EA047476B1 - 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 to a releasable damaging 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.

Next connection:

XXX/ x ξ o on o A ^R , 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., JMed Chem, 1998, 41, 2308-2318 (DOI: 10.1021/jm970765q), where its biological activity is discussed along with the biological activity of a number of related compounds.

Various topoisomerase inhibitors, such as irinotecan and exatecan derivatives and doxorubicin, have been incorporated into antibody-drug conjugates. For example, Daiichi Sankyo's DS-8201a 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:

In Burke, P.J. et al., Bioconjugate Chem., 2009, 20, 1242-1250 conjugates are disclosed

which are linked 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)

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Brief description of the invention

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

with a linker for attaching a unit representing a ligand, wherein the linker is attached via a cleavable bond to an amino acid residue. The unit representing a ligand is preferably an antibody. The present invention also provides A* with an attached linker and intermediate compounds for synthesizing them, as well as a releasable damaging element.

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

and its salts and solvates, wherein R ^L is a linker for attachment to a unit representing a ligand, which is selected from (ia)

where Q represents

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

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

- 2 047476 (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.

In a second aspect of the present invention there is provided a process for producing a compound according to the first aspect of the present invention, comprising at least one of the process steps set out 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, wherein L is a ligand (i.e., targeting agent) unit, D ^L is a drug-linker unit represented by formula III

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

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

r ^l1 kr ^L2 su slb' o

where R ^L1 and R ^L2 are as defined in the first aspect; and p is an integer from 1 to 20.

Accordingly, the conjugates comprise 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 attached). The ligand unit, described more fully below, is a targeting agent that binds to a target moiety. The ligand unit may, for example, specifically bind to a cellular component (cell-binding agent) or to other target molecules of interest. Accordingly, the present invention also provides methods of treating, for example, various cancers and an autoimmune disease. These methods encompass the use of conjugates, wherein the ligand unit is a targeting agent that specifically binds to a target molecule. The ligand unit can be, for example, a protein, polypeptide, or peptide, such as an antibody, an antigen-binding fragment of an antibody, or another binding agent, such as an Fc fusion protein.

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

A fourth aspect of the present invention provides the use of the conjugate of the third aspect of the present invention in the manufacture of a medicinal product for the treatment of proliferative

- 3 047476 disease. In a fourth aspect, there is also provided a conjugate according to 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 or not a candidate compound is capable of treating a proliferative condition in any particular cell type. For example, assays that can be usefully used to assess the activity provided by a particular compound are described in the examples below.

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

DOS(1): X = none, Y = SI?

ADC (2): X = ΝΗ,-CHACO). Y = CH _;

ADC (3) X = MH _G (CH^C=O)Y = CHj

ADC (4): X = NH ₇ -(CH _? M ^C =°)

ADC (5) X = NH _r (CH,MC=O). Y=Cl^

ADC (6): X = MHp-(CH;} ₅ -(C=O).Y«CH,

ADC (7): X = NH-,-(CH _? >^C=O) Y= (C-O|-MJCH _? CH,0} _; η DAR and it was concluded that the decrease in cytotoxicity of ADCs (1) and (2) may be due to steric hindrance of the drug release moiety at the site of action of degradative enzymes in tumor cells. This document explains the importance of positioning the peptide moiety away from the large drug release moiety. In contrast, in the present invention, the peptide moiety is attached directly to the large drug release moiety.

The fifth aspect of the present invention is compound A

In some embodiments, compound A is present as a single enantiomer or in an enantiomerically enriched form.

Compound A and conjugates containing A* may exhibit lower toxicity and higher efficacy compared to other known drug units and conjugates. Thus, compound A and conjugates containing A* may exhibit an improved therapeutic window. Therefore, compound A may be particularly suitable as a drug unit, in particular for use in the treatment of cancer.

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

where Q is as defined in the first aspect.

In further general aspects, the present invention provides:

(i) the use of a conjugate comprising A* attached to a unit representing a ligand via a cleavable bond in the manufacture of a medicinal product for the treatment of a proliferative disease such as cancer;

(ii) a conjugate comprising A* attached to a unit representing a ligand via a cleavable bond, for use in the treatment of a proliferative disease such as cancer;

(iii) a method of conservative treatment, such as the treatment of cancer, comprising administering a conjugate comprising A* attached to a unit representing a ligand via a cleavable bond;

(iv) the use of a conjugate with a unit representing a ligand that releases A in the manufacture of a medicinal product for the treatment of a proliferative disease such as cancer;

(v) a conjugate with a unit representing a ligand that releases A, for use

- 4 047476 in the treatment of a proliferative disease such as cancer;

(vi) a method of conservative treatment, such as treatment of cancer, comprising administering a conjugate with a unit which is a ligand that releases A; and (vii) a conjugate with a unit which is a ligand that releases A.

Definitions

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

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

All the atoms of the ring may be carbon atoms, as in carboarylene groups, in which case the group is phenylene (C ₆ ).

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

N1: pyrrole (azole) (C ₅ ), pyridine (azine) (C ₆ );

O ₁ : furan (oxol) (C ₅ );

S ₁ : thiophene (thiol) (C ₅ );

N1O1: oxazole (C ₅ ), isoxazole (C ₅ ), isoxazine (C ₆ );

N2O1: oxadiazole (furazan) (C ₅ );

N3O1: oxatriazole (C ₅ );

N1S1: thiazole (C ₅ ), isothiazole (C ₅ );

N2: imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C ₆ ), pyrimidine (1,3-diazine) (C ₆ ) (for example, cytosine, thymine, uracil), pyrazine (1,4-diazine) (C ₆ ) and

N3: triazole (C5), triazine (C6).

_C1-4 alkyl: As used herein, the term _C1-4 alkyl refers to a monovalent moiety derived by the removal of a hydrogen atom from a carbon atom of a hydrocarbon compound containing from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). As used herein, the term C1 _-n- alkyl refers to a monovalent moiety derived by the removal of 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, fully 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 tert-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 2-propynyl (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, i.e., a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, wherein the moiety contains from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, groups derived from saturated monocyclic hydrocarbon compounds:

cyclopropane (C3) and cyclobutane (C4) and unsaturated monocyclic hydrocarbon compounds:

cyclopropene (C3) and cyclobutene (C4).

Link marking:

in the formula

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The superscript ^C(O) and ^NH notation denotes the group to which the atoms are bonded. For example, the NH group is shown bonded to a carbonyl (which is not part of the fragment illustrated), and the carbonyl is shown bonded to an NH group (which is not part of the fragment illustrated).

Salts

It may be convenient or desirable to prepare, purify and/or process the active compound into a corresponding salt, such as 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 (e.g., -COOH can be ^-COO- ), then a salt can be formed with a suitable cation. 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 ^Ca2+ and Mg2 ⁺ , and other cations such as Al ⁺³ . Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4) and substituted ammonium ions (e.g., 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(CH3)4.

If the compound is cationic or contains a functional group that can be cationic (e.g., _-NH2 can be _-NH3 ), then a salt can be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, anions 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, anions derived from the following organic acids: 2-acetoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucoheptone, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucinous, 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, anions derived from the following polymeric acids: tannic acid, carboxymethylcellulose.

Solvates

It may be convenient or desirable to prepare, purify and/or process a corresponding solvate of the active compound. The term solvate is used herein in its generally accepted sense and refers to a complex of a solute (e.g., an active compound, a salt of the active compound) and a solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, e.g., a monohydrate, a dihydrate, a trihydrate, etc.

Isomers

Certain compounds of the present invention may exist in one or more specific geometric, optical, enantiomeric, diastereomeric, epimeric, atropisomeric, stereoisomeric, tautomeric, conformational, or anomeric forms, including, without limitation, cis and trans forms; E and Z forms; c, t and r forms; endo and exo forms; R, S and meso forms; D and L forms; 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 forms; bath, armchair, twist, envelope - and half-armchair shapes, and their combinations, hereinafter in this document collectively referred to as isomers (or isomeric forms).

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

The term stereoisomers refers to compounds that have identical chemical composition 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 high

- 6 047476 cosensitive analytical procedures such as electrophoresis and chromatography.

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

The 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. It is intended that 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, 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 denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and l or (+) and (-) are used to denote the sign of rotation of the compound by plane-polarized light, where (-) or l means the compound is levorotatory. A compound with the prefix (+) 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 may occur when there is no stereoselection or stereospecificity in a chemical reaction or process. The terms racemic mixture and racemate refer to an equimolar mixture of two enantiomer species that lack optical activity.

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

It should be noted that, except for 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 merely in the positions of the atoms in space). For example, reference to a methoxy group, -OCH3, should not be construed as a reference to its structural isomer, the hydroxymethyl group, -CH2OH. Likewise, reference to orthochlorophenyl should not be construed as a reference to its structural isomer, meta-chlorophenyl. However, reference to a class of structures may well include structural isomeric forms within that class (e.g. C _1-7 alkyl includes n-propyl and isopropyl; butyl includes n-, iso-, sec-, and 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/enthiol, N-nitroso/hydroxyazo and nitro/acinitro.

_ζ ο _x on n ⁺ _ch O' —cc ^z c=c c=c

I\/\ _n ⁺ /\keto enol enolate

The term tautomer or tautomeric form refers to structural isomers of different energies that are interconvertible due to a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversion species by proton migration, such as keto-enol and imine-enamine isomerization. Valence tautomers include interconversion species through rearrangement 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 may be in any isotopic form, including ¹ H, 2 H (D), and 3 H (T); C may be in any isotopic form, including ¹² C, ¹³ C, and ¹⁴ C; O may be in any isotopic form, including ¹⁶ O and ¹⁸ O, etc.

Examples of isotopes that can be incorporated into 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 , ^13C , ^14C , ^15N , ^18F , ^31P , ^32P , ^35S , ^36Cl and ^125I . Various isotopically labeled compounds of the present invention, for example, those compounds into which radioactive isotopes such as ^3H , ^13C and ^14C are incorporated. Such isotopically labeled compounds can be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including drug or substrate tissue distribution assays, or in the treatment of patients using radioactive substances. The deuterium-labeled or deuterium-substituted therapeutic compounds of the present invention may have improved

- 7 047 476 DMPK (drug metabolism and pharmacokinetics) properties with respect to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes, such as deuterium, may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or decreased dosage requirements. The 18F-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 carrying out the procedures disclosed in the schemes or in the examples and preparation methods described below, by replacing a reagent that is not an 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 advantages resulting from greater metabolic stability, such as increased in vivo half-life, or decreased dosage requirements, or increased therapeutic index. It is understood that deuterium is considered a substituent in this context. The concentration of such a heavier isotope, particularly deuterium, can be determined using an isotopic 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 particular compound includes all such isomeric forms, including (in whole or in part) racemic and other mixtures thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic methods) of such isomeric forms are either known in the art or are readily developed by adapting the methods set forth herein or known methods in a known manner.

A link that represents a ligand

The ligand unit may be any and includes a protein, a polypeptide, a peptide, and a non-peptide agent that specifically binds to a target molecule. In some embodiments, the ligand unit may be a protein, a polypeptide, or a 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 cell-binding molecule or substance that is capable of specifically binding to a target.

The terms "specifically binds" and "specific binding" refer to the binding of an antibody or other protein, polypeptide, or peptide to a target molecule (e.g., an antigen). Typically, the antibody or other molecule binds with an affinity of at least about 1 x 10 ⁷ M ^-1 and binds to the target molecule with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the target 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 moiety is a cell-binding agent that binds to an extracellular target on a 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, a polypeptide, or a 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 may be any and includes peptides and agents other than peptides. They may include antibodies or an antibody fragment that contains at least one binding site, lymphokines, hormones, hormone mimetics, vitamins, growth factors, molecules that carry out nutrient transport, or any other molecule or substance that binds to a cell.

Peptides

In one embodiment, the cell binding agent is a linear or cyclic peptide comprising 4-30, preferably 6-20 contiguous amino acid residues.

In one embodiment, the cell binding agent comprises a peptide that binds integrin α _γ β ₆ . The peptide may be selective for α _γ β ₆ over XYS.

In one embodiment, the cell binding agent comprises a polypeptide

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A20FMDV-Cys. A20FMDV-Cys is characterized by the sequence navpnlrgdlqvlaqkvartc.

Alternatively, a variant of the A20FMDV-Cys sequence may be used, wherein one, two, three, four, five, six, seven, eight, nine or ten amino acid residues are replaced by another amino acid residue. In addition, the polypeptide may be characterized by the sequence NAVXXXXXXXXXXXXXXXRTC.

Antibodies

The term antibody is used herein in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, polyspecific antibodies (e.g., bispecific antibodies), multivalent antibodies, and antibody fragments, provided that 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, C, Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, ^5th Ed., Garland Publishing, New York). A target antigen typically has multiple binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a distinct structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds the antigen of a target of interest or a portion thereof, wherein such targets include, but are not limited to, a cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. An immunoglobulin may be an immunoglobulin molecule of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Immunoglobulins may be derived from any species, including those from humans, mice, or rabbits.

Antibody fragments comprise a portion 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-binding fragments of any of the foregoing that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

As used herein, the term monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being 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 the 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 obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody 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 produced by recombinant DNA techniques (see US 4,816,567). Monoclonal antibodies can also be isolated from antibody phage 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 include, but are not limited to, 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 prostatic epithelial antigen)

- 9 047476 (4) 0772P (CA125, MUC16) (5) MPF (MPF, MSLN, SMR, megakaryocyte-potentiating factor, mesothelin) (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, 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, cDNA RIKEN 2700050C12, cDNA of the RIKEN gene 2700050C12) (9) ETBR (endothelin receptor type B) (10) MSG783 (RNF124, hypothetical protein FLJ20315) (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six-transmembrane prostate epithelial antigen 2, six-transmembrane prostate protein) (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, cation channel 5, transient receptor potential-acting, 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 (immunoglobulin beta-associated), B29) (16) FcRH2 (IFGP4, IRtA4, SPAP1A (SH2 domain-containing anchor protein phosphatase 1a), SPAP1B, SPAP1C) (17) HER2 (ErbB2) (18) NCA (CEACAM6) (19) MDP (DPEP1) (20) IL20R-alpha (IL20Ra, ZCYTOR7) (21) Brevican (BCAN, VENAB) (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5) (23) ASLG659 (B7h) (24) PSCA (prostate stem cell antigen precursor) (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), immunoglobulin alpha-associated protein, B-cell specific, which covalently interacts with Ig beta (CD79B) and forms a complex with Ig M molecules on the surface, transmitting a signal involved in B-cell differentiation), pI: 4.84, MW: 25028, TM: 2 [P], gene, chromosome: 19q13.2).

(29) CXCR5 (Burkitt's lymphoma receptor 1, a protein G-associated 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 (ligand-gated ion channel 5 of the purinergic receptor P2X, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability); 422 aa), pI: 7.63, MW: 47206, TM: 1 [P], gene, chromosome: 17p13.3).

