Azetidine benzodiazepine dimers and conjugates containing them for treating cancer

Abstract

A compound of formula IV: a drug linker and a conjugate comprising the compound, and use of the conjugate in treating cancer.

Description

Technical Field

[0001] The present invention relates to azetidinone and benzodiazepine (ABD) dimer, a conjugate comprising the dimer, and a prodrug linker for preparing the conjugate. Background Art

[0002] Pyrrolobenzodiazepine (PBD) dimers have been shown to be cytotoxic compounds.

[0003] For example, SG2000 (SJG-136):

[0004]

[0005] (Gregson, SJ et al., Chem. Commun., 1999, 797-798. doi: 10.1039 / A809791G; Gregson, S. et al., J. Med. Chem., 44, 737-748 (2001); Alley, MC et al., Cancer Research, 64, 6700-6706 (2004); and Hartley, JA et al., Cancer Research, 64, 6693-6699 (2004)) has been involved in clinical trials as an independent agent, for example, NCT02034227 studied its use in the treatment of acute myeloid leukemia and chronic lymphocytic leukemia (see: https: / / www.clinicaltrials.gov / ct2 / show / NCT02034227).

[0006] Dimeric PBD compounds carrying C2 aryl substituents and endo-unsaturation, such as SG2202 (ZC-207), are disclosed in WO 2005 / 085251:

[0007]

[0008] And in WO2006 / 111759, the bisulfite of the PBD compound, such as SG2285 (ZC-423):

[0009]

[0010] These compounds have been shown to be highly useful cytotoxic agents (Howard, PW et al., Bioorg. Med. Chem. (2009), doi: 10.1016 / j.bmcl.2009.09.012).

[0011] Dimeric PBD compounds with linker groups for attachment to cell binding agents such as antibodies are described in WO2011 / 130598. The linker in these compounds is attached to one of the available N10 positions and is typically cleaved by the action of an enzyme on the linker group. WO 2014 / 057074 and WO 2015 / 052322 describe specific PBD dimer conjugates attached to one monomer via the N10 position.

[0012] At a relatively early stage in the development of PBD as a molecule of interest, the following compound was reported in 1997 (Bose, DS et al., Tetrahedron Letters, 38(33), 5839-5842, 1997; doi: 10.1016 / S0040-4039(97)01297-5):

[0013]

[0014] It has been synthesized and will be evaluated as a potential DNA binding ligand and cytotoxic agent. There are no further publications for this compound, so it appears that they were either unstable or inactive in the tests. Summary of the invention

[0015] The first aspect of the present invention provides a compound of formula IV:

[0016]

[0017] and salts and solvates thereof, wherein:

[0018] R 2 and R 2’ is H;

[0019] R 6 and R 9 is independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halogen;

[0020] wherein R and R' are independently selected from optionally substituted C 1-12 Alkyl, C 3-20 Heterocyclic and C 5-20 Aryl;

[0021] and

[0022] (a)R 7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halogen;

[0023] R 7’is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halogen; or

[0024] (b)R 7 and R 7 'Together form a group which is: (i) -O-(CH2) n -O-, wherein n is 7 to 16; or (ii) -O-(CH2CH2O) m -, wherein m is 2 to 5;

[0025] R″ is C 3-12 Alkylene, the chain may be separated by one or more heteroatoms, such as O, S, NR N2 (where R N2 H or C 1-4 alkyl), and / or aromatic rings, such as benzene or pyridine;

[0026] Y and Y' are selected from O, S or NH;

[0027] R 6’ and R 9’ Selected from R 6 and R 9 The same group;

[0028] and

[0029] (ia)R 10 and R 11 together to form a double bond between the N atom and the C atom to which they are bound; or

[0030] (ib)R 10 H and R 11 Selected from OH and OR A , where R A C 1-4 Alkyl; or (ic) R 10 and R 11 All are H;

[0031] and

[0032] (ii-a)R 20 and R 21 together to form a double bond between the N atom and the C atom to which they are bound; or

[0033] (ii-b)R 20 H and R 21 Selected from OH and OR B , where R B C 1-4 Alkyl; or (ii-c) R 20 and R 21Both are H.

[0034] A second aspect of the present invention includes a compound having Formula I:

[0035]

[0036] and salts and solvates thereof, wherein:

[0037] Y, Y', R", R 2 , R 2’ , R 6 , R 6’ , R 7 , R 7’ , R 9 and R 9’ As defined in the first aspect of the present invention;

[0038] R 11b Selected from OH, OR A , where R A C 1-4 Alkyl; and

[0039] R L is a linker for connecting to a cell binding agent selected from:

[0040] (iiia):

[0041]

[0042] in

[0043] Q is:

[0044] Where Q X is such that Q is an amino acid residue, a dipeptide residue or a tripeptide residue;

[0045] X is:

[0046]

[0047] Where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5;

[0048] G L is a linker for attachment to a Ligand unit; and

[0049] (iiib):

[0050]

[0051] Where R L1 and R L2are independently selected from H and methyl, or together with the carbon atoms to which they are attached form cyclopropylene or cyclobutylene; and e is 0 or 1;

[0052] and:

[0053] (a)R 30 and R 31 together to form a double bond between the N atom and the C atom to which they are bound; or

[0054] (b)R 30 H and R 31 Selected from OH and OR B , where R B C 1-4 alkyl;

[0055] (c)R 30 and R 31 All are H; or

[0056] (d) R 31 OH or OR B , where R B C 1-4 Alkyl and R 30 Selected from:

[0057] (i)

[0058] (ii)

[0059] (iii) Where R Z Selected from:

[0060] (zi)

[0061] (z-ii) OC(=O)CH3;

[0062] (z-iii) NO2;

[0063] (z-iv)OMe;

[0064] (zv) glucuronide;

[0065] (z-vi)NH-C(=O)-X1-NHC(=O)X2-NH-C(=O)-R ZC , wherein -C(=O)-X1-NH- and -C(=O)-X2-NH- represent natural amino acid residues and R ZC Selected from Me, OMe, CH2CH2OMe and (CH2CH2O)2Me.

[0066] A third aspect of the present invention provides a conjugate of formula II:

[0067] L-(D L ) p (II)

[0068] Wherein L is a ligand unit (i.e., a targeting agent), D L A drug linker unit of formula I':

[0069]

[0070] Where R 2 , R 2’ , R 6 , R 7 , R 9 , R 11b ,Y,R”,Y',R 6’ , R 7’ , R 9’ , R 30 and R 31 As defined in the second aspect of the present invention;

[0071] R LL is a linker for connecting to a cell binding agent selected from:

[0072] (iiia):

[0073]

[0074] wherein Q and X are as defined in the first aspect and G LL is a linker attached to the Ligand unit; and

[0075] (iiib):

[0076]

[0077] Where R L1 and R L2 As defined in the first aspect;

[0078] wherein p is an integer from 1 to 20.

[0079] The Ligand unit described more fully below is a targeting agent that binds to a target moiety. The Ligand unit can, for example, specifically bind to a cellular component (cell binding agent) or other target molecule of interest. The Ligand unit can 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.

[0080] The fourth aspect of the present invention provides the use of the conjugate of the third aspect of the present invention in the manufacture of a medicament for treating a proliferative disease. The fourth aspect also provides the conjugate of the third aspect of the present invention for treating a proliferative disease. The fourth aspect also provides a method for treating a proliferative disease, comprising administering a therapeutically effective amount of the conjugate of the second aspect of the present invention to a patient in need thereof.

[0081] One of ordinary skill in the art can readily determine whether a candidate conjugate treats a proliferative condition of any particular cell type. For example, assays that can be conveniently used to assess the activity provided by a particular compound are described in the Examples below.

[0082] The fifth aspect of the present invention provides the synthesis of the conjugate of the third aspect of the present invention, which comprises conjugating the compound of the second aspect of the present invention (drug linker) to a Ligand unit.

[0083] The compound of formula IV is the warhead released from the conjugate of the third aspect.

[0084] definition

[0085] Substituents

[0086] As used herein, the phrase "optionally substituted" refers to a parent group which may be unsubstituted or may be substituted.

[0087] Unless otherwise indicated, the term "substituted" as used herein refers to a parent group carrying one or more substituents. The term "substituent" is used in the conventional sense herein and refers to a chemical moiety that is covalently attached to a parent group or, where appropriate, is fused to a parent group. A variety of substituents are well known, and their formation and methods of introduction into various parent groups are also well known.

[0088] Examples of substituents are described in more detail below.

[0089] C 1-12 Alkyl: As used herein, the term "C 1-12 "Alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). As used herein, the term "C 1-4 "Alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). Thus, the term "alkyl" includes the subclasses alkenyl, alkynyl, cycloalkyl, etc. discussed below.

[0090] 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).

[0091] Examples of saturated straight chain alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), n-butyl (C4), n-pentyl (amyl) (C5), n-hexyl (C6), and n-heptyl (C7).

[0092] Examples of saturated branched alkyl groups include isopropyl (C3), isobutyl (C4), sec-butyl (C4), tert-butyl (C4), isopentyl (C5), and neopentyl (C5).

[0093] C 2-12 Alkenyl: As used herein, the term "-C 2-12 "Alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds.

[0094] 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-methylethenyl, -C(CH3)=CH2), butenyl (C4), pentenyl (C5), and hexenyl (C6).

[0095] C 2-12 Alkynyl: As used herein, the term "C 2-12 "Alkynyl" refers to an alkyl group having one or more carbon-carbon triple bonds.

[0096] Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (-C≡CH) and 2-propynyl (propargyl, -CH2-C≡CH).

[0097] C 3-12 Cycloalkyl: As used herein, the term "C 3-12 "Cycloalkyl" refers to an alkyl group which is also a cyclic group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, said moiety having from 3 to 7 carbon atoms, inclusive of 3 to 7 ring atoms.

[0098] Examples of cycloalkyl groups include, but are not limited to, those derived from:

[0099] Saturated monocyclic hydrocarbon compounds:

[0100] Cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C7), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7), and methylcyclohexane (C7);

[0101] Unsaturated monocyclic hydrocarbon compounds:

[0102] Cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcyclobutene (C6), methylcyclopentene (C6), dimethylcyclopentene (C7), and methylcyclohexene (C7); and

[0103] Saturated polycyclic hydrocarbons:

[0104] Norcarane (C7), norpinane (C7), norbornane (C7).

[0105] C 3-20 Heterocyclyl: As used herein, the term "C 3-20 "Heterocyclyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, said moiety having 3 to 20 ring atoms, of which 1 to 10 are ring heteroatoms. Preferably, each ring has 3 to 7 ring atoms, of which 1 to 4 are ring heteroatoms.

[0106] In this context, a prefix (such as C 3-20 , C 3-7 , C 5-6 , etc.) refers to the number of ring atoms or a range of the number of ring atoms, whether carbon atoms or heteroatoms. For example, as used herein, the term "C 5-6 "Heterocyclyl" refers to a heterocyclyl radical having 5 or 6 ring atoms.

[0107] Examples of monocyclic heterocyclic groups include, but are not limited to, heterocyclic groups derived from:

[0108] N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoxazole) (C5), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7);

[0109] O1: ethylene oxide (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxole (dihydrofuran) (C5), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7);

[0110] S1: thiirane (C3), thietane (C4), thiolane (tetrahydrothiophene) (C5), thiazane (tetrahydrothiopyran) (C6), thiepane (C7);

[0111] O2: dioxolane (C5), dioxane (C6) and dioxepane (C7);

[0112] O3: trioxane (C6);

[0113] N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C6);

[0114] N1O1: tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), dihydroisoxazole (C5), morpholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (C6);

[0115] N1S1: Thiazoline (C5), Thiazolidine (C5), Thiomorpholine (C6);

[0116] N2O1: Oxadiazine (C6);

[0117] O1S1: oxathiole (C5) and oxathiane (thioxane) (C6); and

[0118] N1O1S1: Oxathiazine (C6).

[0119] Examples of the substituted monocyclic heterocyclic group include heterocyclic groups derived from sugars in a cyclic form, for example, furanose (C5) such as arabinofuranose, lyxofuranose, ribofuranose and xylofuranose; and pyranose (C6) such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose and talopyranose.

[0120] C5-20 Aryl: As used herein, the term "C 5-20 "Aryl" refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, the moiety having 3 to 20 ring atoms. As used herein, the term "C 5-7 "Aryl" refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, the moiety having 5 to 7 ring atoms and as used herein, the term "C 5-10 "Aryl" refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, said moiety having 5 to 10 ring atoms. Preferably, each ring has 5 to 7 ring atoms.

[0121] In this context, a prefix (such as C 3-20 , C 5-7 , C 5-6 , C 5-10 , etc.) refers to the number of ring atoms or a range of the number of ring atoms, whether carbon atoms or heteroatoms. For example, as used herein, the term "C 5-6 "Aryl" refers to an aromatic radical having 5 or 6 ring atoms.

[0122] The ring atoms can be all carbon atoms, as in "carboaryl".

[0123] Examples of carboaryl groups include, but are not limited to, groups derived from benzene (i.e., phenyl) (C6), naphthalene (C 10 )、azulene (C 10 ), anthracene (C 14 ), Philippines (C 14 ), naphthacene (C 18 ) and pyrene (C 16 )

[0124] Examples of aryl groups including fused rings in which at least one ring is aromatic include, but are not limited to, those derived from indan (e.g., 2,3-dihydro-1H-indene) (C9), indene (C9), isoindene (C9), tetralin (1,2,3,4-tetralin (C9), 10 ), dihydroacenaphthene (C 12 ), fluorene (C 13 ), phenalene (C 13 ), acephenanthrene (C 15 ) and aceanthrene (C 16 ) group.

[0125] Alternatively, the ring atoms may include one or more heteroatoms, as in "heteroaryl". Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:

[0126] N1: pyrrole (azole) (C5), pyridine (azine) (C6);

[0127] O1: Furan (oxacyclopentadiene) (C5);

[0128] S1: Thiophene (thiolene) (C5);

[0129] N1O1: oxazole (C5), isoxazole (C5), isoxazine (C6);

[0130] N2O1: Oxadiazole (furazan) (C5);

[0131] N3O1: Oxatriazole (C5);

[0132] N1S1: Thiazole (C5), isothiazole (C5);

[0133] N2: imidazole (1,3-oxadiazole) (C5), pyrazole (1,2-oxadiazole) (C5), pyridazine (1,2-oxadiazine) (C6), pyrimidine (1,3-oxadiazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1,4-oxadiazine) (C6);

[0134] N3: triazole (C5), triazine (C6); and,

[0135] N4: Tetrazole (C5).

