EA046016B1 - COMPOUNDS AND CONJUGATES BASED ON THEM - Google Patents

Description

The present invention relates to targeting conjugates containing a specific topoisomerase inhibitor, and compounds useful in their synthesis, as well as a released target element.

BACKGROUND OF THE INVENTION

Topoisomerase inhibitors

Topoisomerase inhibitors are chemical compounds that block the action of topoisomerase (topoisomerase I and II), which is a type of enzyme that controls changes in DNA structure by catalyzing the breaking and repair of the phosphodiester backbone of DNA strands during the normal cell cycle.

The following connection:

I ^h2n ^X^L-\ o

T JL\o he o

^AR in racemic form was disclosed in EP 0296597 (Example 63). It is also disclosed (as compound 34 in racemic form) in Sugimori, M., et al., J Med Chem, 1998, 41, 2308-2318 (DOI: 10.1021/jm970765q), where its biological activity is discussed along with the biological activity of the series related compounds.

Various topoisomerase inhibitors, such as irinotecan and exatecan derivatives and doxorubicin, have been included in antibody-drug conjugates. For example, DS-8201a from Daiichi Sankyo is in clinical trials.

where the antibody is Her2 (Takegawa, N., et al., Int J Cancer, 2017, 141, 1682-1689 (DOI: 10.1002/ijc.30870). This ADC releases an exatecan derivative:

it

Burke, P.J., et al., Bioconjugate Chem., 2009, 20, 1242-1250, discloses conjugates:

which are connected via an amino group to the following structures:

which contain the PABC (para-aminobenzyloxycarbonyl) group.

Sacituzumab govitecan (IMMU-132) from Immunomedics is in clinical trials (Cardillo, T.M., et al., Bioconjugate Chem, 2015, 26(5), 919-931, DOI:

10.1021/acs.bioconjchem.5b00223)

- 1 046016

Brief description of the invention

In a general aspect, the present invention provides a conjugate containing the following topoisomerase inhibitor derivative (A*, drug unit):

with a linker for attaching a ligand unit, where the linker is attached via a cleavable bond to an amino acid residue. The ligand unit is preferably an antibody. The present invention also provides A* with an attached linker and intermediates for their synthesis, as well as a releasable lethal element.

In a first aspect of the present invention there is provided a compound of formula I

and salts and solvates thereof, wherein RL is a linker for attachment to a ligand unit selected from (ia):

Where

Q represents

wherein Q ^X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; X represents

where a equals 0-5, b1 equals 0-16, b2 equals 0-16, c1 equals 0 or 1, c2 equals 0 or 1, d equals 0-5, where at least b1 or b2 equals 0 (i.e. only one of b1 and b2 may not be equal to 0), and at least c1 or c2 is equal to 0 (ie, only one of c1 and c2 may not be equal to 0);

G ^l is a linker for attachment to a ligand unit;

(ib):

- 2 046016 where R ^l1 and R ^L2 are independently selected from H and methyl or, together with the carbon atom to which they are bonded, form a cyclopropylene or cyclobutylene group; and e equals 0 or 1.

In a second aspect of the present invention, there is provided a method for preparing a compound according to the first aspect of the present invention, comprising at least one of the method steps set forth below.

In a third aspect, the present invention provides conjugates of formula IV:

L-(D ^L ) _P (IV) or a pharmaceutically acceptable salt or solvate thereof, where L represents a ligand unit (i.e., a targeting agent), DL represents a drug-linker unit represented by formula III:

R ^LL is a linker attached to a ligand unit selected from (ia'):

where Q and X are as defined in the first aspect, and G ^LL is a linker attached to a ligand unit; and (ib'):

where R ^l1 and RL ² are as defined in the first aspect; and p is an integer from 1 to 20.

Accordingly, the conjugates contain a ligand unit covalently linked to at least one drug unit (A*) via a linker unit (i.e., a ligand unit with one or more drug-linker units). The ligand unit, more fully described below, is a targeting agent that binds to the target moiety. The ligand unit may, for example, specifically bind to a cellular component (cell binding agent) or other target molecules of interest. Accordingly, the present invention also provides methods for treating, for example, various types of cancer and autoimmune disease. These methods include the use of conjugates, wherein the ligand moiety is a targeting agent that specifically binds to the target molecule. The ligand unit may be, for example, a protein, a polypeptide, or a peptide such as an antibody, an antigen binding fragment of an antibody, or other binding agent such as an Fc fusion protein.

Drug load is represented by p, the number of drug units per ligand (eg antibody) unit. The drug loading may range from 1 to 20 drug units (D) per ligand unit (eg, Ab or mAb). For compositions, p is the average drug loading value for the conjugates in the composition, and p ranges from 1 to 20.

In a fourth aspect of the present invention, there is provided the use of a conjugate according to the third aspect of the present invention in the manufacture of a medicament for the treatment of a proliferative disease. The fourth aspect also provides a conjugate of the third aspect of the present invention for use in the treatment of a proliferative disease.

One skilled in the art will readily be able to determine whether a candidate compound is or is not capable of treating a proliferative condition in any particular cell type. For example, assays that can be successfully used to evaluate the activity provided by a particular compound are described in the examples below.

B Nakada, et al., Bioorg Med Chem Lett, 26(2016), 1542-1545 (DOI: 10.1016/j.bmcl.2016.02.020) reviews the ADC series:

- 3 046016

ADC (1): X = none ¥= CH,

ADC |2I X = N<-C4-;C=O, '-' = CH ADCi3i.X = M- -(CH ,; _; -<C=O| ¥=CH ADC |4| Χ = :-, -.C=O|t'=CHADC I5l X = nC<h'-* _i -(C=Oi Y = C4 adci6i κ = Ν-.-ΰΗ ·,->:=ϋι y = ch ADC t7i X - "CH :.-.1=01 Y = iC =Oi-! JH-iC H CH O>. i DAR ......... .......

and concluded that the reduction in cytotoxicity of ADCs (1) and (2) may be due to steric hindrance of the drug release moiety at the site exposed to degradative enzymes in tumor cells. This document explains the importance of positioning the peptide group at a distance from the large releasable drug moiety. In contrast, in the present invention, the peptide group is attached directly to the large releasable drug moiety.

The fifth aspect of the present invention is Compound A:

as a single enantiomer or in an enantiomerically enriched form.

The sixth aspect of the present invention is a compound of formula VI:

where Q is as defined in the first aspect.

Definitions

C _5-6 arylene: As used herein, the term C _5-6 arylene refers to a divalent moiety obtained by removing two hydrogen atoms from an aromatic ring atom of an aromatic compound.

In this context, prefixes (eg C _5-6 ) indicate the number of ring atoms or a range of numbers of ring atoms, whether carbon atoms or heteroatoms.

All ring atoms may be carbon atoms, as in carboarylene groups, in which case the group is phenylene (C6).

Alternatively, the ring atoms may include one or more heteroatoms, as in heteroarylene groups. Examples of heteroarylene groups include, but are not limited to, those derived from:

N ₁ : pyrrole (azole) (C5), pyridine (azine) (C6);

Οι: furan (oxol) (C5);

S1: thiophene (thiol) (C5);

N1O1: oxazole (C5), isoxazole (C5), isoxazine (C6);

N2O ₁ : oxadiazole (furazan) (C5);

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) ( eg cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6); And

N ₃ : triazole (C ₅ ), triazine (C ₆ ).

C _1-4 alkyl: As used herein, the term C _1-4 alkyl refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound containing 1 to 4 carbon atoms, which may be aliphatic or alicyclic.

- 4 046016 sky, and which may be saturated or unsaturated (eg, partially unsaturated, completely unsaturated). As used herein, the term C1 _-n alkyl refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound containing from 1 to n carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, completely unsaturated). Thus, the term alkyl includes the subclasses alkenyl, alkynyl, cycloalkyl, etc., discussed below.

Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl ( _C2 ), propyl (C3), and butyl (C4).

Examples of saturated linear alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl ( _C3 ) and n-butyl ( _C4 ).

Examples of saturated branched alkyl groups include isopropyl ( _C3 ), isobutyl ( _C4 ), sec-butyl (C4) and t-butyl (C4).

C _2-4 alkenyl: As used herein, the term C _2-4 alkenyl refers to an alkyl group containing one or more carbon-carbon double bonds.

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) and butenyl (C4).

C _2-4 alkynyl: As used herein, the term C _2-4 alkynyl refers to an alkyl group containing one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (-C+CH) and 2propynyl (propargyl, -CH ₂ -C=CH).

C _3-4 cycloalkyl: As used herein, the term C _3-4 cycloalkyl refers to an alkyl group that is also a cyclyl group, that is, a monovalent moiety obtained by removing a hydrogen atom from the alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, where the fragment contains from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, those derived from the saturated monocyclic hydrocarbon compounds: cyclopropane (C ₃ ) and cyclobutane (C ₄ ) and the unsaturated monocyclic hydrocarbon compounds: cyclopropene (C ₃ ) and cyclobutene (C4).

Labeling connections: in formula

o Labeling with superscripts ^C( = ^o) and ^NH denotes the group to which the atoms are bonded. For example, an NH group is shown associated with a carbonyl (which is not part of the illustrated fragment), and a carbonyl is shown associated with an NH group (which is not part of the illustrated fragment).

Salts

It may be convenient or desirable to prepare, purify and/or treat an appropriate salt of the active compound, for example a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).

For example, if the compound is anionic or contains a functional group that can be anionic (eg, -COOH can be ^-COO- ), then a salt with the appropriate cation can be formed. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na ⁺ and K ⁺ , alkaline earth metal cations such as Ca ²⁺ and Mg2 ⁺ , and other cations such as Al ⁺³ . Examples of suitable organic cations include, but are not limited to, ammonium ion (ie, NH4 ⁺ ) and substituted ammonium ions (eg, NH3R ⁺ , NH2R2 ⁺ , NHR3 ⁺ , NR4 ⁺ ). Examples of some suitable substituted ammonium ions are those derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine and tromethamine, as well as amino acids such as lysine and arginine. An example of a commonly encountered quaternary ammonium ion is N(OH3) ₄ ⁺ .

If the compound is cationic or contains a functional group that can be cationic (eg, -NH2 can be -NH3), then a salt with a suitable anion can be formed. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic, acetic, ascorbic, aspartic, ben

- 5 046016 zoic, camphorsulfonic, cinnamic, citric, edethic, ethanesulfonic, ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycol, hydroxmaleic, hydroxynaphthalene carbonic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mu zinc, oleic, oxalic , palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethylcellulose.

Solvates

It may be convenient or desirable to prepare, purify and/or process the corresponding solvate of the active compound. The term solvate is used herein in its conventional sense and refers to a complex of a solute (eg, an active compound, a salt of an active compound) and a solvent. If the solvent is water, the solvate may for convenience be referred to as a hydrate, for example, monohydrate, dihydrate, trihydrate and the like.

Isomers

Certain compounds of the present invention may exist in one or more specific geometric, optical, enantiomeric, diastereomeric, epimeric, atropomeric, stereoisomeric, tautomeric, conformational or anomeric forms, including without limitation cis and trans forms; E- and Z-shapes; c-, t- and r-forms; endo- and exoforms; R-, S- and meso-forms; D- and L-shapes; d- and l-forms; (+) and (-)-forms; keto, enol and enolate forms; syn- and anti-forms; synclinal and anticlinal forms; α- and β-forms; axial and equatorial shapes; bath, chair, twist, envelope and half-chair forms, and combinations thereof, hereinafter collectively referred to as isomers (or isomeric forms).

The term chiral refers to molecules that have the property of not matching their mirror image when superimposed, while the term achiral refers to molecules that match their mirror image when superimposed.

The term stereoisomers refers to compounds that have identical chemical compositions but differ in the arrangement of atoms or groups in space.

The term diastereomer refers to a stereoisomer with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties and reactivity. Mixtures of diastereomers can be separated using highly sensitive analytical procedures such as electrophoresis and chromatography.

The term enantiomers refers to two stereoisomers of a compound that are non-matching mirror images of each other.

Stereochemical definitions and conventions used herein generally follow the information given in P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including without limitation diastereomers, enantiomers and atropisomers, as well as mixtures thereof, such as racemic mixtures, are intended to form part of the present invention. Many organic compounds exist in optically active forms, i.e. they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule relative to its chiral center(s). The prefixes d and 1 or (+) and (-) are used to indicate the sign of rotation of a plane-polarized light connection, where (-) or 1 means that the connection is left-handed. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A particular stereoisomer may also be called an enantiomer, and a mixture of such isomers is often called a mixture of enantiomers. A 50:50 mixture of enantiomers is called a racemic mixture or racemate and can occur in the absence of stereoselection or stereospecificity during a chemical reaction or process. The terms racemic mixture and racemate refer to an equimolar mixture of two types of enantiomers lacking optical activity.

The term enantiomerically enriched form refers to a sample of a chiral substance where the enantiomer ratio is greater than 50:50 but less than 100:0.

It should be noted that, with the exception of the tautomeric forms discussed below, the term isomers as used herein specifically excludes structural (or constitutional) isomers (i.e., isomers that differ in the bonds between atoms and not just in the position of the atoms in space). For example, a reference to a methoxy group, -OCH3, should not be construed as a reference to its structural isomer, the hydroxymethyl group, -CH2OH. Likewise, a link to ortho

- 6 046016 chlorophenyl should not be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structural isomeric forms found within that class (e.g., C _1-7 αlkyl includes n-propyl and isopropyl; butyl includes n-, iso-, sec-tert-butyl; methoxyphenyl includes ortho-, meta- and para-methoxyphenyl).

The above exception does not apply to tautomeric forms, such as keto, enol and enolate forms, as in the case of the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/enediamine, nitroso/ oxime, thioketone/ethiol, Nnitroso/hydroxyazo and nitro/acy-nitro.

V , _Z ^O \ , ^{OH N} \ ,0' θ ·»— Q — Q .......θ — θ

I\/\ _n ⁺ /\keto enol enolate

The term tautomer or tautomeric form refers to structural isomers having different energies that are interconvertible due to a low energy barrier. For example, protic tautomers (also known as prototropic tautomers) include proton migration interconversion species such as keto-enol and imine-enamine isomerization. Valence tautomers involve types of interconversion through the reorganization of some of the bonding electrons.

It should be noted that the term isomer specifically includes compounds with one or more isotopic substitutions. For example, H can be in any isotopic form, including 1H, ^2H (D), and ^3H (T); C can be in any isotopic form, including ¹² C, ¹³ C and ¹⁴ C; O can be in any isotopic form, including ¹⁶ O and ¹⁸ O, etc.

Examples of isotopes that may be included in the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine and iodine, such as, but not limited to, ^2H (deuterium, D), ^3H (tritium), 11C , ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F, ³¹ P, ³² P, ³⁵ S, ³⁶ Cl and ¹²⁵ I. Various isotopically labeled compounds of the present invention, for example compounds in which radioactive isotopes such as ³ H are included, ¹³ C and ¹⁴ C. Such isotopically labeled compounds may be useful in metabolic studies, reaction kinetics studies, detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including drug distribution assays or substrate in tissue, or in the treatment of patients using radioactive substances. The deuterium-labeled or deuterium-substituted therapeutic compounds of the present invention may have improved DMPK (drug metabolism and pharmacokinetics) distribution, metabolism, and elimination (ADME) properties. Substitution with heavier isotopes such as deuterium may provide certain therapeutic benefits resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. The ¹⁸ F labeled compound may be useful for PET or SPECT studies. The isotopically labeled compounds of the present invention and their prodrugs can generally be prepared by following the procedures disclosed in the Schemes or in the Examples and Preparation Methods described below by substituting a non-isotopically labeled reagent with a readily available isotopically labeled reagent. In addition, substitution with heavier isotopes, particularly deuterium (i.e., ^2H or D), may provide certain therapeutic benefits resulting from greater metabolic stability, such as increased in vivo half-life, or reduced dosage requirements, or increased therapeutic index. It is clear that deuterium is considered a substituent in this context. The concentration of such a heavier isotope, in particular deuterium, can be determined using the isotope enrichment factor. In the compounds of the present invention, any atom not specifically designated as a particular isotope is considered to be any stable isotope of that atom.

Unless otherwise indicated, reference to a specific compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for preparing (eg, asymmetric synthesis) and separating (eg, fractional crystallization and chromatographic methods) such isomeric forms are either known in the art or can be easily developed by adapting the methods set forth herein or known methods in a known manner.

Ligand unit

The ligand unit can be any one and includes a protein, a polypeptide, a peptide and a non-peptide agent that specifically binds to the 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. Such ligand units may include antibodies or an antibody fragment that contain at least one binding site for a target molecule, lymphokines, hormones, growth factors, or any other molecule or substance that binds

- 7 046016 with a cell that are capable of specifically binding to the target.

The terms specifically bind and specific binding refer to the binding of an antibody or other protein, polypeptide or peptide to a given molecule (eg, antigen). Typically, an antibody or other molecule binds with an affinity of at least about 1x107 M ^-1 and binds to a given molecule with an affinity that is at least twice that of its binding to a nonspecific molecule (eg, BSA , casein), different from the given molecule, or a closely related molecule.

Examples of ligand units include those 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 extracellular target on the cell. Such a cell-binding agent may be a protein, a polypeptide, a peptide, or a non-peptide 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 may also be an antibody or an antigen binding fragment of an antibody. Thus, in one embodiment, the present invention provides an antibody-drug conjugate (ADC).

Cell binding agent

The cell binding agent can be anything and includes peptides and agents other than peptides. These may include antibodies or an antibody fragment that contain at least one binding site, 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 containing 4-30, preferably 6-20 contiguous amino acid residues.

In one embodiment, _the cell-binding agent includes a peptide that binds _αγβ6 integrin. The peptide may be selective for α,,β ₆ over XYS.

In one embodiment, the cell binding agent comprises an A20FMDV-Cys polypeptide. A20FMDV-Cys is characterized by the sequence NAVPNLRGDLQVLAQKVARTC. Alternatively, a sequence variant A20FMDV-Cys may be used where one, two, three, four, five, six, seven, eight, nine or ten amino acid residues are replaced by another amino acid residue. In addition, the polypeptide may be characterized by the sequence NAVXXXXXXXXXXXXXXXXRTC.

Antibodies

The term antibody is used herein in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), multivalent antibodies, and antibody fragments, provided 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 produced by the immune system that has the ability to recognize and bind to a specific antigen. (Janeway S., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). The target antigen typically has multiple binding sites, also called epitopes, recognized by CDRs on numerous antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen can have more than one corresponding antibody. The antibody comprises 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 a portion thereof, such targets including, but not limited to, a cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. An immunoglobulin can be an immunoglobulin molecule of any type (eg, IgG, IgE, IgM, IgD and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. Immunoglobulins can be obtained from any species, including human, mouse, or rabbit origin.

Antibody fragments contain part of a full-length antibody, typically its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and scFv fragments; diabodies; linear antibodies; fragments produced in a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementarity determining region) and epitope

- 8 046016 binding fragments of any of the above, which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single chain antibody molecules and polyspecific antibodies formed from antibody fragments.

As used herein, the term monoclonal antibody refers to an antibody derived from a population of substantially homogeneous antibodies, i.e. the individual antibodies that make up the population are identical, except for possible naturally occurring mutations that may be present in minute quantities. Monoclonal antibodies are highly specific, being directed to a single antigenic site. Moreover, unlike polyclonal antibody preparations, which contain different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized uncontaminated by other antibodies. The modifier monoclonal characterizes the antibody as being derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies to be used in the present invention can be produced by the hybridoma method first described in Kohler et al. (1975) Nature 256:495, or can be obtained by methods using recombinant DNA (see US 4816567). Monoclonal antibodies can 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).

Monoclonal antibodies described herein specifically include chimeric antibodies, humanized antibodies and human antibodies.

Examples of cell binding agents include those described for use in WO 2007/085930, which is incorporated herein.

Tumor-associated antigens and cognate antibodies for use in embodiments of the present invention are listed below and described in more detail on pages 14-86 of WO 2017/186894, which is incorporated herein.

(1) BMPR1B (bone morphogenetic protein receptor type IB).

(2) E16 (LAT1, SLC7A5).

(3) STEAP1 (six transmembrane segment prostate epithelial antigen).

(4) 0772P (CA125, MUC16).

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin).

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute transporter family 34 (sodium phosphate), member 2, sodium-dependent phosphate transporter 3b type II).

(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).

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN gene cDNA 2700050C12).

(9) ETBR (endothelin type B receptor).

(10) MSG783 (RNF124, hypothetical protein of FLJ20315).

(11) STEAP2 (HGNC8639, IPCA-1, PCANAP1, STAMP I, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane segment prostate epithelial antigen 2, prostate protein glands with six transmembrane segments).