(32) CD72 (CD72 B-cell differentiation antigen, 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 leucine-rich repeat (LRR) type I 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 receptor for the immunoglobulin Fc domain that contains Ig-like and C2-type ITAM domains and may play a role in B-cell differentiation; 429 aa, pI: 5.28, MW: 46925, TM: 1 [P], gene, chromosome: 1q21-1q22) (35) IRTA2 (immunoglobulin superfamily translocation-associated receptor 2, a putative immunoreceptor with a possible role in B-cell development and lymphomagenesis; gene dysregulation through translocation occurs in some B-cell malignancies

- 10 047476 formations); 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) (54) CLEC14A (lectin C-type family 14, member A; Genbank accession number NM175060) (55) GRP78 - HSPA5 (heat shock protein 5 with molecular 70 kDa (glucose-regulated protein, 78 kDa) (56) CD70 (CD70 molecule) L08096 (57) specific stem cell antigens.

For example:

5T4 (see list item (63) below)

CD25 (see list item (48) above)

CD32

LGR5/GPR49 prominin/ CD 133 (58) ASG-5 (59) ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase 3) (60) PRR4 (proline-rich protein 4 (lacrimal gland)) (61) GCC - GUCY2C (guanylate cyclase 2C (heat-stable enterotoxin receptor) (62) Liv-1 - SLC39A6 (solute carrier family 39 (zinc transporter), member 6) (63) 5T4, trophoblast glycoprotein, TPBG - TPBG (trophoblast glycoprotein) (64) CD56 -NCMA1 (neural cell adhesion molecule 1) (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, prostate tumor target - 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, invariant chain class II) (75) claudins - CL (claudins) (76) EGFR (epidermal growth factor receptor) (77) Her3 (ErbB3) - ERBB3 (homology 3 viral oncogene in erythroblastic leukemia v-erb-b2 (avian))

- 11 047476 (78) RON - MST1R (macrophage-stimulating receptor 1 (c-met-related tyrosine kinase)) (79) EPHA2 (EPH 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 (Dickkopf homolog 1 (Xenopus laevis) (84) CD52 (CD52 molecule) (85) CS1 - SLAMF7 (SLAM family member 7) (86) endoglin - ENG (endoglin) (87) annexin A1 - ANXA1 (annexin A1) (88) V-CAM (CD106) - VCAM1 (vascular endothelial cell adhesion molecule 1)

Additional tumor-associated antigen and cognate antibodies of interest include:

(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 methods

The conjugates of the present invention may be used in a method of therapy. Also provided is a method of treatment comprising administering to a subject in need of treatment a therapeutically effective amount of a conjugate of formula IV. The term therapeutically effective amount means an amount sufficient to demonstrate a benefit to the patient. Such benefit may be at least a decrease 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 dose decisions, is within the competence of general practitioners and other physicians.

The conjugate may be administered alone or in combination with other treatments, 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 in the present invention may contain, in addition to the active ingredient, i.e. the conjugate of formula IV, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials commonly known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral or may be by injection, such as intradermal, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in the form of a tablet, capsule, powder or liquid. A tablet may contain a solid carrier or 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. A capsule may contain a solid carrier such as gelatin.

For intravenous, intradermal or subcutaneous injection or injection into the lesion, the active ingredient will be in the form of an aqueous solution suitable for parenteral administration, which does not contain pyrogens and is characterized by a suitable pH, isotonicity and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic media such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included if necessary.

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 (e.g., histiocytoma, glioma, astrocytoma, osteoma), cancers (e.g., lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, colon cancer), and ovarian cancer (e.g., ovarian cancer, esophageal cancer, esophageal cancer, gastrointestinal cancer, and esophageal cancer).

- 12 047476 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 (e.g. of connective tissues) and atherosclerosis. Other cancers of interest include, but are not limited to, malignancies of the blood system such as types of leukemia and lymphomas such as non-Hodgkin's lymphoma and its subtypes such as DLBCL, marginal zone cell lymphoma, mantle cell lymphoma and follicular lymphoma, Hodgkin's lymphoma, AML and other cancers of B- or T-cell origin. Any cell type can 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 (eg, Crohn's disease), anaphylaxis, allergic reaction, Sjogren's syndrome, type 1 diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine insufficiency, Schmidt syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, atrophy stomach, chronic hepatitis, lupus hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, 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 rhinitis, Goodpasture's syndrome, Chagas disease, sarcoidosis, rheumatic fever, asthma, habitual abortion, 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's arteritis, polymyalgia rheumatoid arthritis, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sumpter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan syndrome, Kawasaki disease, dengue fever, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, persistent erythema protuberans, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochromic cyclitis, Fuchs cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft-versus-host disease, graft rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulinemia, Evans syndrome, and autoimmune gonadal insufficiency.

In some embodiments, the autoimmune disease is a disorder associated with B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture syndrome, rheumatoid arthritis, and type 1 diabetes), Th1 lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjogren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft-versus-host disease), or Th2 lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn syndrome, systemic sclerosis, or chronic graft-versus-host disease). 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 chemotherapeutic 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, spindle poison plant alkaloids, cytotoxic/antineoplastic 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 Pham.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS# 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS# 391210-10-9, Pfizer), cisplatin (cis-diamine, di

- 13 047476 chloroplatinum(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)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]N.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 cremaphor), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, II), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa and cyclophosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamin; acetogenins (in particular, bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, calicheamicin gamma II, calicheamicin omega II (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A; bisphosphonates such as clodronate; esperamicin; and the neocarzinostatin chromophore and related chromoprotein chromophores of the enediyne antibiotics, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-E-norleucine, morpholinodoxorubicin, Cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, nemorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, 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, thiamiprine, thioguanine; Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; Androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; Adrenal suppressants such as aminoglutethimide, mitotane, trilostane; Folate supplement such as frolinic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Eniluracil; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside (Ara-C); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts thereof

- 14 047476 slots and derivatives of any of the above.

Also included within the definition of 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®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestane, fadrozole, RIVISOR® (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, particularly oligonucleotides that downregulate gene expression in signaling pathways involved in cell proliferation impairment, such as PKCalpha, Raf and H-Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes, such as VEGF expression inhibitors (e.g. ANGIOZYME®) and HER2 expression inhibitors; (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 (AVASTEST®, Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the above.

Also included in the definition of chemotherapeutic agent are therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (AVASTEST®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idee), pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTEST®, 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, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, recivizumab, rovelizumab, sibrotuzumab, siplizumab, sontuzumab, takatuzumab tetraxetane, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleuquin, tucuzituzumab, umavizumab, urtoxazumab and visilizumab.

Compositions

While it is possible to administer (e.g. administer) the conjugate alone, it is often preferable to present it as a composition or formulation.

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

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

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

Information on suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical literature sources. 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 widely known to those skilled in the art.

- 15 047476 of this field of technology, for example, carriers, diluents, auxiliary substances, etc. In the case of formulation in the form of discrete units (for example tablets, etc.), each unit contains a given amount (dose) of the active compound.

As used herein, the term pharmaceutically acceptable refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are considered by careful medical evaluation to be suitable for use in contact with the tissues of a designated subject (e.g., a human) without causing undue 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 the other ingredients of the formulation.

The compositions may be prepared by any of the methods commonly known in the pharmaceutical field. Such methods include the step of bringing the active compound into contact with a carrier, which consists of one or more auxiliary ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into contact with carriers (e.g., liquid carriers, finely divided solid carriers, etc.) and then shaping the product as needed.

The formulation may be prepared to provide fast or slow release; immediate, delayed, sustained or extended release; or a combination thereof.

Formulations suitable for parenteral administration (e.g. by injection) include aqueous or non-aqueous isotonic, pyrogen-free sterile liquids (e.g. solutions, suspensions) in which the active ingredient is dissolved, suspended or otherwise present (e.g. in a liposome or other microparticle). Such liquids may additionally contain other pharmaceutically acceptable ingredients such as antioxidants, buffers, preservatives, stabilizing agents, bacteriostats, suspending agents, thickening agents and solutes which render the formulation isotonic with the blood (or other appropriate biological fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyhydric alcohols, glycerol, vegetable oils and the like. Examples of suitable isotonic vehicles for use in such formulations include sodium chloride injection, Ringer's solution or Ringer's lactate injection. Typically, the concentration of the active ingredient in the liquid is from about 1 to about 10 μg/ml, such as from about 10 to about 1 μg/ml. The formulations may be presented in single-dose or multi-dose hermetically 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 before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Dose

It will be appreciated by those skilled in the art that appropriate doses of the conjugates and compositions containing the conjugates may vary from patient to patient. Determination of the optimal dose will generally involve balancing the therapeutic benefit against any risk or deleterious side effects. The dose level selected will depend on a variety of factors, including but not limited to the activity of the particular compound, the route of administration, the time of administration, the rate of elimination of the compound, the duration of treatment, other drugs, compounds and/or materials used in combination, the severity of the condition and the species, sex, age, weight, condition, general health and medical history of the patient. The amount of compound and the route of administration will ultimately be left to the judgment of the physician, veterinarian or clinician, but will generally be adjusted to achieve local concentrations at the site of action that provide the desired effect without causing significant harmful or deleterious side effects.

Administration may be in a single dose, continuously, or intermittently (e.g., in divided doses at appropriate intervals) over the course of treatment. Methods for determining the most effective routes and doses of administration are well known to those skilled in the art and will vary depending on the formulation used for therapy, the purpose of therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations may be performed using a 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 about 1 mcg to about 10 mg) per day based on kilogram of body weight of the subject. In the case where the active compound is a salt, ester, amide, prodrug, or the like, the amount administered is based on the parent compound and thus the actual weight applied is increased proportionally.

The dose values described above may apply to the conjugate or the effective co

- 16 047476 amount of compound that is released after 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 to be treated, as defined above, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the discretion of the treating physician. The molecule is suitably administered to the patient in a single treatment or over a series of treatment courses. Depending on the type and severity of the disease, an amount of the molecule of from about 1 μg/kg to 100 mg/kg or more represents the initial dose of the candidate for administration to the patient, whether, for example, by one or more separate administrations or by continuous infusion. With repeated administrations over several days or longer, depending on the condition, the treatment is extended until the desired suppression of the symptoms of the disease occurs. Other dosing regimens may be applicable. The progress of this therapy can be readily monitored by conventional techniques and assays.

Drug loading

Drug loading (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 obtained by conjugation reactions can be characterized by conventional methods such as UV, reversed phase HPLC, HIC, mass spectrometry, ELISA analysis and electrophoresis. The quantitative distribution of ADC in terms of p can also be determined. By ELISA, the average p value in a certain ADC preparation can be determined (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 discernible by antibody-antigen binding and the detection limit of ELISA. In addition, the use of ELISA assays to detect antibody-drug conjugates does not provide a determination of the site of attachment of drug moieties 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 a specific value obtained for ADCs loaded with another drug, can be accomplished by methods such as reversed-phase HPLC 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 a few cysteine thiol groups, or may contain only one or a few 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, less than the theoretical maximum number of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, multiple lysine residues that are unreactive with the drug linker. Only the most reactive lysine groups are reactive with the amine-reactive linker reagent. In addition, only the most reactive cysteine thiol groups are reactive with the thiol-reactive linker reagent. In general, antibodies do not contain many or any free and reactive cysteine thiol groups that can be attached to a drug moiety. Most cysteine thiol residues in antibody compounds exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP under partially or completely reducing conditions. The loading (drug/antibody ratio) of the ADC can be controlled in several different ways, including (i) limiting the molar excess of drug linker relative to antibody, (ii) limiting the time or temperature of the conjugation reaction, and (iii) partial or limiting reducing conditions for modification of the cysteine thiol group.

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 (Troth's reagent), converting the amine to a thiol. Reactive thiol groups can be introduced into an antibody (or antibody fragment) by

- 17 047476 by means of engineering one, two, three, four or more cysteine residues (e.g. by producing mutant antibodies containing one or more non-native cysteine amino acid residues). US 7,521,541 describes engineering antibodies by introducing reactive amino acids that represent cysteine.

Cysteine amino acids can be engineered at reactive sites of the antibody and also in such a way that they do not form intrachain or intermolecular disulfide bonds (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; US 7,521,541; US 7,723,485; WO 2009/052249). The thiol groups of the engineered cysteine residues are capable of reacting with linkers of the drug of the present invention (i.e., Formula I) that contain thiol-reactive electrophilic groups such as maleimide or alpha-haloamides to form cysteine engineered antibody ADCs. The location of the drug-representing unit can thus be designed, controlled and known. Drug loading can be controlled since the thiol groups of the engineered cysteine typically react with drug-linker reagents in high yield. Engineering an IgG antibody to introduce a cysteine amino acid by substitution at a single site on the heavy or light chain provides two new cysteine residues on a symmetrical antibody. Drug loadings close to 2 can be achieved with near homogeneity of the ADC conjugation product.

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

Thus, antibody-drug conjugate compositions of the present invention may comprise mixtures of antibody-drug conjugates wherein the antibody comprises one or more drug moieties, and wherein the drug moieties may be attached to the antibody at different amino acid residues.

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

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

General routes of synthesis

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

where RL* represents -QH due to addition to the compound of formula 3 o

formula ³ pi l or its activated version.

This 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 a compound of formula 4

- 18 047476

where RL* ^prot is -Q-Prot ^N , where Prot ^N is a protecting group for the amino group.

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

with compound A5 using the Friedlander reaction.

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

by converting the group representing the fluorine atom into an amino group, for example by treatment with NH ₄ OH.

Compounds of formula 6 can be synthesized by coupling RL* ^prot . OH with compound A3.

Compounds of formula I, wherein RL is represented by formula Ia or Ib, can be synthesized from compound 1 by a coupling reaction with the compound RL-OH or its activated form.

Protective groups for the amino group

Protecting groups for the amino group are well known to those skilled in the art. Particular attention is drawn 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), pages 696871.

Additional preferences

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

Q ^X .

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, wherein Cit is citrulline.

In one embodiment, Q comprises 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 comprises naturally occurring amino acids. If the linker is a linker that is labile to cathepsin cleavage, the dipeptide is a site of action for cathepsin-mediated cleavage. Thus, the dipeptide is a recognition site for cathepsin.

In one embodiment, Q is selected from ^NH -Phe-Lys- ^C=O , ^NH -Val-Ala- ^C = ^O , ^NH -Val-Lys- ^C = ^{O NH} -Ala-Lys- ^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 ,

- 19 047476 ^NH -Gly-Val- ^C = ^O ;

where Cit represents citrulline.

Preferably Q is selected from

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

NH-Val-Lys-C*

NH-Ala-Lys- ^CO 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 the tripeptide may be any combination of naturally occurring amino acids and unnatural amino acids. In some embodiments, the tripeptide comprises naturally occurring amino acids. If the linker is a linker that is labile to cathepsin cleavage, the tripeptide is the site of action for cathepsin-mediated cleavage. Thus, the tripeptide is a recognition site for cathepsin. Tripeptide linkers of particular interest are

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

N ^H -Glu-Val-Cit- ^C = ^O ,

N ^H -aGlu-Val-Ala- ^C = ^O ,

N ^H -aGlu-Val-Cit- ^C = ^O .

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

N ^H -Gly-Gly-Phe-Gly ^C = ^O and

N ^H -Gly-Phe-Gly-Gly ^CO In some embodiments, the tetrapeptide is

N ^H -Gly-Gly-Phe-Gly ^C = ^O .

