EA045942B1 - AZETHIDOBENZODIAZEPINE DIMERS AND CONJUGATES CONTAINING THEM FOR USE IN THE TREATMENT OF CANCER - Google Patents

Description

The invention relates to azetidobenzodiazepine (ABD) dimers, conjugates containing said dimers and drug precursor linkers used to prepare such conjugates.

State of the art

Pyrrolobenzodiazepine (PBD) dimers have been shown to be cytotoxic compounds. For example, SG2000 (SJG-136):

(Gregson, S.J., et al., CHem. Commun., 1999, 797-798. doi: 10.1039/A809791G; (Gregson, S., et al., J. Med. CHem., 44, 737-748 (2001 ); Alley, M.C., et al., Cancer ResearCH, 64, 6700-6706 (2004); and Hartley, J.A., et al., Cancer ResearCH, 64, 6693-6699 (2004)) have been studied in clinical trials as a separate agent, such as NCT02034227, studying its use in the treatment of acute myeloid leukemia and chronic lymphocytic leukemia (see: https://www.clinicaltrials.gov/ct2/show/NCT02034227).

Dimeric PBD compounds containing C2 aryl substituents near the endo-unsaturation, such as SG2202 (ZC-207), are described in WO 2005/085251:

These compounds have been shown to be very useful cytotoxic agents (Howard, P.W., et al., Bioorg. Med. CHem. (2009), doi: 10.1016/j.bmcl.2009.09.012).

Dimeric PBD compounds containing linker groups for binding to a cell binding agent such as an antibody are described in WO 2011/130598. The linker in such compounds is attached to one of the available N10 positions, and is usually cleaved by an enzyme at this linker group. WO 2014/057074 and WO 2015/052322 describe two specific dimeric PBD conjugates linked through the N10 position in one monomer.

At a relatively early stage in the development of PBDs as molecules of interest, it was described in 1997 (Bose, D.S., et al., Tetrahedron Letters, 38(33), 5839-5842, 1997; doi: 10.1016/S0040-4039(97)01297-5 ) that the following connection:

synthesized and to be evaluated as a potential DNA-binding ligand and cytotoxic agent. No further publications have been made about this compound, so it appears that they were either unstable for testing or inactive.

Description of the invention

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

- 1 045942

and its salts and solvates, where:

R ² and R ² represent H;

R ⁶ and R ⁹ are independently selected from H, R, OH, OR, SH, SR, NH ₂ , NHR, NRR', nitro, Me3Sn and halogen;

wherein R and R' are independently selected from optionally substituted C _1-12 alkyl, C _3-20 heterocyclyl and C _6-20 aryl groups; or (a) R ⁷ is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halogen; R ⁷ ' is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me ₃ Sn and halogen; or (b) R ⁷ and R ⁷ ' together form a group which is: (i) -O-(CH ₂ ) _p -O-, where n is from 7 to 16; or (ii) -O-(CH ₂ CH ₂ O) _m -, where m is from 2 to 5;

R represents a C _3-12 alkylene group, the chain of which may be interrupted by one or more heteroatoms, e.g. O, S, NRN ² (where RN ² represents H or C _1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;

Y and Y' are selected from O, S or NH;

R ⁶ and R ⁹ are selected from the same groups as R ⁶ and R ⁹ , respectively; or (ia) R ¹⁰ and R ¹¹ together form a double bond between the N and C atoms to which they are bonded; or (ib) R ¹⁰ represents H, and R ¹¹ is selected from OH and ORA, and R ^A represents C _1-4 alkyl; or (ic) both R ¹⁰ and R ¹¹ are H; or (ii-a) R ²⁰ and R ²¹ together form a double bond between the N and C atoms to which they are bonded; or (ii-b) R ²⁰ represents H, and R ²¹ is selected from OH and OR ^B , and ^RB represents C _1-4 alkyl; or (ii-c) both R ²⁰ and R ²¹ are H.

The second aspect of this invention includes a compound of formula I:

and its salts and solvates, where:

Y, Y', R, R ² , R ² ', R ⁶ , R ⁶ ', R ⁷ , R ⁷ ', R ⁹ and R ⁹ ' are as defined in the first aspect of the present invention;

R ^11b is selected from OH, OR ^A , where R ^A represents C _1-4 alkyl; And

R ^l is a linker for binding to a cell binding agent which is selected from:

(iiia):

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where Q represents:

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

X represents:

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

G ^l represents a linker for binding to a ligand unit; And

(iiib):

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; or:

(a) R ³⁰ and R ³¹ together form a double bond between the N and C atoms to which they are bonded; or (b) R ³⁰ is H and R ³¹ is selected from OH and OR ^B , wherein ^RB is C _1-4 alkyl;

(c) R ³⁰ and R ³¹ are both H; or (d) R ³¹ is OH or OR ^B , wherein ^RB is C _1-4 alkyl, and R ³⁰ is selected from:

wherein R ^Z is selected from:

N.

Ν' (z-i)

- 3 045942 (z-ii) OS(=O)СНз;

(z-iii) NO2;

(z-iv) OMe;

(z-v) glucuronide;

(z-vi) NH-C(=O)-X1-NHC(=O)X ₂ -NH-C(=O)-R ^ZC , where -C(=O)-X1-NH- and -C( =O)-X2-NH- are natural amino acid residues, and R ^ZC is selected from Me, OMe, CH2CH ₂ OMe and (CH ₂ CH ₂ O) ₂ Me.

In a third aspect of the present invention there are provided conjugates of formula II:

L - (D ^l ) _p (II) where L represents the ligand unit (i.e. the targeting agent), DL represents the

rum aspect of this invention;

R ^ll is a linker for binding to a cell binding agent which is selected from:

(iiia):

where Q and X are as defined in the first aspect, and G ^LL is a linker associated with a ligand unit; And

(iiib):

where R ^l1 and R ^L2 are as defined in the first aspect; where p is an integer from 1 to 20.

The ligand unit, described in more detail below, is a targeting agent that binds to the target moiety. The ligand unit may, for example, specifically bind to a cellular component (cell binding agent) or other desired target molecules. The ligand unit may be, for example, a protein, polypeptide, or peptide such as an antibody, an antigen binding fragment of an antibody, or other binding agent such as an Fc fusion protein.

The fourth aspect of the present invention provides the use of a conjugate according to the third aspect of the present invention in the manufacture of a medicament for the treatment of a proliferative disease. The fourth aspect also provides a conjugate of the third aspect of the present invention for use in the treatment of a proliferative disease. A fourth aspect also provides a method of treating a proliferative disease, comprising administering a therapeutically effective amount of a conjugate of the second aspect of the invention to a patient in need thereof.

Those skilled in the art can readily determine whether a potential conjugate is or is not suitable for treating a proliferative condition for any particular cell type. For example, assays that can be conveniently used to assess the activity provided by con

- 4 045942 specific connection are described below in the examples.

A fifth aspect of the present invention provides the synthesis of a conjugate according to the third aspect of the present invention, comprising conjugating a compound (drug compound linker) according to the second aspect of the present invention with a ligand unit.

The compounds of formula IV are the active groups released by the conjugates of the third aspect.

Definitions

Deputies

The expression "optionally substituted" in this context refers to the parent group, which may be unsubstituted or which may be substituted.

Unless otherwise indicated, the term substituted in this context refers to the parent group which contains one or more substituents. The term substituent in this context is used in its ordinary sense and refers to a chemical moiety that is covalently attached or, if appropriate, fused to the parent group. Many substituents are known, and methods for preparing them and introducing them into various starting groups are also well known.

Examples of substituents are described in more detail below.

C _1-12 alkyl: The term C _1-12 alkyl as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound containing from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (eg, partially unsaturated, completely unsaturated). Term C1. ₄ alkyl in this context refers to a monovalent moiety obtained by removing 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). 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), butyl ( _C4 ), pentyl (C5), hexyl ( _C6 ) and heptyl (C7).

Examples of saturated linear alkyl groups include, but are not limited to, methyl ( _C1 ), ethyl ( _C2 ), n-propyl (C3), n-butyl ( _C4 ), n-pentyl (amyl) (C5), n -hexyl (C ₆ ) and n-heptyl (C ₇ ).

Examples of saturated branched alkyl groups include iso-propyl ( _C3 ), iso-butyl ( _C4 ), sec-butyl (C4), t-butyl (C4), iso-pentyl (C5) and neo-pentyl (C5).

C _2-12 alkenyl: The term C _2-12 alkenyl as used herein 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(CH ₃ )=CH ₂ ), butenyl (C ₄ ), pentenyl (C5) and hexenyl (C ₆ ).

C _2-12 alkynyl: The term C _2-12 alkynyl as used herein 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 _3-12 cycloalkyl: The term C _3-12 cycloalkyl as used herein refers to an alkyl group that is also a cyclic group; that is, to a monovalent fragment obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, said fragment containing from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, those derived from:

saturated monocyclic hydrocarbon compounds:

cyclopropane (C ₃ ), cyclobutane (C ₄ ), cyclopentane (C ₅ ), cyclohexane (C ₆ ), cycloheptane (C ₇ ), methylcyclopropane (C ₄ ), dimethylcyclopropane (C ₅ ), methylcyclobutane (C ₅ ), dimethylcyclobutane ( C ₆ ), methylcyclopentane (C ₆ ), dimethylcyclopentane (C ₇ ) and methylcyclohexane (C ₇ );

unsaturated monocyclic hydrocarbon compounds:

cyclopropene (C ₃ ), cyclobutene (C ₄ ), cyclopentene (C ₅ ), cyclohexene (C ₆ ), methylcyclopropene (C ₄ ), dimethylcyclopropene (C ₅ ), methylcyclobutene (C ₅ ), dimethylcyclobutene (C ₆ ), methylcyclopentene (C6 ), dimethylcyclopentene (C7) and methylcyclohexene (C7); and saturated polycyclic hydrocarbon compounds: norcarane (C7), norpinane (C7), norbornane (C7).

C _3-20 heterocyclyl: The term C _3-20 heterocyclyl as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, said moiety containing from 3 to 20 ring atoms, of which 1 to 10 are ring atoms heteroatoms. Preferably, each ring contains from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.

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In this context, prefixes (eg, C _3-20 , C _3-7 , C5-6, etc.) indicate the number of ring atoms or a range of numbers of ring atoms, whether carbon atoms or heteroatoms. For example, the term C _5-6 heterocyclyl as used herein refers to a heterocyclyl group containing 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limited to, groups derived from:

N ₁ : aziridine (C ₃ ), azetidine (C ₄ ), pyrrolidine (tetrahydropyrrole) (C ₅ ), pyrroline (for example, 3-pyrrole, 2,5-dihydropyrrole) (C ₅ ), 2H-pyrrole or 3-pyrrole (isopyrrole , isoazole) ( _C5 ), piperidine ( _C6 ), dihydropyridine ( _C6 ), tetrahydropyridine ( _C6 ), azepine (C7);

О ₁ : oxirane ( _C3 ), oxetane ( _C4 ), oxolane (tetrahydrofuran) ( _C5 ), oxole (dihydrofuran) ( _C5 ), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6) , oxpein (C7);

S1: thiirane (C ₃ ), thietane (C ₄ ), thiolane (tetrahydrothiophene) (C ₅ ), thiane (tetrahydrothiopyran) (C ₆ ), thiepane (C7);

O2: dioxolane (C5), dioxane (C6) and dioxepane (C7);

O ₃ : trioxane (C ₆ );

N ₂ : imidazolidine (C ₅ ), pyrazolidine (diazolidine) (C ₅ ), imidazoline (C ₅ ), pyrazoline (dihydropyrazole) (C ₅ ), piperazine (C ₆ );

N1O1: tetrahydrooxazole (C ₅ ), dihydrooxazole (C ₅ ), tetrahydroisoxazole (C ₅ ), dihydroisoxazole (C ₅ ), morpholine (C ₆ ), tetrahydrooxazine (C ₆ ), dihydrooxazine (C ₆ ), oxazine (C ₆ );

N1S1: thiazoline (C ₅ ), thiazolidine (C ₅ ), thiomorpholine (C ₆ );

N2O1: oxadiazine (C ₆ );

O1S1: oxathiol (C ₅ ) and oxathiane (thioxane) (C ₆ ); And

N1O1S1: oxathiazine (C ₆ ).

Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides in cyclic form, for example furanoses (C5) such as arabinofuranose, lyxofuranose, ribofuranose and xylofuranose, and pyranoses ( _C6 ) such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose , idopyranose, galactopyranose and talopyranose.

C _6-20 aryl: The term C _6-20 aryl as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, said moiety containing from 6 to 20 ring atoms. The term C _6-7 aryl as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, said moiety containing 6 to 7 ring atoms, and the term C _6-10 aryl as used herein refers to a monovalent a fragment obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, said fragment containing from 6 to 10 ring atoms. Preferably, each ring contains from 6 to 7 ring atoms.

In this context, prefixes (eg, C _6-7 , C _6-10, etc.) mean the number of ring atoms or a range of numbers of ring atoms, whether carbon atoms or heteroatoms. For example, the term C ₆ aryl in this context refers to an aryl group containing 6 ring atoms.

All ring atoms may be carbon atoms, as in carboaryl groups. Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) ( _C6 ), naphthalene ( _C10 ), azulene ( _C10 ), anthracene ( _C14 ), phenanthrene ( _C14 ) , naphthacene (C ₁₈ ) and pyrene (C ₁₆ ).

Examples of aryl groups that contain fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (for example, 2,3-dihydro-N-indene) (C ₉ ), indene ( _C9 ), isoindene ( _C9 ), tetralin (1,2,3,4tetrahydronaphthalene (C10), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (C15) and aceanthrene (C16).

The ring atoms may contain one or more heteroatoms in the case of heteroaryl groups. Examples of monocyclic heteroaryl groups include, but are not limited to, groups derived from:

N ₁ : 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) (C ₅ ), pyrazole (1,2-diazole) (C ₅ ), pyridazine (1,2-diazine) (C ₆ ), pyrimidine (1,3-diazine) ( C ₆ ) (for example, cytosine, thymine, uracil), pyrazine (1,4-diazine) (C ₆ );

N3: triazole (C5), triazine (C6); And

N ₄ : tetrazole (C ₅ ).

Examples of heteroaryl that contain fused rings include, but are not limited to:

C ₉ (with 2 fused rings), derived from benzofuran (O1), isobenzofuran (O1), indole (N1), isoindole (N1), indolysine (N1), indoline (N1), isoindoline (N1), purine ( _N4 ) (e.g. adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzoisoxazole (N1O1), benzodioxole ( _O2 ), benzofurazan (N2O1), benzotriazole (N3), benzothiofuran (S1), benzothiazole (N1S1), benzothiadiazole (N2S);

C ₁₀ (with 2 fused rings), derived from chromene (O1), isochromene (O1), chromane (O1), isochromane (O1), benzodioxane ( _O2 ), quinoline (N1), isoquinoline (N1), quinolysine (N1 ), benzoxazine (N1O1), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinolysine (N2), cinnoline (N2), phthalazine (N2), naphthyridine (N2), pteridine (N4);

C11 (with 2 fused rings), derived from benzodiazepine (N2);

C ₁₃ (with 3 fused rings), derived from carbazole (N1), dibenzofuran (O1), dibenzothiophene (S1), carboline (N2), perimidine (N2), pyridoindole (N2); And

C ₁₄ (with 3 fused rings), derived from acridine (N1), xanthene (O1), thioxanthene (S1), oxanthrene ( _O2 ), phenoxathiine (O1S1), phenazine (N2), phenoxazine (N1O1), phenothiazine (N1S1 ), tianthrene ( _S2 ), phenanthridine (N1), phenanthroline (N2), phenazine (N2).

The above groups, alone or as part of another substituent, may themselves be optionally substituted by one or more groups selected from themselves and additional substituents listed below.

Halogen: -F, -Cl, -Br and -I.

Hydroxy: -OH.

Ether: -OR, where R is an ether substituent, for example, a C _1-7 alkyl group (also referred to as a C _1-7 alkoxy group, which is discussed below), a C _3-20 heterocyclyl group (also referred to as a C _{3- 20} heterocyclyloxy group), or a C _6-20 aryl group (also referred to as a C _6-20 aryloxy group), preferably a C _1-7 alkyl group.

Alkoxy: -OR, where R represents an alkyl group, for example, a C _1-7 alkyl group. Examples of C _1-7 alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), -O(n-propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy) and -O(tBu) (tert-butoxy).

Acetal: -CH(OR1)( ^OR2 ), where R ¹ and R ² independently represent acetal substituents, for example a C1-7 alkyl group, a C3-20 heterocyclyl group or a C6-20 aryl group, preferably a C1-7 alkyl group , or in the case of a cyclic acetal group, R ¹ and R ² together with the two oxygen atoms to which they are attached and the carbon atoms to which they are attached form a heterocyclic ring containing from 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, -CHMc, -CH(OEt)2, and -CH(OMe)(OEt).

Hemiacetal: -CH(OH)(OR1), where R ¹ represents a hemiacetal substituent, for example a C _1-7 alkyl group, a C3-20 heterocyclyl group or a C6-20 aryl group, preferably a C1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, -CH(OH)(OMe) and -CH(OH)(OEt).

Ketal: -CR(OR1)( ^OR2 ), where ^R1 and ^R2 are as defined for acetals, and R is a ketal substituent other than hydrogen, e.g., a _C1-7 alkyl group, _C3-20 a heterocyclyl group or a C _6-20 aryl group, preferably a C _1-7 alkyl group. Examples of ketal groups include, but are not limited to, -C(Me)(OMe) ₂ , -C(Me)(OEt) ₂ , -C(Me)(OMe)(OEt), -C(Et)(OMe) 2, -C(Et)(OEt)2n -C(Et)(OMe)(OEt).

Hemiketal: -CR(OH)(OR1), where R ¹ is as defined for hemiacetals and R is a hemiketal substituent other than hydrogen, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or C6 -20 aryl group, preferably a C1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, -C(Me)(OH)(OMe), -C(Et)(OH)(OMe), -C(Me)(OH)(OEt) and -C(Et )(OH)(OEt).

Oxo (keto, -on): = O.

Thione (thioketone)^.

Imino (umuh):=NR, where R represents a substituent on an imino group, for example hydrogen, a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group, preferably hydrogen or a C _1-7 alkyl group. Examples of ester groups include, but are not limited to, =NH, =NMe, =NEt, and =NPh.

Formyl (carbaldehyde, carboxaldehyde): -C(=O)H.

Acyl (keto): -C(=O)R, where R is an acyl substituent, e.g. _C1-7 alkyl group (also referred to as _C1-7 alkylacyl or _C1-7 alkanoyl), _C3-20 heterocyclyl group (also referred to as C3-20 heterocyclyl acyl) or a C6-20 aryl group (also referred to as C6-20 arylacyl), preferably a C _1-7 alkyl group. Examples of acyl groups include, but are not limited to, -C(=O)CH ₃ (acetyl), -C(=O)CH ₂ CH ₃ (propionyl), -C(=O)C(CH ₃ ) ₃ (tert -butyryl) and -C(=O)Ph (benzoyl, phenon).

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Carboxy (carboxylic acid): -C(=O)OH.

Thiocarboxy (thiocarboxylic acid): -C(=S)SH.

Thiolocarboxy (thiolocarboxylic acid): -C(=O)SH.

Thionocarboxy (thionocarboxylic acid): -C(=S)OH.

Imidic acid: -C(=NH)OH.

Hydroxamic acid: -C(=NOH)OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): -C(=O)OR, where R is an ester substituent, e.g. a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group group, preferably a C _1-7 alkyl group. Examples of ester groups include, but are not limited to, -C(=O)OCH ₃ , -C(=O)OCH ₂ CH ₃ , -C(=O)OC(CH ₃ ) ₃ and -C(=O)OPh .

Acyloxy (reverse ester): -OC(=O)R, where R is an acyloxy substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group, preferably a C _1-7 alkyl group. Examples of acyloxy groups include, but are not limited to, OC(=O)CH ₃ (acetoxy), -OC(=O)CH ₂ CH ₃ , -OC(=O)C(CH ₃ ) ₃ , -OC(=O) Ph and -OC(=O)CH ₂ Ph.

Hydroxycarboyloxy: -OC(=O)OR, where R is an ester substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group, preferably a C _1-7 alkyl group. Examples of ester groups include, but are not limited to, -OC(=O)OCH ₃ , -OC(=O)OCH2CH3, -OC(=O)OC(CH3)3, and -OC(=O)OPh.