[0136] Examples of heteroaryl groups containing fused rings include, but are not limited to:

[0137] C9 (having 2 fused rings) derived from benzofuran (O1), isobenzofuran (O1), indole (N1), isoindole (N1), indolizine (N1), indoline (N1), isoindole (N1), purine (N4) (e.g., adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazole (N1O1), benzisoxazole (N1O1), benzodioxole (O2), benzofurazan (N2O1), benzotriazole (N3), benzothiofuran (S1), benzothiazole (N1S1), benzothiadiazole (N2S);

[0138] C 10 (having 2 fused rings) derived from benzopyran (O1), isobenzopyran (O1), chroman (O1), isochroman (O1), benzoxane (O2), quinoline (N1), isoquinoline (N1), quinolizine (N1), benzoxazine (N1O1), benzodiazine (N2), pyridopyridine (N2), quinoxaline (N2), quinazoline (N2), cinnoline (N2), phthalazine (N2), naphthyridine (N2), pteridine (N4);

[0139] C 11 (with 2 fused rings), which are derived from benzodiazepines (N2);

[0140] C 13 (having 3 fused rings) derived from carbazole (N1), dibenzofuran (O1), dibenzothiophene (S1), carboline (N2), perylene (N2), pyridoindole (N2); and,

[0141] C 14 (having 3 fused rings), which are derived from acridine (N1), xanthene (O1), thioxanthene (S1), dibenzo-p-dioxin (oxanthrene) (O2), oxathane (O1S1), phenazine (N2), phenoxazine (N1O1), phenothiazine (N1S1), thianthrene (S2), phenanthridine (N1), phenanthroline (N2), phenazine (N2).

[0142] The above groups, whether alone or as part of another substituent, may themselves be optionally substituted with one or more groups selected from themselves and the further substituents listed below.

[0143] Halogen: -F, -Cl, -Br, and -I.

[0144] Hydroxyl group: -OH.

[0145] Ether: -OR, where R is an ether substituent, for example, C 1-7 Alkyl (also called C 1-7 Alkoxy, discussed below), C 3-20 Heterocyclic group (also called C 3-20 Heterocyclic oxy) or C 5-20 Aryl (also called C 5-20 Aryloxy), preferably C 1-7 alkyl.

[0146] Alkoxy: -OR, where R is an alkyl group, for example, C 1-7 Alkyl. C 1-7 Examples of alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n-propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy).

[0147] Acetal: -CH(OR 1 )(OR 2 ), where R 1 and R 2 are independently acetal substituents, for example, C 1-7 Alkyl, C3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 alkyl, 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 atom to which they are attached, form a heterocycle having 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, -CH(OMe)2, -CH(OEt)2, and -CH(OMe)(OEt).

[0148] Hemiacetal: -CH(OH)(OR 1 ), where R 1 is a hemiacetal substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Examples of hemiacetal groups include, but are not limited to, -CH(OH)(OMe) and -CH(OH)(OEt).

[0149] Ketal: -CR(OR 1 )(OR 2 ), where R 1 and R 2 As defined for acetals, and R is a ketal substituent other than hydrogen, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 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)2, and -C(Et)(OMe)(OEt).

[0150] Hemiketal: -CR(OH)(OR 1 ), where R 1 As defined for a hemiacetal, and R is a hemiketal substituent other than hydrogen, e.g., C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 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).

[0151] Oxo (ketone, -ketone): =O.

[0152] Thione (thione)(thioketone):=S.

[0153] Imino (imine): =NR, where R is an imino substituent, for example, hydrogen, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably hydrogen or C 1-7 Examples of ester groups include, but are not limited to, =NH, =NMe, =NEt, and =NPh.

[0154] Formyl (aldehyde, formaldehyde): -C(=O)H.

[0155] Acyl (keto): -C(=O)R, where R is an acyl substituent, for example, C 1-7 Alkyl (also called C 1-7 Alkyl acyl or C 1-7 Alkanoyl), C 3-20 Heterocyclic group (also called C 3-20 Heterocyclic acyl) or C 5-20 Aryl (also called C 5-20 Aryl acyl), preferably C 1-7 Alkyl. Examples of acyl groups include, but are not limited to, -C(=O)CH3 (acetyl), -C(=O)CH2CH3 (propionyl), -C(=O)C(CH3)3 (tert-butyryl), and -C(=O)Ph (benzoyl, phenone).

[0156] Carboxyl (carboxylic acid): -C(=O)OH.

[0157] Thiocarboxy (thiocarboxylic acid): -C(=S)SH.

[0158] Thiol carboxyl (thiol carboxylic acid): -C(=O)SH.

[0159] Thioketocarboxyl (thioketocarboxylic acid): -C(=S)OH.

[0160] Imidic acid: -C(=NH)OH.

[0161] Hydroxamic acid: -C(=NOH)OH.

[0162] Ester (carboxylate, ester of carboxylic acid, oxycarbonyl): -C(=O)OR, where R is an ester substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Examples of ester groups include, but are not limited to, -C(=O)OCH3, -C(=O)OCH2CH3, -C(=O)OC(CH3)3, and -C(=O)OPh.

[0163] Acyloxy (reverse ester): -OC(=O)R, where R is an acyloxy substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Examples of acyloxy groups include, but are not limited to, -OC(=O)CH3 (acetoxy), -OC(=O)CH2CH3, -OC(=O)C(CH3)3, -OC(=O)Ph, and -OC(=O)CH2Ph.

[0164] Oxycarbonyloxy: -OC(=O)OR, where R is an ester substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Examples of ester groups include, but are not limited to, -OC(=O)OCH3, -OC(=O)OCH2CH3, -OC(=O)OC(CH3)3, and -OC(=O)OPh.

[0165] Amino group: -NR 1 R 2 , where R 1 and R 2 are independently amino substituents, for example, hydrogen, C 1-7 Alkyl (also called C 1-7 Alkylamino or di-C 1-7 Alkylamino), C 3-20 Heterocyclic or C 5-20 Aryl, preferably H or C 1-7 alkyl, or, in the case of a "cyclic" amino group, R 1 and R 2 Together with the nitrogen atom to which they are attached, they form a heterocyclic ring having 4 to 8 ring atoms. Amino groups can be primary (-NH2), secondary (-NHR 1 ) or tert-NHR 1 R 2 ), and in cationic form, may be quaternary (- + NR 1 R 2 R 3 ). Examples of amino groups include, but are not limited to, -NH2, -NHCH3, -NHC(CH3)2, -N(CH3)2, -N(CH2CH3)2, and -NHPh. Examples of cyclic amino groups include, but are not limited to, aziridine, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholino, and thiomorpholino.

[0166] Acylamino (carbamoyl, carbamoyl, aminocarbonyl, carboxyamide): -C(=O)NR 1 R 2 , where R 1 and R 2 is independently an amino substituent, as defined for amino. Examples of acylamino groups include, but are not limited to, -C(=O)NH2, -C(=O)NHCH3, -C(=O)N(CH3)2, -C(=O)NHCH2CH3, and -C(=O)N(CH2CH3)2, as well as groups in which R 1 and R 2 Acylamino groups which, together with the nitrogen atom to which they are attached, form a heterocyclic structure, for example piperidinylcarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl and piperazinylcarbonyl.

[0167] Thioamido (thiocarbamoyl): -C(=S)NR 1 R 2 , where R 1 and R 2 is independently an amino substituent, as defined for amino. Examples of acylamino groups include, but are not limited to, -C(=S)NH2, -C(=S)NHCH3, -C(=S)N(CH3)2, and -C(=S)NHCH2CH3.

[0168] Acylamido (acylamino): -NR 1 C(=O)R 2 , where R 1 is an amide substituent, for example, hydrogen, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably hydrogen or C 1-7 Alkyl, and R 2 is an acyl substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably hydrogen or C 1-7 Alkyl. Examples of acylamide groups include, but are not limited to, -NHC(=O)CH3, -NHC(=O)CH2CH3, and -NHC(=O)Ph. 1 and R 2 Together they may form a ring structure, such as, for example, in succinimidyl, maleimidyl and phthalimidyl groups:

[0169]

[0170] Aminocarbonyloxy: -OC(=O)NR 1 R 2 , where R 1and R 2 is independently an amino substituent, as defined for amino.Examples of aminocarbonyloxy include, but are not limited to, -OC(=O)NH2, -OC(=O)NHMe, -OC(=O)NMe2, and -OC(=O)NEt2.

[0171] Urea group: -N(R 1 )CONR 2 R 3 , where R 2 and R 3 are independently amino substituents, as defined for amino groups, and R 1 is a ureido substituent, for example, hydrogen, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably hydrogen or C 1-7 Examples of urea groups include, but are not limited to, -NHCONH2, -NHCONHMe, -NHCONHEt, -NHCONMe2, -NHCONEt2, -NMeCONH2, -NMeCONHMe, -NMeCONHEt, -NMeCONMe2, and -NMeCONEt2.

[0172] Guanidine: -NH-C(=NH)NH2.

[0173] Tetrazolyl: A five-membered aromatic ring with four nitrogen atoms and one carbon atom,

[0174]

[0175] Imino: =NR, where R is an imino substituent, for example, hydrogen, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably H or C 1-7 Examples of imino groups include, but are not limited to, =NH, =NMe, and =NEt.

[0176] Amidine (amidino): -C(=NR)NR2, wherein each R is an amidine substituent, for example, hydrogen, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably H or C 1-7 Examples of amidine groups include, but are not limited to, -C(=NH)NH2, -C(=NH)NMe2, and -C(=NMe)NMe2.

[0177] Nitro: -NO2.

[0178] Nitroso: -NO.

[0179] Azide: -N3.

[0180] Cyano (nitrile, carbonitrile): -CN.

[0181] Isocyano: -NC.

[0182] Cyanamide: -OCN.

[0183] Isocyanate: -NCO.

[0184] Thiocyanato (thiocyanate): -SCN.

[0185] Isothiocyanate (isothiocyanate): -NCS.

[0186] Sulfhydryl (thiol, mercapto): -SH.

[0187] Thioether (sulfide): -SR, where R is a thioether substituent, for example, C 1-7 Alkyl (also called C 1-7 Alkylthio), C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Alkyl. C 1-7 Examples of alkylthio groups include, but are not limited to, -SCH3 and -SCH2CH3.

[0188] Disulfide: -SS-R, where R is a disulfide substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Alkyl (also referred to herein as C 1-7 C 1-7 Examples of alkyl disulfide groups include, but are not limited to, -SSCH3 and -SSCH2CH3.

[0189] Sulfonium compound (sulfinyl, sulfoxide): -S(=O)R, where R is a sulfonium compound substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Examples of sulfonium groups include, but are not limited to, -S(=O)CH3 and -S(=O)CH2CH3.

[0190] Sulfone (sulfonyl): -S(=O)2R, where R is a sulfone substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7Alkyl groups, including, for example, fluorinated or perfluorinated C 1-7 Examples of sulfone groups include, but are not limited to, -S(=O)2CH3 (methylsulfonyl, mesyl), -S(=O)2CF3 (trifluoromethanesulfonyl), -S(=O)2CH2CH3 (ethylsulfonyl), -S(=O)2C4F9 (nonafluorobutanesulfonyl), -S(=O)2CH2CF3 (trifluoroethanesulfonyl), -S(=O)2CH2CH2NH2 (tauryl), -S(=O)2Ph (phenylsulfonyl, benzenesulfonyl), 4-methylphenylsulfonyl (toluenesulfonyl), 4-chlorophenylsulfonyl (chlorobenzenesulfonyl), 4-bromophenylsulfonyl (bromobenzenesulfonyl), 4-nitrophenylsulfonyl (nitrobenzenesulfonyl), 2-naphthalenesulfonate (naphthalenesulfonyl), and 5-dimethylamino-naphthalene-1-ylsulfonate (dansyl).

[0191] Sulfinic acid (sulfinyl group): -S(=O)OH, -SO2H.

[0192] Sulfonic acid (sulfo group): -S(=O)2OH, -SO3H.

[0193] Sulfinate (ester of sulfinic acid): -S(=O)OR, where R is a sulfinate substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Examples of sulfinate groups include, but are not limited to, -S(=O)OCH3 (methoxysulfinyl; methylsulfinate) and -S(=O)OCH2CH3 (ethoxysulfinyl; ethylsulfinate).

[0194] Sulfonate (ester of sulfonic acid): -S(=O)2OR, where R is a sulfonate substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Examples of sulfonate groups include, but are not limited to, -S(=O)2OCH3 (methoxysulfonyl; methylsulfonate) and -S(=O)2OCH2CH3 (ethoxysulfonyl; ethylsulfonate).

[0195] Sulfinyloxy: -OS(=O)R, where R is a sulfinyloxy substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Examples of sulfinyloxy groups include, but are not limited to, -OS(=O)CH3 and -OS(=O)CH2CH3.

[0196] Sulfonyloxy: -OS(=O)2R, where R is a sulfonyloxy substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Examples of sulfonyloxy groups include, but are not limited to, -OS(=O)2CH3 (mesylate) and -OS(=O)2CH2CH3 (ethanesulfonate).

[0197] Sulfate: -OS(=O)2OR; where R is a sulfate substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Examples of sulfate groups include, but are not limited to, -OS(=O)2OCH3 and -SO(=O)2OCH2CH3.

[0198] Sulfonamide (sulfonamide; sulfinic acid amide; sulfinamide): -S(=O)NR 1 R 2 , where R 1 and R 2 is independently an amino substituent, as defined for amino. Examples of sulfamoyl groups include, but are not limited to, -S(=O)NH2, -S(=O)NH(CH3), -S(=O)N(CH3)2, -S(=O)NH(CH2CH3), -S(=O)N(CH2CH3)2, and -S(=O)NHPh.

[0199] Sulfonamido (sulfonamide): -S(=O)2NR 1 R 2 , where R 1 and R 2 is independently an amino substituent, as defined for amino. Examples of sulfonylamino groups include, but are not limited to, -S(=O)2NH2, -S(=O)2NH(CH3), -S(=O)2N(CH3)2, -S(=O)2NH(CH2CH3), -S(=O)2N(CH2CH3)2, and -S(=O)2NHPh.

[0200] Sulfonamide: -NR 1 S(=O)2OH, where R 1 is an amino substituent, as defined for amino. Examples of sulfonylamino groups include, but are not limited to, -NHS(=O)2OH and -N(CH3)S(=O)2OH.

[0201] Sulfonamide: -NR 1 S(=O)2R, where R 1is an amino substituent, as defined for amino, and R is a sulfonylamino substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Examples of sulfonylamino groups include, but are not limited to, -NHS(=O)2CH3 and -N(CH3)S(=O)2C6H5.

[0202] Sulfamylamino group: -NR 1 S(=O)R, where R 1 is an amino substituent, as defined for an amino group, and R is a sulfonylamino substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Examples of sulfinylamino groups include, but are not limited to, -NHS(=O)CH3 and -N(CH3)S(=O)C6H5.

[0203] Phosphino: -PR2, where R is a phosphino substituent, e.g., -H, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably -H, C 1-7 Alkyl or C 5-20 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.

[0204] Phosphorus group: -P(=O)2.

[0205] Phosphinyl (phosphine oxide): -P(=O)R2, where R is a phosphinyl substituent, for example, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably C 1-7 Alkyl or C 5-20 Aryl. 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.

[0206] Phosphonic acid (phosphonyl): -P(=O)(OH)2.

[0207] Phosphonate (phosphonyl ester): -P(=O)(OR)2, where R is a phosphonate substituent, for example, -H, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably -H, C 1-7 Alkyl or C5-20 Aryl. Examples of phosphonate groups include, but are not limited to, -P(=O)(OCH3)2, -P(=O)(OCH2CH3)2, -P(=O)(Ot-Bu)2, and -P(=O)(OPh)2.

[0208] Phosphoric acid (phosphonooxy): -OP(=O)(OH)2.