In the above images of peptide residues, NH- represents the N-terminus and ^-C ~° represents the C-terminus of the residue. The C-terminus is linked to the NH of A*. Glu represents a glutamic acid residue, i.e.

aGlu is a glutamic acid residue when linked via the α-chain, i.e.

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

Protective groups for amino acid side chains are widely known in the art and are described in

- 20 047476 in the Novabiochem catalogue and are as described above. GL:

GL can be selected from

(O ¹ - ¹ ' ¹ ) 0 be (G ^L6 ) O o /° \ Ύ-Ν C^ ¹ ' ² ) by (G ¹ - ⁷ ) ^VG— ((T ² ) ξγ (θ“) U (O ¹ - ³ ' ¹ ) s—s 0 (NO ₂ ) where the NCh group is optional (6“) N ₃ (G ^L3 - ² ) s— Y (NO ₂ ) where the NCh group is optional (G ^L1 °) ^Z C^ Η (G ³ · ³ - ³ ) s— ο ₂ ν<Ξ=/ where the group NCh is optional (G ¹ - ¹¹ ) o o V/ (G ¹ - ^{3 4} ) ¢5 ^s O ₂ N-\=/ where the NCh group is optional (G^ ² ) (G ¹ - ⁴ ) Hal Η ί where Hal represents I, Br, Cl (θ“ ³ ) χ /= ^{Ν N} -Y 7 (G ¹ - ³ ) Hal-f N (θ“ ⁴ ) ^Η 2Ν^ \

where Ar is an arylene group, such as phenylene, and X is

C _1-4 alkyl.

In some embodiments, GL is selected from GL ^1-1 and GL ^1-2 . In some of these embodiments, ^GL is ^GL1-1 .

_G LL _:

G ^LL can be selected from

- 21 047476

( _G ^ll 1-1) 0 CBA| ./nA ⁴ o (G ^LL ₈ -i) OBA (G ^ll1 · ² ) “i^X _o ^Ar/ (g ^LL8 · ² ) ,N < ^CBA 4 (C- ¹ - ² ) 0 cbaJ/'' Ν'' * 0 0 (G ^ll 9-1) cba ( _G ^LL 3-l·) cs*p _s >· (G ^LL9 - ² ) SVA (G ^LL3 · ² ) OB*|_ _S K7 (G ^ll1 °) SVA t n (G ² - ¹ - ⁴ ) CBA<___ I \ η λ— N 0 A (qLL·!!) Ύ O Ν _χ AA ⁿ <T Λ O (G^ ⁵ ) 0 CBA<___// (G ^ll12 ) .. -SVA ^Ν ' ^ίί ι ^/ 1 1 Lt ^HN ^A\ /ί X (G ^LL6 ) 0 CBA<___// (G ¹ - ¹ - ¹³ ) X ЧЪ SVA (C- ¹ -') CBAJ___ (G ^LL14 ) H U ^cba Y ⁿ x ₀ A

where Ar is a C _5-6 arylene group, such as phenylene, and X is 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 is 0-5, b1 is 0-16, b2 is 0-16, c1 is 0 or 1, c2 is 0 or 1, d is 0-5, where at least b1 or b2 is 0, and at least c1 or c2 is 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 further 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 can 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

- 22 047476 can be equal to 0, 2, 3, 4, 5 or 8.

Only one of b1 and b2 can be different from 0.

c 1 can be equal to 0 or 1.

c2 can be equal to 0 or 1.

Only one of c1 and c2 can be different from 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 further embodiments, d is 2. In further embodiments, d is 5.

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

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

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

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

In some embodiments, X a equals 1, b2 equals 0, c1 equals 0, c2 equals 1, d equals 2, and b1 can equal 0-8. In some of these embodiments, b2 equals 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 in 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 invention, the enantiomerically enriched form has an enantiomer ratio of greater than 60:40, 70:30, 80:20, or 90:10. In further embodiments, the enantiomer ratio is greater than 95:5, 97:3, or 99:1. In some embodiments, R ^L is selected from

- 23 047476

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

Examples

Silica gel column chromatography was performed using Qingdao Hailang silica gel or Biotage® Isolera™ and fractions were tested for purity by thin layer chromatography (TLC). TLC was performed using Huanghai HSF254 silica gel or Merck Kieselgel 60 F254 silica gel with a fluorescent indicator on a glass plate. TLC was visualized using UV light. Solvents for extraction and chromatography and all chemicals in pure form were purchased and used without further purification from SINOPHARM (China), VWR (US) or Sigma-Aldrich (US), unless otherwise stated. 6,8-Difluoro-3,4-dihydronaphthalene

- 24 047476

1(2H)-on was obtained from Bide Pharmatech Ltd.

Reversed-phase purification was performed on a Waters Prep HPLC system consisting of a Waters 2767, Waters 2545, Waters 515 HPLC pumps, WATERS SFO, WATERS 2424, Acquity QDa with MassLynx software.

The LC/MS analytical conditions were as follows. Electrospray mass spectrometry in positive ion detection mode was performed using a Waters Aquity H-grade SQD2. The mobile phases used were solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). A 5 min gradient was run: the initial composition with 5% B was held for 1 min, then the concentration was increased from 5% B to 95% B over a period of 3 min. The composition was held for 30 s at 95% B, then returned to 5% B over 30 s and held at this value for 84 s. The total gradient run time was 5.0 min. The flow rate was 0.8 mL/min. Columns: Agilent ZORBAX Extend 80A 1.8 µm 2.1x50 mm at 45°C.

Conditions for running the gradient for 3 min: Flow rate was 0.3 ml/min. Detection was performed at 210 nm. Columns: Waters Acquity UPLC® BEH Shield C18, 1.7 μm, 2.1 x 50 mm at 35°C, equipped with a Waters Acquity UPLC® BEH Shield C18 VanGuard, 130A, 1.7 μm, 2.1 mm x 5 mm guard column.

Example 1.

a) 6,8-Difluoro-5-nitro-1-tetralone A2

To 6,8-difluoro-1-tetralone A1 powder (15 g, 82.3 mmol) was added concentrated H2SO4 (90 mL) dropwise at 0 °C. To the resulting mixture was added _KNO3 (8.2 g, 90.1 mmol) portionwise at 0 °C. The reaction mixture was stirred at 0 °C for 2 h. The reaction was quenched with ice water (200 mL) and then extracted with EtOAc (400 mL×3). The combined organic layers were washed with aqueous NaHO3 solution (400 mL) and brine (400 mL), dried over anhydrous _MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether/EtOAc=100:1) to give compound A2 (8.1 g, yield 43%). 1H NMR (400 MHz, _CDCl3 ): δ ppm 6.98 (t, J=10.0 Hz, 1H), 3.01-2.98 (m, 2H), 2.72-2.68 (m, 2H), 2.21-2.05 (m, 2H).

b) 5-Amino-6,8-difluoro-1-tetralone A3

To a mixture of compound A2 (9.1 g, 39.6 mmol) in EtOH/ _H2O (8:1, 270 mL) were added _NH4Cl (6.4 g, 0.12 mol) and iron powder (17.6 g, 0.32 mol). The reaction mixture was stirred at 80 °C for 2 h. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure. The residue was diluted with water (50 mL) and then extracted with EtOAc (200 mL×3). The combined organic layers were washed with brine (200 mL), dried over anhydrous _MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 8:1) to give compound A3 (7.3 g, yield 94%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ ppm 7.04 (t, J=11.6 Hz, 1H), 5.05 (br s, 2H), 2.71-2.2.68 (m, 2H), 2.5 (m, 2H), 2.03-1.98 (m, 2H).

c) 5 - Acetylamino -6,8 - difluoro -1 -tetraalon A4

To a solution of compound A3 (7.3 g, 37 mmol) and Et ₃ N (4.5 g, 44.4 mmol) in DCM (100 mL) was added dropwise Ac2O (4.5 g, 44.4 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM/MeOH=300:1) to give

- 25 047476 units A4 (5.3 g, yield 60%). 1H NMR (400 MHz, CDCI3): δ ppm 6.84 (t, J=10 Hz, 1H), 6.75 (br s, 1H), 2.89-2.86 (m, 2H), 2.66-2.63 (m, 2H), 2.25 (s, 3H), 2.10-2.06 (m, 2H).

d) 5-Acetylamino-6-fluoro-8-amino-1-tetralone A5

To a solution of compound A4 (5.2 g, 21.7 mmol) in DMSO (50 mL) was added 25% aqueous _NH4OH (80 mL) at room temperature. The reaction mixture was stirred at 130 °C for 16 h. The mixture was cooled to room temperature and then extracted with EtOAc (200 mL x 5). The combined organic layers were washed with brine (200 mL), dried over anhydrous _MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM/MeOH=100:1) to give compound A5 (1.5 g, yield 30%) as a brownish solid. 1H NMR (400 MHz, DMSO-d ₆ ): δ ppm 9.16 (s, 1H), 6.42 (d, J=12.4 Hz, 1H), 2.66 (m, 2H), 2.55-2.48 (m, 2H), 2.00 (s, 3H), 1.88-1.85 (m, 2H).

e) (S)-N-(9-Ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 D-EDquinoline^-and^acetamide A7

Compound A5 (150 mg, 0.635 mmol), 168 mg (0.638 mmol) of (D)-4-ethyl-4-hydroxy-7,8-dihydro-1Hpyrano[3,4-D-indolizine-3,6,10-trione A6 and 168 mg (0.668 mmol) of pyridinium p-toluenesulfonate were mixed in 30 ml of anhydrous toluene. Using a Dean-Stark trap, the reaction mixture was heated at 130 °C for 4 h. An aqueous layer was formed under reflux. The solvent was evaporated and the residue was precipitated in 14 ml of acetone and centrifuged to obtain 180 mg of the desired product as a brown solid. The residue on the walls of the flask was washed with acetone and collected to obtain 60 mg of the desired product as a brown solid. The combined yield of crude product A7 was 82%. LCMS (0.1% formic acid/acetonitrile) ESI [M+H]=464; 1H NMR (400 MHz, DMSO-d ₆ ): signals for the desired product, δ ppm 9.77(s, 1H), 7.72(d, J=11.1 Hz, 1H), 7.25 (s, 1H), 5.36 (s, 2H), 5.17(s, 2H), 3.09 (t, J=5.5 Hz, 2H), 2.91 (t, J=5.5 Hz, 2H), 2.22(s, 1H), 2.08 (s, 3H), 1.96 (m, 2H), 1.80 (m, 2H), 0.81 (t, J=7.3 Hz, 3H).

f) (^)-4-Amino-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo (^)pyrano[3',4':6,7]indolizino[1 ,2-b]quinoline-10,13-dione 1 mg of crude compound A7 was dissolved in 0.5 ml HCl (37%) and the reaction was carried out in a sealed tube in a microwave reactor at 100 °C for 1 h. The solvent was evaporated, the residue was dissolved in 1 ml NMP and purified by Prep-HPLC with 0.1% TFA in water as solvent and 0.1% TFA in acetonitrile as solvent B. Fractions containing the desired product were collected and frozen. After lyophilization of the reaction mixture, 28 mg (42%) of the desired product 1 was obtained as an orange solid. LCMS (0.1% formic acid/acetonitrile) ESI [M+H]=422; 1H NMR (400 MHz, DMSO-d ₆ ): δ ppm 7.56 (d, J=12.4 Hz, 1H), 7.14 (s, 1H), 5.34 (s, 2H), 5.10 (s, 2H), 2.99 (t, J=6.1 Hz, 2H), 2.78 (t, J=6.1 Hz, 2 H), 1.95 (t, J=5.8 Hz, 2H), 1.79 (m, 2H), 1.40-1.00 (m, 3H), 0.81 (t, J=7.4 Hz, 3H).

Example 2.

A5 A8 A9

A11

- 26 047476 /L

a) 5,8-Diamino-6-fluoro-1-tetralone A8

A solution of 5-acetylamino-6-fluoro-8-amino-1-tetralone A5 (1.0 g, 4.2 mmol) in internal HCl (50 mL) was heated under reflux for 4 h. The mixture was concentrated under reduced pressure. The residue was slowly added to saturated aqueous _NaHO3 (60 mL). The resulting mixture was extracted with EtOAc (100 mL×3). The combined organic layers were washed with brine (100 mL), dried over anhydrous _MgSO4, and concentrated under reduced pressure to afford compound A8 (0.7 g, 90% yield) as a yellow solid.

(Microwave method) 240 mg of 5-acetylamino-6-fluoro-8-amino-1-tetralone A5 (1.06 mmol) was dissolved in 3 mL of HCl (37%) and reacted in a microwave reactor at 100 °C for 1 h. The mixture was concentrated under reduced pressure. The residue was slowly added to saturated aqueous NaHO ₃ solution (10 mL). The resulting mixture was extracted with EtOAc (15 mL x 3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to give compound A8 (180 mg, yield 87%).

b) 5-Alloc-glycine-8-amino-6-fluoro-1-tetrolαlone A9

To a solution of compound A8 (0.7 g, 3.8 mmol) and Alloc-Gly-OH (0.7 g, 4.2 mmol) in THF (50 mL) were added _Et3N (0.4 g, 4.2 mmol), HOBt (0.6 g, 4.2 mmol) and EDCI (0.9 g, 4.6 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (100 mL) and then washed with saturated aqueous NaHO3 (50 mL) and brine (50 mL). The organic phase was dried over anhydrous _MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (DCM/MeOH=200:1) to give compound A9 (0.52 g, yield 41%) as an off-white solid.

¹ H NMR (400 MHz, DMSO-de): δ ppm 9.15 (s, 1H), 7.53 (t, J=6.0 Hz, 1H), 6.41 (d, J=12.4 Hz, 1H), 5.925.88 (m, 1H), 5.33-5.28 (m, 1H), 5.20-5.17 (m, 1H), 4.51-4.49 (m, 2H), 3.78 (d, J=6.0 Hz, 1H), 2.65 (t, J=6.0 Hz, 1H), 2.55-2.49 (m, 2H), 1, 87-1.84 (m, 2H).

c) Allyl-^)-(2-((9-ethyl-5-fluoro-9-hydroxy-10,13 -dioxo-2,3,9,10,13,15-hexahydro- 1H,12H-ben- so |de|i shrsh yu [3 ',4':6,7]indolizino [1 ,2-b]quinolin-4-yl)amino)-2-oxoethyl)carbamate A10

250 mg (0.746 mmol) of compound A9, 200 mg (0.760 mmol) of (4S)-4-ethyl-4-hydroxy-7,8-dihydro-1Hpyrano[3,4-1]indolizine-3,6,10-trione A6 and 200 mg (0.796 mmol) of pyridinium p-toluenesulfonate were dissolved in 30 ml of anhydrous toluene. Using a Dean-Stark trap, the reaction mixture was heated at 130 °C for 4 h. The solvent was evaporated and the residue was precipitated in acetone to give 250 mg of the desired product as a brown solid after centrifugation and drying in vacuo. The residue on the wall of the flask was washed with acetone and concentrated to give 110 mg of compound A10 as a brown solid. The yield of the crude product was 87%. LCMS (0.1% formic acid/acetonitrile) ESI [M+H]=563; 1H NMR (400 MHz, DMSO-d ₆ ): δ ppm: signals for the desired product, 9.88 (s 1 H), 7.83 (d, J=11 Hz, 1 H), 7.63 (t, J=6.1 Hz, 1 H), 7.33 (s, 1 H), 5.99-5.88 (m, 1 H), 5.44 (s, 2 H), 5.32 (dd, J=6.4 Hz, 1 H), 5.26 (s, 2 H), 5.20 (dd, 1= Hz, 1 H), 4.53 (d, J=5.3 Hz, 2 H), 3.93 (d, J=6 Hz, 2 H), 3.18 (t, J=5.7 Hz, 2 H), 2.97 (t, J=5.3 Hz, 2H), 2.23 (s, 1H), 2.03 (m, 2H), 1.88 (m, 2H), 0.88 (t, J=7.4 Hz, 3H).