Amino: -NR ¹ R ² where R ¹ and R ² independently represent amino group substituents, for example hydrogen, a C1-7 alkyl group (also referred to as a C1-7 alkylamino or di-C1-7 alkylamino), a C3-20 heterocyclyl group or a C6-20 aryl group, preferably an H or C1-7 alkyl group, or in the case of a cyclic amino group, R ¹ and R ² together with the nitrogen atom to which they are attached form a heterocyclic ring containing from 4 to 8 ring atoms. Amino groups can be primary (-NH2), secondary (-NHR ¹ ) or tertiary (-NHR ¹ R ² ), and in cationic form can be quaternary (- ⁺ NR ¹ R ² R ³ ). Examples of amino groups include, but are not limited to, -NH2, -NHCH3, -NHC(CH3) ₂ , -N( _CH3 ) ₂ , -N _{( CH2CH3} ) ₂ and -NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): -C(=O)NR ¹ R ² where R ¹ and R ² independently represent amino group substituents as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=O)NH2, -C(=O)NHCH3, -C(=O)N(CH3)2, -C(=O)NHCH2CH3 and -C( =O)N(CH ₂ CH ₃ ) ₂ , as well as amido groups in which R ¹ and R ² together with the nitrogen atom to which they are attached form a heterocyclic structure, as, for example, in piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl and piperazinocarbonyl.

Thioamido (thiocarbamyl): -C(=S)NR1R ² where R ¹ and R ² independently represent amino group substituents as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=S)NH ₂ , -C(=S)NHCH ₃ , -C(=S)N(CH ₃ ) ₂ u -C(=S)NHCH ₂ CH ₃ .

Acylamido (acylamino): -NR ¹ C(=O)R ² where R ¹ is an amide substituent, for example hydrogen, a C1-7 alkyl group, a C3-20 heterocyclyl group or a C6-20 aryl group, preferably hydrogen or C1 -7 alkyl group, and R ² represents an acyl substituent, for example a C1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group, preferably hydrogen or a C1-7 alkyl group. Examples of acylamide groups include, but are not limited to, -NHC(=O)CH3, -NHC(=O)CH2CH3 and -NHC(=O)Ph. R ¹ and R ² can together form a cyclic structure, as in succinimidyl, maleimidyl and phthalimidyl:

Aminocarbonyloxy: -OC(=O)NR1R ² where R ¹ and R ² independently represent amino group substituents as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, -OC(=O)NH2, -OC(=O)NHMe, -OC(=O)NMe2U -OC(=O)NEt2.

Ureido: -N(R1)CONR ² R ³ where R ² and R ³ independently represent amino group substituents as defined for amino groups, and R ¹ represents a ureido group substituent, eg hydrogen, C1-7 alkyl group, C3 -20 heterocyclyl group or C6-20 aryl group, preferably hydrogen or C _1-7 alkyl group. Examples of ureido groups include, but are not limited to, -NHCONH2, NHCONHMe, -NHCONHEt, -NHCONMe2, -NHCONEt2, -NMeCONH2, NMeCONHMe,

- 8 045942

-NMeCONHEt, -NMeCONMe ₂ and -NMeCONEt ₂ .

Guanidino: -NH-C(=NH)NH2.

Tetrazolyl: a five-membered aromatic ring containing four nitrogen atoms and one carbon atom

kind,

Hmuho:=NR, where R represents a substituent of an imino group, for example hydrogen, a C1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group, preferably an H or a C1-7 alkyl group. Examples of imino groups include, but are not limited to, =NH, =NMe, and =NEt.

Amidine (amidino): -C(=NR)NR ₂ where each R is an amidine substituent, for example hydrogen, a C1-7 alkyl group, a _C3-20 heterocyclyl group or a _C6-20 aryl group, preferably H or C1 -7 alkyl group. Examples of amidine groups include, but are not limited to, -C(=NH)NH ₂ , -C(=NH)NMe ₂ and -C(=NMe)NMe ₂ .

Nitro: -NO ₂ .

Nitroso: -NO.

Azido: -N ₃ .

Cyano (nitrile, carbonitrile): -CN.

Isocyano: -NC.

Cyanato: -OCN.

Isocyanato: -NCO.

Thiocyano (thiocyanato): -SCN.

Isothiocyano (isothiocyanato): -NCS.

Sulfhydryl (thiol, mercapto): -SH.

Thioether (sulfide): -SR, where R is a thioether substituent, for example a C1-7 alkyl group (also referred to as a C1-7 alkylthio group), a _C3-20 heterocyclyl group or a _C6-20 aryl group, preferably C1 -7 alkyl group. Examples of C1-7 alkylthio groups include, but are not limited to, -SCH3 and -SCH2CH3.

Disulfide: -SS-R, where R is a disulfide substituent, for example a C1-7 alkyl group, a _C3-20 heterocyclyl group or a _C6-20 aryl group, preferably a C1-7 alkyl group (also referred to herein as C1 -7 alkyl disulfide). Examples of C1-7 alkyl disulfide groups include, but are not limited to, -SSCH3 and -SSCH2CH3.

Sulfin (sulfinyl, sulfoxide): -S(=O)R, where R is a sulfin substituent, for example a C1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group, preferably a C1-7 alkyl group . Examples of sulfinic groups include, but are not limited to, -S(=O)CH3 and -S(=O)CH2CH3.

Sulfone (sulfonyl): -S(=O) ₂ R, where R is a sulfone substituent, for example a C1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group, preferably a C1-7 alkyl group, including, for example, a fluorinated or perfluorinated C1-7 alkyl group. Examples of sulfone groups include, but are not limited to, -S(=O) ₂ CH ₃ (methanesulfonyl, mesyl), -S(=O) ₂ CF ₃ (triflyl), -S(=O) ₂ CH ₂ CH ₃ ( ezyl), -S(=O) ₂ C ₄ F ₉ (nonaphlyl), -S(=O) ₂ CH ₂ CF ₃ (trezyl), -S(=O) ₂ CH ₂ CH ₂ NH ₂ (tauryl), -S(=O) ₂ Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenyl (nosyl), 2-naphthalene sulfonate (napsil) and 5- dimethylaminonaphthalen-1-ylsulfonate (dansyl).

Sulfinic acid (sulfin): -S(=O)OH, -SO2H.

Sulfonic acid (sulfo): -S(=O) ₂ OH, -SO3H.

Sulfinate (sulfinic acid ester): -S(=O)OR; where R represents a sulfinate substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group, preferably a C _1-7 alkyl group. Examples of sulfinate groups include, but are not limited to, -S(=O)OCH ₃ (methoxysulfinyl; methylsulfinate) and -S(=O)OCH ₂ CH ₃ (ethoxysulfinyl; ethyl sulfinate).

Sulfonate (sulfonic acid ester): -S(=O)2OR; where R is a sulfonate substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group, preferably a C _1-7 alkyl group. Examples of sulfonate groups include, but are not limited to, -S(=O) ₂ OCH ₃ (methoxysulfonyl; methylsulfonate) and -S(=O) ₂ OCH ₂ CH ₃ (ethoxysulfonyl; ethyl sulfonate).

Sulfinyloxy: -OS(=O)R, where R is a sulfinyloxy substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group, preferably a C _1-7 alkyl group. Examples of sulfinyloxy groups include, but are not limited to, -OS(=O)CH3 and -OS(=O)CH2CH3.

Sulfonyloxy: -OS(=O) ₂ R, where R represents a sulfonyloxy substituent, e.g.

- 9 045942 mer, a C1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group, preferably a C1-7 alkyl group. Examples of sulfonyloxy groups include, but are not limited to, -OS(=O) ₂ CH ₃ (mesylate) and -OS(=O) ₂ Ch ₂ CH ₃ (esylate).

Sulfate: -OS(=O)2OR, where R is a sulfate substituent, for example a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group, preferably a C _1-7 alkyl group. Examples of sulfate groups include, but are not limited to, -OS(=O) ₂ OCH ₃ and -SO(=O)2OCH2CH3.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): -S(=O)NR1R ² where R1 and ^R2 independently represent amino group substituents as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, -S(=O)NH2, -S(=O)NH(CH ₃ ), -S(=O)N(CH ₃ ) ₂ , -S(=O)NH (CH2CH3), -S(=O)N(CH2CH3)2 and -S(=O)NHPh.

Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): -S(=O)2NR1R ² where R1 and ^R2 independently represent amino group substituents as defined for amino groups. Examples of sulfonamido groups include, but are not limited to, -S(=O)2NH2, -S(=O) ₂ NH(CH ₃ ), -S(=O) ₂ N(CH ₃ ) ₂ , -S(=O) ₂ NH(CH ₂ CH ₃ ), -S(=O) ₂ N(CH ₂ CH 3)2 and -S(=O) ₂ NHPh.

Sulfamino: -NR1S(=O)2OH, where R1 is an amino group substituent as defined for amino groups. Examples of sulfamino groups include, but are not limited to, -NHS(=O) ₂ OH and -N(CH3)S(=O) ₂ OH.

Sulfonamino: -NR ¹ S(=O)2R, where R ¹ is an amino group substituent as defined for amino groups, and R is a sulfonamino group substituent, for example a C1-7 alkyl group, a _C3-20 heterocyclyl group or a _C6-20 an aryl group, preferably a C _1-7 alkyl group. Examples of sulfonamino groups include, but are not limited to, -NHS(=O) ₂ CH3 and -N(CH3)S(=O)2C6H5.

Sulfinamino: -NR ¹ S(=O)R, where R ¹ is an amino group substituent as defined for amino groups, and R is a sulfinamino group substituent, for example, a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _{6- 20} aryl group, preferably a C _1-7 alkyl group. Examples of sulfinamino groups include, but are not limited to, -NHS(=O)CH3 and -N(CH ₃ )S(=O)C ₆ H ₅ .

Phosphino (phosphine): -PR2, where R is a substituent of a phosphino group, for example -H, a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group, preferably -H, C _{1- 7} alkyl group or C _6-20 aryl group. Examples of phosphino groups include, but are not limited to, -PH2, -P(CH3)2, -P(CH2CH3)2, -P(t-Bu)2 and -P(Ph)2.

Phospho: -P(=O)2.

Phosphinyl (phosphine oxide): -P(=O)R2, where R is a phosphinyl substituent, for example a C1-7 alkyl group, a _C3-20 heterocyclyl group or a _C6-20 aryl group, preferably a C1-7 alkyl group or _{C6 -20} aryl group. Examples of phosphinyl groups include, but are not limited to, -P(=O)(CH3)2, -P(=O)(CH2CH3)2, -P(=O)(t-Bu)2 and -P(=O )(Ph)2.

Phosphonic acid (phosphono): -P(=O)(OH) ₂ .

Phosphonate (phosphonic ester): -P(=O)(OR) ₂ , where R is a phosphonate substituent, for example -H, a C1-7 alkyl group, a _C3-20 heterocyclyl group or a _C6-20 aryl group, preferably -H, a C _1-7 alkyl group or a C _6-20 aryl group. Examples of phosphonate groups include, but are not limited to, -P(=O)(OCH ₃ ) ₂ , -P(=O)(oCH ₂ CH ₃ ) ₂ , -P(=O)(Ot-Bu) ₂ and - P(=O)(OPh) ₂ .

Phosphoric acid (phosphonoxy): -OP(=O)(OH) ₂ .

Phosphate (phosphonoxy ester): -OP(=O)(OR) ₂ where R is a phosphate substituent, for example -H, a C _1-7 alkyl group, a C _3-20 heterocyclyl group or a C _6-20 aryl group group, preferably -H, a C _1-7 alkyl group or a C _6-20 aryl group. Examples of phosphate groups include, but are not limited to, -Or(=O)(OCH ₃ ) ₂ , -OP(=O)(oCH ₂ CH ₃ ) ₂ , -OP(=O)(Ot-Bu) ₂ and - OP(=O)(OPh)2.

Phosphorous acid: -OP(OH) ₂ .

Phosphite: -OP(OR) ₂ where R is a phosphite substituent, for example -H, C _1-7 alkyl group, C 3 _-20 heterocyclyl group or C _6-20 aryl group, preferably -H, C _1-7 alkyl group or a C _6-20 aryl group. Examples of phosphite groups include, but are not limited to, -OP(OCH3) ₂ , -OP(OCH ₂ CH3) ₂ , -OP(Ot-Bu) ₂ and -OP(OPh) ₂ .

Phosphoramidite: -OP(OR1)-NR ² 2, where R1 and R ² are phosphoramidite substituents, for example -H, an (optionally substituted) C1-7 alkyl group, a C _3-20 heterocyclyl group or a C ₆₂₀ aryl group, preferably -H, C _1-7 alkyl group or C _6-20 aryl group. Examples of phosphoramidite groups include, but are not limited to, -OP(OCH ₂ CH3)-N(CH3) ₂ , -OP(OCH ₂ CH3)N(i-Pr) ₂ and -OP(OCH ₂ CH ₂ CN)-N (i-Pr) ₂ .

Phosphoramidate: -OP(=O)(OR1)-NR ² 2, where R ¹ and R ² are phosphoramidate substituents, for example -H, an (optionally substituted) C1-7 alkyl group, a C _3-20 heterocyclyl group or C _{a 6-20} aryl group, preferably -H, a C _1-7 alkyl group or a C _6-20 aryl group. Examples of phosphoramidate groups include, but are not limited to, -OP(=O)(OCH ₂ CH3)-N(CH3) ₂ .

- 10 045942

-OP(=O)(OCH ₂ CH ₃ )-N(i-Pr) ₂ and -OP(=O)(OCH ₂ CH ₂ CN)-N(i-Pr) ₂ .

Alkylene

C _3-12 alkylene: The term C ₃ . ₁₂ alkylene in this context refers to a bidentate moiety produced by the removal of two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms in a hydrocarbon compound containing from 3 to 12 carbon atoms (if not otherwise specified) which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated or completely unsaturated. Thus, the term alkylene includes the subclasses alkenylene, alkynylene, cycloalkylene and

etc., discussed below.

Examples of linear saturated C _3-12 alkylene groups include, but are not limited to, -(CH ₂ ) _n -, where n is an integer from 3 to 12, for example, -CH ₂ CH ₂ CH ₂ - (propylene), - CH2CH2CH2CH2- (butylene), -CH2CH2CH2CH2CH2- (pentylene) and -CH2CH2CH2CH2-CH2CH2CH2- (heptylene).

Examples of branched saturated C _3-12 alkylene groups include, but are not limited to, -CH(CH3)CH2-, -CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH (CH3)CH2 CH2-, -CH( _{CH2CH3 )} -, -CH( _{CH2CH3 ) CH2-} and _-CH2CH ( _{CH2CH3 ) CH2-} .

Examples of linear partially unsaturated C _3-12 alkylene groups (C _3-12 alkenylene and alkynylene groups) include, but are not limited to, -CH=CH-CH ₂ -, -CH ₂ -CH=CH ₂ -, -CH=CH -CH ₂ CH ₂ -, -CH=CH-CH2-CH2-CH2-, -CH=CH-CH=CH--CH=CH-CH=CH-CH2-, -CH=CH-CH=CH-CH2 -CH2-,

-CH=CH-CH ₂ -CH=CH-, -CH=CH-CH ₂ -CH ₂ -CH=CH- and -CH2-C C-CH2-.

Examples of branched partially unsaturated C _3-12 alkylene groups (C _3-12 alkenylene and alkynylene groups) include, but are not limited to, -C( _CH )=CH-, -C( _CH )=CH-CH ₂ -, -CH=CHSN(CH _h )- and -C=C-CH(CH _h )-.

Examples of alicyclic saturated C _3-12 alkylene groups (C _3-12 cycloalkylenes) include, but are not limited to, cyclopentylene (eg, cyclopent-1,3-ylene) and cyclohexylene (eg, cyclohex-1,4-ylene).

Examples of alicyclic partially unsaturated C _3-12 alkylene groups (C _3-12 cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g., 4-cyclopenten-1,3-ylene), cyclohexenylene (e.g., 2-cyclohexene-1,4 -ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).

If the C _3-12 alkylene group is interrupted by a heteroatom, the subscript refers to the number of atoms in the chain, including heteroatoms. For example, the chain -C2H4-O-C2H4- represents a C ₅ group.

If the C _3-12 alkylene group is interrupted by an aromatic ring, then the subscript refers to the number of atoms in the chain itself, including the aromatic ring. For example, the aJCLa chain represents a C ₅ group.

^C(= 2 o ^x 1

A Ά

Connection designations: In the formula θ, the designations written in superscripts are

C(=O) and NH denote the group to which the indicated atoms are bonded. For example, an NH group is shown as bonded to a carbonyl (which is not part of the depicted fragment) and a carbonyl is shown as bonded to an NH group (which is not part of the depicted fragment).

Ligand unit

The ligand unit can be of any kind and includes protein, polypeptide, peptide and non-peptide agent that specifically binds to the target molecule. In some embodiments, the ligand unit may be a protein, polypeptide, or peptide. In some embodiments, the ligand unit may be a cyclic polypeptide. These ligand units may include antibodies or an antibody fragment that contains at least one site that binds to a target molecule, lymphokines, hormones, growth factors or any other cell-binding molecule or substance that can specifically bind to the target.

The terms specifically bind and specific binding refer to the binding of an antibody or other protein, polypeptide or peptide to a given molecule (eg, antigen). Typically, an antibody or other molecule binds with an affinity of at least about 1 x 10 ⁷ M ^-1 , and binds to a given molecule with an affinity that is at least twice as high as its affinity for binding to a nonspecific molecule (eg, BSA, casein ), different from a given molecule or a closely related molecule.

Examples of ligand units include those described for use in WO 2007/085930, incorporated herein.

In some embodiments, the ligand unit 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 implementations

- 11 045942 tion 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 of the present invention, an antibody-drug conjugate (ADC) is provided.

Cell binding agent

The cell binding agent can be of any type and includes peptides and non-peptide compounds. These may include antibodies or an antibody fragment that contains at least one binding site, lymphokines, hormones, hormone mimetics, vitamins, growth factors, nutrient transport molecules, or any other cell-binding molecule or substance.

Peptides

In one embodiment, the cell binding agent is a linear or cyclic peptide containing 4-30, preferably 6-20, consecutive amino acid residues. In this embodiment, it is preferred that one cell-binding agent is associated with one monomeric or dimeric azetidobenzodiazepine compound.

In one embodiment _, the cell binding agent comprises a peptide that binds _αγβ6 integrin. _The peptide may be selective for _αγβ6 over XYS.

In one embodiment, the cell binding agent comprises an A20FMDV-Cys polypeptide. A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC.

Alternatively, a sequence variant A20FMDV-Cys may be used in which one, two, three, four, five, six, seven, eight, nine or ten amino acid residues are replaced by another amino acid residue. Moreover, the polypeptide may have the sequence NAVXXXXXXXXXXXXXXXXRTC.

Antibodies

The term antibody is used in this context in its broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), multivalent antibodies and antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour, of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein created by the immune system that is able to recognize and bind to a specific antigen. (Janeway, S., 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, that are recognized by the complementarity determining regions (CDRs) of various antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. The antibody comprises a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e. a molecule that contains an antigen-binding site that immunospecifically binds to the target's antigen or portion thereof, and such targets include, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any type (eg, IgG, IgE, IgM, IgD and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules. Immunoglobulins can be obtained from any animal species, including humans, mice or rabbits.

Antibody fragments contain part of a full-length antibody, usually its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementarity determining region) and epitope-binding fragments of any of the above that immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The term monoclonal antibody as used herein refers to an antibody derived from a population of essentially homogeneous antibodies, i.e. The individual antibodies that make up the population are identical, except for possible natural mutations that may be present in minute quantities. Monoclonal antibodies are highly specific, directed against a single antigenic site. Additionally, unlike polyclonal antibody preparations, which contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without the admixture of other antibodies. The modifier monoclonal indicates the nature of the antibody as being derived from a substantially homogeneous population of antibodies, and should not be construed as requiring that said antibody be produced by any particular method. For example, monoclonal antibodies suitable for use in this invention can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can be obtained by recombinant DNA methods (see US 4816567). Monoclonal antibodies can also be isolated from phage antibody libraries using 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 in this context include, in particular, chimeric antibodies, humanized antibodies and human antibodies.