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel 5, subfamily M, member 4).

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor) (14) CD21 (CR2 (complement receptor 2), or C3DR (Epstein-Barr virus receptor), or Hs.73792) .

(15) CD79b (CD79B, CD79e, IGb (associated immunoglobulin beta), B29).

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor protein 1a), la) SPAP1B, SPAP1C).

(17) HER2 (ErbB2).

(18) NCA (CEACAM6).

(19) MDP (DPEP1).

(20) IL20R-alpha (IL20Ra, ZCYTOR7).

(21) Brevican (BCAN, BEHAB).

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5).

(23) ASLG659 (B7h).

- 9 046016 (24) PSCA (prostate stem cell precursor antigen).

(25) GEDA.

(26) BAFF-R (B-cell activating factor receptor, BLyS receptor 3, BR3).

(27) CD22 (B cell receptor isoform CD22-B, BL-CaM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814).

(27a) CD22 (CD22 molecule).

(28) CD79a (CD79A, CD79alpha), an immunoglobulin alpha-associated protein, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex with Ig M molecules on the surface, transmits a signal involved in B differentiation -cells), 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 chemokine CXCL13, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and possibly in the development of AIDS, lymphoma, myeloma and leukemia); 372 aa, pI: 8.54, MW: 41959, TM: 7 [P], gene, chromosome: 11q23.3.

(30) HLA-DOB (the beta subunit of the MHC class II molecule (antigen Ia) that binds peptides and presents them to CD4+ T lymphocytes); 273 aa, pI: 6.56, MW: 30820.TM. 1 [P], gene, chromosome: 6p21.3).

(31) P2X5 (P2X purinergic receptor ligand-gated ion channel 5, an extracellular ATP-gated ion channel 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).

(33) LY64 (lymphocyte antigen 64 (RP105), a membrane protein of the type I leucine-rich repeat (LRR) protein family, regulates B cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosus); 661 aa, pI: 6.20, MW: 74147, TM: 1 [P], gene, chromosome: 5q12).

(34) FcRH1 (Fc receptor-like protein 1, a putative immunoglobulin Fc domain receptor that contains Ig-like and C2-type ITAM domains, may play a role in Blymphocyte 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 a possible role in B-cell development and lymphomagenesis; gene dysregulation 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).

(38) SST (somatostatin receptor; note: there are 5 subtypes).

(38.1) SSTR2 (somatostatin receptor 2).

(38.2) SSTR5 (somatostatin receptor 5).

(38 .3) SSTR1.

(38 .4) SSTR3.

(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).

(42) MET (met proto-oncogene; hepatocyte growth factor receptor).

(43) MUC1 (cell surface associated mucin 1).

(44) CA9 (carbonic anhydrase IX).

(45) EGFRvIII (epidermal growth factor receptor (EGFR) transcript variant 3.

(46) CD33 (CD33 molecule).

(47) CD19 (CD19 molecule).

(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).

(52) CT Ag - CTA (cancer-testicular antigens).

(53) CD174 (Lewis Y) - FUT3 (fucosyltransferase 3 (galactoside-3(4)-L-fucosyltransferase, Lewis blood group).

- 10 046016 (54) CLEC14A (type C lectin domain family 14, member A; Genbank accession number NM175060).

(55) GRP78 is HSPA5 (70 kDa heat shock protein 5 (glucose regulated protein, 78 kDa).

(56) CD70 (CD70 molecule) L08096.

(57) stem cell specific antigens.

For example:

5Т4 (see list item (63) below);

CD25 (see list item (48) above);

CD32;

LGR5/GPR49;

prominin/SB 133.

(58) ASG-5.

(59) ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase 3).

(60) PRR4 (proline-rich protein 4 (lacrimal gland)).

(61) GCC - GUCY2C (guanylate cyclase 2C (receptor for thermostable enterotoxin).

(62) Liv-1-SLC39A6 (solute transporter family 39 (zinc transporter), member 6).

(63) 5T4, trophoblast glycoprotein, TPBG - TPBG (trophoblast glycoprotein).

(64) CD56 - NCMA1 (nerve cell adhesion molecule 1).

(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/mindin, target in prostate tumors is mindin/RG-I.

(70) B7-H4 - VTCN1 (V-domain containing inhibitor of T cell activation 1.

(71) PTK7 (PTK7 protein tyrosine kinase 7).

(72) CD37 (CD37 molecule).

(73) CD138 - SDC1 (syndecan 1).

(74) CD74 (CD74 molecule, major histocompatibility complex, class II invariant chain).

(75) claudins - CL (claudins).

(76) EGFR (epidermal growth factor receptor).

(77) Her3 (ErbB3) - ERBB3 (erythroblastic leukemia viral oncogene 3 homolog v-erb-b2 (avian)).

(78) RON - MST1R (macrophage stimulating receptor 1 (c-met-related tyrosine kinase)).

(79) ERNA2 (ERN A2 receptor).

(80) CD20 - MS4A1 (transmembrane domains 4, subfamily A, member 1).

(81) Tenascin C - TNC (tenascin C).

(82) FAP (fibroblast activation protein, alpha).

(83) DKK-1 (homolog of Dickkopf 1 (Xenopus laevis).

(84) CD52 (CD52 molecule).

(85) CS1 - SLAMF7 (member 7 of the SLAM family).

(86) Endoglin - ENG (endoglin).

(87) Annexin A1 - ANXA1 (annexin A1).

(88) V-CAM (CD106) - VCAM1 (vascular endothelial adhesion molecule 1).

Additional tumor-associated 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.

The cell-binding agent may be labeled, for example, to facilitate detection or purification of the agent, either prior to incorporation as a conjugate or as part of a conjugate. The label may be a biotin label. In another embodiment, the cell binding agent may be radiolabeled.

Treatment options

The conjugates of the present invention can 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 IV. The term therapeutically effective amount means an amount sufficient to demonstrate benefit to a patient. Such benefit may be at least a reduction in the intensity of at least one symptom. The actual amount administered, as well as the rate and dynamics of administration, will depend on the nature and severity of the condition being treated. Prescribing treatment, such as deciding on doses,

- 11 046016 is within the competence of general practitioners and other doctors.

The conjugate can be administered alone or in combination with other agents for treatment, either simultaneously or sequentially depending on the condition being treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (administration of active agents including, for example, drugs), surgery, and radiation therapy.

The pharmaceutical compositions of the present invention and for use according to the present invention may contain, in addition to the active ingredient, i.e. conjugate of Formula IV, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials generally known to those skilled in the art. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The specific nature of the carrier or other material will depend on the route of administration, which may be oral or may be by injection, for example, intradermal, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in the form of a tablet, capsule, powder or liquid. The tablet may contain a solid carrier or an adjuvant. Liquid pharmaceutical compositions typically contain a liquid carrier such as water, petroleum products, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included. The capsule may contain a solid carrier such as gelatin.

For intravenous, intradermal, subcutaneous or intralesional injection, the active ingredient will be in the form of an aqueous solution suitable for parenteral administration, which is pyrogen-free and has a suitable pH, isotonicity and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as needed.

The conjugates can be used to treat proliferative disease and autoimmune disease. The term proliferative disease refers to unwanted or uncontrolled cellular proliferation of excess or abnormal cells that is undesirable, such as neoplastic or hyperplastic growth, whether in vitro or in vivo.

Examples of proliferative conditions include, but are not limited to, cellular proliferation of a benign, premalignant, and malignant nature, including, but not limited to, neoplasms and tumors (eg, histiocytoma, glioma, astrocytoma, osteoma), cancers (eg, lung cancer, small cell lung cancer, gastrointestinal cancer, intestinal tract, intestinal cancer, colon cancer, breast carcinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma ), types of leukemia, psoriasis, bone diseases, fibroproliferative disorders (eg connective tissue) and atherosclerosis. Other cancers of interest include, but are not limited to, hematologic malignancies such as leukemias and lymphomas, such as non-Hodgkin's lymphoma and its subtypes such as DLBCL, marginal zone lymphoma, mantle cell lymphoma, and follicular lymphoma, lymphoma Hodgkin's, AML and other types of cancer of B- or T-cell origin. Any cell type may be treated, including but not limited to cells from the lung, gastrointestinal tract (including, for example, intestine, colon), breast (mammary), ovary, prostate, liver (hepatic), kidney ( renal), bladder, pancreas, brain and skin.

Examples of autoimmune disease include the following: rheumatoid arthritis, autoimmune demyelinating diseases (eg, multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune liver 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 insufficiency, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis , autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis , alopecia areata , pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcification, Raynaud's phenomenon, esophageal motility disorder, sclerodactyly and telangiectasia), male and female autoimmune infertility, ankylosing spondylitis, ulcerative colitis, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis , atopic dermatitis, atopic

- 12 046016 rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, antiphospholipid syndrome, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, poultry farmer's lung, toxic epidermal necrolysis, Alport syndrome , alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu arteritis, polymyalgia rheumatoid, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sumpter's syndrome, eczema, lymphomatoid granulomatosis, disease Behçet , Kaplan syndrome, Kawasaki disease, dengue fever, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, persistent elevated erythema, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman syndrome, Felty syndrome, filariasis, cyclitis, chronic cyclitis, heterochromic cyclitis, Fuchs cyclitis, IgA nephropathy, Henoch-Schönlein purpura, graft-versus-host disease, graft rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenström macroglobulinemia, Evans syndrome and autoimmune gonadal failure.

In some embodiments, the autoimmune disease is a disorder involving 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 (eg, atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn syndrome, systemic sclerosis, or chronic disease graft versus host). In general, disorders associated with dendritic cells include disorders associated with Th1 lymphocytes and Th2 lymphocytes. In some embodiments, the autoimmune disorder is a T cell-mediated immunological disorder.

A chemotherapy agent is a chemical compound useful in the treatment of cancer, regardless of its mechanism of action. Classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, plant spindle venom alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in targeted therapy and conventional chemotherapy.

Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, BristolMyers Squibb Oncology, Princeton, NJ), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona -2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen (^)-2-[4-(1,2-diphenylbut-1- enyl)phenoxy-1N.N-dimethylethanamine. NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2, HPPD and rapamycin. Additional examples of chemotherapeutic agents include oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), Sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis ), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016 , Glaxo Smith Kline), lonafarnib (SARASAR™, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer) , tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (without Cremophor), paclitaxel formulations bound to albumin engineered nanoparticles (American Pharmaceutical Partners, Schaumberg, I1), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca) , chlorambucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa and cyclophosphamide (CYTOXAN®, NEOSAR®); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including a synthetic analogue of topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (in particular cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, chlorphosphamide, estramustine,

- 13 046016 ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquin, phenesterol, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (eg calicheamicin, calicheamicin gamma 1I, calicheamicin omega I1 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dinemycin, dinemycin A; bisphosphonates such as clodronate; esperamycin ; as well as neocarcinostatin chromophore and related chromoprotein chromophores of enediin antibiotics, aclacinomysins, actinomycin, outramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carcinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5oxo-b-norley cin, morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin, epirubicin, ezorubicin, idarubicin, nemorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porphyromycin, puromycin, quelamycin, rhodorubicin, strep tonigrine, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as ancytabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepithiostane, testolactone; substances that suppress adrenal function, such as aminoglutethimide, mitotane, trilostane; a folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquon; elfornitine; elliptinium acetate; epothilone; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxtantrone; mopidanmol; nitraerin; pentostatin; phenomet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurine A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepu; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); Novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; SRT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

Also included in the definition of a chemotherapeutic agent are (I) antihormonal agents that act by regulating or suppressing the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; citrate tamoxifene), raloxifene, droloxifene, 4hydroxytamoxifene, trioxifene, keoxifene, LY117018, onapristone and FARESTON® (toremifine citrate); (ii) aromatase inhibitors, which inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestane, fadrozole , RTVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxacitabine (1,3-dioxolane nucleoside analogue of cytosine); (iv) protein kinase inhibitors, such as MEK inhibitors (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, in particular oligonucleotides that suppress the expression of genes in signaling pathways involved in impaired cell proliferation, for example, PKC-alpha, Raf and H-Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes, such as inhibitors of VEGF expression (eg ANGIOZYME®) and inhibitors of HER2 expression; (viii) vaccines, such as gene therapy vaccines, such as ALLOVECTIN®, LEUVECTIN® and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH; (ix) antiangiogenic agents such as bevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the above.

Also included in the definition of a chemotherapeutic agent are therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec), pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia) and the antibody-drug conjugate gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic agents in combination with the conjugates of the present invention include alemtuzumab, apolizumab, azelizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuza mab, daclizumab, eculiza

- 14 046016 mab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, but lovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab , pekfusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resivizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, takatuzumab tetraxetane, tadocizumab, talizumab, tefibazumab , tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab , urtoxazumab and visilizumab.

Compositions

While individual use (eg administration) of the conjugate is possible, it is often preferred to present it as a composition or formulation.

In one embodiment, the composition is a pharmaceutical composition (eg, formulation, preparation, drug) containing a conjugate described herein and a pharmaceutically acceptable carrier, diluent or excipient.

In one embodiment, the composition is a pharmaceutical composition containing at least one conjugate described herein, together with one or more other pharmaceutically acceptable ingredients generally known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents , excipients, adjuvants, excipients, buffers, preservatives, antioxidants, lubricants, stabilizing agents, solubilizing agents, surfactants (eg wetting agents), masking agents, coloring agents, flavoring agents and sweeteners.

In one embodiment, the composition further contains other active agents, such as other therapeutic or prophylactic agents.

Information on suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical literature. See, for example, Handbook of Pharmaceutical Additives. 2nd edition (ed. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences. 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients. 2nd edition, 1994.

Another aspect of the present invention relates to methods for preparing a pharmaceutical composition comprising mixing at least one radiolabeled [ ¹¹ C] conjugate or conjugate-like compound as defined herein with one or more other pharmaceutically acceptable ingredients generally known to those skilled in the art. techniques, e.g. carriers, diluents, auxiliaries, etc. When formulated in discrete units (eg tablets, etc.), each unit contains a predetermined amount (dose) of the active compound.

As used herein, the term pharmaceutically acceptable refers to compounds, ingredients, materials, compositions, dosage forms, and the like that, based on careful medical evaluation, are suitable for use in contact with the tissues of a specified subject (eg, a human) without causing excessive toxicity, irritation, allergic reaction or other problem or complication commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be acceptable in terms of compatibility with other ingredients of the formulation.

The compositions can be prepared by any methods generally known in the pharmaceutical field. Such methods include the step of contacting the active compound with a carrier that consists of one or more auxiliary ingredients. In general, the compositions are prepared by uniformly and directly bringing the active compound into contact with carriers (eg, liquid carriers, finely divided solid carriers, etc.) and then shaping the product as necessary.

A fast or slow release formulation may be prepared; immediate, delayed, delayed or extended release or a combination thereof.

Formulations suitable for parenteral administration (eg, by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (eg, solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise present (eg, in a liposome or other microparticle). Such liquids may further contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizing agents, bacteriostatic agents, suspending agents, thickening agents and solutes that render the formulation isotonic with the blood (or other appropriate body fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyhydric alcohols, glycerin, vegetable oils and the like. Examples of suitable isotonic vehicles for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Typically, the concentration of active ingredient in the liquid is from about 1 ng/ml to about 10 μg/ml, e.g.

- 15 046016 approximately 10 ng/ml to approximately 1 μg/ml. The formulations may be presented in single-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

Dose

One skilled in the art will appreciate that suitable dosages of conjugates and compositions containing conjugates may vary from patient to patient. Determining the optimal dose typically involves balancing the therapeutic benefit against any risk or harmful side effects. The dose level selected will depend on a variety of factors, including, without limitation, the activity of the particular compound, route of administration, time of administration, rate of elimination of the compound, duration of treatment, other drugs, compounds and/or materials used in combination, severity of the condition, and type , gender, age, weight, condition, general health and medical history of the patient. The amount of compound and route of administration will ultimately be left to the discretion of the physician, veterinarian or clinician, however, generally, the dose will be adjusted to achieve local concentration values at the site of action that achieve the desired effect without causing significant harmful or detrimental side effects .

Administration may be administered as a single dose, continuously or intermittently (eg, in divided doses at suitable intervals) throughout the course of treatment. Methods for determining the most effective routes and dosage of administration are well known to those skilled in the art and will vary depending on the formulation used for therapy, the target of therapy, the target cell(s) to be treated, and the subject to be treated. Single or multiple administrations may be carried out using the dose level and schedule selected by the attending physician, veterinarian or clinician.

In general, a suitable dose of the active compound is in the range of about 100 ng to about 25 mg (more typically from about 1 μg to about 10 mg) per day per kilogram of body weight of the subject. In case the active compound is a salt, ester, amide, prodrug or the like, the amount administered is calculated based on the original compound and thus the actual weight applied increases proportionally.

The dosage amounts described above may be applied to the conjugate or the effective amount of the compound that is released upon cleavage of the linker.

For the prevention or treatment of a disease, the appropriate dose of the ADC of the present invention will depend on the type of disease being treated as defined above, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, prior therapy, the patient's medical history, and response to the antibody. , as well as at the discretion of the attending physician. The molecule is suitably administered to a patient either once or over a series of treatments. Depending on the type and severity of the disease, an amount of molecules ranging from about 1 μg/kg to 100 mg/kg or more represents an initial candidate dose to be administered to a patient, whether, for example, through one or more separate administrations or through a continuous infusion. With repeated administrations over several days or longer, depending on the condition, treatment is extended until the desired suppression of disease symptoms occurs. Other dosage schedules may be applicable. The progress of this therapy can be easily monitored using traditional techniques and tests.

Drug load

Drug load (p) is the average amount of drug per ligand unit, which may be a cell-binding agent, such as an antibody.

The average amount of drug per antibody in ADC preparations produced by conjugation reactions can be characterized by conventional methods such as UV, reverse phase HPLC, HIC, mass spectrometry, ELISA and electrophoresis. The quantitative distribution of ADC in terms of p can also be determined. The average p value of a particular ADC preparation can be determined by ELISA (Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al. (2005) Clin. Cancer Res. 11:843-852). However, the distribution of p (drug) values is not distinguishable by antibody-antigen binding and ELISA detection limit. In addition, the use of ELISA to detect antibody-drug conjugates does not identify where drug moieties are attached to the antibody, such as light chain or heavy chain moieties or specific amino acid residues. In some cases, separation, purification and characterization of a homogeneous ADC, where p is the determined value obtained for the ADC loaded with another drug, can be accomplished by methods such as HPLC with

- 16 046016 whelping phase or electrophoresis. Such techniques are also applicable to other types of conjugates.

For some antibody-drug conjugates, the p value may be limited by the number of attachment sites on the antibody. For example, the antibody may contain only one or more cysteine thiol groups, or may contain only one or more sufficiently reactive thiol groups through which a linker can be attached. Higher drug loading may cause aggregation, insolubility, toxicity, or loss of cell penetration for certain antibody-drug conjugates.

Typically, during the conjugation reaction, less than the theoretical maximum number of drug moieties are conjugated to the antibody. The antibody may contain, for example, many lysine residues that do not react with the drug linker. Only the most reactive lysine groups are able to react with the amine-reactive linker reagent. In addition, only the most reactive cysteine thiol groups are able to react with the thiol-reactive linker reagent. In general, antibodies do not contain many or no free and reactive cysteine thiol groups that can be attached to the drug moiety. Most thiol cysteine residues in antibody compounds exist as disulfide bridges and must be reduced in number using a reducing agent such as dithiothreitol (DTT) or TCEP under partially or fully reducing conditions. The loading (drug/antibody ratio) of an ADC can be adjusted in several different ways, including (i) limiting the molar excess of the drug linker relative to the antibody, (ii) limiting the time or temperature of the conjugation reaction, and (iii) partial or limiting reducing conditions for modification of the thiol group of cysteine.

Some antibodies contain reducible interchain disulfide bonds, i.e. cysteine bridges. Antibodies can be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by reacting lysine residues with 2-iminothiolane (Trot's reagent), resulting in the conversion of the amine to a thiol. Reactive thiol groups can be introduced into an antibody (or fragment thereof) by engineering one, two, three, four or more cysteine residues (eg, by generating mutant antibodies containing one or more non-native cysteine amino acid residues). US 7,521,541 describes the construction of antibodies by introducing reactive amino acids that are cysteine.

