl group. Examples of C1-7 alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n- propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy). 35 008188229 18 Oxo (keto, -one): =O. Acyl (keto): -C(=O)R, wherein R is an acyl substituent, for example, a C1-7 alkyl group (also referred to as C1-7 alkylacyl or C1-7 alkanoyl), a C3-20 heterocyclyl group (also referred to as 5 C3-20 heterocyclylacyl), or a C5-20 aryl group (also referred to as C5-20 arylacyl), preferably a C1-7 alkyl group. Examples of acyl groups include, but are not limited to, -C(=O)CH3 (acetyl), -C(=O)CH2CH3 (propionyl), -C(=O)C(CH3)3 (t-butyryl), and -C(=O)Ph (benzoyl, phenone). 10 Carboxy (carboxylic acid): -C(=O)OH. Ester (carboxylate, carboxylic acid ester, oxycarbonyl): -C(=O)OR, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to,15 -C(=O)OCH3, -C(=O)OCH2CH3, -C(=O)OC(CH3)3, and -C(=O)OPh. Amino: -NR1R2, wherein R1 and R2 are independently amino substituents, for example, hydrogen, a C1-7 alkyl group (also referred to as C1-7 alkylamino or di-C1-7 alkylamino), a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably H or a C1-7 alkyl group, or, in the 20 case of a “cyclic” amino group, R1 and R2, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Amino groups may be primary (-NH2), secondary (-NHR1), or tertiary (-NHR1R2), and in cationic form, may be quaternary (-+NR1R2R3). Examples of amino groups include, but are not limited to, -NH2, -NHCH3, -NHC(CH3)2, -N(CH3)2, -N(CH2CH3)2, and -NHPh. Examples of cyclic amino 25 groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino. Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): -C(=O)NR1R2, wherein R1 and R2 are independently amino substituents, as defined for amino groups. Examples of amido 30 groups include, but are not limited to, -C(=O)NH2, -C(=O)NHCH3, -C(=O)N(CH3)2, -C(=O)NHCH2CH3, and -C(=O)N(CH2CH3)2, as well as amido groups in which R1 and R2, together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl. 35 008188229 19 Nitro: -NO2. Cyano (nitrile, carbonitrile): -CN. 5 Hydroxyl protecting group: Hydroxyl protecting groups are well known in the art, for example, in Wuts & Greene 2007. Those groups suitable for use in the present disclosure include substituted methyl ethers, substituted ethyl ethers, methoxy substituted benzyl ethers, silyl ethers and acetates. Of particular relevance are tert-butyldimethylsilyl (TBS)10 and triisopropylsilyl (TIPS). Amine protecting group: Hydroxyl protecting groups are well known in the art, for example, in Wuts & Greene 2007. Those groups suitable for use in the present disclosure include carbamate. Of particular relevance is allyl carbamate (Alloc). 15 Alkylene C3-12 alkylene: The term “C3-12 alkylene”, as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 3 to 12 20 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term “alkylene” includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below. Examples of linear saturated C3-12 alkylene groups include, but are not limited to, -(CH2)n- 25 where n is an integer from 3 to 12, for example, -CH2CH2CH2- (propylene), -CH2CH2CH2CH2- (butylene), -CH2CH2CH2CH2CH2- (pentylene) and -CH2CH2CH2CH-2CH2CH2CH2- (heptylene). Examples of branched saturated C3-12 alkylene groups include, but are not limited to, 30 -CH(CH3)CH2-, -CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH(CH3)CH2CH2-, -CH(CH2CH3)-, -CH(CH2CH3)CH2-, and -CH2CH(CH2CH3)CH2-. Examples of linear partially unsaturated C3-12 alkylene groups (C3-12 alkenylene, and alkynylene groups) include, but are not limited to, -CH=CH-CH2-, -CH2-CH=CH2-, 35 -CH=CH-CH2-CH2-, -CH=CH-CH2-CH2-CH2-, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH2-, - 008188229 20 CH=CH-CH=CH-CH2-CH2-, -CH=CH-CH2-CH=CH-, -CH=CH-CH2-CH2-CH=CH-, and -CH2- C≡C-CH2-. Examples of branched partially unsaturated C3-12 alkylene groups (C3-12 alkenylene and 5 alkynylene groups) include, but are not limited to, -C(CH3)=CH-, -C(CH3)=CH-CH2-, -CH=CH-CH(CH3)- and -C≡C-CH(CH3)-. Examples of alicyclic saturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentylene (e.g. cyclopent-1,3-ylene), and cyclohexylene 10 (e.g. cyclohex-1,4-ylene). Examples of alicyclic partially unsaturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g.4-cyclopenten-1,3-ylene), cyclohexenylene (e.g.2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-15 1,4-ylene). Where the C3-12 alkylene group is interrupted by a heteroatom, the subscript refers to the number of atoms in the chain including the heteroatoms. For example, the chain -C2H4-O- C2H4- would be a C5 group. 20 Where the C3-12 alkylene group is interrupted by an aromatic ring, the subscript refers to the number of atoms directly in the chain including the aromatic ring. For example, the chain would be a C5 group.
Figure imgf000022_0001
25 The symbols and are used interchangably to represent the attachment point of the chemical group. Ligand Unit The Ligand Unit may be of any kind, and include a protein, polypeptide, peptide and a non- 30 peptidic agent that specifically binds to a target molecule. In some embodiments, the Ligand unit may be a protein, polypeptide or peptide. In some embodiments, the Ligand unit may be a cyclic polypeptide. These Ligand units can include antibodies or a fragment of an antibody that contains at least one target molecule-binding site, lymphokines, 008188229 21 hormones, growth factors, or any other cell binding molecule or substance that can specifically bind to a target. The terms “specifically binds” and “specific binding” refer to the binding of an antibody or 5 other protein, polypeptide or peptide to a predetermined molecule (e.g., an antigen). Typically, the antibody or other molecule binds with an affinity of at least about 1x107 M-1, and binds to the predetermined molecule with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or a closely-related molecule. 10 Examples of Ligand units include those agents described for use in WO 2007/085930, which is incorporated herein. In some embodiments, the Ligand unit is a Cell Binding Agent that binds to an extracellular15 target on a cell. Such a Cell Binding Agent can be a protein, polypeptide, peptide or a non- peptidic agent. In some embodiments, the Cell Binding Agent may be a protein, polypeptide or peptide. In some embodiments, the Cell Binding Agent may be a cyclic polypeptide. The Cell Binding Agent also may be antibody or an antigen-binding fragment of an antibody. Thus, in one embodiment, the present disclosure provides an 20 antibody-drug conjugate (ADC). Cell Binding Agent A cell binding agent may be of any kind, and include peptides and non-peptides. These can include antibodies or a fragment of an antibody that contains at least one binding site, 25 lymphokines, hormones, hormone mimetics, vitamins, growth factors, nutrient-transport molecules, or any other cell binding molecule or substance. Peptides In one embodiment, the cell binding agent is a linear or cyclic peptide comprising 4-30, 30 preferably 6-20, contiguous amino acid residues. In this embodiment, it is preferred that one cell binding agent is linked to one monomer or dimer pyrrolobenzodiazepine compound. In one embodiment the cell binding agent comprises a peptide that binds integrin ανβ6. The 35 peptide may be selective for ανβ6 over XYS. 008188229 22 In one embodiment the cell binding agent comprises the A20FMDV-Cys polypeptide. The A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC. Alternatively, a variant of the A20FMDV-Cys sequence may be used wherein one, two, three, four, five, six, seven, eight, nine or ten amino acid residues are substituted with another amino acid 5 residue. Furthermore, the polypeptide may have the sequence NAVXXXXXXXXXXXXXXXRTC. Antibodies The term “antibody” herein is used in the broadest sense and specifically covers 10 monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), multivalent antibodies and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854- 4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of 15 recognizing and binding to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. 20 An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any 25 type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species, including human, murine, or rabbit origin. "Antibody fragments" comprise a portion of a full length antibody, generally the antigen 30 binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody 35 molecules; and multispecific antibodies formed from antibody fragments. 008188229 23 The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being 5 directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” 10 indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant 15 DNA methods (see, US 4816567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450- 459). 20 The monoclonal antibodies herein specifically include chimeric antibodies, humanized antibodies and human antibodies. Examples of cell binding agents include those agents described for use in 25 WO 2007/085930, which is incorporated herein. Modified antibodies suitable for use in the present disclosure include those where cysteines have been inserted in selected sites in antibodies. These are described in Dimasi, N., et al., Molecular Pharmaceutics, 2017, 14, 1501-1516 (DOI: 30 10.1021/acs.molpharmaceut.6b00995) and WO2015/157595. In particular, antibodies which have been modified by insertion of a cysteine after the S239 position (ie. between positions 239 and 240) are of use. In some embodiments, antibodies which have been modified by insertion of a non-natural 35 amino acid at F241 (EU numbering according to Kabat) may be used. A non-natural amino acid as employed herein refers to an amino acid which is other than one of the twenty-one 008188229 24 naturally occurring amino acids. The non-natural amino acids are generally derived from natural amino acids. Derived from a natural amino acid refers to the fact that the non- natural amino acid is based on (or incorporates) or is similar to the structure of natural amino acid, for example the alkylene chain in lysine may be shortened to provide a 3- 5 carbon chain as opposed to the natural 4 carbon chain but the structural relationship or similarity to lysine still exists. Thus, derivatives of natural amino acids include modifications such as incorporating a functional group, lengthening or shortening an alkylene chain, adding one or more substituents to a nitrogen, oxygen, sulfur in a side chain or converting a nitrogen, oxygen or sulfur into a different functional group or a 10 combination of any of the same. Usually the majority of modifications will be the addition of structure in the non-natural amino acid. However, modification may include removed or replacing an atom naturally found in an amino acid. In some embodiments the non-natural amino has a formula (AAII): RX-XAA1-O0-1C(O)-amino-acid-residue (AAII) 15 wherein: RX represents an unsaturated group selected from a: i) C4-9 linear conjugated diene, ii) C5-14 carbocyclyl comprising a conjugated diene, and iii) a 5 to 14 membered heterocyclyl comprising 1, 2 or 3 heteroatoms selected O, N 20 and S, and a conjugated diene, wherein i), ii) and iii) may bear up to five substituents, (such as one, two or three substituents) for example, the substituents are independently selected from C1-3 alkyl, oxo, halogen, sulfo, sulfhydryl, amino, -C1-3alkyleneN3, or -C2- 5alkynyl; 25 and XAA1 represents i) a saturated or unsaturated branched or unbranched C1-8 alkylene chain, wherein at least one carbon (for example 1, 2 or 3 carbons) is replaced by a heteroatom selected from O, 30 N, S(O)0-3, wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, amino, -C1-3alkyleneN3, or -C2-5alkynyl; or ii) together with a carbon from the carbocylcyl or heterocyclyl represents a cyclopropane ring linked to a saturated or 35 unsaturated (in particular saturated) branched or unbranched C1-6 alkylene chain, wherein at least one carbon (for example 1, 2 or 3 carbons) is replaced by a heteroatom selected from 008188229 25 O, N, S(O)0-3, wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, amino, -C1-3alkyleneN3, or -C2-5alkynyl and -O0-1C(O)- is linked through a side chain of an amino acid. 5 The amino acid residue referred to in AAII is as defined for AAI above. In the context of formula AAII, the amino acid residue refers to an amino acid comprising the -NH2 and -COOH groups. The amino acid residue in formula AAII may additionally comprise an R group of a natural amino acid. Alternatively, the amino acid residue in 10 formula AAII may be derived from a natural amino acid but have its natural R group replaced with RX-XAA1-O0-1C(O). In one embodiment the non-natural amino acid is a residue of the structure of formula 15
Figure imgf000027_0001
wherein X2 represents -C-, -C(R’)-, -CH2 or O; R’ represents H or C1-3 alkyl, Ra represents 20 i) a saturated or unsaturated branched or unbranched C1-8 alkylene chain, wherein at least one carbon (for example 1, 2 or 3 carbons) is replaced by a heteroatom selected from O, N, S(O)0-3, wherein said chain is optionally, substituted by one or more groups independently selected from oxo,25 halogen, amino; or ii) together with a carbon from the 5 membered ring represents a cyclopropane ring linked to a saturated or unsaturated (in particular saturated) branched or unbranched C1-6 alkylene chain, wherein at least one carbon (for example 1, 2 or 3 30 carbons) is replaced by a heteroatom selected from O, N, S(O)0-3, wherein said chain is optionally, substituted by one or more groups independently selected from oxo, halogen, amino; 008188229 26 Rb represents H, -OC1-3 alkyl, C1-6alkyl optionally bearing a hydroxyl substituent, -C1-3alkyleneN3, or -C2-5 alkynyl; Rc represents H, -OC1-3 alkyl, C1-6alkyl optionally bearing a hydroxyl substituent, -C1-3 alkyleneN3, or -C2-5 alkynyl; 5 Rd represents H, -OC1-3alkyl, C1-6alkyl optionally bearing a hydroxyl substituent, -C1-3 alkyleneN3, or -C2-5 alkynyl; Re represents H, saturated or unsaturated (in particular saturated) branched or unbranched C1-8 alkylene chain, wherein one or more carbons are optionally replaced by -O- and the chain is optionally10 substituted by one or more halogen atoms (such as iodo), N3 or -C2- 5alkynyl. In one embodiment Ra is -(CH2)mC(O)-, -CH2(CH3)C(O)-, -(CH2)mCH2OC(O)-, -CHCHCH2OC(O)-, or -OCH2CH2COC(O)- and m represents 0 or 1. 15 In one embodiment Rb is H, -OC1-3alkyl, -CH3, -CH(CH3)2, CH2OH, -CH2N3, or –CCH. In one embodiment Rc is H, -OC1-3alkyl, -CH3, -CH(CH3)2, CH2OH, -CH2N3, or –CCH. In one embodiment Rd is H, -OC1-3alkyl, -CH3, -CH(CH3)2, CH2OH, -CH2N3, or –CCH. 20 In one embodiment Re represents H or -CH2OCH2CH2N3. In one embodiment the non-natural amino acid is a residue of the structure of formula (AAIIIa): 25
Figure imgf000028_0001
(AAIIIa) wherein Ra, Rb, Rc, Rd, Re and X2 are defined above. In one embodiment the non-natural amino acid has the structure of formula (AAIIIb): 30 008188229 27 (AAIIIb) wherein Ra, Rb, Rc, Rd, Re and X2 are defined above. In one embodiment the non-natural amino acid has the structure of formula (AAIIIc):
Figure imgf000029_0001
(AAIIIc) 5 wherein Ra, Rb, Rc, Rd, Re are defined above and X2’ is -C- or -CR’ as defined above. Generally compounds, for example formula (AAIII), (AAIIIa), (AAIIIb) and (AAIIIc) will at most contain only one azide group. 10 In one embodiment the non-natural amino acid is selected from the group comprising:
 008188229 29 In some embodiments, the non-natural amino acid is selected from:
Figure imgf000031_0001
Antibodies which have been modified by the insertion of CP2-NNAA are of particular use in the present invention. These are described in as described in WO2019/224340, and Roy 5 et al., MAbs 12 (1): 1684749 (doi:10.1080/19420862.2019.1684749), which are both incorporated herein by reference. In particular, antibodies with this insertion at F241 (EU numbering according to Kabat) may be used, e.g. modified Herceptin. Tumour-associate antigens and cognate antibodies for use in embodiments of the present 10 disclosure are listed below, and are described in more detail on pages 14 to 86 of WO 2017/186894, which is incorporated herein. (1) BMPR1B (bone morphogenetic protein receptor-type IB) (2) E16 (LAT1, SLC7A5) 15 (3) STEAP1 (six transmembrane epithelial antigen of prostate) (4) 0772P (CA125, MUC16) (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin) (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b) 20 (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B) 008188229 30 (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene) (9) ETBR (Endothelin type B receptor) 5 (10) MSG783 (RNF124, hypothetical protein FLJ20315) (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein) (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation10 5 channel, subfamily M, member 4) (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor) (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792) (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29) 15 (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C) (17) HER2 (ErbB2) (18) NCA (CEACAM6) (19) MDP (DPEP1) 20 (20) IL20R-alpha (IL20Ra, ZCYTOR7) (21) Brevican (BCAN, BEHAB) (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5) (23) ASLG659 (B7h) (24) PSCA (Prostate stem cell antigen precursor) 25 (25) GEDA (26) BAFF-R (B cell -activating factor receptor, BLyS receptor 3, BR3) (27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814) (27a) CD22 (CD22 molecule) (28) CD79a (CD79A, CD79alpha), immunoglobulin-associated alpha, a B cell-specific 30 protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation), pI: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19q13.2). (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays35 a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, 008188229 31 (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and 20 presents them to CD4+ T lymphocytes); 273 aa, pI: 6.56, MW: 30820.TM: 1 [P] Gene Chromosome: 6p21.3) (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by 5 extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability); 422 aa), pI: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3). (32) CD72 (B-cell differentiation antigen CD72, Lyb-2); 359 aa, pI: 8.66, MW: 40225, TM: 1 5 [P] Gene Chromosome: 9p13.3). 10 (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis); 661 aa, pI: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12). 15 (34) FcRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation); 429 aa, pI: 5.28, MW: 46925 TM: 1 [P] Gene Chromosome: 1q21-1q22) (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; 20 deregulation of the gene by translocation occurs in some B cell malignancies); 977 aa, pI: 6.88, MW: 106468, TM: 1 [P] Gene Chromosome: 1q21) (36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin); 374 aa) (37) PSMA – FOLH1 (Folate hydrolase (prostate-specific membrane antigen) 1) 25 (38) SST (Somatostatin Receptor; note that there are 5 subtypes) (38.1) SSTR2 (Somatostatin receptor 2) (38.2) SSTR5 (Somatostatin receptor 5) (38.3) SSTR1 (38.4) SSTR3 30 (38.5) SSTR4 AvB6 – Both subunits (39+40) (39) ITGAV (Integrin, alpha V) (40) ITGB6 (Integrin, beta 6) (41) CEACAM5 (Carcinoembryonic antigen-related cell adhesion molecule 5) 35 (42) MET (met proto-oncogene; hepatocyte growth factor receptor) (43) MUC1 (Mucin 1, cell surface associated) 008188229 32 (44) CA9 (Carbonic anhydrase IX) (45) EGFRvIII (Epidermal growth factor receptor (EGFR), transcript variant 3, (46) CD33 (CD33 molecule) (47) CD19 (CD19 molecule) 5 (48) IL2RA (Interleukin 2 receptor, alpha); NCBI Reference Sequence: NM_000417.2); (49) AXL (AXL receptor tyrosine kinase) (50) CD30 - TNFRSF8 (Tumor necrosis factor receptor superfamily, member 8) (51) BCMA (B-cell maturation antigen) - TNFRSF17 (Tumor necrosis factor receptor superfamily, member 17) 10 (52) CT Ags – CTA (Cancer Testis Antigens) (53) CD174 (Lewis Y) - FUT3 (fucosyltransferase 3 (galactoside 3(4)-L-fucosyltransferase, Lewis blood group) (54) CLEC14A (C-type lectin domain family 14, member A; Genbank accession no. NM175060) 15 (55) GRP78 – HSPA5 (heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) (56) CD70 (CD70 molecule) L08096 (57) Stem Cell specific antigens. For example: ^ 5T4 (see entry (63) below) ^ CD25 (see entry (48) above) 20 ^ CD32 ^ LGR5/GPR49 ^ Prominin/CD133 (58) ASG-5 (59) ENPP3 (Ectonucleotide pyrophosphatase/phosphodiesterase 3) 25 (60) PRR4 (Proline rich 4 (lacrimal)) (61) GCC – GUCY2C (guanylate cyclase 2C (heat stable enterotoxin receptor) (62) Liv-1 – SLC39A6 (Solute carrier family 39 (zinc transporter), member 6) (63) 5T4, Trophoblast glycoprotein, TPBG – TPBG (trophoblast glycoprotein) (64) CD56 – NCMA1 (Neural cell adhesion molecule 1) 30 (65) CanAg (Tumor associated antigen CA242) (66) FOLR1 (Folate Receptor 1) (67) GPNMB (Glycoprotein (transmembrane) nmb) (68) TIM-1 – HAVCR1 (Hepatitis A virus cellular receptor 1) (69) RG-1/Prostate tumor target Mindin – Mindin/RG-1 35 (70) B7-H4 – VTCN1 (V-set domain containing T cell activation inhibitor 1 (71) PTK7 (PTK7 protein tyrosine kinase 7) 008188229 33 (72) CD37 (CD37 molecule) (73) CD138 – SDC1 (syndecan 1) (74) CD74 (CD74 molecule, major histocompatibility complex, class II invariant chain) (75) Claudins – CLs (Claudins) 5 (76) EGFR (Epidermal growth factor receptor) (77) Her3 (ErbB3) – ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian)) (78) RON - MST1R (macrophage stimulating 1 receptor (c-met-related tyrosine kinase)) (79) EPHA2 (EPH receptor A2) 10 (80) CD20 – MS4A1 (membrane-spanning 4-domains, subfamily A, member 1) (81) Tenascin C – TNC (Tenascin C) (82) FAP (Fibroblast activation protein, alpha) (83) DKK-1 (Dickkopf 1 homolog (Xenopus laevis) (84) CD52 (CD52 molecule) 15 (85) CS1 - SLAMF7 (SLAM family member 7) (86) Endoglin – ENG (Endoglin) (87) Annexin A1 – ANXA1 (Annexin A1) (88) V-CAM (CD106) - VCAM1 (Vascular cell adhesion molecule 1) 20 An additional tumour-associate antigen and cognate antibodies of interest are: (89) ASCT2 (ASC transporter 2, also known as SLC1A5). ASCT2 antibodies are described in WO 2018/089393, which is incorporated herein by reference. 25 The cell binding agent may be labelled, for example to aid detection or purification of the agent either prior to incorporation as a conjugate, or as part of the conjugate. The label may be a biotin label. In another embodiment, the cell binding agent may be labelled with a radioisotope. 30 Methods of Treatment The compounds of the present disclosure may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a conjugate of formula II. The term “therapeutically effective amount” is an amount sufficient to show benefit to a patient. Such 35 benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and 008188229 34 severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors. A conjugate may be administered alone or in combination with other treatments, either 5 simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs; surgery; and radiation therapy. Pharmaceutical compositions according to the present disclosure, and for use in 10 accordance with the present disclosure, may comprise, in addition to the active ingredient, i.e. a conjugate of formula II, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may15 be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous. Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or 20 vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin. For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, 25 the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required. 30 The Conjugates can be used to treat proliferative disease and autoimmune disease. The term “proliferative disease” pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo. 35 008188229 35 Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian 5 carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Other cancers of interest include, but are not limited to, haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin 10 lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers of B or T cell origin. Examples of autoimmune disease include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic 15 arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves’ disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn’s disease), anaphylaxis, allergic reaction, Sjögren’s syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener’s granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, 20 Schmidt’s syndrome, autoimmune uveitis, Addison’s disease, adrenalitis, thyroiditis, Hashimoto’s thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler’s syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, 25 dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture’s syndrome,30 Chagas’ disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti- phospholipid syndrome, farmer’s lung, erythema multiforme, post cardiotomy syndrome, Cushing’s syndrome, autoimmune chronic active hepatitis, bird-fancier’s lung, toxic epidermal necrolysis, Alport’s syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, 35 Takayasu’s arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter’s syndrome, eczema, lymphomatoid 008188229 36 granulomatosis, Behcet’s disease, Caplan’s syndrome, Kawasaki’s disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman’s syndrome, Felty’s syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, 5 Fuch’s cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom’s macroglobulemia, Evan’s syndrome, and autoimmune gonadal failure. 10 In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture’s syndrome, rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren’s syndrome, Hashimoto’s thyroiditis, Graves’ disease, primary biliary cirrhosis, Wegener’s granulomatosis, tuberculosis, or graft versus host disease), or Th2-lymphocytes 15 (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn’s syndrome, systemic sclerosis, or chronic graft versus host disease). Generally, disorders involving dendritic cells involve disorders of Th1- lymphocytes or Th2-lymphocytes. In some embodiments, the autoimmunie disorder is a T cell-mediated immunological disorder. 20 In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 10 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.01 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administerd ranges from about 0.05 to about 5 mg/kg per 25 dose. In some embodiments, the amount of the Conjugate administerd ranges from about 0.1 to about 5 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 4 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.05 to about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to 30 about 3 mg/kg per dose. In some embodiments, the amount of the Conjugate administered ranges from about 0.1 to about 2 mg/kg per dose. Drug loading The drug loading (p) is the average number of PBD drugs per cell binding agent, e.g. 35 antibody. Where the compounds of the disclosure are bound to cysteines, drug loading may range from 1 to 8 drugs (D) per cell binding agent, i.e. where 1, 2, 3, 4, 5, 6, 7, and 8 008188229 37 drug moieties are covalently attached to the cell binding agent. Compositions of conjgates include collections of cell binding agents, e.g. antibodies, conjugated with a range of drugs, from 1 to 8. Where the compounds of the disclosure are bound to lysines, drug loading may range from 1 to 80 drugs (D) per cell binding agent, although an upper limit of 40, 20, 5 10 or 8 may be preferred. Compositions of conjgates include collections of cell binding agents, e.g. antibodies, conjugated with a range of drugs, from 1 to 80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8. The average number of drugs per antibody in preparations of ADC from conjugation 10 reactions may be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, and electrophoresis. The quantitative distribution of ADC in terms of p may also be determined. By ELISA, the averaged value of p in a particular preparation of ADC may be determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res.11:843-852). However, the 15 distribution of p (drug) values is not discernible by the antibody-antigen binding and detection limitation of ELISA. Also, ELISA assay for detection of antibody-drug conjugates does not determine where the drug moieties are attached to the antibody, such as the heavy chain or light chain fragments, or the particular amino acid residues. In some instances, separation, purification, and characterization of homogeneous ADC where p is a 20 certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates. For some antibody-drug conjugates, p may be limited by the number of attachment sites on 25 the antibody. For example, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached. Higher drug loading, e.g. p >5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates. 30 Typically, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with the Drug Linker. Only the most reactive lysine groups may react with an amine-reactive linker reagent. Also, only the most reactive cysteine thiol groups may react with a thiol-reactive linker reagent. Generally, antibodies do not contain 35 many, if any, free and reactive cysteine thiol groups which may be linked to a drug moiety. Most cysteine thiol residues in the antibodies of the compounds exist as disulfide bridges 008188229 38 and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP, under partial or total reducing conditions. The loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of Drug Linker relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) 5 partial or limiting reductive conditions for cysteine thiol modification. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two 10 reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut’s reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by engineering one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine 15 amino acid residues). US 7521541 teaches engineering antibodies by introduction of reactive cysteine amino acids. Cysteine amino acids may be engineered at reactive sites in an antibody and which do not form intrachain or intermolecular disulfide linkages (Junutula, et al., 2008b Nature Biotech., 20 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; WO2009/052249). The engineered cysteine thiols may react with linker reagents or the drug-linker reagents of the present disclosure which have thiol-reactive, electrophilic groups such as maleimide or alpha-halo amides to form ADC with cysteine engineered antibodies and the PBD drug moieties. The location of the drug moiety can thus be 25 designed, controlled, and known. The drug loading can be controlled since the engineered cysteine thiol groups typically react with thiol-reactive linker reagents or drug-linker reagents in high yield. Engineering an IgG antibody to introduce a cysteine amino acid by substitution at a single site on the heavy or light chain gives two new cysteines on the symmetrical antibody. A drug loading near 2 can be achieved with near homogeneity of30 the conjugation product ADC. Where more than one nucleophilic or electrophilic group of the antibody reacts with a drug- linker intermediate, or linker reagent followed by drug moiety reagent, then the resulting product is a mixture of ADC compounds with a distribution of drug moieties attached to an 35 antibody, e.g.1, 2, 3, etc. Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) may separate compounds in the mixture 008188229 39 by drug loading value. Preparations of ADC with a single drug loading value (p) may be isolated, however, these single loading value ADCs may still be heterogeneous mixtures because the drug moieties may be attached, via the linker, at different sites on the antibody. 5 Thus the antibody-drug conjugate compositions of the disclosure include mixtures of antibody-drug conjugate compounds where the antibody has one or more PBD drug moieties and where the drug moieties may be attached to the antibody at various amino acid residues. 10 In one embodiment, the average number of dimer pyrrolobenzodiazepine groups per cell binding agent is in the range 1 to 20. In some embodiments the range is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4, and 4 to 8. 15 In some embodiments, there is one dimer pyrrolobenzodiazepine group per cell binding agent. Therapeutic Index The Therapeutic Index of a particular drug-linker/conjugate can be calculated by dividing 20 the maximum tolerated single dose (MTD) of a non-targeted ADC in rat, by the minimal effective single dose (MED) of a comparable targeted ADC in mouse. The MED may be the single dose necessary to achieve tumour stasis in an in vivo model at 28 days. General synthetic routes 25 Mitsunobu A key step in the synthesis of compounds (drug-linkers) of formula II (and similar compounds) is the synthesis of compounds of formula IV: Lpre R
Figure imgf000041_0001
from compounds of formula V: 008188229 40 Lpre R O O 9 R O H 8 R NH H V 7 R N 6 R O D by using a Mitsunobu reaction to close the ring. Where R8 in the compound of formula V is of formula Vb, the compound of formula IV 5 would have both R20 and R21 as H due to the Mitsunobu reaction. The reaction may be carried out under conventional conditions, using triphenylphosphine and an azodicarboxylate such as diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD). 10 In particular, for the synthesis of compounds of formula II, R8 should be selected from: (a) OH and -Y’-R”-Hal; (b) 15
Figure imgf000042_0001
. 008188229 41 Further derivation When R8 is (Vc), compounds of formula II may be synthesised from compounds of formula IV by converting RLpre into RL. Typically the precursor of RL (RLpre) will comprise:
Figure imgf000043_0001
5 Where ProtN is an amine protecting group, such as Alloc. The conversion may be carried out by removal of the amine protecting group and reaction with the remainder of the RL group (i.e. HO-C(=O)-X-GL). 10 When R8 is -Y’-R”-Hal, compounds of formula IV where R8 is (Vc) may be synthesised by coupling a compound of formula VIa:
Figure imgf000043_0002
to the compound of formula IV when R8 is -Y’-R”-Hal. 15 When R8 is –OH, and Y’ is O, compounds of formula IV where R8 is (Vc) may be synthesised by coupling a compound of formula VIb:
Figure imgf000043_0003
to the compound of formula IV when R8 is –OH. 20 Compounds of formula V may be synthesised from compounds of formula VII: 008188229 42 Lpre R O O 9 R O OProt 8 R NH H (VII) 7 R N 6 R O D by removal of the hydroxyl protecting group (ProtO) where R8 is selected from: (a) OMe, OCH2Ph, OH and –Y’-R”-Hal; 5 b)
Figure imgf000044_0001
and R6, R7, R9, R11a, R6’, R7’, R9’, Y, R”, Y’, D and D’ are as defined in the first aspect of the10 disclosure. Compounds of the sixth aspect of the disclosure may be made by analogous methods to those described above. 15 008188229 43 Synthesis of Drug Conjugates Conjugates can be prepared as previously described. Antibodies can be conjugated to the Drug Linker compound as described in Doronina et al., Nature Biotechnology, 2003, 21, 778-784). Briefly, antibodies (4-5 mg/mL) in PBS containing 50 mM sodium borate at pH 5 7.4 are reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37 ºC. The progress of the reaction, which reduces interchain disulfides, is monitored by reaction with 5,5’-dithiobis(2-nitrobenzoic acid) and allowed to proceed until the desired level of thiols/mAb is achieved. The reduced antibody is then cooled to 0°C and alkylated with 1.5 equivalents of maleimide drug-linker per antibody thiol. After 1 hour, the reaction is 10 quenched by the addition of 5 equivalents of N-acetyl cysteine. Quenched drug-linker is removed by gel filtration over a PD-10 column. The ADC is then sterile-filtered through a 0.22 μm syringe filter. Protein concentration can be determined by spectral analysis at 280 nm and 329 nm, respectively, with correction for the contribution of drug absorbance at 280 nm. Size exclusion chromatography can be used to determine the extent of antibody15 aggregation, and RP-HPLC can be used to determine the levels of remaining NAC- quenched drug-linker. Further Preferences The following preferences may apply to all aspects of the disclosure as described above, or 20 may relate to a single aspect. The preferences may be combined together in any combination. In some embodiments, R6’, R7’, R9’, and Y’ are selected from the same groups as R6, R7, R9, and Y respectively. In some of these embodiments, R6’, R7’, R9’, and Y’ are the same25 as R6, R7, R9, and Y respectively. In some embodiments, R22 is the same as R2. N10’-C11’ 30 In some embodiments, R20 is H and R21 is H. In some embodiments, R20 is H and R21 is =O. In some embodiments, R20 and R21 together form a nitrogen-carbon double bond between35 the nitrogen and carbon atoms to which they are bound. In some embodiments, R21 is OH or ORA, where RA is C1-4 alkyl and R20 is selected from: 008188229 44
Figure imgf000046_0001
008188229 45
Figure imgf000047_0001
where -C(=O)-X1-NHC(=O)X2-NH- represent a dipeptide. The amino acids in the dipeptide may be any combination of natural amino acids. The dipeptide may be the site of action for cathepsin-mediated cleavage. 5 In one embodiment, the dipeptide, -C(=O)-X1-NHC(=O)X2-NH-, is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, 10 -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit-, 15 -Phe-Arg-, -Trp-Cit- where Cit is citrulline. Preferably, the dipeptide, -C(=O)-X1-NHC(=O)X2-NH-, is selected from: 20 -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-. 008188229 46 Most preferably, the dipeptide, -C(=O)-X1-NHC(=O)X2-NH-, is -Phe-Lys- or -Val-Ala-. Other dipeptide combinations may be used, including those described by Dubowchik et al., 5 Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference. In one embodiment, the amino acid side chain is derivatised, where appropriate. For example, an amino group or carboxy group of an amino acid side chain may be derivatised. 10 In one embodiment, an amino group NH2 of a side chain amino acid, such as lysine, is a derivatised form selected from the group consisting of NHR and NRR’. In one embodiment, a carboxy group COOH of a side chain amino acid, such as aspartic acid, is a derivatised form selected from the group consisting of COOR, CONH2, CONHR 15 and CONRR’. In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed above. The present inventors have established that protected amino acid sequences are cleavable by 20 enzymes. For example, it has been established that a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin. Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog. Additional protecting group strategies are set out25 in Protective Groups in Organic Synthesis, Greene and Wuts. Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality: Arg: Z, Mtr, Tos; 30 Asn: Trt, Xan; Asp: Bzl, t-Bu; Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt; Glu: Bzl, t-Bu; Gln: Trt, Xan; 35 His: Boc, Dnp, Tos, Trt; Lys: Boc, Z-Cl, Fmoc, Z, Alloc; 008188229 47 Ser: Bzl, TBDMS, TBDPS; Thr: Bz; Trp: Boc; Tyr: Bzl, Z, Z-Br. 5 In one embodiment, the side chain protection is selected to be orthogonal to a group provided as, or as part of, a capping group, where present. Thus, the removal of the side chain protecting group does not remove the capping group, or any protecting group functionality that is part of the capping group. 10 In other embodiments of the disclosure, the amino acids selected are those having no reactive side chain functionality. For example, the amino acids may be selected from: Ala, Gly, Ile, Leu, Met, Phe, Pro, and Val. 15 It is particularly preferred in the present disclosure, that if Q comprises a dipeptide, then -C(=O)-X1-NHC(=O)X2-NH- is the same dipeptide. An example of a preferred group is:
Figure imgf000049_0001
. Other preferred R20 groups include: 20
Figure imgf000049_0002
. Dimer link In some embodiments, Y and Y’ are both O. 008188229 48 In some embodiments, R’’ is a C3-7 alkylene group with no substituents. In some of these embodiments, R’’ is a C3, C5 or C7 alkylene. In particular, R’’ may be a C3 or C5 alkylene. In other embodiments, R” is a group of formula: 5
Figure imgf000050_0001
In other embodiments, R” is a group of formula: 10
Figure imgf000050_0002
R6 to R9 In some embodiments, R and R’ are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups, wherein the optional substituents are 15 selected from C1-12 alkyl, C3-20 heterocyclyl, C5-20 aryl, halo, hydroxy, ether, alkoxy, oxo, acyl, carboxy, ester, amino, amido, nitro, and cyano. In some embodiments, R9 is H. 20 In some embodiments, R6 is selected from H, OH, OR, SH, NH2, nitro and halo, and may be selected from H or halo. In some of these embodiments R6 is H. In some embodiments, R7 is selected from H, OH, OR, SH, SR, NH2, NHR, NRR’, and halo. In some of these embodiments R7 is selected from H, OH and OR, where R is 25 selected from optionally substituted C1-7 alkyl, C3-10 heterocyclyl and C5-10 aryl groups. R may be more preferably a C1-4 alkyl group, which may or may not be substituted. A substituent of interest is a C5-6 aryl group (e.g. phenyl). Particularly preferred substituents at the 7- positions are OMe and OCH2Ph. Other substituents of particular interest are dimethylamino (i.e. –NMe2); -(OC2H4)qOMe, where q is from 0 to 2; nitrogen-containing C630 heterocyclyls, including morpholino, piperidinyl and N-methyl-piperazinyl. 008188229 49 These embodiments and preferences apply to R9’, R6’ and R7’ respectively. D and D’ In some embodiments, D and D’ are D1 and D’1 respectively. 5 In some embodiments, D and D’ are D2 and D’2 respectively. R2 When there is a double bond present between C2 and C3, R2 is selected from: 10 (a) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene; (b) C1-5 saturated aliphatic alkyl; (c) C3-6 saturated cycloalkyl; 15
Figure imgf000051_0001
, wherein each of R11, R12 and R13 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R2 group is no more than 5;
Figure imgf000051_0002
(e) , wherein one of R15a and R15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo methyl,20 methoxy; pyridyl; and thiophenyl; and (f)
Figure imgf000051_0003
, where R14 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. 25 When R2 is a C5-10 aryl group, it may be a C5-7 aryl group. A C5-7 aryl group may be a phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, R2 is preferably phenyl. In other embodiments, R2 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl. 008188229 50 When R2 is a C5-10 aryl group, it may be a C8-10 aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position. For example, the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3- 5 yl and quinolin-6-yl may be preferred. The isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred. When R2 is a C5-10 aryl group, it may bear any number of substituent groups. It preferably 10 bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be at any position. Where R2 is C5-7 aryl group, a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond 15 to the remainder of the compound. Therefore, where the C5-7 aryl group is phenyl, the substituent is preferably in the meta- or para- positions, and more preferably is in the para- position. Where R2 is a C8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any 20 number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent). R2 substituents, when R2 is a C5-10 aryl group 25 If a substituent on R2 when R2 is a C5-10 aryl group is halo, it is preferably F or Cl, more preferably Cl. If a substituent on R2 when R2 is a C5-10 aryl group is ether, it may in some embodiments be an alkoxy group, for example, a C1-7 alkoxy group (e.g. methoxy, ethoxy) or it may in some 30 embodiments be a C5-7 aryloxy group (e.g phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itself be further substituted, for example by an amino group (e.g. dimethylamino). If a substituent on R2 when R2 is a C5-10 aryl group is C1-7 alkyl, it may preferably be a C1-435 alkyl group (e.g. methyl, ethyl, propryl, butyl). 008188229 51 If a substituent on R2 when R2 is a C5-10 aryl group is C3-7 heterocyclyl, it may in some embodiments be C6 nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by 5 C1-4 alkyl groups. If the C6 nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom. If a substituent on R2 when R2 is a C5-10 aryl group is bis-oxy-C1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene. 10 If a substituent on R2 when R2 is a C5-10 aryl group is ester, this is preferably methyl ester or ethyl ester. Particularly preferred substituents when R2 is a C5-10 aryl group include methoxy, ethoxy,15 fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl- thiophenyl. Other particularly preferred substituents for R2 are dimethylaminopropyloxy and carboxy. Particularly preferred substituted R2 groups when R2 is a C5-10 aryl group include, but are20 not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4- fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4- cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl. Another possible substituted R2 group is 4-nitrophenyl. R2 groups of particular interest include 4-(4-25 methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl. Other R2 groups When R2 is C1-5 saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of 30 these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched. When R2 is C3-6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl. 35 008188229 52 12 R 13 R When R2 11 is R , each of R11, R12 and R13 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R2 group is no more than 5. In some embodiments, the total number of carbon atoms in the R2 group is no more than 4 or no more than 3. 5 In some embodiments, one of R11, R12 and R13 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl. In other embodiments, two of R11, R12 and R13 are H, with the other group being selected10 from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl. In some embodiments, the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that are not H are methyl. 15 In some embodiments, R11 is H. In some embodiments, R12 is H. In some embodiments, R13 is H. 20 In some embodiments, R11 and R12 are H. In some embodiments, R11 and R13 are H. 25 In some embodiments, R12 and R13 are H.