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

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

(1) BMPR1B (bone morphogenetic protein receptor type IB) (2) E16 (LAT1, SLC7A5) (3) STEAP1 (sixth transmembrane epithelial antigen of the prostate) (4) 0772P (CA125, MUC16) (5) MPF (MPF, MSLN , SMR, megakaryocyte stimulating factor, mesothelin) (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II, sodium-dependent phosphate transporter 3b) (7) Sema 5b (FLJ10372 , KIAA1445, Mm.42015, SEMA5B, SEMAG, Hlog of semaphorin 5b, sema domain 25, of seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B) (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA, gene 2700050C12) (9) ETBR (endothelin type B receptor) (10) MSG783 (RNF124, hypothetical protein FLJ20315) (11) STEAP2 (H GNC8639, IPCA-1, PCANAP1 , STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, sixth transmembrane epithelial prostate antigen 2, sixth transmembrane prostate protein) (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel 5, subfamily M, member 4) (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor) (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d /Epstein-Barr virus receptor) or Hs.73792) (15) CD79b (CD79B, CD79p, IGb (immunoglobulin-associated, beta), B29) (16) FcRH2 (IFGP4, IRtA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C) (17) HER2 (ErbB2) (18) NCA (CEACAM6) (19) MDP(DPEP1) (20) IL20R-alpha (IL20Ra, ZCYTOR7) (21) Brevican (BCAN, BEHAB) (22 ) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5) (23) ASLG659 (B7h) (24) PSCA (prostate stem cell antigen precursor) (25) GEDA (26) BAFF-R (B-cell activating factor receptor, receptor BLyS3, BR3) (27) CD22 (isoform B of B-cell receptor Cd22, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814) (27a) CD22 (CD22 molecule) (28) CD79a (CD79A, CD79alpha ), immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transmits a signal involved in B cell differentiation), pI: 4.84, MW: 25028 TM: 2 [P] Chromosome gene: 19q13.2).

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the chemokine CXCL13, is involved in lymphocyte migration and humoral defense, and plays a role in HIV-2 infection and possibly the development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, pI: 8.54 MM: 41959 TM: 7 [P] Gene chromosome: 11q23.3, (30) HLA-DOB (beta subunit of the MHC class II molecule (antigen Ia), which binds peptides and 20 provides them into CD4+ T lymphocytes); 273 aa, pI: 6.56, MM: 30820.TM: 1 [P] Gene chromosome: 6p21.3) (31) P2X5 (ligand-gated ion channel of the purinergic receptor P2X, ion channel controlled by extracellular ATP, may be involved in synaptic transmission and neurogenesis, defi

- 13 045942 cit may contribute to the pathophysiology of idiopathic detrusor instability); 422 aa), pI: 7.63, MM: 47206 TM: 1 [P] Gene chromosome: 17p13.3).

(32) CD72 (B cell differentiation antigen CD72, Lyb-2); 359 aa, pI: 8.66, MM: 40225, TM: 1 5 [P] Gene chromosome: 9p13.3).

(33) LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich (LRR) 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, MM: 74147 TM: 1 [R] Gene chromosome: 5q12).

(34) FcRH1 (Fc receptor-like protein 1, a putative immunoglobulin Fc domain receptor that contains a C2 type Ig-like domain and an ITAM domain, may play a role in 20 B cell differentiation); 429 aa, pI: 5.28, MM: 46925 TM: 1 [R] Gene chromosome: 1q21-1q22) (35) IRTA2 (immunoglobulin superfamily receptor associated with translocation 2, a putative immunoreceptor with possible involvement in the development of B cells and lymphomagenesis; gene deregulation due to translocation occurs in some B-cell malignancies); 977 aa, pI: 6.88, MM: 106468, TM: 1 [P] Gene chromosome: 1q21) (36) TENB2 (TMEFF2, tomoregulin, TPEF, FIPP1, TR, putative transmembrane 35 proteoglycan associated with the EGF/heregulin family growth factors and follistatin); 374 aa) (37) PSMA - FOLH1 (Folate hydrolase (prostate-specific membrane antigen) 1) (38) SST (somatostatin receptor; note that there are 5 subtypes) (38.1) SSTR2 (somatostatin receptor 2) (38. 2) SSTR5 (somatostatin receptor 5) (38.3) SSTR1 (38.4) SSTR3 (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 (proto -met oncogene; hepatocyte growth factor receptor) (43) MUC1 (cell surface associated mucin 1) (44) CA9 (carbonic anhydrase IX) (45) EGFRvIII (epidermal growth factor receptor (EGFR), transcription 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 Ags - CTA (testicular cancer antigens) (53) CD174 (Lewis Y) - FUT3 (fucosyltransferase 3 (galactoside-3(4)-L-fucosyltransferase, Lewis blood group) (54) CLEC14A (member A of the C-domain lectin family 14 type; Genbank accession number NM175060) (55) GRP78 - HSPA5 (heat shock protein 5 70 kDa (glucose-regulated protein, 78 kDa) (56) CD70 (CD70 molecule) L08096 (57) Stem cell-specific antigens. For example:

5Т4 (see line (63) below)

CD25 (see line (48) below)

CD32

LGR5/GPR49

Prominin/CD 133 (58) ASG-5 (59) ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase 3) (60) PRR4 (proline-rich 4 (lacrymal)) (61) GCC - GUCY2C (guanylate cyclase 2C (heat-stable enterotoxin receptor) (62 ) Liv-1 - SLC39A6 (solute carrier family 6 member 39 (zinc transporter)) (63) 5T4, trophoblast glycoprotein, TPBG- TPBG (trophoblast glycoprotein) (64) CD56 - NCMA1 (nerve cell adhesion molecule 1) (65) CanAg (tumor associated antigen CA242) (66) FOLR1 (folate receptor 1) (67) GPNMB (glycoprotein (transmembrane)nmb)

- 14 045942 (68) TIM-1-HAVCR1 (hepatitis A virus cell receptor 1) (69) RG-1/prostate tumor target Mindin - Mindin/RG-1 (70) B7-H4-VTCN1 (T activation inhibitor 1 -cells containing a V-shaped domain) (71) RTK7 (protein tyrosine kinase 7 RTK7) (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 (homologue 3 of the erythroblastic leukemia viral oncogene v-erb-b2 (avian)) (78) RON - MST1R (macrophage-stimulating receptor 1 (c-met-related tyrosine kinase)) (79) ERNA2 (ERN receptor A2) (80) CD20-MS4A1 (transmembrane 4-domains, subfamily A, member 1) (81) Tenascin C -TNC (tenascin C) (82) FAP (fibroblast activation protein alpha) (83) DKK-1 (Dickkopf 1 homologue (Xenopus laevis) (84) CD52 (CD52 molecule) (85) CS1-SLAMF7 (7 member of the SLAM family ) (86) Endoglin - ENG (endoglin) (87) Annexations A1-ANXA1 (annexin A1) (88) V-CAM (CD106)-VCAM1 (vascular endothelial adhesion molecule type 1)

Additional tumor-associated antigens and cognate antibodies are:

(89) ASCT2 (ASC transporter 2, also known as SLC1A5).

ASCT2 antibodies are described in WO 2018/089393, which is incorporated herein by reference.

The cell-binding agent may be labeled, for example, to facilitate detection or purification of the agent prior to inclusion in or within the conjugate. The tag may be a biotin tag. In another embodiment, the cell-binding agent may be radiolabeled.

Treatment options

The compounds of this invention can be used in a method of therapy. Also provided is a method of treatment comprising administering to a subject in need of treatment a therapeutically effective amount of a conjugate of Formula II. The term therapeutically effective amount is an amount sufficient to provide benefit to the patient. Such a beneficial effect may be a relief of at least one symptom. The actual amount administered, as well as the frequency and schedule of administration, depend on the nature and severity of the pathological condition being treated. Prescribing treatment, such as determining dosage, is the responsibility of general practitioners and other medical personnel.

The conjugate can be administered alone or in combination with other treatments, simultaneously or sequentially, depending on the pathological 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 this invention and for use in accordance with this invention may contain, in addition to the active ingredient, i.e. conjugate of Formula II, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials known to those skilled in the art. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material depends on the route of administration, which may be oral or by injection, for example, dermal, subcutaneous or intravenous.

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

For intravenous, dermal, subcutaneous, or intralesional injection, the active ingredient must be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. If necessary, preservatives, stabilizers, buffers, antioxidants and/or other additives can be added.

These conjugates can be used to treat proliferative diseases and autoimmune diseases.

- 15 045942 moon disease. The term proliferative disease refers to unwanted or uncontrolled cellular proliferation of excess or abnormal cells that are undesirable, such as neoplastic or hyperplastic growth in vitro or in vivo.

Examples of proliferative pathological conditions include, but are not limited to, benign, pre-malignant and malignant cell proliferation, including, but not limited to, neoplasms and tumors (eg, histocytoma, glioma, astrocytoma, osteoma), cancer (eg, lung cancer, small cell lung cancer, gastrointestinal cancer, intestinal cancer, colon cancer, breast carcinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, breast cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemia, psoriasis, bone diseases, fibroproliferative disorders (eg, connective tissue) and atherosclerosis. Other cancers of interest include, but are not limited to, hematologic diseases; malignancies such as leukemias and lymphomas such as non-Hodgkin lymphoma and subtypes such as DLBCL, marginal zone cell lymphoma, mantle cell lymphoma and follicular lymphoma, Hodgkin lymphoma, AML and other B- or T-cell cancers.

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, Gray's disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease ( e.g. Crohn's disease), anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine insufficiency, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, thyroiditis Hashimoto's, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, herpetiformis dermatitis, focal alopecia, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcification, Raynaud's phenomenon, esophageal dyskinesia, sclerodactyly and telangiectasia), autoimmune infertility of men and women, 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, primary miscarriage, antiphospholipid syndrome, farmers' allergic alveolitis, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, poultry farmers' pulmonary allergy, toxic epidermal Necrololysis, alport syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, nodous erythema, gangrenous pyoderma, transfusion reaction, arteritis Tomas, rheumatic polymialgia, temporal arteritis, schistosomosis, giant arteritis, ascaridosis, aspergillosis, sintrome, ecseus Zema, lymphomatoid granulomatosis, Behçet's disease, Kaplan's syndrome, Kawasaki disease, dengue, encephalomyelitis, endocarditis, edomyocardial fibrosis, endophthalmitis, persistently elevated erythema, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuchs cyclitis, IgA nephroma, Henoch-Schonlein purpura, graft-versus-host disease, graft rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenström macroglobulinemia, Evans syndrome, and autoimmune gonadal failure.

In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th1 lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' disease , primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis or graft-versus-host disease) or T2 lymphocytes (eg, atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis or chronic graft-versus-host disease). In general, disorders affecting dendritic cells include disorders of TM lymphocytes or Th2 lymphocytes. In some embodiments, the autoimmune disorder is a T cell-mediated immunological disorder.

In some embodiments, the amount of conjugate administered is from about 0.01 to about 10 mg/kg per dose. In some embodiments, the amount of conjugate administered is from about 0.01 to about 5 mg/kg per dose. In some embodiments, the amount of conjugate administered is from about 0.05 to about 5 mg/kg per dose. In some embodiments, the amount of conjugate administered is from about 0.1 to about 5 mg/kg per dose. In some embodiments, the amount of conjugate administered is from about 0.1 to about 4 mg/kg per dose. In some embodiments, the amount of conjugate administered is from about

- 16 045942

0.05 to about 3 mg/kg per dose. In some embodiments, the amount of conjugate administered is from about 0.1 to about 3 mg/kg per dose. In some embodiments, the amount of conjugate administered is from about 0.1 to about 2 mg/kg per dose.

Drug content

Drug content (p) is the average amount of ABD drugs per cell-binding agent, such as antibody. If the compounds of this invention are associated with cysteines, the drug content may be from 1 to 8 drug units (D) per cell-binding agent, i.e. 1, 2, 3, 4, 5, 6, 7 and 8 drug moieties are covalently linked to the cell-binding agent. Conjugate compositions include aggregates of cell-binding agents, for example, antibodies, conjugated to a drug in an amount of from 1 to 8. If the compounds of this invention are associated with lysines, the drug content can be from 1 to 80 units of drug (D) per cell-binding agent, although an upper limit of 40, 20, 10, or 8 may be preferred. Conjugate compositions include combinations of cell-binding agents, for example, antibodies conjugated to a drug in an amount of 1 to 80, 1 to 40, 1 to 20, 1 to 10, or from 1 to 8.

The average amount of drug per antibody in ADC preparations obtained from conjugation reactions can be determined by standard methods such as UV, reverse-phase HPLC, GIC, mass spectroscopy, enzyme-linked immunosorbent assay, and electrophoresis. It is also possible to quantify the ADC distribution in terms of p. An enzyme-linked immunosorbent assay can determine the mean p value of a particular ADC preparation (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 cannot be determined from antibody-antigen binding and the detection limit of enzyme-linked immunosorbent assays. In addition, enzyme-linked immunosorbent assays for detecting antibody-drug conjugates do not detect the positions at which drug moieties are attached to the antibody, such as heavy chain or light chain moieties of specific amino acid residues. In some cases, separation, purification and characterization of a homogeneous ADC, in which p is a certain value derived from an ADC with a different drug content, can be accomplished using, for example, reverse phase HPLC or electrophoresis. Such technologies are also applicable to other types of conjugates.

For some antibody-drug conjugates, p may be limited by the number of attachment sites in the antibody. For example, an antibody may have only one or more cysteine thiol groups, or may have only one or more sufficiently reactive thiol groups to which a linker can be attached. Higher drug content, such as p>5, may result in aggregation, instability, toxicity, or decreased cellular permeability of some antibody-drug conjugates.

Typically, a conjugation reaction with an antibody conjugates fewer drug moieties than the theoretically possible maximum number. The antibody may contain, for example, multiple lysine residues that do not interact with the drug linker. Only the most reactive lysine groups can react with the amine-reactive linker reagent. Also, only the most reactive cysteine thiol groups can react with the thiol-reactive linker reagent. In general, antibodies do not contain many, if any, free and reactive cysteine thiol groups that can be linked to the drug moiety. Most cysteine thiol residues in the antibodies of these compounds exist as disulfide bridges and must be reduced by a reducing agent such as dithiothreitol (DTT) or TCEP under partial or complete reducing conditions. The drug content (drug/antibody ratio) of the ADC can be controlled in several different ways, including: (i) limiting the molar excess of the drug linker relative to the antibody, (ii) limiting the time or temperature of the conjugation reaction, and (iii) partial or limited reduction conditions for modification cysteine thiol.

Some antibodies contain interchain disulfides capable of reduction, i.e. cysteine bridges. Antibodies can be rendered reactive for conjugation with linker reagents by treating them with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by reacting lysines with 2-iminothiolane (Trout's reagent), converting the amine to a thiol. Reactive thiol groups can be introduced into an antibody (or fragment thereof) by engineering one, two, three, four or more cysteine residues (eg, generating mutant antibodies containing one or more unnatural cysteine amino acid residues). US 7,521,541 describes the construction of antibodies by introducing reactive cysteine amino acids.

Cysteine amino acids can be engineered into antibody reactive sites to

- 17 045942 which 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 7521541; US 7723485; WO2009/052249). Engineered cysteine thiols can react with linker reagents or drug-linker reagents of the present invention that contain thiol-reactive electrophilic groups, such as maleimide or alpha-haloamides, to form ADCs with cysteine-engineered antibodies and ABD drug moieties. In this way, the position of the drug moiety can be designed, controlled and known. The drug content can be controlled because the thiol groups of the engineered cysteine typically react with thiol-reactive linker reagents or drug-linker reagents in high yield. Designing an IgG antibody to introduce a cysteine amino acid through single-site substitution of the heavy or light chain provides two new cysteines in a symmetrical antibody. A drug content of about 2 can be achieved with almost complete homogeneity of the ADC conjugation product.

If more than one nucleophilic or electrophilic group of an antibody reacts with a linker drug intermediate, or with a linker reagent and then with a drug moiety, the resulting product is a mixture of ADC compounds with a distribution of drug moieties attached to the antibody, e.g. etc. Liquid chromatography techniques such as polymer reversed phase (RPP) chromatography and hydrophobic interaction chromatography (HIC) can separate compounds in a mixture based on drug content. It is possible to isolate ADC formulations with a single drug content value (p), but such a single ADC content value may still represent a heterogeneous mixture since drug moieties may be attached via a linker at different sites in the antibody.

Thus, the antibody-drug conjugate compositions of the present invention include mixtures of antibody-drug conjugate compounds in which the antibody has one or more ABD drug moieties and in which the drug moieties may be attached to the antibody at different amino acid residues.

In one embodiment, the average number of dimeric azetidobenzodiazepine groups per cell-binding agent is from 1 to 20. In some embodiments, the range is selected from the ranges 1 to 8, 2 to 8, 2 to 6, 2 to 4, and 4 until 8.

In some embodiments, there is one dimeric azetidobenzodiazepine group per cell-binding agent.

General synthesis methods

A large number of suitable N-ProtN, O-Prot ^O and Y-Prot ^Y protecting groups are described in Greene, TW and Wuts, GM, Protective Groups in Organic Synthesis, ^3rd edition, John Wiley & Sons, Inc., 1999, included into this document by reference.

Synthesis of compounds of formula IV

A possible synthetic step for compounds of the first aspect of this invention, in particular compounds of formula IV, is presented in Scheme 1. It begins with an NW-protected ABD dimer (d1A).

- 18 045942

Scheme 1

The d1A dimer is protected at the N10 position by standard methods to obtain the compound of formula IV. If ProtN is Alloc, then deprotection is carried out using palladium. The resulting compound may be in the form of its carbinolamine or carbinolamine ether, depending on the solvents used, in equilibrium with the imine.

In the case of ABD, the ring stress of the four-membered azetidine ring means that the carbinolamine form is predominant at equilibrium.

An alternative step in the synthesis of compounds of formula IV is presented in Scheme 2. It begins with the ABD monomer protected at the N10 nitrogen (ιη2Ά).

- 19 045942

Scheme 2

The monomer ABD m2A protected at the N10 and Y8 positions is protected at the alcohol group at the C11 position to give m2B. Preferably, Prot ^O is TBS and this protection is accomplished by adding excess TBS-Cl. Subsequent removal of the protective group! Prot ^Y -Y results in dimerizable species (m2Q. If ProtY is a TIPS, then deprotection can be accomplished using LiOAc in DMF and water.

m2C is reacted with the dimeric linker R(X) ₂ to obtain the d2D dimer. Typically Y is O and X is halogen (preferably Br). Here, a double Williamson ether synthesis produces a dimer using TBAI as an additive.

The IO protecting group is removed from the dimeric product to obtain d2E. For example, if Prot ^N is Alloc and Prot ^O is an oxygen protecting group for synthesis, then deprotection is accomplished by using palladium to remove the N10 protecting group, followed by removal of the oxygen protecting group for synthesis. If Prot ^N is Troc and Prot ^O is an oxygen protecting group for synthesis, then deprotection is carried out using a Cd/Pb pair. If Prot ^N is SEM or a similar group, and ProtO is an oxo group, then the oxo group can be removed by reduction, resulting in a protected carbinolamine intermediate, which can then be treated to remove the SEM protecting group, followed by elimination of water. Removing the protecting group! alcohol at the C11 position provides the compound of formula IV. If ProtO is TBS, then alcohol deprotection can occur simultaneously with the aforementioned Alloc N-deprotection using palladium and pyrrolidine in DCM.

The d1A dimer and m2A monomer required for Schemes 1 and 2, respectively, can be synthesized in several ways. One possible route, through oxidative ring closure, is presented in Scheme 3.

- 20 045942

Scheme 3

Compounds 3A, 3B, 3C, 3D and 3E may be dimeric (if the RY group is R linked to the same ABD precursor) or monomeric (if the RY group is a suitable protecting group).

Monomeric compound 3A is a nitrobenzoic acid derivative. Many such derivatives are commercially available, and others can be synthesized by conventional methods (eg, Althuis, T. H. and Hess, H. J., J Medicinal CHem., 20(1), 146-266 (1977)). Often, nitrobenzoic acid is prepared from the ester by hydrolysis of the ester under mild conditions (such as with LiOH). Dimeric compound 3A can be prepared by various methods known in the art (eg, Scheme 3 of WO 00/12508). For example, the corresponding benzoic acid esters can be dimerized to a suitable diol by the Mitsunobo esterification reaction, followed by nitration and hydrolysis. Alternatively, benzoic acid esters can be dimerized to a suitable dihalide via the Williamson ether synthesis reaction. Additional transformations required to obtain monomeric and dimeric compounds 3A are available in the literature.

The azetidine starting material can be synthesized by modifying a similar proline synthesis described in the prior art (eg Scheme 4 of WO 00/12508). Strategies specifically addressing azitidine are also known in the literature (e.g., Bose, DS, et al., Tetrahedron Letters, 38(33), 5839-5842, 1997; doi: 10.1016/S0040-4039(97)01297-5) . For example, a commercially available azetidine-2-carboxylic acid can be protected at the azetidine nitrogen with a suitable protecting group such as Cbz, followed by acid esterification to produce the methyl ester. The ester can be reduced with LiBH ₄ in THF to give Cbz-protected 2-(hydroxymethyl)azetidine. In some approaches, an appropriate protecting group (ProtO), such as TBS, can be added to the alcohol by reaction with TBS-Cl. In other approaches, the alcohol is left without a protecting group. In Scheme 3, ProtO can be either a suitable protecting group or H, a suitable Prot ^O group must be able to withstand NO2 reduction conditions. The nitrogen protecting group is then removed, usually by reduction in H2 gas, to yield the azetidine starting material required in Scheme 3.