Cysteine amino acids can be designed at antibody reactive sites and so that they do not form intrachain or intermolecular disulfide bonds (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Doman et al (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; WO 2009/052249). The thiol groups of engineered cysteine residues are capable of reacting with drug linkers of the present invention (ie, Formula I) that contain thiol-reactive electrophilic groups, such as maleimide or alpha-halogenamides, to form ADCs with antibodies with engineered cysteine residues. The location of the drug unit can thus be designed, controlled and known. Drug loading can be controlled because the thiol groups of the engineered cysteine tend to react with drug-linker reagents in high yield. Designing an IgG antibody to introduce a cysteine amino acid through a single site substitution on the heavy or light chain provides two new cysteine residues on the symmetrical antibody. Drug loadings close to 2 can be achieved with near uniformity of the ADC conjugation product.

In case more than one nucleophilic or electrophilic group of an antibody reacts with drug linkers, the resulting product may be a mixture of ADC compounds with a distribution of drug units attached to the antibody, for example, 1, 2, 3, etc. . Liquid chromatography techniques such as polymer reverse phase chromatography (PLRP) and hydrophobic interaction chromatography (HIC) can separate compounds in a mixture based on drug loading. ADC preparations with a single drug loading value (p) can be isolated, however, such ADCs with a single loading value may still be heterogeneous mixtures since the drug units may be attached via a linker to different sites of the antibody.

Thus, antibody-drug conjugate compositions herein

- 17 046016 of the invention may contain mixtures of antibody-drug conjugates, where the antibody contains one or more drug fragments, and where the drug fragments can be attached to the antibody at different amino acid residues.

In one embodiment, the average amount of drug per cell binding agent is in the range of 1 to 20. In some embodiments, the range is selected from 1 to 10, 2 to 10, 2 to 8, 2 to 6 and from 4 to 10.

In some embodiments, there is one drug per cell binding agent.

Common synthesis routes

Compounds of formula I, where RL is represented by formula Ia, can be synthesized from a compound of formula 2 n

lLAl /O γ formula 2

BEFORE

OH O where R ^l * represents -QH due to addition to the compound of formula 3 o

O ^L formula 3

HO^X or its activated version.

Such a reaction can be carried out under conditions that promote the formation of an amide bond.

Compounds of formula 2 can be synthesized by removing the protecting group from the compound

groups.

Compounds of formula 4 can be synthesized by coupling a compound of formula 5

with compound A3 using the Friedlander reaction.

Compounds of formula 5 can be synthesized from compounds of formula 6

। * N JL A.

R ^ρΓΟίχΧ formula 6

A. COCF3

H by removing the trifluoroacetamide protecting group.

Compounds of formula 6 can be synthesized by coupling R ^L * ^prot -OH with compound 17.

Compounds of formula I, where R ^L is represented by formula Ia or Ib, can be synthesized from compound I11 by coupling reaction with the compound R ^L -OH or an activated form thereof.

Protecting groups for the amino group

Protecting groups for the amino group are widely known to those skilled in the art. Particular attention is paid to the disclosure of suitable protecting groups in Greene's Protecting Groups in Organic Synthesis, fourth edition, John Wiley & Sons, 2007 (ISBN 978-0-471-69754-1), pp. 696-871.

Additional preferences

The following preferences may apply to all aspects of the present invention as described above, or they may apply to a particular aspect. Preferences can be combined together in any combination.

Q ^X

- 18 046016

In one embodiment, Q is an amino acid residue. The amino acid may be a naturally occurring amino acid or a non-naturally occurring amino acid.

In one embodiment, Q is selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg and Trp, where Cit is citrulline.

In one embodiment, Q includes a dipeptide residue. The amino acids in the dipeptide may be any combination of naturally occurring amino acids and unnatural amino acids. In some embodiments, the dipeptide contains naturally occurring amino acids. If the linker is a linker labile to cathepsin cleavage, then the dipeptide is the site of action for cathepsin-mediated cleavage. Thus, the dipeptide represents a recognition site for cathepsin.

In one embodiment, Q is selected from

NH-Phe-LysNH-Val-Ala- ^C NH-Val-Lys- ^C NH-Ala-Lys- ^C NH-Val-Cit- ^C NH-Phe-Cit-C NH-Leu-Cit- ^C NH-Ile- Cit-C' ^NH -Phe-ArgNH-Trp-Cit- ^C NH-Gly-Val- ^C where Cit is citrulline.

Preferably Q is selected from

NH-Phe-Lys- ^C = ^O ,

NH-Val-Ala- ^C = ^O ,

NH-Val-Lys- ^C = ^O ,

NH-Ala-Lys- ^C = ^O and

NH-Val-Cit- ^C = ^O

Most preferably, Q is selected from

NH-Phe-Lys- ^C = ^O ,

NH-Val-Cit- ^C = ^O or

NH-Val-Ala- ^C = ^O

Other dipeptide combinations of interest include

NH-Gly-Gly- ^C = ^O ,

NH-Gly-Val- ^C = ^O ,

NH-Pro-Pro- ^C = ^O and

NH-Val-Glu- ^C = ^O .

Other combinations of dipeptides may be used, including those described in 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 a tripeptide can be any combination of naturally occurring amino acids and unnatural amino acids. In some embodiments, the tripeptide contains naturally occurring amino acids. If the linker is a linker labile to cathepsin cleavage, then the tripeptide is the site of action for cathepsin-mediated cleavage. Thus, the tripeptide represents a recognition site for cathepsin. Tripeptide linkers of particular interest are

NH-Glu-Val-Ala- ^C = ^O ,

NH-Glu-Val-Cit- ^C = ^O ,

NH-aGlu-Val-Ala- ^C = ^O ,

NH-aGlu-Val-Cit- ^C = ^O

In some embodiments, Q is a tetrapeptide residue. The amino acids in the tetrapeptide can be any combination of naturally occurring amino acids and unnatural amino acids. In some embodiments, the tetrapeptide contains naturally occurring amino acids. If the linker is a linker labile to cathepsin cleavage, then the tetrapeptide is the site of action for cathepsin-mediated cleavage. Thus, the tetrapeptide represents a recognition site for cathepsin. Tetrapeptide linkers of particular interest are:

NH-Gly-Gly-Phe-Gly ^C = ^O and

NH-Gly-Phe-Gly-Gly ^C = ^O

- 19 046016

In some embodiments, the tetrapeptide is ^NH -Gly-Gly-Phe- ^GlyC = ^O .

In the above images of peptide residues, NH- represents the N-terminus and - ^CO represents the C-terminus of the residue. The C-terminus is connected to NH from A*.

Glu is a glutamic acid residue, i.e.

aGlu is a glutamic acid residue when bound via the α chain, i.e.:

In one embodiment, the amino acid side chain is chemically protected, if desired. The protecting group for the side chain may be a group discussed above. Protected amino acid sequences are enzymatically cleavable. For example, a dipeptide sequence containing a Lys residue with side chain protection by Boc is cleavable by cathepsin.

Protecting groups for amino acid side chains are well known in the art and are described in the Novabiochem catalog and are as described above.

G ^l

G ^l can be selected from

- 20 046016

(G ¹² ) 0 GY o ( ^GLS ) V (GLV ^| ) ss^ ¹ G (ΝΟ2) where the group ΝΟ2 is optional (0% N ₃ (G ¹⁴² ) s— G (NO2) where the group ΝΟ2 is optional (G ¹¹⁰ ) H (G'% ss^ ri O ₂ n\^=/ where the group ΝΟ2 is optional (GF ¹ ) 0 0 w ( ^GLV4 ) s-s-E m where the group ΝΟ2 is optional ( ^GF2 ) (G ¹⁴ ) __ ABOUT Hal N—1 N* where Hal represents I, Br, C1 (CY ¹³ ) X ( ^G1 ) About Hal—n (G ¹¹⁴ ) ^h ₂ N. Y CT 7

where Ar represents a C _5-6 arylene group, for example phenylene, and X represents a C _1-4 alkyl.

In some embodiments, GL is selected from G ^L1-1 and G ^L1-2 . In some of these embodiments, G ^L is G ^L1-1 .

GLL

G ^ll can be selected from

- 21 046016

(GLL!- ¹ ) ABOUT (Gixs- ¹ ) C.B.A. CBA| Z'' ABOUT (GLL1-2) O AG e st/ ₄ N y (GLL8-2) N,CBA (GLL2) 0 cba| Ν' * V-X o 0 (GLL9-!) £ ^N ₄ ⁴ _N CBA (QLL3-1) (QLL ⁹ - ² ) C.B.A. (GLL3-2) cba| _{_s} ^7 (QLLiO) ---, H (G^- ⁴ ) CBAJ___ i\Hft—N 0 A (GU- ¹¹ ) ^CBA 'X Ύ ( > Nv/^/ ^HO ) H O (G ¹¹³ ) 0 CBA|__A (GIXH) । b / 7 X (Gixe) 0 CBAi___// (G^ ¹³ ) ((E ^L7 ) CBA| (QLL ¹⁴ ) ^H ί sva\/A _o D

where Ar represents a C _5-6 arylene group, for example phenylene, and X represents a C _1-4 alkyl.

In some embodiments, G ^LL is selected from G ^LL1-1 and G ^LL1-2 . In some of these embodiments, G ^LL is G ^LL1-1 .

X

X represents

where a equals 0-5, b1 equals 0-16, b2 equals 0-16, c equals 0 or 1, d equals 0-5, where at least b1 or b2 equals 0, and at least c1 or c2 equals 0 .

a may be 0, 1, 2, 3, 4, or 5. In some embodiments, a is 0-3. In some of these embodiments, a is 0 or 1. In additional embodiments, a is 0.

b1 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b1 is 0-12. In some of these embodiments, b1 is 0-8 and may be 0, 2, 3, 4, 5, or 8.

b2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b2 is 0-12. In some of these embodiments, b2 is 0-8 and may be 0, 2, 3, 4, 5, or 8. Only one of b1 and b2 may not be 0.

c1 can be 0 or 1.

c2 can be 0 or 1.

Only one of c1 and c2 can be other than 0.

d may be 0, 1, 2, 3, 4, or 5. In some embodiments, d is 0-3. In some of these embodiments, d is 1 or 2. In additional embodiments, d is 2. In additional embodiments, d is 5.

- 22 046016

In some embodiments, X a is 0, bl is 0, cl is 1, c2 is 0, and d is 2, and b2 may be 0-8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8.

In some embodiments, X a is l, b2 is 0, cl is 0, c2 is 0 and d is 0, and b1 may be 0-8. In some of these embodiments, b1 is 0, 2, 3, 4, 5, or 8.

In some embodiments, X a is 0, bl is 0, cl is 0, c2 is 0 and d is 1, and b2 may be 0-8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8.

In some embodiments, X b1 is 0, b2 is 0, c1 is 0, c2 is 0, and one of a and d is 0. The other of a and d is 1-5. In some of these embodiments, the other of a and d is 1. In others of these embodiments, the other of a and d is 5.

In some embodiments, X a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 may be 0-8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8.

In some embodiments, RL is represented by Formula Ib. In some embodiments, R ^ll is represented by formula Ib'.

R ^L1 and R ^L2 are independently selected from H and methyl or, together with the carbon atom to which they are bonded, form a cyclopropylene or cyclobutylene group.

In some embodiments, R ^L1 and R ^L2 are both H.

In some embodiments, R ^L1 is H and R ^L2 is methyl.

In some embodiments, R ^L1 and R ^L2 are both methyl.

In some embodiments, R ^L1 and R ^L2 , together with the carbon atom to which they are bonded, form a cyclopropylene group.

In some embodiments, R ^L1 and R ^L2 , together with the carbon atom to which they are bonded, form a cyclobutylene group.

In group Ib, in some embodiments, e is 0. In other embodiments, e is 1, and the nitro group can be at any available position on the ring. In some of these embodiments, it is in the ortho position. In other of these embodiments, it is in the para position.

In some embodiments of the fifth aspect of the present invention, the enantiomerically enriched form has an enantiomer ratio of greater than 60:40, 70:30; 80:20 or 90:10. In additional embodiments, the enantiomer ratio is greater than 95:5, 97:3, or 99:1.

In some embodiments, R ^L is selected from

- 23 046016

In some embodiments, R ^LL is a group derived from the ^RL groups above.

In one embodiment of the first aspect of the present invention, the compound of formula I is

Additional preferences

In some embodiments, a compound of formula I is represented by formula IP

and its salts and solvates, wherein R ^LP is a linker for attachment to a cell binding agent selected from (ia):

where QP represents

- 24 046016

O where Q ^XP is such that QP is an amino acid residue, a dipeptide residue or a tripeptide residue;

XP is

cP where aP equals 0-5, bP equals 0-16, cP equals 0 or 1, dP equals 0-5; GL is a linker for attachment to a ligand unit;

(ib):

where R ^L1 and R ^L2 are independently selected from H and methyl or, together with the carbon atom to which they are bonded, form a cyclopropylene or cyclobutylene group and e is 0 or 1.

aP may be 0, 1, 2, 3, 4, or 5. In some embodiments, aP is 0-3. In some of these embodiments, aP is 0 or 1. In additional embodiments, aP is 0.

bP may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b is 0-12. In some of these embodiments, bP is 0-8 and may be 0, 2, 4, or 8.

cP can be 0 or 1.

dP may be 0, 1, 2, 3, 4, or 5. In some embodiments, dP is 0-3. In some of these embodiments, dP is 1 or 2. In additional embodiments, dP is 2.

In some embodiments, XP ^aP is 0, cP is 1, and dP is 2, and bP can be 0-8. In some of these embodiments, bP is 0, 4, or 8.

The preferences regarding Q ^X indicated above for compounds of formula I may, if necessary, apply to Q ^XP .

The preferences regarding ^GL , ^RL1 , ^RL2 and e mentioned above for compounds of formula I may apply to compounds of formula ^IP .

In some embodiments, the conjugate of formula IV is represented by formula IVP

L-(D ^LP ) _P (IV ^p ) or a pharmaceutically acceptable salt or solvate thereof, wherein L is a ligand (ie, targeting) unit, D ^LP is a drug-linker unit represented by formula IIIP

it

R ^llp is a linker attached to a ligand unit selected from ^(ia ' ^):

where QP and XP are as defined above, and G ^LL is a linker attached to a ligand unit; and (ib'):

o where R ^L1 and R ^L2 are as defined above; and p is an integer from 1 to 20.

- 25 046016

In some embodiments, the compound of Formula I is represented by the formula IP ²

it o and its salts and solvates, where RLP ² is a linker for attachment to a cell binding agent selected from (ia)

ABOUT

where Q represents

o where Q ^X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; XP ² is

where aP2 equals 0-5, b1P2 equals 0-16, b2P2 equals 0-16, cP2 equals 0 or 1, dP2 equals 0-5, where at least b1P2 or b2P2 equals 0 (i.e. only one of b1 and b2 may not equal 0);

GL is a linker for attachment to a ligand unit;

(ib):

where R ^l1 and R ^L2 are independently selected from H and methyl or, together with the carbon atom to which they are bonded, form a cyclopropylene or cyclobutylene group; and e equals 0 or 1.

aP2 may be 0, 1, 2, 3, 4, or 5. In some embodiments, aP2 is 0-3. In some of these embodiments, aP2 is 0 or 1. In additional embodiments, aP2 is 0.

b1P2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b1P2 is 0-12. In some of these embodiments, b1P2 is equal to 08 and may be equal to 0, 2, 3, 4, 5 or 8.

b2P2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b2P2 is 0-12. In some of these embodiments, b2P2 is equal to 08 and may be equal to 0, 2, 3, 4, 5 or 8. Only one of b1P2 and b2P2 may not be equal to 0.

cP2 can be 0 or 1.

dP2 may be 0, 1, 2, 3, 4, or 5. In some embodiments, dP2 is 0-3. In some of these embodiments, dP2 is equal to 1 or 2. In additional embodiments, dP2 is equal to 2. In additional embodiments, dP2 is equal to 5.

In some embodiments, X ^P2 aP2 is 0, b1P2 is 0, cP2 is 1, and dP2 is 2, and b2P2 may be 0-8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8.

In some embodiments, X ^P2 aP2 is 1, b2P2 is 0, cP2 is 0, and dP2 is 0, and b1P2 may be 0-8. In some of these embodiments, b1P2 is 0, 2, 3, 4, 5, or 8.

In some embodiments, X ^P2 aP2 is 0, b1P2 is 0, cP2 is 0, and dP2 is 1, and b2P2 may be 0-8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8.

In some embodiments, X ^P2 b1P2 is 0, b2P2 is 0, cP2 is 0, and one of aP2 and dP2 is 0. The other of aP2 and d is 1-5. In some of these embodiments, the other of aP2 and d is 1. In others of these embodiments, the other of aP2 and dP2 is 5.

Preferences regarding Q ^X indicated above for compounds of formula I, if necessary

- 26 046016 can be applied to Q ^X in formula Ia ^P2 .

The preferences regarding ^GL , ^RL1 , ^RL2 and e mentioned above for compounds of formula I may apply to compounds of formula ^IP2 .

In some embodiments, the conjugate of formula IV is represented by formula IV ^P2 :

L-(D ^LP2 ) _P (IV ^P2 ), or a pharmaceutically acceptable salt or solvate thereof, wherein L is a ligand (ie, targeting) unit, D ^LP2 is a drug-linker unit represented by the formula III ^P2

R ^LLP2 is a linker attached to a ligand unit selected from (ia'):

where Q and X ^P2 are as defined above, and G ^LL is a linker attached to a ligand unit; and (ib'):

where R ^L1 and R ^L2 are as defined above; and p is an integer from 1 to 20.

Examples

general information

Flash chromatography was performed using Biotage® Isolera™ and fractions tested for purity using thin layer chromatography (TLC). TLC was performed using Merck Kieselgel 60 F254 silica gel with a fluorescent indicator on aluminum plates. TLC was visualized using UV light.

Solvents for extraction and chromatography were purchased from VWR U.K. and used without further purification.

All pure chemicals were purchased from Sigma-Aldrich unless otherwise noted. PEGylated reagents were obtained from Quanta biodesign (USA) via Stratech (UK).

LC/MS conditions.

Method A.

Electrospray mass spectrometry in positive ion detection mode was performed using Waters Aquity H-class SQD2.

The mobile phases used were solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The initial formulation of 5% B was held for 25 s, then the concentration was increased from 5% B to 100% B over a period of 1 min 35 s. The composition was held for 50 s at 100% B, then returned to 5% B in 5 s and held at this value for 5 s. The total duration of the gradient run was 3.0 min. The flow rate was 0.8 ml/min. Detection was carried out at 254 nm. Columns: Waters Acquity UPLC® VEN Shield RP18, 1.7 μm, 2.1x50 mm at 50°C, equipped with a Waters Acquity UPLC® VEN Shield RP18 VanGuard precolumn, 130A, 1.7 μm, 2.1x5 mm.

Method B.

HPLC (Waters Alliance 2695) was carried out using a mobile phase of water (A) (0.1% formic acid) and acetonitrile (B) (0.1% formic acid).

The initial formulation of 5% B was held for 25 s, then the concentration was increased from 5% B to 100% B over a period of 1 min 35 s. The composition was held for 50 s at 100% B, then returned to 5% B in 5 s and held at this value for 5 s. The total duration of the gradient run was 3.0 minutes. The flow rate was 0.8 ml/min. Detection wavelength range: 190-800 nm. Columns: Waters Acquity UPLC® VEN Shield RP18, 1.7 μm, 2.1x50 mm at 50°C, equipped with a Waters Acquity UPLC® VEN Shield RP18 VanGuard precolumn, 130A, 1.7 μm, 2.1x5 mm.

- 27 046016

Method C.

HPLC (Waters Alliance 2695) was carried out using a mobile phase of water (A) (0.1% formic acid) and acetonitrile (B) (0.1% formic acid).

The initial composition of 5% B was held for 1 minute, then the concentration was increased from 5% B to 100% B over a period of 9 minutes. The composition was held for 2 minutes at 100% B, then returned to 5% B in 0.10 minutes and held at this value for 3 minutes. The total duration of the gradient run was 15 min. The flow rate was 0.6 ml/min. Detection wavelength range: 190-800 nm. Thermostat temperature: 50°C. Column: ACE Excel 2 C18-AR, 2 µm, 3.0x100 mm.

HPLC terms.

Ultrafast reverse phase high performance liquid chromatography (UFLC) was performed on a Shimadzu Prominence™ apparatus using a Phenomenex™ Gemini NX, 5 µm C18 column (at 50°C) with dimensions: 150x21.2 mm. The eluents used were solvent A ( _H2O with 0.1% formic acid) and solvent B (CH3CN with 0.1% formic acid). All experiments using UFLC were performed under the following gradient elution conditions. The concentration of the original 13% B formulation was increased to 30% B over a period of 3 minutes, then increased to 45% B over 8 minutes and increased again to 100% within 6 minutes, then returned to 13% within 2 min and held for 1 min. The total duration of the gradient run was 20.0 min. The flow rate was 20.0 mL/min, and detection was performed at 254 nm and 223 nm.