[0209] Phosphate (phosphonooxy ester): -OP(=O)(OR)2, where R is a phosphate substituent, e.g., -H, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably -H, C 1-7 Alkyl or C 5-20 Aryl. Examples of phosphate groups include, but are not limited to, -OP(=O)(OCH3)2, -OP(=O)(OCH2CH3)2, -OP(=O)(Ot-Bu)2, and -OP(=O)(OPh)2.

[0210] Phosphorous acid: -OP(OH)2.

[0211] Phosphite: -OP(OR)2, where R is a phosphite substituent, e.g., -H, C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably -H, C 1-7 Alkyl or C 5-20 Examples of phosphite groups include, but are not limited to, -OP(OCH3)2, -OP(OCH2CH3)2, -OP(Ot-Bu)2, and -OP(OPh)2.

[0212] Phosphoramidite: -OP(OR 1 )-NR 2 2, where R 1 and R 2 is a phosphoramidite substituent, for example, -H, (optionally substituted)C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably -H, C 1-7 Alkyl or C 5-20 Examples of phosphoramidite groups include, but are not limited to, -OP(OCH2CH3)-N(CH3)2, -OP(OCH2CH3)-N(i-Pr)2, and -OP(OCH2CH2CN)-N(i-Pr)2.

[0213] Phosphoramide: -OP(=O)(OR 1 )-NR 2 2, where R1 and R 2 is a phosphoramide substituent, for example, -H, (optionally substituted)C 1-7 Alkyl, C 3-20 Heterocyclic or C 5-20 Aryl, preferably -H, C 1-7 Alkyl or C 5-20 Aryl. Examples of phosphoramide groups include, but are not limited to, -OP(=O)(OCH2CH3)-N(CH3)2, -OP(=O)(OCH2CH3)-N(i-Pr)2, and -OP(=O)(OCH2CH2CN)-N(i-Pr)2.

[0214] Alkylene

[0215] C 3-12 Alkylene: As used herein, the term "C 3-12 "Alkylene" refers to a bidentate moiety obtained by removing two hydrogen atoms (two hydrogen atoms removed from the same carbon atom or one hydrogen atom each removed from two different carbon atoms) from a hydrocarbon compound having 3 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and may be saturated, partially unsaturated or fully unsaturated. Thus, the term "alkylene" includes the subclasses alkenylene, alkynylene, cycloalkylene, etc. discussed below.

[0216] Straight chain saturated C 3-12 Examples of alkylene groups include, but are not limited to, -(CH2) n -, wherein n is an integer from 3 to 12, for example, -CH2CH2CH2-(propylene), -CH2CH2CH2CH2-(butylene), -CH2CH2CH2CH2CH2-(pentylene) and -CH2CH2CH2CH-2CH2CH2CH2-(heptylene).

[0217] Branched chain saturated C 3-12 Examples of alkylene groups include, but are not limited to, -CH(CH3)CH2-, -CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH(CH3)CH2-, -CH2CH(CH3)CH2CH2-, -CH(CH2CH3)-, -CH(CH2CH3)CH2-, and -CH2CH(CH2CH3)CH2-.

[0218] Straight chain partially unsaturated C 3-12 Alkylene (C 3-12Examples of alkenylene and alkynylene groups include, but are not limited to, -CH=CH-CH2-, -CH2-CH=CH2-, -CH=CH-CH2-CH2-, -CH=CH-CH2-CH2-, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH2-, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH2-, -CH=CH-CH=CH-CH2-, -CH=CH-CH=CH-CH2-, -CH=CH-CH=CH-CH2-, -CH=CH-CH2-CH=CH-, -CH=CH-CH2-CH=CH-, -CH=CH-CH2-CH=CH-, and -CH2-C≡C-CH2-.

[0219] Branched partially unsaturated C 3-12 Alkylene (C 3-12 Examples of alkenylene and alkynylene groups include, but are not limited to, -C(CH3)=CH-, -C(CH3)=CH-CH2-, -CH=CH-CH(CH3)-, and -C≡C-CH(CH3)-.

[0220] Alicyclic saturated C 3-12 Alkylene (C 3-12 Examples of cycloalkylene groups include, but are not limited to, cyclopentylene (eg, cyclopent-1,3-ylene) and cyclohexylene (eg, cyclohex-1,4-ylene).

[0221] Alicyclic partially unsaturated C 3-12 Alkylene (C 3-12 Examples of cycloalkylene groups include, but are not limited to, cyclopentenylene (e.g., 4-cyclopenten-1,3-yl), cyclohexenylene (e.g., 2-cyclohexen-1,4-yl; 3-cyclohexen-1,2-yl; 2,5-cyclohexadien-1,4-yl).

[0222] Among them C 3-12 Alkylene is interrupted by heteroatoms and the subscript refers to the number of atoms in the chain including the heteroatom. For example, the chain -C2H4-O-C2H4- would be a C5 group.

[0223] Among them C 3-12 The alkylene group is interrupted by an aromatic ring and the subscript refers to the number of atoms directly in the chain including the aromatic ring.

[0224] will be a C5 group.

[0225] Connection mark: In the style In , the superscript labels C(=0) and NH indicate the group to which the atom is bound. For example, an NH group is shown bound to a carbonyl group (which is not part of the depicted portion), and a carbonyl group is shown bound to an NH group (which is not part of the depicted portion).

[0226] Ligand unit

[0227] The ligand unit can be of any kind and includes proteins, polypeptides, peptides, and non-peptide agents that specifically bind to a target molecule. In some embodiments, the ligand unit can be a protein, polypeptide, or peptide. In some embodiments, the ligand unit can be a cyclic polypeptide. These ligand units can include antibodies or antibody fragments containing at least one target molecule binding site, lymphokines, hormones, growth factors, or any other cell binding molecules or substances that can specifically bind to a target.

[0228] The terms "specifically bind" and "specific binding" refer to the binding of an antibody or other protein, polypeptide or peptide to a predetermined molecule (e.g., an antigen). Typically, an antibody or other molecule binds to a predetermined molecule at a specific affinity of at least about 1 x 10 7 M -1 The binding affinity of the binding protein to the predetermined molecule is at least two times greater than the binding affinity to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or a closely related molecule.

[0229] Examples of Ligand units include those agents described in WO 2007 / 085930, incorporated herein.

[0230] In some embodiments, the Ligand unit is a cell binding agent that binds to an extracellular target on a cell. The cell binding agent can be a protein, polypeptide, peptide, or non-peptide agent. In some embodiments, the cell binding agent can be a protein, polypeptide, or peptide. In some embodiments, the cell binding agent can be a cyclic polypeptide. The cell binding agent can also be an antibody or an antigen-binding fragment of an antibody. Therefore, in one embodiment, the present invention provides an antibody-drug conjugate (ADC).

[0231] Cell Binding Agents

[0232] Cell binding agents can be of any kind and include peptides and non-peptides. These cell binding agents can include antibodies or antibody fragments containing at least one binding site, lymphokines, hormones, hormone mimetics, vitamins, growth factors, nutrient transport molecules or any other cell binding molecules or substances.

[0233] Peptides

[0234] In one embodiment, the cell binding agent is a linear or cyclic peptide comprising 4-30, preferably 6-20, consecutive amino acid residues. In this embodiment, it is preferred that a cell binding agent is linked to a monomeric or dimeric azetidinol and benzodiazepine Compound.

[0235] In one embodiment, the cell-binding agent comprises a νβ6 peptide. This peptide has an effect on α ν β6 is more selective than XYS.

[0236] In one embodiment, the cell binding agent includes an A20FMDV-Cys polypeptide. A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC. Alternatively, a variant of the A20FMDV-Cys sequence can 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. In addition, the polypeptide may have the sequence NAVXXXXXXXXXXXXXXXRTC.

[0237] Antibody

[0238] The term "antibody" is used in the broadest sense herein and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), multivalent antibodies, and antibody fragments, as long as they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that are able to recognize and bind to specific antigens. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th edition, Garland Publishing, New York). Target antigens typically have many binding sites, also referred to as epitopes, that are recognized by CDRs on multiple antibodies. Individual antibodies that specifically bind to different epitopes have different structures. Therefore, an antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules containing antigen binding sites that immunospecifically bind to an antigen or a portion thereof of a target, such targets including but not limited to one or more cancer cells that produce autoimmune antibodies associated with autoimmune diseases. Immunoglobulins can belong to any type (e.g., IgG, IgE, IgM, IgD, and IgA), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins can be derived from any species, including human, mouse, or rabbit sources.

[0239] "Antibody fragments" comprise a portion of a full-length antibody, typically its antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and scFv fragments; diabody molecules; linear antibodies; fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions), 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.

[0240] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for the possible presence of a small amount of naturally occurring mutations, the individual antibodies constituting the population are identical. Monoclonal antibodies have a high degree of specificity for a single antigenic site. In addition, in contrast to polyclonal antibody preparations comprising different antibodies for different determinants (epitopes), each monoclonal antibody is for a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" indicates that the feature of an antibody is obtained from a substantially uniform antibody population, and should not be construed as requiring the production of antibodies by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared by the hybridoma method originally described by Kohler et al. (1975) Nature 256:495, or can be prepared by a recombinant DNA method (see US4816567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597, or can be isolated from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459).

[0241] The monoclonal antibodies herein specifically include chimeric antibodies, humanized antibodies and human antibodies.

[0242] Examples of cell binding agents include those described for use in WO 2007 / 085930, which is incorporated herein.

[0243] Tumor-associated antigens and cognate antibodies for use in embodiments of the present invention are listed below and described in more detail on pages 14 to 86 of WO 2017 / 186894, which is incorporated herein.

[0244] (1) BMPR1B (bone morphogenetic protein receptor-IB type)

[0245] (2) E16 (LAT1, SLC7A5)

[0246] (3) STEAP1 (six-transmembrane epithelial antigen of the prostate)

[0247] (4) 0772P (CA125, MUC16)

[0248] (5) MPF (MPF, MSLN, SMR, megakaryocyte enhancer factor, mesothelin)

[0249] (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b)

[0250] (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, 25 sema domains, 7 thrombospondin repeats (type 1 and type 1-like), a transmembrane domain (TM) and a short cytoplasmic domain (semaphorin 5B)

[0251] (8)PSCA hlg(2700050C12Rik,C530008O16Rik,RIKEN cDNA 2700050C12,RIKENcDNA

[0252] 2700050C12 gene)

[0253] (9) ETBR (endothelin type B receptor)

[0254] (10) MSG783 (RNF124, hypothetical protein FLJ20315)

[0255] (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer

[0256] Related gene 1, prostate cancer-associated protein 1, six transmembrane epithelial cells of the prostate

[0257] Antigen 2, six transmembrane prostate protein

[0258] (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation 5 channel, subfamily M, member 4)

[0259] (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor)

[0260] (14) CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein Barr virus receptor) or Hs.73792)

[0261] (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-related β), B29)

[0262] (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain, containing phosphatase anchor protein 1a), SPAP1B, SPAP1C)

[0263] (17) HER2 (ErbB2)

[0264] (18) NCA (CEACAM6)

[0265] (19)MDP(DPEP1)

[0266] (20) IL20R-α (IL20Ra, ZCYTOR7)

[0267] (21) Brevican (BCAN, BEHAB)

[0268] (22)EphB2R(DRT,ERK,Hek5,EPHT3,Tyro5)

[0269] (23)ASLG659(B7h)

[0270] (24) PSCA (prostate stem cell antigen precursor)

[0271] (25)GEDA

[0272] (26) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3)

[0273] (27) CD22 (B cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814)

[0274] (27a)CD22 (CD22 molecule)

[0275] (28) CD79a (CD79A, CD79α), an immunoglobulin-related α, B cell-specific protein that covalently interacts with Igβ (CD79B) and forms a complex with Ig M molecules on the surface, transducing signals involved in B cell differentiation, pI: 4.84, MW: 25028TM: 2[P] gene chromosome: 19q13.2).

[0276] (29) CXCR5 (Burkitt lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine and plays a role in lymphocyte migration and humoral defense.

[0277] 10 plays a role in HIV-2 infection and perhaps in the development of AIDS, lymphoma, myeloma, and leukemia); 372aa, pI:8.54MW:41959TM:7[P] gene chromosome: 11q23.3,

[0278] (30) HLA-DOB (β subunit of MHC class II molecule (Ia antigen), which binds peptides and

[0279] 20 presents them to CD4+ T lymphocytes); 273aa, pI:6.56, MW:30820.TM:1[P] gene chromosome: 6p21.3)

[0280] (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability); 422aa), pI: 7.63,

[0281] MW:47206TM:1[P] gene chromosome: 17p13.3).

[0282] (32) CD72 (B cell differentiation antigen CD72, Lyb-2); 359aa, pI: 8.66, MW: 40225, TM: 15[P] gene chromosome: 9p13.3).

[0283] (33) LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function of which is associated with increased disease activity in patients with systemic lupus erythematosus); 661aa, pI: 6.20, MW: 74147TM: 1 [P] gene chromosome: 5q12).

[0284] (34) FcRH1 (Fc receptor-like protein 1, putative receptor for immunoglobulin Fc domains containing C2-type Ig-like and ITAM domains, which may play a role in the differentiation of B lymphocytes 20); 429aa, pI: 5.28, MW: 46925TM: 1[P] gene chromosome: 1q21-1q22)

[0285] (35) IRTA2 (immunoglobulin superfamily receptor translocation-associated 2, putative immune receptor with a possible role in B-cell development and lymphomagenesis; deregulation of the gene (via translocation) occurs in some B-cell malignancies); 977aa, pI: 6.88, MW: 106468, TM: 1[P] gene chromosome: 1q21)

[0286] (36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative 35-span membrane proteoglycan, EGF / heregulin family involved in growth factors and follistatin); 374aa)

[0287] (37) PSMA–FOLH1 (Folate hydrolase (prostate-specific membrane antigen) 1)

[0288] (38) SST (somatostatin receptor; note: there are 5 subtypes)

[0289] (38.1) SSTR2 (somatostatin receptor 2)

[0290] (38.2) SSTR5 (somatostatin receptor 5)

[0291] (38.3)SSTR1

[0292] (38.4)SSTR3

[0293] (38.5)SSTR4

[0294] AvB6 – two subunits (39+40)

[0295] (39) ITGAV (integrin, αV)

[0296] (40) ITGB6 (integrin, β6)

[0297] (41) CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5)

[0298] (42) MET (met oncogene; hepatocyte growth factor receptor)

[0299] (43)MUC1 (Mucin 1, cell surface associated)

[0300] (44) CA9 (Carbonic Anhydrase IX)

[0301] (45) EGFRvIII (epidermal growth factor receptor (EGFR), transcript variant 3,

[0302] (46)CD33 (CD33 molecule)

[0303] (47)CD19 (CD19 molecule)

[0304] (48) IL2RA (interleukin 2 receptor, α); NCBI reference sequence: NM_000417.2);

[0305] (49) AXL (AXL receptor tyrosine kinase)

[0306] (50) CD30-TNFRSF8 (tumor necrosis factor receptor superfamily, member 8)

[0307] (51) BCMA (B cell maturation antigen)-TNFRSF17 (tumor necrosis factor receptor superfamily, member 17)

[0308] (52) CT Ags–CTA (Cancer Testis Antigen)

[0309] (53) CD174 (Lewis Y)-FUT3 (fucosyltransferase 3 (galactoside 3 (4)-L-fucosyltransferase, Lewis blood type)