Compound 3A is condensed with azetidine starting material to give 3B. Often condensation is carried out by coupling with DCC or through an acid chloride (derived from a carboxylic acid with oxalyl chloride or SOCl ₂ ), or with HOBt in DCM at low temperature.

The nitro group of compound 3B is reduced to the amine (3C) using a standard method such as SnCl ₂ in MeOH or zinc in MeOH/H ₂ O/formic acid (90:5:5), or sodium dithionite, or Raney nickel and hydrazine, or catalytic hydrogenation on palladium on charcoal. The method chosen depends on the requirements of the hydroxyl protecting group.

- 21 045942

The resulting amine is protected once with a suitable protecting group to obtain 3D. The N-ProtN group is preferably a carbamate such as N-Alloc. The nucleophilicity of the amine is reduced when protected using Alloc, so single protection is preferred. This is typically accomplished by reacting with pyridine and one equivalent of allyl chloroformate. If Prot ^O is H, then the 3D compound is equivalent to 3E. If Prot ^O is a protecting group, it is removed under standard conditions to give alcohol 3E. If Prot ^O is an acetate protecting group, it can be removed under slightly alkaline conditions (eg K ₂ CO ₃ ), or if Prot ^O is a silyl ether protecting group such as TBS, it can be removed using TBAF or weak acids.

Oxidative ring closure through an aldehyde or functional equivalent from dimeric 3E leads to the production of d1A (for further reaction according to scheme 1), and from monomeric 3E leads to the production of t2A (for further reaction according to scheme 2). Selective oxidation of the alcohol to the aldehyde can be accomplished by treatment with tetrapropylammonium perruthenate (TPAP) in Nmethylmorpholine N-oxide (NMO) on molecular sieves or by Swern oxidation (with DMSO and oxalyl chloride), or by Dess-Martin oxidation (with DMP), or preferably via radical oxidation of Cu(I)/TEMPO (with copper(I) tetrakisacetonitrile triflate, 1-hydroxy-2,2,6,6tetramethylpiperidine (TEMPO), 1-methylimidazole and 2-(2-pyridyl)pyridine). The latter is preferred because it does not require strictly anhydrous conditions and there is no evidence of excess oxidation to dilactam ABD particles. Aldehyde particles undergo spontaneous closure of the B-ring, in which it is attacked at the singly protected position N10.

An alternative method for preparing the d1A dimer and m2A monomer is shown in Scheme 4. This method uses aldehyde unmasking to mediate ring closure.

Scheme 4

t2L

Compounds 3A, 4B, 4C and 4D may be dimeric (if the RY group is R linked to the same ABD precursor) or monomeric (if the RY group is a suitable protecting group).

Monomeric and dimeric variants of 3A can be prepared by the methods discussed above with respect to Scheme 3. The azetidine starting material is characterized by the presence of a thioacetal at the 2-position (although other masked aldehyde equivalents may be used). Diethylthioacetal-azetidine can be prepared by modification of a similar proline synthesis method (eg, Langley, D.R. & Thurston, D.E., J Organic CHemistry, 52, 91-97 (1987)). Methods specific to azitidine are also known in the literature (e.g., Bose, D.S., et al., Tetrahedron Letters, 38(33), 5839-5842, 1997; doi: 10.1016/S0040-4039(97)01297-5) . For example, Cbz-protected 2-(hydroxymethyl)azetidine can be prepared as described above (for Scheme 3). Behind

- 22 045942 the resulting alcohol is usually re-oxidized to the aldehyde by Dess-Martin oxidation (with DMP) or by IBX in DMSO. The resulting aldehyde is preferably condensed with a thiol such as EtSH with a weak acid catalyst such as TMSCl in a protic solvent to produce a thioacetal. The thioacetal is incompatible with reduction using H2 gas, so the N-protecting group (eg Cbz) is often removed using TMS-I in DCM. The result is the starting material, diethylthioacetal-azetidine.

Direct condensation of 3A with thioacetal-azetidine starting material results in 4B. The nitro group of compound 4B can be reduced to the amine (4C) by the methods discussed above in relation to scheme 3, preferably by the method using tin (II) chloride (SnCl ₂ in MeOH) or zinc in a mixture of MeOH/I ₂ O/formic acid (90: 5:5). The reduction is preferably not carried out by direct hydrogenation due to the incompatibility of the thioacetal group. The amino is protected once with a suitable amine protecting group, such as Alloc, by reaction with the appropriate chloroformate or acid chloride. The N-ProtN moiety of compound 4D is preferably a carbamate, such as N-Alloc, since these species promote one-shot protection.

Selective unmasking of the thioacetal to the aldehyde leads to spontaneous cyclization of the B-ring as a result of its attack at the singly protected position N10. Typically, unmasking is mediated by mercury (II), for example, HgCl ₂ with CaCO ₃ in a mixture of acetonitrile: water. For dimeric 4D this leads to the formation of d1A (for further reaction according to scheme 1), and for monomeric 4D this leads to the formation of t2A (for further reaction according to scheme 2).

Dimeric or monomeric thioacetal 4B (according to Scheme 4) can be synthesized by an alternative method shown in Scheme 5. This method leads to the production of thioacetal in situ.

Scheme 5

Synthetic strategies for preparing monomeric and dimeric 3A compounds are discussed above for Scheme 3. Compound 3A is condensed with a commercially available azetidine-2-carboxylic acid to give 5B. The method for converting 5B to 4B is similar to the method for synthesizing the thioacetal-azetidine starting material from azetidine-2-carboxylic acid (described for Scheme 4).

Compound 5C is reduced with a hydride reducing agent, usually LiBH ₄ , to secondary alcohol 5D. 5D is then re-oxidized to the aldehyde (5E), often using hypervalent iodine compounds (e.g. IBX or DMP). The thioacetal is prepared in situ, preferably using EtSH under acidic conditions, to give compound 4B. It can then be used in the reactions of Scheme 4 to obtain the desired ABD compounds.

Synthesis of compounds of formula I

A possible synthetic step for compounds of the second aspect of this invention, in particular compounds of formula I, is shown in Scheme 6. It begins with two cyclized ABD monomers: m2A with a ProtN-protected N10 position and m6A with RL attached to the N10 nitrogen.

The compound of formula I can exist in equilibrium between an imine and a carbinolamine or carbinolamine ether form, depending on the solvent used (similar to the equilibrium shown in Scheme 1 for formula IV).

In some embodiments, ProtN may be equivalent to the R ³⁰ substituent of formula I (described in embodiment (d) i, ii and iii of the second aspect of this invention).

- 23 045942

Scheme 6

The starting material m2A can be prepared according to Schemes 3, 4 and 5. For those compounds in which ProtN is the equivalent of the carbamate linker groups R ³⁰ , compound m2A is prepared via isocyanate (i.e. in the same way as m6A - discussed below ).

m2A and m6A can be dimerized at position Y8 using a dimeric linker R using the same strategy as described for Scheme 2. The alcohol at position C11 is protected with a ^ProtO group, ^ProtO preferably being RBS, and introduced by reaction with TBS- Cl. Subsequent removal of the Prot ^Y group, where Prot ^Y is TIPS, can be carried out with LiOAc in DMF and water to obtain m2C and m6B, respectively.

Then m2C and m6B are reacted with R(X) ₂ to produce the d6C dimer. Typically Y is O and X is halogen (preferably Br). In this case, the addition of TBAI may cause double Williamson ether synthesis to form a dimer. Alternative dimerization strategies are also known in the art, for example, Mitsunobu esterification.

In some embodiments, the I10 protecting group is removed from the non-linker ABD to produce an asymmetric d6D dimer. Different deprotection strategies are described for Schemes 1 and 2. In those

- 24 045942 cases where ProtN is Alloc, deprotection can be carried out with palladium.

In other embodiments, the N10 protecting group is not removed. d6C is converted directly to the compound of formula I by removal of Prot ^O.

Deprotection of the alcohol at the C11 position provides an asymmetric compound of formula I. If ProtO is TBS, then deprotection of the alcohol can be accomplished simultaneously with deprotection of Alloc at the N10 position using palladium and pyrrolidine in DCM.

A possible synthesis of the m6A monomer (required for Scheme 6) and an alternative method for preparing compounds of formula I are presented in Scheme 7.

Scheme 7

Compounds 7A, 7B, 7C and 7D may be dimeric (if the R ^YY group is R linked to an ABD precursor protected at the N10 position) or monomeric (if the R ^YY group is a suitable protecting group). Monomeric compound 7A is equivalent to monomeric 3C and can be synthesized in the same way (as shown in Scheme 3).

Amine 7A is converted to isocyanate 7B. Strategies for producing isocyanate are described in detail in the prior art (eg WO 2005/023814). Typically, phosgene is used, preferably triphosgene, under alkaline conditions; It is safer and easier to use solid triphosgene crystals than toxic phosgene gas. The reaction should be carried out in an anhydrous and non-hydroxyl organic solvent, which is preferably non-polar. Suitable solvents include anhydrous DCM and anhydrous toluene. The reaction can be carried out at room temperature and the progress of the reaction can be conveniently monitored by infrared spectroscopy at about 2265 cm ^-1 .

Carbamate 7C is obtained from isocyanate by attack with RL-OH. The formation of the carbamate is often carried out by a one-pot process in which the isocyanate is produced by reacting triphosgene with TEA in DCM and RL-OH is added directly to the reaction mixture. This approach shortens the time the isocyanate is present before carbamate is formed, which reduces the likelihood of adverse reactions.

The ProtO protecting group is removed by a suitable method to produce the secondary alcohol 7D, usually under acidic conditions (eg acetic acid in a THF:water solvent).

Oxidative ring closure of 7D via an aldehyde or functional equivalent can be accomplished by treatment with tetrapropylammonium perruthenate (TPAP) to N-methylmorpholine

- 25 045942

N-oxide (NMO) on molecular sieves or via Swern oxidation (DMSO and oxalyl chloride), or preferably via Cu(I)/TEMPO radical oxidation (copper(I) tetrakisacetonitrile triflate, 1-hydroxy-2,2,6,6- tetramethylpiperidine (TEMPO), 1-methylimidazole and 2-(2-pyridyl)pyridine). The result is m6A (for the next reaction in Scheme 6) for the monomeric variant 7D (i.e. if R ^YY =Prot ^Y ), or d7E is obtained for the dimeric variant 7D (i.e. if R ^YY =R associated with No. 0-protected ABD).

Removal of the nitrogen protecting group (ProtN), usually Alloc is removed using palladium, results in the asymmetric compound of formula I.

An alternative method for preparing monomer m6A (Scheme 6) and dimer d7E (Scheme 7) is presented in Scheme 8.

Scheme 8

Compounds 8A, 8B and 8C may be dimeric (if the R ^YY group is R linked to an ABD precursor protected at the N10 position) or monomeric (if the R ^YY group is a suitable protecting group, Prot ^Y ). 8A is converted to isocyanate 8B, which in turn is reacted with R ^L - OH to attach RL via a carbamate. The preferred strategy is similar to the method described for Scheme 7.

Selective unmasking of thioacetal 8C to the aldehyde leads to spontaneous cyclization of the B-ring as a result of its attack at the singly protected position N10. Typically, unmasking is mediated by mercury (II), for example, HgCl ₂ with CaCO3 in a mixture of acetonitrile: water. Cyclization leads to the formation of monomeric m6A (from monomeric 8C) and dimeric d7E (from dimeric 8C). Compound m6A can be reacted according to Scheme 6, and d7E - according to Scheme 7 to obtain compounds of formula I.

The starting materials for Scheme 7 can be prepared in the same manner as shown in Scheme 3 (conversion of monomer 3A to 3C) for monomer 7A (ie if R ^YY is Prot ^Y ). Likewise, the starting materials for Scheme 8 can be prepared in the same manner as shown in Scheme 4 (conversion of monomer 3A to 4C) for monomer 8A (ie if R ^YY is Prot ^Y ).

The method for preparing dimeric 7A and 8A (i.e. if R ^YY is R linked to an ABD precursor singly protected at position N10) is presented in Scheme 9.

- 26 045942

Scheme 9

Dimer variants 3C and 4C are prepared by the methods described in Schemes 3, 4 and 5. 3C and 4C protect at only one N10 position to produce asymmetric dimers ABD 7A and 8A, respectively. This is achieved by adding one equivalent of a protecting reagent, typically allyl chloroformate if ProtN is Alloc, followed by purification to remove unprotected or doubly protected products.

Synthesis of conjugates of formula II

A possible step in the synthesis of conjugates according to the third aspect of this invention, in particular, the drug linker unit (DL) of formula II, involves the binding of the linker to the ligand unit, which leads to the conversion of the RL group (for compounds of formula I) to the R ^LL group (for compounds formula I').

Conjugates can be prepared as described previously. Antibodies can be conjugated to a drug linker compound as described in Doronina et al., Nature Biotechnology, 2003, 21, 778-784). Briefly, antibodies (4-5 mg/ml) in PBS containing 50 mM sodium borate at pH 7.4 were reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37°C. The progress of the reaction in which interchain disulfides are reduced is monitored by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) and the reaction is continued until the required thiol/mAb ratio is achieved. The reduced antibody is then cooled to 0°C and alkylated using 1.5 equivalents of maleimide drug linker per antibody thiol. After 1 hour, the reaction is quenched by adding 5 equivalents of N-acetylcysteine. The quenched drug linker is removed by gel filtration on a PD-10 column. The ADC is then sterile filtered through a 0.22 µm syringe filter. Protein concentration can be determined by spectral analysis at 280 nm and 329 nm, respectively, corrected for the contribution of drug absorption at 280 nm. Size exclusion chromatography can be used to determine the extent of antibody aggregation, and RP-HPLC can be used to determine the content of residual NAC-quenched drug linker.

Synthesis of macrocyclic implementation options

In some embodiments of the first, second and third aspects of this invention, the substituents R ⁷ and R ⁷ together may form a group that is: (i) -O-(CH ₂ ) _n -O-, where n is from 7 to 16, or (ii) -O-(CH ₂ CH ₂ O) _m -, where m is from 2 to 5, to obtain a macrocyclic dimer ABD.

Various strategies can be used to introduce the R7-R ⁷ linker, as shown below in Scheme 10. Based on d2D, where both R ⁷ and R ⁷ are -OR, the R group can be removed by adding BBr ₃ to DCM to expose the alcohol. A dibromoalkane substitution reaction at the base, such as the substitution reaction for 1,7-dibromoheptane in K ₂ CO ₃ , results in the formation of a macrocyclic product as a result of its attack on both alcohol groups at the C7 position.

An alternative method starting with d2D involves replacing the alcohol at the C7 position with nbromoalc-1-ene. The result is two alkienyl chains with terminal unsaturation that can readily undergo ring-closing metathesis (RCM). For example, substitution can be performed using 5-bromopent-1-ene and RCM using Grubbs-II catalyst. Macrocyclization via RCM usually has high yield.

The preferred method for preparing the macrocycle starts with a dimeric 3A or ester precursor where both R ⁷ and R ⁷ are -OR. Removal of the R group and substitution with a dibromoalkane (using the same conditions as above) results in the formation of a macrocyclic compound. Then, according to schemes 3 or 4, ABD can be obtained.

- 27 045942

Scheme 10

The resulting product can be used for reactions according to Scheme 2 or Scheme 6 to obtain compounds of formula IV and I, respectively.

Additional information on the transformations necessary to obtain such macrocyclic products is available in the literature (Donnell, A.F., Zhang, Y., Stang, E.M., Wei, D.D., Tebben, A.J., Perez, H.L., Schroeder, G.M., Pan, C. Rao, C., Borzilleri, R.M., Vite, G.D., Gangwar, S., Macrocyclic pyrrolobenzodiazepine dimers as antibody-drug conjugate payloads, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016Zj.bmcl .2017.10.028 and WO 2016/209951).

Synthesis of options for the implementation of secondary amines

Compounds in which the N10-C11 group is -NH-CH2- (ie secondary amines) can be synthesized by modification of the methods described above.

In particular, reductive amination of compound 3B* can provide a modified version of t2A or d1A for use in the following steps:

ZV*

Compounds 3B* can be synthesized from the precursor alcohol by oxidation, and the precursor alcohol can be prepared by steps similar to those used for the synthesis of 3B.

Synthesis of drug conjugates

Conjugates can be prepared as described previously. Antibodies can be conjugated to a drug linker compound as described in Doronina et al., Nature Biotechnology, 2003, 21, 778-784). Briefly, antibodies (4-5 mg/ml) in PBS containing 50 mM sodium borate at pH 7.4 were reduced with tris(carboxyethyl)phosphine hydrochloride (TCEP) at 37°C. The progress of the reaction in which interchain disulfides are reduced is monitored by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) and the reaction is continued until the required thiol/mAb ratio is achieved. The reduced antibody is then cooled to 0°C and alkylated using 1.5 equivalents of maleimide drug linker per antibody thiol. After 1 hour, the reaction is quenched by adding 5 equivalents of N-acetylcysteine. The quenched drug linker is removed by gel filtration on a PD-10 column. The ADC is then sterile filtered through a 0.22 µm syringe filter. Protein concentration can be determined by spectral analysis at 280 nm and 329 nm, respectively, corrected for the contribution of drug absorption

- 28 045942 at 280 nm. Size exclusion chromatography can be used to determine the extent of antibody aggregation, and RP-HPLC can be used to determine the content of residual NAC-quenched drug linker.

Brief description of graphic materials

The figure shows the effect on the growth of a tumor cell line when treated with a control drug or conjugate of this invention.

Additional preferences

The following preferences may apply to all aspects of this invention described above, or they may apply to one aspect. Preferences can be combined with each other in any combination.

R ⁶ and R ⁹ are selected from the same groups as R ⁶ and R ⁹ , respectively. In some embodiments, R ⁶ , R ⁷ , R ⁹ and Y' are the same as R ⁶ , R ⁷ , R ⁹ and Y , respectively.

Dimer bond

In some embodiments, both Y and Y' are O.

In some embodiments, R is an unsubstituted C _3-7 alkylene group. In some of these embodiments, R'' is C ₃ , C ₅ or C ₇ alkylene. In particular, R may represent C ₃ or C ₅ alkylene.

In other embodiments, R is a group of the formula:

where r is 1 or 2.

The phenylene group may be replaced by a pyridylene group.

R ⁶ -R ⁹

In some embodiments ^{, R9} is H.

In some embodiments, R ⁶ is selected from H, OH, OR, SH, NH ₂ , nitro, and halogen, and may be selected from H or halogen. In some of these embodiments, R ⁶ is H.

In some embodiments, R ⁷ is selected from H, OH, OR, SH, SR, NH2, NHR, NRR', and halogen. In some such embodiments, R ⁷ is selected from H, OH, and OR, where R is selected from optionally substituted C _1-7 alkyl, C _3-10 heterocyclyl, and C _5-10 aryl groups. More preferably, R may represent a C _1-4 alkyl group, which may or may not be substituted. A suitable substituent is a C _5-6 aryl group (eg phenyl). Particularly preferred substituents at the 7-position are OMe and OCH ₂ Ph. Other particularly useful substituents are dimethylamino (ie -NMe ₂ ); -(OC ₂ H ₄ )OMe, where q is from 0 to 2; nitrogen-containing _C6 heterocyclyls, including morpholino, piperidinyl and N-methylpiperazinyl.

Said embodiments and preferences relate to R ⁹ , R ⁶ and R ⁷ , respectively.

In other embodiments, R ⁷ and R ⁷ together form the group -O-(CH ₂ ) _p -O-, where n is from 7 to 16. The value of n can be at least 7, 8, 9, 10 or 11. The value n can be no more than 16, 15, 14 or 13.

In other embodiments, R ⁷ and R ⁷ ' together form the group -O-(CH ₂ CH ₂ O) _Ш -, where m is from 2 to 5. The value of m can be at least 2, 3 or 4. The value of m can be be no more than 5, 4 or 3.

R ¹⁰ , R ¹¹ , R ²⁰ , R ²¹ (formula IV)

In some embodiments, R ¹⁰ and R ¹¹ together form a double bond between the N and C atoms to which they are bonded. In some such embodiments, R ²⁰ and R ²¹ together form a double bond between the N and C atoms to which they are bonded. In other such embodiments, both R ²⁰ and R ²¹ are H.

In some embodiments, R ¹⁰ is H, and R ¹¹ is selected from OH and ORA, where RA is C1-4 alkyl. In some such embodiments, R ²⁰ is H, and R ²¹ is selected from OH and OR ^B , wherein ^RB is C1-4 alkyl. In other such embodiments, both R ²⁰ and R ²¹ are H.