Figure imgf000054_0001
An R2 group of particular interest is: . When R2
Figure imgf000054_0002
is , one of R15a and R15b is H and the other is selected from: 30 phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, 008188229 53 methoxy; pyridyl; and thiophenyl. In some embodiments, the group which is not H is optionally substituted phenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted. 5 When R2 is
Figure imgf000055_0001
, R14 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted. In some embodiments, R14 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some10 of these embodiments, R14 is selected from H and methyl. When there is a single bond present between C2 and C3,
Figure imgf000055_0002
, where R16a and R16b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a 15 group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R16a and R16b is H, the other is selected from nitrile and a C1-4 alkyl ester. In some embodiments, R2 is H. 20 In some embodiments,
Figure imgf000055_0003
. In some embodiments, it is preferred that R16a and R16b are both H. In other embodiments, it is preferred that R16a and R16b are both methyl. 25 In further embodiments, it is preferred that one of R16a and R16b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted. In these further embodiment, it may be further preferred that the group which is not H is selected from methyl and ethyl. 30 008188229 54 R22 When there is a double bond present between C2’ and C3’, R22 is selected from: (a) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 5 alkylene; (b) C1-5 saturated aliphatic alkyl; (c) C3-6 saturated cycloalkyl; (d)
Figure imgf000056_0001
, wherein each of R31, R32 and R33 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of10 carbon atoms in the R22 group is no more than 5; R25b (e)
Figure imgf000056_0002
, wherein one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl; and (f)
Figure imgf000056_0003
, where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 15 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. When R22 is a C5-10 aryl group, it may be a C5-7 aryl group. A C5-7 aryl group may be a phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl. In 20 some embodiments, R22 is preferably phenyl. In other embodiments, R22 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl. When R22 is a C5-10 aryl group, it may be a C8-10 aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be bound to the PBD core 25 through any available ring position. For example, the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may be preferred. The isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be30 preferred. 008188229 55 When R22 is a C5-10 aryl group, it may bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred. The substituents may be any position. 5 Where R22 is C5-7 aryl group, a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably β or γ to the bond to the remainder of the compound. Therefore, where the C5-7 aryl group is phenyl, the substituent is preferably in the meta- or para- positions, and more preferably is in the para-10 position. Where R22 is a C8-10 aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the 15 proximal and distal rings or both (if more than one substituent). R22 substituents, when R22 is a C5-10 aryl group If a substituent on R22 when R22 is a C5-10 aryl group is halo, it is preferably F or Cl, more preferably Cl. 20 If a substituent on R22 when R22 is a C5-10 aryl group is ether, it may in some embodiments be an alkoxy group, for example, a C1-7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C5-7 aryloxy group (e.g phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itself be further substituted, for example by an amino group (e.g. 25 dimethylamino). If a substituent on R22 when R22 is a C5-10 aryl group is C1-7 alkyl, it may preferably be a C1-4 alkyl group (e.g. methyl, ethyl, propryl, butyl). 30 If a substituent on R22 when R22 is a C5-10 aryl group is C3-7 heterocyclyl, it may in some embodiments be C6 nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by C1-4 alkyl groups. If the C6 nitrogen containing heterocyclyl group is piperazinyl, the said35 further substituent may be on the second nitrogen ring atom. 008188229 56 If a substituent on R22 when R22 is a C5-10 aryl group is bis-oxy-C1-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene. If a substituent on R22 when R22 is a C5-10 aryl group is ester, this is preferably methyl ester 5 or ethyl ester. Particularly preferred substituents when R22 is a C5-10 aryl group include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl- thiophenyl. Other particularly preferred substituents for R22 are dimethylaminopropyloxy10 and carboxy. Particularly preferred substituted R22 groups when R22 is a C5-10 aryl group include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4- fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4- 15 cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl. Another possible substituted R22 group is 4-nitrophenyl. R22 groups of particular interest include 4-(4- methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl. 20 Other R22 groups When R22 is C1-5 saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched. 25 When R22 is C3-6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl. When R22 is
Figure imgf000058_0001
, each of R31, R32 and R33 are independently selected from H, 30 C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R22 group is no more than 5. In some embodiments, the total number of carbon atoms in the R22 group is no more than 4 or no more than 3. 008188229 57 In some embodiments, one of R31, R32 and R33 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl. In other embodiments, two of R31, R32 and R33 are H, with the other group being selected 5 from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl. In some embodiments, the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that are not H are methyl. 10 In some embodiments, R31 is H. In some embodiments, R32 is H. In some embodiments, R33 is H. 15 In some embodiments, R31 and R32 are H. In some embodiments, R31 and R33 are H. 20 In some embodiments, R32 and R33 are H. 2
Figure imgf000059_0001
An R 2 group of particular interest is: . R25b When R22 is
Figure imgf000059_0002
, one of R25a and R25b is H and the other is selected from: phenyl, 25 which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the group which is not H is optionally substituted phenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted. 30 When R22 is
Figure imgf000059_0003
, R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from 008188229 58 halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted. In some embodiments, R24 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R24 is selected from H and methyl. 5 When there is a single bond present between C2’ and C3’, R22 is H or
Figure imgf000060_0001
, where R26a and R26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is10 H, the other is selected from nitrile and a C1-4 alkyl ester. In some embodiments, R22 is H. In some embodiments,
Figure imgf000060_0002
. 15 In some embodiments, it is preferred that R26a and R26b are both H. In other embodiments, it is preferred that R26a and R26b are both methyl. 20 In further embodiments, it is preferred that one of R26a and R26b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted. In these further embodiment, it may be further preferred that the group which is not H is selected from methyl and ethyl. 25 In some embodiments of the first aspect of the present disclosure, DL is of formula I’-a, I’-b, I’-c or I’-d: 008188229 59 I'-a I'-b I'-c I'-d where the dotted line represents the possible presence of a double bond between C2 and C3 and C2’ and C3’; where there is no double bond between C2 and C3 and C2’ and C3’, R2a and R22a are the same and are selected from: 008188229 60 (a) H; and
Figure imgf000062_0001
; where there is a double bond between C2 and C3 and C2’ and C3’ R2a and R22a are the 5 same and are selected from: 10
Figure imgf000062_0002
; R1a is selected from methyl and benzyl; 15 R20, R21 and RLL are as defined above. These embodiments and preferences also apply to the second, fifth and sixth aspects of the disclosure, as appropriate. 20 GL GL may be selected from:
Figure imgf000062_0003
008188229 61
Figure imgf000063_0001
008188229 62
Figure imgf000064_0001
Figure imgf000065_0001
where CBA represents the point of connection to the Ligand/Cell Binding Agent, Ar represents a C5-6 arylene group, e.g. phenylene, and X1 represents C1-4 alkyl.
Figure imgf000065_0004
In some embodiments, GLL is selected from GLL1-1 and GLL1-2. In some of these 5 embodiments, GLL is GLL1-1. In other embodiments, GLL is GLL1-1A. GLL1-1A may be formed by a Diels-Alder reaction between GL1-1 and a spirocyclopropyl- cyclopentadiene of formula:
Figure imgf000065_0003
Figure imgf000065_0002
Such a group can be incorporated into the antibody via the addition of a 10 linker or by incorporating a non-natural amino acid into the polypeptide sequence, as described in WO2019/224340, which is incorporated herein by reference). 008188229 64 X X is:
Figure imgf000066_0001
, where a = 0 to 5, b = 0 to 16, c = 0 or 1, d = 0 to 5. 5 a may be 0, 1, 2, 3, 4 or 5. In some embodiments, a is 0 to 3. In some of these embodiments, a is 0 or 1. In further embodiments, a is 0. b may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b is10 0 to 12. In some of these embodiments, b is 0 to 8, and may be 0, 2, 4 or 8. c may be 0 or 1. d may be 0, 1, 2, 3, 4 or 5. In some embodiments, d is 0 to 3. In some of these 15 embodiments, d is 1 or 2. In further embodiments, d is 2. In some embodiments of X, a is 0, c is 1 and d is 2, and b may be from 0 to 8. In some of these embodiments, b is 0, 4 or 8. 20 Q In one embodiment, Q is an amino acid residue. The amino acid may a natural amino acids or a non-natural amino acid. In one embodiment, Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp,25 where Cit is citrulline. In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a 30 cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin. In one embodiment, Q is selected from: CO-Phe-Lys-NH, 008188229 65 CO-Val-Ala-NH, CO-Val-Lys-NH, CO-Ala-Lys-NH, CO-Val-Cit-NH, 5 CO-Phe-Cit-NH, CO-Leu-Cit-NH, CO-Ile-Cit-NH, CO-Phe-Arg-NH, and CO-Trp-Cit-NH; 10 where Cit is citrulline. Preferably, Q is selected from: CO-Phe-Lys-NH, CO-Val-Ala-NH, 15 CO-Val-Lys-NH, CO-Ala-Lys-NH, CO-Val-Cit-NH. Most preferably, Q is selected from CO-Phe-Lys-NH, CO-Val-Cit-NH and CO-Val-Ala-NH. 20 Other dipeptide combinations of interest include: CO-Gly-Gly-NH, CO-Pro-Pro-NH, and CO-Val-Glu-NH. 25 Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference. In some embodiments, Q is a tripeptide residue. The amino acids in the tripeptide may be 30 any combination of natural amino acids and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide then is a recognition site for cathepsin. Tripeptide linkers of particular interest are: 35 NH-Glu-Val-Ala-C=O NH-Glu-Val-Cit-C=O 008188229 66 NH-αGlu-Val-Ala-C=O NH-αGlu-Val-Cit-C=O In some embodiments, Q is a tetrapeptide residue. The amino acids in the tetrapeptide 5 may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tetrapeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the tetrapeptide is the site of action for cathepsin-mediated cleavage. The tetrapeptide then is a recognition site for cathepsin. Tetrapeptide linkers of particular interest are: 10 NH -Gly-Gly-Phe-Gly C=O; and NH -Gly-Phe-Gly-Gly C=O. In some embodiments, the tetrapeptide is: NH -Gly-Gly-Phe-Gly C=O. 15 In the above representations of peptide residues, NH- represents the N-terminus, and -C=O represents the C-terminus of the residue. Glu represents the residue of glutamic acid, i.e.:
Figure imgf000068_0001
20 αGlu represents the residue of glutamic acid when bound via the α-chain, i.e.:
Figure imgf000068_0002
. In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed below. Protected amino 25 acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin. 008188229 67 Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog, and as described above. In some embodiments of the present disclosure, the C11 substituent may be in the 5 following stereochemical arrangement relative to neighbouring groups:
Figure imgf000069_0001
10 In other embodiments, the C11 substituent may be in the following stereochemical arrangement relative to neighbouring groups:
Figure imgf000069_0002
. Examples 20 General Information Reaction progress was monitored by LCMS using the short run conditions outlined below. Extraction and chromatography solvents (HPLC grade) were bought from VWR, U.K. All other chemicals were purchased from Aldrich (standard reagent grades). Visualization of TLC was achieved with UV light or iodine vapor unless otherwise stated. Extraction and 25 chromatography solvents were bought and used without further purification from VWR UK. All fine chemicals were purchased from Sigma-Aldrich or TCI Europe unless otherwise stated. Pegylated reagents were obtained from Quanta Biodesign US via Stratech UK, or Purepeg. Column chromatography was performed on an Isolera (Biotage) automated system using normal phase SNAP Ultra cartridges purchased from Biotage. 30 The LC/MS conditions were as follow: The analytical LC/MS conditions (for reaction monitoring and purity determination) were as follows: Positive mode electrospray mass spectrometry was performed using a Shimadzu Nexera®/Prominence® LCMS-2020. Mobile phases used were solvent A (H2O with 0.1% 35 formic acid) and solvent B (CH3CN with 0.1% formic acid). 008188229 68 Gradient for routine 3-minute run (method A): Initial composition 5% B held over 25 seconds, then increased from 5% B to 100% B over a 1 minute 35 seconds’ period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 5 seconds and held there for 5 seconds. The total duration of the gradient run was 3.0 minutes. 5 Gradient for 15-minute run (method B): Initial composition 5% B held over 1 minute, then increased from 5% B to 100% B over a 9 minutes period. The composition was held for 2 minutes at 100% B, then returned to 5% B in 10 seconds and held there for 2 minutes 50 seconds. The total duration of the gradient run was 15.0 minutes. Flow rate was 0.8 10 mL/minute (for 3-minute run) and 0.6 mL/minute (for 15-minute run). Detection was at 254 nm. Columns: Waters Acquity UPLC® BEH Shield RP181.7µm 2.1 x 50 mm at 50 °C fitted with Waters Acquity UPLC® BEH Shield RP18 VanGuard Pre-column, 130A, 1.7µm, 2.1 mm x 5 mm (routine 3-minute run); and ACE Excel 2 C18-AR, 2 µ, 3.0 x 100mm fitted with Waters Acquity UPLC® BEH Shield RP18 VanGuard Pre-column, 130A, 1.7µm, 2.1 mm x15 5 mm (15-minute run). Gradient for 15-minute run (method 20-60): Initial composition 20% B held over 1.25 minute, then increased from 20% B to 60% B over a 9.35 minutes period, then increased from 60% B to 100% B over a 30 seconds period. The composition was held for 2 minutes 20 at 100% B, then returned to 5% B in 30 seconds and held there for 2 minutes. The total duration of the gradient run was 15.0 minutes. Flow rate was 0.5 mL/minute. Detection was at 223 nm. Columns: Waters Acquity UPLC® BEH Shield RP181.7µm 2.1 x 50 mm at 50 °C. 25 General Experimental for Examples 2, 4 and 7 only Flash chromatography was performed using a Biotage Isolera One™ using gradient elution on SNAP Ultra™ columns starting from either 88% hexane/EtOAc or 99.9% DCM/MeOH until all UV active components (detection at 214 and 254 nm) eluted from the column. The gradient was manually held whenever substantial elution of UV active material was 30 observed. Fractions were checked for purity using thin-layer chromatography (TLC) using Merck Kieselgel 60 F254 silica gel, with fluorescent indicator on aluminum plates. Visualization of TLC was achieved with UV light or iodine vapor unless otherwise stated. Extraction and chromatography solvents were bought and used without further purification from VWR UK. All fine chemicals were purchased from Sigma-Aldrich or TCI Europe35 unless otherwise stated. Pegylated reagents were obtained from Quanta Biodesign US via Stratech UK. 008188229 69 The LC-MS conditions were as follows: positive mode electrospray mass spectrometry was performed using a Waters Acquity UPLC® (ultra-high-performance liquid chromatography) fitted with an SQ2 mass detector. Mobile phases used were solvent A (H2O with 0.1% 5 formic acid) and solvent B (CH3CN with 0.1% formic acid) at a flow rate of 0.8 mL/min. The column was a Waters Acquity UPLC® BEH Shield RP181.7 µm 2.1 mm x 50 mm fitted with a Waters Acquity UPLC® BEH Shield RP18 VanGuard pre-column, 130 Å, 1.7 µm, 2.1 mm x 5 mm at 50 °C. Method 1: Gradient started with initial composition 5% B held over 25 seconds, then 10 increased from 5% B to 100% B over a 1 min 35 seconds period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 5 seconds and held there for 5 seconds. The total duration of the gradient run was 3.0 min with a sample injection volume of 2 µL and detection at 223 nm and 254 nm. Method 2: Gradient started with initial composition 25% B held over 25 seconds, then 15 increased from 25% B to 100% B over a 1 min 35 seconds period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 5 seconds and held there for 5 seconds. The total duration of the gradient run was 3.0 min with a sample injection volume of 2 µL and detection at 223 nm and 254 nm. Method 3: Gradient started with initial composition 5% B held over 1 min 25 seconds, then 20 increased from 5% B to 100% B over a 9 min 35 second period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 10 seconds and held there for 2 min. The total duration of the gradient run was 15.0 min with a sample injection volume of 2 µL and detection at 223 nm and 254 nm. 25 The preparative HPLC conditions were as follows: reverse-phase UPLC was carried out on a Shimazdzu Prominence® machine using a Phenomenex® Gemini NX 5µm C18 column 150 mm x 21.2 mm fitted with a Phenomenex® Gemini SecurityGuard PREP Cartridge NX C18 15 mm x 21.2 mm at 50 °C. Eluents used were solvent A (H2O with 0.1% formic acid) and solvent B (CH3CN with 0.1% formic acid). All UPLC experiments were performed with 30 gradient conditions: initial composition 15% B increased to 55% B over a 15-min period then increased to 100% B over 2 min, held for 1 min at 100% B, then returned to 13% B in 0.1 min and held there for 1.9 min. The total duration of the gradient run was 20.0 min. Flow rate was 20.0 mL/min and with detection at 254 and 280 nm. 35 008188229 70 Example 1
Figure imgf000072_0001
008188229 71 8 8 8 9 a) tert-butyl (11S,11aS)-8-((6-(bromomethyl)pyridin-2-yl)methoxy)-11-((tert- butyldimethylsilyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H- benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (2) 5 tert-butyl (11S,11aS)-11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methylene-5- oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (1, 2 g, 4.07 mmol), α,α′-dibromo-2,6-lutidine (4.3 g, 16.2 mmol, 4 eq) and potassium carbonate (2.25 g, 16.2 mmol, 4 eq) were suspended in acetone (25 mL, 12.5 V). The reaction mixture was allowed to stir at 50°C for 2h when the reaction was deemed 10 complete by TLC. The reaction mixture was filtered through cotton wool and washed with ethyl acetate. The volatiles were removed under vacuum. The residue was purified by chromatography (50g Ultra, Biotage, gradient 15% to 100% EtOAc in heptane; desired product elution at 55% EtOAc; dimer at 85%). The pure fractions were pooled and the solvent removed under vacuum to give a colorless oil, crystallising as a white foam (2, 2.09 15 g, 3.09 mmol, 76% Yield). LC-MS (Method A) 1.82 min, ES+ 676.30 (M+H) m/z. 008188229 72 b) tert-butyl (11S,11aS)-8-((6-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert- butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2- methoxyphenoxy)methyl)pyridin-2-yl)methoxy)-11-((tert-butyldimethylsilyl)oxy)-7- 5 methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2- a][1,4]diazepine-10(5H)-carboxylate (4) Phenol 3 allyl ((S)-1-(((S)-1-((4-((((2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4- methylenepyrrolidine-1-carbonyl)-5-hydroxy-4- methoxyphenyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1- 10 oxobutan-2-yl)carbamate (1.39 g, 1.75 mmol) and bromopyridyl 2 (1.3 g, 1.93 mmol, 1.1 eq) were dissolved in acetone (10 mL, 7.2 V). Potassium carbonate (289 mg, 2.09 mmol, 1.2 eq) and tetrabutylammonium iodide (64 mg, 0.17 mmol, 0.1 eq) were added and the reaction was stirred for 4 hours at 50 °C when completion was observed. The mixture was dried under vacuum. The residue was diluted in DCM and purified by flash chromatography 15 (Biotage SNAP Ultra 25 g.15/85 to 100/0 EtOAc/EtOH 4/1 in Hexane; elution around 55%). The pure fractions were pooled. The solvent was evaporated and (4, 1.98 g, 1.42 mmol, 82%) was obtained as a white solid. LC-MS (Method A) 2.01 min, ES+ m/z 1390.95 [M+H]. 20 c) tert-butyl (11S,11aS)-8-((6-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2- (hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2- methoxyphenoxy)methyl)pyridin-2-yl)methoxy)-11-((tert-butyldimethylsilyl)oxy)-7- methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-25 a][1,4]diazepine-10(5H)-carboxylate (5) p-Toluenesulfonic acid (161 mg, 0.85 mmol, 0.6 eq) was added to a solution of TBS- protected primary alcohol 4 (1.963 g, 1.41 mmol) in tetrahydrofuran (20 mL) and water (1 mL). The reaction was stirred at 30 °C for 2 h, and then at 40 °C for 30 min at which point completion was observed. The mixture was partitioned between ethyl acetate (100 mL) and 30 water (100 mL). The organic layer was washed with brine (50 mL) and dried over magnesium sulfate, followed by evaporation to dryness under vacuum. The residue was diluted in DCM and purified by flash chromatography (Biotage Ultra 50g EtOAc/EtOH 4:1 / Hex 15/85 to 70/30). The pure fractions were pooled. The solvent was evaporated to give 5 (1.672 mg, 1.31 mmol, 93% Yield) as a white solid. 35 LC-MS (Method A) 1.77 min, ES+ m/z 1276.05 [M+H]; 008188229 73 d) tert-butyl (11S,11aS)-8-((6-((((S)-10-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methylene-5- oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8- yl)oxy)methyl)pyridin-2-yl)methoxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2- 5 methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine- 10(5H)-carboxylate (6) Triphenylphosphine (459 mg, 1.75 mmol, 3 eq) followed by diisopropyl azodicarboxylate (252 µL, 1.28 mmol, 2.2 eq) was added to as solution of primary alcohol 5 (744 mg, 0.583 mmol) in THF (21 mL, 28 V). The reaction mixture was stirred at 40 °C for 40 min. The 10 volatiles were removed under vacuum. The residue was dissolved in DCM and purified by chromatography (Biotage Ultra 100 g EtOAc/EtOH 4:1 / Hex 15/85 up to 65/35 in 10 CV; elution from 45% to 53%). The cleanest fractions were pooled and the solvent was removed by rotoevaporation under vacuum, followed by hard vacuum to give 6 (543 mg, 0.353 mmol, 74% Yield) as a white solid. LC-MS (Method B) 8.87 min, ES+ m/z 1257.55 15 [M+H]; e) tert-butyl (11S,11aS)-11-((tert-butyldimethylsilyl)oxy)-8-((6-((((S)-10-(((4-((2S,5S)-37- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo- 10,13,16,19,22,25,28,31-octaoxa-3,6,34-20 triazaheptatriacontanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methylene-5-oxo- 2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8- yl)oxy)methyl)pyridin-2-yl)methoxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a- tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (8) Tetrakis(triphenylphosphine)palladium(0) (2.7 mg, 0.0023 mmol, ) was added to a mixture 25 of Alloc carbamate 6 (297 mg, 0.236 mmol) and pyrrolidine (188 μL, 2.36 mmol, 10 eq) in dichloromethane (10 mL, 33 V). The reaction mixture was stirred under argon at room temperature for 30 minutes and was found complete by LCMS. Aqueous ammonium chloride (30 mL, 6 mass%) was added and the mixture was stirred vigorously. The mixture was then decanted in a biotage phase separation cartridge. The DCM layer was 30 evaporated to dryness under vacuum. The residue was dissolved in chloroform (20 mL) and the solvent removed by rotoevaporation under vacuum at 35°C. This cycle was repeated a second time, followed by drying under hard vacuum (3 mbar, on rotoevaporator). Chloroform (10.00 mL) was added, followed by Maleimide-PEG8- CH2CH2COOH (140 mg, 0.236 mmol, 1.0 eq) and EDCI (50 mg, 0.260 mmol, 1.1 eq). The 35 reaction was allowed to proceed at room temperature for 3h when completion was observed by LCMS. The volatiles were removed by rotoevaporation and the crude residue 008188229 74 was purified by chromatography (25g Ultra, Biotage, gradient 35/65 to 60/40 of 10% MeOH in DCM / DCM in 10CV; hold at elution around 60%). The fractions were analysed by TLC (7% MeOH in DCM). The pure fractions were pooled. The solvent was removed by evaporation to give 8 (336 mg, 0.192 mmol, 81% Yield) as a yellow foam. 5 LC-MS (Method B) 8.26 min, ES+ m/z 1748.00 [M+H]; f) 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35- trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl (S)- 7-methoxy-8-((6-((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-10 benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)pyridin-2-yl)methoxy)-2- methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine- 10(5H)-carboxylate, 9 A cold (5 °C) mixture of TFA/water 95/5 (2.5 mL) was added to the Boc-TBS protected starting material 8 (326 mg, 0.186 mmol) and the reaction mixture was allowed to stir at 0 15 °C for 1h, when completion was observed by LCMS. Water (5 mL) and DCM (10 mL) were added, followed by a saturated solution of potassium phosphate dibasic (20 mL). Further solid potassium phosphate dibasic was added until the pH reached 5 to 6. The DCM layer was separated through a Biotage decantation cartridge, and the volatiles removed under vacuum. 20 The crude residue was purified by chromatography (25g Ultra, Biotage, gradient 35/65 to 100/0 of 10% MeOH in DCM / DCM in 12 CV; hold at elution around 90%). The fractions were analysed by TLC (10% MeOH in DCM). The purest fractions were pooled. The solvent was removed by evaporation to give 9 (137.4 mg, 0.090 mmol, 48.6% Yield; 91 %25 pure) as a yellow foam. LC-MS (Method 20-60) 8.86 min, ES+ m/z 1515.69 [M+H]; 1H NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H), 8.15 (s, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.92 (t, J = 7.7 Hz, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 4.4 Hz, 1H), 7.64 – 7.42 (m, 4H), 7.38 (s, 1H), 7.21 – 7.04 (m, 4H), 30 6.99 (s, 2H), 6.96 (s, 2H), 5.28 (q, J = 13.0 Hz, 2H), 5.21 – 5.02 (m, 5H), 5.02 – 4.80 (m, 2H), 4.36 (s, 2H), 4.27 – 3.94 (m, 5H), 3.83 (d, J = 18.5 Hz, 6H), 3.78 – 3.64 (m, 2H), 3.59 (t, J = 7.3 Hz, 4H), 3.54 – 3.40 (m, 28H), 3.36 (s, 2H), 3.14 (q, J = 5.8 Hz, 2H), 3.09 – 2.96 (m, 2H), 2.93 – 2.75 (m, 2H), 2.49 – 2.24 (m, 4H), 2.05 – 1.85 (m, 1H), 1.26 (s, 4H), 0.83 (dd, J = 13.9, 6.0 Hz, 6H). 35 008188229 75 Example 2