NMR method.

Proton NMR chemical shift values were measured on the delta scale at 400 MHz using a Bruker AV400. The following abbreviations were used: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; br, wide. Interaction constants are given in Hz.

Synthesis of key intermediates

a) ^(5,6,7,8-tetrahydronaphthalene-1-yl)acetamide (I2).

5,6,7,8-Tetrahydronaphthalene-1-amine I1 (8.54 g, 58.0 mmol) was dissolved in dichloromethane (80 ml). Triethylamine (18 ml, 129 mmol) was added and the mixture was cooled to 0°C. Acetic anhydride (11.5 mL, 122 mmol) was added dropwise and the reaction mixture was warmed to room temperature after addition was complete. and stirred for 45 min, immediately after which LCMS indicated completion of the reaction. The mixture was diluted with CH ₂ Cl ₂ , washed with H2O, sat. NaHCO ₃ , 10% citric acid, the organic phase was dried over MgSO ₄ and concentrated in vacuo. The off-white solid was triturated with 1:3 Et ₂ O/isohexane to give I2 (10.8 g, 57.1 mmol, 98% yield) as a white solid, which was used without further purification. LC/MS (method A): retention time 1.44 minutes (ES+) mass/charge 190 [M+H] ⁺ .

b) ^(4-nitro-5,6,7,8-tetrahydronaphthalene-1-yl)acetamide (I3).

Na(5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I2 (1.00 g, 5.2840 mmol) was added portionwise to sulfuric acid (15 mL, 281 mmol) at -5°C. Sodium nitrate (450 mg, 5.2945 mmol) was added to the reaction mixture in portions and stirred for 30 minutes at -5°C, immediately after which LCMS indicated no further reaction. The reaction mixture was poured onto ice with external cooling, the aqueous mixture was extracted with CH ₂ Cl ₂ , the organic phase was dried over MgSO ₄ and purified with Isolera (10-80% EtOAc in isohexane) to obtain a mixture of N-(4-hutpo-5 ,6,7,8tetrahydronaphthalen-1-yl)acetamide I3 and ^(2-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (956 mg, 4.0811 mmol, 77% yield) as white/yellow solid. LC/MS (method A): retention time 1.53 min (ES ⁺ ) mass/charge 235 [M+H] ⁺ .

c) ^(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)acetamide (I4).

Na(4-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I3 (1.01 g, 4.31 mmol) was dissolved in acetone

- 28 046016 (30 ml). Magnesium sulfate in water (3.9 mL, 5.9 mmol, 1.5 mol/L) was added and the mixture was cooled to 0°C. Potassium permanganate (2.07 g, 13.0 mmol) was added to the reaction mixture in portions and the mixture was heated to room temperature. and stirred for 50 min, immediately after which TLC indicated completion of the reaction. The reaction mixture was filtered through Celite, the solids were washed with CHCl ₃ and the resulting organic mixture was washed with H ₂ O, brine, dried over MgSO ₄ and purified with Isolera (20-50% EtOAc in iso-hexane) to obtain the mixture N(4-nitro-8-oxo-5,6,7,8tetrahydronaphthalen-1-yl)acetamide I4 and N(2-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (709 mg, 2.86 mmol, 66%) as a white/yellow solid. LC/MS (method A): retention time 1.44 min (ES+) mass/charge 190 [M+H] ⁺ .

d) 8-Amino-5-nitro-3,4-dihydronaphthalene-1 (2H)-one (I5).

Mixture of N(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I4 and N-(2-hhtpo-8-okco5,6,7,8-tetrahydronaphthalen-1-yl )acetamide (709 mg, 2.8561 mmol) and 6 N. hydrochloric acid (7 ml) was stirred at 80°C for 2.5 hours, after which LCMS indicated completion of the reaction. The reaction mixture was cooled in an ice bath and 6 N. NaOH solution was added until the pH value became basic. The aqueous mixture was extracted with CH ₂ Cl ₂ and the organic phase was dried over MgSO ₄ and concentrated in vacuo. Purification with Isolera (0-50% EtOAc in iso-hexane) yielded 8-amino-5-nitro-3,4-dihydronaphthalene-1(2H)-one I5 (320 mg, 1.552 mmol, 54% yield) as a yellow/orange solid. LC/MS (method A): retention time 1.54 min (ES ⁺ ) mass/charge 207 [M+H] ⁺ .

e) 2,2,2-Trifluoro-N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)acetamide (I6).

8-Amino-5-nitro-3,4-dihydronaphthalene-1(2H)-one I5 (430 mg, 2.0854 mmol) was dissolved in dichloromethane (20 ml). Pyridine (340 μl, 4.20 mmol) was added and the mixture was cooled to 0°C. Trifluoroacetic anhydride (590 μL, 4.197 mmol) was added and stirred for 30 min, immediately after which LCMS indicated completion of the reaction. The mixture was diluted with CH2Cl2, washed with H2O, the organic phase was dried over MgSO ₄ and concentrated in vacuo to give 2,2,2-trifluoro-N(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalene -1-yl)acetamide I6 (630 mg, 2.0846 mmol, >99% yield) as a yellow solid, which was used without further purification. LC/MS (method A): retention time 1.86 min (ES ⁺ ) mass/charge 301X [M-H] ^- .

f) N(4-Amino-8-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)-2,2,2-trifluoroacetamide (I7).

Zinc (2.73 g, 41.7 mmol) was suspended in methanol (80 ml), formic acid (4 ml) and water (4 ml) and the mixture was cooled to 0°C. 2,2,2-Trifluoro-N-(4-nitro-8-oxo-5,6,7,8tetrahydronaphthalen-1-yl)acetamide I6 (568 mg, 2.0865 mmol) was added in portions and the mixture was stirred at 0° C for 30 min, immediately after which LCMS indicated completion of the reaction. The reaction mixture was filtered, the filtrate was diluted with EtOAc and washed with sat. NaHCO3. The organic phase was dried over MgSO ₄ and concentrated in vacuo to give H-(4-amino-8-oxo-5.6.7.8tetrahydronaphthalene-1-yl)-2,2,2-trifluoroacetamide I7 (568 mg, 2.0865 mmol, yield >99%) as a yellow solid, which was used without further purification. LC/MS (method A): retention time 1.65 min (ES ⁺ ) mass/charge 273 [M+H] ⁺ .

g) N(4-Acetamido-8-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)-2,2,2-trifluoroacetamide (I8).

Na(8-amino-4-oxotetralin-5-yl)-2,2,2-trifluoroacetamide I7 (568 mg, 2.0865 mmol) was dissolved in dichloromethane (20 ml). Triethylamine (580 μL, 4.16 mmol) was added followed by acetyl chloride (297 mL, 4.173 mmol) and the mixture was stirred for 30 min, immediately after which LCMS indicated completion of the reaction. The reaction mixture was diluted with CH ₂ Cl ₂ , washed with H ₂ O, the organic phase was dried over MgSO ₄ and concentrated in vacuo to give N(8-acetamido-4-oxotetralin-5yl)-2,2,2-trifluoroacetamide I8 (655 mg, 2.084 mmol, >99% yield) as a yellow solid, which was used without further purification. LC/MS (method A): retention time 1.55 min (ES ⁺ ) mass/charge 315 [M+H] ⁺ .

h) N(4-Amino-5-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)acetamide (I9).

Na(8-acetamido-4-oxotetralin-5-yl)-2,2,2-trifluoroacetamide I8 (2.77 g, 8.81 mmol) was dissolved in methanol (240 ml) and water (17 ml). Potassium carbonate (4.88 g, 35.3 mmol) was added and the mixture was stirred for 1.5 h at 50°C, immediately after which LCMS indicated completion of the reaction. The reaction mixture was cooled, concentrated in vacuo, dissolved in 10% MeOH in CH2Cl2 and washed with H2O. The organic phase was dried over MgSO4 and purified by chromatography using Isolera (2-15% MeOH in CH ₂ Cl ₂ ) to give 7V-(8-amino-1-oxotetralin-5-yl)acetamide I9 (1.20 g, 5 .50 mmol, yield 62.3%) as a yellow solid. LC/MS (method A): retention time 0.98 min (ES ⁺ ) mass/charge 219 [M+H] ⁺ .

- 29 046016

i) ^)-N-(9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-III,12H-benzo[de]pyrano[3',4 ':6,7]indolizino[1,2-b]quinolin-4-yl)acetamide (I10).

^-(8-amino-1-oxotetralin-5-yl)acetamide I9 (641 mg, 2.94 mmol, 1.0 eq.), ^)-4-ethyl-4-hydroxy7,8-dihydro-1H- pyrano[3,4-f]indolysine-3,6,10(4H)-trione A3 (840 mg, 3.19 mmol, 1.1 eq.) and PPTS (740 mg, 2.95 mmol, 1.0 eq.) was dissolved in toluene (60 ml) and stirred at reflux for 3 hours, immediately after which LCMS indicated that I9 was consumed. The reaction mixture was cooled and concentrated in vacuo. The resulting solids were triturated with acetonitrile and then acetone to give I10 as a brown solid with a minor trace of TsOH (1.26 g, 96%). LC/MS (method A): retention time 1.32 min. (ES+) mass/charge 447 [M+H] ⁺ .

j) ^)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7 ]indolizino[1,2-b]quinoline-10,13-dione (I11).

^)-Н-(9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-III, 12Hbenzo^e^irano^'L'^^indolizino^^ -Qinoline-yl-cetamide (I10) (1.26 g, 2.83 mmol, 1.0 eq.) was dissolved in hydrochloric acid (6 mol/l) in H ₂ O (12 ml) and the mixture was stirred for 5 hours at 80°C, immediately after which LCMS indicated that I10 was consumed.The reaction mixture was diluted with H ₂ O and concentrated in vacuo to give ^)-4-amino-9-ethyl-9-hydroxy-1, 2,3,9,12,15hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione I11 (1.51 g, 2.85 mmol, 90 wt.%, 101% yield) as a red crystalline solid. LC/MS (method A): retention time 1.36 min. (ES ⁺ ) mass/charge 405 [M+H] ⁺ .

Alternative route for synthesis of I11

IPC, purity control and analysis method for this synthesis

- 30 046016

a) 5-Bromo-8-nitrotetralin-1-one (I13).

A solution of potassium nitrate (1.15 eq., 13.83 g) dissolved in sulfuric acid (conc., 5.0 rel. vol., 160 ml) was added (addition time 4-12 hours, maintaining temperature below 10 °C) to a solution of 5bromotetralin-1-one (I12) (1.0 eq., 26.77 g) in sulfuric acid (conc., 5.0 rel. vol., 160 ml) under a nitrogen atmosphere. After completion of the reaction, the reaction mixture was transferred to a flask containing water (36 rel. vol., 1.15 L), adjusting the transfer rate to maintain the temperature below 10°C. The resulting solid was filtered, washed three times with water (4.0 rel. vol., 128 ml) and then dried at ~40°C for 24 hours. The dry residue was dissolved in a mixture of acetone (2.5 rel. vol., 80 ml ) and water (0.38 rel.

vol., 12.2 ml), heated to ~75°C, and then cooled to ~20°C. The resulting solid was removed by filtration. The solvent was replaced with ethanol by distillation and the volume of the solution was reduced to 2.0 rel. about. (64 ml). The solution was cooled to ~25°C and the resulting solid was collected by filtration. The solid was washed with ethanol (1.0 Rel. vol., 32 ml), then dried under vacuum at 40°C to obtain 5-bromo-8-nitrotetralin-1-one I13 (15.36 g, 40%) as brown solid; RT 14.0 min.

Method 1. IPC method,

-1-purity and analysis for bromine-8-

Device Thermo U-3000 Column ACE Excel 3 C18-PFP (3.0 mm x 150 mm) Thermostat 40°C Mobile phase A: 10 mM ammonium formate in water, pH=3.5 In: ACN Gradient program Time (min) A% B% 0.0 90 10 20.0 10 90 23.0 10 90 24.0 90 10 30.0 90 10 Re-equilibration time: 6 min Flow rate 1.0 ml/min Detector UV 220 nm Diluent ACN

b) N(8-nitro-1-oxotetralin-5-yl)acetamide (I14)

Solution of bromo-8-nitrotetralin-1-one (I13) (1.0 eq., 18.0 g, 90.6% w/w), acetamide (1.2 eq., 4.72 g), tris(dibenzylideneacetone)dipalladium(0) (0.01 eq., 0.61 g) and potassium phosphate (1.4 eq., 19.8 g) in dioxane (15 rel. vol., 270 ml) in atmosphere nitrogen was heated to ~70°C. After completion of the reaction, the solution was cooled to ~20°C, and also diluted with dioxane (5 rel. vol., 90.0 ml) and filtered. The solvent was replaced with ethanol and the volume was reduced to a total reaction volume of 3 rel. about. (54.0 ml). The solution was cooled to ~20°C and the resulting solid was collected by filtration and washed with MTBE (methyl tert-butyl ether) (1.0 Rel. vol., 18.0 ml). The solid was dried under vacuum at 40°C to obtain N(8-nitro-1-oxotetralin-5-yl)acetamide I14 (10.0 g, 60.6%) as a dark yellow solid; RT 8.86 min.

c) N(8-amino-1-oxotetralin-5-yl)acetamide (I15)

Palladium hydroxide on carbon (20% w/w, 0.15 eq., 5.25 g) was added to a solution of N-(8-hutpo-1oxotetralin-5-yl)acetamide (I14) (1.0 eq. , 32.6 g) in methanol (40 rel. vol., 1250 ml). The reaction mixture was placed under a hydrogen atmosphere at ~40 psi. inch and a temperature of ~40°C for 8 hours. Hydrogen was removed and replaced with nitrogen, and the catalyst was removed by filtration through cellulose, washing the cellulose with methanol (4.0 rel. vol., 130 ml). The volume of the solution was reduced to 4.0 rel. about. by distillation and then diluted with MTBE (4 Rel. vol. 130 ml). The resulting solid was collected by filtration, washed with MTBE (2 rel. vol., 65 ml) and dried under vacuum at 40°C to obtain N(8-amino-1-oxotetralin-5-yl)acetamide I15 (21, 1 g, 77.8%) as a gray-green solid; RT 5.44 min.

d) 5,8-Diaminotetralin-1-one (I16)

A solution of Na(8-amino-1-oxotetralin-5-yl)acetamide (I15) (1.0 eq., 10.0 g) in hydrochloric acid (5 M, 6.0 rel. vol., 60 ml) was kept at ~90°C for 3 hours. The temperature was lowered to 25°C and sodium hydroxide (2 M, 4.0 rel. vol., 40 ml) was added until a pH value of 10.0 was reached. while maintaining the temperature at 25°C. The resulting solid was collected by filtration and washed with water (2.0 rel. vol., 20 ml). The wet precipitate was dissolved in tetrahydrofuran (60 rel. vol., 600 ml) and filtered. The solution was concentrated to 5.0 rel. about. and heptane (20 rel. vol., 200 ml) was added. The solution was concentrated to 10.0 rel. about. and additional heptane was added (20 rel. vol., 200 ml), and then the volume was again reduced to 10.0 rel. about. The resulting solid was collected by filtration and washed with heptane (5.0 rel. vol., 50 ml). The solid was dried under

- 31 046016 under vacuum at 40°C for 17 hours to obtain 5,8-diaminotetralin-1-one (I16) (6.90 g, 82.7%) as a yellow solid; 1H NMR (400 MHz DMSO-d ₆ ) δ ppm 1.82 (m, 2H), 2.38 (t, J=2.0 Hz, 2H), 2.47 (t, J=2.0 Hz, 2H), 6.34 (d, J=2.0 Hz, 1H), 6.68 (d, J=2.0 Hz, 1H); RT 3.90.

e) ^)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H, 1311-benzo[de]pyrano [3',4': 6,7 ]indolizino[1,2-b]quinoline-10,13-dione (I11).

Solution of 5,8-diaminotetralin-1-one (I16) (1.0 eq., 5.0 g), ^)-4-ethyl-4-hydroxy-7,8-dihydro-1Hpirano[3,4-£ ]indolizine-3,6,10-trione (A3) (1.06 eq., 7.9 g) and pyridinium para-toluenesulfonate (1.0 eq., 7.2 g) in toluene (50.0 rel. vol., 250 ml) was kept at 120°C for 15 hours. The volume of the solution was reduced to 2.0 rel. about. and then diluted with acetonitrile (20 rel. vol., 100 ml) and water (20 rel. vol., 100 ml). The resulting slurry was filtered and the solid was washed with aqueous acetonitrile (1:1, 20 rel. vol., 100 ml). The solid was suspended in aqueous methanol (water:MeOH 3:1, 40 rel. vol., 200 ml), filtered and washed with aqueous methanol (1:1, 20 rel. vol., 100 ml). The solid was suspended in water (60 Rel. vol., 300 ml) at 50°C, filtered and washed with water (10 Rel. vol., 50 ml). The solid was suspended in aqueous acetonitrile (water:acetonitrile, 1:3, 40 rel. vol., 200 ml) at 30°C, filtered and washed with aqueous acetonitrile (water: acetonitrile, 1:3, 5 rel. vol., 50 ml) and then dried under vacuum at 40°C to obtain ^)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15hexahydro-10H,13H-benzo[de]pyrano[ 3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (I11) as a white solid (5.0 g, 43.7%); RT 5.13.

Synthesis of I18.

a) tert-Butyl-^)-(2-((2-((1-((2-((4-amino-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)amino) -2-oxo-ethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (I17)

Boc-GGFG-OH (227 mg, 0.52 mmol) and EEDQ (157 mg, 0.634 mmol) were solubilized in CH ₂ Cl ₂ (25 ml) and the mixture was stirred for 15 min until the peptide went into solution. Compound I16 (100 mg, 0.56747 mmol) was then added and the mixture was left to stir until the reaction was complete. Based on LVMC results after 1 hour, the reaction appeared to be 90% complete. The mixture became thicker as the product settled. The mixture was left for another hour before vacuum drying. The crude material was taken up in Et ₂ O (50 ml). The solid was filtered and then taken up in CH ₂ Cl ₂ (50 ml) for further purification. The solid was filtered and dried to give product I17 (273 mg, 0.459 mmol, 80.9% yield) as a gray solid. Analysis data: LCMS 3 min: ES ⁺ =1.46 min, mass/charge 595.7 [M+H] ⁺ .

b) ^)-2-(2-(2-Aminoacetamido)acetamido)^-(2-((^)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13 ,15hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-2-oxoethyl)-3phenylpropanamide (I18)

Aniline I17 (450 mg, 1.045 mmol), lactone A5 (280 mg, 1.064 mmol) and pyridinium n-toluenesulfonate (273 mg, 1.086 mmol) were solubilized in toluene (20 ml) and the mixture was heated to 150 °C (high reflux ). MeOH (4 mL) was added to assist solubilization of the mixture. After 7 hours, the crude reaction mixture was brought to dryness in vacuo.

The crude product was purified by silica gel chromatography (CHCl ₃ /MeOH, 100% to 65:35) to give product I18 (259 mg, 0.359 mmol, 78.1 yield). Analysis data: LCMS 3 min: ES ⁺ =1.17 min, mass/charge 722.8 [M+H] ⁺ .

An alternative method for the synthesis of I16.

a) 5-Fluoro-8-nitrotetralin-1-one (I20)

- 32 046016

5-Fluorotetralin-1-one I19 (4.7 g, 29 mmol) was solubilized in 1/2 sulfuric acid (120 mL) in a 3-neck round bottom flask. The mixture was stirred until the solid dissolved and then cooled to 0-5°C. In a dropping funnel, potassium nitrate (3 g, 29.6730 mmol) was dissolved in the remaining half of sulfuric acid (120 ml) at 0-5°C. Slowly add SM to the mixture, ensuring the solution remains cool (45 min). Stir at 0-5°C until the reaction is complete. The reaction mixture was then quenched with water (250 ml) and left to stir at 0-5°C. The solid was filtered and washed with water (50 ml). The solid was dried in a vacuum oven for 2 hours at 50°C. The crude solid was suspended in Et ₂ O overnight and then cooled to 0°C and filtered. The wet cake was washed with additional cold Et ₂ O (50 ml) and left to dry in a vacuum oven at 50°C to give pure product I20 (5.5 g, 26 mmol, 92% yield) as a light pink fine powder. LCMS (Method B): ES ⁺ =1.55 min, mass/charge 210.1 [M+H] ⁺ .

b) 5-Amino-8-nitrotetralin-1-one (I21)

Compound I20 (2.7 g, 13 mmol) was solubilized in CH3CN (2.5 ml) and NH4OH (21 wt%) in H ₂ O (8 ml, 40 mmol) added to a sealed pressure-resistant tube and heated to 185°C. Once complete, the mixture was transferred to a round bottom flask and vacuumed. The crude material was purified by silica gel column chromatography (CHCl ₃ /MeOH; 100 to 99:1) to give pure product I21 (1.1 g, 5.3 mmol, 41% yield) as a black solid. LCMS (Method B): ES ⁺ =1.34 min, mass/charge 207.1 [M+H] ⁺ .

c) 5,8-Diaminotetralin-1-one (I16)

Compound I21 (1.35 g, 6.55 mmol) was dissolved in a mixture of methanol (20 ml), H ₂ O (1 ml) and formic acid (1 ml) at 0°C. Zinc (8.5 g, 130 mmol) was added slowly, ensuring the temperature was maintained below 40°C. To speed up the completion of the reaction, an additional small amount of formic acid/H ₂ O (0.5 ml) was added. The reaction mixture was filtered and the filtrate was diluted with EtOAc and CH ₂ Cl ₂ followed by vacuum. The crude material was dry introduced into a silica gel column chromatography system (CHCl ₃ /EtOAc; 100 to 7:3, then CHCl ₃ /MeOH; 99:1 to 98:2) to give pure product I16 (1.015 g, 5.760 mmol, yield 88.0%). LCMS (Method B): ES ⁺ =0.2 min, mass/charge not observed.