- 27 047476

d) (9H-Fluoren-9-yl)methyl (2-((2-((^)-1-((2-((^)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-

2,3,9,1(),13,15-hexahydro-1H,12H-benzoAl|pyrano[3',4':6,7|indolizino[1,2-b|quinolin-4-yl)amino) -2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate A12

A11 was synthesized as follows.

Fmoc-GGF (500 mg, 0.997 mmol, synthesized by the synthetic route using standard peptide solution) and 276 mg (1.50 mmol) of pentafluorophenol were dissolved in 20 mL of NMP. To this suspension, 0.33 mL of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (1.8 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction progress was monitored by LC-MS.

mg (0.089 mmol) of compound A10, 103 mg (0.0887 mmol) of Pd( _PPh3 ) ₄ and 145 μl (0.899 mmol) of triethylsilane were dissolved in 2 ml of NMP. To the mixture was added 4 ml (0.2 mmol) of acid-activated solution of A11. The reaction progress was monitored by LC-MS. The reaction mixture was precipitated with ether (2 x 15 ml vials) and centrifuged to give compound A12. The solid was air-dried and used without further purification.

e) ^)-2-(2-(2-Aminoacetamido)acetamido)-N-(2-((^)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-

2,3.9.10.1.3.15-hexahydro-1H.12H-benzo[pyrano[3'.4':6.7|indolizino[1.2-b|quinolin-4-yl)amino)-2oxoethyl)-3-phenylpropanamide A13

Crude compound A12 was dissolved in 2 mL of NMP, 2 mL of 20% 4-methylpiperidine (3.0 mmol) was added. The reaction mixture was stirred at room temperature and the reaction progress was monitored by LC-MS. After completion of the reaction, the reaction mixture was purified by Prep-HPLC with 0.1% TFA in water as solvent A and 0.1% TFA in acetonitrile as solvent B. Fractions containing the desired product were collected and frozen/lyophilized to yield 23 mg (35%) of compound A13 as a yellow solid.

LCMS (0.1% formic acid/acetonitrile) ESI [M+H|=741; 'H NMR (400 MHz, DMSO-d ₆ ): δ ppm 9.74 (s, 1H), 8.51 (t, J=5.5 Hz, 1H), 8.43 (t, J=5.5 Hz, 1H), 8.30 (d, J=8.2 Hz, 1H), 7.91 (br, s, 2H+H+), 7.76 ( d, J=11 Hz, 1H), 7.26 (s, 1H), 7.21-7.15 (m, 4H), 7.14-7.07 (m, 1H), 5.37 (s, 2H), 5.21 (s, 2H), 4.55 (m, 1H), 3.98 (m, 2H), 3.82 (dd, J=16.8, 5 .6 Hz, 1H), 3.64 (dd, J=16.8, 5.6 Hz, 1H), 3.48 (m, 2H), 3.11 (t, J=5.6 Hz, 2H), 3.05 (dd, J=13.9, 4.4 Hz, 1H), 2.91 (t, J=5.3 Hz, 2H), 2.73 (dd, J=13 ,8, 9.9 Hz, 1H), 1.96 (m, 2H), 1.80 (m, J=7.4 Hz, 2H), 0.81 (t, J=7.4 Hz, 3H).

j) 1-(3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-M-(2-((2-((^)-1-((2 -((^)-9-ethyl-5fluoro-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)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2oxoethyl)amino)- 2-oxoethyl)-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-amide 2 mg (0.020 mmol) of compound A13 and 15 mg (0.022 mmol) of Mal-PEG8-NHS ester A14 were dissolved in 1 ml of NMP and 14 μl (0.10 mmol) of TEA were added to the solution. The reaction mixture was stirred at room temperature. During the course of the reaction, reactions were monitored by LC/MS. After complete consumption of the amine, the reaction mixture was filtered and purified by Prep-HPLC with 0.1% TFA in water as solvent A and 0.1% TFA in acetonitrile as solvent B. Fractions containing the desired product was collected/frozen/lyophilized to yield 14 mg (53%) of the desired product as a yellow solid.

LCMS (0.1% formic acid/acetonitrile) ESI [M+H|=1315; 'H NMR (400 MHz, DMSO-d ₆ ): δ ppm 9.64 (s, 1H), 8.43 (t, J=5.6 Hz, 1H), 8.12-8.06 (m, 2H), 7.94 (t, J=4.6 Hz, 2H), 7.76 (d, J=11 Hz, 1H), 7.26 (s, 1H), 7.21-7.15 (m, 4 H), 7.14-7.07 (m, 1H), 6.93 (s, 2H), 5.37 (s, 2H), 5.20 (s, 2H), 4.51-4.46 (m, 1H), 3.95 (m, 2H), 3.72 (d, J=6.0 Hz, 1H), 3.68 (d, J=6.0 Hz, 2H), 3.60 (d, J=5.6 Hz, 2H), 3.44-3.41 (m, overlaps with the peak for PEG and H ₂ O), 3.29 (t, J=6.0 Hz, 2H), 3.14-3.00 (m, 5 H), 2.91 (t, J=6.1 Hz, 2H), 2.78 (m, 1H), 2.31 (t, J=6.5 Hz, 2H), 2.26 (t, J=7.2 Hz, 2H), 1.96 (m, 2H), 1.80 (m, 2H), 0.81 (t, J=7.2 Hz, 3H).

General information for example 3.

Flash chromatography was performed using Biotage® Isolera™ and fractions were checked for purity by 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 and used without further purification from VWR U.K. All chemicals were purchased from Sigma-Aldrich unless otherwise stated. PEGylated reagents were obtained from Quanta biodesign (USA) via Stratech (UK).

The analytical conditions for LC/MS were as follows. Electrospray mass spectrometry in positive ion detection mode was performed using a 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). A gradient was run over 3 min: the initial formulation with 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 formulation was held for 50 s at 100% B, then

- 28 047476 was spun to 5% B in 5 s and held at this value for 5 s. The total gradient run time was 3.0 min. The flow rate was 0.8 mL/min. Detection was performed at 254 nm. Columns: Waters Acquity UPLC® BEH Shield RP18, 1.7 μm, 2.1 x 50 mm at 50 °C, equipped with a Waters Acquity UPLC® BEH Shield RP18 VanGuard, 130A, 1.7 μm, 2.1 mm x 5 mm guard column.

Example 3.

a) An alternative synthesis of ^)-N9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15hexahydro- 1H,12H-benzo ^]pyrano[3 ',4':6,7]indolizino[1 ^-^quinolin^-yl^cetamide A7

Compound A5 (136 mg, 0.57569 mmol) and trione A6 (167 mg, 0.63 mmol) were dissolved in toluene (20 mL) before addition of pyridin-1-ium 4-methylbenzenesulfonate (149 mg, 0.59 mmol) and the mixture was stirred at reflux for 3.5 h. LCMS indicated the reaction was complete. The reaction mixture was concentrated in vacuo and triturated with MeCN to give compound A7 (220 mg, 0.4746 mmol, 82.45% yield) as a beige solid, which was used without further purification. The MeCN washes were concentrated in vacuo and purified by isolera chromatography (0-5% MeOH in _CH2Cl2 ) to give _{an additional} 20 mg of compound A7 after isolera purification (0-5% MeOH in _CH2Cl2 ) as a brown solid. LCMS: RT=1.41 min, 464.5 [M+H] ⁺ .

b) An alternative synthesis of (^)-4-amino-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro10H, 13H-benzo(^)pyrano[3',4':6,7]indolizino[1',2-b]quinoline-10,13-dione!

Compound A7 (220 mg, 0.474 mmol) was dissolved in 5 M HCl _aq (15 mL, 75 mmol, 5 mol/L) and the mixture was stirred for 4 h at 80 °C, at which point LCMS indicated that all starting material had been consumed. The reaction mixture was concentrated in vacuo to give compound 1.2HCl (235 mg, 0.475 mmol, 100.2% yield) as a red solid. The product was used as crude material in the next step. LCMS: RT = 1.49 min, mass not reported.

c) In situ formation of [(2R)-2-[(2-nitrophenyl)disulfanyl]propyl]carbonyl chloride A16 (2R)-2-[(3-Nitro-2-pyridyl)disulfanyl]propan-1-ol A15 (14 mg, 0.057 mmol) was dissolved in CH ₂ Cl ₂ (0.5 mL, 8 mmol). Pyridine (5.0 μL, 0.062 mmol) was added, followed by triphosgene (6 mg, 0.020 mmol) and the reaction mixture was stirred under argon for 30 min, after which LCMS (quenched with Et ₂ NH) indicated completion of the reaction. LCMS: RT = 1.94 min, 346.4 [M+Et ₂ NH]+

d) (R)-2-((3-Nitropyridin-2-yl)disulfanyl)propyl(^)-9-ethyl-5-fluoro-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 3

In a separate flask _, compound 1.2HCl (22 mg, 0.044 mmol) was dissolved in _CH2Cl2 (1 mL, 15.60 mmol, 100 wt%), DIPEA (45 μL, 0.258 mmol), and pyridine (22 μL, 0.272 mmol).

The reaction mixture with chloroformate was added to the aniline solution and the mixture was stirred for 30 min, after which LCMS indicated that the chloroformate was consumed, with no presence of compound 3 observed. Additional triphosgene was added to the reaction mixture and stirred for 20 min, after which LCMS indicated the presence of a small amount of product. Additional triphosgene was added and the mixture was stirred for 1 h, after which

- 29 047476 of which LCMS indicated that compound 3 was the major component. The reaction mixture was concentrated in vacuo and purified by isolera chromatography (0-4% MeOH in CH2Cl2) followed by reverse phase isolera chromatography (0-60% eluent B in eluent A) to afford, after freeze-drying, pure compound 3 (8 mg, 0.01153 mmol, 25.91% yield) as a yellow solid.

Eluent A = 0.01% HCO2H in _H2O

Eluent B=0.01% HCO2H in MeCN LCMS: RT=1.95 min, 694.6 [M+H]+.

Example 4.

a) 5-Fmoc-alanine-6-fluoro-8-amino-1-tetralone A17

164 mg (0.84 mmol) of 5,8-diamino-6-fluoro-1-tetralone A8 were dissolved in 6 mL of THF and 315 mg (1.01 mmol, 1.2 equiv) of Fmoc-Ala-OH and 138 mg of HOAt (1.01 mmol, 1.2 equiv) were added to the solution. Then 275 μL (1.24 mmol) of EDCI and 142 μL (1.02 mmol) of _Et3N were added to the solution. The reaction mixture was stirred at room temperature. The reaction progress was monitored by LC/MS. After 4 h, the reaction mixture was placed in a freezer. The reaction mixture was treated with 50 mL EtOAc/50 mL _H2O followed by washing the organic layer with _H2O , then brine and then dried over _Na2SO4 . The crude product was purified by _silica gel column.

- 30 047476 lem with dichloromethane/methanol to obtain 260 mg of the desired product. LCMS ESI [M+H]=488.93; according to calculations 488.20

b) A18

210 mg of 5-Fmoc-alanine-6-fluoro-8-amino-1-tetralone A17 (0.43 mmol), 114 mg of trione A6 (0.43 mmol) and 109 mg of pyridinium p-toluenesulfonate (0.43 mmol) were dissolved in 30 ml of anhydrous toluene. The reaction mixture was heated in an oil bath equipped with a Dean-Stark trap at 130 °C for 4 h to obtain an aqueous layer under reflux. The solution was decanted and dried under reduced pressure to obtain 270 mg of the desired product. The solvent was evaporated from the solution and dissolved in 0.5 ml of NMP and precipitated with 14 ml of diethyl ether. Centrifugation gave a brown solid, which was repeatedly washed with ether. The resulting solid was dried to yield an additional 30 mg of crude product. The entire amount of crude desired product (300 mg, 97% yield) was used without further purification. LCMS ESI [M+H]=716.01; calculated 715.26

c) A19

220 mg (0.31 mmol) of A18 was dissolved in 2 mL of NMP and 150 μL (1.28 mmol) of 4-methylpiperidine was added to the solution. The reaction mixture was stirred at room temperature and the reaction progress was monitored by LC-MS. After completion of the reaction, the reaction mixture was purified with 0.1% TFA in water/0.1% TFA in acetonitrile. Fractions containing the desired product were collected, pooled and then frozen to obtain 42 mg (yield 28%) of the desired product after lyophilization. LCMS ESI [M+H] = 493.23; according to calculations 493.19

d) A20 mg (0.046 mmol) of A19 was dissolved in 0.5 mL of NMP. To the above solution were added 35 mg (0.11 mmol) of Boc-Val-NHS and 20 μL (0.12 mmol) of DIPEA. The reaction mixture was stirred at room temperature and the reaction progress was monitored by LC-MS. After completion of the reaction, the product was precipitated with ether and washed twice with ether. The residue was dried under air to give 32 mg (yield 99%) of a brown solid. LCMS ESI [M+H]=693.67; according to calculation 692.31

e) A21

Crude A20 was treated with 0.1 mL TFA in 0.3 mL DCM and the reaction progress was monitored by LC-MS. After completion of the reaction, DCM and trifluoroacetic acid were removed in vacuo. The residue was dried in vacuo overnight to give 27 mg (98% yield) of crude product. LCMS ESI [M+H]=592.04; calculated 592.26. 1H NMR (DMSO-d ₆ ): δ ppm 10.07 (s, 1H), 8.78 (d, J=6.9 Hz, 1H), 8.10 (d, J=4.1 Hz, 3H), 7.82 (d, J=11.0 Hz, 1H), 7.32 (s, 1H), 6.53 (s, br, 1H), 5 .43 (s, 2H), 5.27 (s, 2H), 4.67 (q, J=6.7 Hz, 1H), 4.67 (q, J=7.0 Hz, 1H), 3.63 (q, J=5.2 Hz, 1H), 3.17 (t, J=5.9 Hz, 2H), 2.96 (t, J=5.7 Hz, 2H), 2.14-2.07 (m, 1H), 2.05-1.94 (m, 2H), 1.87 (p, J=7.3 Hz, 2H), 1.46 (d, J=7.1 Hz, 3H), 0.96 (dd, J=6.8, 4.2 Hz, 6 H), 0.88 (t, J=7.3 Hz, 3H).

f) A14 mg (0.017 mmol) of Mal-PEG8-NHS A14 was dissolved in 1 ml of NMP. To the above solution were added 10.3 mg (0.017 mmol) of crude A21 and 12 μl (0.0094 mmol) of DIPEA. The reaction progress was monitored by LC-MS. After consumption of the starting material A21, the reaction mixture was acidified with 8 μl of TFA and then purified with 0.1% TFA in water/0.1% TFA in acetonitrile to obtain 11 mg of the desired product (yield 54%) after lyophilization. LCMS ESI [M+H]=1166.09; according to calculations 1165.52 ¹ H NMR (DMSO-d6): δ ppm 9.86 (s, 1H), 8.26 (d, J=6.7 Hz, 1H), 8.00 (t, J=5.5 Hz, 1H), 7.90 (d, J=8.7 Hz, 1H), 7.80 (d, J=11 Hz, 1H), 7.32 (s, 1H), 7.00 (s, 2H), 5.43 (s, 2H), 5.26 (s, 2H), 4.54 (q, J=6.7 Hz, 1H), 4.26 (dd, J=8.2, 6.7 Hz, 1H), 3.81-3.48 (m, overlaps with the peak for H ₂ O), 3.35 (t, J=6.0 Hz, 2H), 3.20-3.10 (m, 4H), 2.96 (t, 2H), 2.40 (t, J=6.3 Hz, 1H), 2.32 (m, 2H), 2.06-1.93 (m, 3H), 1.93-1.80 (m, 2H), 1.41 (d, J=7.1 Hz, 3H), 0.91-0.80 (m, 9H).