Figure imgf000077_0001
5 008188229 76 OAllyl NO2 O Alloc H O O N NH O N H O AllocO OAlloc OAlloc O O O O H O H N O O N H H N OMe MeO N O O 19 OAllyl NO 2 O Alloc H O O N NH O N H O AllocO OAlloc OAlloc O O O O H O N O O N H H N OMe MeO N O O 20 O H NO 2 O H O O N NH 2 O N H O H O O H O H O O O O H O N O O N H H N OMe MeO N O O 21 O O O N N H 8 O O H NO 2 O O H O O N NH O N H O H O O H O H O O O O H O N O O N H H N OMe MeO N O O 22 008188229 77 a) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl(S)-7-methoxy-2-methyl-5-oxo-8- ((triisopropylsilyl)oxy)-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)- carboxylate (11) 5 Diisopropyl azodicarboxylate (2.2 equiv., 0.26 mL, 0.26 g, 1.31 mmol) was added to a stirred solution of 10 (1 equiv., 500 mg, 0.5966 mmol) and triphenylphosphine (3.00 equiv., 472 mg, 1.79 mmol) in THF (10 mL). The reaction was heated at 40 °C for 2 hours, at which point a satisfactory amount of product was formed by LC-MS. The volatiles were removed under vacuum and the residue was purified by Biotage Isolera. The pure fractions 10 were pooled and concentrated under vacuum to give 11 (418 mg, 85% yield) as a white solid (LC-MS (method 1) 2.18 min, ES+ m/z 821 [M+H]). b) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl(S)-8-hydroxy-7-methoxy-2-methyl-5-oxo-15 11,11a-dihydro-1H- pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (12) Lithium acetate dihydrate (2 equiv., 100 mg, 0.976 mmol) was added to a stirred solution of 11 (1 equiv., 400 mg, 0.4877 mmol) in N, N-dimethylformamide (10 mL) and H2O (3 mL). The reaction mixture was stirred at 40 °C for 3 hours at which point analysis by LC-MS revealed 50% conversion to desired product. The heat was switched off and the mixture 20 stirred for 4 days where LC-MS revealed satisfactory conversion to desired product. The mixture was partitioned between water (50 mL) and EtOAc (50 mL) and the layers separated. The aqueous phase was extracted with EtOAc (3 x 25 mL) and the combined organic layers washed with 10% citric acid (30 mL), brine (40 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by Isolera gave desired 25 product 12 (175 mg, 54% yield) as a white solid (LC-MS (method 1) 1.35 min, ES+ m/z 664 [M+H]). c) (S)-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)(4- hydroxy-5-methoxy-2-nitrophenyl)methanone (14) 30 Lithium acetate dihydrate (1.2 equiv., 1.06 g, 10.36 mmol) was added to a stirred solution of 13 (1.0 equiv., 5 g, 8.64 mmol) in N,N-dimethylformamide (90 mL) and H2O (30 mL). The reaction mixture was allowed to stir for 3 hours at which point analysis by LC-MS revealed presence of starting material. An additional equivalent of LiOAc was added and the mixture was heated at 40 °C for 16 hours, at this point analysis by LC-MS revealed reaction 35 completion. The mixture was partitioned between water (100 mL) and EtOAc (76 mL) and the layers separated. The aqueous phase was extracted with EtOAc (3 x 50 mL) and the 008188229 78 combined organic layers washed with 10% citric acid (60 mL), brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product 14 as an orange solid (3.65 g, 100% yield) which was carried through to next step without further analysis or purification (LC-MS (method 1) 1.84 min, ES+ m/z 424 [M+H]). 5 d) (S)-(4-((3-(bromomethyl)benzyl)oxy)-5-methoxy-2-nitrophenyl)(2-(((tert- butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)methanone (15) Potassium carbonate (1.05 equiv., 1.25 g, 9.07 mmol) was added to a stirred solution of 14 (1.0 equiv., 3.65 g, 8.64 mmol) and 1,3-bis(bromomethyl)benzene (4 equiv., 9.40 g, 34.5 10 mmol) in N,N-dimethylformamide (90 mL). The reaction mixture was allowed to stir for 3 hours heated at 60 °C at which point analysis by LC-MS revealed presence of starting phenol (5%). In an effort to consume remaining starting material the mixture was left at 60 °C for 16 hours, at which point analysis by LC-MS revealed reaction completion albeit with TBDMS group cleavage, LC-MS Rt = 1.55 min, ES+ 493 (M+H). The mixture was 15 partitioned between water (100 mL) and EtOAc (76 mL) and the layers separated. The aqueous phase was extracted with EtOAc (3 x 50 mL) and the combined organic layers washed with brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product which was dissolved in DCM (80 mL) and treated with TBDMSCl (1.5 equiv., 1.95 g, 13.0 mmol) and imidazole (1.5 equiv., 1.76 g, 25.9 mmol). After stirring for 1 hour, 20 conversion to original desired product was observed. The reaction mixture was diluted with DCM (50 mL) washed with brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product which was treated with ether (200 mL) and the precipitate removed by filtration. The filtrate was evaporated in vacuo and the resulting residue purified by Isolera to give 15 as an orange foam (1.64 g, 31% yield, LC-MS 2.16 min, ES+ 25 m/z 607 [M+H]). Product contained some chlorine exchanged material (LC-MS (method 1) 2.14 min, ES+ m/z 562 [M+H]) arising from the brine wash. e) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl(S)-8-((3-((4-((S)-2-(((tert-30 butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-2- methoxy-5-nitrophenoxy)methyl)benzyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a- dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (16) Potassium carbonate (2.0 equiv., 676 mg, 4.89 mmol) was added to a stirred solution of 12 (1 equiv., 1.62 g, 2.44 mmol), tetrabutylammonium iodide (0.1 equiv., 90 mg, 0.24 mmol) 35 and 15 (1.48 g, 2.44 mmol) in N,N-dimethylformamide (100 mL). The reaction mixture was allowed to stir for 3 hours heated at 60 °C, at which point analysis by LC-MS revealed 008188229 79 completion of reaction. The mixture was partitioned between water (100 mL) and EtOAc (76 mL) and the layers separated. The aqueous phase was extracted with EtOAc (3 x 50 mL) and the combined organic layers washed with brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product which was purified by Isolera to give 5 16 as a white foam (2.14 g, 74% yield, LC-MS (method 1) 2.10 min, ES+ m/z 1189 [M+H]). f) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl(S)-8-((3-((5-amino-4-((S)-2-(((tert- butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-2-10 methoxyphenoxy)methyl)benzyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H- pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (17) Zinc (20 equiv., 2.36 g, 36.0 mmol) and water (4 mL) were added to a stirred solution of 16 (1 equiv., 2.14 g, 1.80 mmol) in ethanol (40 mL) and ethyl acetate (40 mL) at room temperature. The mixture was treated dropwise with a 10% solution of formic acid in 15 ethanol (10 mL). An exotherm to ~25.5 °C was observed during the addition, when this subsided the addition was stopped (~5 mL 10% solution of formic acid in ethanol added) and progress assessed by LC-MS. Reaction was deemed complete without TBS cleavage. The mixture was filtered through celite (pad washed with 50 mL ethyl acetate) and the filtrate extracted with NaHCO3 (4 x 50 mL). The combined organic layers were washed with 20 brine (70 mL), dried (MgSO4), filtered and evaporated in vacuo to provide crude 17 (2.03 g, 97% yield). LC-MS (method 1) 2.06 min, ES+ m/z 1159 [M+H]. g) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl(S)-8-((3-((5-((((4-(((2S,3R,4S,5S,6S)-6-25 ((allyloxy)carbonyl)-3,4,5-tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)- 3-nitrobenzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4- methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-2-methoxyphenoxy)methyl)benzyl)oxy)-7- methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine- 10(5H)-carboxylate (18) 30 Triphosgene (0.36 equiv., 187 mg, 0.631 mmol) was added to a stirred solution of 17 (1 equiv., 2.03 g, 1.75 mmol) and pyridine (2.20 equiv., 0.305 g, 0.312 mL, 3.86 mmol) in dichloromethane (100 mL) at room temperature. The reaction mixture was stirred under argon for 10 minutes at which point analysis by LC-MS (sampled in MeOH) showed complete conversion to methyl carbamate (LC-MS 2.11 min, ES+ m/z 1218 [M+H]). The 35 mixture was treated with pyridine (1.50 equiv., 0.208 g, 0.213 mL, 2.63 mmol) followed by 17a (1 equiv., 1.12 g, 1.75 mmol) and dibutyltin dilaurate (0.2 equiv., 0.221 g, 0.210 mL, 008188229 80 0.350 mmol) and allowed to stir for 18 hours, analysis by LC-MS showed good conversion to desired product. The mixture was diluted with DCM (50 mL) and washed with saturated ammonium chloride (2 x 60 mL), water (60 mL), brine (60 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by Isolera gave the pure 5 carbamate 18 as a yellow foam (2.17 g, 68% yield). LC-MS (method 1) 2.23 min, ES+ m/z 1823 [M+H]. h) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl(S)-8-((3-((5-((((4-(((2S,3R,4S,5S,6S)-6- ((allyloxy)carbonyl)-3,4,5-tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-10 3-nitrobenzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)-4-methyl-2,3-dihydro- 1H-pyrrole-1-carbonyl)-2-methoxyphenoxy)methyl)benzyl)oxy)-7-methoxy-2-methyl- 5-oxo-11,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (19) Acetic acid (15 mL) was added to a stirred solution of 18 (1 equiv.,2.17 g, 1.19 mmol) in tetrahydrofuran (5 mL) and water (5 mL). The mixture was allowed to stir for 20 hours at 15 which point analysis by LC-MS revealed complete conversion to desired product. The reaction mixture was added drop-wise to a stirred saturated aqueous solution of sodium bicarbonate (300 mL) where vigorous effervescence was observed. The aqueous layer was extracted with EtOAc (3 x 80 mL) and the combined organic layers washed with brine (100 mL), dried (MgSO4), filtered and solvent removed by evaporation in vacuo to provide 20 crude material which was purified by Isolera to give 19 as a white solid (1.74 g, 84% yield). LC-MS (method 1) 1.94 min, ES+ m/z 1709 [M+H]. i) 4-(((2S,3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5- tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-nitrobenzyl(11S,11aS)-8-25 ((3-((((S)-10-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-oxo- 5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8- yl)oxy)methyl)benzyl)oxy)-11-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H- pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (20) 30 Stahl TEMPO (0.3 equiv., 0.3 mL of a 0.2M solution in CH3CN, 0.0527 mmol) and tetrakisacetonitrile copper(I) triflate (0.3 equiv., 20 mg, 0.053 mmol) were added to a solution of 19 (1 equiv, 300 mg, 0.176 mmol) in dichloromethane (20 mL) at room temperature. The mixture (red-brown) was heated to 35 °C under air balloon overnight (colour changed to dark green). LC-MS showed completion of reaction and the mixture was 35 evaporated under reduced pressure, the residue was purified by Biotage Isolera to provide 008188229 81 pure ring-closed compound 20 as a white solid (267 mg, 89% yield). LC-MS (method 1) 1.92 min, ES+ m/z 1707 [M+H]. j) (2S,3S,4S,5R,6S)-6-(4-((((11S,11aS)-8-((3-((((S)-10-(((4-((S)-2-((S)-2-amino-3- 5 methylbutanamido)propanamido)benzyl)oxy)carbonyl)-7-methoxy-2-methyl-5-oxo- 5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8- yl)oxy)methyl)benzyl)oxy)-11-hydroxy-7-methoxy-2-methyl-5-oxo-5,10,11,11a- tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10-carbonyl)oxy)methyl)-2- nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (21) 10 Tetrakis(triphenylphosphine)palladium(0) (0.1 equiv., 18 mg, 0.0157 mmol) was added to a stirred solution of 20 (1 equiv, 267 mg, 0.157 mmol) and pyrrolidine (5.25 equiv., 58 mg, 68 µL, 0.822 mmol) in dichloromethane (5 mL) at room temperature. The reaction was allowed to stir under argon for 2.5 hours at which point analysis by LC-MS revealed complete consumption of starting material. The solvent was removed by evaporation in vacuo, 15 triturated with diethyl ether, then evaporated in vacuo (with high vac overnight) to provide the crude product 21 (208 mg, 100% yield) which was used in the next step without purification. LC-MS (method 1) 1.07 min, ES+ m/z 1330 [M+H]. k) (2S,3S,4S,5R,6S)-6-(4-((((11S,11aS)-8-((3-((((S)-10-(((4-((2S,5S)-37-(2,5-dioxo-2,5-20 dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31- octaoxa-3,6,34-triazaheptatriacontanamido)benzyl)oxy)carbonyl)-7-methoxy-2- methyl-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8- yl)oxy)methyl)benzyl)oxy)-11-hydroxy-7-methoxy-2-methyl-5-oxo-5,10,11,11a- tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10-carbonyl)oxy)methyl)-2-25 nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (22) A solution of mal-amido-peg8-TFP ester (1.2 equiv., 142 mg, 0.188 mmol) and pyridine (2.5 equiv., 31 mg, 32 µL, 0.391 mmol) in dichloromethane (5 mL) was added to a sample of 21 (208 mg, 0.156 mmol). The reaction mixture was stirred under argon and progress monitored by LC-MS. Additional TFP ester (58 mg) was added after 2 days. Reaction 30 complete at 254 nm after 6 days total stirring. The solvent was removed by evaporation in vacuo and the resulting residue purified by Isolera to give crude product as a yellow foam (101 mg). Further purification by preparative HPLC gave 21 (39 mg, 13% yield) as a white solid. LC-MS (method 1) 1.42 min, ES+ m/z 1905 [M+H]. Example 3
Figure imgf000084_0001
008188229 83
Figure imgf000085_0001
008188229 84 a) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl (2-((S)-2-(hydroxymethyl)pyrrolidine-1- carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (I2) I1 (1.0 g, 1.1 mmol) was dissolved in a mixture of acetic acid (5 mL), tetrahydrofuran (1 5 mL), methanol (1 mL) and water (2 mL). The resulting solution was stirred at 25°C for 60 mins. The solvent was removed under reduced pressure and the residue taken up in ethyl acetate (25 mL) and washed successively with water (20 mL), saturated NaHCO3 (20 mL) and brine (15 mL). The organic phase was dried (MgSO4) and evaporated to leave a yellow oil which was purified by column (gradient ethyl acetate / heptane, 75/25 to 100/0 v/v) to10 leave the product as a white solid. LC/MS rt 2.13 min; m/z (827.1) M+1. b) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl (S)-7-methoxy-5-oxo-8- ((triisopropylsilyl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-15 10(5H)-carboxylate (I3) triphenylphosphine (0.64 g, 2.4 mmol) and diisopropyl azodicarboxylate (0.36 g, 1.7 mmol) were stirred at room temperature in THF (5 mL) for 30 mins after which a white precipitate had formed. A solution of I2 (0.67 g, 0.81 mmol) in THF (5 mL) was added and the resulting mixture stirred for 2 hrs. The reaction mixture was evaporated to dryness and the 20 residue purified by column (gradient methanol / DCM, 1/99 to 3/97 v/v) to leave the product as a white solid. LC/MS rt 2.13 min; m/z (809.0) M+1. c) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl (S)-8-hydroxy-7-methoxy-5-oxo-2,3,11,11a-25 tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (23) lithium acetate dihydrate (60 mg, 0.5878 mmol) was added to a solution of 3 (0.47 g, 0.58 mmol) in DMF (0.5 mL) containing 1 drop of water. The resulting mixture was stirred at room temperature for 5hrs. The solvent was removed under reduced pressure and the residue purified by column (gradient methanol / DCM, 5/95 to 8/92 v/v) to leave the product30 as a white solid. LC/MS rt 1.55min; m/z (252.7) M+1. d) tert-butyl (11S,11aS)-8-((5-(((S)-10-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo- 2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-35 yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a- tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (25) 008188229 85 Potassium carbonate (75 mg, 0.53 mmol) was added to a solution of 24 (0.35 g, 0.54 mmol) and 23 (0.32 g, 0.49 mmol) in acetone (5 mL) and stirred at 55 °C for 24hrs. The inorganics were removed by filtration, the filtrate evaporated under reduced pressure and the residue purified by column chromatography (gradient methanol / dichloromethane, 5/95 5 to 10/90 v/v) to leave the product as a white solid, 0.54 g (94%). LC/MS rt 1.94 min m/z (1169.3) M+H. e) tert-butyl (11S,11aS)-8-((5-(((S)-10-(((4-((S)-2-((S)-2-amino-3- methylbutanamido)propanamido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo-10 2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8- yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a- tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (26) Pd(Ph3P)4 (26 mg, 0.022 mmol) was added to a solution of 3 (0.54 g, 0.46 mmol) and pyrrolidine (48 μL, 0.57 mmol) in chloroform (10 mL) and stirred at room temperature for 30 15 min. The resulting mixture was washed with saturated ammonium chloride (10 mL), dried (biotage SPE) and evaporated under reduced pressure. The resulting solid was placed under high vacuum for 3hrs then used in the next step without further purification, 0.49 g (98%). LC/MS rt 1.47 min m/z (1085.2) M+H. 20 f) tert-butyl (11S,11aS)-8-((5-(((S)-10-(((4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol- 1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34- triazaheptatriacontanamido)benzyl)oxy)carbonyl)-7-methoxy-5-oxo-2,3,5,10,11,11a- hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5- oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-25 a][1,4]diazepine-10(5H)-carboxylate (27) EDCI.HCl (53 mg, 0.27 mmol) was added to a solution of 26 (200 mg, 0.18 mmol) and Mal- PEG8-OH (131 mg, 0.22 mmol) in chloroform (10 mL, 124.6 mmol) and stirred at room temperature for 60 min. The reaction mixture was washed with water (10 mL), dried (biotage SPE) and evaporated to dryness. The resulting solid was purified by column 30 chromatography (gradient methanol / dichloromethane, 5/95 to 10/90 v/v) to leave the product as a white solid, 240 mg (78%). LC/MS rt 1.81 min m/z (1659.8) M+H. g) 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35- trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl (S)-35 7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2- 008188229 86 a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-5-oxo-2,3,11,11a-tetrahydro-1H- benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (28) 27 (240 mg, 0.14467 mmol) was added to a mixture of TFA (450 μL) and water (50 μL) at 0 °C and the resulting solution stirred at this temperature for 2 hrs. DCM (10 mL) and water 5 (10 mL) were added, followed by solid sodium hydrogen carbonate until the aqueous phase became basic (pH8). The organic phase was removed, dried (biotage SPE) and evaporated to leave a yellow oil which was purified by prep HPLC (15 to 60% MeCN / water + 0.1% formic acid over 13 min) to leave the product as a white solid, 110 mg (52%). LC/MS rt 1.58 min (1457.5) M+H. 10 Example 4
Figure imgf000088_0001
008188229 87 Compound 29 is described in Tiberghien et al., Org. Process Res. Dev.2018, 22, 9, 1241- 1256. a) Allyl (S)-7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H- 5 benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (30) Diisopropyl azodicarboxylate (4.17 mL, 19.3 mmol) was added to a solution of 29 (5 g, 9.64 mmol) and triphenylphosphine (7.55 g, 28.9 mmol) in tetrahydrofuran (100 mL). The reaction was heated at 40°C for 40 min, at which point a satisfactory amount of product was formed by LCMS. The solvent was removed under vacuum, and the residue was 10 purified by chromatography: (100g Ultra, Biotage, EtOAc in Hexane, gradient from 20% to 50%. Elution around 50% upwards). The purest fractions were pooled and concentrated under vacuum to give 30 (4.8g, 9.64 mmol, 100% Yield, some contamination with DIAD). LC-MS (method 2) 2.02 min, ES+ m/z 501.20 [M+H]. 15 b) allyl (S)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H- benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (31) TIPS protected phenol 30 (4.8 g, 9.64 mmol) was dissolved in DMF (40.0 mL) at 25°C. A solution of lithium acetate (1.02 g, 10.0 mmol) in water (2.0 mL) was added. The reaction was allowed to proceed at 25°C for 2h at which point completion was observed by LCMS. 20 The reaction mixture was partitioned between ethyl acetate (200 mL) and aqueous 1M ammonium chloride (100 mL). The organic phase was washed with brine (70 mL) and dried over magnesium sulfate. The volatiles were removed under vacuum, and the residue was purified by chromatography: (100g Ultra, Biotage, EtOAc in Hexane, gradient from 50% to 100%. Elution around 90% upwards). The pure fractions were pooled and concentrated25 under vacuum to give 31 (1.83g, 5.31 mmol, 55% Yield). LC-MS (method 2) 1.30 min, ES+ m/z 344.95 [M+H]. c) Diallyl 8,8'-((1,3-phenylenebis(methylene))bis(oxy))(11aS,11a'S)-bis(7-methoxy-2- methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-30 carboxylate) (32) 1,3-bis(bromomethyl)benzene (67.5 mg, 0.255 mmol) was added to a mixture of 31 (160 mg, 0.465 mmol) and potassium carbonate (74 mg, 0.535 mmol) in acetone (5.0 mL). The mixture was heated at 55°C for 18h, at which point LCMS showed completion of the reaction. The mixture was evaporated to dryness. The residue was loaded on a 10g Ultra 35 Biotage column and purified by chromatography (gradient EtOAc / Hexane 30/70 up to 100% EtOAc). The fractions were pooled and the volatiles were removed under vacuum to 008188229 88 give the product 32 (100 mg, 0.126 mmol, 54% Yield). LC-MS (method 2) 1.68 min, ES+ m/z 791.25 [M+H]. d) (11aS,11a'S)-8,8'-((1,3-phenylenebis(methylene))bis(oxy))bis(7-methoxy-2-methyl- 5 1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5-one) (33) Bis-Alloc-protected 32 (100 mg, 0.126 mmol) was dissolved in a mixture of dichloromethane (5 mL) and pyrrolidine (23 μL, 0.278 mmol). The atmosphere was purged with argon. A catalytic amount of tetrakis(triphenylphosphine)palladium(0) (5.84 mg, 0.00505 mmol) was added and the reaction allowed to proceed for 15 min, when LCMS10 showed completion. The reaction mixture was partitioned between DCM (10 mL) and 1M aqueous ammonium chloride (10 mL). The organic layer was decanted through an isolera cartridge, and evaporated to dryness. The residue was loaded on a 10g Ultra Biotage column and purified twice by chromatography (gradient DCM/MeOH 4/1 in DCM, from 0% up to 35%). The pure 15 fractions were pooled and the volatiles were removed under vacuum to give 33 (30 mg, 0.048 mmol, 38% Yield). LC-MS (method 2) 1.54 min, ES+ m/z 623.4 [M+H].