[0310] (54) CLEC14A (C-type lectin domain family 14, member A; Genbank accession number NM175060)

[0311] (55) GRP78–HSPA5 (heat shock 70 kDa protein 5 (glucose regulated protein, 78 kDa)

[0312] (56)CD70 (CD70 molecule) L08096

[0313] (57) Stem cell specific antigens. For example:

[0314] 5T4 (see entry (63) below)

[0315] CD25 (see above item (48))

[0316] CD32

[0317] LGR5 / GPR49

[0318] ·Prominin / CD133

[0319] (58)ASG-5

[0320] (59) ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase 3)

[0321] (60) PRR4 (proline rich 4 (tear gland))

[0322] (61) GCC–GUCY2C (Guanylate cyclase 2C (heat-stable enterotoxin receptor)

[0323] (62) Liv-1–SLC39A6 (solute carrier family 39 (zinc transporter), member 6)

[0324] (63) 5T4, trophoblast glycoprotein, TPBG – TPBG (trophoblast glycoprotein)

[0325] (64) CD56–NCMA1 (neural cell adhesion molecule 1)

[0326] (65) CanAg (tumor-associated antigen CA242)

[0327] (66) FOLR1 (Folate receptor 1)

[0328] (67) GPNMB (glycoprotein (transmembrane) nmb)

[0329] (68) TIM-1–HAVCR1 (Hepatitis A virus cellular receptor 1)

[0330] (69) RG-1 / prostate tumor target Mindin – Mindin / RG-1

[0331] (70) B7-H4–VTCN1 (V-set domain containing T cell activation inhibitor 1

[0332] (71) PTK7 (PTK7 protein tyrosine kinase 7)

[0333] (72)CD37 (CD37 molecule)

[0334] (73) CD138-SDC1 (syndecan 1)

[0335] (74) CD74 (CD74 molecule, major histocompatibility complex, class II invariant chain)

[0336] (75) Claudins (Integral membrane link proteins) – CLs (Claudins)

[0337] (76) EGFR (epidermal growth factor receptor)

[0338] (77) Her3 (ErbB3) – ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian))

[0339] (78)RON-MST1R (macrophage stimulatory 1 receptor (c-met-related tyrosine kinase))

[0340] (79) EPHA2 (EPH receptor A2)

[0341] (80) CD20–MS4A1 (4 transmembrane domains, subfamily A, member 1)

[0342] (81) Tenascin C (Tenascin C)–TNC (Tenascin C)

[0343] (82) FAP (fibroblast activation protein, α)

[0344] (83) DKK-1 (Dickkopf 1 homolog (Xenopus laevis))

[0345] (84)CD52 (CD52 molecule)

[0346] (85) CS1-SLAMF7 (SLAM family member 7)

[0347] (86) Endoglin-ENG (Endoglin)

[0348] (87) Annexin A1-ANXA1 (Annexin A1)

[0349] (88) V-CAM (CD106) - VCAM1 (vascular cell adhesion molecule 1)

[0350] Additional tumor-associated antigens and cognate antibodies of interest are:

[0351] (89)ASCT2 (ASC transporter 2, also known as SLC1A5).

[0352] ASCT2 antibodies are described in WO 2018 / 089393, which is incorporated herein by reference.

[0353] The cell binding agent may be labeled prior to incorporation as a conjugate or as part of a conjugate, for example to aid detection or purification of the agent. The label may be a biotin label. In another embodiment, the cell binding agent may be labeled with a radioactive isotope.

[0354] Treatment

[0355] The compounds of the present invention can be used in therapeutic methods. A therapeutic method is also provided, which comprises administering a therapeutically effective amount of a conjugate of Formula II to a subject in need of treatment. The term "therapeutically effective amount" is an amount sufficient to show benefit to the patient. Such benefit can be at least an improvement in at least one symptom. The actual amount administered and the rate and time course of administration will depend on the nature and severity of the subject to be treated. Treatment prescriptions, such as decisions on dosage, are within the responsibility of general practitioners and other doctors.

[0356] The conjugates may be administered alone or in combination with other treatments (simultaneously or sequentially, depending on the condition to be 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).

[0357] According to the present invention and the pharmaceutical composition used according to the present invention, in addition to the active ingredient (i.e., the conjugate of formula II), a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art may also be included. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other materials will depend on the route of administration, which may be oral or by injection, such as skin, subcutaneous or intravenous injection.

[0358] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. Tablets can contain solid carriers or adjuvants. Liquid pharmaceutical compositions usually contain liquid carriers, such as water, petroleum, animal or vegetable oils, mineral oils or synthetic oils. Physiological saline solutions, glucose or other sugar solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be contained. Capsules can contain solid carriers such as gelatin.

[0359] For intravenous, cutaneous or subcutaneous injection or injection at the painful part, the active ingredient will have the form of an acceptable aqueous solution for parenteral administration, which is pyrogen-free and has suitable pH, isotonicity and stability. The relevant technical personnel in this area are fully able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as required.

[0360] The conjugates may be used to treat proliferative diseases and autoimmune diseases.The term "proliferative disease" relates to unwanted or uncontrolled cell proliferation of excessive or abnormal cells, which is undesirable, such as neoplastic or hyperplastic growth (whether in vitro or in vivo).

[0361] Examples of proliferative conditions include, but are not limited to, benign, premalignant, and malignant cell proliferations, including, but not limited to, neoplasms and tumors (e.g., histiocytomas, gliomas, astrocytomas, osteomas), cancers (e.g., lung cancer, small cell lung cancer, gastrointestinal cancer, intestinal cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcomas, osteosarcomas, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g., fibroproliferative disorders of connective tissue), and atherosclerosis. Other cancers of interest include, but are not limited to, hematology; malignancies such as leukemias and lymphomas, such as non-Hodgkin's lymphoma, and subtypes such as DLBCL, marginal zone, mantle layer and follicular, Hodgkin's lymphoma, AML, and other cancers of B or T cell origin.

[0362] Examples of autoimmune diseases include the following: rheumatoid arthritis, autoimmune demyelinating diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine eye diseases, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune liver disease, inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergy, Sjögren's syndrome, type I diabetes, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis thyroiditis), autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcification, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), autoimmune infertility in men and women, ankylosing spondylitis, ulcerative colitis, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, allergic dermatitis, atopic rhinitis, Goodpasture's syndrome syndrome), Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, antiphospholipid syndrome, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki'sdisease), dengue fever, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, metachronous cyclitis, Fuchs' cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft-versus-host disease, transplant rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan's syndrome, and autoimmune gonadal failure.

[0363] 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), a disorder of 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 a disorder of Th2 lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, conjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft-versus-host disease). In general, disorders involving dendritic cells involve a disorder of Th1 lymphocytes or Th2 lymphocytes. In some embodiments, the autoimmune disorder is a T cell-mediated immunological disorder.

[0364] In some embodiments, the amount of the conjugate administered is in the range of about 0.01 mg / kg / dose to about 10 mg / kg / dose. In some embodiments, the amount of the conjugate administered is in the range of about 0.01 mg / kg / dose to about 5 mg / kg / dose. In some embodiments, the amount of the conjugate administered is in the range of about 0.05 mg / kg / dose to about 5 mg / kg / dose. In some embodiments, the amount of the conjugate administered is in the range of about 0.1 mg / kg / dose to about 5 mg / kg / dose. In some embodiments, the amount of the conjugate administered is in the range of about 0.1 mg / kg / dose to about 4 mg / kg / dose. In some embodiments, the amount of the conjugate administered is in the range of about 0.05 mg / kg / dose to about 3 mg / kg / dose. In some embodiments, the amount of the conjugate administered is in the range of about 0.1 mg / kg / dose to about 3 mg / kg / dose. In some embodiments, the amount of the conjugate administered is in the range of about 0.1 mg / kg / dose to about 2 mg / kg / dose.

[0365] Drug loading

[0366] The drug loading (p) is the average number of ABD drugs per cell binding agent (e.g., antibody). In the case of a compound of the invention conjugated to cysteine, the drug loading may be in the range of 1 to 8 drugs (D) per cell binding agent, wherein 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalently attached to the cell binding agent. The composition of the conjugate includes a collection of cell binding agents (e.g., antibodies) conjugated to a range of drugs (1 to 8). In the case of a compound of the invention conjugated to lysine, the drug loading may be in the range of 1 to 80 drugs (D) per cell binding agent, but an upper limit of 40, 20, 10, or 8 may be preferred. The composition of the conjugate includes a collection of cell binding agents (e.g., antibodies) conjugated to a range of drugs (1 to 80, 1 to 40, 1 to 20, 1 to 10, or 1 to 8).

[0367] The average number of drugs per antibody in the preparation of the ADC from the conjugation reaction can be characterized by conventional methods such as UV, reversed-phase HPLC, HIC, mass spectrometry, ELISA assays and electrophoresis. The quantitative distribution of ADCs in terms of p can also be determined. By ELISA, the average value of p in a specific preparation of ADC can be determined (Hamblett et al. (2004) Clin. Cancer Res. 10: 7063-7070; Sanderson et al. (2005) Clin. Cancer Res. 11: 843-852). However, the distribution of p (drug) values ​​cannot be distinguished by the antibody-antigen binding and detection limits of ELISA. In addition, the ELISA assay for detecting antibody-drug conjugates does not determine where the drug moiety is connected to the antibody, such as a heavy chain or light chain fragment, or a specific amino acid residue. In some cases, the separation, purification and characterization of homogeneous ADCs with a certain value of p and ADCs with other drug loadings can be achieved by means such as reversed-phase HPLC or electrophoresis. Such technology is also applicable to other types of conjugates.

[0368] For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, the antibody may have only one or a few cysteine ​​thiol groups, or may have only one or a few sufficiently reactive thiol groups available for attachment of a linker. Higher drug loadings, such as p>5, may result in aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates.

[0369] Typically, during the conjugation reaction, a drug moiety less than the theoretical maximum is conjugated to the antibody. The antibody may contain, for example, many lysine residues that do not react with the drug linker. Only the most reactive lysine groups can react with amine-reactive linker reagents. In addition, only the most reactive cysteine ​​thiol groups can react with thiol-reactive linker reagents. Typically, antibodies do not contain many (if any) free and reactive cysteine ​​thiol groups that can be connected to the drug moiety. Most of the cysteine ​​thiol residues in the antibody of the compound exist as disulfide bridges and must be reduced under partial or complete reducing conditions with a reducing agent such as dithiothreitol (DTT) or TCEP. The loading amount (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 conjugation reaction time or temperature, and (iii) partial or limiting reducing conditions for cysteine ​​thiol modification.

[0370] Some antibodies have reducible interchain disulfides, i.e. cysteine ​​bridges. Can be made reactive for conjugation with linker reagents by treating antibodies with reducing agents such as DTT (dithiothreitol). Each cysteine ​​bridge will therefore theoretically form two reactive thiol nucleophiles. Other nucleophilic groups can be introduced into antibodies by the reaction of lysine and 2-iminothiolane (Traut's reagent), which causes amines to be converted into thiols. Reactive thiol groups (e.g., preparing mutant antibodies comprising one or more non-natural cysteine ​​amino acid residues) can be introduced into antibodies (or their fragments) by engineering one, two, three, four or more cysteine ​​residues. US 7521541 teaches that antibodies are engineered by introducing reactive cysteine ​​amino acids.

[0371] Cysteine ​​amino acids can be engineered at reactive sites in antibodies and this does 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). The engineered cysteine ​​thiol can be reacted with a linker reagent or a drug-linker reagent of the invention having a thiol-reactive electrophilic group (such as maleimide or α-haloamide) to form an ADC having a cysteine ​​engineered antibody and an ABD drug moiety. The position of the drug moiety can therefore be designed, controlled and known. The drug loading can be controlled because the engineered cysteine ​​thiol group typically reacts with a thiol-reactive linker reagent or a drug linker reagent in high yield. Engineering IgG by introducing a cysteine ​​amino acid by substitution at a single site in the heavy or light chain resulted in two new cysteines on the symmetric antibody. Drug loadings of approximately 2 can be achieved by near homogeneity of the conjugated product ADC.

[0372] In case more than one nucleophilic or electrophilic group of an antibody is reacted with a drug-linker intermediate or linker reagent followed by a drug moiety reagent, then the resulting product is a mixture of ADC compounds with distributions of drug moieties attached to the antibody, e.g., 1, 2, 3, etc. Liquid chromatography methods such as polymer reverse phase (PLRP) and hydrophobic interaction (HIC) can separate the compounds in the mixture according to drug loading values. Preparations of ADCs with a single drug loading value (p) can be separated, however, these single loading value ADCs can still be heterogeneous mixtures because the drug moieties can be attached at different sites on the antibody via the linker.

[0373] Thus, the antibody-drug conjugate compositions of the invention include mixtures of antibody-drug conjugate compounds wherein the antibody has one or more ABD drug moieties and wherein the drug moieties may be attached to the antibody at different amino acid residues.

[0374] In one embodiment, each cell-binding agent is a dimer of azetidinone and benzodiazepine. The average number of groups ranges from 1 to 20. In some embodiments, the range is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4, and 4 to 8.

[0375] In some embodiments, there is one dimer azetidinone benzodiazepine per cell-binding agent. Group.

[0376] General synthetic route

[0377] A large number of suitable N-Prot N 、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, which is incorporated herein by reference.

[0378] Synthesis of compounds of formula IV

[0379] One possible procedure for the synthesis of compounds of the first aspect of the invention (in particular, compounds of formula IV) is shown in Scheme 1. This starts from the N10 protected ABD dimer (d1A).

[0380]

[0381] Dimer d1A is deprotected at the N10 position by standard methods to give compounds of formula IV. N In the case of Alloc, deprotection is carried out using palladium. Depending on the solvent used, the resulting compound can be in its carbinolamine or carbinolamine ether form, in equilibrium with the imine.

[0382] In the case of ABD, the ring strain of the four-membered azetidine ring means that the carbinolamine form predominates in the equilibrium.

[0383] An alternative procedure for the synthesis of compounds of Formula IV is shown in Scheme 2. This starts with the N10 nitrogen protected ABD monomer (m2A).

[0384]

[0385] The ABD monomer m2A protected at the C11 alcohol position at the N10 and Y8 positions is protected to obtain m2B. Preferably, Prot O The solution was TBS and protection was achieved by adding excess TBS-Cl. Y Subsequent deprotection of the -Y protecting group provides a dimerizable species (m2C). Y In the case of TIPS, deprotection can be achieved using LiOAc in DMF and water.

[0386] m2C is reacted with a dimer linker R"(X)2 to give the dimer d2D. Typically, Y is O and X is a halogen (preferably Br). In this case, the dimer is formed using a bis-Williamson ether synthesis using TBAI additive.

[0387] Remove the N10 protecting group from the dimer product to yield d2E. For example, if Prot N For Alloc and Prot O If Prot is an oxygen protecting group for synthesis, then deprotection is performed using palladium to remove the N10 protecting group and then eliminate the oxygen protecting group for synthesis. N For Troc and Prot O For the oxygen protecting group used in the synthesis, a Cd / Pb pair was used for deprotection. N is SEM or similar group, and Prot O If Prot is an oxo group, then the oxo group can be removed by reduction, which produces a protected carbinolamine intermediate, which can then be treated to remove the SEM protecting group followed by elimination of water. Removal of the alcohol protecting group at C11 provides a compound of formula IV. O Alcohol deprotection can occur simultaneously with the Alloc N deprotection described above using palladium and pyrrolidine in DCM for TBS.