In some such embodiments, both R ¹⁰ and R ¹¹ are H. In some such embodiments, R ²⁰ and R ²¹ together form a double bond between the N and C atoms to which they are bonded. In other such embodiments, R ²⁰ is H and R ²¹ is selected from OH and OR ^B , wherein ^RB is C _1-4 alkyl.

In some embodiments, RA is methyl. In some embodiments, ^RB is methyl.

In some embodiments, only one of the pairs R ¹⁰ and R ¹¹ and R ²⁰ and R ²¹ are both H. In other embodiments, none of the pairs R ¹⁰ and R ¹¹ and R ²⁰ and R ²¹ are both H. do not represent H.

- 29 045942

In some embodiments, R ¹⁰ , R ¹¹ , R ²⁰ , and R ²¹ are all H.

N10'-C11' (formulas I and I*)

In some embodiments, R ³⁰ and R ³¹ together form a double bond between the N and C atoms to which they are bonded.

In some embodiments, R ³⁰ is H, and R ³¹ is selected from OH and OR ^B , wherein ^RB is C _1-4 alkyl. In some of these embodiments, ^RB is methyl.

In some embodiments, R ³⁰ is H and R ³¹ is H.

In some embodiments, ^R31 is OH or ^ORB , wherein ^RB is _C1-4 alkyl, and ^R30 is selected from:

- 30 045942

-C(=O)-X ₁ -NHC(=O)X2-NH- means dipeptide. The amino acids in the dipeptide can be any combination of naturally occurring amino acids. The dipeptide may be a site of action for cathepsin-mediated cleavage.

In one embodiment, the dipeptide -C(=O)-X ₁ -NHC(=O)X2-NH- is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys- , -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit-, -Phe-Arg-, -Trp-Cit-, where Cit is citrulline.

Preferably, the dipeptide -C(=O)-X ₁ -NHC(=O)X2-NH- is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, - Val-Cit-.

Most preferably, the dipeptide -C(=O)-X ₁ -NHC(=O)X2-NH- is -Phe-Lys- or -Val-Ala-.

Other dipeptide combinations may be used, including those described in DubowCHik et al., Bioconjugate CHemistry, 2002, 13,855-869, incorporated herein by reference.

In one embodiment, the amino acid side chain is derivatized, unless it is

- 31 045942 bypassed. For example, the amino group or carboxy group of an amino acid side chain may be derivatized.

In one embodiment, the NH2 amino group of a side chain of an amino acid, such as lysine, is a derivatized form selected from the group consisting of NHR and NRR'.

In one embodiment, the carboxy group COOH of a side chain of an amino acid, such as aspartic acid, is a derivatized form selected from the group consisting of COOR, CONH2, CONHR, and CONRR'.

In one embodiment, the amino acid side chain is chemically protected, if desired. The side chain protecting group may be a group described above. The authors of this invention have found that protected amino acid sequences can be cleaved by enzymes. For example, it has been found that a dipeptide sequence containing a Lys residue with a Boc-protected side chain is cleaved by cathepsin.

Protecting groups for amino acid side chains are well known in the art and are described in the Novabiochem catalogue. Additional protecting group strategies are outlined in Protective Groups in Organic Synthesis, Greene and Wuts.

The following are possible side chain protecting groups for those amino acids that contain a reactive functional group in the side chain:

Arg: Z, Mtr, Tos;

Asn: Trt, Xan;

Asp: Bzl, t-Bu;

Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;

Glu: Bzl, t-Bu;

Gln: Trt, Xan;

His: Boc, Dnp, Tos, Trt;

Lys: Boc, Z-Cl, Fmoc, Z, Alloc;

Ser: Bzl, TBDMS, TBDPS;

Thr: Bz;

Trp: Boc;

Tyr: Bzl, Z, Z-Br.

In one embodiment, side chain protection is selected that is orthogonal to the group present as or as part of the cap group, if present. Thus, removal of a side chain protecting group does not remove the cap group or any functional protecting group that is part of the cap group.

In other embodiments of the present invention, amino acids are selected that do not have reactive functional side groups. For example, amino acids can be selected from: Ala, Gly, Ile, Leu, Met, Phe, Pro and Val.

In this invention, it is particularly preferred that if Q contains a dipeptide, then -C(=O)-X1NHC(=O)X2-NH- is the same dipeptide. An example of a preferred group is:

Other preferred R ³⁰ groups include:

And

R ^11b (formulas I and I*)

In some embodiments, R ^11b is OH.

In some embodiments, R ^11b is ORA, where RA is C _1-4 alkyl. In some such embodiments, RA is methyl.

- 32 045942

Additional formulas

In some embodiments of the first aspect of the present invention, formulas IVa, IVb, or IVc are provided:

where R ^1a is selected from methyl and benzyl;

R ¹⁰ , R ¹¹ , R ²⁰ and R ²¹ are as defined above.

In some embodiments of the second aspect of the present invention, formulas Ia, Ib, or Ic are provided:

- 33 045942

where R ^1a is selected from methyl and benzyl;

R ³⁰ , R ³¹ , RL and R ^11b are as defined above.

These embodiments and preferences also relate to the third aspect of the present invention.

Linker ( ^Rl )

In some embodiments, R ^L has formula IIIa.

In some embodiments, R ^LL has formula IIIa'.

G ^l

G ^l can be selected from

- 34 045942

(G ^L2 ) O (G ^L6 ) 0 O / \ -N .X x/X ⁷⁰ (O ³ ' ¹ ) (G ^L7 ) In g— s—s ^z / Ψ ( _NO2 ) where the NO ₂ group is optional (G ^{L3 2} ) S— Η (G ^L8 ) <4? ( _NO2 ) where the NO2 group is optional (G ^{L3 3} ) (G ^L9 ) N ₃ s— / </ , ^N ο ₂ ν-λ=ζ/ where the group ΝΟ2 is optional (G ^L3 - ⁴ ) S— _02N d where the NO2 group is optional

where Ar represents a C _5-6 arylene group, for example phenylene.

In some embodiments, GL is selected from GL ^1-1 and GL ^1-2 . In some of such embodiments, GL is G ^Lb1 .

^Gll

G ^ll can be selected from:

- 35 045942

( _Gll ^1-1 ) ABOUT CBAg O ( ^GLL6 ) ABOUT CBAj ( ^{Gll1 2} ) o ΪΙ ^Ar g CBAg / N y ^X o ( ^GLL7 ) NVA^ ( ^GLL2 ) ABOUT O (G ^ll s-1) SVA A Ύ ⁿ c^ ₍ ^GLL 3-i) CBA< X H ( ^{gLL8 2} ) _N NEA ( ^{gLL3 2} ) (G ^LL9 -i) % NVA ( ^GLL4 ) CBA «___ 1 \ H 7/ ^N 0 X (G ^{LL9 2} ) < SVA ( ^GLL5 ) p ^CBA |__ 4

where Ar represents a C5-6 arylene group, for example phenylene.

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

X

X represents:

where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5.

a can be 0, 1, 2, 3, 4, or 5. In some embodiments, a is between 0 and 3. In some such embodiments, a is 0 or 1. In additional embodiments, a is 0.

b can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some implementations, b is between 0 and 12. B In some such embodiments, b is between 0 and 8, and may be 0, 2, 4, or 8.

- 36 045942 s can be 0 or 1.

d may be 0, 1, 2, 3, 4, or 5. In some embodiments, d is between 0 and 3. In some such embodiments, d is 1 or 2. In additional embodiments, d is 2.

In some implementations of group X, a is 0, c is 1, and d is 2, and b can be from 0 to 8. In some such implementations, b is 0, 4, or 8.

Q

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

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

In one embodiment, Q 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 contains naturally occurring amino acids. If the linker is a cathepsin-movable linker, then the dipeptide represents the site of action for cathepsin-mediated cleavage. In this case, the dipeptide represents the recognition site for cathepsin.

In one embodiment, Q is selected from:

^C0 -Phe-Lys- ^NH , ^-Vai-Ala-™, ^-Val-Lys-™ ^-Ala-Lys-™, ^-Val-Cit-™, ^co -Phe-Cit- ^NH , ^-Leu -Cit-™ ^-Ile-Cit-™ ^C0 -Phe-Arg- ^NH and ^-Trp-Cit-™; where Cit represents citrulline.

Preferably, Q is selected from:

^C0 -Phe-Lys- ^NH , ^-Vai-Ala-™, ^-Val-Lys-™ ^-Ala-Lys-™, ^-Val-Cit-™ Most preferably, Q is selected from ^CO -Phe-Lys- ^NH , CO-Val-Cit-NH and CO-Val-Ala-NH. Other promising dipeptide combinations include:

^C0 -Gly-Gly·™ ^co -Pro-Pro- ^NH and ^-Val-Glu-™

Other dipeptide combinations may be used, including those described in DubowCHik et al., Bioconjugate CHemistry, 2002, 13,855-869, incorporated herein by reference.

In some embodiments, Q ^X is a tripeptide residue. The amino acids in a tripeptide can be any combination of naturally occurring amino acids and unnatural amino acids. In some embodiments, the tripeptide contains naturally occurring amino acids. If the linker is a cathepsin-movable linker, then the tripeptide represents the site of action for cathepsin-mediated cleavage. In this case, the tripeptide is a recognition site

- 37 045942 research for cathepsin. Particularly promising tripeptide linkers are:

^co -Glu-Val-Ala- ^{NH co} -Glu-Yal-Cit·*® ^-aGlu-Val-Ala- ^{1411 co} -aGlu-Val-Cit- ^NH

In one embodiment, the amino acid side chain is chemically protected, if desired. The side chain protecting group may be a group described below. Protected amino acid sequences can be cleaved by enzymes. For example, a dipeptide sequence containing a Lys residue with a Boc-protected side chain is cleaved by cathepsin.

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

In some embodiments, RL has formula IIIb. In some embodiments, R ^LL has formula IIIb'.

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.

In some embodiments, both R ^L1 and RL ² are H.

In some embodiments, R ^L1 is H and RL ² is methyl.

In some embodiments, both R ^L1 and RL ² are 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 some embodiments, in group IIIb, e is 0. In other embodiments, e is 1 and the nitro group can be at any available position on the ring. In some of these embodiments, it is in the ortho position. In other such embodiments, it is in the para position.

In one specific embodiment, the second aspect of the present invention includes a compound of formula Id:

where Q is selected from:

(a) -CH2-;

(b) -C3H6-; And

^(With)

In one specific embodiment of the third aspect of the present invention, the drug linker (DL) has the formula (Id'):

- 38 045942

o o where Q is selected from:

(a) -CH2-;

(b) -C3H6-; And

(With) ·

In some embodiments of this invention, the C11 substituent may be in the following stereochemical position relative to adjacent groups:

In other embodiments, the C11 substituent may be in the following stereochemical position relative to adjacent groups:

Examples

general information

Manual flash chromatography was performed using Merck Kieselgel 60 F254 silica gel. Solvents for extraction and chromatography were purchased and used without further purification from Fisher Scientific, UK. All chemicals were purchased from Aldrich, Lancaster, or BDH. Automated flash chromatography was performed on a Biotage Isolera 1™ using a gradient elution starting with 88% hexane in EtOAc or 99.9% DCM in MeOH until all UV active components were completely eluted from the column (detection at 214 and 254 nm). When significant elution of UV active material was observed, the gradient was maintained manually. The purity of the fractions was checked by thin layer chromatography (TLC) using Merck Kieselgel 60 F254 silica gel with a fluorescent indicator on aluminum plates. TLC visualization was performed using UV light or iodine vapor unless otherwise stated. Solvents for extraction and chromatography were purchased and used without further purification from VWR, UK. All chemicals were purchased from Sigma-Aldrich or TCI Europe unless otherwise stated. PEGylated reagents were purchased from Quanta Biodesign, USA, through Stratech, UK.

The following are the LC/MS conditions:

Electrospray ionization mass spectrometry in positive mode was performed on a Waters Aquity H-class instrument. Solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid) were used as mobile phases.

LCMS for 3 min: the initial composition of 5% B was maintained for 0.25 min, then increased from 5% B to 100% B in 2 min. The specified composition was kept for 0.50 minutes. at 100% B, then returned to 5% B for 0.05 min. and held for 0.05 min. The total gradient passage time was 3 min. Flow rate 0.8 ml/min. Detection at 254 nm. Columns: Waters Acquity UPLC® VEN Shield RP18 1.7 μm 2.1x50 mm at 50°C, equipped with a Waters Acquity UPLC® VEN Shield RP18 VanGuard precolumn, 130A, 1.7 μm, 2.1 mmx5 mm.

- 39 045942

LCMS for 15 min: the initial composition of 5% B was maintained for 1 min, then increased from 5% B to 100% B in 9 min. This composition was kept for 2 minutes at 100% B, then returned to 5% B in 0.10 minutes and kept for 3 minutes. The total gradient passage time was 15 minutes. Flow rate 0.6 ml/min. Detection wavelength range: 190-800 nm. Oven temperature: 50°C. Column: ACE Excel 2 C18-AR, 2 µm, 3.0x100 mm.

Preparative HPLC:

Reversed-phase ultrafast high-performance liquid chromatography (UHPLC) was performed on a Shimazdzu Prominence® instrument using a Phenomenex® Gemini NX, 5 µm, C18 column (at 50 °C), dimensions: 150 x 21.2 mm. Solvent A (H2O with 0.1% formic acid) and solvent B (CH3CN with 0.1% formic acid) were used as eluents. All UHPLC experiments were performed using the following gradient conditions: an initial content of 13% B was increased to 60% B in 15 min, then increased to 100% B in 2 min. This composition was held for 1 min at 100% B, then returned to 13% B in 0.1 min and held for 1.9 min. The total duration of the gradient cycle was 20.0 minutes. The flow rate was 20.0 mL/min, and detection was performed at 254 and 280 nm.

Example 1

a) 1-((benzyloxy)carbonyl)azetidine-2-carboxylic acid (2)

DO)-Azetidine-2-carboxylic acid 1 (3 g, 29.674 mmol) and sodium bicarbonate (6.3 g, 75 mmol) were solubilized in H2O (25 ml, 1387.75 mmol) and Na(benzyloxycarbonyl)succinimide was added dropwise (8.5 g, 34 mmol) in THF (25 ml, 307 mmol, 100; wt%). After stirring at room temperature for 12 hours, the mixture was left to separate the two phases. The aqueous phase was washed with diethyl ether (50 ml), cooled in an ice bath and then acidified to pH 2 with concentrated HCl. The aqueous layer was extracted with ethyl acetate (2x50 ml) and the combined organics were dried

- 40 045942 tracts (MgSO ₄ ), and the excess solvent was evaporated in vacuo to obtain the crude product as a clear oily substance. The crude material was used without purification in the next step. LCMS for 3 min: ER ⁺ =1.34 min, m/z 258.2 [M + Na] ⁺

b) (1-benzyl-2-methyl-^)-azetidine-1,2-dicarboxylate (3)

In a dry round bottom flask, (2S)-1-benzyloxycarbonyl azetidin-2-carboxylic acid 2 (6.98 g, 29.7 mmol) was solubilized in MeOH (65 mL) and sulfuric acid (3 mL) was added. The mixture was heated to reflux and left to stir overnight. The mixture was allowed to cool to room temperature and quenched with Net ₃ (to pH 7), then stirred for 1 hour. The methanol was removed in vacuo. The residue was dissolved in EtOAc, washed with H2O and brine, then dried with MgSO ₄ and filtered. The organics were removed in vacuo to give crude product 3 (8.004 g, 32.11 mmol) as a clear oil. LCMS for 3 min: ER ⁺ =1.53 min, m/z without ionization.

c) Benzyl-^)-2-(hydroxymethyl)azetidine-1-carboxylate (4)

O1-benzyl-O2-methyl-(2S)-azetidine-1,2-dicarboxylate 3 (7.6 g, 30 mmol) was solubilized in THF (75 ml, 922 mmol), cooled to 0°C and added LiBH ₄ ( 1 g, 45 mmol). The mixture was left to warm to room temperature and stirred for another hour, after which the reaction was complete. The reaction mixture was cooled to 0°C, then quenched with H2O and 1 M HCl. Volatiles were removed in vacuo. The residue was dissolved in EtOAc and washed with brine (2x50 ml), dried with MgSO ₄ , filtered and the solvent was removed on a rotary evaporator under reduced pressure. Purification by silica gel column chromatography (Hex/EtOAc, 100% to 1:2) provided product 4 as a clear oil (4.076 g, 60% yield over 3 steps). LCMS for 3 min: ER ⁺ =1.36 min, m/z 222.3 [M+H] ⁺ .

d) Benzyl-^)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carboxylate (5)

Benzyl-(2S)-2-(hydroxymethyl)azetidine-1-carboxylate 4 (4.0766 g, 18.425 mmol) was solubilized in dry CH ₂ Cl ₂ (20 ml, 312.0 mmol) and cooled the mixture to 0°C, then imidazole (2.508 g, 36.84 mmol) and TBS-Cl (4.16 g, 27.6 mmol) were added. The mixture was left to warm to room temperature and left to stir. LCMS showed that the reaction was complete in 5 min. Organics were washed with saturated NH ₄ Cl solution, water, brine, dried with MgSO ₄ , filtered and devolatilized in vacuo. Purification by silica gel column chromatography (Hex/EtOAc, 100% to 9:1) gave product 5 (6.90 g, not completely dry, quantified). LCMS for 3 min: ER ⁺ =2.15 min, m/z 336.9 [M+H] ⁺ .

e) ^)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine (6)

Palladium on carbon (10%) (100 mg, 0.93 mmol) was treated dropwise with EtOAc (5 ml) and the resulting slurry was added to a suspension of compound 5 (6.9027 g, 20.57 mmol) in EtOH (100 ml) at room temperature in a Parr hydrogenation reactor. The reaction mixture was treated with H2 gas at 20 psi (138 kPa), then the reactor was emptied under vacuum (repeated 3 times). The reactor was then brought to 38 psi (262 kPa) H ₂ and shaken for 1 hour. During this time, the pressure dropped to ~30 psi (207 kPa) and the reactor was again adjusted to 40 psi .inch (276 kPa) and shaken for another hour. No further decrease in pressure was observed and the reaction was considered complete. Completion of the reaction was confirmed using LC-MS. The mixture was filtered through celite and the filtrate was evaporated in vacuo to give crude product 6 as a brown oil (3.761 g, 90% yield). LCMS for 3 min: ER ⁺ =1.70 min, m/z without ionization.

f) (^)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidin-1 -yl)(4-(6-(4-((2R)-2-(((tert-butyldimethylsilyl)oxy )methyl)cyclobutan-1-carbonyl)-2-methoxy-5-nitrophenoxy)hexyl)-5-methoxy-2-nitrophenyl)methanone (8)

DCC (3.8 g, 18 mmol) was added to a solution of compound 7 (3.9 g, 7.9 mmol) and HOBt (2.3 g, 17 mmol) in CH ₂ Cl ₂ (200 ml) at 0°C . The cold bath was removed and the reaction was allowed to continue for 30 min at room temperature, after which a solution of compound 6 (3.65 g, 18 mmol) and triethylamine (3.2 ml, 23 mmol) in CH ₂ Cl ₂ (200 ml) was quickly added ) at -10°C in an argon atmosphere. The reaction mixture was left stirring at room temperature and monitored by LC/MS. After 2 minutes the reaction was complete. The solid was removed by filtration through celite and the organic phase was washed with cold 0.1 M aqueous HCl to a measured pH of 2. The organic phase was then washed with water, then saturated aqueous sodium bicarbonate, brine, dried with MgSO ₄ , filtered and evaporated at reduced pressure. Purification by column chromatography on silica gel (Nχ/ΕΏΑ^ΟΗ ₂ Π ₂ , from 100% to 1:2:1) gave product 8 (5.9 g, 87% yield). The product contained a small amount of an impurity, a mono-bound product (the impurity was not separated by chromatography). LCMS for 3 min: ER ⁺ =2.35 min, m/z 862.2 [M+H] ⁺ .

g) ((Pentan-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis((^)-2-(((tert-butyldimethylsilyl)oxy) methyl)azetidin-1-yl)methanone) (9)

Zinc (4.65 g, 71.1 mmol) was slowly added to a solution of compound 8 (2.45 g, 2.85 mmol) in MeOH/NiO/formic acid 90:5:5 (66 ml). The exotherm was controlled using an ice bath to maintain the temperature of the reaction mixture below 40°C. Solids removed upon completion