008188229 89 Example 5
Figure imgf000091_0001
Compound I4 is Compound 19-4 of WO 2012/65964 (see page 253). 5 a) Allyl (S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-8-((triisopropylsilyl)oxy)-11,11a- dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (I5) Diisopropyl azodicarboxylate (2.2 equiv., 2.22 mL, 11.3 mmol) was added to a stirred solution of I4 (1 equiv., 3.14 g, 5.14 mmol) and triphenylphosphine (3.00 equiv., 4.04 g, 10 15.42 mmol) in THF (50 mL). The reaction was heated at 40 °C for 40 minutes, at which point the reaction was complete as judged by TLC (Product Rf = ~0.60, 1:1 hexane/EtOAc). The volatiles were removed under vacuum and the residue was purified by flash chromatography. The pure fractions were pooled and concentrated under vacuum to give I5 (4.33 g, >100% yield including TPO) as a white solid (ES+ m/z 593 [M+H]). 008188229 90 b) Allyl (S)-8-hydroxy-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H- pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate (34) Lithium acetate dihydrate (1 equiv., 524 mg, 5.14 mmol) was added to a stirred solution of 5 I5 (1 equiv., assumed 3.05 g, 5.14 mmol) in N, N-dimethylformamide (15 mL) and H2O (3 mL). The reaction mixture was stirred at 40 °C for 2 hours at which point analysis by LC- MS revealed completion of reaction. The mixture was partitioned between water (50 mL) and EtOAc (50 mL) and the layers separated. The aqueous phase was extracted with EtOAc (3 x 25 mL) and the combined organic layers washed with 10% citric acid (30 mL), 10 brine (40 mL), dried (MgSO4), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography gave desired product 34 (1.64 g, 73% yield) as a white solid (ES+ m/z 437 [M+H]). c) Allyl (S)-8-((5-iodopentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-15 dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (35) Monomer 34 (400 mg, 0.91 mmol) was solubilised in DMF (5 mL).1,5-Diiodopentate (680 μL, 4.5 mmol) and K2CO3 (194 mg, 0.91 mmol) were added and the mixture heated to 60oC. Upon completion the mixture was diluted with EtOAc (100 mL) and washed with H2O (2x50 mL) and brine (50 mL) before being dried (MgSO4), filtered and evaporated under 20 reduced pressure. The crude product was purified by silica gel chromatography (gradient elution: 100% hexane to 8:2 v/v hexane/EtOAc) to afford pure product 35 (417 mg, 85% yield). LC/MS 2.12 min (ES+) m/z 707.20 [M+H]+. d) allyl (11aS)-8-((5-(((3aR)-5-((allyloxy)carbonyl)-8-methoxy-2-(4-methoxyphenyl)-10-25 oxo-3,3a,4,5,10,10a-hexahydrobenzo[b]cyclopenta[e]azepin-7-yl)oxy)pentyl)oxy)-7- methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2- a][1,4]diazepine-10(5H)-carboxylate (36) Monomer 35 (100 mg, 0.158 mmol) and monomer 34 (69 mg, 0.158 mmol) were solubilised in dry DMF (2 mL) under argon. K2CO3 (33 mg, 0.158 mmol) was added and the 30 mixture heated to 60oC. Upon completion the mixture was diluted with EtOAc (100 mL) and washed with H2O (2x50 mL) and brine (50 mL) before being dried (MgSO4), filtered and evaporated under reduced pressure. The crude product was purified by silica gel chromatography (gradient elution: 27:68:5 Hex/EtOAc/MeOH) to afford pure product 36 (179 mg). Analytical data: ES+ = 2.19 min, m/z 1130.40 [M+H]+ . 35 008188229 91 e) (11aS)-7-methoxy-8-((5-(((3aR)-8-methoxy-2-(4-methoxyphenyl)-10-oxo- 3,3a,4,5,10,10a-hexahydrobenzo[b]cyclopenta[e]azepin-7-yl)oxy)pentyl)oxy)-2-(4- methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5-one (37) 5 Tetrakis(triphenylphosphine)palladium (0) (13 mg, 0.014 mMol) was added to a solution of 36 (179 mg, 0.19 mmol) and pyrrolidine (78 L, 0.95 mMol) in DMF (3 mL). The mixture was allowed to stir at room temperature for 20 min, at which point the reaction had gone to completion (as monitored by LC/MS). The reaction mixture was diluted with CH2Cl2 (50 mL) and the organic phase was washed with H2O (2 x 50 mL) until complete pyrrolidine 10 removal. The organic phase was dried over MgSO4, filtered and excess solvent removed by rotary evaporation under reduced pressure. The crude material was purified by silica gel column chromatography (CHCl3/MeOH ; 100% to 95:3) to afford pure product 37 (86 mg, 58% yield). Analytical data: ES+ = 1.21 min, m/z 868.35 [M+H]+. 15 Example 6
Figure imgf000093_0001
008188229 92 a) allyl (S)-8-((3-((((S)-10-((allyloxy)carbonyl)-7-methoxy-2-(4-methoxyphenyl)-5-oxo- 5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8- yl)oxy)methyl)benzyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-11a-methyl-5-oxo-11,11a- dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (38) 5 Monomer 34 (200 mg, 0.46 mMol) was solubilised in DMF (4 mL).1,3- bis(bromomethyl)benzene (60.5 mg, 0.29 mMol) and K2CO3 (97 mg, 0.46 mmol) were added and the mixture heated to 60oC. Upon completion the mixture was diluted with EtOAc (100 mL) and washed with H2O (2x50 mL) and brine (50 mL) before being dried (MgSO4), filtered and evaporated under reduced pressure. The crude product was purified 10 by silica gel chromatography (gradient elution: 100% hexane to 8:2 v/v hexane/EtOAc) to afford pure product 38 (240 mg). Analytical Data: LC/MS 2.12 min (ES+) m/z 707.20 [M+H]+. b) (11aS)-7-methoxy-8-((3-((((3aR)-8-methoxy-2-(4-methoxyphenyl)-10-oxo-15 3,3a,4,5,10,10a-hexahydrobenzo[b]cyclopenta[e]azepin-7-yl)oxy)methyl)benzyl)oxy)- 2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepin- 5-one (39) Tetrakis(triphenylphosphine)palladium (0) (117 mg, 0.014 mMol) was added to a solution of 38 (240 mg, 0.24 mmol) and pyrrolidine (101 µL, 1.2 mMol) in CH2Cl2 (5 mL). The mixture 20 was allowed to stir at room temperature for 20 min, at which point the reaction had gone to completion (as monitored by LC/MS). The reaction mixture was diluted with CH2Cl2 (50 mL) and the organic phase was washed with H2O (2 x 50 mL) until complete pyrrolidine removal. The organic phase was dried over MgSO4, filtered and excess solvent removed by rotary evaporation under reduced pressure. The crude material was purified by silica gel25 column chromatography (CHCl3/MeOH ; 100% to 95:3) to afford pure product 39 (8.16 mg). Analytical data: ES+ = 1.21 min, m/z 868.35 [M+H]+ .
Example 7
Figure imgf000095_0001
Figure imgf000097_0001
008188229 96
Figure imgf000098_0001
Compound 40 is described in Tiberghien et al., Org. Process Res. Dev.2018, 22, 9, 1241- 5 1256 (a) [(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-(4-methoxyphenyl)-2,3- dihydropyrrol-1-yl]-(5-methoxy-2-nitro-4-triisopropylsilyloxy-phenyl)methanone (41) Trifluoromethanesulfonic anhydride (8.87 mL, 51.7 mmol) was added to a solution of 40 10 (20.0 g, 34.4 mmol) and 2,6-dimethylpyridine (8.1 mL, 69 mmol) in dry toluene (120 mL) at -40°C under argon. The solution was stirred at -35°C for 30 min at which point completion was indicated by LCMS and TLC (EtOAc / hexane 1/3). The solution was diluted with ethyl acetate (100 mL) and water (100 mL). The organic layer was washed further with 0.02 N HCl (100 mL), followed by saturated bicarbonate (100 mL), and brine (50 mL). The 15 organics were dried with magnesium sulfate and concentrated down to around 50 mL under vacuum. The solution was diluted with toluene (100 mL). The solution underwent two vacuum/argon cycles, followed by addition of potassium phosphate dibasic (36.7 g, 206 mmol, 98.0 mass%), 4-methoxyphenylboronic acid (7.01 g, 44.7 mmol), tetrakis(triphenylphosphine)palladium(0) (800 mg, 0.689 mmol), and water (30 mL). The 20 reaction was stirred under argon at room temperature for 1h, at which point TLC and LCMS indicated reaction completion. The mixture was diluted with ethyl acetate (100 mL) and washed with water (100 mL) and brine (50 mL). The organics were dried over magnesium sulfate and concentrated under vacuum. 008188229 97 The residue was dry-loaded on silica and purified by filtration over a bed of silica (gradient EtOAc/Hexane 10/90 up 25/75). The purer fractions were pooled and concentrated under vacuum to give 41 (20.0 g, 29.8 mmol, 86.6% Yield) as a dark yellow foam. LC-MS (method 2) 2.66 min, ES+ m/z 672.0 [M+H]. 5 b) (2-amino-5-methoxy-4-triisopropylsilyloxy-phenyl)-[(2S)-2-[[tert- butyl(dimethyl)silyl]oxymethyl]-4-(4-methoxyphenyl)-2,3-dihydropyrrol-1- yl]methanone (42) Zinc (39.7 g, 606 mmol) was added to mixture of water (5.50 mL), acetic acid (5.50 mL, 10 95.9 mmol) and ethanol (88.0 mL) at 0°C. A solution of 41 (A, 11.0 g, 16.4 mmol) in ethanol (60 mL) was added slowly at 5°C, followed by further stirring at 5°C. After 1h30, about 20% of hydroxylamine side product was observed. A further 20g of zinc was added, together with another 5 mL of acid and water. The reaction was allowed to warm up to room temperature slowly over 3h, at which point completion was observed. The solids were15 removed by filtration over a bed of celite. The solution was partitioned between water (300 mL) and ethyl acetate (300 mL), separated, and washed again with water (300 mL), followed by aqueous sodium bicarbonate (150 mL). The organic phase was dried over magnesium sulfate and the volatiles were removed under vacuum. The residue was purified by flash chromatography (340g Ultra Biotage, gradient ethyl acetate / hexane 8/92 20 to 24/76 in 6 CV. Elution around 24/76) to give 42 (9.3 g, 15 mmol, 89% Yield) as a pale yellow foam. LC-MS (method 2) 2.49 min, ES+ m/z 642.5 [M+H]. c) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl-25 butanoyl]amino]propanoyl]amino]phenyl]methyl N-[2-[(2S)-2-[[tert- butyl(dimethyl)silyl]oxymethyl]-4-(4-methoxyphenyl)-2,3-dihydropyrrole-1-carbonyl]- 4-methoxy-5-triisopropylsilyloxy-phenyl]carbamate (43) Triphosgene (1.46 g, 4.87 mmol, 99 mass%) was added to a stirred solution of 3 (8.70 g, 13.6 mmol) in dry dichloromethane (87.0 mL, 99.5 mass%) at -10°, followed by dry 30 triethylamine (4.18 mL, 29.8 mmol, 99.5 mass%). The mixture was allowed to warm up to room temperature. Solid allyl N-[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo- ethyl]carbamoyl]-2-methyl-propyl]carbamate (5.38 g, 14.3 mmol) was added in one portion, followed by triethylamine (2.85 mL, 20.3 mmol) and dichloromethane (87.0 mL). The reaction mixture was allowed to stir for 4h at room temperature. Full dissolution of the 35 starting material was observed. Completion was observed by LCMS and TLC (ethyl acetate / hexane 1/2). The solution was washed with 0.15 N HCl (500 mL), followed by 008188229 98 saturated hydrogen carbonate (200 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was dried overnight under vacuum to give 43 (13.87 g, 13.28 mmol, 97.8% Yield) as a solid orange foam. LC-MS (method 2) 2.47 min, ES+ m/z 1045.4 [M+H]. 5 d) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl N-[2-[(2S)-2-(hydroxymethyl)-4-(4- methoxyphenyl)-2,3-dihydropyrrole-1-carbonyl]-4-methoxy-5-triisopropylsilyloxy- phenyl]carbamate (44) 10 p-Toluenesulfonic acid (2.24 g, 12.7 mmol) was added to a solution of 43 (13.3 g, 12.7 mmol) in a mixture of acetic acid (2.66 mL), methanol (13.3 mL), water (4.00 mL) and 2- methyltetrahydrofuran (80.0 mL) and stirred at room temperature. The reaction was found to be progressing rapidly and complete after 1h by LCMS. The reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (100 15 mL), followed by saturated aqueous hydrogen carbonate (50 mL), and brine (50 mL). The organics were dried over magnesium sulphate. The volatiles were removed under vacuum. The residue was purified with a first chromatography (340 g ultra, EtOAc/Hexanes 35/65 up to 100/0 in 6 CV. Elution from 90/10); The pure fractions were pooled and the volatiles removed under vacuum to give 44 (11.8 g, 12.7 mmol, 99.6% Yield) as a yellow solid.20 LC-MS (method 2) 2.12 min, ES+ m/z 931.1 [M+H]. e) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-2-methoxy-8-(4- methoxyphenyl)-11-oxo-3-triisopropylsilyloxy-6a,7-dihydro-6H-pyrrolo[2,1-25 c][1,4]benzodiazepine-5-carboxylate (45) Diisopropyl azodicarboxylate (5.70 mL, 28.4 mmol) was added to a solution of 44 (12.0 g, 12.9 mmol) and triphenylphosphine (9.00 g, 34.1 mmol) in tetrahydrofuran (200 mL). The reaction was heated at 40°C for 2h, at which point a satisfactory amount of product was formed by LCMS. The volatiles were removed under vacuum and the residue was purified 30 by chromatography (340g Ultra, Biotage, EtOAc/Hexane, gradient from 35% to 75%. Elution around 65% upwards). The pure fractions were pooled and concentrated under vacuum to give 45 (8.50 g, 9.32 mmol, 72.2% Yield) as a pale yellow solid (85% pure by LC). LC-MS (method 2) 2.15 min, ES+ m/z 913.1 [M+H]. 35 008188229 99 f) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-hydroxy-2-methoxy-8-(4- methoxyphenyl)-11-oxo-6a,7-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5- carboxylate (46) 5 Lithium acetate (1.3 g, 20 mmol) was added to a solution of 45 (8.40 g, 9.21 mmol) in DMF (42.0 mL, 543 mmol) and water (1.7 mL, 94 mmol). The solution was stirred at 40°C for 1h and was then partitioned between 2-MeTHF (250 mL) and water (400 mL). The organics were washed with brine (150 mL) and dried over magnesium sulfate. The solution was concentrated under vacuum. The residue was purified by chromatography (100g Ultra, 10 gradient 50/50 EtOAc / Hexane up 100% EtOAc). The pure fractions were concentrated and dried under vacuum. The solids were taken up in diethylether (100 mL), collected by filtration and dried to give 46 (5.0 g, 6.6 mmol, 72% Yield) as an off-white powder. LC-MS (method 2) 1.53 min, ES+ m/z 756.7 [M+H]. 15 g) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3- (bromomethyl)phenyl]methoxy]-2-methoxy-8-(4-methoxyphenyl)-11-oxo-6a,7- dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate (47) and 20 [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3-[[(6aS)-5-[[4-[[(2S)-2- [[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]-2-methoxy-8-(4- methoxyphenyl)-11-oxo-6a,7-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepin-3-25 yl]oxymethyl]phenyl]methoxy]-2-methoxy-8-(4-methoxyphenyl)-11-oxo-6a,7-dihydro- 6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate (47A) 1,3-bis(bromomethyl)benzene (0.698 g, 2.64 mmol) was added to a mixture of 46 (1.00 g, 1.32 mmol) and potassium carbonate (603 mg, 4.36 mmol) in acetone (10.0 mL). The mixture was heated at 50°C for 1.5h, at which point LCMS showed conversion of the 30 phenol to the mono-alkylated and bis-alkylated products. The mixture was decanted, and the supernatant was evaporated to dryness. The residue was loaded on a 10g silica samplet, dried under vacuum, and loaded on top of a 50g Ultra Biotage column. The mixture was purified by chromatography (gradient EtOAc / Hexane 25/75 up to 100% EtOAc, followed by EtOAc / EtOH 90/10). The fractions were pooled and the volatiles were 35 removed under vacuum to give the product 47 (462 mg, 0.492 mmol, 37.2% Yield), and 47A (531 mg, 0.329 mmol, 49.8% Yield), as pale yellow solids. 008188229 100 47 LC-MS (method 2) 1.85 min, ES+ m/z 940.6 [M+H]. 47A LC-MS (method 2) 1.93 min, ES+ m/z 1615.3 [M+H]. h) [(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-(4-methoxyphenyl)-2,3- 5 dihydropyrrol-1-yl]-(4-hydroxy-5-methoxy-2-nitro-phenyl)methanone (48) TIPS protected phenol 41 (10.0 g, 14.9 mmol) was dissolved in a mixture of ethyl acetate (20.0 mL) and DMF (20.0 mL) at 40°C. A solution of lithium acetate (1.20 g, 18.2 mmol) in water (3.0 mL) was added. The reaction was allowed to proceed at 40°C for 1h at which point completion was observed by LCMS. The reaction mixture was partitioned between 2- 10 MeTHF (200 mL) and 2% citric acid in water (200 mL). The organic phase was washed with brine (70 mL) and dried over magnesium sulfate. The volatiles were removed under vacuum. The solid residue was precipitated by addition of diethyl ether (10 mL) and hexane (60 mL). The product was collected by decantation and drying under vacuum to give 48 (7.10 g, 13.8 mmol, 92.6% Yield) as a yellow powder. 15 LC-MS (method 2) 1.97 min, ES+ m/z 515.7 [M+H]. i) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3-[[4-[(2S)-2-[[tert- butyl(dimethyl)silyl]oxymethyl]-4-(4-methoxyphenyl)-2,3-dihydropyrrole-1-carbonyl]-20 2-methoxy-5-nitro-phenoxy]methyl]phenyl]methoxy]-2-methoxy-8-(4- methoxyphenyl)-11-oxo-6a,7-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5- carboxylate (49) Bromobenzyl 47 (332 mg, 0.354 mmol) was added to a mixture of phenol 48 (191 mg, 0.371 mmol) and potassium carbonate (58.8 mg, 0.425 mmol) in acetone (5.00 mL). The 25 mixture was heated at 55°C for 2h, at which point LCMS showed complete conversion. The mixture was decanted, and the supernatant was evaporated to dryness. The residue was loaded on a 25g Ultra Biotage column. The mixture was purified by chromatography (gradient EtOAc / Hexane 25/75 up to 100% EtOAc; elution around 90%). The fractions were pooled and the volatiles were removed under vacuum to give the product 49 (380 mg,30 0.277 mmol, 78.3% Yield), as a yellow solid. LC-MS (method 2) 2.20 min, ES+ m/z 1373.4 [M+H]. j) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3-[[5-amino-4-[(2S)-2-[[tert-35 butyl(dimethyl)silyl]oxymethyl]-4-(4-methoxyphenyl)-2,3-dihydropyrrole-1-carbonyl]- 2-methoxy-phenoxy]methyl]phenyl]methoxy]-2-methoxy-8-(4-methoxyphenyl)-11- 008188229 101 oxo-6a,7-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate (50) A mixture of formic acid (0.500 mL, 12.7 m), water (0.500 mL) and ethanol (5.00 mL, 85 mmol) was added to a solution of 49 (500 mg, 0.364 mmol) in tetrahydrofuran (5.00 mL) at 5°C (ice bath). Zinc powder was added in portion (2.00 g, 30.5 mmol) and the ice bath was 5 removed. After 1h30, about 20% of hydroxylamine side product was observed. A further 1g of zinc was added, together with another 0.25 mL of acid and water. The reaction was allowed to carry on to completion over another 1.5h. The solids were removed by filtration over a bed of celite. The solution was partitioned between water (100 mL) and ethyl acetate (150 mL), separated, and washed with aqueous 10 sodium bicarbonate (150 mL). The organic phase was dried over magnesium sulfate and the volatiles were removed under vacuum. The residue 50 was found pure enough to be used directly in the next step (489 mg, 0.364 mmol, 100% Yield) LC-MS (method 2) 2.16 min, ES+ m/z 1344.3 [M+H]. 15 k) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3-[[5- (allyloxycarbonylamino)-4-[(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-(4- methoxyphenyl)-2,3-dihydropyrrole-1-carbonyl]-2-methoxy-20 phenoxy]methyl]phenyl]methoxy]-2-methoxy-8-(4-methoxyphenyl)-11-oxo-6a,7- dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate (51) Allyl chloroformate (44.1 μL, 0.401 mmol) was added to a solution of 50 (490 mg, 0.365 mmol) and pyridine (36.0 μL, 0.44 mmol) in dichloromethane (10.0 mL) at -10°C (acetone/ice bath). The solution was then allowed to warm up to room temperature. 25 The solution was partitioned between water (40 mL) and DCM (40 mL) and decanted through a phase separation cartridge. The volatiles were removed under vacuum to give 51 (521 mg, 0.365 mmol, 100% Yield) which was deemed pure enough to be used as such in the next step. LC-MS (method 2) 2.24min, ES+ m/z 1428.0 [M+H]. 