[0388] The dimer d1A and monomer m2A required for Schemes 1 and 2, respectively, can be synthesized by several routes. One possible route via oxidative ring closure is shown in Scheme 3.

[0389]

[0390] Compounds 3A, 3B, 3C, 3D and 3E may be dimers (wherein the group R Y represents R”) or a monomer (where the group R Y represents a suitable protecting group).

[0391] Monomer 3A is a nitrobenzoic acid derivative. Many such derivatives are commercially available and other derivatives can be synthesized by conventional methods (e.g., Althuis, TH and Hess, HJ, J Medicinal Chem., 20 (1), 146-266 (1977)). Typically, nitrobenzoic acid is derived from an ester, obtained by ester hydrolysis under mild conditions (e.g., using LiOH). Dimer 3A can be prepared by various strategies disclosed in the prior art (e.g., Scheme 3 of WO 00 / 12508). For example, suitable benzoic acid esters can be dimerized by Mitsunobo etherification, followed by nitration and hydrolysis with respect to suitable diols. Alternatively, benzoic acid esters can be dimerized by Williamson ether synthesis with respect to suitable dihalides. Additional conversions required to obtain monomers and dimer 3A can be obtained in the literature.

[0392] The azetidine starting material can be synthesized by modification of the comparable proline synthesis disclosed in the prior art (e.g., Scheme 4 of WO 00 / 12508). Strategies particularly relevant to azetidine 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, commercially available azetidine-2-carboxylic acid can be protected at the nitrogen of the azetidine by a suitable protecting group such as Cbz and then acidic esterified to give the methyl ester. The ester can be reduced with LiBH4 in THF to give the Cbz-protected 2-(hydroxymethyl)azetidine. In some methods, a suitable protecting group (Prot O ), such as TBS, is added to the alcohol. In other methods, the alcohol is not protected. In Scheme 3, Prot O Can represent a suitable protecting group or H-suitable Prot O The group should be able to withstand NO2 reducing conditions.Next, the nitrogen protecting group is removed, typically by reduction under H2 gas, to produce the desired azetidine starting material in Scheme 3.

[0393] Compound 3A is condensed with an azetidine starting material to give 3B. The condensation is usually achieved by DCC coupling or by an acyl chloride (formed from a carboxylic acid with oxalyl chloride or SOCl2) or using HOBt in DCM at low temperature.

[0394] The nitro group of 3B is reduced to the amine (3C) using standard procedures such as SnCl2 in MeOH, or zinc in MeOH / H2O / formic acid (90:5:5), or sodium dithionite, or Raney nickel and hydrazine, or catalytic hydrogenation over palladium / charcoal. The method chosen depends on the need for a hydroxy protecting group.

[0395] The resulting amine is monoprotected with a suitable protecting group to give 3D. N The group is preferably a carbamate, such as N-Alloc. The nucleophilicity of the amine is reduced after protection with Alloc, so single protection is advantageous. Typically, this is achieved by reaction with pyridine and one equivalent of allyl chloroformate. When Prot O When Prot is H, 3D is equal to 3E. O When Prot is a protecting group, it is removed under standard conditions to give alcohol 3E. O If the protecting group is an acetate group, it can be removed under weak alkaline conditions (such as K2CO3), or if Prot O If it is a silyl ether protecting group, such as TBS, it can be removed by using TBAF or a weak acid.

[0396] Oxidative ring closure by aldehyde or functional equivalent affords d1A from dimer 3E for further reaction according to Scheme 1 and m2A from monomer 3E for further reaction according to Scheme 2. Selective alcohol-aldehyde oxidation can be achieved by exposure to tetrapropylammonium perruthenate (TPAP) in N-methylmorpholine N-oxide (NMO) over molecular sieves or by Swern oxidation (using DMSO and oxalyl chloride) or by Dess-Martin oxidation (using DMP) or preferably by Cu(I) / TEMPO free radical oxidation (using tetraacetonitrile copper(I) trifluoromethanesulfonate, 1-hydroxy-2,2,6,6-tetramethyl-piperidine (TEMPO), 1-methylimidazole and 2-(2-pyridyl)pyridine). The latter is advantageous because strict anhydrous conditions are not required and there is no evidence of overoxidation of the ABD bislactam species. The aldehyde species undergo spontaneous B ring closure, including attack of the singly protected N10 position.

[0397] An alternative route to obtain dimer d1A and monomer m2A is shown in Scheme 4. This route uses aldehyde unmasking to mediate the ring closure.

[0398]

[0399] Compounds 3A, 4B, 4C and 4D may be dimers (wherein the group R Y represents R”) or a monomer (where the group R Yrepresents a suitable protecting group).

[0400] 3A monomer and dimer variants can be generated by the strategy discussed above for Scheme 3. The azetidine starting material has a thioacetal at the 2 position (but other masked aldehyde equivalents can be used). Diethylthioacetal azetidine can be prepared by modification of a similar proline synthesis strategy (e.g., Langley, DR & Thurston, DE, J Organic Chemistry, 52, 91-97 (1987)). Routes specifically related to azetidine 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, Cbz-protected 2-(hydroxymethyl)azetidine can be prepared as described above (for Scheme 3). The alcohol is then reoxidized to an aldehyde, typically by Dess-Martin oxidation (using DMP) or IBX in DMSO. Preferably, the resulting aldehyde is condensed with a thiol such as EtSH using a weak acid catalyst such as TMSCl in a protic solvent to obtain a thioacetal. Thioacetal is incompatible with H2 gas reduction, so the N protecting group (e.g., Cbz) is usually removed using TMS-I in DCM. This produces a diethylthioacetal azetidine starting material.

[0401] Direct condensation of 3A with the thioacetal azetidine starting material affords 4B. The nitro group of 4B can be reduced to the amine (4C) by the methods discussed above for Scheme 3, preferably by the tin(II) chloride method (SnCl2 in MeOH) or zinc in MeOH / H2O / formic acid (90:5:5). Due to the incompatibility of the thioacetal group, reduction is preferably not performed by direct hydrogenation. The amine is monoprotected with a suitable amine protecting group such as Alloc by reaction with the corresponding chloroformate or acyl chloride. The N-Prot of 4D N The group is preferably a carbamate, such as N-Alloc, since these species facilitate monoprotection.

[0402] Selective demasking of the aldehyde by the thioacetal leads to spontaneous cyclization of the B ring due to attack on the singly protected N10 position. Typically, the demasking is mediated by mercury(II), such as HgCl2 and CaCO3 in acetonitrile:water. For dimer 4D, this provides d1A (for further reaction according to Scheme 1), and for monomer 4D, this provides m2A (for further reaction according to Scheme 2).

[0403] Dimeric or monomeric thioacetal 4B (according to Scheme 4) can be synthesized by an alternative route, shown in Scheme 5. This route generates the thioacetal in situ.

[0404]

[0405] The synthetic strategy to achieve monomer and dimer 3A is discussed above with respect to Scheme 3. Condensation of 3A with commercially available azetidine-2-carboxylic acid affords 5B. The route from 5B to 4B follows a similar approach to the synthesis of the thioacetal azetidine starting material from azetidine-2-carboxylic acid (as discussed with respect to Scheme 4).

[0406] 5C is reduced to secondary alcohol 5D by a hydride reducing agent, typically by LiBH4. 5D is then reoxidized to aldehyde (5E), typically by a hypervalent iodine species (e.g., IBX or DMP). The thioacetal is generated in situ, preferably using EtSH under acidic conditions, to provide compound 4B. This can be further reacted according to Scheme 4 to give the desired ABD species.

[0407] Synthesis of compounds of formula I

[0408] One possible procedure for synthesizing the compounds of the second aspect of the invention (specifically, compounds of Formula I) is shown in Scheme 6. This starts with two cyclized ABD monomers: one with Prot N m2A with protected N10 position and R L Additional N10 nitrogen to m6A.

[0409] Depending on the solvent used, compounds of Formula I may exist in equilibrium between the imine and either the carbinolamine or carbinolamine ether forms (similar to the equilibrium shown in Scheme 1 for Formula IV).

[0410] In some embodiments, Prot N Can be equivalent to R of Formula I 30 Substituents (described by options (d) i, ii and iii of the second aspect of the invention).

[0411]

[0412] The m2A starting material can be prepared according to Schemes 3, 4 and 5. N Equivalent to R 30 For compounds with a carbamate linker, m2A is prepared via isocyanate (i.e., the same route as m6A - discussed below).

[0413] Using a similar strategy as described for Scheme 2, m2A and m6A can be dimerized about the Y8 position using a dimer linker R". The alcohol at the C11 position is represented by Prot O Protection, where Prot O TBS is preferred and introduced by reaction with TBS-Cl.Y Subsequent removal of the group, where Prot Y For TIPS, LiOAc in DMF and water can be used to generate m2C and m6B, respectively.

[0414] m2C and m6B are further reacted with R”(X)2 to give the dimer d6C. Typically, Y is O and X is a halogen (preferably Br). In this case, the TBAI additive can drive the bis-Williamson ether synthesis to form the dimer. Alternative strategies for dimerization are also known in the art, such as by Mitsonobu etherification.

[0415] In some embodiments, the N10 protecting group is removed from the non-linker ABD to give the asymmetric dimer d6D. Various deprotection strategies are discussed in relation to Schemes 1 and 2. N In the case of Alloc, palladium can be used for deprotection.

[0416] In other embodiments, the N10 protecting group is not removed. O d6C is directly converted to the compound of formula I.

[0417] Removal of the alcohol protecting group at C11 provides an asymmetric compound of Formula I. O If TBS is used, alcohol deprotection can occur simultaneously with Alloc N10 deprotection using palladium and pyrrolidine in DCM.

[0418] A possible synthesis of monomer m6A (required for Scheme 6) and an alternative route to compounds of Formula I are shown in Scheme 7.

[0419]

[0420] Compounds 7A, 7B, 7C and 7D may be dimers (wherein the group R YY represents R" attached to a monoprotected ABD precursor at position N10) or a monomer (wherein the group R YY represents a suitable protecting group). Monomer 7A is identical to monomer 3C and can be synthesized by a similar route (as shown in Scheme 3).

[0421] The amine of 7A is converted to isocyanate 7B. Strategies for isocyanate formation are described in detail in the prior art (e.g., WO 2005 / 023814). Typically, phosgene is used, preferably triphosgene under alkaline conditions; solid triphosgene crystals are safer and easier to handle than toxic phosgene gas. The reaction should be carried out in an anhydrous and non-hydroxylic organic solvent, preferably a non-polar solvent. Suitable solvents include anhydrous DCM and anhydrous toluene. The reaction can be carried out at room temperature and is characterized by infrared spectroscopy at about 2265cm-1 Conveniently monitor.

[0422] Carbamate 7C is synthesized by R L The attack of -OH is by isocyanate formation. The carbamate formation is usually achieved by a one-pot process, in which the isocyanate is formed by triphosgene and TEA in DCM and R L The -OH is added directly to the reaction mixture. This approach reduces the residence time of the isocyanate before the carbamate is formed, thus reducing the chance of side reactions.

[0423] Remove Prot by appropriate method O Protecting groups are provided to provide secondary alcohols 7D, typically under acidic conditions (eg, acetic acid in THF:water solvent).

[0424] Oxidative ring closure of 7D with an aldehyde or functional equivalent can be achieved by exposure to tetrapropylammonium perruthenate (TPAP) in N-methylmorpholine N-oxide (NMO) over molecular sieves or by Swern oxidation (DMSO and oxalyl chloride) or preferably by Cu(I) / TEMPO radical oxidation (tetraacetonitrile copper(I) trifluoromethanesulfonate, 1-hydroxy-2,2,6,6-tetramethyl-piperidine (TEMPO), 1-methylimidazole and 2-(2-pyridyl)pyridine). This is true for the monomeric 7D variant (i.e., where R YY =Prot Y ) to give m6A (for further reaction in Scheme 6), or for the dimeric 7D variant (i.e., in which R YY =R" linked to N10 protected ABD) to give d7E.

[0425] Nitrogen protecting group (Prot N Removal of ) (Alloc which is typically removed with palladium) provides the asymmetric compound of formula I.

[0426] Alternative routes to monomeric m6A (Scheme 6) and dimeric d7E (Scheme 7) are shown in Scheme 8.

[0427]

[0428] Compounds 8A, 8B and 8C may be dimers (wherein the group R YY represents R" attached to a monoprotected ABD precursor at the N10 position) or a monomer (wherein the group R YY represents a suitable protecting group Prot Y ). 8A is converted to isocyanate 8B, which then reacts with R L -OH reacts to attach R via carbamate L The preferred strategy is similar to that discussed with respect to Scheme 7.

[0429] Selective demasking of the aldehyde by the thioacetal 8C results in spontaneous cyclization of the B ring due to attack of the singly protected N10 position. Typically, demasking is mediated by mercury (II), such as HgCl2 and CaCO3 in acetonitrile:water. Cyclization provides monomer m6A (from monomer 8C) and dimer d7E (from dimer 8C). m6A can be reacted according to Scheme 6, and d7E can be reacted according to Scheme 7 to produce compounds of Formula I.

[0430] For monomer 7A (i.e., where R YY For Prot Y ), the starting materials of Scheme 7 can be obtained by a route similar to Scheme 3 (monomers 3A to 3C). Similarly, for monomer 8A (i.e., where R YY For Prot Y ), the starting materials of Scheme 8 can be obtained by a route similar to Scheme 4 (monomers 3A to 4C).

[0431] The route to obtain dimers 7A and 8A (i.e., where R YY is R″ attached to the ABD precursor singly protected at the N10 position) is shown in Scheme 9.

[0432]

[0433] Dimeric variants of 3C and 4C were generated by the strategies discussed in Schemes 3, 4, and 5. 3C and 4C were protected at only one N10 position to generate asymmetric ABD dimers 7A and 8A, respectively. This was accomplished by adding one equivalent of the protecting agent (when Prot N In the case of Alloc, it is typically allyl chloroformate), and subsequent purification to remove unprotected or doubly protected products.

[0434] Synthesis of the conjugate of formula II

[0435] Synthesis of the conjugate of the third aspect of the invention (specifically, the component drug linker unit (D L One possible step of )) involves attaching a linker to the Ligand unit, thereby attaching the group R L (Compound according to formula I) is converted into a group R LL (Compound according to formula I').

[0436] Conjugates can be prepared as previously described. Antibodies can be conjugated to drug linker compounds 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 to reduce interchain disulfides was monitored by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) and allowed to proceed until the desired level of sulfhydryl / mAb was achieved. The reduced antibody was then cooled to 0°C and alkylated with 1.5 equivalents of maleimide drug-linker per antibody sulfhydryl. After 1 hour, the reaction was quenched by adding 5 equivalents of N-acetylcysteine. The quenched drug-linker was removed by gel filtration on a PD-10 column. The ADC was then sterile filtered through a 0.22 μm syringe filter. Protein concentration can be determined by spectroscopic analysis at 280 nm and 329 nm, respectively, correcting for the contribution of drug absorbance at 280 nm. The extent of antibody aggregation can be determined using size exclusion chromatography, and the level of remaining NAC-quenched drug-linker can be determined using RP-HPLC.