Example 5.

- 31 047476 mg (0.037 mmol) of A21 and 14 mg (0.045 mmol) of Ma1-caproyl-Na2B A22 were dissolved in 0.5 mL of NMP and 12 μL (0.068 mmol) of DIPEA were added to this solution. The reaction mixture was stirred at room temperature and the reaction progress was monitored by LC-MS. After completion of the reaction, the reaction mixture was quenched with 12 μL of trifluoroacetic acid and purified by prep-HPLC with 0.1% TFA in water/0.1% TFA in acetonitrile to give 10 mg (34% yield) of the desired product after lyophilization. LCMS ESI [M+H]=785.88; according to calculations 785.33. 1H NMR (DMSO-d6): δ ppm 9.86 (s, 1H), 8.23 (d, J=6.7 Hz, 1H), 7.84 (d, J=8.7 Hz, 1H), 7.80 (d, J=11 Hz, 1H), 7.32 (s, 1H), 6.98 (s, 2H), 6.55-6.50 (m, 1H), 5.43 (s, 2H), 5.26 (s, 2H), 4.53 (q, J=7.0 Hz, 1H), 4.22 (dd, J=8.7, 6.7 Hz, 1H), 3.16 (t, J=6.0 Hz, 2H), 2.96 (t, 2H), 2.22- 2.07(m, 3 H), 2.04-1.94 (m, 3H), 1.93-1.81 (m, 2H), 1.49-1.43 (m, 4 H), 1.40 (d, J=7.1 Hz, 3H), 1.15 (q, J=7.5 Hz, 2H), 0.92-0.82 (m, 9H).

Example 6.

OH 0.0365 mg A13 (0.0365 mmol) and 13 mg Mal-caproyl-NHS A22 (0.04217 mmol) were dissolved in 0.5 mL NMP and 10 μL DIPEA were added to the reaction mixture. The reaction mixture was stirred at room temperature and the reaction progress was monitored by LC-MS. After completion of the reaction, the reaction mixture was purified by prep-HPLC with 0.1% TFA/ACN to give 9 mg (26%) of the desired product after lyophilization. LCMS (0.1% formic acid/acetonitrile) ESI [M+H] = 933.29; calculated 933.36. 1H NMR (DMSO-de): δ ppm 9.70 (s, 1H), 8.49 (d, J=5.8 Hz, 1H), 8.15 (d, J=8.0 Hz, 1H), 8.05 (t, J=5.7 Hz, 1H), 8.01 (t, J=5.7 Hz, 1H), 7.82 (d, J=11.0 Hz, 1H), 7.32 (s, 1H), 7.26 (s, 2H), 7.24 (s, 2H), 7.21-7.15 (m, 1H), 6.98 (s, 2H), 6.56 (br, 1H), 5.43 (s, 2H), 5.26 (s, 2H), 4.58-4.52 (m, 1H), 4.02 (dt, J=16.9, 6.0 Hz, 2H), 3.76 (dd, J=16.7, 5.9 Hz, 1H), 3.65 (d, J=5.7 Hz, 2H), 3.60 (dd, J=16.7, 5.4 Hz, 1H), 3.34 (t, J=7.1 Hz, 2H), 3.1 7 (t, J=5.7 Hz, 2H), 3.10 (dd, J=13.7, 4.3 Hz, 1H), 2.97 (t, J=5.4 Hz, 2H), 2.84 (dd, J=13.7, 9.7 Hz, 1H), 2.08 (t, J=7.5 Hz, 2H), 2.02 (t, J=5.7 Hz, 2H), 1.87 (dq, J=7.3 Hz, 2H), 1.44 (dt, J=7.3 Hz, 4 H), 1.201.12 (m, 2H), 0.88 (t, J=7.3 Hz, 3H).

Example 7 Conjugation

Classical conjugation

Trastuzumab-derived anti-HER2 antibodies and negative control NIP228 antibodies were used as full-length antibodies to generate ADCs. Antibody reconstitution was performed by mixing antibodies with 50 mM tris-(2-carboxyethyl)-phosphine (TCEP) in 1x PBS, 1 mM EDTA, pH 7.2 at 37°C and the reaction mixture was shaken for 1 h. The reconstituted antibodies were then used for conjugation using a 5 molar excess of compound 2 in dimethyl sulfoxide (Sigma-Aldrich). The buffer volume was adjusted to achieve a final concentration of 10% DMSO for the conjugation solution. Conjugation was performed at room temperature with shaking for 1 h. This method was used to generate:

Her2-2 conjugate, Nip228-2 conjugate, Her2-4 conjugate, Nip228-4 conjugate, Her2-5 conjugate, Nip228-5 conjugate, Her2-6 conjugate, Nip228-6 conjugate.

Conjugation with construction

Herceptin antibodies and Nip228 antibodies engineered 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: 510.1021/acs.molpharmaceut.6b00995). These antibodies were prepared using 50 mM tris-(2-carboxyethyl)-phosphine (TCEP) and reconstituted with 50 mM in PBS 1x, 1 mM EDTA, pH 7.2 at 37°C with shaking for 3 h. Uncapped antibodies were dialyzed against conjugation buffer (PBS 1x, 1 mM EDTA, pH 7.2) at 4°C overnight. The reconstituted antibodies were then

- 32 047476 was used for oxidation using 20 molar excess of 50 mM dehydroascorbic acid (dhAA) at room temperature with shaking for 4 h. The reduced antibodies were then used for conjugation using 8 molar excess of the antibody payload prepared in 100% dimethyl sulfoxide (final DMSO concentration was 10% Sigma-Aldrich). Conjugation was performed at room temperature with shaking for 1 h. This method was used to prepare Her2*-2 conjugate, Nip228*-2 conjugate.

Cleaning

Following conjugation, ADC was purified by ceramic hydroxyapatite (CHT) HPLC to remove free base 2 and other contaminants. Cleanup was performed using a 5 mL Bio-Scale Mini CHT Type II column with a 40 μm cartridge (Bio-Rad) and an AKTA Pure system (GE Healthcare). ADC was diluted 1:3 in purified water prior to loading. After loading and washing with two column volumes of buffer A, ADC was eluted using a linear gradient of 50% buffer B over 30 min. (Buffer A: 10 mM sodium phosphate buffer, pH 7.0; Buffer B: 10 mM sodium phosphate/2 M sodium chloride, pH 7.0). SEC was used to characterize fractions containing ADC. Fractions were concentrated to approximately 1 mg/mL ADC. SEC was used to analyze the monomer, aggregate, and fragment content of ADC. Data collection and method were performed using MassHunter software (Agilent). ADC was filtered using a 0.22 mm syringe filter (Pall Corporation) to remove potential endotoxin contamination. ADC aliquots were stored at -80°C for further use.

The Her2-2 conjugate had a DAR of 8.0, while the Nip228-2 conjugate had a DAR of 7.79.

The Her2-4 conjugate had a DAR of 8.0, while the Nip228-4 conjugate had a DAR of 7.88.

The Her2-5 conjugate had a DAR of 8.0, while the Nip228-5 conjugate had a DAR of 8.0.

The Her2-6 conjugate had a DAR of 7.91, while the Nip228-6 conjugate had a DAR of 8.0.

The Her2*-2 conjugate had a DAR of 2.0, and the Nip228*-2 conjugate had a DAR of 2.0.

Example 8. Additional conjugation A solution of mM tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (40 mol. equiv./antibody, 11.2 μM, 1.12 mL) to 20 mL of a solution of antibody (Herceptin, engineered to contain a cysteine inserted between positions 239 and 240) (42 mg, 280 nM) in a reconstitution buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA), giving a final antibody concentration of 2.1 mg/mL. The reduction mixture was allowed to react at room temperature for 16 h (or until complete reduction as determined by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. To remove any excess reducing agent by spin filter centrifugation, the reduced antibody was buffer exchanged with reoxidation buffer containing PBS and 1 mM EDTA. A 50 mM solution of dehydroascorbic acid (DHAA, 30 mol equiv/antibody, 7.0 μM, 141 μL) in DMSO was added to 22 mL of the buffer exchanged reconstituted antibody (35.2 mg, 235 nM) and the reoxidation mixture was allowed to react for 2 h and 30 min at room temperature with gentle (60 rpm) shaking at an antibody concentration of 1.6 mg/mL (or additional DHAA was added and the reaction mixture was allowed to react for a longer time until the cysteine thiol groups were completely reoxidized, re-forming interchain cysteine disulfide bonds as determined by UHPLC). The reoxidation mixture was then sterilizing filtered. Compound 3 was added as a solution in DMSO (20 mol eq/antibody, 2.2 μM, in 1.29 mL DMSO) to 10.5 mL of this reoxidized antibody solution (16.8 mg, 112 nM), the pH was adjusted with 1.16 mL 1 M sodium bicarbonate to give a final DMSO concentration of 10% (v/v) and a final sodium bicarbonate concentration of 10% (v/v). The solution was allowed to react at room temperature for 2 h with gentle shaking. The conjugation reaction was then quenched by the addition of N-acetylcysteine (11 μM, 112 μL at 100 mM), purified and buffer exchanged with pH 6.0 buffer containing 25 mM histidine and 205 mM sucrose using a 50 mL Amicon Ultracell 50 kDa MWCO spin filter, sterilize-filtered, and analyzed. UHPLC analysis of an intact Her2*-3 conjugate sample using a Shimadzu Prominence system equipped with a Sepax Proteomix HIC butyl-No. 5 4.6 x 35 mm, 5 μm column, eluting with a gradient of pH 7.4 buffer containing 25 mM sodium phosphate, 1.5 M ammonium sulfate, and 20% acetonitrile (v/v) in 25 mM sodium phosphate buffer pH 7.4, with detection at 214 and 330 nm (specific

- 33 047476 for compound 3), demonstrated the presence of unconjugated and conjugated antibody attached to one or two molecules of compound 3, corresponding to a drug/antibody ratio (DAR) of 1.48 molecules of compound 3 per antibody.

UHPLC analysis of the Her2*-3 conjugate sample using a Shimadzu Prominence system equipped with a Tosoh Bioscience TSKgel SuperSW mAb HTP column, 4 μm, 4.6 x 150 mm (with a 4 μm, 3.0 x 20 mm guard column), eluting at a flow rate of 0.3 ml/min with 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 monomer purity of 98%. UHPLC SEC analysis showed that the final Her2*-3 conjugate concentration was 1.38 mg/mL in 8.6 mL, and the resulting mass of Her2*-3 conjugate was 11.9 mg (yield 71%).

Example 9. In vitro testing of cytotoxicity of test compounds

Killing of human tumor cell lines was assessed in vitro using the protocol recommended in the CELLTITER-GLO® kit (Promega, Madison, WI). Briefly, ^3x103 cells in 80 µl RPMI+10% FBS were added to the inner wells of a white-walled 96-well plate (Corning® Costar®, Fisher Scientific, Waltham, MA). The following cell lines were tested: A549, HCT116, and SKBR3. Test compounds were diluted to 5Ax from a stock solution (125 µg/ml) in RPMI+10% FBS. Treatments were then serially diluted 1:10 in RPMI+10% FBS. Twenty ml of this series were added to the cells in triplicate, resulting in a 9-point dose curve of test compound ranging from 25 mM at the highest concentration to 2.5 x 10 ^-7 mM at the lowest. Controls consisting of DMSO (vehicle medium) and medium alone were also included. The plates were incubated at 37°C, 5% CO ₂ for 72 h. At the end of the incubation period, 100 ml of substrate solution (Promega, Madison, WI) was added to each well. Luminescence was measured with an EnVision Multilabel plate reader (Perkin Elmer, Waltham, MA). Data were analyzed and plotted using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA).

Exatecan:

were included in the analysis for comparison with compound 1.

IC50 (nM) Exatecan Connection 1 A549 2,449 0.2484 SKBR3 0,181 0,09575 NST116 0.9956 0,1644

Example 10. In vitro testing of ADC cytotoxicity

For in vitro cytotoxicity testing of ADCs, the same protocol was used as for small molecules. Human breast cancer cell lines SKBR-3 (ATCC) and NCI-N87 (ATCC), which express HER2, were used for in vitro cytotoxicity assays. MDA-MB-468 breast cancer cell lines (ATCC), which do not express HER2, were used as a negative control. Five-fold serial dilutions of each ADC (starting at 300 μg/mL) were added to each well in triplicate. ADC-treated cells were cultured for six days. At the end of the incubation period, 100 mL of substrate solution (Promega, Madison, WI) was added to each well. Luminescence was measured using an EnVision Multilabel plate reader (Perkin Elmer, Waltham, MA). Data were analyzed and graphs were generated using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA)._______

ECso (mcg/ml) Neg2-2 NIP228-2 Neg2*-2 NIP228*-2 SKBR3 0,0004781 84.91 0,002179 10.06 NCI-N87 0,001003 -77610 0,01878 - 10637 MDA-MB-468 2,849 -275569 - 137570 466,0

Example 11. Additional in vitro cytotoxicity testing of ADCs

The concentration and viability of cells from subconfluent culture (80-90% confluency) in a T75 flask were measured by trypan blue staining and counted using a LUNA-II™ automated cell counter. Cells were diluted to 2 x 10 ⁵ /mL and dispensed (50 µL/well) into 96-well flat-bottomed plates.