30 l) [4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3-[[5- (allyloxycarbonylamino)-4-[(2S)-2-(hydroxymethyl)-4-(4-methoxyphenyl)-2,3- dihydropyrrole-1-carbonyl]-2-methoxy-phenoxy]methyl]phenyl]methoxy]-2-methoxy-35 8-(4-methoxyphenyl)-11-oxo-6a,7-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5- carboxylate (52) 008188229 102 p-Toluenesulfonic acid (0.064 g, 0.36 mmol) was added to a solution of 51 (521 mg, 0.365 mmol) in a mixture of acetic acid (0.5 mL, 9 mmol), methanol (3.00 mL), water (1 mL) and 2-methyltetrahydrofuran (7 mL) and stirred at room temperature. The reaction was found to be progressing rapidly and complete after 3h by TLC (EtOAc). 5 The reaction mixture was diluted with 2-MeTHF (30 mL) and washed with water (30 mL), followed by saturated aqueous hydrogen carbonate (30 mL). The volatiles were removed under vacuum. The residue was purified with a first chromatography (25 g ultra, DCM versus DCM/MeOH 4/1; gradient from 10/90 up to 35/65 up to 50/50 in 6 CV. Elution from 25/75); The pure fractions were pooled and the volatiles removed under vacuum to give 5210 (360 mg, 0.274 mmol, 75.1% Yield over 3 steps) as a white solid. LC-MS (method 2) 1.89 min, ES+ m/z 1313.0 [M+H]. m) Allyl (6aS)-3-[[3-[[(6aS)-5-[[4-[[(2S)-2-[[(2S)-2-(allyloxycarbonylamino)-3-methyl- butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]-2-methoxy-8-(4-15 methoxyphenyl)-11-oxo-6a,7-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepin-3- yl]oxymethyl]phenyl]methoxy]-2-methoxy-8-(4-methoxyphenyl)-11-oxo-6a,7-dihydro- 6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate (53) Diisopropyl azodicarboxylate (0.120 mL, 0.597 mmol) was added to a solution of 52 (355 mg, 0.270 mmol) and triphenylphosphine (214 mg, 0.812 mmol) in tetrahydrofuran (7.10 20 mL). The reaction was heated at 40°C for 2h, at which point a satisfactory amount of product was formed by LCMS. Half of the THF was removed by evaporation, and hexane (20 to 30 mL) was added. The supernatant was removed, and the solid residue was purified by chromatography: (25g Ultra, Biotage, EtOAc/EtOH 4/1 in Hexane, gradient from 10% to 95%. Elution around 50% upwards). The pure fractions were pooled and 25 concentrated under vacuum to give 53 (250 mg, 0.193 mmol, 71.4% Yield) as a pale- yellow solid (93% pure by LC) LC-MS (method 2) 1.93 min, ES+ m/z 1296.0 [M+H]. n) [4-[[(2S)-2-[[(2S)-2-amino-3-methyl-30 butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3-[[(6aS)-2-methoxy-8-(4- methoxyphenyl)-11-oxo-5,6,6a,7-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-3- yl]oxymethyl]phenyl]methoxy]-2-methoxy-8-(4-methoxyphenyl)-11-oxo-6a,7-dihydro- 6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate (54) Alloc-protected 53 (240 mg, 0.185 mmol) was dissolved in a mixture of dichloromethane 35 (4.80 mL) and pyrrolidine (156 μL, 1.86 mmol). The atmosphere was purged with argon. A 008188229 103 catalytic amount of tetrakis(triphenylphosphine)palladium(0) (4.31 mg, 0.00371 mmol) was added and the reaction allowed to proceed for 30 min, when LCMS showed completion. The reaction mixture was partitioned between DCM (10 mL) and 1M aqueous ammonium chloride (10 mL). The organic layer was decanted through an isolera cartridge, and the 5 volatiles were removed under vacuum to give crude 54 (209 mg, 0.186 mmol, 100% Yield). LC-MS (method 1) 1.60 min, ES+ m/z 1127.4 [M+H]. o) [4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1- yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop10 anoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (6aS)-3-[[3- [[(6aS)-2-methoxy-8-(4-methoxyphenyl)-11-oxo-5,6,6a,7-tetrahydropyrrolo[2,1- c][1,4]benzodiazepin-3-yl]oxymethyl]phenyl]methoxy]-2-methoxy-8-(4- methoxyphenyl)-11-oxo-6a,7-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5- carboxylate (55) 15 Mal-amido-peg8-acid (123 mg, 0.203 mmol,) and 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (39.5 mg, 0.204 mmol) were added to a solution of 54 (209 mg, 0.185 mmol) in dichloromethane (4 mL) at room temperature. The reaction mixture was stirred for 3h when completion was observed by LCMS. The DCM was removed by evaporation and the residue was purified by flash chromatography (25g Ultra, DCM / 20 DCM/MeOH 80/20; gradient from 15/85 up to 40/60 in 14 CV). The pure fractions were pooled and evaporated. Further purification by preparative reverse phase chromatography, and freeze-drying gave 55 (177.3 mg, 0.1042 mmol, 56.2% Yield) as a yellow solid. Purity 98.7%. LC-MS (method 1) 1.95 min, ES+ m/z 1702.0 [M+H]; (method 3) 7.82 min, ES+ m/z 1702.0 25 [M+H].1H NMR (400 MHz, DMSO-d6) δ 9.90 (s, 1H), 8.16 (d, J = 6.9 Hz, 1H), 8.00 (t, J = 5.7 Hz, 1H), 7.87 (d, J = 8.6 Hz, 1H), 7.63 – 7.51 (m, 3H), 7.50 – 7.31 (m, 10H), 7.27 – 7.16 (m, 2H), 7.15 – 7.08 (m, 2H), 7.00 (s, 2H), 6.95 – 6.86 (m, 4H), 6.56 (d, J = 6.6 Hz, 1H), 6.43 (s, 1H), 5.16 – 5.02 (m, 4H), 5.01 – 4.81 (m, 2H), 4.39 (t, J = 7.1 Hz, 1H), 4.26 – 4.00 (m, 4H), 3.81 (s, 3H), 3.76 (s, 6H), 3.69 (s, 3H), 3.66 – 3.53 (m, 6H), 3.53 – 3.43 (m, 30 28H), 3.37 (t, J = 5.9 Hz, 2H), 3.30 – 3.19 (m, 2H), 3.15 (q, J = 5.8 Hz, 2H), 2.84 – 2.70 (m, 2H), 2.43 (dt, J = 22.8, 6.6 Hz, 2H), 2.33 (t, J = 7.3 Hz, 2H), 2.03 – 1.87 (m, 1H), 1.29 (d, J = 7.1 Hz, 3H), 0.85 (dd, J = 15.1, 6.8 Hz, 6H). 008188229 104 Example 8 Herceptin and R347 antibodies engineered to have cysteine inserted between the 239 and 240 positions were produced following the methods described in Dimasi, N., et al., Molecular Pharmaceutics, 2017, 14, 1501-1516 (DOI: 5 10.1021/acs.molpharmaceut.6b00995). HerC239i-9 ADC (ConjA) HerC239i (30 mg, 200 nmol) was loaded onto solid support and reduced, reoxidised, conjugated to Compound 9, purified, released from the resin and formulated into 25 mM 10 Histidine, 200 mM Sucrose pH 6.0 buffer containing 0.02% Tween-80 according to patent # WO2012140433. UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample 15 of ConjA at 214 nm and 330 nm (Compound 9 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of compound 9, consistent with a drug-per-antibody ratio (DAR) of 1.87 molecules of compound 9 per antibody. 20 UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 µm 4.6 x 150 mm column (with a 4 µm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjA at 280 nm shows a monomer purity of 96%. UHPLC SEC analysis gives a 25 concentration of final ConjA at 1.82 mg/mL in 11.6 mL, obtained mass of ConjA is 21.1 mg (70% yield). R347C239i-9 ADC (ConjB) R347C239i (300 mg, 2 µmol) was loaded onto solid support and reduced, reoxidised, 30 conjugated to Compound 9, purified, released from the resin and formulated into 25 mM Histidine, 200 mM Sucrose pH 6.0 buffer containing 0.02% Tween-80 according to patent # WO2012140433. UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 35 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjB at 214 nm and 330 nm (Compound 9 specific) shows unconjugated light chains 008188229 105 and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 9, consistent with a drug-per-antibody ratio (DAR) of 1.86 molecules of Compound 9 per antibody. 5 UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 µm 4.6 x 150 mm column (with a 4 µm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjB at 280 nm shows a monomer purity of 97%. UHPLC SEC analysis gives a 10 concentration of final ConjB at 2.19 mg/mL in 109 mL, obtained mass of ConjB is 238.9 mg (80% yield). HerC239i-22 ADC (ConjC) A 10 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline pH 15 7.4 (PBS) was added (80 molar equivalent/antibody, 14.9 micromoles, 1.49 mL) to a 10 mL solution of HerC239i (28 mg, 187 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 3.0 mg/mL. The reduction mixture was allowed to react at room temperature for 3 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. The 20 reduced antibody was buffer exchanged, via spin filter centrifugation using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 30 molar equivalent/antibody, 5.6 micromoles, 112 µL) in DMSO was added and the reoxidation mixture was allowed to react for 17 hours at room temperature with gentle 25 (60 rpm) shaking at an antibody concentration of 3 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). The reoxidation mixture was then sterile- filtered and diluted in a conjugation buffer containing PBS and 1 mM EDTA for a final antibody concentration of ~ 3 mg/mL. Compound 22 was added as a DMSO solution (10 30 molar equivalent/antibody, 1.87 micromoles, in 0.93 mL DMSO) to ~ 8.4 mL of this reoxidised antibody solution (28 mg, 187 nanomoles) for a 10% (v/v) final DMSO concentration. The solution left to react at room temperature for 1 hour at room temperature with gentle shaking, then the conjugation was quenched by addition of N- acetyl cysteine (9.33 micromoles, 93 ^L at 100 mM) for 30 min at room temperature, then 35 purified by spin filtration into PBS using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile-filtered and analysed. 008188229 106 UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjC at 280 nm and 330 nm (Compound 22 specific) shows unconjugated light chains 5 and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 22, consistent with a drug-per-antibody ratio (DAR) of 1.77 molecules of Compound 22 per antibody. UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel10 SuperSW mAb HTP 4 µm 4.6 x 150 mm column (with a 4 µm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjC at 280 nm shows a monomer purity of 99%. SEC-UHPLC analysis gave a concentration of final ConjC at 2.24 mg/mL in 8.5 mL, obtained mass of ConjC is 19.03 mg15 (68% yield). HerC239i-28 ADC (ConjD) A 1 M solution of DL-dithiothreitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added (100 molar equivalent/antibody, 33.3 micromoles, 33 ^L) to a 7.5 mL solution of 20 HerC239i (50 mg, 333 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 5.0 mg/mL. The reduction mixture was allowed to react at room temperature for 4 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. The reduced antibody was buffer exchanged, via spin filter centrifugation, into a reoxidation 25 buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 20 molar equivalent/antibody, 6.67 micromoles, 133.3 µL) in DMSO was added and the reoxidation mixture was allowed to react for 16 hours at room temperature with gentle (60 rpm) shaking at an antibody concentration of 4.0 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the 30 cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). The reoxidation mixture was then sterile-filtered and diluted to 2.2 mg/mL in a conjugation buffer containing PBS and 1 mM EDTA. Compound 28 was added as a DMSO solution (10 molar equivalent/antibody, 1.33 micromoles, in 1.0 mL DMSO) to 9.0 mL of this reoxidised antibody solution (20 mg, 133 nanomoles) for a 10% (v/v) final DMSO concentration and a 35 final antibody concentration of 2.0 mg/mL. The solution was mixed for 1.5 hours at room temperature, then the conjugation was quenched by addition of N-acetyl cysteine (6.67 008188229 107 micromoles, 66.7 ^L at 100 mM) for 0.5 h at room temperature, then purified on an AKTA™ Start FPLC using a GE Healthcare HiLoadTM 26/600 column packed with Superdex 200 PG, eluting with 2.6 mL/min PBS. Fractions corresponding to ConjD monomer peak were pooled, concentrated using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile- 5 filtered and analysed. UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjD at 214 nm and 330 nm (Compound 28 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single 10 molecule of Compound 28, consistent with a drug-per-antibody ratio (DAR) of 1.70 molecules of Compound 28 per antibody. UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 µm 4.6 x 150 mm column (with a 4 µm 3.0 x 20 mm guard column) 15 eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjD at 280 nm shows a monomer purity of > 99%. UHPLC SEC analysis gives a concentration of final ConjD at 1.72 mg/mL in 8.2 mL, obtained mass of ConjD is 14.10 mg (71% yield). 20 Her2-28 ADC (ConjE) A 10 mM solution of Tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (10 molar equivalent/antibody, 3.33 micromoles, 333 ^L) to a 22.5 mL solution of Trastuzumab (50 mg, 333 nanomoles) in reduction buffer containing 30 25 mM Histidine and 30 mM Arginine pH 6.8 and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 2.2 mg/mL. The reduction mixture was heated at +37 °C for 3.5 hours (or until full reduction is observed by UHPLC) in an incubated orbital shaker with gentle (60 rpm) shaking. After cooling down to room temperature, Compound 28 was added as a DMSO solution (20 molar equivalent/antibody, 6.67 30 micromoles, in 2.5 mL DMSO) to 22.5 mL of this reduced antibody solution (50 mg, 333 nanomoles) for a 10% (v/v) final DMSO concentration. The solution was mixed for 16 hours at room temperature, then the conjugation was quenched by addition of N-acetyl cysteine (33.3 micromoles, 333 ^L at 100 mM) for 1 hour at room temperature, then purified on an AKTA™ Start FPLC using a GE Healthcare HiLoadTM 26/600 column packed with 35 Superdex 200 PG, eluting with 2.6 mL/min PBS. Fractions corresponding to ConjE 008188229 108 monomer peak were pooled, concentrated using a 15mL Amicon Ultracell 30KDa MWCO spin filter, sterile-filtered and analysed. UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 5 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjE at 214 nm and 330 nm (Compound 28 specific) shows a mixture of unconjugated light chains, light chains attached to a single molecule of Compound 28, unconjugated heavy chains and heavy chains attached to up to three molecules of Compound 28, consistent with a drug-per-antibody ratio (DAR) of 7.44 molecules of Compound 28 per10 antibody. UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 µm 4.6 x 150 mm column (with a 4 µm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium 15 phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjE at 280 nm shows a monomer purity of 89%. UHPLC SEC analysis gives a concentration of final ConjE at 1.28 mg/mL in 8.7 mL, obtained mass of ConjE is 11.09 mg (22% yield). 20 HerC239i-55 ADC (ConjF) A 10 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (80 molar equivalent/antibody, 16 micromoles, 1.6 mL) to a 10 mL solution of HerC239i (30 mg, 200 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 3.0 mg/mL. 25 The reduction mixture was allowed to react at room temperature for 3.5 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. The reduced antibody was buffer exchanged, via spin filter centrifugation using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent. A 50 mM solution of dehydroascorbic acid 30 (DHAA, 30 molar equivalent/antibody, 6 micromoles, 120 µL) in DMSO was added and the reoxidation mixture was allowed to react for 17 hours at room temperature with gentle (60 rpm) shaking at an antibody concentration of 3 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). The reoxidation mixture was then sterile-filtered and 35 diluted in a conjugation buffer containing PBS and 1 mM EDTA and 33% (v/v) propylene glycol for a final antibody concentration of ~ 2 mg/mL. Compound 55 was added as a 008188229 109 DMSO solution (15 molar equivalent/antibody, 3 micromoles, in 1.5 mL DMSO) to ~ 13.5 mL of this reoxidised antibody solution (30 mg, 200 nanomoles) for a 10% (v/v) final DMSO concentration. The solution left to react at room temperature for 20 hours at room temperature with gentle shaking, then the conjugation was quenched by addition of N- 5 acetyl cysteine (15 micromoles, 150 ^L at 100 mM) for 30 min at room temperature. Then the conjugation mixture was then purified on an AKTA™ Start FPLC using a GE Healthcare HiLoadTM 26/600 column packed with Superdex 200 PG, eluting with 2.6 mL/min PBS. High monomeric purity fractions corresponding to ConjF peak were pooled, diluted 3-5x with 1 M ammonium sulfate, 25 mM potassium phosphate pH 6.0 HIC Buffer A 10 and loaded onto a 5 mL Hydrophobic Interaction Chromatography (HIC) HiTrap Butyl HP column, eluting with 0 ^100% 25 mM potassium phosphate pH 6.0 HIC Buffer B over 100 mL (20 CV) at 5 mL/min. Drug-to-antibody ratio = 2 (DAR 2) fractions were pooled and diluted 3-5x with 10 mM sodium phosphate pH 6.0 CHT Buffer A and loaded onto a 5 mL Bio-Scale Mini CHT ceramic hydroxyapatite 40 µm Type II cartridge, eluting with 0 ^100%15 10 mM sodium phosphate, 1 M sodium chloride pH 6.0 CHT Buffer B over 125 mL (25 CV) at 5 mL/min. High monomeric purity fractions were pooled and buffer exchanged into PBS via spin filtration using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile-filtered and analysed. UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 20 mm x 2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjF at 280 nm and 330 nm (Compound 55 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 55, consistent with a drug-per-antibody ratio (DAR) of 1.91 molecules of Compound 55 per antibody. 25 UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 µm 4.6 x 150 mm column (with a 4 µm 3.0 x 20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of 30 ConjF at 280 nm shows a monomer purity of 96%. SEC-UHPLC analysis gave a concentration of final ConjF at 0.87 mg/mL in 2.0 mL, obtained mass of ConjF is 1.74 mg (6% yield). Example 9 - PBD Cytotoxicity Assay 35 The potency of the PBD molecules were measured via in vitro cytotox assays in the carcinoma cell line NCI-N87. 008188229 110 Solid PBD material was dissolved in DMSO to a 2 mM stock solution, from which eight serial dilutions were made at a 1:10 ratio in DMSO and stored at -20°C until use. Adherent NCI-N87 cells were washed with D-PBS and detached with Trypsin-EDTA, cell 5 density and viability were then determined in duplicate by Trypan blue exclusion assay using an automated cell counter (LUNA-II™). Cell suspension was diluted to 1 x 105 cells/ml in growth media (RPMI 1640 with Glutamax + 10% (v/v) HyClone™ Fetal Bovine Serum) and vortexed before dispensing 2mL per well into sterile 3 mL polypropylene plates. Warhead dilutions were then dispensed into the appropriate wells at 10 µl/well and 10 mixed by repeat pipetting. For control wells 10 µl of DMSO was dispensed onto 2 mL cell suspension, and thoroughly mixed.100µl of each sample was then aliquoted into 2 replicate wells of a sterile flat 96-well microplate and incubated in a 37°C CO2-gassed (5%) incubator. At the end of the incubation period time (7 days), cell viability was measured by CellTiter 96TM Aqueous One (MTS) assay, which was dispensed at 20μl/well and incubated 15 for 4 hours at 37°C, 5%CO2. Plates were then read on an EnVisionTM Multi-label Plate Reader (Perkin Elmer) using absorbance at 490 nm. Cell survival percentage was calculated from the mean absorbance of the 2 replicate wells for each sample, compared to the mean absorbance in the two control wells treated with20 DMSO only (100%). The IC50 was determined by fitting each data set to sigmoidal dose- response curves with a variable slope using the non-linear curve fit algorithm on the GraphPad Prism software (San Diego, CA). All the experiments in this report were carried out and tested in three independent 25 experiments. Data are reported as the mean of the three independent replicates.