[0437] Synthesis of macrocyclic embodiments

[0438] In some embodiments of the first, second and third aspects of the invention, R 7 and R 7 The substituents may together form a group, said group being: (i) -O-(CH2) n -O-, wherein n is 7 to 16, or (ii) -O-(CH2CH2O) m -, wherein m is 2 to 5, to obtain a macrocyclic ABD dimer.

[0439] Various strategies can be used to introduce R 7 -R 7 ' linker, as shown in Scheme 10 below. Starting from d2D, where R 7 and R 7 ' represents -OR, and the R group can be removed by adding BBr3 in DCM to reveal the alcohol. Substitution reactions of dibromoalkanes in bases (such as 1,7-dibromoheptane with K2CO3) give macrocyclic products through the attack of two C7 alcohols.

[0440] An alternative route starting from d2D involves substitution of the C7 alcohol with n-bromoalkane-1-ene. This provides two terminally unsaturated alkenyl chains that can readily undergo ring-closing metathesis (RCM). For example, substitution can be achieved using 5-bromopent-1-ene and RCM using Grubs-II catalyst. Macrocyclization achieved by RCM is generally high yielding.

[0441] The preferred route to the macrocycle starts from the dimer 3A or an ester precursor, where R 7 and R 7' All represent -OR. Removal of the R group and substitution with a dibromoalkane (using similar conditions as above) provides the macrocyclic compound. ABD can then be obtained according to Schemes 3 or 4.

[0442]

[0443] The resulting product can be reacted via Scheme 2 or Scheme 6 to obtain compounds of Formula IV and I, respectively.

[0444] Additional transformation details required to obtain such macrocyclic products are available in the literature (Donnell, AF, Zhang, Y., Stang, EM, Wei, DD, Tebben, AJ, Perez, HL, Schroeder, GM, Pan, C., Rao, C., Borzilleri, RM, Vite, GD, Gangwar, S., Macrocyclic pyrrolobenzodiazepinedimers as antibody-drug conjugate payloads, Bioorganic & Medicinal Chemistry Letters (2017), doi: https: / / doi.org / 10.1016 / j.bmcl.2017.10.028 and WO 2016 / 209951).

[0445] Synthesis of Secondary Amine Embodiments

[0446] Compounds in which the N10-C11 group is -NH-CH2- (i.e., secondary amines) can be synthesized by modification of the above procedure. Specifically, reductive amination of compound 3B* can produce modified versions of m2A or d1A for use in additional steps:

[0447]

[0448] Compound 3B* can be synthesized by oxidation from a precursor alcohol which can be obtained by steps similar to those used to synthesize 3B.

[0449] Synthesis of drug conjugates

[0450] Conjugates can be prepared as previously described. Antibodies can be conjugated to drug linker compounds 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 to reduce interchain disulfides was monitored by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) and allowed to proceed until the desired level of sulfhydryl / mAb was achieved. The reduced antibody was then cooled to 0°C and alkylated with 1.5 equivalents of maleimide drug-linker per antibody sulfhydryl. After 1 hour, the reaction was quenched by adding 5 equivalents of N-acetylcysteine. The quenched drug-linker was removed by gel filtration on a PD-10 column. The ADC was then sterile filtered through a 0.22 μm syringe filter. Protein concentration can be determined by spectroscopic analysis at 280 nm and 329 nm, respectively, correcting for the contribution of drug absorbance at 280 nm. The extent of antibody aggregation can be determined using size exclusion chromatography, and the level of remaining NAC-quenched drug-linker can be determined using RP-HPLC. BRIEF DESCRIPTION OF THE DRAWINGS

[0451] Figure 1 Shown are the effects on the growth of tumor cell lines when treated by a control or by a conjugate of the invention. DETAILED DESCRIPTION

[0452] Other Preferences

[0453] The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect.The preferences may be combined together in any combination.

[0454] R 6’ and R 9’ Selected from R 6 and R 9 In some embodiments, R 6’ , R 7’ , R 9’ and Y' are respectively 6 , R 7 , R 9 Same as Y.

[0455] Dimer connection

[0456] In some embodiments, Y and Y' are both O.

[0457] In some embodiments, R" is an unsubstituted C 3-7Alkylene. In some of these embodiments, R" is C3, C5 or C7 alkylene. Specifically, R" can be C3 or C5 alkylene.

[0458] In other embodiments, R" is a group of the formula:

[0459]

[0460] Where r is 1 or 2.

[0461] The phenylene group may be replaced by a pyridylene group.

[0462] R 6 To R 9

[0463] In some embodiments, R 9 It's H.

[0464] In some embodiments, R 6 is selected from H, OH, OR, SH, NH2, nitro and halogen, and may be selected from H or halogen. In some of these embodiments, R 6 It's H.

[0465] In some embodiments, R 7 is selected from H, OH, OR, SH, SR, NH2, NHR, NRR' and halogen. In some of these embodiments, R 7 is selected from H, OH and OR, wherein R is selected from optionally substituted C 1-7 Alkyl, C 3-10 Heterocyclic and C 5-10 Aryl. R may more preferably be C 1-4 Alkyl, which may or may not be substituted. The substituent of interest is C 5-6 Aryl (e.g., phenyl). Particularly preferred substituents at position 7 are OMe and OCH2Ph. Other substituents of particular interest are dimethylamino (i.e., –NMe2); -(OC2H4) q OMe, wherein q is 0 to 2; nitrogen-containing C6 heterocyclic groups including morpholino, piperidinyl and N-methyl-piperazinyl.

[0466] These embodiments and preferences are respectively applicable to R 9’ , R 6’ and R 7’ .

[0467] In other embodiments, R 7 and R 7’ Together they form -O-(CH2) n-O-, wherein n is 7 to 16. n may be at least 7, 8, 9, 10 or 11. N may be at most 16, 15, 14 or 13.

[0468] In other embodiments, R 7 and R 7’ Together they form -O-(CH2CH2O) m -, wherein m is 2 to 5. m may be at least 2, 3 or 4. m may be at most 5, 4 or 3.

[0469] R 10 , R 11 , R 20 , R 21 (Formula IV)

[0470] In some embodiments, R 10 and R 11 Together, they form a double bond between the N atom and the C atom to which they are bound. In some of these embodiments, R 20 and R 21 Together, they form a double bond between the N atom and the C atom to which they are bound. In other of these embodiments, R 20 and R 21 Both are H.

[0471] In some embodiments, R 10 H and R 11 Selected from OH and OR A , where R A C 1-4 In some of these embodiments, R 20 H and R 21 Selected from OH and OR B , where R B C 1-4 In other of these embodiments, R 20 and R 21 Both are H.

[0472] In some embodiments, R 10 and R 11 are H. In some of these embodiments, R 20 and R 21 Together, they form a double bond between the N atom and the C atom to which they are bound. In other of these embodiments, R 20 H and R 21 Selected from OH and OR B , where R B C 1-4 alkyl.

[0473] In some embodiments, R A In some embodiments, R B It is methyl.

[0474] In some embodiments, R 10 and R 11 For and R 20 and R 21 Only one of the pairs is both H. In other embodiments, R 10 and R 11 For and R 20 and R 21 None of the pairs are both H.

[0475] In some embodiments, R 10 , R 11 , R 20 and R 21 All are H.

[0476] N10'-C11' (Formula I and I*)

[0477] In some embodiments, R 30 and R 31 Together they form a double bond between the N atom and the C atom to which they are bound.

[0478] In some embodiments, R 30 H and R 31 Selected from OH and OR B , where R B C 1-4 In some of these embodiments, R B It is methyl.

[0479] In some embodiments, R 30 H and R 31 For H.

[0480] In some embodiments, R 31 OH or OR B , where R B C 1-4 Alkyl and R 30 Selected from:

[0481]

[0482]

[0483] -C(=O)-X1-NHC(=O)X2-NH- represents a dipeptide. The amino acids in the dipeptide can be any combination of natural amino acids. The dipeptide can be the action site of cathepsin-mediated cleavage.

[0484] In one embodiment, the dipeptide -C(=O)-X1-NHC(=O)X2-NH- is selected from:

[0485] -Phe-Lys-,

[0486] -Val-Ala-,

[0487] -Val-Lys-,

[0488] -Ala-Lys-,

[0489] -Val-Cit-,

[0490] -Phe-Cit-,

[0491] -Leu-Cit-,

[0492] -Ile-Cit-,

[0493] -Phe-Arg-,

[0494] -Trp-Cit-,

[0495] Where Cit is citrulline.

[0496] Preferably, the dipeptide -C(=O)-X1-NHC(=O)X2-NH- is selected from:

[0497] -Phe-Lys-,

[0498] -Val-Ala-,

[0499] -Val-Lys-,

[0500] -Ala-Lys-,

[0501] -Val-Cit-.

[0502] More preferably, the dipeptide -C(=O)-X1-NHC(=O)X2-NH- is -Phe-Lys- or -Val-Ala-.

[0503] Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, which is incorporated herein by reference.

[0504] In one embodiment, the amino acid side chains are derivatized, where appropriate. For example, the amino or carboxyl groups of the amino acid side chains may be derivatized.

[0505] In one embodiment, the amino group NH2 of a side chain amino acid such as lysine is in a derivatized form selected from the group consisting of NHR and NRR'.

[0506] In one embodiment, the carboxyl group COOH of a side chain amino acid such as aspartic acid is a derivatized form selected from the group consisting of COOR, CONH2, CONHR and CONRR'.

[0507] In one embodiment, when appropriate, the amino acid side chain is chemically protected. The side chain protecting group can be a group as discussed above. The inventors have confirmed that the protected amino acid sequence can be cleaved by enzymes. For example, it has been confirmed that the dipeptide sequence of the Lys residue containing the Boc side chain protection can be cleaved by cathepsin.

[0508] Protecting groups for amino acid side chains are well known in the art and are described in the Novabiochem catalog. Additional protecting group strategies are described in Protective Groups in Organic Synthesis, Greene and Wuts.

[0509] Possible side chain protecting groups for those amino acids having reactive side chain functionalities are shown below:

[0510] Arg: Z, Mtr, Tos;

[0511] Asn: Trt, Xan;

[0512] Asp: Bzl, t-Bu;

[0513] Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;

[0514] Glu: Bzl, t-Bu;

[0515] Gln: Trt, Xan;

[0516] His: Boc, Dnp, Tos, Trt;

[0517] Lys: Boc, Z-Cl, Fmoc, Z, Alloc;

[0518] Ser: Bzl, TBDMS, TBDPS;

[0519] Thr: Bz;

[0520] Trp: Boc;

[0521] Tyr: Bzl, Z, Z-Br.

[0522] In one embodiment, the side chain protection is selected so as to be orthogonal to the group provided as a capping group (if present) or a portion thereof. Thus, removal of the side chain protecting group does not remove the capping group, or any protecting group functional group that is a part of the capping group.

[0523] In other embodiments of the invention, the selected amino acids are those that do not have reactive side chain functional groups. For example, the amino acids may be selected from: Ala, Gly, Ile, Leu, Met, Phe, Pro and Val.

[0524] It is particularly preferred in the present invention that if Q comprises a dipeptide, then -C(=O)-X1-NHC(=O)X2-NH- are the same dipeptide. Examples of preferred groups are:

[0525]

[0526] Other preferred R 30 Groups include:

[0527]

[0528] R 11b (Formula I and I*)

[0529] In some embodiments, R 11b It's OH.

[0530] In some embodiments, R 11b Yes OR A , where R A It is C 1-4 In some of these embodiments, R A It's methyl.

[0531] Other formula

[0532] In some embodiments of the first aspect of the present invention, it has formula IVa, IVb or IVc:

[0533]

[0534] Where R 1a is selected from methyl and benzyl;

[0535] R 10 , R 11 , R 20 and R 21 As defined above.

[0536] In some embodiments of the second aspect of the invention, it has Formula Ia, Ib or Ic:

[0537] Where R 1a is selected from methyl and benzyl;

[0538] R 30 , R 31 , R L and R 11b As defined above.

[0539] These embodiments and preferences also apply to the third aspect of the present invention.

[0540] Connector (R L )

[0541] In some embodiments, R L It has the formula IIIa.

[0542] In some embodiments, R LL It has the formula IIIa'.

[0543] G L

[0544] G L Can be selected from

[0545]

[0546]

[0547] Where Ar represents C 5-6 Arylene groups, such as phenylene groups.

[0548] In some embodiments, G L Selected from G L1-1 and G L1-2 In some of these embodiments, G L It's G L1-1 .

[0549] G LL

[0550] G LL You can choose from:

[0551]

[0552] Where Ar represents C 5-6 Arylene groups, such as phenylene groups.

[0553] In some embodiments, G LL Selected from G LL1-1 and G LL1-2 In some of these embodiments, G LL It's G LL1-1 .

[0554] X

[0555] X is:

[0556]

[0557] Wherein a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5.

[0558] a can be 0, 1, 2, 3, 4, or 5. In some embodiments, a is 0 to 3. In some of these embodiments, a is 0 or 1. In other embodiments, a is 0.

[0559] b can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b is from 0 to 12. In some of these embodiments, b is from 0 to 8, and can be 0, 2, 4, or 8.

[0560] c can be 0 or 1.

[0561] d can be 0, 1, 2, 3, 4, or 5. In some embodiments, d is from 0 to 3. In some of these embodiments, d is 1 or 2. In other embodiments, d is 2.

[0562] In some embodiments of X, a is 0, c is 1 and d is 2, and b can be 0 to 8. In some of these embodiments, b is 0, 4 or 8.

[0563] Q

[0564] In one embodiment, Q is an amino acid residue. The amino acid can be a natural amino acid or an unnatural amino acid.

[0565] In one embodiment, Q is selected from the group consisting of: Phe, Lys, Val, Ala, Cit, Leu, He, Arg and Trp, wherein Cit is citrulline.

[0566] In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide can be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. In the case where the linker is a cathepsin-labile linker, the dipeptide is the action site of cathepsin-mediated cleavage. The dipeptide is then a recognition site for cathepsin.

[0567] In one embodiment, Q is selected from:

[0568] CO -Phe-Lys- NH ,

[0569] CO -Val-Ala-NH ,

[0570] CO -Val-Lys- NH ,

[0571] CO -Ala-Lys- NH ,

[0572] CO -Val-Cit- NH ,

[0573] CO -Phe-Cit- NH ,

[0574] CO -Leu-Cit- NH ,

[0575] CO -Ile-Cit- NH ,

[0576] CO -Phe-Arg- NH ,as well as

[0577] CO -Trp-Cit- NH ;

[0578] Where Cit is citrulline.

[0579] Preferably, Q is selected from:

[0580] CO -Phe-Lys- NH ,

[0581] CO -Val-Ala- NH ,

[0582] CO -Val-Lys- NH ,

[0583] CO -Ala-Lys- NH ,

[0584] CO -Val-Cit- NH .

[0585] Most preferably, Q is selected from CO -Phe-Lys- NH , CO -Val-Cit- NH andCO -Val-Ala- NH .

[0586] Other dipeptide combinations of interest include:

[0587] CO -Gly-Gly- NH ,

[0588] CO -Pro-Pro- NH ,as well as

[0589] CO -Val-Glu- NH .

[0590] Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, which is incorporated herein by reference.