Example 1.

a) Allyl-(^)-3-methyl-1-oxo-1-((^)-1-oxo-1-((5-oxo-4-(2,2,2-trifluoroacetamido)-5,6 ,7,8-tetra-hydronaphthalene-1-yl)amino)propan-2-yl)amino)butan-2-yl)carbamate (A1)

DCC (6.54 g, 31.7 mmol) and HOPO (3.36 g, 30.2 mmol) were added to a solution of alloc-Val-Ala-OH (9.09 g, 31.7 mmol) and I7 (7 .85 g, 28.8 mmol) in CH ₂ Cl ₂ (300 ml) at 25°C. The resulting mixture was left to stir overnight. The white solid that formed during the reaction was

- 33 046016 filtered and washed with cold CH2C12. The filtrate was washed with water (150 ml) and saline (150 ml). The organic layer was dried over MgSO ₄ , filtered and evaporated. The crude product was purified by silica gel chromatography (Hex/EtOAc, 60:40). The isolated product A1 contained an impurity in the form of co-eluted DCU (21.1 g, 140% yield). LC/MS (Method B): ES ⁺ =1.81 min, mass/charge 527.6 [M+H] ⁺ .

b) Allyl-(^)-1-((^)-1-((4-amino-5-oxo-5,6,7,8-tetrahydronaphthalene-1-yl)amino)-1-oxopropan-2yl) amino)-3 -methyl-1-oxobutan-2-yl)carbamate (A2)

Protected aniline A1 (18 g, 34.19 mmol) was solubilized in a 10:1 mixture of MeOH and H2O (165 ml) and K ₂ CO ₃ (10 g, 72.36 mmol) was added. The mixture was stirred at 50°C until the reaction was complete. The mixture was vacuumed until almost dry and the residue was taken up in CH2Cl2 and washed with H2O and brine, then dried over _MgSO4 , filtered and evaporated. The crude product was purified by silica gel chromatography (CHCl ₃ /MeOH, 100% to 7:3). The isolated product A2 contained a co-eluted impurity (10.71 g, 73% yield). LC/MS (Method B): ES ⁺ =1.46 min, mass/charge 431.7 [M+H] ⁺ .

c) Allyl-(^)-1-((^)-1-((^)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro- 1H,12H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3- methylbutan-2-yl)carbamate (A4)

Aniline A2 (450 mg, 1.045 mmol), lactone A3 (280 mg, 1.064 mmol) and pyridinium n-toluenesulfonate (273 mg, 1.086 mmol) were solubilized in toluene (20 ml) and the mixture was heated to 130 °C (high reflux ). To promote solubilization of the mixture, a few drops of MeOH were periodically added. After 7 hours, the crude reaction mixture was brought to dryness in vacuo. The crude product was purified by silica gel chromatography (CHCl ₃ /MeOH, 100% to 95:5) to give product A4 (360 mg, 52.3% yield). LC/MS (Method B): ES ⁺ =1.51 min, mass/charge 658.8 [M+H] ⁺ .

d) Allyl-^)-2-amino-H-(^)-1-((^)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15- hexahydro-

1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide ( A5)

Excess piperidine (642 μl) was added to a solution of A4 (543 mg, 0.82 mmol) and PdP(Ph ₃ ) ₄ (89 mg, 0.08 mmol) in CH ₂ Cl ₂ (15 ml). The mixture was left to stir at room temperature for 20 minutes, at which time the reaction was complete (as monitored by LC/MS). The reaction mixture was diluted with CH ₂ Cl ₂ (25 ml) and the organic phase was washed with H ₂ O (25 ml) and brine (25 ml). The organic phase was dried over MgSO4, filtered and excess solvent was removed by rotary evaporation under reduced pressure to give crude product A5, which was used without further purification in the next step. LC/MS (Method B): ES ⁺ =1.15 min, mass/charge 574.6 [M+H] ⁺ .

e) 1-(3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-((S)-1-(((S)-1-(( (S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexαhydro-1H,12H-benzo[de]pyrano[3',4':6, 7]indolizino[1,2-b]quinolin4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15 ,18,21,24-octaoxaheptacosane-27-amide (1).

Pyridine (83 µl, 1.03 mmol) and Mal-dPEG8-OTFP (767 mg, 1.03 mmol) were added to a solution of crude A5 (calculated amount 1.03 mmol) in dry CH ₂ Cl ₂ (50 ml) in an atmosphere argon. The reaction mixture was stirred overnight and, since the reaction was not complete, 0.5 eq. Mal-dPEG8-OTFP as an attempt to speed up the reaction. The reaction mixture was diluted with CH ₂ Cl ₂ (25 ml) and the organic phase was washed with H2O (2x50 ml) and brine, then dried over MgSO4, filtered and excess solvent was removed by rotary evaporation under reduced pressure. . The crude material was purified by reverse phase HPLC (H2O/CH3CN +0.05% FA gradient) and freeze-dried to give 1 (1.189 g, 31% yield after 2 steps). LC/MS (Method B): ES ⁺ =1.43 min, mass/charge 1149.3 [M+H] ⁺ . LC/MS (Method C): ES ⁺ =5.37 min, mass/charge 1149.4 [M+H] ⁺ .

- 34 046016

Example 2.

6-(2,5-Dioxo-2,5-dihydro-1 H-pyrrole-1 -un)-N-((S)-1 -(((S)-1 -(((S)-9- 3Tun-9-rugpoKCu-10,13-dioxo2,3,9,10,13,15-hexahydro-III,12H-benzo[de]pyrano[3',4':6,7]ivdolysino[1,2- b]quinolin-4-yl)amino)-1oxopropan-2-yl)amino)-3 -methyl-1-oxobutan-2-yl)hexanamide (2)

Mal-caproic acid (56 mg, 0.26 mmol) and EDCI.HCl (51 mg, 0.26 mmol) were added to a solution of crude A5 (calculated amount 0.26 mmol) in dry CH ₂ Cl ₂ (20 ml) in argon atmosphere. The reaction mixture was stirred overnight and, since the reaction was not complete, an additional 0.5 eq was added. Mal-caproic acid and EDCI.HCl. The reaction mixture was diluted with

CH ₂ Cl ₂ (25 ml) and the organic phase were washed with H ₂ O (2x50 ml) and brine, then dried over MgSO ₄ , filtered and excess solvent was removed by rotary evaporation under reduced pressure. The crude material was purified by silica gel column chromatography (CHCl ₃ /MeOH 95:5) to give 2 (31.6 mg, 20% yield after 2 steps). LC/MS (Method B): ES ⁺ =1.56 minutes, mass/charge 767.8 [M+H] ⁺ . LC/MS (Method C) 15 min: ES ⁺ =6.05 min, mass/charge 767.8 [M+H] ⁺ .

Example 3.

^)-2-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)acetamido)^-(^)-1 -((^)-9-ethyl-9-hydroxy-10,13dioxo- 2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4yl)amino)-1 -oxopropan-2-yl)-3-methylbutanamide (3).

Azido-dPEG ₃ -acid (77.5 mg, 0.31 mmol) and EDCI.HCl (60 mg, 0.31 mmol) were added to a solution of crude A5 (calculated amount 0.31 mmol) in dry CH ₂ Cl ₂ ( 20 ml) in an argon atmosphere. The reaction mixture was stirred overnight and, since the reaction was not complete, an additional 0.5 eq was added. azido-dPEG ₃ -OH and EDCI.HCl. The reaction mixture was diluted with CH ₂ Cl ₂ (25 ml) and the organic phase was washed with H ₂ O (2x50 ml) and brine, then dried over MgSO4, filtered and excess solvent was removed by rotary evaporation under reduced pressure by rotary evaporation at low pressure. The crude material was purified by preparative HPLC and the fractions were freeze-dried to give pure 3 (92.2 mg, 24.7% yield after 2 steps). LC/MS (Method B): ES ⁺ =1.69 min, mass/charge 789.9 [M+H] ⁺ . LC/MS (Method C): ES ⁺ =6.68 min, mass/charge 790.0 [M+H] ⁺ .

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Example 4.

N-((S)-1 -(((S)-1 -(((8)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro - 1H,12Hbenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl -1oxobutan-2-yl)-4,7,10,13,16-pentaoxanonedec-18-ynamide (4).

Propargyl-dPEG ₅ -acid (56 mg, 0.19 mmol) and EDCI.HCl (37 mg, 0.19 mmol) were added to a solution of crude A5 (calculated amount 0.19 mmol) in dry CH ₂ Cl ₂ (10 ml ) in an argon atmosphere. The reaction mixture was stirred overnight and, since the reaction was not complete, an additional 0.5 eq was added. propargyl-dPEG ₅ -OH and EDCI.HCl. The reaction mixture was diluted with CH ₂ Cl ₂ (25 ml) and the organic phase was washed with H2O (2x50 ml) and brine, then dried over MgSO ₄ , filtered and excess solvent was removed by rotary evaporation under reduced pressure. pressure. The crude material was purified by preparative HPLC and the fractions were freeze-dried to give pure 4 (22 mg, 16.7% yield after 2 steps). LC/MS (Method B): ES ⁺ =1.54 minutes, mass/charge 860.9 [M+H] ⁺ . LCMS (Method C): ES ⁺ =5.57 min, mass/charge 860.9 [M+H] ⁺ .

Example 5.

^)-2-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetamido)^-(^)-1-((^)-9 -ethyl-9hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-III,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2b ]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (5).

PM-acetic acid-OSu (64 mg, 0.19 mmol) was added to a solution of crude A5 (calculated amount 0.19 mmol) in dry CH ₂ Cl ₂ (10 ml) under argon. The reaction did not proceed, so DIPEA (51 μl, 0.28 mmol) was added. The reaction mixture was stirred until the reaction was complete. The mixture was diluted with CH ₂ Cl ₂ (25 ml) and the organic phase was washed with H2O (2x50 ml) and brine, then dried over MgSO ₄ , filtered and excess solvent was removed by rotary evaporation under reduced pressure. . The crude material was purified by preparative HPLC and the fractions were freeze-dried to give pure 5 (2.5 mg, 1.6% yield after 2 steps). LC/MS (Method B): ES ⁺ =1.54 min, mass/charge 787.7 [M+H] ⁺ . LC/MS (Method C): ES ⁺ = 5.61 min, mass/charge 787.8 [M+H] ⁺ .

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Example 6.

^)-2-((3-nitropyridin-2-yl)disulfanyl)propyl-(^)-9-ethyl-9-hydroxy-10,13-dioxo2,3,9,10,13,15-hexahydro-1H ,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbamate (6).

(i) (2R)-2-[(3-nitro-2-pyridyl)disulfanyl]propan-1-ol A6 (25 mg, 0.1015 mmol, 1.0 eq.) was dissolved in dichloromethane (1 ml). Pyridine (8.5 µl, 0.11 mmol, 1.0 eq) was added, then triphosgene (11 mg, 0.0370685 mmol, 0.33 eq) was added and the mixture was stirred under Ar for 45 min, immediately whereupon LCMS (quenching with Et ₂ NH) indicated the formation of the corresponding carbamate.

(ii) ^)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6, 7]indolizino[1,2-b]quinoline-10,13-dione (I11) (43 mg, 0.09026 mmol, 1.0 eq.) was dissolved in dichloromethane (2 ml), H^diisopropylethylamine (42 μl, 0.241 mmol, 2.7 eq.) and pyridine (25 μl, 0.309 mmol, 3.4 eq.). The reaction mixture from step (i) was added and the mixture was stirred for 30 minutes, immediately after which LCMS indicated completion of the reaction. The reaction mixture was concentrated in vacuo and purified by Isolera chromatography (0-4% MeOH in CH ₂ Cl ₂ ) to give 6 (22 mg, 0.03256 mmol, 36% yield, QC=96.8%) as yellow solid matter. LC/MS (Method B): RT=1.86 min, 676.6 [M+H]+.

Example 7.

6-(2,5-Dioxo-2,5-dihydro-III-pyrrol-1-yl)^-(2-((2-((^)-1-((2-((^)-9- ethyl-9-hydroxy-10,13dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-4yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide (7).

Compound I18 (259 mg, 0.3588 mmol) was solubilized in CH ₂ Cl ₂ (25 ml). The starting material was completely insoluble, so DMA (1 ml) was added. Since no improvement was observed, DIPEA (68 μl, 0.390 mmol) was added and all solid went into solution. Maleimide-caproic acid (69 mg, 0.358 mmol) was added and the mixture was left to stir at room temperature. overnight, at which time LCMS analysis indicated completion of the reaction. The reaction mixture was quenched with MeOH (2 ml) and vacuumed to dryness. The crude product was purified by preparative HPLC and then freeze-dried to give compound 7 as a brownish-yellow solid (38.2 mg, 11% yield). Analysis data: LCMS 3 min: ES ⁺ =1.47 min, mass/charge 916.2 [M+H]+. LCMS 15 min: ES ⁺ =5.46 min, mass/charge 916.1 [M+H]+.

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Example 8.

1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)^-(2-((2-((^)-1-((2-(( ^)-9-ethyl-9hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-III,12H-benzo[de]pyrano[3',4':6,7]indolizino [1,2^quinolineAyl^mino^-oxoethyl^mino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2oxoethyl)-3,6,9,12,15, 18,21,24-octaoxaheptacosane-27-amide (8).

Compound I18 (70 mg, 0.096 mmol) was solubilized in CH ₂ Cl ₂ (5 ml). The starting material was completely insoluble, so DMA (0.5 ml) was added. Since no improvement was observed, DIPEA (19 µl, 0.106 mmol) was added and all solid went into solution. Mal-dPEG ₈ -OH (63 mg, 0.106 mmol) and EDCI.HCl (19 mg, 0.099 mmol) were added and the mixture was left to stir at room temperature. overnight, after which LCMS analysis indicated completion of the reaction. The reaction mixture was quenched with MeOH (2 ml) and vacuumed to dryness. The crude product was purified by preparative HPLC and then freeze-dried to give 8 as a brownish-yellow solid (30 mg, 24% yield). LCMS 3 min: ES ⁺ =1.44 min, mass/charge 1297.6 [M+H] ⁺ .

Example 9 Alternative Synthesis Method 1

^)-4-Amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]ivdolisino [1,2-b]quinoline-10,13-dione I11 (371 mg, 0.779 mmol, 1.0 eq.) was dissolved in dichloromethane (30 ml). Added A^diisopropylethylamine (69 µl, 0.396 mmol, 0.51 eq) and (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]propanoylamino]-3-methylbutanoyl]amino]propane acid (664 mg, 0.871 mmol, 1.1 eq.) in A^dimethylacetamide (10 ml), then EDCI.HCl (226 mg, 1.18 mmol, 1.5 eq.) was added and the mixture was stirred for 2 h , immediately after which LCMS indicated sufficient conversion, but the reaction was noted to have stopped. The reaction mixture was heated to 30°C and stirred for 30 min, LCMS indicated no change, so CH ₂ Cl ₂ was removed in vacuo and Et ₂ O was added to the resulting DMA solution. The precipitated oil was collected, Et ₂ O was removed in vacuo and the deposition process was repeated. The combined precipitates were purified by HPLC (10-60% B in A for 13 min) to give 1 (200 mg, 0.174 mmol, 98% purity, 22% yield) as a yellow residue after freeze-drying. LC/MS (method A): retention time 1.44 min (ES+) mass/charge 1149 [M+H] ⁺ .

^1H NMR (600 MHz, chloroform-d) δ 8.81 (s, 1H), 7.83 (s, 2H), 7.48 (s, 1H), 7.18 (dd, J=18.7 , 7.5 Hz, 2H), 6.69 (s, 2H), 6.43 (s, 1H), 5.68 (d, J=16.1 Hz, 1H), 5.27 (d, J =16.1 Hz, 1H), 5.03 (d, J=18.4 Hz, 1H), 4.90 (d, J=18.4 Hz, 1H), 4.75 (p, J=7 ,2 Hz, 1H), 4.32 (dd, J=7.4, 5.8 Hz, 1H), 4.05 (s, 1H), 3.83 (t, J=7.2 Hz, 3H ), 3.783.68 (m, 3H), 3.68-3.57 (m, 31H), 3.53 (t, J=5.1 Hz, 3H), 3.40 (q, J=5, 3 Hz, 2H), 3.06-2.91 (m, 3H), 2.84 (dt, J=16.3, 6.2 Hz, 1H), 2.63 (ddd, J=14.8 , 8.5, 4.2 Hz, 1H), 2.57-2.44 (m, 4H), 2.30 (dq, J=13.4, 6.7 Hz, 1H), 2.10 ( p, J=6.4 Hz, 3H), 1.91 (ddt, J=16.8, 14.3, 7.2 Hz, 3H), 1.54 (d, J=7.1 Hz, 3H ), 1.02 (dd, J=15.5, 6.9 Hz, 10H).

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Example 10. Alternative method for the synthesis of A2

Allyl-(^)-1 -(((S)-1 -((4-amino-5-oxo-5,6,7,8-tetrahydronaphthalene-1 -yl)amino)-1-oxopropan-2yl)amino )-3-methyl-1-oxobutan-2-yl)carbamate (A2)

EDCI.HCl (7.71 g, 31.2 mmol) was added to a solution of alloc-Val-Ala-OH (8.49 g, 31.2 mmol) in CH ₂ Cl ₂ (200 ml) and stirred for 15 min or until solubilization is achieved. I16 (5 g, 28.3 mmol) was then added and the resulting mixture was left stirring until the reaction was complete. Volatiles were removed under reduced pressure. The crude product was taken up in Et ₂ O (50 ml) and the mixture was sonicated for 3 minutes. The solid was filtered and reabsorbed in CH ₂ Cl ₂ (50 ml), sonicated for 3 min and filtered again to give pure product A2 as a gray solid ES ⁺ =1.47 min, wt/charge 431.5[ M+H] ⁺ .

Example 11.

substances (12.21 g, yield 79%). LC/MS (Method B):

a) (9H-Fluoren-9-yl)methyl-K2-( 1 -(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-3 -oxo-

7,10,13,16,19,22,25,28-octaoxa-4-azagentriacontan-31 -oiu'i)-N5-((S)- 1 -(((S)-1 -((^) -9-ethyl-9-hydroxy10,13-dioxo-2,3,9,10,13,15-hexahydro-III,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)^-glutamate (A7) EDCI.HCl (0.10 mmol, 1.2 eq.) was added to a solution of A5 (0.087 mmol, 1.0 eq.) and Mal-PEG ₈ -Glu-OH (0.10 mmol, 1.2 eq.) in DCM (5 ml) and the resulting mixture stirred at room temperature overnight. The reaction mixture was evaporated to dryness and purified by column (8-12% MeOH/DCM) to give the product as a white solid. Yield=80 mg (63%). LC/MS (Method B) rt 1.66 min mass/charge (1456.2) M+H.

b) N2-( 1 -(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-3-oxo-7,10,13,16,19,22,25,28- octaoxa-4-azagentriacontane-31 -o iu'i)-N5-((S)- 1 -(((S)-1 -((^)-9-ethyl-9-hydroxy-10,13-dioxo- 2,3,9,10,13,15-hexahydro1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1 -oxopropan-2-yl)amino)-3methyl-1-oxobutan-2-yl)^-glutamine (9).