- 41 045942 by filtration through celite and washed the organic phase with water and brine, then dried with MgSO ₄ , filtered and removed volatiles under reduced pressure. Crude 9 (2.28 g, quantitative) was used as such in the next step. LCMS for 3 min: ER ⁺ =2.32 min, m/z 802.3 [M+H] ⁺ .

h) Diallyl-((pentane-1,5-diylbis(oxy))bis(6-(^)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidin-1-carbonyl)-4-methoxy- 3,1-phenylene))dicarbamate (10)

Compound 9 (2.23 g, 2.78 mmol) was solubilized in CH ₂ Cl ₂ (50 ml) under argon. The mixture was cooled to -78°C, then pyridine (0.99 ml, 12.3 mmol) and allyl chloroformate (0.738 ml, 2.49 mmol) were added. The reaction mixture was left stirring at -78°C for 10 minutes, then the mixture was allowed to warm to room temperature. After 15 minutes the reaction was complete. The organics were washed with saturated CuSO ₄ , H ₂ O, brine, then dried with MgSO ₄ , filtered and devolatilized under reduced pressure. Crude product 10 (1.47 g, 1.52 mmol, quantitative) was used as such in the next step. LCMS for 3 min: ER ⁺ =2.53 min, m/z 970.3 [M+H] ⁺ .

i) Diallyl-((pentane-1,5-diylbis(oxy))bis(6-(^)-2-(hydroxymethyl)azetidine-1-carbonyl)-4-methoxy-3,1-phenylene))dicarbamate (eleven)

Compound 10 (1.47 g, 1.52 mmol) was solubilized in 3:1:1 Η^/ΤΓΦ/υι^όκηι acid (16 ml) and the reaction mixture was left to stir over the weekend. The mixture was extracted with CH ₂ Cl ₂ and washed with saturated NaHCO ₃ solution, H ₂ O and brine, then dried with MgSO ₄ , filtered and devolatilized under reduced pressure. Purification by silica gel column chromatography (Hex/EtOAc, 100% to 1:1) provided product 11 (859 mg, 76.5% yield) as a clear oil. LCMS for 3 min: ER ⁺ =1.75 min, m/z 742.0 [M+H] ⁺ .

j) Diallyl-7,7'-(pentane-1,5-diylbis(oxy))(10aS,10a'S)-bis(10-hydroxy-6-methoxy-4-oxo-1,2,10, 10a-tetrahydroazeto [ 1,2-a]benzo [e] [ 1,4]diazepine-9(4H)-carboxylate) (12)

Compound 11 (850 mg, 1.14 mmol) was solubilized in CH ₂ Cl ₂ (60 ml). 1-Hydroxy-2,2,6,6-tetramethylpiperidine was added sequentially; 1-methylimidazole; 2-(2-pyridyl)pyridine (0.7 ml, 1140 mmol, 0.2 mol/L) and copper(I) tetrakisacetonitrile triflate (55 mg, 0.145 mmol) and stirred the mixture at 35°C under pressure from 2 cylinders with air. The reaction mixture was left stirring overnight, then evacuated to dryness on a rotary evaporator. Purification by column chromatography on silica gel (OTS3/MeCH, 100% to 95:5) gave product 12 (346 g, 0.47 mmol, 41% yield). LCMS for 3 min: ER ⁺ =1.48 min, m/z 737.9 [M+H] ⁺ .

k) (10aS,10a'S)-7,7'-(pentane-1,5-diylbis(oxy))bis(6-methoxy-1,10a-dihydroazeto[1,2-a]benzo[e][1, 4] diazepine CH^SH-on) (Ex1)

Compound 12 (335 mg, 0.45 mmol) was solubilized in CH ₂ Cl ₂ (20 ml) in a flask under argon. Pyrrolidine (650 μl, 7.8 mmol) and Pd(PPh ₃ ) ₄ (50 mg, 0.004 mmol) were added sequentially and the mixture was left to stir at room temperature until the reaction was complete. The organics were washed with saturated NH ₄ Cl, H ₂ O and brine, then dried with MgSO ₄ , filtered and devolatilized under reduced pressure. Then purified by chromatography on an Isolera system (CH ₂ Cl ₂ /(CH ₂ Cl ₂ +10%MeOH), from 92:7 to 10:90. Two fractions containing the product were isolated, but they were not pure enough. The fractions were combined and re-purified manual chromatography, and isolated the pure product Ex1 (146 mg, 0.27 mmol, 24% yield): LCMS for 3 min: ER ⁺ = 1.32 min, m/z 533.8 [M+H] ⁺ LCMS for 15 min: ER ⁺ =4.83 min, m/z 533.9 [M+H] ⁺ .

- 42 045942

Example 2

a) ('(b)-('2-('('(tert-butyldimethylsilyl)oxy)methyl)azetidin-1-yl)('5-methoxy-2-nitro-4-('(triisopropylsilyl)oxy )phenyl)methanone (13)

DCC (4.021 g, 19.49 mmol) was added to a solution of 5-methoxy-2-nitro-4-triisopropyl lyloxybenzoic acid 13 (6 g, 16.24 mmol) and HOPO (1.984 g, 17.86 mmol) in CH ₂ Cl ₂ (100 ml) at 0°C. The cold bath was removed and the reaction was allowed to continue for 30 min at room temperature, after which a solution of [(2S)-azetidin-2-yl]methoxy-Ίret-butyldimethylsilane 6 (3.761 g, 18.68 mmol) and triethylamine (3 .39 ml, 33.5 mmol) in CH ₂ Cl ₂ (100 ml) at -10 ° C in an argon atmosphere. The reaction mixture was left stirring at room temperature and monitored by LC/MS. After 2 minutes the reaction was complete. The solid was removed by filtration through celite and the organic phase was washed with cold 0.1 M aqueous HCl to a measured pH of 2. The organic phase was then washed with water, then saturated aqueous sodium bicarbonate, brine, dried with MgSO ₄ , filtered and evaporated at reduced pressure.

Purification by silica gel column chromatography (Hex/EtOAc, 100% to 1:1) gave product 14 (8.6737 g, 96.63% yield). LCMS for 3 min: ER ⁺ =2.44 min, m/z 554.2 [M+H] ⁺ .

b) ^)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)azetidin-1-yl)methanone (15)

Zinc (10 g, 152.9 mmol) was slowly added to a solution of compound 14 (8.6737 g, 15.69 mmol) in MeOH/I ₂ O/formic acid 90:5:5 (200 ml). The exotherm was controlled by using an ice bath to maintain the temperature of the reaction mixture below 40°C. Once complete, remove solids by filtration through celite and wash the organic phase with water and brine, then dry with MgSO ₄ , filter and remove volatiles under reduced pressure. Crude 15 (7.6343 g, 14.6 mmol, 93.05% yield) was used as such in the next step. LCMS for 3 min: ER ⁺ =2.42 min, m/z 524.4 [M+H] ⁺ .

c) allyl-^)-(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-4-methoxy-5-((tri

- 43 045942 isopropylsilyl)oxy)phenyl)carbamate (16)

Compound 15 (7.6343 g, 14.60 mmol) was solubilized in CH2C12 (100 ml) under argon. The mixture was cooled to -78°C, then pyridine (2.6 ml, 32 mmol) and allyl chloroformate (1.7 ml, 16 mmol) were added. The reaction mixture was left stirring at -78°C for 10 minutes, after which it was allowed to warm to room temperature. After 15 minutes the reaction was completed. The organics were washed with saturated CuSO ₄ , H ₂ O, brine, then dried with MgSO ₄ , filtered and devolatilized under reduced pressure.

Crude product 16 (8.9129 g, 14.69 mmol, quantitative) was used as such in the next step. LCMS for 3 min: ER ⁺ =2.53 min, m/z 608.2 [M+H] ⁺ .

d) Allyl-^)-(2-(2-(hydroxymethyl)azetidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (17)

Compound 16 (8.9129 g, 14.69 mmol) was solubilized in 3:1:1 H ₂ O/THF/acetic acid (80 ml) and the reaction mixture was allowed to stir over the weekend. The mixture was extracted with CH ₂ Cl ₂ and washed with saturated NaHCO ₃ solution, H ₂ O and brine, then dried with MgSO ₄ , filtered and devolatilized under reduced pressure. Purification by silica gel column chromatography (Hex/EtOAc, 100% to 1:1) provided product 17 (5.5572 g, 76.80% yield) as a clear oil. LCMS for 3 min: ER ⁺ =1.97 min, m/z 494.0 [M+H] ⁺ .

e) Allyl-(10aS)-10-hydroxy-6-methoxy-4-oxo-7-((mriisopropylsilyl)oxy)-1,2,10,10a-tetrahydro azeto[1,2-a]benzo[e] [1,4]diazepine-9(4H)-carboxylate (18).

Compound 17 (5.5572 g, 11.28 mmol) was solubilized in CH ₂ Cl ₂ (40 ml). 1-Hydroxy-2,2,6,6-tetramethylpiperidine was added sequentially; 1-methylimidazole; 2-(2-pyridyl)pyridine (6 ml, 1 mmol) and copper(I) tetrakisacetonitrile triflate (425 mg, 1.1279 mmol) and stirred the mixture at 35°C under air pressure from 2 cylinders. The reaction mixture was left stirring overnight, then evacuated to dryness on a rotary evaporator. Purification by column chromatography on silica gel (CHCl3/MeOH, 100% to 97:3) gave product 18 (5.3835 g, 10.97 mmol, 97.27% yield) as a light orange foam. LCMS for 3 min: ER ⁺ =2.00 min, m/z 491.8 [M+H] ⁺ .

f) Allyl-(10αS)-10-((tert-butyldimethylsilyl)oxy)-6-methoxy-4-oxo-7-((triisonropylsilyl)oxy)-1,2,10,10a-tetrahydroazeto[1,2- a]benzo[e][1,4]diazepine-9(4H)-carboxylate (19)

Compound 18 (5.3835 g, 10.97 mmol) was solubilized in CH ₂ Cl ₂ (50 ml) and the mixture was cooled to 78°C. 2,6-lutidine (2.55 mL, 21.9 mmol) and TBS-OTf (3.78 mL, 16.4 mmol) were then added. The mixture was left for 10 minutes, then the cooling bath was removed and the mixture was allowed to warm to room temperature. Organics were washed with H2O and brine, then dried with MgSO4, filtered and devolatilized under reduced pressure. Purification by column chromatography on silica gel (CHC1 ₃ /MeOH, 100% to 95:5) gave product 19 (6.8532 g, quantitative). LCMS for 3 min: ER ⁺ =2.47 min, m/z 606.0 [M+H] ⁺ .

g) Allyl-(10aS)-10-((tert-butyldimethylsilyl)oxy)-7-hydroxy-6-methoxy-4-oxo-1,2,10,10a-tetrahydrazeto[1,2-a]benzo[ e][1,4]diazepine-9(4H)-carboxylate (20) Compound 19 (6.8 g, 14 mmol) was solubilized in DMF (10 ml). LiOAc-2H2O (1.4 g, 14 mmol) and H2O (3 ml or as much as possible) were added. When the solution became clear again, a few drops of water were added. The described process was repeated until the reaction was completed. The organics were diluted with CHCl ₃ and washed with citric acid (pH 3), H2O and brine, then dried with MgSO ₄ , filtered and devolatilized under reduced pressure. Purification by silica gel column chromatography ( _CHC13 /MeOH, 100% to 95:5) provided product 20 (5.2885 g, 11.79 mmol, 85% yield) as a yellow oil. LCMS for 3 min: ER ⁺ =1.86 min, m/z 449.8 [M+H] ⁺ .

h) Diallyl-7,7'-(propane-1,3-diylbis(oxy))(10αS,10a'S)-bis(10-((tert-butyldimethylsilyl)oxy)-6methoxy-4-oxo-1,2, 10,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepine-9(4H)-carboxylate) (21)

1,3-Dibromopropane (204.9 mg, 1.015 mmol) and compound 20 (1 g, 2.030 mmol) were solubilized in CH2C12 (50 ml) under argon. K2CO3 (280 mg, 2.026 mmol) and TBAI (149 mg, 0.2 mmol) were added sequentially and the mixture was left to stir at 40°C until the reaction was complete. The mixture was left to stir overnight, but the reaction was not complete and an impurity was formed. Organics were washed with H2O and brine, then dried with _MgSO4 , filtered and devolatilized under reduced pressure. After purification by column chromatography on silica gel (CHC1 ₃ /MeOH, from 100% to 97:3), product 21 (482 mg, 0.471 mmol, 46.50% yield) was obtained, contaminated with an integral impurity (R _t = 9.95 min at LCMS in 15 minutes). LCMS for 15 min: ER ⁺ =9.86 min, m/z 938.3 [M+H] ⁺ .

i) (10αS,10α'S)-7,7'-(propane-1,3-diylbis(oxy))bis(6-methoxy-1,10a-dihydroαzeto[1,2-a]benzo[e][1, 4] diazepin-4(2H)-one) (Ex2A)

Compound 21 (482 mg, 0.5143 mmol) was solubilized in CH2C12 (20 ml) in a flask under argon. Pyrrolidine (786 μl, 9.44 mmol) and Pd(PPh ₃ ) ₄ (54 mg, 0.046 mmol) were added successively and the mixture was left to stir at room temperature until complete. The organics were washed with saturated NH ₄ Cl, H ₂ O and brine, then dried with MgSO ₄ , filtered and devolatilized under reduced pressure. Then purified by chromatography on an Isolera system (CH ₂ C1 ₂ /(CH ₂ C1 ₂ +10%MeOH), from 98:2 to 30:70. Two fractions containing the product, but with insufficient purity, were isolated. The fractions were combined and purified again by chromatography on the Isolera system (same solvent system) and isolated the pure product Ex2A (35.1 mg, 0.135 mmol, 13.5% yield): LCMS for 3 min: ER ⁺ = 1.23 min, m/z 505.8 [ M+H]+.

j) Diallyl-7,7'-((1,3-phenylenebis(methylene))bis(oxy))(10aS,10a'S)-bis(10-((tert-butyldimethylsilyl)oxy)-6-methoxy-4 -oxo-1,2,10,10a-tetrahydroazeto [1,2-a]benzo[e] [1,4]diazepine-9(4H)-carboxylate) (22)

1,3-Bis(bromomethyl)benzene (267.9 mg, 1.011 mmol) and compound 20 (1 g, 2.030 mmol) were solubilized in DMF (5 ml) under argon. K ₂ CO ₃ (280 mg, 2.026 mmol) and TBAI (749 mg, 2.027 mmol) were added sequentially and the mixture was left to stir at 40° C. until complete. The mixture was left to stir overnight, but the reaction was not complete and an impurity was formed. The mixture was diluted with CH2C12 and washed with H2O and brine, then dried with MgSO ₄ , filtered and devolatilized under reduced pressure. Purification by silica gel column chromatography ( _CHC13 /MeOH, 100% to 97:3) gave product 22 (467 mg, 0.43 mmol, 42.47% yield) + 398 mg mixed fractions. LCMS for 3 min: ER ⁺ =2.30 min, m/z 1000.5 [M+H]+.

k) (10aS,10a'S)-7,7'-((1,3-phenylenebis(methylene))bis(oxy))bis(6-methoxy-1,10a-dihydroazeto[1,2-a] benzo[e ][1,4]diazepin-4(2H)-one) (Ex2B)

Compound 22 (455 mg, 0.419 mmol) was solubilized in CH2C12 (20 ml) in a flask under argon. Pyrrolidine (600 μl, 7.2 mmol) and Pd(PPh ₃ ) ₄ (48 mg, 0.041 mmol) were added sequentially and the mixture was left to stir at room temperature until complete. The organics were washed with saturated NH ₄ Cl, H ₂ O and brine, then dried with MgSO ₄ , filtered and devolatilized under reduced pressure. Purified by chromatography on an Isolera system (CH ₂ C1 ₂ /(CH ₂ C1 ₂ +10%MeOH), from 98:2 to 30:70. Two fractions were isolated containing the product, but with insufficient purity. The fractions were combined and purified again by chromatography on Isolera system (same solvent system) and isolated the pure product Ex2B (214.5 mg, 0.378 mmol, 90.5% yield) as a white solid. LCMS for 3 min: ER ⁺ = 1.38 min, m/z 567.8 [M+H]+.

- 45 045942

Example 3

a) Allyl-(5-((5-(5-amino-4-((^)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-2methoxyphenoxy)pentyl)oxy)- 2-((^)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-4methoxyphenyl)carbamate (23)

Compound 9 (1.192 g, 1.488 mmol) was solubilized in CH2C12 (250 ml) under argon. The mixture was cooled to -78°C, then pyridine (0.241 ml, 2.98 mmol) and allyl chloroformate (0.158 ml, 1.484 mmol) were added. The reaction mixture was left stirring at -78°C for 10 minutes, then allowed to warm to room temperature. After 15 minutes the reaction was complete. The organics were washed with saturated CuSO ₄ , H ₂ O, brine, then dried with MgSO ₄ , filtered and devolatilized under reduced pressure. Purification by column chromatography on silica gel (CHC^/MeOH) gave a mixture of mono- and bis-Alloc, which was further purified on a second column (Hex/EtOAc) to obtain pure product 23 (499.2 g, 37.9% yield of 50 % possible). LCMS for 3 min: ER ⁺ =2.41 min, m/z 886.6 [M+H] ⁺ .

b) Allyl-(5-((5-(5-((((4-(^)-2-(^)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanes to)benzyl )oxy)carbonyl)amino)-4-(^)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)2-methoxyphenoxy)pentyl)oxy)-2-(^)-2- (((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)4-methoxyphenyl)carbamate (24)

Triphosphene (68.8 mg, 0.232 mmol) was added in one portion to a mixture of compound 23 (620 mg, 0.7 mmol) and TEA (203 μl, 1.46 mmol) in CH ₂ Cl ₂ (50 ml) at 0°C . The ice bath was removed and after 15 min Alloc-Val-Ala-PAB-OH (275 mg, 0.728 mmol) was added in one portion as a fine powder, followed by additional TEA (73 μL, 0.524 mmol) and dibutyltin dilaurate.

- 46 045942 (39.6 µl, 0.07 mmol). The reaction mixture was left stirring at 37°C for 4 hours, then stirred at room temperature overnight. Organics were washed with H2O, saturated NH ₄ Cl solution and brine, then dried with MgSO ₄ , filtered and devolatilized under reduced pressure. Purification by silica gel column chromatography (CHC^/MeOH) provided pure product 24 (414 g, 45.9% yield). LCMS for 3 min: ER ⁺ =2.43 min, m/z 1289.5 [M+H] ⁺ .

c) Allyl-(5-((5-(5-((((4-(^)-2-(^)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanes to)benzyl )oxy)carbonyl)amino)-4-(^)-2-(hydroxymethyl)azetidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-(^)-2-(hydroxymethyl)azetidine-1 -carbonyl)-4-methoxyphenyl)carbamate (25)

Compound 24 (414 mg, 0.32 mmol) was solubilized in a 3:1:1 mixture of ECE/THF/acetic acid (10 ml) and the reaction mixture was allowed to stir over the weekend. The mixture was extracted with CH ₂ Cl ₂ and washed with saturated NaHCO ₃ solution, H ₂ O and brine, then dried with MgSO ₄ , filtered and devolatilized under reduced pressure. Purification by silica gel column chromatography (CHCls/MeOH, 100% to 94:6) gave product 25 (326 mg, 95.7% yield). LCMS for 3 min: ER ⁺ =1.80 min, m/z 1060.1 [M+H] ⁺ .

d) Allyl-(10aS)-7-((5-(((10S,10aS)-9-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl) amino)-3-methylbutane amido)propanamido)benzyl)oxy)carbonyl)-10-hydroxy-6-methoxy-4-oxo-1,2,4,9,10,10a-hexahydro azeto[1,2-a] benzo[e][1,4]diazepin-7-yl)oxy)pentyl)oxy)-10-hydroxy-6-methoxy-4-oxo-1,2,10,10atetrahydroazeto[1,2-a]benzo[ e][1,4]diazepine-9(4H)-carboxylate (26)

Compound 25 (202.4 mg, 0.3 mmol) was solubilized in CH ₂ Cl ₂ (20 ml). 1-Hydroxy-2,2,6,6-tetramethylpiperidine was added sequentially; 1-methylimidazole; 2-(2-pyridyl)pyridine (0.4 ml, 0.03 mmol) and copper(I) tetrakisacetonitrile triflate (11 mg, 0.03 mmol) and stirred the mixture at 35°C under air pressure from 2 cylinders. The reaction mixture was left stirring overnight, then evaporated to dryness on a rotary evaporator. Purification by column chromatography on silica gel (CHC^/MeOH, 100% to 97:3) gave product 26 (313 mg, 0.19 mmol, 64.5% yield). LCMS for 3 min: ER ⁺ =1.59 min, m/z 1057.1 [M+H] ⁺ .

e) 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl-(10S,10aS)-10-hydroxy-6-methoxy-7-((5-( (^)-6-methoxy-4-oxo-1,2,4,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepin-7-yl)oxy)pentyl)oxy)- 4-oxo-1,2,10,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepine-9(4H)-carboxylate) (27)

Compound 26 (195 mg, 0.184 mmol) was solubilized in CH ₂ Cl ₂ (10 ml) in a flask under argon. Pyrrolidine (262 μl, 3.15 mmol) and Pd(PPh ₃ ) ₄ (21 mg, 0.018 mmol) were added sequentially and the mixture was left to stir at room temperature until complete. The organics were washed with saturated NH4Cl, H2O and brine, then dried with MgSO4, filtered and devolatilized under reduced pressure. Purification by chromatography on an Isolera system (CH ₂ Cl ₂ /(CH ₂ Cl ₂ +10%MeOH), 98:2 to 30:70, gave product 27 (141 mg, 0.16 mmol, 87.7% yield) LCMS for 3 min: ER ⁺ =1.23 min, m/z 870.9 [M+H] ⁺ .

f) 4-((2S,5S)-37-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo -10, 13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl-(10S,10aS)-10-hydroxy-6-methoxy-7-((5- ((^)-6-methoxy-4-oxo-1,2,4,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepin-7-yl)oxy)pentyl)oxy) -4-oxo-1,2,10,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepine-9(4H)-carboxylate (Ex3)

The reaction was carried out in a glove box. Compound 27 (70 mg, 0.080 mmol) was solubilized in CH ₂ Cl ₂ (10 ml) in a flask under argon at room temperature. Mal-dPEG8-OH (50 mg, 0.084 mmol) and EDCI-HCl (15.4 mg, 0.080 mmol) were added and the mixture was stirred until complete. Organics were washed with H ₂ O and brine, then dried with MgSO ₄ , filtered and devolatilized under reduced pressure. After purification by chromatography on an Isolera system (CH ₂ Cl ₂ /(CH ₂ Cl ₂ +10%MeOH), from 98:2 to 30:70, a product with impurities was obtained. After additional purification on an Isolera reverse-phase system, a pure compound Ex3 was obtained (4 mg, 0.027 mmol, yield 3.4%) and several fractions with impurities (22 mg).LCMS for 3 min: ER ⁺ =1.51 min, m/z 1445.6 [M+H] ⁺ .