[0591] In some embodiments, Q X is a tripeptide residue. The amino acids in the tripeptide can be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. When the linker is a cathepsin labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide is then a recognition site for cathepsin. Particularly interesting tripeptide linkers are:

[0592] CO -Glu-Val-Ala- NH

[0593] CO -Glu-Val-Cit- NH

[0594] CO -αGlu-Val-Ala- NH

[0595] CO -αGlu-Val-Cit- NH

[0596] In one embodiment, the amino acid side chains are chemically protected when appropriate. The side chain protecting group can be a group as discussed below. The protected amino acid sequence can be cleaved by an enzyme. For example, a dipeptide sequence containing a Lys residue protected by a Boc side chain can be cleaved by a cathepsin.

[0597] Protecting groups for amino acid side chains are well known in the art and are described in the Novabiochem catalogue and as described above.

[0598] In some embodiments, R L Has the formula IIIb.

[0599] In some embodiments, R LL Has the formula IIIb'.

[0600] R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form cyclopropylene or cyclobutylene.

[0601] In some embodiments, R L1 and R L2 Both are H.

[0602] In some embodiments, R L1 is H and R L2 It's methyl.

[0603] In some embodiments, R L1 and R L2 All are methyl.

[0604] In some embodiments, R L1 and R L2 Together with the carbon atom to which they are attached they form a cyclopropylene group.

[0605] In some embodiments, R L1 and R L2 Together with the carbon atom to which they are attached they form a cyclobutylene group.

[0606] In group IIIb, in some embodiments, e is 0. In other embodiments, e is 1 and the nitro group can be in any available position of the ring. In some of these embodiments, it is in the ortho position. In other of these embodiments, it is in the para position.

[0607] In a specific embodiment, the second aspect of the invention comprises compounds of formula Id:

[0608]

[0609] Where Q is selected from:

[0610] (a) -CH2-;

[0611] (b) -C3H6-; and

[0612] (c)

[0613] In a specific embodiment, the third aspect of the invention, the drug linker (D L ) has the formula (Id'):

[0614]

[0615] Where Q is selected from:

[0616] (a) -CH2-;

[0617] (b) -C3H6-; and

[0618] (c)

[0619] In some embodiments of the invention, the C11 substituent may be in the following stereochemical arrangement relative to adjacent groups:

[0620]

[0621] In other embodiments, the C11 substituent may be in the following stereochemical arrangement relative to adjacent groups:

[0622]

[0623] Example

[0624] General information

[0625] Manual flash chromatography was performed using Merck Kieselgel 60F254 silica gel. Extraction and chromatography solvents were purchased from Fisher Scientific, UK and used without further purification. All chemicals were purchased from Aldrich, Lancaster or BDH.

[0626] Using Biotage Isolera 1 TM Automated flash chromatography was performed using a gradient elution starting from 88% hexane / EtOAc or 99.9% DCM / MeOH until all UV active components were eluted from the column (detected at 214nm and 254nm). Whenever a large amount of elution of UV active material was observed, the gradient was manually maintained. The purity of the fractions was checked with thin layer chromatography (TLC) using Merck Kieselgel 60F254 silica gel using a fluorescent indicator on an aluminum plate. Unless otherwise stated, visualization of TLC was achieved with the aid of UV light or iodine vapor. Extraction and chromatographic solvents were purchased from VWR UK and used without further purification. All fine chemicals were purchased from Sigma-Aldrich or TCI Europe, unless otherwise stated. Pegylation reagents were obtained from Quanta biodesign US by Stratech UK.

[0627] LC / MS conditions were as follows:

[0628] Positive mode electrospray mass spectrometry was performed using Waters Aquity H-class. The mobile phases used were solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid).

[0629] LCMS 3min: Initial composition 5% B, hold 0.25min, then increase from 5% B to 100% B over a 2min period. Hold composition at 100% B for 0.50min, then return to 5% B over 0.05min and hold for 0.05min. Total gradient run time equals 3min. Flow rate 0.8mL / min. Detection at 254nm. Column: Waters Acquity BEH Shield RP18 1.7μm 2.1x50mm, 50℃, equipped with Waters Acquity BEH ShieldRP18 VanGuard precolumn, 130A, 1.7μm, 2.1mm x 5mm.

[0630] LCMS 15 min: Initial composition 5% B, hold 1 min, then increase from 5% B to 100% B over a 9 min period. The composition was held at 100% B for 2 min, then returned to 5% B over 0.10 min and held for 3 min. Total gradient run time equals 15 min. Flow rate 0.6 mL / min. Wavelength detection range: 190 to 800 nm. Oven temperature: 50°C. Column: ACE Excel2 C18-AR, 2μ, 3.0x100 mm.

[0631] Preparative HPLC:

[0632] Reversed-phase ultrafast high performance liquid chromatography (UFLC) in Shimazdzu Use on the machine Gemini NX 5μC18 posts (at 50 ℃) are carried out, and the column size is: 150x21.2mm. The eluent used is solvent A (HO with 0.1% formic acid) and solvent B (CHCN with 0.1% formic acid). All UFLC experiments are carried out using gradient conditions: initial composition 13%B is increased to 60%B through 15 minutes period, then increased to 100%B through 2 minutes. Composition is kept at 100%B for 1 minute, then returned to 13%B in 0.1 minute and kept 1.9 minutes here. The total duration of the gradient operation is 20.0 minutes. Flow velocity is 20.0 ml / min and detects at 254nm and 280nm.

[0633] Example 1

[0634]

[0635] a) 1-((Benzyloxy)carbonyl)azetidine-2-carboxylic acid (2)

[0636] (2S)-azetidine-2-carboxylic acid 1 (3 g, 29.674 mmol) and sodium bicarbonate (6.3 g, 75 mmol) were dissolved in H2O (25 mL, 1387.75 mmol) and N-(benzyloxycarbonyl)succinimide (8.5 g, 34 mmol) in THF (25 mL, 307 mmol, 100 mass %) was added dropwise. After stirring at room temperature for 12 h, the two phases were separated. 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 organic extracts were dried (MgSO4), and the excess solvent was evaporated in vacuo to give the crude product as a clear oil. The crude material was used in the next step without purification. LCMS 3 min:ES + =1.34min,m / z 258.2[M+Na] + .

[0637] b) (S)-Azetidine-1,2-dicarboxylic acid 1-benzyl 2-methyl ester (3)

[0638] In a dry round-bottom flask, (2S)-1-benzyloxycarbonylazetidine-2-carboxylic acid 2 (6.98 g, 29.7 mmol) was dissolved in MeOH (65 mL) and sulfuric acid (3 mL) was added. The mixture was heated to reflux and stirred overnight. The mixture was cooled to room temperature and quenched with Net3 (to pH = 7), then stirred for 1 h. Methanol was removed in vacuo. The residue was dissolved in EtOAc, washed with H2O and brine, then dried over MgSO4 and filtered. The organics were removed in vacuo to give the crude product 3 (8.004 g, 32.11 mmol) as a clear oil. LCMS 3 min: ES + =1.53min, m / z no ionization

[0639] c) (S)-2-(Hydroxymethyl)azetidine-1-carboxylic acid benzyl ester (4)

[0640] (2S)-azetidine-1,2-dicarboxylic acid O1-benzyl ester O2-methyl ester 3 (7.6g, 30mMol) was dissolved in THF (75mL, 922mmol), cooled to 0°C and LiBH4 (1g, 45mMol) was added. The mixture was allowed to warm to room temperature and stirred for another hour, at which point the reaction was complete. The reaction mixture was cooled to 0°C and then quenched with H2O and 1M HCl. Volatiles were removed in vacuo. The residue was dissolved in EtOAc and washed with brine (2x50mL), dried with MgSO4, filtered, and the solvent was removed by rotary evaporation under reduced pressure. Purified by silica gel column chromatography (Hex / EtOAc, 100% to 1:2) to give product 4 (4.076g, 60% yield over 3 steps) as a clear oil. LCMS 3min:ES + =1.36min,m / z 222.3[M+H] + .

[0641] d) (S)-Benzyl 2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carboxylate (5)

[0642] (2S)-2-(Hydroxymethyl)azetidine-1-carboxylic acid benzyl ester 4 (4.0766 g, 18.425 mmol) was dissolved in anhydrous CH2Cl2 (20 mL, 312.0 mmol) and the mixture was cooled 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 warmed to room temperature and stirred. LCMS showed that the reaction was complete within 5 min. The organic matter was washed with saturated NH4Cl, water, brine, dried with MgSO4, filtered and volatiles were removed in vacuo. Purification by silica gel column chromatography (Hex / EtOAc, 100% to 9:1) gave the product 5 (6.90 g, not completely dried, quantitative). LCMS 3min:ES + =2.15min,m / z 336.9[M+H] + .

[0643] e) (S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine (6)

[0644] 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 5 (6.9027 g, 20.57 mmol) in EtOH (100 mL) at room temperature in a Parr hydrogenation bottle. The reaction mixture was subjected to H2 gas at 20 psi, and the bottle was then evacuated under vacuum (repeated 3 times). The bottle was then inflated to 38 psi H2 and vibrated for 1 hour. During this period, the pressure was reduced to ~30 psi and the bottle was inflated to 40 psi again and vibrated for another 1 hour. No further reduction in pressure was observed and the reaction was considered complete. This was confirmed by LC-MS. The mixture was filtered through diatomaceous earth and the filtrate was evaporated in vacuo to provide the crude product 6 (3.761 g, 90% yield) as a brown oil. LCMS 3 min:ES + =1.70min, m / z no ionization.

[0645] f)((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidin-1-yl)(4-(6-(4-((2R)-2-(((tert-butyldimethylsilyl)oxy)methyl)cyclobutane-1-carbonyl)-2-methoxy-5-nitrophenoxy)hexyl)-5-methoxy-2-nitrophenyl)methanone (8)

[0646] DCC (3.8 g, 18 mmol) was added to a solution of 7 (3.9 g, 7.9 mMol) and HOBt (2.3 g, 17 mMol) in CH2Cl2 (200 mL) at 0°C. The cold bath was removed and the reaction was allowed to proceed for 30 min at room temperature, at which time a solution of 6 (3.65 g, 18 mMol) and triethylamine (3.2 mL, 23 mmol) in CH2Cl2 (200 mL) was quickly added under argon at -10°C. The reaction mixture was stirred at room temperature and monitored by LC / MS. After 2 min, the reaction was complete. The solids were removed by filtration on diatomaceous earth and the organic phase was washed with cold 0.1 M HCl aqueous solution until a pH of 2 was measured. The organic phase was then washed with water, followed by saturated sodium bicarbonate aqueous solution, brine, dried over MgSO4, filtered and vacced down. Purification by silica gel column chromatography (Hex / EtOAc / CH2Cl2, 100% to 1:2:1) gave product 8 (5.9 g, 87% yield). The product was contaminated with some mono-coupling products (the impurities were not separated during chromatography). LCMS 3 min: ES + =2.35min,m / z 862.2[M+H] + .

[0647] g)((Pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis(((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidin-1-yl)methanone)(9)

[0648] Zinc (4.65 g, 71.1 mmol) was slowly added to a solution of 8 (2.45 g, 2.85 mmol) in a 90:5:5 mixture of MeOH / H2O / formic acid (66 mL). An ice bath was used to control the resulting exotherm to maintain the temperature of the reaction mixture below 40°C. Upon completion, the solids were removed by filtration over diatomaceous earth and the organic phase was washed with water and brine, then dried over MgSO4, filtered and the volatiles removed under reduced pressure. The crude material 9 (2.28 g, quantitative) was used as is in the next step. LCMS 3 min: ES + =2.32min,m / z 802.3[M+H] + .

[0649] h)((Pentane-1,5-diylbis(oxy))bis(6-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-4-methoxy-3,1-phenylene))diallyl dicarbamate (10)

[0650] Under argon atmosphere, compound 9 (2.23 g, 2.78 mmol) was dissolved in CH2Cl2 (50 mL). 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 reactant was stirred at -78 °C for 10 min, then allowed to warm to room temperature. The reaction was complete after 15 min. The organic matter was washed with saturated CuSO4, H2O, brine, then dried with MgSO4, filtered and volatiles were removed under reduced pressure. The crude product 10 (1.47 g, 1.52 mMol, quantitative) was used as is in the next step. LCMS 3 min: ES + =2.53min,m / z 970.3[M+H] + .

[0651] i) ((pentane-1,5-diylbis(oxy))bis(6-((S)-2-(hydroxymethyl)azetidine-1-carbonyl)-4-methoxy-3,1-phenylene))dicarbamic acid diallyl ester (11)

[0652] Compound 10 (1.47 g, 1.52 mMol) was dissolved in a 3:1:1 mixture (16 mL) of H2O / THF / acetic acid and the reaction was stirred over the weekend. The mixture was extracted with CH2Cl2 and washed with saturated NaHCO3, H2O and brine, then dried with MgSO4, filtered and volatiles were removed under reduced pressure. Purification by silica gel column chromatography (Hex / EtOAc, 100% to 1:1) gave product 11 (859 mg, 76.5% yield) as a clear oil. LCMS 3 min:ES + =1.75min,m / z 742.0[M+H] + .

[0653] j) Diallyl 7,7'-(pentane-1,5-diylbis(oxy))(10aS,10a'S)-bis(10-hydroxy-6-methoxy-4-oxo-1,2,10,10a-tetrahydroazetidin[1,2-a]benzo[e][1,4]diazepine -9(4H)-carboxylate)(12)

[0654] Compound 11 (850 mg, 1.14 mMol) was dissolved in CH2Cl2 (60 mL). 1-Hydroxy-2,2,6,6-tetramethyl-piperidine; 1-methylimidazole; 2-(2-pyridyl)pyridine (0.7 mL, 1140 mmol, 0.2 mMl / L) and trifluoromethanesulfonic acid tetraacetonitrile copper (I) (55 mg, 0.145 mMol) were then added and the mixture was stirred at 35 ° C with 2 balloons of air pressed in. The reactants were stirred overnight and then evacuated to dryness in a rotary evaporator. Purification by silica gel column chromatography (CHCl3 / MeOH, 100% to 95:5) gave product 12 (346 g, 0.47 mMol, 41% yield). LCMS 3min:ES + =1.48min,m / z 737.9[M+H] + .

[0655] k) (10aS,10a'S)-7,7'-(pentane-1,5-diylbis(oxy))bis(6-methoxy-1,10a-dihydroazetidin[1,2-a]benzo[e][1,4]diazepine -4(2H)-Keto)(Ex1)

[0656] Under argon, compound 12 (335 mg, 0.45 mmol) was dissolved in CH2Cl2 (20 mL) in a flask. Pyrrolidine (650 μL, 7.8 mMol) and Pd(PPh3)4 (50 mg, 0.004 mMol) were subsequently added, and the mixture was stirred at room temperature until the reaction was complete. The organic matter was washed with saturated NH4Cl, H2O and brine, then dried with MgSO4, filtered and volatiles were removed under reduced pressure. Purification was performed by isolera chromatography (CH2Cl2 / (CH2Cl2+10%MeOH) 92:7 to 10:90. Two fractions containing the product were separated but the purity was insufficient. The fractions were combined and repurified by manual chromatography, and the pure product Ex1 (146 mg, 0.27 mMol, 24% yield) was isolated. LCMS 3 min:ES + =1.32min,m / z 533.8[M+H] + . LCMS15min:ES + =4.83min,m / z 533.9[M+H] + .