A stock solution (1 ml) of antibody-drug conjugate (ADC) (20 μg/ml) was prepared

- 34 047476 by diluting filter-sterilized ADC in cell culture medium. A set of 8x 10-fold dilutions of the ADC stock solution were prepared in a 24-well plate by serially transferring 100 µl into 900 µl of cell culture medium. The ADC dilutions were added (50 µl/well) to 4 replicate wells of a 96-well plate containing 50 µl of the previously seeded cell suspension. Control wells received 50 µl of cell culture medium. The 96-well plate containing the cells and ADC 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 using the MTS assay. MTS (Promega) was added (20 µl/well) to each well and incubated for 4 h at 37°C in a CO2-saturated incubator. The absorbance in the wells was measured at 490 nm. The percentage of surviving cells was calculated from the average absorbance of the 4 ADC-treated wells compared to the average absorbance of the 4 untreated control wells (100%). IC ₅₀ was determined from the dose-response data using GraphPad Prism using a nonlinear 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. ___________________________________________________

ECso (mcg/ml) Neg2*-3 NCI-N87 0,09328 MDA-MB-468 -0.9772

Example 12. In vivo studies in mouse xenograft models (JIMT-1)

Mice

On study day 1, female SCID mice (Fox Chase SCID®, CB17/Icr-Prkdcscid/IcoIcrCrl, Charles River) were 10 weeks old and weighed between 17.3 and 26.3 g. Animals were provided ad libitum access to water (reverse osmosis, 1 ppm Cl) and a modified NIH 31 diet and an irradiated Lab Diet® diet containing 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. Mice were housed on irradiated Enrich-o'cobs™ Laboratory Animal Bedding in fixed microisolators under a 12-hour light cycle at 20-22°C (68-72°F) and 40-60% humidity. Charles River Research strictly adheres to the Guide for the Care and Use of Laboratory Animals regarding housing, breeding, surgical procedures, feeding and water management, and veterinary care. The animal care and use program at Charles River Research is accredited by the Association for Assessment and Accreditation of Laboratory Animal Science International (AAALAC), ensuring that accepted standards for the care and use of laboratory animals are met.

Tumor cell culture

JIMT-1 human breast cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 100 units/ml sodium penicillin G, 100 μg/ml streptomycin sulfate, 25 μg/ml gentamicin, and 2 mM glutamine. Cell cultures were maintained in tissue culture flasks in a humidified incubator at 37°C with 5% CO2 and 95% air.

In vivo implantation and tumor growth

JIMT-1 tumor cells used for implantation were harvested at logarithmic growth phase and resuspended in 50% Matrigel® Matrix (Corning®) in phosphate-buffered saline (PBS) at a concentration of 1 x 10 ⁸ cells/mL. Each experimental mouse was injected subcutaneously in the right flank with 1 x 10 ⁷ JIMT-1 cells (0.1 mL of cell suspension), and tumor growth was monitored when the average size approached the target range of 150-250 mm ³ . Tumors were measured twice weekly in two directions using calipers, and the volume was calculated using the formula ζ ₃ x ^{× 1}

Tumor volume (mm ^j ) = —-— where w=width and I=length of the tumor in mm. Tumor mass can be calculated based on the assumption that 1 mg is equivalent to 1 mm ³ of tumor volume.

Twenty-one days after tumor implantation, on a day designated as study day 1, animals with individual tumor volumes ranging from 172-221 mm ³ were assigned to nine groups (n=8), with the median tumor volume per group being 199-202 mm ³ .

Processing

Treatment was initiated on day 1 in nine groups of female SCID mice (n=8) with subcutaneously injected JIMT-1 xenografts (172-221 ^mm3 ). Each test agent was evaluated

- 35 047476 was administered at a dose of 3 mg/kg intravenously (i.v.) as a single injection on day 1 (qdx 1). The vehicle-treated group served as a control for tumor engraftment and growth.

Tumors were measured twice weekly until the end of the study on day 78. Each mouse was euthanized when the tumor reached an endpoint volume of 1000 mm ³ or on the last day of the study, whichever came first. For each mouse, time to endpoint (TTE) was calculated using the following equation:

logio (volume at end point) - b

TTE = m

where TTE is expressed in days, endpoint volume is expressed in ^mm3 , b is the intercept, and m is the slope of the line obtained from a linear regression of the log-transformed tumor growth data set. Treatment outcome was determined based on the percentage tumor growth reduction (%TGD), defined as the percentage reduction in the median TTE of the treatment group compared to the control group of mice, with statistically significant differences between groups considered at P<0.05, using log-rank survival analysis.

Treatment efficacy can also be determined based on tumor volumes in animals remaining in the study on the final day. MTV(n) was defined as the median tumor volume on the final day of the study among the remaining number of animals (n) whose tumor volume did not reach the endpoint volume.

Treatment efficacy can also be determined based on the incidence and magnitude of regression responses observed during the study. Treatment can induce partial regression (PR) or complete regression (CR) of tumor in an animal. A PR response is defined as tumor volume that is 50% or less of day 1 tumor volume on three consecutive measurements during the study and is equal to or greater than 13.5 ^mm3 on one or more of the three measurements. A CR response is defined as tumor volume that is less than 13.5 ^mm3 on three consecutive measurements during the study. An animal with a CR response is further classified as tumor free survivor (TFS) at the end of the study. Animals are monitored for the occurrence of regression response.

Results

All regimens were well tolerated. The median TTE for controls was 39.4 days, with the maximum possible TGD found to be 38.6 days (98%) for the 78-day study.

Group η Remedy Median TTE value T-S % THD Statistical significance 1 8 Carrier medium 39.4 ___ ___ ___ 2 8 Neg2-2 78,0 38.6 98 3 8 Neg2-4 78,0 38.6 98 *** 4 8 Neg2-6 78,0 38.6 98 5 8 Neg2-5 78,0 38.6 98 *** 6 8 NIR228-2 56.9 17,5 44 *** 7 8 ΝΙΡ228-4 49.9 10,5 27 8 8 ΝΙΡ228-6 60.2 20.8 53 *** 9 8 ΝΙΡ228-5 45.9 6.5 16 *

- 36 047476

Group Remedy MTV (η) Day 78 Regression Median value (BW), lowest level Fatal outcomes PR CR TFS TR NTR 1 Carrier medium ___ 0 0 0 -1.5%, day 33 0 0 2 Neg2-2 255 (8) 3 5 1 -3.7%, day 50 0 0 3 Neg2-4 226 (8) 4 4 0 -1.0%, day 4 0 0 4 Neg2-6 550 (7) 6 2 0 -5.0%, day 40 0 0 5 Neg2-5 365 (8) 3 5 0 -10.4%, day 75 0 0 6 NIP228-2 — 0 0 0 -0.6%, day 5 0 0 7 NIP228-4 — 1 0 0 -3.0%, day 43 0 0 8 NIP228-6 — 1 0 0 -1.0%, day 4 0 0 9 NIP228-5 — 0 0 0 -4.8%, day 43 0 0

Four trastuzumab-ADCs achieved a maximum TGD of 98%, with each demonstrating both partial and complete regressions.

Example 12. In vivo studies in mouse xenograft models (NCI-N87)

Mice:

On study day 1, female SCID mice (Fox Chase SCID®, CB17/Icr-Prkdcscid/IcoIcrCrl, Charles River) were twelve weeks old and weighed between 15.9 and 26.4 g. Animals were provided ad libitum access to water (reverse osmosis, 1 ppm Cl) and a modified NIH 31 diet and an irradiated Lab Diet® diet containing 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. Mice were housed on irradiated Enrich-o'cobs™ Laboratory Animal Bedding in fixed microisolators under a 12-hour light cycle at 20-22°C (68-72°F) 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, monitoring of feeding and water, and provision of veterinary care.

The CR Research Animal Care and Use Program is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), ensuring that accepted standards for the care and use of laboratory animals are met.

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 flasks in a humidified incubator at 37°C with 5% _CO2 and 95% air.

In vivo implantation and tumor growth

NCI-N87 tumor cells used for implantation were harvested at logarithmic growth phase and resuspended in 50% Matrigel® Matrix (Corning®) in phosphate-buffered saline (PBS) at a concentration of 1 x 10 ⁸ cells/mL. Each experimental mouse was injected subcutaneously in the right flank with 1 x 10 ⁷ NCI-N87 cells (0.1 mL of cell suspension), and tumor growth was monitored when the average size approached the target range of 150-250 mm ³ . Tumors were measured twice weekly in two directions using calipers, and the volume was calculated using the formula:

w ^{2 x}

Tumor volume (mm ^j ) = —-— where w is the width and l is the length of the tumor in mm. The tumor mass can be calculated based on the assumption that 1 mg is equivalent to 1 mm ³ of tumor volume.

Forty days after tumor implantation, on a day designated as study day 1, animals with individual tumor volumes ranging from 144-256 mm ³ were assigned to nine groups (n=8), with the median tumor volume per group being 190-192 mm ³ .

Processing

Treatment began on day 1 in nine groups of female SCID mice (n=8) bearing subcutaneously injected NCI-N87 xenografts (190-192 ^mm3 ). Each test drug was evaluated at 3 mg/kg intravenously (iv) as a single injection on day 1 (qdx1). The vehicle group served as a control for tumor engraftment and growth.

Tumors were measured twice weekly until the end of the study on day 59. Each mouse was subjected to

- 37,047,476 euthanasia when the tumor reached the endpoint volume of 800 mm ³ or on the last day of the study, whichever came first. Tumor progression was slow and all evaluable animals remained in the study on the last day of the study. Because no animal reached the endpoint tumor volume, percent growth inhibition (% TGI) of tumor on the last day of the study was used to assess efficacy. MTV(n), the median tumor volume for the number of animals, n, on the last day (day 59), was determined for each group to calculate total tumor volume. % TGI was defined as the difference between the MTV of the designated control group (Group 1) and the MTV of the drug group, expressed as a percentage of the MTV of the control group:

% TGI [1-(MTVdrug treatment/MTV control] X 100

Treatment efficacy can also be determined from tumor volumes in animals remaining in the study on the final day and from the incidence and magnitude of regression responses. MTV(n) was defined as the median tumor volume on the final day (day 59) among the remaining evaluable animals, n.

Treatment may induce partial regression (PR) or complete regression (CR) of tumor in an animal. A PR response was defined as tumor volume that was 50% or less of its day 1 volume on three consecutive measurements during the study, and was equal to or greater than 13.5 ^mm3 on one or more of the three measurements. A CR response was defined as tumor volume that was less than 13.5 ^mm3 on three consecutive measurements during the study. Animals were assessed for a PR or CR event only once during the study, and were only assessed as CR if both PR and CR criteria were met.

Results

All regimens were well tolerated. Control tumors were characterized by slow, progressive growth but did not reach the endpoint of 800 ^mm3 at study completion. The magnitude of tumor growth inhibition was assessed on the last day of the study (day 59). ____________________________,_________________ _____________________________,

Group 1 2 3 4 5 6 7 8 9 Group η Remedy 8 Medium-nost 8 Neg2-2 8 Neg2-4 8 Neg2-6 8 Neg2-5 8 ΝΙΡ228-2 8 ΝΙΡ228-4 8 ΝΙΡ228-6 8 ΝΙΡ228-5 Remedy MTV (η) Day 59 [tel 550 (8) 3(8) 1(8) 5(8) 4(8) 446 (8) 405 (8) 493 (8) 466 (8) Regression % Sta TGI val* 22 *** 100 *** 22 *** 22 *** 19 ns 26 ns 10 ns 15 ns Average tistic 1IMOSTB Lethal PR CR TR NTR 1 Carrier medium 0 0 -7.0%, day 52 0 0 2 Neg2-2 1 7 -10.1%, day 52 0 0 3 Neg2-4 0 8 -10.3%, day 56 0 0 4 Neg2-6 2 6 -10.6%, day 56 0 0 5 Neg2-5 0 8 -7.6%, day 59 0 0 6 NIR228-2 0 0 -9.3%, day 56 0 0 7 ΝΙΡ228-4 0 0 -10.0%, day 59 0 0 8 ΝΙΡ228-6 0 0 -6.4%, day 59 0 0 9 NIR228-5 0 0 -5.8%, day 59 0 0

All trastuzumab-ADC treatment groups had statistically significant TGI at day 59 compared with the vehicle treatment group (P< 0.001).

- 38 047476

Statement of the essence of the invention

l. Compound of formula I

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

where Q represents

where 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, bl is 0-16, b2 is 0-16, cl is 0 or 1, c2 is 0 or 1, d is 0-5, where at least bl or b2 is 0 and at least cl or c2 is 0;

GL is a linker for attachment to a unit that is a ligand;

(ib):

about _Ι_1 Γ-,Ι-2

R to R

C>

e lb where R ^l1 and RL ² 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.

2. The compound according to claim 1, wherein RL is represented by formula Ia.

3. The compound according to claim 2, wherein Q is an amino acid residue.

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

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

6. The compound according to item 5, where Q is selected from

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

-Phe-Lys- ^C=O , -Val-Ala- ^C=O , -Val-Lys- ^C=O , Ala-Lys- ^C=O ,

-Val-Cit-Phe-CitC=O

-Leu-CitC=O

C=O,

-Ile-Cit- ^C=O ,

-Phe-Arg- ^C=O , -Trp-Cit- ^C=O and -Gly-Val- ^C=O .

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

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

- 39 047476

9. The compound according to claim 8, wherein Q is selected from -Glu-V al-Ala-', '-Glii-Val-Cn-',

-aGlii-V al-Ala-' and

AaGlu-Val-Cn-'.

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

11. The compound according to claim 10, where Q is selected from

-Gly-Gly-Plie-Gly' and

-Gly-Phe-Gly-Gly'.

12. The compound according to claim 11, wherein Q is NH-Gly-Gly-Phe-Gly' ^-0

13. A compound according to any one of paragraphs 2-12, where a is equal to 0-3.

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

15. The compound according to item 13, where a is equal to 0.

16. A connection according to any one of paragraphs 2-15, where b1 is equal to 0-8.

17. The connection according to item 16, where b1 is equal to 0.

18. The compound according to item 16, where b1 is equal to 2.

19. The compound according to item 16, where b1 is equal to 3.

20. The compound according to item 16, where b1 is equal to 4.

21. The compound according to item 16, where b1 is equal to 5.

22. The compound according to item 16, where b1 is equal to 8.

23. A compound according to any one of paragraphs 2-15 and 17, where b2 is equal to 0-8.

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

25. The compound according to item 23, where b2 is equal to 2.

26. The compound according to item 23, where b2 is equal to 3.

27. The compound according to item 23, where b2 is equal to 4.

28. The compound according to item 23, where b2 is equal to 5.

29. The compound according to item 23, where b2 is equal to 8.

30. A connection according to any one of claims 2 to 29, where c1 is equal to 0.

31. A compound according to any one of claims 2 to 29, where c1 is equal to 1.

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

33. A compound according to any one of claims 2 to 30, where c2 is equal to 1.

34. A compound according to any one of claims 2 to 33, where d is 0 to 3.

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

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

37. A compound according to any one of claims 2 to 33, where d is equal to 5.

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

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

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

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

42. A compound according to any one of claims 2 to 12, where a is 0, b1 is 0, c1 is 0, c2 is 0, d is 1, and b2 is 0-8.

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

44. A compound according to any one of claims 2 to 12, wherein b1 equals 0, b2 equals 0, c1 equals 0, c2 equals 0, one of a and d equals 0 and the other of a and d equals 1 to 5.