Figure imgf000112_0001
Example 10 - ADC cytotoxicity method MTS The concentration and viability of cells from a sub-confluent (80-90% confluency) T75 flask 30 are measured by trypan blue staining, and counted using the LUNA-II™ Automated Cell Counter. Cells were diluted to 2x105/ml, dispensed (50 µl per well) into 96-well flat-bottom plates. 008188229 111 A stock solution (1 ml) of antibody drug conjugate (ADC) (20 µg/ml) was made by dilution of filter-sterilised ADC into cell culture medium. A set of 8x 10-fold dilutions of stock ADC were made in a 24-well plate by serial transfer of 100 µl into 900 µl of cell culture medium. 5 ADC dilution was dispensed (50 µl per well) into 4 replicate wells of the 96-well plate, containing 50 µl cell suspension seeded the day previously. Control wells received 50 µl cell culture medium. The 96-well plate containing cells and ADCs was incubated at 37C in a CO2-gassed incubator for the exposure time. 10 At the end of the incubation period, cell viability was measured by MTS assay. MTS (Promega) was dispensed (20 µl per well) into each well and incubated for 4 hours at 37C in the CO2-gassed incubator. Well absorbance was measured at 490 nm. Percentage cell survival was calculated from the mean absorbance in the 4 ADC-treated wells compared to the mean absorbance in the 4 control untreated wells (100%). IC50 was determined from 15 the dose-response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose-response curve with variable slope. ADC incubation times were 4 days with MDA-MB-468 and 7 days for NCI-N87. MDA-MB- 468 and NCI-N87 were cultured in RPMI 1640 with Glutamax + 10% (v/v) HyClone™ Fetal20 Bovine Serum.
Figure imgf000113_0001
Example 11 - Xenograft Testing NCI-N87 Xenografted Mice 25 Female severe combined immune-deficient mice (Fox Chase SCID®, C.B-17/Icr-Prkdcscid, Charles River) were: A. ten weeks old with a body weight (BW) range of 16.9 to 21.9 grams on Day 1 of the study; B. eight weeks old with a body weight (BW) range of 16.5 to 21.6 grams on Day 1 of the 30 study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl), and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fibre. The mice were housed on irradiated Enricho’cobs ™ Laboratory Animal 008188229 112 Bedding in static micro-isolators on a 12-hour light cycle at 20–22°C (68–72°F) and 40– 60% humidity. CR Discovery Services specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and 5 use program at CR Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals. Tumour Cell Culture 10 Human NCI-N87 gastric carcinoma lymphoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin G sodium, 100 μg/mL streptomycin sulfate and 25 μg/mL gentamicin. The cells were grown in tissue culture flasks in a humidified incubator at 37 °C, in an atmosphere of 5% CO2 and 95% air. 15 In Vivo Implantation and Tumour Growth The NCI-N87 cells used for implantation were harvested during log phase growth and Re- suspended in phosphate buffered saline (PBS) containing 50% Matrigel™ (BD Biosciences). On the day of tumour implant, each test mouse was injected subcutaneously 20 in the right flank with 1 x 107 cells (0.1 mL cell suspension), and tumour growth was monitored as the average size approached the target range of 100 to 150 mm3. Fourteen days later, designated as Day 1 of the study, mice were sorted according to calculated tumour size into groups each consisting of ten animals with individual tumour volumes ranging from: 25 A.108 to 172 mm3 and group mean tumour volumes of 131 mm3; B.108 to 144 mm3 and group mean tumour volumes of 113-115 mm3. Tumours were measured in two dimensions using calipers, and volume was calculated using the formula: 30 Tumour Volume
Figure imgf000114_0001
where w = width and l = length, in mm, of the tumour. Tumour weight may be estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumour volume. Treatment 35 Treatment began on Day 1 in groups of 10 mice (n=10) with established subcutaneous NCI-N87 tumours (108–144 mm3). 008188229 113 A.ConjA (1 mg/kg) was administered intravenously once on Day 1 (qd x 1). A vehicle- treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 85. B. ConjD (4 mg/kg) was administered intravenously once on Day 1 (qd x 1). A vehicle- 5 treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 79. Each mouse was euthanized when its tumour reached the endpoint volume of 800 mm3 or on the final day, whichever came first. The time to endpoint (TTE) was calculated for each mouse. 10 Endpoint and Tumor Growth Delay (TGD) Analysis Tumors were measured using calipers twice per week, and each animal was euthanized when its tumor reached the endpoint volume of 800 mm3 or at the end of the study, whichever came first. Animals that exited the study for tumor volume endpoint were 15 documented as euthanized for tumor progression (TP), with the date of euthanasia. The time to endpoint (TTE) for analysis was calculated for each mouse by the following equation: where TTE is expressed in days, endpoint volume is expressed in mm3, b is the intercept, 20 and m is the slope of the line obtained by linear regression of a log-transformed tumor growth data set. The data set consisted of the first observation that exceeded the endpoint volume used in analysis and the three consecutive observations that immediately preceded the attainment of this endpoint volume. The calculated TTE is usually less than the TP date, the day on which the animal was euthanized for tumor size. Animals with tumors that 25 did not reach the endpoint volume were assigned a TTE value equal to the last day of the study. In instances in which the log-transformed calculated TTE preceded the day prior to reaching endpoint or exceeded the day of reaching tumor volume endpoint, a linear interpolation was performed to approximate the TTE. Any animal classified as having died from NTR (non-treatment-related) causes due to accident (NTRa) or due to unknown 30 etiology (NTRu) were excluded from TTE calculations (and all further analyses). Animals classified as TR (treatment-related) deaths or NTRm (non-treatment-related death due to metastasis) were assigned a TTE value equal to the day of death. Treatment outcome was evaluated from tumor growth delay (TGD), which is defined as the increase in the median time to endpoint (TTE) in a treatment group compared to the control group: 35 TGD = T – C, 008188229 114 expressed in days, or as a percentage of the median TTE of the control group: where: 5 T = median TTE for a treatment group, and C = median TTE for the designated control group. Tumour growth inhibition Tumor growth inhibition (TGI) analysis evaluates the difference in median tumor volumes10 (MTVs) of treated and control mice. For this study, the endpoint for determining TGI was: A. Day 26 B. Day 19 , which was the last day that all evaluable control mice remained in the study. The MTV (n), the median tumor volume for the number of animals, n, on the day of TGI analysis, was 15 determined for each group. Percent tumor growth inhibition (%TGI) was defined as the difference between the MTV of the designated control group and the MTV of the drug- treated group, expressed as a percentage of the MTV of the control group:
Figure imgf000116_0001
The data set for TGI analysis included all animals in a group, except those that died due to 20 treatment-related (TR) or non-treatment-related (NTR) causes prior to the day of TGI analysis. MTV and Criteria for Regression Responses Treatment efficacy may be determined from the tumor volumes of animals remaining in the 25 study on the last day. The MTV (n) was defined as the median tumor volume on the last day of the study in the number of animals remaining (n) whose tumors had not attained the endpoint volume. Treatment efficacy may also be determined from the incidence and magnitude of regression responses observed during the study. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR 30 response, the tumor volume was 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm3 for one or more of these three measurements. In a CR response, the tumor volume was less than 13.5 mm3 for three consecutive measurements during the course of the study. 008188229 115 Animals were scored only once during the study for a PR or CR event and only as CR if both PR and CR criteria were satisfied. An animal with a CR response at the termination of a study was additionally classified as a tumor-free survivor (TFS). Animals were monitored for regression responses. 5 Toxicity Animals were weighed daily on Days 1–5, then twice per week until the completion of the study. The mice were observed frequently for overt signs of any adverse, treatment-related (TR) side effects, and clinical signs were recorded when observed. Individual body weight 10 was monitored as per protocol, and any animal with weight loss exceeding 30% for one measurement or exceeding 25% for three consecutive measurements was euthanized as a TR death. Group mean body weight loss was also monitored according to CR Discovery Services protocol. Acceptable toxicity was defined as a group mean body weight (BW) loss of less than 20% during the study and no more than 10% TR deaths. Dosing was 15 suspended in any group where mean weight loss exceeded acceptable limits. If group mean body weight recovered to acceptable levels, then dosing was modified to lower levels and/or reduced frequency then resumed. Deaths were classified as TR if it was attributable to treatment side effects as evidenced by clinical signs and/or necropsy. A TR classification was also assigned to deaths by unknown causes during the dosing period or within 14 20 days of the last dose. A death was classified as non-treatment-related (NTR) if there was no evidence that death was related to treatment side effects. NTR deaths are further categorized as follows: NTRa describes deaths due to accidents or human error; NTRm is assigned to deaths thought to result from tumor dissemination by invasion and/or metastasis based on necropsy results; NTRu describes deaths of unknown causes that 25 lack available evidence of death related to metastasis, tumor progression, accident or human error. It should be noted that treatment side effects cannot be excluded from deaths classified as NTRu. Statistical and Graphical Analyses 30 GraphPad Prism 8.0 for Windows was used for all statistical analysis and graphical presentations. Study groups experiencing toxicity beyond acceptable limits (>20% group mean body weight loss or greater than 10% treatment-related deaths) or having fewer than five evaluable observations, were not included in the statistical analysis. The logrank test was employed to assess the significance of the difference between the overall survival 35 experiences of two groups. The logrank test analyzes the individual TTEs for all animals in a group, except those lost to the study due to NTR death. Statistical analyses of the 008188229 116 differences between Day 19 median tumor volumes (MTVs) of control and treated groups were accomplished using the Mann-Whitney U-test. For statistical analyses, two-tailed tests were conducted at significance level P = 0.05. Prism summarizes test results as not significant (ns) at P > 0.05, significant (symbolized by “*”) at 0.01 < P ≤ 0.05, very 5 significant (“**”) at 0.001 < P ≤ 0.01, and extremely significant (“***”) at P ≤ 0.001. Because tests of statistical significance do not provide an estimate of the magnitude of the difference between groups, all levels of significance were described as either significant or not significant within the text of this report. 10 Assay A
Figure imgf000118_0001
Figure imgf000118_0002
The regimen resulted in a significant overall survival difference versus controls (Group 1 vs.4; P < 0.001, Mann-Whitney). On Day 26, the MTV for Group 4 was 196 mm3 which 15 corresponded to significant TGI of 63% and attained the 60% threshold for potential therapeutic activity Assay B
Figure imgf000118_0003
Figure imgf000118_0004
20 008188229 117 MTV(10) was 117 mm3, or a significant 75% TGI (P < 0.001, Mann-Whitney). Two animals survived the study and the median TTE was 55.3 days. All documents and other references mentioned above are herein incorporated by5 reference.
008188229 118 EMBODIMENTS OF DISCLOSURE 1. A conjugate of formula I: L - (DL)p (I) 5 wherein L is a Ligand unit (i.e., a targeting agent), p is from 1 to 20, DL is a Drug Linker unit of formula I’: I'
Figure imgf000120_0001
RLL is a linker for connection to the Ligand Unit, which is
Figure imgf000120_0004
, 10 wherein Q is:
Figure imgf000120_0002
, where QX is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; X is: 15
Figure imgf000120_0003
, where a = 0 to 5, b = 0 to 16, c = 0 or 1, d = 0 to 5; 008188229 119 GLL is a linker connected to the Ligand Unit; D represents either group D1 or D2:
Figure imgf000121_0001
D1 D2 ; the dotted line indicates the optional presence of a double bond between C2 and C3; 5 when there is a double bond present between C2 and C3, R2 is selected from the group consisting of: (ia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene; 10 (ib) C1-5 saturated aliphatic alkyl; (ic) C3-6 saturated cycloalkyl;
Figure imgf000121_0002
, wherein each of R11, R12 and R13 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R2 group is no more than 5;
Figure imgf000121_0003
15 (ie) , wherein one of R15a and R15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (if)
Figure imgf000121_0004
, where R14 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from20 halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2 and C3, R2 is selected from H, OH, F, diF
Figure imgf000121_0005
, where R16a and R16b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl 008188229 120 groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R16a and R16b is H, the other is selected from nitrile and a C1-4 alkyl ester; D’ represents either group D’1 or D’2:
Figure imgf000122_0001
D'1 D'2 5 wherein the dotted line indicates the optional presence of a double bond between C2’ and C3’; when there is a double bond present between C2’ and C3’, R22 is selected from the group consisting of: 10 (iia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene; (iib) C1-5 saturated aliphatic alkyl; (iic) C3-6 saturated cycloalkyl; 15 (iid)
Figure imgf000122_0002
, wherein each of R31, R32 and R33 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R22 group is no more than 5; R25b (iie)
Figure imgf000122_0003
, wherein one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl,20 methoxy; pyridyl; and thiophenyl; and (iif)
Figure imgf000122_0004
, where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2’ and C3’, 008188229 121 26a * R 26b R22 is selected from H, OH, F, diF and R , where R26a and R26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester; 5 R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR’, nitro, Me3Sn and halo; where R and R’ are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups; R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR’, nitro, Me3Sn and halo; 10 R″ is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine; Y and Y’ are selected from O, S, and NH; R6’, R7’, and R9’ are selected from the same groups as R6, R7 and R9 respectively; 15 and either: (a) R20 is H and R21 is H; (b) R20 is H and R21 is =O; (c) R20 and R21 together form a nitrogen-carbon double bond between the nitrogen20 and carbon atoms to which they are bound; or (d) R21 is OH or ORA, where RA is C1-4 alkyl and R20 is selected from:
Figure imgf000123_0001
(i) ;
Figure imgf000123_0002
(ii) ; 008188229 122 NO2 Z R O O (iii) , where RZ is selected from:
Figure imgf000124_0001
(z-i) ; (z-ii) OC(=O)CH3; (z-iii) NO2; 5 (z-iv) OMe;
Figure imgf000124_0003
NH- and -C(=O)-X2-NH- represent natural amino acid residues and RZC is selected from Me, OMe, CH2CH2OMe, and (CH2CH2O)2Me. 10 2. A conjugate according to statement 1, wherein both Y and Y’ are O. 3. A conjugate according to either statement 1 or statement 2, wherein R’’ is C3-7 alkylene. 15 4. A conjugate according to either statement 1 or statement 2, wherein R” is either a group of formula: (a)
Figure imgf000124_0002
008188229 123 where r is 1 or 2; or (b)
Figure imgf000125_0001
5 5. A conjugate according to any one of statements 1 to 4, wherein R9 is H. 6. A conjugate according to any one of statements 1 to 5, wherein R6 is H. 10 7. A conjugate according to any one of statements 1 to 6, wherein R7 is selected from H, OH and OR. 8. A conjugate according to statement 7, wherein R7 is a C1-4 alkyloxy group. 15 9. A conjugate according to statement 8, wherein R7 is a methoxy group. 10. A conjugate according to any one of statements 1 to 9, wherein D is D1, there is a double bond between C2 and C3, and R2 is a C5-7 aryl group. 20 11. A conjugate according to statement 10, wherein R2 is phenyl. 12. A conjugate according to any one of statements 1 to 9, wherein D is D1, there is a double bond between C2 and C3, and R2 is a C8-10 aryl group. 25 13. A conjugate according to any one of statements 10 to 12, wherein R2 bears one to three substituent groups. 14. A conjugate according to any one of statements 10 to 13, wherein the substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-30 piperazinyl, morpholino and methyl-thiophenyl. 15. A conjugate according to any one of statements 1 to 9, wherein D is D1, there is a double bond between C2 and C3, and R2 is a C1-5 saturated aliphatic alkyl group. 008188229 124 16. A conjugate according to statement 15, wherein R2 is methyl, ethyl or propyl. 17. A conjugate according to any one of statements 1 to 9, wherein D is D1, there is a double bond between C2 and C3, and
Figure imgf000126_0001
saturated cycloalkyl group. 5 18. A conjugate according to statement 17, wherein R2 is cyclopropyl. 19. A conjugate according to any one of statements 1 to 9, wherein D is D1, there is a double bond between C2 and C3, and R2 is a group of formula: 10
Figure imgf000126_0002
. 20. A conjugate according to statement 19, wherein the total number of carbon atoms in the R2 group is no more than 4. 15 21. A conjugate according to statement 20, wherein the total number of carbon atoms in the R2 group is no more than 3. 22. A conjugate according to any one of statements 19 to 21, wherein one of R11, R12 and R13 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-320 alkenyl, C2-3 alkynyl and cyclopropyl. 23. A conjugate according to any one of statements 19 to 21, wherein two of R11, R12 and R13 are H, with the other group being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl. 25 24. A conjugate according to any one of statements 1 to 9, wherein D is D1, there is a double bond between C2 and C3, and R2 is a group of formula:
Figure imgf000126_0003
. 30 25. A conjugate according to statement 24, wherein R2 is the group: 008188229 125 * . 26. A conjugate according to any one of statements 1 to 9, wherein D is D1, there is a double bond between C2 and C3, and R2 is a group of formula: 5
Figure imgf000127_0001
. 27. A conjugate according to statement 26, wherein R14 is selected from H, methyl, ethyl, ethenyl and ethynyl. 10 28. A conjugate according to statement 27, wherein R14 is selected from H and methyl. 29. A conjugate according to any one of statements 1 to 9, wherein D is D1, there is a single bond between C2 and C3, and R2 is H. 15 30. A conjugate according to any one of statements 1 to 9, wherein D is D1, there is a single bond between C2 and C3, R2 is
Figure imgf000127_0002
and R16a and R16b are both H. 31. A conjugate according to any one of statements 1 to 9, wherein D is D1, there is a single bond between C2 and C3, R2 is
Figure imgf000127_0003
, and R16a and R16b are both methyl. 20 32. A conjugate according to any one of statements 1 to 9, wherein D is D1, there is a single bond between C2 and C3, R2 is
Figure imgf000127_0004
, one of R16a and R16b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted. 25 33. A conjugate according to any one of statements 1 to 32, wherein D’ is D’1, there is a double bond between C2’ and C3’, and R22 is a C5-7 aryl group. 008188229 126 34. A conjugate according to statement 33, wherein R22 is phenyl. 35. A conjugate according to any one of statements 1 to 32, wherein D’ is D’1,there is a 5 double bond between C2’ and C3’, and R22 is a C8-10 aryl group. 36. A conjugate according to any one of statements 33 to 35, wherein R22 bears one to three substituent groups. 10 37. A conjugate according to any one of statements 33 to 36, wherein the substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl- piperazinyl, morpholino and methyl-thiophenyl. 38. A conjugate according to any one of statements 1 to 32, wherein D’ is D’1, there is15 a double bond between C2’ and C3’, and R22 is a C1-5 saturated aliphatic alkyl group. 39. A conjugate according to statement 38, wherein R22 is methyl, ethyl or propyl. 40. A conjugate according to any one of statements 1 to 32, wherein D’ is D’1, there is20 a double bond between C2’ and C3’, and R22 is a C3-6 saturated cycloalkyl group. 41. A conjugate according to statement 40, wherein R22 is cyclopropyl. 42. A conjugate according to any one of statements 1 to 32, wherein D’ is D’1, there is25 a double bond between C2’ and C3’, and R22 is a group of formula:
Figure imgf000128_0001
. 43. A conjugate according to statement 42, wherein the total number of carbon atoms in the R22 group is no more than 4. 30 44. A conjugate according to statement 43, wherein the total number of carbon atoms in the R22 group is no more than 3. 008188229 127 45. A conjugate according to any one of statements 42 to 44, wherein one of R31, R32 and R33 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl. 5 46. A conjugate according to any one of statements 42 to 44, wherein two of R31, R32 and R33 are H, with the other group being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl. 47. A conjugate according to any one of statements 1 to 32, wherein D’ is D’1, there is10 a double bond between C2’ and C3’, and R22 is a group of formula:
Figure imgf000129_0001
. 48. A conjugate according to statement 47, wherein R22 is the group:
Figure imgf000129_0002
. 15 49. A conjugate according to any one of statements 1 to 32, wherein D’ is D’1, there is a double bond between C2’ and C3’, and R22 is a group of formula:
Figure imgf000129_0003
. 20 50. A conjugate according to statement 49, wherein R24 is selected from H, methyl, ethyl, ethenyl and ethynyl. 51. A conjugate according to statement 50, wherein R24 is selected from H and methyl. 25 52. A conjugate according to any one of statements 1 to 32, wherein D’ is D’1, there is a single bond between C2’ and C3’, and R22 is H. 53. A conjugate according to any one of statements 1 to 32, wherein D’ is D’1, there is a single bond between C2’ and C3’,