1-Methylpyrrolidine (200 µl) was added to a solution of A7 (0.06 mmol) in DMF (0.8 ml) and stirred at room temperature for 10 min. The solvent was removed in vacuo and the residue was purified by preparative HPLC (30% MeCN/water + 0.05% formic acid for 8.5 min). Fractions containing the product were freeze-dried to obtain the product as an off-white solid. Yield=23 mg (30%). LC/MS (Method B) rt 1.43 min mass/charge (1278.4) M+H.

Example 12 Conjugation

Antibody representing Herceptin-C2391

Herceptin antibodies designed to contain a cysteine inserted between positions 239 and 240 were prepared according to the methods described in Dimasi, N., et al., Molecular Pharmaceutics, 2017, 14, 1501-1516 (DOI: 5

10.1021/acs.molpharmaceut.6b00995).

ConjA mM solution of DL-dithiothreitol (DTT) in phosphate buffered saline pH 7.4 (PBS) was added (150 molar equivalents/antibody, 40 micromolar, 800 μl) to 10 ml of Herceptin-C239i antibody solution (40 mg, 267 nmol), in reconstitution buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA), at a final antibody concentration of

- 39 046016 giving 4.0 mg/ml. The reduction mixture was allowed to react at room temperature for 4 hours 45 minutes (or until complete reduction as determined by UHPLC) in an orbital shaker with gentle shaking (60 rpm). To remove all excess reducing agent, the reduced antibody was buffer exchanged with a reoxidation buffer containing PBS and 1 mM EDTA by spin filter centrifugation. A 50 mM solution of dehydroascorbic acid (DHAA, 20 molar equivalents/antibody, 5.33 μmol, 106.7 μl) in DMSO was added and the reoxidation mixture was allowed to react for 16 h at room temperature on low (60 rpm). min) shaking at an antibody concentration of 4 mg/ml (or additional DHAA was added and the reaction mixture was left for a longer time until the cysteine thiol groups were completely reoxidized to re-form interchain cysteine disulfide bonds, as determined by UHPLC). The reoxidation mixture was then sterilized filtered and diluted in conjugation buffer containing PBS and 1 mM EDTA to obtain a final antibody concentration of 3.6 mg/mL. Compound 1 was added as a solution in DMSO (10 molar equivalents/antibody, 1.33 μmol, in 0.55 ml DMSO) to 5.0 ml of this reoxidized antibody solution (20 mg, 133 nmol) to obtain the final concentration of DMSO, component 10% (v/v). The solution was stirred for 2 h at room temperature, then the conjugation reaction was quenched by the addition of N-acetylcysteine (6.67 μmol, 67 μl at 100 mM), then purified by spin filtration in PBS using an Amicon Ultracell 30 kDa MWCO spin filter with a volume of 15 ml, subjected to sterilization filtration and analyzed.

UHPLC analysis of the reconstituted ConjA sample using a Shimadzu Prominence system equipped with a Thermo Scientific MAbPac 50x2.1 mm column, eluting with a water-acetonitrile gradient, with detection at 214 and 330 nm (specific for compound 1), demonstrated the presence of unconjugated light chains and mixture unconjugated heavy chains and heavy chains attached to one molecule of Compound 1, which corresponded to a drug/antibody ratio (DAR) of 1.89 molecules of Compound 1 per antibody.

UHPLC analysis of ConjA sample using a Shimadzu Prominence system equipped with a Tosoh Bioscience TSKgel SuperSW mAb HTP column, 4 µm, 4.6 x 150 mm (with 4 µm pre-column, 3.0 x 20 mm), eluting at a flow rate of 0.3 ml/min s using filter-sterilized SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride, and 10% isopropanol (v/v) with detection at 280 nm demonstrated a monomeric purity of 98%. UHPLC SEC analysis showed that the final ConjA concentration was 2.14 mg/mL in 6.5 mL and the mass of the resulting ConjA was 13.9 mg (70% yield).

ConjA* mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate buffered saline pH 7.4 (PBS) was added (10 molar equivalents/antibody, 400 nanomoles, 40 μl) to 2.4 ml of antibody solution, which is trastuzumab (6 mg, 40 nanomoles), in reconstitution buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA), at a final antibody concentration of 2.5 mg/ml. The reduction mixture was allowed to react at room temperature for 16 hours (or until complete reduction as determined by UHPLC) in an orbital shaker with gentle shaking (60 rpm). The reduced antibody solution was buffer exchanged (to remove any excess reducing agent) with a conjugation buffer containing PBS and 1 mM EDTA by spin filter centrifugation to obtain a final antibody concentration of 2.0 mg/ml. Compound 1 was added as a solution in DMSO (20 molar equivalents/antibody, 400 nM, in 0.15 mL DMSO) to 1.35 mL of this reduced antibody solution (3 mg, 20 nM) to give a final DMSO concentration of 10% (v/v). The solution was mixed for 2 h at room temperature, then the conjugation reaction was quenched by adding N-acetylcysteine (2 micromolar, 20 μl at 100 mM), then purified by centrifugation using a 15 ml Amicon Ultracell 30 kDa MWCO spin filter, subjected to sterilization filtered and analyzed.

UHPLC analysis of the reconstituted ConjA* sample using a Shimadzu Prominence system equipped with a Thermo Scientific MAbPac 50x2.1 mm column, eluting with a gradient of water and acetonitrile, with detection at 214 and 330 nm (specific for compound 1), demonstrated the presence of a mixture of unconjugated light chains , light chains attached to one molecule of Compound 1, unconjugated heavy chains, and heavy chains attached to up to three molecules of Compound 1, which corresponded to a drug/antibody ratio (DAR) of 7.89 molecules of Compound 1 per antibody.

UHPLC analysis of a ConjA* sample using a Shimadzu Prominence system equipped with a Tosoh Bioscience TSKgel SuperSW mAb HTP column, 4 µm, 4.6 x 150 mm (with 4 µm pre-column, 3.0 x 20 mm), eluting at a flow rate of 0.3 ml/min using filter-sterilized SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol

- 40 046016 (v/v), detected at 280 nm, demonstrated a monomer purity of 98.5%. UHPLC SEC analysis showed the final ConjA* concentration to be 2.02 mg/mL in 1.25 mL and the mass of the resulting ConjA* to be 2.5 mg (84% yield).

ConjB mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate buffered saline pH 7.4 (PBS) was added (10 molar equivalents/antibody, 3.56 micromolar, 356 μl) to 11.1 ml of antibody solution , which is trastuzumab (53.4 mg, 356 nanomoles), in reconstitution buffer containing PBS pH 7.4 and 1 mM ethylenediaminetetraacetic acid (EDTA), at a final antibody concentration of 4.84 mg/ml. The reduction mixture was allowed to react at 37°C for 1 hour 30 minutes (or until complete reduction as determined by UHPLC) in an orbital shaker with gentle shaking (60 rpm). Compound 2 was added as a solution in DMSO (15 molar equivalents/antibody, 5.1 micromolar, in 1.2 ml DMSO) to 10.5 ml of this reduced antibody solution (50.8 mg, 339 nanomolar) to obtain the final concentration of DMSO , constituting 10% (vol./vol.). The solution was mixed for 1 hour 30 minutes at room temperature, then the conjugation reaction was quenched by adding N-acetylcysteine (25.4 micromolar, 254 μl at 100 mM), then purified by AKTA™ Start FPLC using a GE Healthcare HiLoad™ 26 column. 600 filled with Superdex 200 PG, eluting at a flow rate of 2.6 ml/min with PBS. Fractions corresponding to the ConjB monomer peak were pooled, concentrated, and buffer exchanged to pH 6.0 buffer containing 25 mM histidine and 205 mM sucrose using an Amicon Ultracell 50 kDa MWCO 15 mL spin filter, sterile filtered, and analyzed.

UHPLC analysis of the reconstituted ConjB sample using a Shimadzu Prominence system equipped with a Thermo Scientific MAbPac 50x2.1 mm column, eluting with a gradient of water and acetonitrile, with detection at 214 and 330 nm (specific for compound 2), demonstrated the presence of a mixture of unconjugated light chains, light chains attached to one molecule of Compound 2, unconjugated heavy chains, and heavy chains attached to up to three molecules of Compound 2, which corresponded to a drug/antibody ratio (DAR) of 7.93 molecules of Compound 2 per antibody.

UHPLC analysis of a ConjA* sample using a Shimadzu Prominence system equipped with a Tosoh Bioscience TSKgel SuperSW mAb HTP column, 4 µm, 4.6 x 150 mm (with 4 µm pre-column, 3.0 x 20 mm), eluting at a flow rate of 0.3 ml/min using filter-sterilized SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v), with detection at 280 nm, demonstrated a monomeric purity of 98.9% . UHPLC SEC analysis showed the final ConjB concentration to be 2.4 mg/mL in 16 mL and the mass of the resulting ConjB to be 38.4 mg (84% yield).

ConjC mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate buffered saline pH 7.4 (PBS) was added (40 molar equivalents/antibody, 11.2 micromolar, 1.12 ml) to 20 ml of antibody solution , which is Herceptin-C239i (42 mg, 280 nanomoles), in reconstitution buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA), at a final antibody concentration of 2.1 mg/ml. The reduction mixture was allowed to react at room temperature for 16 hours (or until complete reduction as determined by UHPLC) in an orbital shaker with gentle shaking (60 rpm). To remove all excess reducing agent, the reduced antibody was buffer exchanged with a reoxidation buffer containing PBS and 1 mM EDTA by spin filter centrifugation. A 50 mM solution of dehydroascorbic acid (DHAA, 30 molar equivalents/antibody, 7.0 micromolar, 141 μl) in DMSO was added to 22 ml of this reconstituted buffer-exchanged antibody (35.2 mg, 235 nanomolar) and the mixture for reoxidation allowed to react for 2 hours and 30 minutes at room temperature with gentle shaking (60 rpm) at an antibody concentration of 1.6 mg/ml (or additional DHAA was added and the reaction mixture was left for a longer time until complete re-oxidation of cysteine thiol groups with re-formation of interchain cysteine disulfide bonds, determined by UHPLC). The re-oxidation mixture was then subjected to sterilizing filtration. Compound 6 was added as a DMSO solution (20 molar equivalents/antibody, 2.3 micromolar, in 1.36 ml DMSO) to 11.0 ml of this reoxidized antibody solution (17.6 mg, 117 nanomolar), pH adjusted with using 1.22 ml 1 M sodium bicarbonate to obtain a final concentration of DMSO of 10% (v/v) and 10% (v/v) 1 M sodium bicarbonate. The solution was left to react at room temperature for 2 hours with gentle shaking. The conjugation reaction was then quenched by the addition of N-acetylcysteine (12 microM, 117 μl at 100 mM), then purified and buffer exchanged with a pH 6.0 buffer containing 25 mM histidine and 205 mM sucrose using an Amicon Ultracell 50 spin filter. kDa MWCO 15 ml, under

- 41 046016 were subjected to sterilizing filtration and analyzed.

UHPLC analysis of an intact Conj C sample using a Shimadzu Prominence system equipped with a Sepax Proteomix HIC butyl-No. 5 4.6x35 mm, 5 µm column, eluting with a gradient of pH 7.4 buffer containing 25 mM sodium phosphate, 1.5 M sulfate ammonium, and 20% acetonitrile (v/v) in 25 mM sodium phosphate buffer pH 7.4, with detection at 214 and 330 nm (specific for compound 6), demonstrated the presence of unconjugated and conjugated antibody attached to one or two molecules of compound 6, which corresponded to a drug/antibody ratio (DAR) of 1.42 molecules of compound 6 per antibody.

UHPLC analysis of ConjC sample using a Shimadzu Prominence system equipped with a Tosoh Bioscience TSKgel SuperSW mAb HTP column, 4 µm, 4.6 x 150 mm (with 4 µm pre-column, 3.0 x 20 mm), eluting at a flow rate of 0.3 ml/min s using filter-sterilized SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride, and 10% isopropanol (v/v), detected at 280 nm, demonstrated monomer purity of 98%. UHPLC SEC analysis showed the final ConjC concentration to be 1.06 mg/mL in 10.1 mL and the mass of the resulting ConjC to be 10.7 mg (61% yield).

Example 13 In vitro analysis.

The solid test material was dissolved in DMSO to produce a 2 mM stock solution, from which eight serial dilutions were made in DMSO at a ratio of 1:10 and stored at -20°C until use.

Adherent NCI-N87 cells were washed with D-PBS and separated with trypsin-EDTA, then cell density and viability were determined in duplicate by a trypan blue exclusion assay using an automated cell counter (LUNAII™). The cell suspension was diluted to 1x105 cells/mL in growth medium (RPMI 1640 with Glutamax + 10% (v/v) HyClone™ fetal bovine serum) and vortexed before dispensing at 2 mL per well into sterile polypropylene plates containing well volume 3 ml. The target dilutions were then added to the appropriate wells at a rate of 10 μl/well and mixed by repeated pipetting. In the case of control wells, 10 μl of DMSO was added to a 2 ml cell suspension and mixed thoroughly. Then, 100 μl of each sample was aliquoted into duplicate wells of a sterile 96-well flat-bottom microplate and incubated at 37°C in a CO2 (5%) incubator. At the end of the incubation period (7 days), cell viability was measured using the CellTiter 96™ aqueous One (MTS) assay, added at 20 μl/well and incubated for 4 hours at 37°C, 5% CO ₂ . The plates were then read using an EnVision™ multichannel plate reader (Perkin Elmer) using absorbance at 490 nm.

The percentage of surviving cells was calculated based on the average absorbance of 2 replicate wells for each sample compared to the average absorbance of two control wells treated with DMSO alone (100%). IC ₅₀ was determined by fitting each data set to variable-slope sigmoidal dose-response curves using a nonlinear fitting algorithm in GraphPad Prism software (San Diego, CA).

All experiments described in this report were performed and tested in three independent experiments. Data are given as the average of three independent replicates.

1C ₅₀ (nM) Ill 0.3854

Example 14 In vitro ADC assay.

The concentration and viability of cells from a subconfluent culture (8090% confluent) in the T75 mattress were measured by trypan blue staining and counted using a LUNA-II™ automated cell counter. Cells were diluted to ^2x105 /ml and distributed (50 µl per well) into 96-well flat-bottom plates.

A stock solution (1 mL) of antibody-drug conjugate (ADC) (20 μg/mL) was prepared by diluting filter-sterilized ADC in cell culture medium. A set of 8x 10-fold dilutions of the ADC stock solution was prepared in a 24-well plate by serially transferring 100 μL into 900 μL cell culture medium. Dilutions of ADC were added (at a rate of 50 μl per well) into 4 replicate wells in a 96-well plate containing 50 μl of the cell suspension previously seeded there. 50 μl of cell culture medium was added to control wells. A 96-well plate containing cells and ADCs was incubated at 37°C in a CO ₂ -saturated incubator for the exposure period.

At the end of the incubation period, cell viability was measured by MTS assay. MTS (Promega) was added (at a rate of 20 μl per well) to each well and incubated for 4 h at 37°C in a CO ₂ -saturated incubator. Absorbance in the wells was measured at 490 nm. The percentage of surviving cells was calculated based on the average absorbance of 4 ADC-treated wells, according to

- 42 046016 compared to the average absorbance of 4 control wells without treatment (100%). IC ₅₀ was determined from dose-response data using GraphPad Prism using a non-linear fitting algorithm: sigmoidal dose-response curves with variable slope.

Incubation periods with ADC were 4 days for 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™ fetal bovine serum. NCI-N87 is a Her2-expressing cell line, and MDA-MB-468 is a Her2-negative cell line.

ECzo (mcg/ml) NCI-N87 MDA-MB-468 ConjA 0.1176 >10 ConjA* 0.01634 >10 ConjB 0.01857 >10 ConjC 0.1452 >10

Example 15 In Vivo ADC Assay

Methods and materials

Mice.

At Day 1 of the study, female severe combined immunodeficiency mice (Fox Chase SCID™, CB17/Icr-Prkdcscid/IcoIcrCrl, Charles River) were eight weeks old and had a body weight (BW) of 14.5–20.0 g. Animals were provided with ad libitum access to water (reverse osmosis treated, 1 ppm Cl) and a modified and irradiated NIH 31™ laboratory diet containing 18.0% total protein, 5.0% total fat, and 5.0% total fiber. . Mice were housed on irradiated Enrich-o'cobsTM laboratory animal bedding in fixed microisolators under a 12-hour light cycle at 20-22°C and 40-60% humidity. The CR Research Service strictly follows the recommendations of the Guide for the Care and Use of Laboratory Animals regarding housing, breeding, surgical procedures, control of feeding and watering, and the provision of veterinary care. The animal care and use program at CR Research Services is accredited by the International Association for the Assessment and Accreditation of Laboratory Animal Research (AAALAC), ensuring compliance with accepted standards for the care and use of laboratory animals.

Tumor cell culture

NCI-N87 human gastric carcinoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin sulfate, and 25 μg/ml gentamicin. Cells were grown in tissue culture mats in a humidified incubator at 37°C under an atmosphere of 5% CO ₂ and 95% air.

In vivo implantation and tumor growth

NCI-N87 cells used for implantation were harvested at logarithmic growth phase and resuspended in phosphate-buffered saline (PBS) containing 50% Matrigel™ (BD Biosciences). On the day of tumor implantation, each experimental mouse was given 1 x 10 ⁷ cells (0.1 ml cell suspension) by subcutaneous injection into the right flank, and tumor growth was monitored as the average size approached the target range of 100-150 mm ³ . Twelve days later, on a day designated as study day 1, mice were divided into fourteen groups based on calculated tumor size, with seven groups designated for efficacy assessment (n=10) and seven groups designated for sample collection (n=3). each group consisted of animals with individual tumor volumes ranging from 108-172 mm ³ and group average tumor volumes ranging from 120-124 mm ³ . Tumors were measured in two directions using a caliper, and the volume was calculated using the formula:

Tumor volume (mm ³ )=(w ² x1)/2, where w = width and l = tumor length in mm. Tumor weight can be calculated based on the assumption that 1 mg is equivalent to 1 mm ³ tumor volume.

Therapeutics

ConjA* was stored protected from light at 4°C. Sterile PBS was used to dose the vehicle control group.

Treatment

On day 1 of the study, female SCID mice carrying established NCIN87 xenografts were divided into groups. An aliquot of the stock solution was diluted with PBS to the appropriate concentration. The drug was administered intravenously by injection into the tail vein once on day 1. The dose volume was 0.2 ml per 20 g of body weight (10 ml/kg) and was recalculated relative to

- 43 046016 body weight for each individual animal.

Mice in group 1 received vehicle PBS and served as a control group. Group 2 received ConjA* at a dose of 4 mg/kg.

Tumors were measured using calipers twice a week, and each animal was euthanized when its tumor reached the end point volume of 800 mm ³ or at the end of the study (day 68), whichever came first. Animals that left the study due to reaching the tumor volume endpoint were recorded as euthanized due to tumor progression (TP) and the date of euthanasia was indicated.

Response criteria in the form of regression

The effectiveness of the treatment can be determined based on the frequency of occurrence and magnitude of regression responses observed during the study. The treatment may cause partial regression (PR) or complete regression (CR) of the tumor in the animal. For a PR response, tumor volume was 50% or less of its volume on day 1 on three consecutive measurements throughout the study, and was equal to or greater than 13.5 mm ³ on one or more of these three measurements. For a CR response, the tumor volume was less than 13.5 mm ³ on three consecutive measurements throughout the study. Animals were assessed for a PR or CR event only once during the study, and were assessed as CR only if criteria for both PR and CR were met. An animal with a CR response at study completion was further classified as a tumor-free survivor (TFS). Animals were monitored for response occurrence in the form of regression.

Toxicity

Animals were weighed daily on days 1–5, then twice weekly until completion of the study. Mice were observed frequently for significant signs of any treatment-related (TR) adverse events, and clinical symptoms were recorded when observed. The body weight of each individual animal was monitored according to the protocol, and any animal with a body weight loss that exceeded 30% in one measurement or exceeded 25% in three consecutive measurements was euthanized and recorded as death due to TR. Group mean weight loss was also monitored according to CR Research Services protocol. Tolerable toxicity was defined as a group mean body weight (BW) loss of less than 20% during the study and the presence of no more than 10% deaths due to TR.

results

In fig. 1 shows graphs of average tumor growth, where

Medium carrier • ConjA* ♦

Mice in group 1 received PBS vehicle intravenously with qdx1 and served as a control group. The median TTE for group 1 was 24.8 days. In all control animals, tumors reached the end point of 800 mm ³ .

Group 2 received ConjA* at a dose of 4 mg/kg IV qdx1. CR was observed in all 10 mice, which were further classified as TFS at study completion.

In the treatment group, the maximum weight loss was -9.5% on day 50 of the study.

No deaths due to TR were observed.