Example 4

ConjA (Her2-Ex3) mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate buffered saline at pH 7.4 (PBS) was added (50 molar equivalents/antibody, 7.6 micromolar, 762.7 µl) to a 20.8 ml solution of tratuzumab (22.9 mg, 153 nanomoles) in a reducing buffer containing 30 mM histidine/histidine-HCl, 30 mM arginine, pH 6.8, and 1 mM ethylenediaminetetraacetic acid (EDTA), with a final antibody concentration 1.1 mg/ml. The reducing mixture was allowed to react at 37°C for 2 hours (or until complete reduction according to UHPLC data) on an orbital shaker with gentle (60 rpm) shaking. In the reduced antibody solution, the buffer was changed (to completely remove excess reducing agent) by centrifugation on a centrifugal filter with a conjugation buffer containing 30 mM histidine/histidine-HCl mixture, 30 mM arginine and 1 mM EDTA, to a final antibody concentration of 1.1 mg/ml. Ex3 was added as a solution in DMSO (12.5 mo

- 47 045942 lar equivalents/antibody, 1.9 micromoles, in 2.1 ml DMSO) to 18.6 ml of the resulting reduced antibody solution (20.5 mg, 136 nanomoles) to a final DMSO concentration of 10% (v/v). ). The solution was stirred for 17 hours at room temperature, then conjugation was quenched by the addition of N-acetylcysteine (8.5 micromolar, 68 μl at 100 mM), then purified by centrifugation on a centrifugal filter using a 30 kDa Amicon Ultracell centrifugal filter with a volume of 15 ml, subjected to sterilization filtration and analyzed.

UHPLC analysis on a Shimadzu Prominence system, which was performed using a Thermo Scientific MAbPac 50 mm x 2.1 mm column, eluting with a gradient of water and acetonitrile on the reduced ConjA sample at 214 nm and 330 nm (specific for compound SG3931), showed a mixture of unconjugated light chains, light chains attached to one SG3931 molecule, unconjugated heavy chains and heavy chains attached to up to three SG3931 molecules, consistent with a drug-to-antibody ratio (DAR) of 7.32 SG3931 molecules per antibody.

UHPLC analysis on a Shimadzu Prominence system, which was carried out using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 µm 4.6 x 150 mm column (with a 4 µm 3.0 x 20 mm guard column), eluting at a rate of 0.3 ml/min, filtered through a sterilizing filter SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on the ConjA sample at 280 nm showed 94.2% monomer purity. SEC UHPLC analysis showed a final ConjA concentration of 1.29 mg/mL in 5.8 mL, resulting in a mass of 7.5 mg of ConjA (37% yield).

Example 5 Cytotoxicity Assay

The efficacy of the resulting molecules was measured in an in vitro cytotoxicity assay in the NCI-N87 carcinoma cell line.

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

Adherent NCI-N87 cells were washed with D-PBS and separated with trypsin-EDTA, then cell density and viability were measured in duplicate using trypan blue exclusion assay and an automatic cell counter (LUNA-II™). The cell suspension was diluted to 1x10 ⁵ cells/ml in growth medium (RPMI 1640 with Glutamax + 10% (v/v) HyClone™ fetal bovine serum) and vortexed, then dispensed 2 ml per well into sterile polypropylene plates with a volume of 3 ml. Then diluted solutions of the active agent in an amount of 10 μl/well were introduced into the corresponding wells and mixed several times in a pipette. In control wells, 10 μl of DMSO was applied to 2 ml of cell suspension and mixed thoroughly. A 100 µL aliquot of each sample was then taken into 2 replicate wells of a sterile flat 96-well microplate and incubated in a 37°C incubator with CO2 gas (5%). At the end of the incubation period (7 days), cell viability was measured using the CellTiter 96 Aqueous One (MTS) assay by dispensing 20 μl per well and incubating for 4 hours at 37°C, 5% CO ₂ . The plates were then read on an EnVision multichannel plate reader (Perkin Elmer) using absorbance at 490 nm.

The percentage of cell survival was calculated from the average absorbance of 2 replicate wells for each sample, compared with the average absorbance of two control wells treated with DMSO alone (100%). The IC ₅₀ value was determined by fitting each data set to sigmoidal variable dose response curves using the nonlinear curve smoothing algorithm in GraphPad Prism software (San Diego, CA).

All experiments in this report were performed and tested in three independent replicates. Data are recorded as the mean of three independent replicates.

1C ₅₀ (nM) Ex2A 70.11 Ex1 2,202 Ex2B 4.035

Example 6 ADC Cytotoxicity Assay

Cell concentration and viability from subconfluent (80-90% confluent) cell culture in a T75 flask were measured by trypan blue staining and counted using a LUNA-II™ automatic cell counter. Cells were diluted to 2x10 ⁵ /ml, distributed (50 µl per well) into 96-well flat-bottomed plates.

A stock solution (1 mL) of experimental antibody-drug conjugate (ADC) (20 μg/mL) was prepared by diluting filter-sterilized ADC in cell culture medium. A series of 8x 10-fold dilutions of the original ADC were carried out in a 24-well plate by serially transferring 100 µl into 900 µl cell culture medium. Diluted ADC was dispensed (50 μl per well) in 4 replicates into wells of a 96-well plate containing 50 μl of cell suspension, high

- 48 045942 announced earlier. Control wells were filled with 50 μl of cell culture medium. A 96-well plate containing cells and ADCs was incubated at 37°C in a CO2-saturated incubator for exposure time.

At the end of the incubation period, cell viability was measured using the MTS assay. MTS (Promega) was placed (20 μl per well) in each well and incubated for 4 h at 37°C in a CO2 incubator. Absorbance in the wells was measured at 490 nm. Percent cell survival was calculated from the average absorbance in 4 ADC-treated wells compared to the average absorbance in 4 control, untreated wells (100%). IC ₅₀ was determined from dose response data using GraphPad Prism using a non-linear curve smoothing algorithm: variable slope sigmoidal dose response curve.

ADC incubation times were 4 days for MDA-MB-468 and 7 days for NCI-N87. MDA-MB-468 and NCI-N87 were grown in RPMI 1640 with Glutamax + 10% (v/v) HyClone™ fetal bovine serum.

EC ₅₀ values were determined by fitting the data to a variable slope sigmoidal dose response curve using GraphPad Prism v6.05 software (GraphPad, San Diego, CA). ________________________________

EC50 (µg/ml) NCI-N87 MDA-MB-468 ConjA 0.002285 15.71

Example 7 Xenograft Test

NCI-N87 xenograft mice

Female mice with severe combined immunodeficiency (Fox CHase SCID®, C.B17/Icr-Prkdcscid, Charles River) were eight weeks old, with a body weight (BW) range of 16.5 to 21.6 g on day 1 of the study. Animals were provided with water ad libitum (reverse osmosis, 1 ppm Cl) and modified NIH 31 chow, as well as irradiated Lab Diet® chow consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% unrefined fiber. Mice were housed on Enricho'cobs™ laboratory animal bedding in static microisolators on a 12-hour light cycle at 20-22°C (68-72°F) and 40-60% humidity. CR Discovery Services specifically adheres to the recommendations of the Guide for the Care and Use of Laboratory Animals with respect to restraint, housing, surgical procedures, and food and fluid management, as well as veterinary care. CR Discovery Services' animal care and use program is accredited by the International Association for the Accreditation and Accreditation of Laboratory Animal Care (AAALAC), ensuring compliance with accepted standards for the care and use of laboratory animals.

Tumor cell culture

NCI-N87 human gastric carcinoma-lymphoma cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml sodium penicillin G, 100 μg/ml streptomycin sulfate, and 25 μg/ml gentamicin. Cells were grown in tissue culture flasks in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air.

In Vivo implantation and tumor growth

NCI-N87 cells used for implantation were harvested at logarithmic growth phase and resuspended in phosphate-buffered saline (PBS) containing 50% Matrigel™ (BD Biosciences). On the day of tumor implantation, each experimental mouse was given a subcutaneous injection of 1 x 107 cells (0.1 ml of cell suspension) into the right flank and tumor growth was monitored at an average size approaching the required range of 100 to 150 mm ³ . Fourteen days later, on the day designated as study day 1, the mice were divided into groups according to the calculated tumor size, and each group consisted of ten animals with a tumor size of each of them ranging from 108 to 144 mm ³ , and an average tumor volume of group was 115 mm ³ .

Tumors were measured in two directions using a caliper and the volume was calculated using the formula:

Tumor volume (mm ³ ) = ^w * ^? where w=width and ^length of tumor in mm. Tumor mass can be estimated by assuming that 1 mg is equivalent to a tumor volume of 1 mm ³ .

Treatment

Treatment began on day 1 in groups of 10 mice (n=10) with developed subcutaneous NCI-N87 tumors (108-144 mm ³ ). ConjA (4 mg/kg) was administered intravenously as a single dose on day 1 (qdx1). The vehicle-treated group was used as a control group for efficacy analysis. Tumors were measured twice a week until the end of the study on day 79. Each mouse was euthanized

- 49 045942 reaching the final tumor volume of 800 mm ³ or until the last day, whichever comes first. Time to end point (TTE) was calculated for each mouse.

The results are presented in Fig. 1, which shows the change in normalized tumor growth (control; ♦ - ConjA).

Endpoint and Tumor Growth Delay (TGD) Analysis

Tumors were measured with calipers twice a week, and each animal was euthanized when its tumor volume reached a final value of 800 mm ³ or at the end of the study (day 79), whichever occurred first. Animals withdrawn from the study based on final tumor volume were recorded as euthanized due to tumor progression (TP), with the date of euthanization indicated. Time to end point (TTE) for the assay for each mouse was calculated using the following equation:

|1о£1й(volume at end point) - b

TTE - ------------------------- m where TTE is expressed in days, end point volume is expressed in ^mm3 , b is intercept, and m represents the slope of the line obtained by linear regression of the log-transformed tumor growth data set. The data set consisted of the first observation that exceeded the endpoint volume used for analysis and three consecutive observations immediately preceding the achievement of that endpoint volume. The calculated TTE value is usually less than the TP date, the day the animal was euthanized based on tumor size. Animals with tumors that did not reach volume at the end point were assigned a TTE value equal to the last day of the study (day 79). In cases in which the log-transformed estimated TTE value preceded or exceeded the day of tumor volume endpoint, linear interpolation was performed to approximate the TTE. Animals classified as dying due to NTR (non-treatment related), accidental (NTRa) or unknown etiology (NTRu) were excluded from TTE calculations (and all further analyses). Animals whose death was classified as TR (treatment-related) or NTRm (non-treatment-related, due to metastasis) were assigned a TTE value equal to the day of death. Treatment outcome was assessed by tumor growth delay (TGD), which was defined as an increase in median time to end point (TTE) in the experimental group compared to the control group:

expressed in days, or as a percentage of the median TTE in the control group:

T-C %TGD = ------- x 100

C where T=median TTE for the experimental group, and

C=median TTE for the indicated control group.

Tumor growth inhibition

The tumor growth inhibition (TGI) assay evaluates the difference between median tumor volumes (MTV) of experimental and control mice. For this study, the end point for determining TGI was day 19, which was the last day at which all evaluable control mice remained in the study. For each group, the MTV value (n), the median tumor volume for a certain number of animals, n, on the day of TGI analysis was determined. Percent tumor growth inhibition (%TGI) was defined as the difference between the MTV in the indicated control group and the MTV in the drug-treated group, expressed as a percentage of the MTV in the control group:

x 100 = x 100

The TGI analysis data set included all animals in the group except those that died from treatment-related (TR) or non-treatment-related (NTR) causes before the day of TGI analysis.

MTV and regression response criteria

The effectiveness of treatment can be determined by the tumor volume of the animals remaining in the study on the last day. The MTV (n) value was defined as the median tumor volume on the last day of the study in the specified number of remaining animals (n) whose tumors did not reach the endpoint volume. The effectiveness of treatment can also be determined by the frequency and degree of regression response observed during the study. Treatment can cause partial regression (PR) or complete regression (CR) of the tumor in the animal. For a PR response _, the tumor volume was 50% or less of the volume on day 1 for three consecutive measurements throughout the study, and

MTV

- 50 045942 was exactly or more than 13.5 mm ³ for one or more of the specified three measurements. For CR response, tumor volume was less than 13.5 mm ³ for three consecutive measurements throughout the study. Animals were assessed for a PR or CR event only once throughout the study, and only a CR score was given if both PR and CR criteria were met. Animals with a CR response at the end of the study were further classified as tumor-free survival (TFS) animals. Animals were observed for regression response.

Toxicity

Animals were weighed daily on days 1–5, then twice a week until completion of the study. Mice were frequently observed for obvious signs of any adverse treatment-related (TR) side effects, and clinical signs were recorded during observation. Individual body weight was monitored according to protocol, and any animal with a weight loss greater than 30% for a single measurement or greater than 25% for three consecutive measurements was euthanized, recording the death as TR. Group mean weight loss was also monitored according to the CR Discovery Services protocol. Acceptable toxicity was defined as a group mean body weight (BW) loss of less than 20% during the study and no more than 10% TR deaths. Dosing was stopped in any group where the mean weight loss exceeded acceptable limits. If group mean body weight was restored to an acceptable level, dosing was adjusted to lower levels and/or frequency reduced and then resumed. Deaths were classified as TR if they were associated with treatment side effects as determined by clinical signs and/or autopsy. TR classification was also assigned to deaths due to unknown causes during the dosing period or within 14 days of the last dose. Deaths were classified as not treatment related (NTR) if there was no evidence that the death was related to treatment side effects. NTR deaths were further classified as follows: NTRa describes death due to accident or human error; NTRm describes death presumed to be due to tumor spread through invasion and/or metastasis, as determined by autopsy; NTRu describes death from unknown causes when there is no available evidence of death due to metastasis, tumor progression, accident, or human error. It should be noted that treatment side effects cannot be excluded from deaths classified as NTRu.

Statistical and graphical analysis

GraphPad Prism 8.0 for Windows was used for all statistical analyzes and graphical presentations. Treatment arms that experienced toxicity exceeding acceptable limits (mean group weight loss >20% or >10% treatment-related deaths) or had fewer than five evaluable observations were not included in the statistical analysis. The log-rank test was used to assess the significance of the difference between the overall survival experience of the two groups. The log-rank test allows analysis of individual TTEs for all animals in the group, except those excluded from the study due to NTR death. Statistical analysis of differences between median tumor volumes (MTV) on day 19 in the control and experimental groups was performed using the Mann-Whitney U test. For statistical analysis, two-tailed tests were performed at a significance level of P = 0.05. Prism summarizes test results as not significant (ns) at P>0.05, significant (indicated by *) at 0.01<P<0.05, very significant (**) at 0.001<P<0.01, and extremely significant (***) at P<0.001.

Because tests of statistical significance cannot assess the magnitude of differences between groups, all significance levels are described throughout the text of this report as significant or non-significant.

η Median TTE shh %TGD MTV (η), day 79 Carrier 10 24.8 — - 466(10) ConjA 4 mg/kg 10 79.0 54.2 219 32 (9) PR CR TFS lower limit B.W. TR NTRm NTR Carrier 0 0 0 -2.0 (2) 0 0 0 ConjA 6 4 0 -1.9 (2) 0 0 0

On day 19, the MTV(10) value for ConjA-treated animals was 32 mm ³ , or a significant TGI of 93% (P < 0.001, Mann-Whitney test). Nine animals survived in the study and the estimated median TTE was 79.0 days; this demonstrates the highest possible significant TGD value of 219% (P<0.001, log-rank test). The MTV(9) value at day 79 was 320 mm ³ and six PRs and four CRs were observed.

All documents and other references mentioned above are incorporated into this document by

- 51 045942 links.

Summary of the invention

1. Compound of formula IV:

and its salts and solvates, where:

R ² and R ² represent H;

R ⁶ and R ⁹ are independently selected from H, R, OH, OR, SH, SR, NH ₂ , NHR, NRR', nitro, Me3Sn and halogen;

wherein R and R' are independently selected from optionally substituted C _1-12 alkyl, C _3-20 heterocyclyl and C _6-20 aryl groups; or (a) R ⁷ is selected from H, R, OH, OR, SH, SR, NH ₂ , NHR, NRR', nitro, Me ₃ Sn and halogen; R7' is selected from H, R, OH, OR, SH, SR, NH ₂ , NHR, NRR', nitro, Me ₃ Sn and halogen; or (b) R ⁷ and R ⁷ together form a group which is:

(i) -O-(CH ₂ ) _n -O-, where n is from 7 to 16; or (ii) -O-(CH ₂ CH ₂ O)m-, where m is from 2 to 5;

R represents a C _3-12 alkylene group, the chain of which may be interrupted by one or more heteroatoms, e.g. O, S, NRN ² (where RN ² represents H or C _1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;

Y and Y' are selected from O, S or NH;

R ⁶ and R ⁹ are selected from the same groups as R ⁶ and R ⁹ , respectively; or (ia) R ¹⁰ and R ¹¹ together form a double bond between the N and C atoms to which they are bonded; or (ib) R ¹⁰ is H and R ¹¹ is selected from OH and ORA, wherein RA is C1-4 alkyl; or (ic) both R ¹⁰ and R ¹¹ are H; or (ii-a) R ²⁰ and R ²¹ together form a double bond between the N and C atoms to which they are bonded; or (ii-b) R ²⁰ represents H, and R ²¹ is selected from OH and OR ^B , and ^RB represents C _1-4 alkyl; or (ii-c) both R ²⁰ and R ²¹ are H.

2. The compound according to claim 1, characterized in that both Y and Y' are O.

3. A compound according to claim 1 or 2, characterized in that R represents C _3-7 alkylene.

4. A compound according to claim 1 or 2, characterized in that R represents a group of the formula:

where r is 1 or 2.

5. A compound according to any one of claims 1-4, characterized in that R ⁹ represents H.

6. A compound according to any one of claims 1 to 5, characterized in that R ⁶ represents H.

7. A compound according to any one of claims 1 to 6, characterized in that R ⁷ is selected from H, OH and OR, and R ⁷ is selected from H, OH and OR.

8. The compound according to claim 7, characterized in that R ⁷ represents a C1-4 alkyloxy group, and R ⁷ represents a C1-4 alkyloxy group.

9. A compound according to any one of claims 1 to 8, characterized in that R ² is the same group as R ² , R ⁶ is the same group as R ⁶ , R ⁷ is the same group as R ⁷ , R ⁹ is the same group as R ⁹ , and Y' is the same group as Y.