45. The compound of claim 41, wherein the other of a and d is 1 or 5.

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

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

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

- 40 047476

(O ¹ ·!- ¹ ) O A (θ“) 0 I (G ^L % At ⁷ (+’) Br—\ (b ¹ - ² ) O ά/Υ (θ“) V ABOUT (O ¹ - ³ ' ¹ ) S—S ____/ (G ^L9 ) N ₃ V (No ₂ ) where the group NCh is optional (G ¹ - ³ · ² ) s— (G ^L1 °) \ d H ^x t ^/ (NO ₂ ) where the group NCh is optional (G ¹ - ³ · ³ ) A + (G ¹ - ¹¹ ) Λ s—s ^z * d 0 ₂ νΧξ=/ where the group NCh is o w optional (G^· ⁴ ) S— do ₂ n-\=/ (G^ ² ) where the group NCh is optional (G ¹ - ⁴ ) and°. Hal N—| H » (G ³ - ¹³ ) X.__/ ^N ' \\ N-_ h where Hal represents I, Bg, C1 (<+) About Hal— «T») h ₂ n _x oA

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

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

50. The compound according to claim 48, wherein G ^L is G ^L1-1 .

51. The compound according to claim 1, wherein R ^L is represented by formula Ib.

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

53. The 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 simultaneously represent methyl.

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

- 41 047476 cyclopropylene group.

56. The 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. A compound according to any one of claims 51 to 56, where e is 0.

58. A compound according to any one of claims 51 to 56, where e is equal to 1.

59. Conjugate of formula IV

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

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

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

where R ^L1 and R ^L2 are as defined in any one of claims 1 and 52-56; and p is an integer from 1 to 20. 60. The conjugate of claim 59, where G ^LL is selected from

- 42 047476

0 CBAj 0 (G ^ll1 · ² ) 0 -Ar - ^x o (C- ¹ - ² ) 0 CBA< N ^x °'Ύ'^ * V-Ax 0 0 (GLL ³ ' ¹ ) ((54.3.2-, ^CBA |_ _S ^~7 ( _G LL-4) CBA<___ ! \ H /7 ^N 0 7 (<?“) 0 CBA*__Λ °h ( _G LL6) p // (G ^LL7 ) CBA*___ 7

(GLL8-1) CBA A ( _G LL ₈ -2) N zCBA ( _G LL9-1) ⁴ _N CBA _(G LL9-2) 5. ( _G LL10) CBA H ( _G LL11) ^cba Z λ Ύ ( ) ^H <J/ ) O ( _G LL12) , _g SVA 1 Lt ^ΗΝ ^χίΑ / 7 X Ah CBA ( _G LL14) ^H Ί

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

61. The conjugate of claim 60, wherein G ^LL is selected from G ^LL1-1 and G ^LL1-2 .

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

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

64. The conjugate according to any one of claims 59-62, wherein the unit representing the ligand 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) to (89):

(1) BMPR1B;

(2) E16;

(3) STEAP1;

(4) 0772P;

(5) MPF;

(6) Napi3b;

(7) Sema 5b;

(8) PSCA hlg;

- 43 047476 (9) ETBR;

(10) MSG783;

(11) STEAP2;

(12) TrpM4;

(13) CRYPTOCURRENCY;

(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) SEACAM5;

(42) MET;

(43) MUC1;

(44) CA9;

(45) EGFRvIII;

(46) CD33;

(47) CD19;

(48) IL2RA;

(49) AXL;

(50) CD30 - TNFRSF8;

(51) BCMA-TNFRSF17;

(52) CTAg - STA;

(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;

- 44 047476 (66) FOLR1;

(67) GPNMB;

(68) TIM-1-HAVCR1;

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

(7O) 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-MS4A1;

(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).

67. The conjugate of any one of claims 64-66, wherein the antibody or antibody fragment is a cysteine engineered antibody.

68. The conjugate of any one of claims 64-61, wherein the drug loading (p) for drugs (D) with respect to the antibody (Ab) is an integer from 1 to about 10.

69. The conjugate of claim 62, wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

70. A mixture of conjugates according to any one of claims 64-63, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is from about 1 to about 10.

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

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

73. A conjugate or mixture according to any one of claims 59-70 or a pharmaceutical composition according to claim 66 for use in the treatment of a proliferative disease in a subject.

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

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

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

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

78. The method according to 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 of claims 59-70 in a method for the manufacture of a medicinal product for the treatment of a proliferative disease.

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

81. Compound A:

82. The compound according to claim 81 in the form of a single enantiomer or in enantiomerically enriched form.

- 45 047476

83. Compound of formula VI

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

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, which is selected from:

(ia):

where Q is where 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 unit that is a ligand;

(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.

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

P1-3. The compound according to item P1-2, wherein Q is an amino acid residue.

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

P1-5. The compound according to item P1-2, wherein Q is a dipeptide residue.

P1-6. The compound according to item P1-5, where Q is selected from ^NH -Phe-Lys- ^C=O , ^NH -Val-Ala- ^C=O , ^NH -Val-Lys- ^C=O , ^NH Ala-Lys- ^C =O,

- 46 047476 ^NH -Val-Cit- ^C = ^O , ^NH -Phe-Cit- ^< '-Leii-Cil'-Ile-Cn-·, '-Phe-Arg-', '-Trp-Cil-' and '-Gly-Val-'.

P1-7. The compound according to item P1-6, wherein Q is selected from '-Phe-Lys-', '-Val-Cil-' and '-Val-Ala-'.

P1-8. The compound according to item P1-2, wherein Q is a tripeptide residue.

P1-9. The compound according to item P1-8, where Q is selected from '-Glu-V al-Ala-', '-Glu-Val-Cil-'.

'-uGlii-V al-Ala-' and '-aGlii-V al-Cil-'.

P1-10. The compound according to item P1-2, wherein Q is a tetrapeptide residue.

P1-11. The compound according to item P1-10, wherein Q is selected from '-Gly-Gly-Phe-Gly' and '-Gly-Phe-Gly-Gly'

P1-12. The compound according to claim P1-11, wherein Q is '''-Gly-Gly-Phe- ^GlyCO .

P1-13. A connection according to any of P1-2 through P1-12, where a is 0-3.

P1-14. The connection according to item P1-13, where a is equal to 0 or 1.

P1-15. Connection according to item P1-13, where a is equal to 0.

P1-16. Connection according to any of items P1-2 through P1-15, where b1 equals 0-8.

P1-17. Connection according to item P1-16, where b1 equals 0.

P1-18. Connection according to item P1-16, where b1 equals 2.

P1-19. The connection according to item P1-16, where b1 equals 3.

P1-20. Connection according to item P1-16, where b1 equals 4.

P1-21. Connection according to item P1-16, where b1 equals 5.

P1-22. Connection according to item P1-16, where b1 equals 8.

P1-23. A compound according to any one of paragraphs P1-2 through P1-15 and P1-17, where b2 is equal to 0-8.

P1-24. Connection according to item P1-23, where b2 equals 0.

P1-25. The connection according to item P1-23, where b2 is equal to 2.

P1-26. The connection according to item P1-23, where b2 equals 3.

P1-27. The connection according to item P1-23, where b2 equals 4.

P1-28. The connection according to item P1-23, where b2 is equal to 5.

P1-29. The connection according to item P1-23, where b2 is equal to 8.

P1-30. Connection according to any of items P1-2 through P1-29, where c equals 0.

P1-31. A connection according to any of items P1-2 through P1-29, where c equals 1.

P1-32. A connection according to any of items P1-2 through P1-31, where d is 0-3.

P1-33. The compound according to item P1-32, where d is equal to 1 or 2.

P1-34. The connection according to item P1-32, where d equals 2.

P1-35. A compound of any one of P1-2 through P1-12, where a equals 0, b1 equals 0, c equals 1, d equals 2, and b2 equals 0-8.

P1-36. The connection according to item P1-35, where b2 is equal to 0, 2, 3, 4, 5 or 8.

P1-37. A compound of any one of P1-2 through P1-12, where a equals 1, b2 equals 0, c equals 0, d equals 0, and b1 equals 0-8.

P1-38. The connection according to item P1-37, where b1 is equal to 0, 2, 3, 4, 5 or 8.

P1-39. A compound of any one of P1-2 through P1-12, where a equals 0, b1 equals 0, c equals 0, d equals 1, and b2 equals 0-8.

P1-40. The compound according to item P1-39, where b2 is equal to 0, 2, 3, 4, 5 or 8.

P1-41. A compound of any one of P1-2 through P1-12, where b1 equals 0, b2 equals 0, c equals 0, one of a and d equals 0, and the other of a and d equals 1-5.

P1-42. The compound of paragraph P1-41, where the other of a and d is equal to 1 or 5.

P1-43. A connection according to any one of items P1-2 through P1-42, where GL is selected from

- 47 047476

O k (G ²⁶ ) 0 o M (G ^L1 - ² ) (G ^L7 ) Br— (G ¹ - ² ) 0 AY 0 (G ¹ - ⁸ ) (G ²³ ' ¹ ) s—s (θ% ^N 3>- A- (NO ₂ ) where the group NCh is optional (G ^L3 ' ² ) S— ___/ (G ^L1 °) 0 H I ⁷ (NO ₂ ) where the group NCh is optional (G ²³ ' ³ ) (G ²¹¹ ) O s—s) III \ <Hn o ₂ n-\^=/ where the NCh group is optional /i / w (G ²³ ' ⁴ ) S—S^/ / (G ²¹² ) /Ч Ο ₂ ν\Ξ=/ where the group NCh is optional (6“) /__ ^on| |N (G ²³³ ) XZ ^N = ^N \\ /Ук where Hal represents I, Bg, C1 (G ²⁵ ) Haik N (G ²¹⁴ ) ^H2N X _O A

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

P1-44. The compound according to item P1-43, where GL is selected from G ^L1-1 and G ^L1-2 .

P1-45. The compound according to paragraph P1-43, where G ^L is G ^L1-1 .

P1-46. The compound according to item P1-1, where R ^L is represented by formula Ib.

P1-47. The compound according to item 4 P1-6, where R ^L1 and R ^L2 simultaneously represent H.

P1-48. The compound according to claim P1-46, wherein R ^L1 is H and R ^L2 is methyl.

P1-49. The compound according to claim P1-46, wherein R ^L1 and R ^L2 simultaneously represent methyl.

P1-50. The compound according to claim P1-46, where R ^L1 and R ^L2 together with the carbon atom to which they are bonded form a cyclopropylene group.

P1-51. The compound according to claim P1-46, where R ^L1 and R ^L2 together with the carbon atom to which they are bonded are

- 48 047476 form a cyclobutylene group.

P1-52. A connection according to any of items v46 through P1-51, where e equals 0.

P1-53. A connection according to any of P1-46 through P1-51, where e equals 1.

P1-54. Conjugate of formula IV

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

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

ABOUT

il , , G la QX ^x where Q and X are as defined in any one of paragraphs P1-1 through P1-42, and G ^LL is a linker attached to a unit that is a ligand; and (ib'):

where R ^L1 and R ^L2 are as defined in any one of paragraphs P1-1 and P1-47 through P1-51; and p is an integer from 1 to 20.

P1-55. The conjugate according to item P1-54, wherein G ^LL is selected from

- 49 047476

( _G ^LL1 -l·) 0 (G^s- ¹ ) CBA A (G ^ll1 · ² ) 0 (G ^LL8 · ² ) N,CBA (G ^LL2 ) 0 O (G^ ⁹ - ¹ ) *N CBA _(G L ^l 3-1) “ps'' ⁴ _(g l ^l 9- ² ) nA J^CBA (g ^LL3 · ² ) (G ^ll1 °) CBA aza H (G ^ll - ⁴ ) CBA<___ I \ n /7 ^N 0 A (G ^LL11 ) ^CBA λ ^H <J ) 0 (C- ¹ - ⁵ ) About CBA|__Π (G ^LL12 ) .CBA ⁿ AD\__/i X (G ^LL6 ) ABOUT // (G ^LL13 ) X /~ ^N CBA (G ^LL7 ) SVA| (G ^LL14 ) ^H -1

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

P1-56. Conjugate according to item P1-55, wherein G ^LL is selected from G ^LL1-1 and G ^LL1-2

P1-57. The conjugate of P1-56, wherein G ^LL is G ^LL1-1 .

P1-58. A conjugate according to any one of paragraphs P1-54 to P1-57, wherein the ligand unit is an antibody or an active fragment thereof.

P1-59. The conjugate according to item P1-58, wherein the antibody or antibody fragment is an antibody or antibody fragment to a tumor-associated antigen.

P1-60. The conjugate of paragraph P1-59, 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) 0772P;

(5) MPF;

(6) Napi3b;

(7) Sema 5b;

(8) PSCA hlg;

(9) ETBR;

(10) MSG783;

- 50 047476 (11) STEAP2;

(12) TrpM4;

(13) CRYPTOCURRENCY;

(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 - STA;

(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;

- 51 047476 (68) TIM-1-HAVCR1;

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

(7O) 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-MS4A1;

(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).

P1-61. The conjugate of any one of paragraphs P1-58 to P1-60, wherein the antibody or antibody fragment is a cysteine engineered antibody.

P1-62. The conjugate of any one of items P1-58 to P1-61, wherein the drug loading (p) for the drugs (D) with respect to the antibody (Ab) is an integer from 1 to about 10.

P1-63. The conjugate according to item P1-62, where p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

P1-64. A mixture of conjugates according to any one of paragraphs P1-58 to P1-63, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is from about 1 to about 10.

P1-65. A conjugate or mixture according to any of paragraphs P1-54 to P1-64 for use in therapy.

P1-66. A pharmaceutical composition comprising a conjugate or mixture according to any one of items P154 to P1-64 and a pharmaceutically acceptable diluent, carrier or excipient.

P1-67. A conjugate or mixture according to any one of paragraphs P1-54 to P1-64 or a pharmaceutical composition according to paragraph P1-66 for use in the treatment of a proliferative disease in a subject.

P1-68. A conjugate, mixture or pharmaceutical composition according to paragraph P1-67, wherein the disease is cancer.

P1-69. Use of a conjugate or mixture according to any of paragraphs P1-54 to P1-64 or a pharmaceutical composition according to paragraph P1-66 in a method of conservative treatment.

P1-70. A method of conservative treatment, including administering to a patient a pharmaceutical composition according to paragraph P1-66.

P1-71. The method according to paragraph P1-70, wherein the method of conservative treatment is intended for the treatment of cancer.

P1-72. The method according to paragraph P1-71, wherein the patient is administered a chemotherapeutic agent in combination with a conjugate.

P1-73. Use of a conjugate or mixture according to any of paragraphs P1-54 to P1-64 in a method for the manufacture of a medicinal product for the treatment of a proliferative disease.

P1-74. A method of treating a mammal suffering from a proliferative disease, comprising administering an effective amount of a conjugate or mixture according to any of paragraphs P1-54 to P1-64 or a pharmaceutical composition according to paragraph P1-66.

P1-75. Connection A

A

P1-76. The compound according to item P1-75 in the form of a single enantiomer or in an enantiomerically enriched form.

- 52 047476

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

P2-1. Compound of formula I

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

(ia) where Q is

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

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

(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.

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

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

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

P2-5. The compound according to paragraph P2-2, wherein Q is a dipeptide residue.