Summary of the invention

1. Compound of formula I

and salts and solvates thereof, wherein RL is a linker for attachment to a ligand unit selected from (ia)

where Q represents

- 44 046016 where Q ^x is such that Q represents an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;

X represents

where a equals 0-5, b1 equals 0-16, b2 equals 0-16, c1 equals 0 or 1, c2 equals 0 or 1, d equals 0-5, where at least b1 or b2 equals 0 and at least c1 or c2 equals 0;

GL is a linker for attachment to a ligand unit; (ib)

wherein R ^L1 and R ^L2 are independently selected from H and methyl or, together with the carbon atom to which they are bonded, form a cyclopropylene or cyclobutylene group; and e equals 0 or 1.

2. A compound according to claim 1, wherein R ^L is represented by formula Ia.

3. A compound according to claim 2, where Q is an amino acid residue.

4. A compound according to claim 3, wherein Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg and Trp.

5. A compound according to claim 2, wherein Q is a dipeptide residue.

6. The compound according to claim 5, where Q is selected from ^NH -Phe-Lys- ^C = ^O , ^NH -Val-Ala- ^C '°, ^NH -Val-Lys- ^C -°,

NH-Ala-Lys- ^C =°,

NH-Val-Cit-C ⁰ , ^NH -Phe-Cit- ^< =°_

NH-Leu-Cit- ^C =°,

NH-Ile-Cit-C· ⁰ , ^NII -Phe-Arg- ^<0

NH-Trp-Cit-C ⁰ and ^NH -Gly-Val- ^C0

7. The compound according to claim 6, where Q is selected from ^NH -Phe-Lys- ^C =°, ^NH -Val-Cit-C· ⁰ and NH-Val-Ala-C· ⁰ .

8. A compound according to claim 2, wherein Q is a tripeptide residue.

9. The connection according to claim 8, where Q is selected from

NH-Glu-Val-Ala-C· ⁰ , NH-Glu-Val-Cit-C· ⁰ , NH-aGlu-Val-Ala-C· ⁰ and NH-aGlu-Val-Cit-C· ⁰

10. A compound according to claim 2, wherein Q is a tetrapeptide residue.

11. The compound according to claim 10, where Q is selected from NH-Gly-Gly-Phe-GlyC· ⁰ and NH-Gly-Phe-Gly-GlyC· ⁰

12. The compound according to claim 11, where Q is NH-Gly-Gly-Phe-GlyC· ⁰ .

13. The connection according to any of paragraphs 2-12, where a equals 0-3.

14. The connection according to claim 13, where a is 0 or 1.

15. Connection according to claim 13, where a equals 0.

16. Connection according to any of paragraphs 2-15, where b1 equals 0-8.

17. Connection according to item 16, where b1 equals 0.

18. Connection according to claim 16, where b1 equals 2.

19. Connection according to claim 16, where b1 equals 3.

20. Connection according to claim 16, where b1 equals 4.

21. Connection according to claim 16, where b1 equals 5.

22. Connection according to claim 16, where b1 equals 8.

23. The connection according to any of paragraphs 2-15 and 17, where b2 is equal to 0-8.

24. Connection according to item 23, where b2 is equal to 0.

25. Connection according to claim 23, where b2 equals 2.

26. Connection according to claim 23, where b2 equals 3.

27. Connection according to item 23, where b2 equals 4.

28. Connection according to item 23, where b2 equals 5.

29. Connection according to item 23, where b2 equals 8.

30. Connection according to any one of paragraphs 2-29, where c1 equals 0.

31. The connection according to any of paragraphs 2-29, where c1 equals 1.

32. The connection according to any one of paragraphs 2-31, where c2 is equal to 0.

33. The connection according to any one of claims 2-30, where c2 equals 1.

34. The connection according to any one of paragraphs 2-33, where d is equal to 0-3.

- 45 046016

35. The connection according to claim 34, where d is 1 or 2.

36. The connection according to claim 34, where d is equal to 2.

37. The connection according to any one of paragraphs 2-33, where d is equal to 5.

38. A compound as claimed in any one of claims 2 to 12, wherein a is 0, b1 is 0, c1 is 1, c2 is 0, and d is 2, and b2 is 0-8.

39. The connection according to item 38, where b2 is equal to 0, 2, 3, 4, 5 or 8.

40. A compound as claimed in any one of claims 2 to 12, wherein a is 1, b2 is 0, c1 is 0, c2 is 0, and d is 0, and b1 is 0-8.

41. The connection according to claim 40, where b1 is 0, 2, 3, 4, 5 or 8.

42. The connection according to any one of claims 2 to 12, wherein a is 0, b1 is 0, c1 is 0, c2 is 0, and d is 1, and b2 is 0-8.

43. The connection according to item 42, where b2 is equal to 0, 2, 3, 4, 5 or 8.

44. A compound as claimed in any one of claims 2 to 12, wherein b1 is 0, b2 is 0, c1 is 0, c2 is 0, one of a and d is 0, and the other of a and d is 1-5.

45. The compound of claim 44, wherein the other of a and d is 1 or 5.

46. A compound as claimed in any one of claims 2 to 12, wherein a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 is 0-8.

47. The connection according to item 46, where b1 is equal to 0, 2, 3, 4, 5 or 8.

48. The connection according to any one of claims 2 to 47, where GL is selected from

O ((N ABOUT o ,°L N ((E ¹ - ² ) (G') Vg— (G ¹² ) O ( ^GLS ) Y (G ¹ - ³ - ¹ ) S—s^ ¹ ¢- (NO2) where the NO2 group is optional (G ¹ - ⁹ ) N ₃ (G ¹² ) s—s^ ¹ (NO2) where the NO2 group is optional (G ¹¹⁰ ) ^ZCK / Η (G'N S— o ₂ n\^/ where the NO2 group is optional ((E ¹¹ ) oh oh w (G ¹ - ³ - ⁴ ) S— m where the NO2 group is optional (G ¹¹² ) (G ¹⁴ ) __ ABOUT Hal J where Hal represents I, Br, C1 (G ¹¹³ ) xV ^N \ \\ /X/ (G'N About Hal— (G ¹¹⁴ ) _H2N \ ^O5 '

where Ar represents a ^^arylene group and X represents C _1- ^k^.

49. The connection according to claim 48, where GL is selected from GL ^1-1 and GL ^1-2 .

50. A compound according to claim 48, wherein GL is GL ^1-1 .

51. A compound according to claim 1, wherein RL is represented by formula Ib.

52. The compound according to claim 51, where R ^L1 and R ^L2 simultaneously represent H.

- 46 046016

53. A compound according to claim 51, wherein R ^L1 is H and R ^L2 is methyl.

54. The compound according to claim 51, wherein R ^L1 and R ^L2 are both methyl.

55. A compound according to claim 51, wherein R ^L1 and R ^L2 together with the carbon atom to which they are bonded form a cyclopropylene group.

56. A compound according to claim 51, wherein R ^L1 and R ^L2 together with the carbon atom to which they are bonded form a cyclobutylene group.

57. The connection according to any one of paragraphs 51-56, where e equals 0.

58. The connection according to any one of paragraphs 51-56, where e equals 1.

59. Conjugate of formula IV

L-(D ^L ) _P (IV), or a pharmaceutically acceptable salt or solvate thereof, where L represents a ligand unit (i.e., a targeting agent), D ^L represents a drug-linker unit represented by formula III

R ^ll is a linker attached to a ligand unit selected from (ia'):

wherein Q and X are as defined in any one of claims 1 to 47, and G ^LL is a linker attached to a ligand unit; and (ib'):

where R ^L1 and R ^L2 are as defined in any of paragraphs 1 and 52-56; and p is an integer from 1 to 20.

60. The conjugate according to claim 59, where G ^LL is selected from

- 47 046016

(G ^IJM ) 0 CBA$ _N A ^x o (G ¹ - ¹¹ - ² ) About Vl Ar J. CBA| N u ^{h about} (GLL2) 0 CBA| /Χ'ΊΨ V-k 0 0 (GLL3-!) sirH ( ^GLL3-2 ) (G^- ⁴ ) CBA____ A. 0 (G^ ⁵ ) 0 CBA|___// (G^ ⁶ ) 0 •4 (G^ ⁷ ) CBAj

(GU-s-i) C.B.A. (G^s- ² ) N,CBA DH* ( ^GLL9-1 ) ^N ⁴ _N CBA ( ^GLL9-2 ) ^N /nA CBA (GLLW) ,-- _v n—4 i-^-4 H (GLL11) <J\O (G^ ¹² ) Li ^HN \^x /7 X (GLL13) H ⁿ -n CBA (QLL ¹⁴ ) H 4 cba\/A _o A

where Ar represents a C _5-6 arylene group and X represents a C _1-4 alkyl.

61. The conjugate according to claim 60, where G ^LL is selected from G ^LL1-1 and G ^LL1-2 .

62. The conjugate according to claim 61, wherein G ^LL is G ^LL1-1 .

63. The conjugate according to any one of claims 59 to 62, wherein the ligand moiety is a cell binding agent.

64. The conjugate according to any one of claims 59 to 62, wherein the ligand unit is an antibody or an active fragment thereof.

65. The conjugate of claim 64, wherein the antibody or antibody fragment is an antibody or antibody fragment to a tumor-associated antigen.

66. The conjugate of claim 65, wherein the antibody or antibody fragment is an antibody that binds to one or more tumor-associated antigens or cell surface receptors selected from (1)-(89):

(1) BMPR1B;

(2) E16;

(3) STEAP1;

(4) 0772Р;

(5) MPF;

(6) Napi3b;

(7) Sema 5b;

(8) PSCA hlg;

(9) ETBR;

(10) MSG783;

(11) STEAP2;

(12) TrpM4;

(13) CRIPTO;

(14)CD21;

(15) CD79b;

- 48 046016 (16) FcRH2;

(17) HER2;

(18) NCA;

(19) MDP;

(20) Ш20И-alpha;

(21) brevican;

(22) EphB2R;

(23) ASLG659;

(24) PSCA;

(25) GEDA;

(26) BAFF-R;

(27) CD22;

(28) CD79a;

(29) CXCR5;

(30) HLA-DOB;

(31) P2X5;

(32) CD72;

(33) LY64;

(34) FcRH1;

(35) IRTA2;

(36) TENB2;

(37) PSMA - FOLH1;

(38) SST;

(38 .1) SSTR2;

(38 .2) SSTR5;

(38 .3) SSTR1;

(38 .4)SSTR3;

(38 .5) SSTR4;

(39) ITGAV;

(40)ITGB6;

(41) CEACAM5;

(42) MET;

(43) MUC1;

(44) CA9;

(45) EGFRvIII;

(46) CD33;

(47) CD19;

(48) IL2RA;

(49) AXL;

(50) CD30 - TNFRSF8;

(51) BCMA - TNFRSF17;

(52) CT Ag - CTA;

(53) CD174 (Lewis Y) - FUT3;

(54) CLEC14A;

(55) GRP78-HSPA5;

(56) CD70;

(57) stem cell specific antigens;

(58)ASG-5;

(59) ENPP3;

(60) PRR4;

(61) GCC - GUCY2C;

(62) Liv-1-SLC39A6;

(63) 5T4;

(64) CD56 - NCMA1;

(65) CanAg;

(66) FOLR1;

(67) GPNMB;

(68) TIM-1 - HAVCR1;

(69) RG-1/mindin, prostate tumor target is mindin/RG-I;

(70) B7-H4 - VTCN1;

(71) PTK7;

(72) CD37;

- 49 046016 (73) CD138 - SDC1;

(74) CD74;

(75) claudins - CL;

(76) EGFR;

(77) Her3;

(78) RON - MST1R;

(79) EPHA2;

(80) CD20 - MS4Al;

(81) tenascin C - TNC;

(82) FAP;

(83) DKK-1;

(84) CD52;

(85) CS1 - SLAMF7;

(86) endoglin - ENG;

(87) annexin A1 - ANXA1;

(88) V-CAM (CD106) - VCAM1;

(89) ASCT2(SLC1A5).

67. The conjugate according to any one of claims 64 to 66, wherein the antibody or antibody fragment is an antibody constructed with cysteine.

68. The conjugate according to any one of claims 64 to 67, wherein the drug load (p) for the drugs (D) on the antibody (Ab) is an integer from 1 to about 10.

69. The conjugate according to claim 68, where p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

70. The mixture of conjugates according to any one of claims 64 to 69, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is from about 1 to about 10.

71. Conjugate or mixture according to any one of claims 59-70 for use in therapy.

72. A pharmaceutical composition containing a conjugate or mixture according to any one of claims 59 to 70 and a pharmaceutically acceptable diluent, carrier or excipient.

73. A conjugate or mixture according to any one of claims 59 to 70 or a pharmaceutical composition according to claim 72 for use in the treatment of a proliferative disease in a subject.

74. The conjugate, mixture or pharmaceutical composition according to claim 73, wherein the disease is cancer.

75. Use of a conjugate or mixture according to any one of claims 59-70 or a pharmaceutical composition according to claim 72 in a method of conservative treatment.

76. A method of conservative treatment, including administering to the patient a pharmaceutical composition according to claim 72.

77. The method according to claim 76, wherein the conservative treatment method is for treating cancer.

78. The method of claim 77, wherein the patient is administered a chemotherapeutic agent in combination with a conjugate.

79. Use of a conjugate or mixture according to any one of claims 59 to 70 in a method for the manufacture of a medicinal product for the treatment of a proliferative disease.

80. A method of treating a mammal suffering from a proliferative disease, comprising administering an effective amount of a conjugate or mixture according to any one of claims 59 to 70 or a pharmaceutical composition according to claim 72.

81. Compound A

as a single enantiomer or in an enantiomerically enriched form. 82. Compound of formula VI

where Q is as described in any of claims 1, 3 and 12.

- 50 046016

Statement of the essence of the invention from the 1st application, on the basis of which priority is claimed (P1)

P1 -1. Compound of formula I

and salts and solvates thereof, wherein R ^L is a linker for attachment to a cell binding agent selected from:

(ia):

where Q represents

where Q ^X is such that Q is an amino acid residue, a dipeptide residue or a tripeptide residue; X represents

where a equals 0-5, b equals 0-16, c equals 0 or 1, d equals 0-5;

G ^l is a linker for attachment to a ligand unit; (ib):

wherein R ^L1 and R ^L2 are independently selected from H and methyl or, together with the carbon atom to which they are bonded, form a cyclopropylene or cyclobutylene group; and e equals 0 or 1.

P1-2. The compound according to claim P1-1, where R ^L is represented by formula Ia.

P1-3. A compound according to claim P1-2, where Q is an amino acid residue.

P1-4. The compound according to claim P1-3, where Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg and Trp.

P1-5. The compound according to claim P1-2, where Q is a dipeptide residue.

P1-6. The connection according to claim P1-5, where Q is selected from: ^NH -Phe-Lys- ^C =°, ^NH -Val-Ala- ^C =°, ^NH -Val-Lys- ^C =°, ^NH AlaLys- ^C =°, ^NH -Val-Cit- ^C =°, ^NH -Phe-Cit- ^C =°, ^NH -Leu-Cit- ^C =°, ^NH -Ile-Cit- ^C =°, ^NH -Phe-Arg- ^C =°, ^NH -Trp-Cit- ^C =° and ^NH Gly-Val- ^C =°

P1-7. The conjugate according to claim P1-6, where Q is selected from ^NH -Phe-Lys- ^C =°, ^NH -Val-Cit- ^C =° and ^NH -Val-Ala- ^C =°.

P1-8. The compound according to claim P1-2, where Q is a tripeptide residue.

P1-9. The connection according to any one of paragraphs. from P1-2 to P1-8, where a equals 0-3.

P1-10. Connection according to clause P1-9, where a is 0 or 1.

P1 -11. Connection according to item P1-9, where a equals 0.

P1-12. The connection according to any one of paragraphs. from P1-2 to P1-11, where b equals 0-8.

P1-13. Connection according to paragraph P1-12, where b equals 0.

P1-14. Connection according to paragraph P1-12, where b equals 4.

P1-15. Connection according to paragraph P1-12, where b equals 8.

P1-16. The connection according to any one of paragraphs. from P1-2 to P1-15, where c equals 0.

P1-17. The connection according to any one of paragraphs. from P1-2 to P1-15, where c equals 1.

P1-18. The connection according to any one of paragraphs. P1-2 to P1-17, where d is 0-3.

P1-19. Connection according to item P1-18, where d is 1 or 2.

P1-20. Connection according to item P1-19, where d equals 2.

P1-21. The connection according to any one of paragraphs. P1-2 through P1-8, where a is 0, c is 1 and d is 2, and b is 0, 4 or 8.

P1-22. The connection according to any one of paragraphs. P1-2 to P1-21, where G ^L is selected from

- 51 046016

((G ¹ - ¹ ) ABOUT ((G ¹ - ² ) (G ¹² ) o άν ABOUT ((Г ³ ' ¹ ) S— ¢(NO2) where the NO ₂ group is optional ((G ³ - ² ) s— (NO2) where the NO2 group is optional ((G ³ - ³ ) S— /Х ο ₂ ν<Ξ=/ where the group ΝΟ2 is optional ((G ³ - ⁴ ) S-S^H where the NO2 group is optional (G ¹⁴ ) O ^Ha1 nH where Hal represents I, Br, C1 (G ^L5 ) Hal—

(G ¹⁶ ) A ⁰ (With ³ E ^Vg— ( ^GIX ) V (G ^L9 ) N ₃ (Gll ^(l ) H (G ³ - ³³ ) (G ¹¹² ) (G ¹¹³ ) 4'^ (G ³ - ³⁴ ) h ₂ n.

where Ar represents a C _5-6 arylene group and X represents a C _1-4 alkyl.

P1-23. Connection according to item P1-22, where GL is selected from G ^L1-1 and G ^L1-2 .

P1-24. The compound according to claim P1-22, where G ^L is G ^L1-1 .

P1-25. The compound according to claim P1-1, where R ^L is represented by formula Ib.

P1-26. The compound according to paragraph P1-25, where R ^L1 and R ^L2 simultaneously represent H.

P1-27. A compound according to claim P1-25, wherein R ^L1 is H and R ^L2 is methyl.

P1-28. The compound according to claim P1-25, where R ^L1 and R ^L2 are both methyl.

P1-29. A compound according to claim P1-25, wherein R ^L1 and R ^L2 together with the carbon atom to which they are bonded form a cyclopropylene group.

P1-30. A compound according to claim P1-25, where R ^L1 and R ^L2 together with the carbon atom to which they are bonded form a cyclobutylene group.

P1-31. The connection according to any one of paragraphs. from P1-25 to P1-30, where e equals 0.

P1-32. The connection according to any one of paragraphs. from P1-25 to P1-30, where e equals 1.

P1-33. Conjugate of formula IV:

L-(D ^L ) _P (IV), or a pharmaceutically acceptable salt or solvate thereof, where L represents a ligand unit (i.e., a targeting agent), D ^L represents a drug-linker unit represented by formula III

- 52 046016

R ^ll is a linker attached to a ligand unit selected from (ia'):

where Q and X are as defined in any of paragraphs. P1-1 to P1-21, and G ^LL is a linker attached to a ligand unit; and (ib'):

where R ^L1 and R ^L2 are as defined in any of paragraphs P1-1 and P1-25 to P1-30; and p is an integer from 1 to 20.

P1-34. Conjugate according to paragraph P1-33, where G ^LL is selected from ___________________________

(GLL11) 0 (G^s-i) C.B.A. A ^N' CBAi___ / ^n'xo CBA|___ f ^Ar y (GLL8-2) ^CBA _(G L ^L2 ) CBAi___ 0 tV (G^ ⁹ -!) N -k 0 0 C.B.A. (QLL3-!) _With VA|_. (G^ ⁹ - ² ) C.B.A. (GLL3-2) (GLLW) ^p ^CBA weld s ¢(2 H (G^*) CBAJ___ H N (GLMi) O 0 ^HN ( ^H \_ v (GL _L 5) CBAS___ 0 <4 (GLM ² ) 1 HN X (G ¹¹⁶ ) ^CBA j . (GLL1 ³ ) X J1~ _N CBA (G ¹¹ - ⁷ ) CBAS ( ^G1 ^ ⁴ ) H CBA^y ^N X

where Ar represents an arylene group and X represents C _1-4 αlkyl.

P1-35. The conjugate according to claim P1-34, where G ^LL is selected from G ^LL1-1 and G ^LL1-2 .

P1-36. The conjugate according to claim P1-35, where G ^LL is G ^LL1-1 .

P1-37. The conjugate according to any one of paragraphs. P1-33 to P1-36, wherein the cell-binding agent is

- 53 046016 is an antibody or its active fragment.

P1-38. The conjugate of claim P1-37, wherein the antibody or antibody fragment is an antibody or antibody fragment to a tumor-associated antigen.