Figure imgf000129_0004
and R26a and R26b are both H. 30 008188229 128 54. A conjugate according to any one of statements 1 to 32, wherein D’ is D’1, there is 26a * R 26b a single bond between C2’ and C3’, R22 is R , and R26a and R26b are both methyl. 55. A conjugate according to any one of statements 1 to 32, wherein D’ is D’1, there is 26a * R 26b 5 a single bond between C2’ and C3’, R22 is R , one of R26a and R26b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted. 56. A conjugate according to any one of statements 1 to 55, wherein R20 is H and R21 is10 H. 57. A conjugate according to any one of statements 1 to 55, wherein R20 is H and R21 is =O. 15 58. A conjugate according to any one of statements 1 to 55, wherein R20 and R21 together form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound. 59. A conjugate according to any one of statements 1 to 55, wherein R21 is OH or ORA20 and R20 is selected from:
Figure imgf000130_0001
008188229 129
Figure imgf000131_0001
008188229 130 60. The conjugate according to any one of statements 1 to 59, wherein -C(=O)-X1-NHC(=O)X2-NH-, is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, and -Val-Cit-. 5 61. The conjugate according to statement 60, wherein -C(=O)-X1-NHC(=O)X2-NH-, is selected from: -Phe-Lys-, and -Val-Ala-. 62. The conjugate according to any one of statements 59 to 61 wherein RZC is selected from CH2CH2OMe, and (CH2CH2O)2Me. 10 63. The conjugate according to statement 62 wherein RZC is (CH2CH2O)2Me. 64. A conjugate according to any one of statements 1 to 63, wherein R6’ is the same group as R6, R7’ is the same group as R7, R9’ is the same group as R9 and Y’ is the same15 group as Y. 65. A conjugate according to statement 1, wherein DL is of formula I’-a, I’-b, I’-c or I’-d:
Figure imgf000132_0001
008188229 131 I'-c I'-d where the dotted line represents the possible presence of a double bond between C2 and C3 and C2’ and C3’; where there is no double bond between C2 and C3 and C2’ and C3’, R2a and R22a are the same and are selected from: 5 (a) H; and
Figure imgf000133_0001
; where there is a double bond between C2 and C3 and C2’ and C3’ R2a and R22a are the same and are selected from: 10
Figure imgf000133_0002
008188229 132 * (f) ; O O (g) ; and N N (h) ; R1a is selected from methyl and benzyl; 5 R20, R21 and RLL are as defined above. RL and R11b are as defined in statement 1. 66. A conjugate according to any one of statements 1 to 65, wherein Q is an amino acid residue selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp. 10 67. A conjugate according to any one of statements 1 to 65, wherein Q is a dipeptide residue selected from: CO-Phe-Lys-NH, CO-Val-Ala-NH, 15 CO-Val-Lys-NH, CO-Ala-Lys-NH, CO-Val-Cit-NH, CO-Phe-Cit-NH, CO-Leu-Cit-NH, 20 CO-Ile-Cit-NH, CO-Phe-Arg-NH, and CO-Trp-Cit-NH. 68. A conjugate according to statement 67, wherein Q is selected from CO-Phe-Lys-NH,25 CO-Val-Cit-NH and CO-Val-Ala-NH. 69. A conjugate according to any one of statements 1 to 65, wherein Q is a tripeptide residue selected from: NH-Glu-Val-Ala-C=O 30 NH-Glu-Val-Cit-C=O NH-αGlu-Val-Ala-C=O NH-αGlu-Val-Cit-C=O. 008188229 133 70. A conjugate according to any one of statements 1 to 65, wherein Q is a tetrapeptide linkers selected from: NH -Gly-Gly-Phe-Gly C=O; and 5 NH -Gly-Phe-Gly-Gly C=O. 71. A conjugate according to any one of statements 1 to 70, wherein a is 0 to 3. 72. A conjugate according to statement 71, wherein a is 0. 10 73. A conjugate according to any one of statements 1 to 72, wherein b is 0 to 12. 74. A conjugate according to statement 73, wherein b is 0 to 8. 15 75. A conjugate according to any one of statements 1 to 74, wherein c is 0. 76. A conjugate according to any one of statements 1 to 74, wherein c is 1. 77. A conjugate according to any one of statements 1 to 76, wherein d is 0 to 3. 20 78. A conjugate according to statement 77, wherein d is 2. 79. A conjugate according to any one of statements 1 to 70, wherein a is 0, c is 1 and d is 2, and b is from 0 to 8. 25 80. A conjugate according to statement 79, wherein b is 0, 4 or 8. 81. A conjugate according to any one of statements 1 to 80, wherein GLL is selected from:
Figure imgf000135_0001
008188229 134
Figure imgf000136_0001
008188229 135
Figure imgf000137_0001
where CBA represents the point of connection to the Ligand Unit, Ar represents a C5-6 arylene group, e.g. phenylene and X1 represents C1-4 alkyl. 82. A conjugate according to statement 81, wherein Ar is a phenylene group. 5 83. A conjugate according to either statement 81 or statement 82, wherein GLL is selected from GLL1-1 and GLL1-2. 84. A conjugate according to statement 83, wherein GLL is GLL1-1. 10 85. A conjugate according to either statement 81 or statement 82, wherein GLL is GLL1- 1A. 86. A conjugate according to any one of statements 1 to 85, wherein the Ligand Unit is 15 an antibody or an active fragment thereof. 87. The conjugate according to statement 86, wherein the antibody or antibody fragment is an antibody or antibody fragment for a tumour-associated antigen. 20 88. The conjugate according to statement 87 wherein the antibody or antibody fragment is an antibody which binds to one or more tumor-associated antigens or cell- surface receptors selected from (1)-(89): (1) BMPR1B; (2) E16; 25 (3) STEAP1; (4) 0772P; (5) MPF; (6) Napi3b; (7) Sema 5b; 30 (8) PSCA hlg; (9) ETBR; (10) MSG783; 008188229 136 (11) STEAP2; (12) TrpM4; (13) CRIPTO; (14) CD21; 5 (15) CD79b; (16) FcRH2; (17) HER2; (18) NCA; (19) MDP; 10 (20) IL20R-alpha; (21) Brevican; (22) EphB2R; (23) ASLG659; (24) PSCA; 15 (25) GEDA; (26) BAFF-R; (27) CD22; (28) CD79a; (29) CXCR5; 20 (30) HLA-DOB; (31) P2X5; (32) CD72; (33) LY64; (34) FcRH1; 25 (35) IRTA2; (36) TENB2; (37) PSMA – FOLH1; (38) SST; (38.1) SSTR2; 30 (38.2) SSTR5; (38.3) SSTR1; (38.4)SSTR3; (38.5) SSTR4; (39) ITGAV; 35 (40) ITGB6; (41) CEACAM5; 008188229 137 (42) MET; (43) MUC1; (44) CA9; (45) EGFRvIII; 5 (46) CD33; (47) CD19; (48) IL2RA; (49) AXL; (50) CD30 - TNFRSF8; 10 (51) BCMA - TNFRSF17; (52) CT Ags – CTA; (53) CD174 (Lewis Y) - FUT3; (54) CLEC14A; (55) GRP78 – HSPA5; 15 (56) CD70; (57) Stem Cell specific antigens; (58) ASG-5; (59) ENPP3; (60) PRR4; 20 (61) GCC – GUCY2C; (62) Liv-1 – SLC39A6; (63) 5T4; (64) CD56 – NCMA1; (65) CanAg; 25 (66) FOLR1; (67) GPNMB; (68) TIM-1 – HAVCR1; (69) RG-1/Prostate tumor target Mindin – Mindin/RG-1; (70) B7-H4 – VTCN1; 30 (71) PTK7; (72) CD37; (73) CD138 – SDC1; (74) CD74; (75) Claudins – CLs; 35 (76) EGFR; (77) Her3; 008188229 138 (78) RON - MST1R; (79) EPHA2; (80) CD20 – MS4A1; (81) Tenascin C – TNC; 5 (82) FAP; (83) DKK-1; (84) CD52; (85) CS1 - SLAMF7; (86) Endoglin – ENG; 10 (87) Annexin A1 – ANXA1; (88) V-CAM (CD106) - VCAM1; (89) ASCT2 (SLC1A5). 89. The conjugate of any one of statements 86 to 88 wherein the antibody or antibody15 fragment is a cysteine-engineered antibody. 90. The conjugate according to any one of statements 1 to 89 wherein p is an integer from 1 to 8. 20 91. The conjugate according to statement 90, wherein p is 1, 2, 3, or 4. 92. A compound with the formula II: II
Figure imgf000140_0001
and salts and solvates thereof, 25 wherein D, R2, R6, R7, R9, Y, R”, Y’, D’, R22, R6’, R7’, R9’, R20 and R21 (including the presence or absence of double bonds between C2 and C3 and C2’ and C3’ respectively) are as defined in any one of statements 1 to 65; RL is a linker for connecting to a Ligand unit, which is: 008188229 139 O H L N G Q X IIIa , where Q and X are as defined in any one of statements 1 and 66 to 80 and GL is a linker for connecting to a Ligand unit. 5 93. A compound according to statement 92, wherein GL is selected from:
Figure imgf000141_0001
008188229 140
Figure imgf000142_0001
where Ar represents a C5-6 arylene group, e.g. phenylene, and X1 represents C1-4 alkyl. 94. A compound according to statement 93, wherein Ar is a phenylene group. 5 95. A compound according to either statement 93 or statement 94, wherein GL is selected from GL1-1 and GL1-2. 96. A compound according to statement 95, wherein GL is GL1-1. 10 97. A composition comprising a mixture of conjugates according to any one of statements 1 to 91, wherein the average p in the mixture of conjugate compounds is about 1 to about 8. 98. The conjugate according to any one of statements 1 to 91, for use in therapy. 15 99. A pharmaceutical composition comprising the conjugate of any one of statements 1 to 91, and a pharmaceutically acceptable diluent, carrier or excipient. 008188229 141 100. The conjugate according to any one of statements 1 to 91 or the pharmaceutical composition according to statement 99, for use in the treatment of a proliferative disease in a subject. 5 101. The conjugate for use according to statement 100, wherein the disease treated is cancer. 102. Use of a conjugate according to any one of statements 1 to 91 or a pharmaceutical10 composition according to statement 99 in a method of medical treatment. 103. A method of medical treatment comprising administering to a patient the pharmaceutical composition of statement 99. 15 104. The method of statement 103, wherein the method of medical treatment is for treating cancer. 105. The method of statement 104, wherein the patient is administered a chemotherapeutic agent, in combination with the conjugate. 20 106. Use of a conjugate according to any one of statements 1 to 91 in a method of manufacture of a medicament for the treatment of a proliferative disease. 107. A method of treating a mammal having a proliferative disease, comprising 25 administering an effective amount of a conjugate according to any one of statements 1 to 91 or a pharmaceutical composition according to statement 99. 108. A method of synthesis of a conjugate according to any one of statements 1 to 91 comprising conjugating a compound according to any one of statements 92 to 96 with a30 Ligand Unit. 109. A method of making a compound of formula IV: 008188229 142 Lpre R O O 9 R 8 R N H IV 7 R N 6 R O D from a compound of formula V:
Figure imgf000144_0001
using the Mitsunobu reaction; 5 where R8 is selected from: (a) OMe, OCH2Ph, OH, OProtO and –Y’-R”-Hal; (b)
Figure imgf000144_0002
(c) 008188229 143 20 R 9' 21 R R N Y' Y H R'' Vc N 7' R 6' D' O R where D, R2, R6, R7, R9, Y, R”, Y’, D’, R22, R6’, R7’, R9’, R20 and R21 are as defined in any one of statements 1 to 65; Hal is a halogen; 5 RLpre is a precursor to RL; and ProtO is a hydroxyl protecting group. 110. The method according to statement 109, where Hal is Br. 10 111. The method according to either statement 109 or statement 110, where RLpre is:
Figure imgf000145_0001
, where Q is as defined in any one of statements 1 and 66 to 70 and ProtN is an amine protecting group. 15 112. The method according to statement 111, where ProtN is a carbamate. 113. The method according to statement 112, where ProtN is Alloc. 114. The method according to any one of statements 109 to 113, where ProtO is a silyl20 ether. 115. The method according to statement 114, where ProtO is TIPS. 116. The method according to any one of statement 109 to 115, wherein the reaction is25 carried out using triphenylphosphine and an azodicarboxylate. 008188229 144 117. The method according to statement 116, wherein the azodicarboxylate is diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD). 118. A compound of formula VI: VI 5
Figure imgf000146_0001
D, R2, R6, R7, R9, Y, R”, Y’, D’, R6’, R7’, R9’ and R22 (including the presence or absence of double bonds between C2 and C3 and C2’ and C3’ respectively) are as defined in any one of statements 1 to 65. 10 119. A compound according to statement 118, which is selected from:
Figure imgf000146_0002

Claims

008188229 145 CLAIMS 1. A conjugate of formula I: L - (DL)p (I) 5 wherein L is a Ligand unit (i.e., a targeting agent), p is from 1 to 20, DL is a Drug Linker unit of formula I’: I'
Figure imgf000147_0001
RLL is a linker for connection to the Ligand Unit, which is
Figure imgf000147_0004
, 10 wherein Q is:
Figure imgf000147_0002
, where QX is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; X is: 15
Figure imgf000147_0003
, where a = 0 to 5, b = 0 to 16, c = 0 or 1, d = 0 to 5; 008188229 146 GLL is a linker connected to the Ligand Unit; D represents D1:
Figure imgf000148_0001
D1 ; the dotted line indicates the optional presence of a double bond between C2 and C3; 5 when there is a double bond present between C2 and C3, R2 is selected from the group consisting of: (ia) C5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene; 10 (ib) C1-5 saturated aliphatic alkyl; (ic) C3-6 saturated cycloalkyl;
Figure imgf000148_0002
, wherein each of R11, R12 and R13 are independently selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R2 group is no more than 5;
Figure imgf000148_0003
15 (ie) , wherein one of R15a and R15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (if)
Figure imgf000148_0004
, where R14 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from20 halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2 and C3, R2 is selected from H, OH, F, diF
Figure imgf000148_0005
, where R16a and R16b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl 008188229 147 ester; or, when one of R16a and R16b is H, the other is selected from nitrile and a C1-4 alkyl ester; D’ represents D’1:
Figure imgf000149_0001
D'1 5 wherein the dotted line indicates the optional presence of a double bond between C2’ and C3’; when there is a double bond present between C2’ and C3’, R22 is selected from the group consisting of: (iia) C5-10 aryl group, optionally substituted by one or more substituents selected from the 10 group comprising: halo, nitro, cyano, ether, carboxy, ester, C1-7 alkyl, C3-7 heterocyclyl and bis-oxy-C1-3 alkylene; (iib) C1-5 saturated aliphatic alkyl; (iic) C3-6 saturated cycloalkyl; (iid)
Figure imgf000149_0002
, wherein each of R31, R32 and R33 are independently selected from H, 15 C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R22 group is no more than 5; R25b (iie)
Figure imgf000149_0003
, wherein one of R25a and R25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and 20 (iif)
Figure imgf000149_0004
, where R24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2’ and C3’, *
Figure imgf000149_0005
R22 is selected from H, OH, F, diF and R , where R26a and R26b are independently 25 selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are 008188229 148 optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R26a and R26b is H, the other is selected from nitrile and a C1-4 alkyl ester; R6 and R9 are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR’, nitro, Me3Sn and halo; 5 where R and R’ are independently selected from optionally substituted C1-12 alkyl, C3-20 heterocyclyl and C5-20 aryl groups; R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR’, nitro, Me3Sn and halo; R″ is a C3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NRN2 (where RN2 is H or C1-4 alkyl), and/or aromatic rings, e.g. benzene or10 pyridine; Y and Y’ are selected from O, S, and NH; R6’, R7’, and R9’ are selected from the same groups as R6, R7 and R9 respectively; and either: 15 (a) R20 is H and R21 is H; (b) R20 is H and R21 is =O; (c) R20 and R21 together form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (d) R21 is OH or ORA, where RA is C1-4 alkyl and R20 is selected from:
Figure imgf000150_0001
20 (i) ;
Figure imgf000150_0002
(ii) ; 008188229 149 NO2 Z R O O (iii) , where RZ is selected from:
Figure imgf000151_0001
(z-i) ; (z-ii) OC(=O)CH3; (z-iii) NO2; 5 (z-iv) OMe;
Figure imgf000151_0003
NH- and -C(=O)-X2-NH- represent natural amino acid residues and RZC is selected from Me, OMe, CH2CH2OMe, and (CH2CH2O)2Me. 10 2. A conjugate according to claim 1, wherein: (a) both Y and Y’ are O; and/or (b) R’’ is (b-i) C3-7 alkylene; or 15 (b-ii) a group of formula:
Figure imgf000151_0002
(b-iii) a group of formula: 008188229 150 N r r , where r is 1 or 2. 3. A conjugate according to either claim 1 or claim 2, R9 and R6 are both H. 5 4. A conjugate according to any one of claims 1 to 3, wherein R7 is a C1-4 alkyloxy group. 5. A conjugate according to any one of claims 1 to 4, there is a double bond between C2 and C3, and R2 is selected from: 10 (a) optionally substituted phenyl, wherein the optional substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl; (b) optionally substituted C8-10 aryl group, wherein the optional substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl,15 morpholino and methyl-thiophenyl; (c) methyl, ethyl or propyl; (d) cyclopropyl; (e) a group of formula
Figure imgf000152_0001
, wherein the total number of carbon atoms in the R2 group is no more than20 4;
Figure imgf000152_0002
(g) a group of formula:
Figure imgf000152_0003
, wherein R14 is selected from H and methyl. 25 6. A conjugate according to any one of claims 1 to 4, there is a single bond between
Figure imgf000152_0004
and: 008188229 151 (a) R16a and R16b are both H; or (b) R16a and R16b are both methyl; or (c) one of R16a and R16b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted. 5 7. A conjugate according to any one of claims 1 to 6, there is a double bond between C2’ and C3’, and R22 is selected from: (a) optionally substituted phenyl, wherein the optional substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino10 and methyl-thiophenyl; (b) optionally substituted C8-10 aryl group, wherein the optional substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl; (c) methyl, ethyl or propyl; 15 (d) cyclopropyl; (e) a group of formula
Figure imgf000153_0001
, wherein the total number of carbon atoms in the R22 group is no more than 4;
Figure imgf000153_0002
20 (g) a group of formula:
Figure imgf000153_0003
, wherein R24 is selected from H and methyl. 8. A conjugate according to any one of claims 1 to 6, there is a single bond between 26a * R 26b C2’ and C3’, R22 is R and: 25 (a) R26a and R26b are both H; or (b) R26a and R26b are both methyl; or (c) one of R26a and R26b is H, and the other is selected from C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
008188229 152 9. A conjugate according to any one of claims 1 to 8, wherein: (a) R20 is H and R21 is H; (b) R20 is H and R21 is =O; or (c) R20 and R21 together form a nitrogen-carbon double bond between the nitrogen and 5 carbon atoms to which they are bound. 10. A conjugate according to any one of claims 1 to 9, wherein R6’ is the same group as R6, R7’ is the same group as R7, R9’ is the same group as R9 and Y’ is the same group as Y. 10 11. A conjugate according to claim 1, wherein DL is of formula I’-a, I’-b, I’-c or I’-d:
Figure imgf000154_0001
008188229 153 I'-d where the dotted line represents the possible presence of a double bond between C2 and C3 and C2’ and C3’; where there is no double bond between C2 and C3 and C2’ and C3’, R2a and R22a are the same and are selected from: 5 (a) H; and
Figure imgf000155_0001
; where there is a double bond between C2 and C3 and C2’ and C3’ R2a and R22a are the same and are selected from: 10 15
Figure imgf000155_0002
008188229 154 R1a is selected from methyl and benzyl; R20, R21 and RLL are as in either claim 1 or claim 9; and RL and R11b are as defined in claim 1. 5 12. A conjugate according to any one of claims 1 to 11, wherein Q is: (a) an amino acid residue selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp; (b) a dipeptide residue selected from CO-Phe-Lys-NH, CO-Val-Cit-NH and CO-Val-Ala-NH; (c) a tripeptide residue selected from: NH-Glu-Val-Ala-C=O, NH-Glu-Val-Cit-C=O, NH-αGlu-Val- Ala-C=O and NH-αGlu-Val-Cit-C=O; 10 (d) a tetrapeptide linkers selected from: NH -Gly-Gly-Phe-Gly C=O and NH -Gly-Phe-Gly-Gly C=O. 13. A conjugate according to any one of claims 1 to 12, wherein a is 0, c is 1 and d is 2, and b is from 0 to 8. 15 14. A conjugate according to any one of claims 1 to 13, wherein GLL is selected from:
Figure imgf000156_0001
008188229 155
Figure imgf000157_0001
where CBA represents the point of connection to the Ligand Unit, Ar represents a C5-6 arylene group, e.g. phenylene and X1 represents C1-4 alkyl. 15. A conjugate according to claim 14, wherein GLL is GLL1-1. 5 16. A conjugate according to any one of claims 1 to 15, wherein the Ligand Unit is an antibody or an active fragment thereof. 17. The conjugate according to any one of claims 1 to 17 wherein p is an integer from 110 to 8.
008188229 156 18. A compound with the formula II: II
Figure imgf000158_0001
and salts and solvates thereof, wherein D, R2, R6, R7, R9, Y, R”, Y’, D’, R22, R6’, R7’, R9’, R20 and R21 (including the presence 5 or absence of double bonds between C2 and C3 and C2’ and C3’ respectively) are as defined in any one of claims 1 to 11; RL is a linker for connecting to a Ligand unit, which is:
Figure imgf000158_0002
, where Q and X are as defined in any one of claims 1, 12 and 13 and GL is a linker for10 connecting to a Ligand unit. 19. A compound according to claim 18, wherein GL is selected from:
Figure imgf000158_0003
008188229 157
Figure imgf000159_0001
where Ar represents a C5-6 arylene group, e.g. phenylene and X1 represents C1-4 alkyl. 20. A compound according to claim 19, wherein GLL is GLL1-1.
008188229 158 21. A composition comprising a mixture of conjugates according to any one of claims 1 to 17, wherein the average p in the mixture of conjugate compounds is about 1 to about 8. 22. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 5 17, and a pharmaceutically acceptable diluent, carrier or excipient. 23. A method of making a compound of formula IV:
Figure imgf000160_0001
from a compound of formula V: 10
Figure imgf000160_0002
using the Mitsunobu reaction; where R8 is selected from: (a) OMe, OCH2Ph, OH, OProtO and –Y’-R”-Hal; (b) 15
Figure imgf000160_0003
; and 008188229 159 (c)
Figure imgf000161_0001
are as defined in any one of statements 1 to 11; 5 Hal is a halogen; RLpre is a precursor to RL; and ProtO is a hydroxyl protecting group. 24. The method according to claim 23, where RLpre is:
Figure imgf000161_0002
10 , where Q is as defined in either claim 1 or claim 12 and ProtN is an amine protecting group. 25. A compound of formula VI: 15
Figure imgf000161_0003
D, R2, R6, R7, R9, Y, R”, Y’, D’, R6’, R7’, R9’ and R22 (including the presence or absence of double bonds between C2 and C3 and C2’ and C3’ respectively) are as defined in any one of claims 1 to 11. 20 26. A compound according to claim 25, which is selected from:
Figure imgf000162_0001
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