P2-6. The connection according to item P2-5, where Q is selected from

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

NH

-Phe-Lys- ^C=O -Val-Ala- ^C=O -Val-Lys- ^C=O Ala-Lys- ^C=O , -Val-Cit- ^C=O , -Phe-Cit- ^C=O , -Leu-Cit- ^C=O , -Ile-Cit- ^C=O ,

-Phe-Arg-Trp-Cit-C

-Gly-Val- ⁽

C=O

P2-7. The compound according to item P2-6, where Q is selected from ^NH -Phe-Lys- ^C=O ,

- 53 047476

NH-V al-ίΊΐ-' and

NH-Val-Ala-'.

P2-8. The compound according to paragraph P2-2, wherein Q is a tripeptide residue.

P2-9. The compound according to item P2-8, where Q is selected from '-Glii-V al-Ala-', '-Glu-Val-Cn-·.

NH-u.Glii-V al-Ala-' and '-uGlii-Val-Cn-'.

P2-10. The compound according to item P2-2, wherein Q is a tetrapeptide residue.

P2-11. The connection according to item P2-10, where Q is selected from

NH-Gly-Gly-Phe-Gly' and

NH-Gly-Phe-Gly-Gly'.

P2-12. The connection according to item P2-11. where Q is

NH-Gly-Gly-Phe-Gly'.

P2-13. Connection according to any of items P2-2 through P2-12, where a equals 0-3.

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

P2-15. Connection according to item P2-13, where a is equal to 0.

P2-16. Connection according to any of items P2-2 through P2-15, where bl is 0-8.

P2-17. The connection according to item P2-16, where bl is equal to 0.

P2-18. The connection according to item P2-16, where bl is equal to 2.

P2-19. The connection according to item P2-16, where bl is equal to 3.

P2-20. The connection according to item P2-16, where bl is equal to 4.

P2-21. The connection according to item P2-16, where bl is equal to 5.

P2-22. The connection according to item P2-16, where bl is equal to 8.

P2-23. A connection according to any of P2-2 through P2-l5 and P2-l7, where b2 is 0-8.

R2-24. The connection according to item R2-23, where b2 is equal to 0.

R2-25. The connection according to item R2-23, where b2 equals 2.

R2-26. The connection according to item R2-23, where b2 equals 3.

R2-27. The connection according to R2-23, where b2 is equal to 4.

R2-28. The connection according to item R2-23, where b2 equals 5.

R2-29. The connection according to item R2-23, where b2 equals 8.

P2-30. Connection according to any of items P2-2 through P2-29, where c equals 0.

P2-3l. A connection according to any of items P2-2 through P2-29, where c equals l.

P2-32. A connection according to any of items P2-2 through P2-3l, where d is 0-3.

P2-33. The connection according to item P2-32, where d is equal to l or 2.

P2-34. The connection according to item P2-32, where d equals 2.

P2-35. A compound of any one of P2-2 through P2-l2, where a equals 0, bl equals 0, c equals l, d equals 2, and b2 equals 0-8.

P2-36. The compound according to paragraph P2-35, where b2 is equal to 0, 2, 3, 4, 5, or 8.

P2-37. A compound of any one of P2-2 through P2-l2, where a equals l, b2 equals 0, c equals 0, d equals 0, and bl equals 0-8.

P2-38. The compound according to paragraph P2-37, where bl is equal to 0, 2, 3, 4, 5 or 8.

P2-39. A compound of any one of P2-2 through P2-l2, where a equals 0, bl equals 0, c equals 0, and d equals l, and b2 equals 0-8.

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

P2-4l. A compound of any one of P2-2 through P2-l2, where bl equals 0, b2 equals 0, c equals 0, one of a and d equals 0, and the other of a and d equals l-5.

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

P2-43. A connection according to any one of items P2-2 through P2-42, where GL is selected from

- 54 047476

( ^GLM ) (<T ⁶ ) o ?-{ Ύ~ν (G ^l1 · ² ) by (<T ⁷ ) ^Bg -U <<T ² ) brY 0 (Article ⁸ ) U (G ^L3 -') S— (ST ⁹ ) N ₃ u (NO ₂ ) where the group NCh is optional (G ^L3 - ² ) s— (G ^L1 °) /(2^ U n (NO ₂ ) where the group NCh is optional (<T ³ ' ³ ) (ST ¹¹ ) O s— ___/ //7 \ O 0 ₂ νΧξ=/ where the group NCh is optional 1 / V/ <<Τ ^3-4 ) S S (G ¹¹² ) <do ₂ n-\=/ where the group NCh is optional (CT ⁴ ) /__^0 ^Hi ίΗ (Article ¹³ ) \\ Ah 7 where Hal represents I, Bg, C1 (G ¹³ ) Ha.-f (<t ¹⁴ ) η ₂ ν. ϊ

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

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

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

P2-46. The compound according to item P2-1, where R ^L is represented by formula Ib.

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

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

P2-49. The compound according to claim P2-46, wherein R ^L1 and R ^L2 simultaneously represent methyl.

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

- 55 047476

P2-51. The compound according to paragraph P2-46, where R ^L1 and R ^L2 together with the carbon atom to which they are bonded form a cyclobutylene group.

P2-52. A connection according to any of P2-46 through P2-51, where e equals 0.

P2-53. A connection according to any of P2-46 through 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, wherein L is a ligand (i.e., targeting agent) unit, D ^L is a drug-linker unit represented by formula III

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

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

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

P2-55. Conjugate according to item P2-54, where G ^LL is selected from ______________________________

( _G L ^L1 -l·) About CBAg ^x o (G^ ⁸ · ¹ ) CBA ((^-2) O X ^ ^A X y ^x o (G^s- ² ) .NV ^BA O SVA| hK \__L o O (G^ ⁹ - ¹ ) CBA (G ^LL3-1 ) solrX (G ^L L9-2) N-X CBA (G ^LL3 · ² ) SVAR)'·/ (G ^ll1 °) CBA IT ^N- \H (G ^LL · ⁴ ) SVA<___ O (G ^LLU ) ^H <Y \ N--\ J < ^H w

- 56 047476

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

P2-56. The conjugate according to item P2-55, wherein G ^LL is selected from G ^LL1-1 and G ^LL1-2 .

P2-57. The conjugate of P2-56, wherein G ^LL is G ^LL1-1 .

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

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

P2-60. The conjugate of paragraph 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) 0772P;

(5) MPF;

(6) Napi3b;

(7) Sema 5b;

(8) PSCA hlg;

(9) ETBR;

(10) MSG783;

(11) STEAP2;

(12) TrpM4;

(13) CRYPTOCURRENCY;

(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;

- 57 047476 (38 .1) SSTR2;

(38 .2) SSTR5;

(38 .3) SSTR1;

(38 .4) SSTR3;

(38 .5) SSTR4;

(39) ITGAV;

(40) ITGB6;

(41) SEACAM5;

(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 - STA;

(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, target in prostate tumor - mindin/RG-I;

(7O) 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-MS4A1;

(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 of any one of paragraphs P2-58 to P2-60, wherein the antibody or antibody fragment is a cysteine engineered antibody.

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

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

- 58 047476

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

P2-65. A conjugate or mixture according to any of paragraphs P2-54 to P2-64 for use in therapy.

P2-66. A pharmaceutical composition comprising a conjugate or mixture according to any one of items P254 to P2-64 and a pharmaceutically acceptable diluent, carrier or excipient.

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

P2-68. A conjugate, mixture or pharmaceutical composition according to paragraph P2-67, wherein the disease is cancer.

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

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

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

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

P2-73. Use of a conjugate or mixture according to any of paragraphs P2-54 to P2-64 in a method for the manufacture of a medicinal product for the treatment of a proliferative disease.

P2-74. A method of treating a mammal suffering from a proliferative disease, comprising administering an effective amount of a conjugate or mixture according to any of paragraphs P2-54 to P2-64 or a pharmaceutical composition according to paragraph 66.

P2-75. Connection A

A

P2-76. The compound according to item P2-75 in the form of a single enantiomer or in an enantiomerically enriched form.

Claims (21)

1. Compound of formula I and salts thereof, wherein R ^L is a linker for attachment to an antibody or antigen-binding fragment of an antibody, which is selected from: (ia): where Q is: (a) an amino acid residue selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg and Trp; or (b) a dipeptide residue selected from ^NH -Phe-Lys- ^C = ^O , ^NH -Val-Ala- ^C = ^O , ^NH -Val-Lys- ^C = ^O , ^NH -Ala-Lys- ^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 or - 59 047476 (c) a tripeptide residue selected from ^NH -Glu-Val-Ala- ^C= °', ^NH -Glu-Val-Cit- ^C =°, ^NH -aGlu-Val-Ala- ^C ° and ^NH -aGlu-Val-Cit- ^C =°; or (d) a tetrapeptide residue selected from ^NH -Gly-Gly-Phe-Gly ^C =° and ^NH -Gly-Phe-Gly-Gly ^C =°, where ^NH- represents the N-terminus and ^-C ° represents the C-terminus of the residue; X represents where a is 0-5, bl is 0-16, b2 is 0-16, cl is 0 or 1, c2 is 0 or 1, d is 0-5, where at least b1 or b2 is 0 and at least c1 or c2 is 0; G ^l is a linker for attachment to the antibody or its active fragment, where G ^L is selected from - 60 047476 (G ^L3-1 ) s—s 0 (NOz) where the NO 2 group is optional (G ^L9 ) N ₃ (G ^L3 - ² ) 0 (NOz) where the NO 2 group is optional (G ^L1 °) H (G ^L3 - ³ ) S— <H _N ο ₂ νΛ^=/ where the NO 2 group is optional (G ^L11 ) o o W (G ^L3 - ⁴ ) S— where the NO 2 group is optional (G ^L12 ) ”^^0 (G ^M ) O Hal N—| H * where Hal is I, Br, Cl (G ^L13 ) /ί )=* X (G ^L5 ) O Hal—# n (G ^L14 ) H ₂ N Ϊ 0 where Ar is phenylene and X' is C _1-4 alkyl; (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. 2. The compound according to claim 1, wherein R ^L is represented by formula Ia. 3. The compound according to item 2, where a is equal to: (a) 0-3; or (b) 0 or 1; or (c) 0. 4. The compound according to item 2 or 3, where b1 is equal to: (a) 0-8; or (b) 0; or (c) 2; or (d) 3; or (e) 4; or (f) 5; or (g) 8. 5. A connection according to any of paragraphs 2, 3, where b2 is equal to: - 61 047476 (a) 0-8; or (b) 0; or (c) 2; or (d) 3; or (e) 4; or (f) 5; or (g) 8. 6. A connection according to any of paragraphs 2-5, where: (i) c1 is equal to: (a) 0 or (b) 1 and (ii) c2 is: (a) 0 or (b) 1; where at least one of c1 and c2 is equal to 0. 7. A connection according to any one of paragraphs 2-6, where d is equal to: (a) 0-3; or (b) 1 or 2; or (c) 2; or (d) 5. 8. A connection according to any of paragraphs 2-7, where: (a) a is 0, b1 is 0, c1 is 1, c2 is 0, d is 2, b2 is 0, 2, 3, 4, 5, or 8; or (b) a is 1, b2 is 0, c1 is 0, c2 is 0, d is 0 and b1 is 0, 2, 3, 4, 5, or 8; or (c) a is 0, b1 is 0, c1 is 0, c2 is 0, d is 1 and b2 is 0, 2, 3, 4, 5, or 8; or (d) b1 equals 0, b2 equals 0, c1 equals 0, c2 equals 0, one of a and d equals 0, and the other of a and d equals 1 or 5; or (e) a equals 1, b2 equals 0, c1 equals 0, c2 equals 1, d equals 2, and b1 equals 0, 2, 3, 4, 5, or 8. 9. A compound according to any one of claims 1 to 8, where GL is selected from GL ^1-1 and GL ^1-2 . 10. The compound according to claim 1, wherein RL is represented by formula Ib and: (a) R ^L1 and R ^L2 simultaneously represent H; or (b) R ^L1 represents H and R ^L2 represents methyl; or (c) R ^L1 and R ^L2 simultaneously represent methyl; or (d) where R ^L1 and R ^L2 together with the carbon atom to which they are bonded form a cyclopropylene group, or (e) where R ^L1 and R ^L2 together with the carbon atom to which they are bonded form a cyclobutylene group. 11. The compound according to claim 1, which is 12. Conjugate of formula IV L - (D ^L )p (IV) or a pharmaceutically acceptable salt thereof, wherein L is an antibody or an active fragment thereof, D ^L is a drug-linker unit represented by formula III R ^ll is a linker attached to the antibody or active fragment thereof, selected from (ia'): - 62 047476 where Q and X are as defined in any one of claims 1 to 8, and G ^LL is a linker attached to the antibody or active fragment thereof, where G ^LL is selected from ( _G ^LL1 -i) CBAT___ 0 _n A (GLL8- ¹ ) OBA A ^x o (G ^ll1 - ² ) OBAS___ O Ar j. Y (G ^LL8 - ² ) < ^{CBA N} / ⁴ nA ^x o (G ^LL2 ) CBA|___ ζΥ (G ^LL9-1 ) A ⁴ N CBA (G ^LL3_1 ) ^CBA j_ (G ^LL9 - ² ) Ά'” CBA (G ^LL3 - ² ) (G ^ll1 °) T™ ^CBA |_. s ^z_H H (G ^LL ' ⁴ ) SWAT___ 1 D ^Z H •N^ (G ^LL11 ) ^H <J y o (G ^LL5 ) SWAT___ 0 (G ^LL12 ) CBA X' (G ^L “) CBA|__ 0 (G ^ll13 ) HN-N ^χ, Α_γ CBA (G ^LL7 ) СВАТ___ (G ^LL14 ) H cba^N^q- where Ar is phenylene and X' is C _1-4 alkyl; and (ib') where R ^L1 and R ^L2 are as defined in either paragraph 1 or paragraph 10; and p is an integer from 1 to 20. 13. The conjugate of claim 12, wherein G ^LL is selected from G ^LL1-1 and G ^LL1-2 14. The conjugate of claim 12, wherein Q is a dipeptide residue that is ^NH Val-Ala- ^C “ ^O , a is 0, b1 is 0, c1 is 1, c2 is 0, d is 2, b2 is 8, and G ^ll is G ^LL1-1 , where ^NH- is the N-terminus and ^-CO is the C-terminus of the residue. 15. The conjugate of any one of claims 12 to 14, wherein the drug loading (p) for drugs (D) in relation to the antibody (Ab) is an integer from 1 to 10. 16. A pharmaceutical composition comprising a conjugate according to any one of claims 12-15 and a pharmaceutically acceptable diluent, carrier or excipient. 17. Use of a conjugate according to any one of claims 12-15 or a pharmaceutical composition according to claim 16 for treating a proliferative disease in a subject. 18. The use according to claim 17, wherein the disease is cancer. - 63 047476 19. Use of a conjugate according to any of claims 12-15 or a pharmaceutical composition according to claim 16 for conservative treatment, where the conservative treatment is intended for the treatment of cancer. 20. A method of conservative treatment, comprising administering to a patient a pharmaceutical composition according to claim 16, where the method of conservative treatment is intended for the treatment of cancer. 21. Compound of formula VI where Q is as defined in paragraph 1.