P1-39. The conjugate of claim P1-38, wherein the antibody or antibody fragment is an antibody that binds to one or more tumor-associated antigens or cell surface receptors selected from (1)-(88):

(1) BMPR1B;

(2) E16;

(3) STEAP1;

(4) 0772Р;

(5) MPF;

(6) Napi3b;

(7) Sema 5b;

(8) PSCA hlg;

(9) ETBR;

(10)MSG783;

(11) STEAP2;

(12) TrpM4;

(13) CRIPTO;

(14) CD21;

(15) CD79b;

(16) FcRH2;

(17) HER2;

(18) NCA;

(19) MDP;

(20) IL20R-alpha;

(21) brevican;

(22) EphB2R;

(23) ASLG659;

(24) PSCA;

(25) GEDA;

(26) BAFF-R;

(27) CD22;

(28) CD79a;

(29) CXCR5;

(30) HLA-DOB;

(31) P2X5;

(32) CD72;

(33) LY64; (34) FcRH1;

(35) IRTA2;

(36) TENB2;

(37) PSMA-FOLH1;

(38) SST;

(38 .1) SSTR2;

(38 .2) SSTR5;

(38 .3) SSTR1;

(38 .4)SSTR3;

(38 .5) SSTR4;

(39) ITGAV;

(40) ITGB6;

(41) CEACAM5;

(42) MET;

(43) MUC1;

(44) CA9;

(45) EGFRvIII;

(46) CD33;

(47) CD19;

(48) IL2RA;

(49) AXL;

(50) CD30 - TNFRSF8;

(51) BCMA-TNFRSF17;

(52) CT Ag - CTA;

- 54 046016 (53) CD174 (Lewis Y) - FUT3;

(54) CLEC14A;

(55) GRP78-HSPA5;

(56) CD70;

(57) stem cell specific antigens;

(58)ASG-5;

(59) ENPP3;

(60) PRR4;

(61) GCC - GUCY2C;

(62) Liv-1 - SLC39A6;

(63) 5T4;

(64) CD56 - NCMA1;

(65) CanAg;

(66) FOLR1;

(67) GPNMB;

(68) TIM-1 - HAVCR1;

(69) RG-1/mindin, prostate tumor target is mindin/RG-I;

(70) B7-H4 - VTCN1;

(71) PTK7;

(72) CD37;

(73) CD138-SDC1;

(74) CD74;

(75) claudins - CL;

(76) EGFR;

(77) Her3;

(78) RON - MST1R;

(79) EPHA2;

(80) CD20 - MS4Al;

(81) tenascin C - TNC;

(82) FAP;

(83) DKK-1;

(84) CD52;

(85) CS1 - SLAMF7;

(86) endoglin - ENG;

(87) Al annexation - ANXA1;

(88) V-CAM (CD106) - VCAM1;

(89) ASCT2 (SLC1A5).

P1-40. The conjugate according to any one of paragraphs. P1-37 to P1-39, wherein the antibody or antibody fragment is an antibody constructed with a cysteine.

P1-41. The conjugate according to any one of paragraphs. P1-37 to P1-40, wherein the drug load (p) for the drugs (D) on the antibody (Ab) is an integer from 1 to about 10.

R1-42. The conjugate according to paragraph P1-41, where p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

R1-43. A mixture of conjugates according to any one of paragraphs. P1-33 to P1-42, wherein the average drug loading per antibody in the mixture of antibody-drug conjugate compounds is from about 1 to about 10.

R1-44. Conjugate or mixture according to any one of paragraphs. P1-33 to P1-43 for therapeutic use.

P1-45. A pharmaceutical composition containing a conjugate or mixture according to any one of paragraphs. P1-33 to P1-43 and a pharmaceutically acceptable diluent, carrier or excipient.

P1-46. Conjugate or mixture according to any one of paragraphs. P1-33 to P1-43 or the pharmaceutical composition of claim P1-45 for use in the treatment of a proliferative disease in a subject.

R1-47. The conjugate or mixture according to claim P1-46, wherein the disease is cancer.

R1-48. Use of a conjugate or mixture according to any one of paragraphs. from P1-33 to P1-43 or the pharmaceutical composition according to paragraph P1-45 in the method of conservative treatment.

P1-49. A method of conservative treatment, including administering to the patient a pharmaceutical composition according to claim P1-45.

P1-50. The method according to claim P1-49, wherein the conservative treatment method is intended for the treatment of cancer.

P1-51. The method according to claim P1-50, where the patient is administered a chemotherapeutic agent in combination with a conjugate.

R1-52. Use of a conjugate or mixture according to any one of paragraphs. P1-33 to P1-43 in a method for manufacturing a medicament for treating a proliferative disease.

R1-53. A method of treating a mammal suffering from a proliferative disease, comprising administering an effective amount of a conjugate or mixture according to any one of claims. from P1-33 to P1-43 or the pharmaceutical composition according to paragraph P1-45.

P1-54. Connection A:

And in the form of a separate enantiomer or in an enantiomerically enriched form.

Statement of the invention from the 2nd application, on the basis of which priority is claimed (P2)

and salts and solvates thereof, wherein RL is a linker for attachment to a cell binding agent selected from:

^(ia):

where Q represents

wherein Q ^X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;

X represents

where a is 0-5, b1 is 0-16, b2 is 0-16, c is 0 or 1, d is 0-5, where at least b1 or b2 is 0;

G ^l is a linker for attachment to a ligand unit;

(ib):

wherein R ^L1 and R ^L2 are independently selected from H and methyl or, together with the carbon atom to which they are bonded, form a cyclopropylene or cyclobutylene group; and e equals 0 or 1.

R2-2. The compound according to claim P2-1, where R ^L is represented by formula Ia.

P2-3. The compound according to claim P2-2, where Q is an amino acid residue.

P2-4. The compound according to claim P2-3, where Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg and Trp.

P2-5. The compound according to claim P2-2, where Q is a dipeptide residue.

P2-6. Compound according to item P2-5, where Q is selected from: ^NH -Phe-Lys- ^C=O , ^NH -Val-Ala- ^C=O , ^NH -Val-Lys- ^C=O , ^NH -AlaLys- ^C=O , ^NH -Val-Cit- ^C=O , ^NH -Phe-Cit- ^C=O , ^NH -Leu-Cit- ^C=O , ^NH -Ile-Cit- ^C=O , ^NH -Phe-Arg- ^C=O , ^NH -Trp-Cit- ^C=O and ^NH -Gly-Val- ^C=O

P2-7. The compound according to item P2-6, where Q is selected from ^NH -Phe-Lys- ^C “ ^O , ^NH -Val-Cit- ^C “ ^O and ^NH -Val-Ala- ^C “ ^O .

P2-8. The compound according to claim P2-2, where Q is a tripeptide residue.

P2-9. The compound according to claim P2-8, where Q is selected from ^NH -Glu-Val-Ala- ^C “ ^O , ^NH -Glu-Val-Cit- ^C “ ^O , ^NH -aGlu-ValAla- ^C=O and ^NH -aGlu- Val-Cit- ^C=O

P2-10. The compound according to claim P2-2, where Q is a tetrapeptide residue.

- 56 046016

P2-11. The compound according to claim P2-10, where Q is selected from ^NH -Gly-Gly-Phe-Gly ^C = ^O and ^NH -Gly-Phe-Gly-Gly ^C = ^O

P2-12. The compound according to claim P2-11, where Q is N-Gly-Gly-Phe-Gly ^CO .

P2-13. The connection according to any one of paragraphs. from P2-2 to P2-12, where a equals 0-3.

P2-14. The connection according to clause P2-13, where a is 0 or 1.

P2-15. Connection according to clause P2-13, where a equals 0.

R2-16. The connection according to any one of paragraphs. from P2-2 to P2-15, where bl equals 0-8.

R2-17. Connection according to item P2-16, where bl equals 0.

R2-18. Connection according to item P2-16, where bl equals 2.

R2-19. Connection according to item P2-16, where bl equals 3.

R2-20. Connection according to item P2-16, where bl equals 4.

R2-21. Connection according to item P2-16, where bl equals 5.

R2-22. Connection according to item P2-16, where bl equals 8.

R2-23. The connection according to any one of paragraphs. from P2-2 to P2-l5 and P2-l7, where b2 equals 0-8.

R2-24. Connection according to clause P2-23, where b2 equals 0.

R2-25. Connection according to paragraph P2-23, where b2 equals 2.

R2-26. Connection according to item P2-23, where b2 equals 3.

R2-27. Connection according to item P2-23, where b2 equals 4.

R2-28. Connection according to item P2-23, where b2 equals 5.

R2-29. Connection according to item P2-23, where b2 equals 8.

R2-30. The connection according to any one of paragraphs. from P2-2 to P2-29, where c equals 0.

P2-3l. The connection according to any one of paragraphs. from P2-2 to P2-29, where c equals l.

R2-32. The connection according to any one of paragraphs. from P2-2 to P2-3l, where d equals 0-3.

R2-33. Connection according to clause P2-32, where d equals l or 2.

R2-34. Connection according to clause P2-32, where d equals 2.

R2-35. The connection according to any one of paragraphs. P2-2 through P2-l2, where a is 0, bl is 0, c is 1, and d is 2, and b2 is 0-8.

R2-36. Connection according to item P2-35, where b2 is equal to 0, 2, 3, 4, 5 or 8.

R2-37. The connection according to any one of paragraphs. P2-2 through P2-l2, where a equals l, b2 equals 0, c equals 0, and d equals 0, and bl equals 0-8.

R2-38. Connection according to item P2-37, where bl is equal to 0, 2, 3, 4, 5 or 8.

R2-39. The connection according to any one of paragraphs. P2-2 through P2-l2, where a is 0, bl is 0, c is 0, and d is 1, and b2 is 0-8.

R2-40. Connection according to paragraph P2-39, where b2 is equal to 0, 2, 3, 4, 5 or 8.

P2-4l. The connection according to any one of paragraphs. P2-2 through P2-l2, where bl is 0, b2 is 0, c is 0, one of a and d is 0, and the other of a and d is l-5.

R2-42. The connection according to paragraph P2-41, where the other of a and d is equal to l or 5.

R2-43. The connection according to any one of paragraphs. from P2-2 to P2-42, where GL is selected from

- 57 046016

(SP ¹ ) About a: (G ¹⁴ ') ABOUT y-N (G ¹¹² ) (SD Vg— (G ¹² ) ABOUT •h'Y o (SP V ((Г ³ ' ¹ ) S—s ^X (NO2) where the NO2 group is optional (G ¹ - ⁹ ) N ₃ ((G ³ - ² ) _s _ _s * (NO2) where the NO2 group is optional (ST ¹⁰ ) n ((G ³ - ³ ) S— ο ₂ νΛ^=/ where the group ΝΟ2 is optional (ST ¹¹ ) oh oh ((G ³ - ⁴ ) ..d where the NO2 group is optional (ST ¹² ) (G ¹⁴ ) ι__O Hal N—1 N* where Hal represents I, Br, C1 (G ⁴ - ¹³ ) γ Ζ ^Ν ^Ν χχ ( ^G1 ) About Hal— (G ¹¹⁴ ) η ₂ ν. \ ABOUT

where Ar represents a C _5-6 arylene group and X represents a C _1-4 alkyl.

R2-44. Connection according to item P2-43, where GL is selected from G ^L1-1 and G ^L1-2 .

R2-45. The compound according to claim P2-43, where G ^L is G ^L1-1 .

R2-46. The compound according to claim P2-1, wherein R ^L is represented by formula Ib.

R2-47. The compound according to paragraph P2-46, where R ^L1 and R ^L2 simultaneously represent H.

R2-48. The compound of claim P2-46, wherein R ^L1 is H and R ^L2 is methyl.

R2-49. The compound according to claim P2-46, where R ^L1 and R ^L2 are both methyl.

P2-50. A compound according to claim P2-46, wherein R ^L1 and R ^L2 together with the carbon atom to which they are bonded form a cyclopropylene group.

R2-51. A compound according to claim P2-46, wherein R ^L1 and R ^L2 together with the carbon atom to which they are bonded form a cyclobutylene group.

R2-52. The connection according to any one of paragraphs. from P2-46 to P2-51, where e equals 0.

R2-53. The connection according to any one of paragraphs. from P2-46 to P2-51, where e equals 1.

R2-54. Conjugate of formula IV

L-(D ^L ) _P (IV), or a pharmaceutically acceptable salt or solvate thereof, where L represents a ligand unit (i.e., a targeting agent), D ^L represents a drug-linker unit represented by formula III

- 58 046016

R ^ll is a linker attached to a ligand unit selected from (ia'):

where Q and X are as defined in any of paragraphs. P2-1 to P2-42, and G ^LL is a linker attached to a ligand unit; and (ib'):

where R ^L1 and R ^L2 are as defined in any of paragraphs P2-1 and P2-47 to P2-51; and p is an integer from 1 to 20.

R2-55. Conjugate according to paragraph P2-54, where G ^LL from

(GLu-i) 0 CBAj ABOUT ( ^GLL8 ' ¹ ) C.B.A. ( ^GLL1 ' ² ) O .Ar _z CBA| N y ^x o ( ^GLL8 ' ² ) N,CBA ( ^Gu - ² ) 0 0 (GLL ⁹ - ¹ ) AA ⁴ _N CBA (qLL ³ - ¹ ) _(Gl ^l9 - ² ) N ⁴ NA CBA ( ^GLL3-2 ) cba|_ _s )-7 (GLLW) ~rF --- _v As H (GLL- ⁴ ) CBAS___ 0 ( ^GLL11 ) ^H <J ) /~-< ^N » O (GLL5) 0 CBAS___// ( ^GLL12 ) X (GLL6) 0 ^CBA j // (GLLB) xJLn CH C.B.A. (G ^U G CBA^ (GLL14) ^H h. CBAy-^oA

where Ar represents a C _5-6 arylene group, and X represents a C _1-4 alkyl. R2-56. The conjugate according to claim P2-55, where G ^LL is selected from G ^LL1-1 and G ^LL1-2 .

- 59 046016

R2-57. The conjugate according to claim P2-56, where G ^LL is G ^LL1-1 .

R2-58. The conjugate according to any one of paragraphs. P2-54 to P2-57, wherein the ligand unit is an antibody or an active fragment thereof.

R2-59. The conjugate of claim P2-58, wherein the antibody or antibody fragment is an antibody or antibody fragment to a tumor-associated antigen.

R2-60. The conjugate of claim P2-59, wherein the antibody or antibody fragment is an antibody that binds to one or more tumor-associated antigens or cell surface receptors selected from (1) to (89):

(1) BMPR1B;

(2) E16;

(3) STEAP1;

(4) 0772Р;

(5) MPF;

(6) Napi3b;

(7) Sema 5b;

(8) PSCA hlg;

(9) ETBR;

(10) MSG783;

(11) STEAP2;

(12) TrpM4;

(13) CRIPTO;

(14) CD21;

(15) CD79b;

(16) FcRH2;

(17) HER2;

(18) NCA;

(19) MDP;

(20) IL20R-alpha;

(21) brevican;

(22) EphB2R;

(23) ASLG659;

(24) PSCA;

(25) GEDA;

(26) BAFF-R;

(27) CD22;

(28) CD79a;

(29) CXCR5;

(30) HLA-DOB;

(31) P2X5;

(32) CD72;

(33) LY64;

(34) FcRH1;

(35) IRTA2;

(36) TENB2;

(37) PSMA - FOLH1;

(38) SST;

(38 .1) SSTR2;

(38 .2) SSTR5;

(38 .3) SSTR1;

(38 .4) SSTR3;

(38 .5) SSTR4;

(39) ITGAV;

(40)ITGB6;

(41) CEACAM5;

(42) MET;

(43) MUC1;

(44) CA9;

(45) EGFRvIII;

(46) CD33;

(47)CD19;

(48) IL2RA;

(49) AXL;

- 60 046016 (50) CD30 - TNFRSF8;

(51) BCMA - TNFRSF17;

(52) CT Ag - CTA;

(53) CD174 (Lewis Y) - FUT3;

(54) CLEC14A;

(55) GRP78 - HSPA5;

(56) CD70;

(57) stem cell specific antigens;

(58)ASG-5;

(59) ENPP3;

(60) PRR4;

(61) GCC - GUCY2C;

(62) Liv-1-SLC39A6;

(63) 5T4;

(64) CD56 - NCMA1;

(65) CanAg;

(66) FOLR1;

(67) GPNMB;

(68) TIM-1 - HAVCR1;

(69) RG-1/mindin, prostate tumor target is mindin/RG-I;

(70) B7-H4 - VTCN1;

(71) PTK7;

(72) CD37; (73) CD138 - SDC1;

(74) CD74;

(75) claudins - CL;

(76) EGFR;

(77) Her3;

(78) RON - MST1R;

(79) EPHA2;

(80) CD20 - MS4Al;

(81) tenascin C - TNC;

(82) FAP;

(83) DKK-1;

(84) CD52;

(85) CS1 - SLAMF7;

(86) endoglin - ENG;

(87) annexations A1 - ANXA1;

(88) V-CAM (CD106) - VCAM1;

(89) ASCT2 (SLC1A5).

P2-61. The conjugate according to any one of paragraphs. P2-58 to P2-60, wherein the antibody or antibody fragment is an antibody constructed with a cysteine.

R2-62. The conjugate according to any one of paragraphs. P2-58 to P2-61, wherein the drug load (p) for the drugs (D) on the antibody (Ab) is an integer from 1 to about 10.

R2-63. Conjugate according to paragraph P2-62, where p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

R2-64. A mixture of conjugates according to any one of paragraphs. P2-58 to P2-63, wherein the average drug loading per antibody in the antibody-drug conjugate mixture is from about 1 to about 10.

R2-65. Conjugate or mixture according to any one of paragraphs. from P2-54 to P2-64 for use in therapy.

R2-66. A pharmaceutical composition containing a conjugate or mixture according to any one of paragraphs. P2-54 to P2-64 and a pharmaceutically acceptable diluent, carrier or excipient.

R2-67. Conjugate or mixture according to any one of paragraphs. P2-54 to P2-64 or the pharmaceutical composition of claim P2-66 for use in the treatment of a proliferative disease in a subject.

R2-68. The conjugate, mixture or pharmaceutical composition according to claim P2-67, wherein the disease is cancer.

R2-69. Use of a conjugate or mixture according to any one of paragraphs. from P2-54 to P2-64 or the pharmaceutical composition according to paragraph P2-66 in the method of conservative treatment.

R2-70. A method of conservative treatment, including administering to the patient a pharmaceutical composition according to paragraph P2-66.

R2-71. The method according to claim P2-70, wherein the conservative treatment method is intended for the treatment of cancer.

R2-72. The method according to claim P2-71, where the patient is administered a chemotherapeutic agent in combination with a conjugate.

-

Claims (5)

R2-73. Use of a conjugate or mixture according to any one of paragraphs. P2-54 to P2-64 in a method for manufacturing a medicinal product for treating a proliferative disease. R2-74. A method of treating a mammal suffering from a proliferative disease, comprising administering an effective amount of a conjugate or mixture according to any one of claims. from P2-54 to P2-64 or the pharmaceutical composition according to paragraph P2-66. R2-75. Compound A as a single enantiomer or in an enantiomerically enriched form. CLAIM 1. A compound which is 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)propanamido)^-(^)-1 -(((S)-1 -( ((8)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro1H,12H-benzo[de]pyrano [3',4':6,7 ]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3 methyl-1-oxobutan-2-yl)-3,6,9,12,15 ,18,21,24-octaoxaheptacosane-27-amide. 2. Conjugate of formula IV L - (D ^L )i (IV), or a pharmaceutically acceptable salt thereof, where L is an antibody or an antigen-binding fragment of an antibody, DL is a drug-linker unit represented by formula III RLL is a linker attached to said antibody or antigen binding fragment of an antibody, wherein said linker is represented by the formula (Ia') where Q is a dipeptide residue which is ^NH -Val-Ala- ^C =°, where ^NH is the N-terminus and - ^C =° is the C-terminus of said residue, X is where a equals 0, b1 equals 0, c1 equals 1, c2 equals 0, d equals 2, b2 equals 8; and GLL is a linker attached to said antibody or antigen binding fragment of an antibody that is o NVA< N'p is an integer from 1 to 20. 3. The conjugate according to claim 2, wherein the drug load (p) for the drugs (D) on the antibody (Ab) is an integer from 1 to 10. 4. A pharmaceutical composition containing the conjugate according to claim 2 or 3 and a pharmaceutically acceptable diluent, carrier or excipient. 5. Use of the conjugate of claim 2 or 3 or the pharmaceutical composition of claim 4 for the treatment of cancer in a subject. -