10. A compound according to any one of claims 1 to 9, characterized in that R ¹⁰ and R ¹¹ together form a double bond between the N and C atoms to which they are bonded.

11. A compound according to any one of claims 1 to 9, characterized in that R ¹⁰ represents H and R ¹¹ is selected from OH and ORA.

12. The compound according to claim 11, characterized in that RA is methyl.

13. A compound according to any one of claims 1 to 9, characterized in that both R ¹⁰ and R ¹¹ are H.

14. A compound according to any one of claims 1 to 13, characterized in that R ²⁰ and R ²¹ together form a double bond between the N and C atoms to which they are bonded.

- 52 045942

15. A compound according to any one of claims 1 to 13, characterized in that R ²⁰ represents H and R ²¹ is selected from OH and OR ^B .

16. The compound according to claim 14, characterized in that R ^B represents methyl.

17. A compound according to any one of claims 1 to 13, characterized in that both R ²⁰ and R ²¹ are H.

18. A compound according to claim 1, having the formula IVa, IVb or IVc:

where R ^1a is selected from methyl and benzyl. 19. Compound of formula I:

and its salts and solvates, where:

Y, Y', R, R ² , R ² ', R ⁶ , R ⁶ ', R ⁷ , R ⁷ ', R ⁹ and R ⁹ ' are as defined in any one of claims 1 to 18;

R ^11b is selected from OH, ORA, where R ^A represents C _1-4 alkyl; And

R ^l is a linker for binding to a cell binding agent which is selected from:

(iiia):

- 53 045942

where Q represents:

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

X represents:

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

G ^l represents a linker for binding to a ligand unit; And

(iiib):

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; or:

(a) R ³⁰ and R ³¹ together form a double bond between the N and C atoms to which they are bonded; or (b) R ³⁰ is H and R ³¹ is selected from OH and OR ^B , wherein ^RB is C _1-4 alkyl; or (c) both R ³⁰ and R ³¹ are H; or (d) R ³¹ is OH or OR ^B , wherein ^RB is C _1-4 alkyl, and R ³⁰ is selected from:

where R ^Z is selected from:

- 54 045942 (z-ii) OS(=O)СНз;

(z-iii) NO2;

(z-iv) OMe;

(z-v) glucuronide;

(z-vi) NH-C(=O)-X ₁ -NHC(=O)X ₂ -NH-C(=O)-R ^zc , where -C(=O)-X1-NH- and -C (=O)-X2-NH- are natural amino acid residues, and R ^ZC is selected from Me, OMe, CH2CH ₂ OMe and (CH ₂ CH ₂ O) ₂ Me.

20. The compound according to claim 19, characterized in that R ³⁰ and R ³¹ together form a double bond between the N and C atoms to which they are bonded.

21. The compound according to claim 19, characterized in that R ³⁰ represents H, and R ³¹ is selected from OH and OR ^B , where ^RB represents C _1-4 alkyl.

22. The compound according to claim 19, characterized in that both R ³⁰ and R ³¹ are H.

23. The compound according to claim 19, characterized in that R ³¹ is OH or ORA, and R ³⁰ is selected from:

- 55 045942

R30d O-\ °\/o φ _R 3° ^{e 02} 0^,0 R30 ^f ο^^,ο R30g he Η °'/, χ^.·' ° ^Η η„ ί 1 ο xy ^ΟΗ R30h ο ^Χι χΝ /R ^ZC ΥΤΥ th ^Χ2 Υ ° ο °ϊ°

24. The compound according to claim 23, characterized in that -C(=O)-X1-NHC(=O)X ₂ -NH- is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys -, -Ala-Lys- and -Val-Cit-.

25. The compound according to claim 23, characterized in that -C(=O)-X1-NHC(=O)X ₂ -NH- is selected from: -Phe-Lys- and -Val-Ala-.

26. The compound according to any one of claims 23-25, characterized in that R ^ZC is selected from CH ₂ CH ₂ OMe and (CH ₂ CH ₂ O) ₂ Me.

27. The compound according to claim 26, characterized in that R ^ZC is (CH ₂ CH ₂ O) ₂ Me.

28. A compound according to claim 19, having the formula Ia, Ib or Ic:

- 56 045942

where R ^1a is selected from methyl and benzyl.

29. A compound according to any one of claims 19 to 28, characterized in that R ^11b represents OH.

30. A compound according to any one of claims 19-29, characterized in that R ^11b represents ORA, where R ^A represents C _1-4 alkyl.

31. The compound according to claim 30, characterized in that R ^A represents methyl.

32. A compound according to any one of claims 19 to 31, characterized in that RL has formula IIIa and Q is an amino acid residue selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg and Trp.

33. A compound according to any one of claims 19 to 31, characterized in that R ^L has formula IIIa, and Q is a dipeptide residue selected from:

CO-Phe-Lys-NH,

CO-Val-Ala-NH, ^CO -Val-Lys- ^NH , ^CO -Ala-Lys- ^NH ,

CO-Val-Cit-NH,

CO-Phe-Cit- ^NH ,

CO-Leu-Cit- ^NH ,

CO-Ile-Cit- ^NH , CO-Phe-Arg-NH and ^CO -Trp-Cit- ^NH .

34. The compound according to claim 33, characterized in that Q is selected from ^CO -Phe-Lys- ^NH , ^CO -Val-Cit- ^NH and ^CO -Val-Ala- ^NH .

35. The compound according to any one of claims 19-31, characterized in that RL has formula IIIa, and Q is pre

- 57 045942 is a tripeptide residue selected from:

^CO -Glu-Val-Ala- ^NH , ^CO -Glu-Val-Cit- ^NH , ^CO -aGlu-Val-Ala- ^NH and ^CO -aGlu-Val-Cit- ^NH

36. A compound according to any one of claims 19 to 35, characterized in that RL has formula IIIa and a is from 0 to 3.

37. The connection according to claim 36, characterized in that a is equal to 0.

38. A compound according to any one of claims 19 to 37, characterized in that RL has formula IIIa and b is from 0 to 12.

39. The connection according to claim 38, characterized in that b is equal to from 0 to 8.

40. A compound according to any one of claims 19 to 39, characterized in that RL has formula IIIa and d is from 0 to 3.

41. The connection according to claim 38, characterized in that d is equal to 2.

42. The compound according to any one of claims 19 to 35, characterized in that RL has formula IIIa, and a is equal to 0, c is equal to 1, and d is equal to 2, and b is equal to from 0 to 8.

43. The connection according to claim 42, characterized in that b is equal to 0, 4 or 8.

44. A compound according to any one of claims 19 to 43, characterized in that RL has formula IIIa and GL is selected from:

(O ¹ ' ¹ ) k (G ^L4 ) Q> Hal N—I H ⁵ where Hal = I, Br, Cl (G ^{L1 2} ) o Vl Ar j. / ^N У ^x o (G ^L5 ) About Hal—fl °—I (G ^L2 ) O (G ^L6 ) O \.z Λ o <.

- 58 045942

optional optional optional where group where group where group NO2 is group NO2 is optional is is

where Ar represents a C5-6 arylene group.

45. The compound according to claim 44, characterized in that Ar represents a phenyl group.

46. Connection according to claim 44 or 45, characterized in that GL is selected from GL ^1-1 and GL ^1-2 .

47. The compound according to claim 46, characterized in that GL is G ^L1-1 .

48. A compound according to any one of claims 19 to 31, characterized in that RL has formula IIIb and both R ^L1 and R ^L2 are H.

49. A compound according to any one of claims 19 to 31, characterized in that R ^L has formula IIIb, R ^L1 represents H, and R ^L2 represents methyl.

50. A compound according to any one of claims 19 to 31, characterized in that R ^L has formula IIIb and both R ^L1 and R ^L2 are methyl.

51. A compound according to any one of claims 19 to 31, characterized in that R ^L has formula IIIb, and R ^L1 and R ^L2 together with the carbon atom to which they are bonded form a cyclopropylene group.

52. A compound according to any one of claims 19 to 31, characterized in that R ^L has formula IIIb, and R ^L1 and R ^L2 together with the carbon atom to which they are bonded form a cyclobutylene group.

53. A compound according to any one of claims 19-31 and 48-52, characterized in that R ^L has formula IIIb and e is equal to 0.

54. A compound according to any one of claims 19-31 and 48-52, characterized in that R ^L has formula IIIb and e is equal to 1.

55. The compound according to claim 54, characterized in that the nitro group is in the para position.

56. A compound according to claim 19, characterized in that said compound has the formula Id:

- 59 045942

where Q is selected from:

(a) -CH2-;

(b) -C3H6-; And

(With)

57. Conjugate of formula II:

where L represents a ligand unit (i.e., a targeting agent), D ^L represents a linker unit of a drug compound of formula I':

are as defined in where Y, Y', R, R ² , R ² ', R ⁶ , R ⁶ ', R ⁷ , R ⁷ ', R ⁹ and R ⁹ ' any of claims 1 to 18;

R ^11b , R ³⁰ and R ³¹ are as defined in any of paragraphs 19-27 and 29-31;

R ^ll is a linker for binding to a cell binding agent which is selected from:

(IIIa):

where Q and X are as defined in any of claims 19 and 32-43, and G ^LL is a linker associated with a ligand unit; And

(111b):

where R ^L1 and R ^L2 are as defined in any of paragraphs 19 and 48-52; where p is an integer from 1 to 20.

58. The conjugate according to claim 57, characterized in that G ^LL is selected from:

- 60 045942

where Ar represents a C _5-6 arylene group.

59. The conjugate according to claim 58, characterized in that Ar represents a phenylene group.

Conjugate according to claim 58 or 59, characterized in that G ^LL is selected from g ^LL1-1 and G ^ll1-2

61. The conjugate according to claim 60, characterized in that G ^LL is G ^ll-1 .

62. The conjugate according to claim 57, characterized in that D ^L has the formula (Id'):

O O where Q is selected from:

- 61 045942

63. The conjugate according to any one of claims 57-62, characterized in that the ligand unit is an antibody or an active fragment thereof.

64. The conjugate according to claim 63, characterized in that the antibody or antibody fragment is an antibody or antibody fragment to a tumor-associated antigen.

65. The conjugate according to claim 64, characterized in that the antibody or antibody fragment is an antibody that binds to one or more tumor-associated antigens or cell surface receptors selected from (1)-(88):

(1) BMPR1B;

(2) E16;

(3) STEAP1;

(4) 0772Р;

(5) MPF;

(6) Napi3b;

(7) Sema 5b;

(8) PSCA hlg;

(9) ETBR;

(10)MSG783;

(11) STEAP2;

(12) TrpM4;

(13) CRIPTO;

(14) CD21;

(15) CD79b;

(16) FcRH2;

(17) HER2;

(18) NCA;

(19) MDP;

(20) IL20R-alpha;

(21) Brevican;

(22) EphB2R;

(23) ASLG659;

(24) PSCA;

(25) GEDA;

(26) BAFF-R;

(27) CD22;

(28) CD79a;

(29) CXCR5;

(30) HLA-DOB;

(31) P2X5;

(32) CD72;

(33) LY64;

(34) FcRHl;

(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;

- 62 045942 (51) BCMA-TNFRSF17;

(52) CT Ags-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/Prostate Tumor Target Mindin - Mindin/RG-1;

(70) B7-H4-VTCN1;

(71) PTK7;

(72) CD37;

(73) CD138-SDC1;

(74) CD74;

(75) Claudins - CLs;

(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).

66. The conjugate according to any one of claims 63-65, characterized in that the antibody or antibody fragment is an engineered antibody with cysteine substitutions.

67. The conjugate according to any one of claims 57-66, characterized in that p is an integer from 1 to 8.

68. The conjugate according to claim 67, characterized in that p is equal to 1, 2, 3 or 4.

69. A composition containing a mixture of conjugates according to any one of claims 57-68, characterized in that the average p value in said mixture of conjugate compounds is from about 1 to about 8.

70. Conjugate according to any one of claims 57-68 for use in therapy.

71. A pharmaceutical composition containing the conjugate according to any one of claims 57-68 and a pharmaceutically acceptable diluent, carrier or excipient.

72. The conjugate of any one of claims 57 to 68 or the pharmaceutical composition of claim 71 for use in treating a proliferative disease in a subject.

73. A conjugate for use according to claim 72, characterized in that the disease to be treated is cancer.

74. Use of a conjugate according to any one of claims 57-68 or a pharmaceutical composition according to claim 71 in a method of medical treatment.

75. A method of medical treatment, including administering to a patient a pharmaceutical composition according to claim 71.

76. The method according to claim 75, characterized in that the medical treatment method is for the treatment of cancer.

77. The method according to claim 76, characterized in that the patient is administered a chemotherapeutic agent in combination with said conjugate.

78. Use of a conjugate according to any one of claims 57-68 in a method for the production of a medicinal product

-

Claims (11)

for the treatment of proliferative disease. 79. A method of treating a mammal having a proliferative disease, comprising administering an effective amount of a conjugate according to any one of claims 57 to 68 or a pharmaceutical composition according to claim 71. CLAIM 1. A compound of formula IV and its salts or solvates, where: R ² and R ² ' represent H; R ⁶ and R ⁹ are independently selected from H, R, OH, OR, SH, SR, NH ₂ , NHR, NRR', nitro, Me3Sn and halogen; where R and R' are independently selected from C _1-12 alkyl groups, C _3-20 heterocyclyl groups containing from 1 to 4 heteroatoms selected from O, N and S, C _6-16 carboaryl groups and C _5-14 heteroaryl groups ; or (a) R ⁷ is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halogen; R ⁷ ' is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halogen; or (b) R ⁷ and R ⁷ ' together form a group which is: (i) -O-(CH ₂ ) _p -O-, where n is from 7 to 16; or (ii) -O-(CH ₂ CH ₂ O) _t -, where m is from 2 to 5; R represents a C _3-12 alkylene group, the chain of which may be interrupted by one or more heteroatoms selected from O, S, NRN ² (where RN ² represents H or C ₁ . ₄ alkyl), and/or aromatic rings selected from benzene or pyridine; Y and Y' are selected from O, S or NH; R ⁶ ' and R ⁹ ' are selected from the same groups as R ⁶ and R ⁹ , respectively; or (ia) R ¹⁰ and R ¹¹ together form a double bond between the N and C atoms to which they are bonded; or (ib) R ¹⁰ represents H, and R ¹¹ is selected from OH and ORA, and R ^A represents C _1-4 alkyl; or (ic) both R ¹⁰ and R ¹¹ are H; or (ii-a) R ²⁰ and R ²¹ together form a double bond between the N and C atoms to which they are bonded; or (ii-b) R ²⁰ represents H, and R ²¹ is selected from OH and OR ^B , and ^RB represents C _1-4 alkyl; or (ii-c) both R ²⁰ and R ²¹ are H. 2. The connection according to claim 1, characterized in that a) both Y and Y' are O; and/or b) R represents C _3-7 alkylene; or c) R represents a group of the formula: ’ where r is 1 or 2; and/or d) R ⁹ is H, R ⁶ is H, R ⁷ and R ⁷ ' are independently C _1-4 alkyloxy; or e) R ⁶ ' is the same group as R ⁶ , R ⁷ is the same group as R ⁷ , R ⁹ ' is the same group as R ⁹ , and Y' is the same group as Y. 3. A compound according to claim 1, having the formula IVa, IVb or IVc: - 64 045942 R ⁹ ' are as defined according to any one of claims 1 to 3; and its salts or solvates, where: Y, Y', R, ^R2 , ^R2 ', ^R6 , ^R6 ', ^R7 , ^R7 ', ^R9 and where ^R1a is selected from methyl and benzyl 4. Compound of formula I: R ^11b is selected from OH, ORA, where R ^A represents C _1-4 alkyl; And RL is a linker for binding to an antibody or antigen binding fragment of an antibody that is selected from: (iiia): where Q represents: where Q ^X is such that Q is an amino acid residue, a dipeptide residue or a tripeptide residue; - 65 045942 X represents: where a=0-5, b=0-16, c=0 or 1, d=0-5; G ^l is a linker for binding to an antibody or an antigen-binding fragment of an antibody; And (iiib): 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 equal to 0 or 1; or: (a) R ³⁰ and R ³¹ together form a double bond between the N and C atoms to which they are bonded; or (b) R ³⁰ is H and R ³¹ is selected from OH and OR ^B , wherein ^RB is C _1-4 alkyl; (c) R ³⁰ and R ³¹ are both H; or (d) R ³¹ is OH or OR ^B , wherein ^RB is C _1-4 alkyl, and R ³⁰ is selected from: (i) (ii) (iii) g where R ^Z is selected from: R ^z (zi) (z-ii) OC(=O)CH ₃ ; (z-iii) NO2; (z-iv) OMe; (z-v) glucuronide; (z-vi) NH-C(=O)-Xi-NHC(=O)X2-NH-C(=O)-R ^zc , where -C(=O)-Xi-NH- and -C(= O)-X2-NH- are natural amino acid residues, and R ^ZC is selected from Me, OMe, CH ₂ CH ₂ OMe and (CH ₂ CH ₂ O) ₂ Me. 5. A compound according to claim 4, having the formula Ia, Ib or Ic: - 66 045942 where R ^1a is selected from methyl and benzyl. 6. A compound according to claim 4 or 5, characterized in that RL has formula IIIa, and: a) Q is a dipeptide residue selected from: CO-Phe-Lys-NH, CO-Val-Ala-NH, ^CO -Val-Lys- ^NH , ^CO -Ala-Lys- ^NH , CO-Val-Cit-NH, ^CO -Phe-Cit- ^NH , ^CO -Leu-Cit- ^NH , ^CO -Ile-Cit- ^NH , CO-Phe-Arg-NH and '-Trp-Cit-N ¹ ; and/or b) R ^l has formula IIIa, and a is 0, c is 1, and d is 2, and b is from 0 to 8, with b being preferably 0, 4 or 8. 7. A compound according to any one of claims 4-6, characterized in that RL has formula IIIa, and GL is selected from: - 67 045942 (G ^L1 - ¹ ) O o (G ^L4 ) O Hal N—1 H ⁵ where Hal = 1, Br, Cl (G ^L1 - ² ) o Y / Ar e /^ ^{N Y x} o (G ^L5 ) 0 Hal—У °4 (G ^L2 ) О V—о о (G ^L6 ) 0 0 ,°4 'γ-Ν > (G ^L3 ' ¹ ) S— (G ^L7 ) ^Br —У'' IN (NO ₂ ) where the NO2 group is optional (G ^L3 - ² ) (b where optional S—s'”* ¹¹ 5 IO ₂ ) the NO ₂ group is optional (G ^L8 ) (G ^L3 - ³ ) s— ο ₂ νΛ=ζ/ where the group ΝΟ ₂ is optional (G ^L9 ) ^Ν > - 68 045942 where the NO2 group is optional where Ar represents a C _5-6 arylene group. 8. A compound according to claim 4, characterized in that said compound has the formula Id: where Q is selected from: (a) -CH2-; (b) -C3H6-; And (With) 9. Conjugate of formula II: L - (D ^L ) _P (II) where L represents an antibody or an antigen-binding fragment of an antibody, DL represents a linker unit of a drug compound of formula I': where Y, Y', R, R ² , R ² ', R ⁶ , R ⁶ ', R ⁷ , R ⁷ , R ⁹ and R ⁹ ' are as defined in any one of claims 1, 2; R ^11b , R ³⁰ and R ³¹ are as defined in claim 4; R ^ll is a linker for binding to an antibody or antigen binding fragment of an antibody that is selected from: (iiia): - 69 045942 where Q and X are as defined in claims 4 and 6, and G ^LL is a linker associated with an antibody or an antigen-binding fragment of an antibody; And (iiib): _{L1 l2} ' where R ^l1 and R ^L2 are as defined in claim 4; where p is an integer from 1 to 20. 10. The conjugate according to claim 9, characterized in that G ^LL is selected from: (G ^LL1 - ¹ ) O cba$ O (G ^LL6 ) O CBAj (G ^LL1 -2) o Vl ^Ar g CBAg / N y ^X O (G ^LL7 ) CBAi____ (G ^LL2 ) o CBAj >0 |\Γ ° o (G ^LL8 - ¹ ) CBA (G ^LL3 - ¹ ) CBA< H (G ^ll 8-2) _N CBA (G ^LL3 - ² ) (G ^LL9 - ¹ ) A ^N ₄ % CBA (G ^LL4 ) CBAi___ g \ H λ—N O Y (G ^ll 9-2) A' ⁷ ' CBA (G ^LLS ) O CBA^___ °h where Ar represents a C _5-6 arylene group. 11. The conjugate according to claim 9, characterized in that D ^L has the formula (Id ¹ ): -