[0657] Example 2

[0658]

[0659] a)((S)-(2-(((tert-butyldimethylsilyl)oxy)methyl)azetidin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (13)

[0660] At 0°C, DCC (4.021 g, 19.49 mmol) was added to a solution of 5-methoxy-2-nitro-4-triisopropylsilyloxy-benzoic acid 13 (6 g, 16.24 mmol) and HOPO (1.984 g, 17.86 mMol) in CH2Cl2 (100 mL). The cold bath was removed and the reaction was allowed to proceed at room temperature for 30 min, at which point a solution of [(2S)-azetidin-2-yl]methoxy-tert-butyl-dimethyl-silane 6 (3.761 g, 18.68 mmol) and triethylamine (3.39 mL, 33.5 mmol) in CH2Cl2 (100 mL) was quickly added under argon at -10°C. The reaction mixture was stirred at room temperature and monitored by LC / MS. After 2 min, the reaction was complete. The solids were removed by filtration on celite and the organic phase was washed with cold 0.1M HCl aqueous solution until the pH was measured to be 2. The organic phase was then washed with water, followed by saturated sodium bicarbonate aqueous solution, brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by silica gel column chromatography (Hex / EtOAc, 100% to 1:1) gave the product 14 (8.6737 g, 96.63% yield). LCMS 3 min: ES + =2.44min,m / z 554.2[M+H] + .

[0661] b) (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)azetidin-1-yl)methanone (15)

[0662] Zinc (10 g, 152.9 mMol) was slowly added to a solution of 14 (8.6737 g, 15.69 mMol) in a 90:5:5 mixture (200 mL) of MeOH / H2O / formic acid. The resulting exotherm was controlled using an ice bath to maintain the temperature of the reaction mixture below 40°C. Upon completion, the solids were removed by filtration over diatomaceous earth and the organic phase was washed with water and brine, then dried over MgSO4, filtered and the volatiles removed under reduced pressure. The crude material 15 (7.6343 g, 14.6 mMol, 93.05% yield) was used as is in the next step. LCMS 3 min:ES + =2.42min,m / z 524.4[M+H] + .

[0663] c) (S)-(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamic acid allyl ester (16)

[0664] Under argon atmosphere, compound 15 (7.6343 g, 14.60 mMol) was dissolved in CH2Cl2 (100 mL). 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 reactants were stirred at -78 °C for 10 min, then allowed to warm to room temperature. The reaction was complete after 15 min. The organic matter was washed with saturated CuSO4, H2O, brine, then dried with MgSO4, filtered and volatiles were removed under reduced pressure. The crude product 16 (8.9129 g, 14.69 mMol, quantitative) was used as is in the next step. LCMS 3 min: ES + =2.53min,m / z 608.2[M+H] + .

[0665] d) (S)-(2-(2-(Hydroxymethyl)azetidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamic acid allyl ester (17)

[0666] Compound 16 (8.9129 g, 14.69 mMol) was dissolved in a 3:1:1 mixture (80 mL) of H2O / THF / acetic acid and the reaction was stirred over the weekend. The mixture was extracted with CH2Cl2 and washed with saturated NaHCO3, H2O and brine, then dried over MgSO4, filtered and volatiles were removed under reduced pressure. Purification by silica gel column chromatography (Hex / EtOAc, 100% to 1:1) gave product 17 (5.5572 g, 76.80% yield) as a clear oil. LCMS 3 min: ES + =1.97min,m / z494.0[M+H] + .

[0667] e) (10aS)-10-hydroxy-6-methoxy-4-oxo-7-((triisopropylsilyl)oxy)-1,2,10,10a-tetrahydroazetidin[1,2-a]benzo[e][1,4]diazepine -9(4H)-carboxylic acid allyl ester (18).

[0668] Compound 17 (5.5572 g, 11.28 mMol) was dissolved in CH2Cl2 (40 mL). 1-Hydroxy-2,2,6,6-tetramethyl-piperidine; 1-methylimidazole; 2-(2-pyridyl)pyridine (6 mL, 1 mMol) and trifluoromethanesulfonic acid tetraacetonitrile copper (I) (425 mg, 1.1279 mMol) were then added and the mixture was stirred at 35 ° C with 2 balloons of air pressed in. The reactants were stirred overnight and then evacuated to dryness in a rotary evaporator. Purification by silica gel column chromatography (CHCl3 / MeOH, 100% to 97:3) gave product 18 (5.3835 g, 10.97 mMol, 97.27% yield) as a light orange foam. LCMS 3 min:ES + =2.00min,m / z 491.8[M+H] + .

[0669] f) (10aS)-10-((tert-butyldimethylsilyl)oxy)-6-methoxy-4-oxo-7-((triisopropylsilyl)oxy)-1,2,10,10a-tetrahydroazetidin[1,2-a]benzo[e][1,4]diazepine -9(4H)-Carboxylic acid allyl ester (19)

[0670] Compound 18 (5.3835 g, 10.97 mMol) was dissolved in CH2Cl2 (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 subsequently added. The mixture was allowed to stand for 10 min, then the cooling bath was removed and allowed to warm to room temperature. The organic matter was washed with H2O and brine, then dried with MgSO4, filtered and the volatiles were removed under reduced pressure. Purification by silica gel column chromatography (CHCl3 / MeOH, 100% to 95:5) gave product 19 (6.8532 g, quantitative). LCMS 3min:ES + =2.47min,m / z 606.0[M+H] + .

[0671] g) (10aS)-10-((tert-butyldimethylsilyl)oxy)-7-hydroxy-6-methoxy-4-oxo-1,2,10,10a-tetrahydroazetidin[1,2-a]benzo[e][1,4]diazepine -9(4H)-carboxylic acid allyl ester (20)

[0672] Compound 19 (6.8 g, 14 mMol) was dissolved in DMF (10 mL). LiOAc.2H2O (1.4 g, 14 mmMl) 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 process was repeated until the reaction was complete. The organic matter was diluted with CHCl3 and washed with citric acid solution (pH = 3), H2O and brine, then dried with MgSO4, filtered and volatiles were removed under reduced pressure. Purified by silica gel column chromatography (CHCl3 / MeOH, 100% to 95:5) to obtain product 20 (5.2885 g, 11.79 mMol, 85% yield) as a yellow oil. LCMS 3min:ES + =1.86min,m / z449.8[M+H] + .

[0673] h) Diallyl 7,7'-(propane-1,3-diylbis(oxy))(10aS,10a'S)-bis(10-((tert-butyldimethylsilyl)oxy)-6-methoxy-4-oxo-1,2,10,10a-tetrahydroazetidin[1,2-a]benzo[e][1,4]diazepine -9(4H)-carboxylate)(21)

[0674] Under argon atmosphere, 1,3-dibromopropane (204.9 mg, 1.015 mMol) and compound 20 (1 g, 2.030 mMol) were dissolved in CH2Cl2 (50 mL). K2CO3 (280 mg, 2.026 mMol) and TBAI (149 mg, 0.2 mMol) were subsequently added and the mixture was stirred at 40 ° C until the reaction was complete. The mixture was stirred overnight, but the reaction did not proceed to completion but formed impurities. The organic matter was washed with H2O and brine, then dried with MgSO4, filtered and volatiles were removed under reduced pressure. Purified by silica gel column chromatography (CHCl3 / MeOH, 100% to 97:3) to obtain product 21 (482 mg, 0.471 mMol, 46.50% yield), which was contaminated with inseparable impurities (room temperature = 9.95 min, on LCMS, 15 min). LCMS 15min:ES + =9.86min,m / z 938.3[M+H] + .

[0675] i) (10aS, 10a'S)-7,7'-(propane-1,3-diylbis(oxy))bis(6-methoxy-1,10a-dihydroazetidin[1,2-a]benzo[e][1,4]diazepine -4(2H)-Keto)(Ex2A)

[0676] Under argon, compound 21 (482 mg, 0.5143 mMol) was dissolved in CH2Cl2 (20 mL) in a flask. Pyrrolidine (786 μL, 9.44 mMol) and Pd(PPh3)4 (54 mg, 0.046 mMol) were then added and the mixture was stirred at room temperature until the reaction was complete. The organics were washed with saturated NH4Cl, H2O and brine, then dried with MgSO4, filtered and the volatiles were removed under reduced pressure. Purification was performed by isolera chromatography (CH2Cl2 / (CH2Cl2+10%MeOH) 98:2 to 30:70. Two fractions containing the product were separated but the purity was insufficient. The fractions were combined and re-purified by isolera chromatography (same solvent system), and the pure product Ex2A (35.1 mg, 0.135 mMol, 13.5% yield) was isolated. LCMS 3min:ES + =1.23min,m / z 505.8[M+H] + .

[0677] 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-tetrahydroazetidin[1,2-a]benzo[e][1,4]diazepine -9(4H)-carboxylate)(22)

[0678] Under argon atmosphere, 1,3-bis(bromomethyl)benzene (267.9 mg, 1.011 mMol) and compound 20 (1 g, 2.030 mMol) were dissolved in DMF (5 mL). K2CO3 (280 mg, 2.026 mMol) and TBAI (749 mg, 2.027 mMol) were subsequently added and the mixture was stirred at 40 ° C until the reaction was complete. The mixture was stirred overnight, but the reaction was not complete and impurities were formed. The mixture was diluted with CH2Cl2 and washed with H2O and brine, then dried with MgSO4, filtered and volatiles were removed under reduced pressure. Purification by silica gel column chromatography (CHCl3 / MeOH, 100% to 97:3) gave product 22 (467 mg, 0.43 mMol, 42.47% yield) + 398 mg mixed fractions. LCMS 3min:ES + =2.30min,m / z1000.5[M+H] + .

[0679] k) (10aS, 10a'S)-7,7'-((1,3-phenylenebis(methylene))bis(oxy))bis(6-methoxy-1,10a-dihydroazetidin[1,2-a]benzo[e][1,4]diazepine -4(2H)-Keto)(Ex2B)

[0680] Under argon, compound 22 (455 mg, 0.419 mMol) was dissolved in CH2Cl2 (20 mL) in a flask. Pyrrolidine (600 μL, 7.2 mMol) and Pd(PPh3)4 (48 mg, 0.041 mMol) were subsequently added and the mixture was stirred at room temperature until the reaction was complete. The organic matter was washed with saturated NH4Cl, H2O and brine, then dried with MgSO4, filtered and volatiles were removed under reduced pressure. Purification was performed by isolera chromatography (CH2Cl2 / (CH2Cl2+10%MeOH) 98:2 to 30:70. Two fractions containing the product were separated but the purity was insufficient. The fractions were combined and re-purified by isolera chromatography (same solvent system) and isolated as pure product Ex2B (214.5 mg, 0.378 mMol, 90.5% yield) as a white solid. LCMS 3min:ES + =1.38min,m / z 567.8[M+H] + .

[0681] Example 3

[0682]

[0683] a)(5-((5-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-4-methoxyphenyl)carbamic acid allyl ester (23)

[0684] Under argon atmosphere, compound 9 (1.192 g, 1.488 mMol) was dissolved in CH2Cl2 (250 mL). 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 reactant was stirred at -78 °C for 10 min, then allowed to warm to room temperature. The reaction was complete after 15 min. The organic matter was washed with saturated CuSO4, H2O, brine, then dried with MgSO4, filtered and volatiles were removed under reduced pressure. Purification by silica gel column chromatography (CHCl3 / MeOH) gave a mixture of mono- and di-alloc, which was further purified with a second column (Hex / EtOAc) to give pure product 23 (499.2 g, 37.9% yield out of 50% possible). LCMS 3min:ES + =2.41min,m / z 886.6[M+H] + .

[0685] b) Allyl) (5-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-4-methoxyphenyl)carbamate (24)

[0686] At 0 ℃, triphosgene (68.8mg, 0.232mMol) is added once to 23 (620mg, 0.7mMol) and TEA (203μL, 1.46mMol) in CH2Cl2 (50mL) mixture.Remove ice bath, and after 15min, add once as fine powder Alloc-Val-Ala-PAB-OH (275mg, 0.728mMol), then add more TEA (73μL, 0.524mMol,) and dibutyltin dilaurate (39.6μL, 0.07mMol).Reactant mixture is stirred at 37 ℃ for 4h, then stirred at room temperature overnight.By organic matter H2O, saturated NH4Cl and salt water washing, then use MgSO4 drying, filter and remove volatile matter under reduced pressure. Purification by silica gel column chromatography (CHCl3 / MeOH) gave pure product 24 (414 g, 45.9% yield). LCMS 3min:ES +=2.43min,m / z 1289.5[M+H] + .

[0687] c) Allyl) (5-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)azetidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(hydroxymethyl)azetidine-1-carbonyl)-4-methoxyphenyl)carbamate (25)

[0688] Compound 24 (414 mg, 0.32 mMol) was dissolved in a 3:1:1 mixture (10 mL) of H2O / THF / acetic acid and the reaction was stirred over the weekend. The mixture was extracted with CH2Cl2 and washed with saturated NaHCO3, H2O and brine, then dried over MgSO4, filtered and volatiles were removed under reduced pressure. Purification by silica gel column chromatography (CHCl3 / MeOH, 100% to 94:6) gave product 25 (326 mg, 95.7% yield). LCMS 3min:ES + =1.80min,m / z 1060.1[M+H] + .

[0689] d)(10aS)-7-((5-(((10S,10aS)-9-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)benzyl)oxy)carbonyl)-10-hydroxy-6-methoxy-4-oxo-1,2,4,9,10,10a-hexahydroazetidin[1,2-a]benzo[e][1,4]diazepine -7-yl)oxy)pentyl)oxy)-10-hydroxy-6-methoxy-4-oxo-1,2,10,10a-tetrahydroazetidin[1,2-a]benzo[e][1,4]diazepine -9(4H)-Carboxylic acid allyl ester (26)

[0690] Compound 25 (202.4 mg, 0.3 mMol) was dissolved in CH2Cl2 (20 mL). 1-Hydroxy-2,2,6,6-tetramethyl-piperidine; 1-methylimidazole; 2-(2-pyridyl)pyridine (0.4 mL, 0.03 mMol) and trifluoromethanesulfonate tetraacetonitrile copper (I) (11 mg, 0.03 mMol) were then added and the mixture was stirred at 35 ° C with 2 balloons of air pressed in. The reactants were stirred overnight and then evacuated to dryness in a rotary evaporator. Purification by silica gel column chromatography (CHCl3 / MeOH, 100% to 97:3) gave product 26 (313 mg, 0.19 mMol, 64.5% yield). LCMS 3min:ES + =1.59min,m / z1057.1[M+H] + .

[0691] e) 4-((S)-2-((S)-2-amino-3-methylbutyrylamino)propionylamino)benzyl(10S,10aS)-10-hydroxy-6-methoxy-7-((5-(((S)-6-methoxy-4-oxo-1,2,4,10a-tetrahydroazetidin[1,2-a]benzo[e][1,4]diazepine -7-yl)oxy)pentyl)oxy)-4-oxo-1,2,10,10a-tetrahydroazetidin[1,2-a]benzo[e][1,4]diazepine -9(4H)-carboxylate)(27)

[0692] Under argon, compound 26 (195 mg, 0.184 mMol) was dissolved in CH2Cl2 (10 mL) in a flask. Pyrrolidine (262 μL, 3.15 mMol) and Pd(PPh3)4 (21 mg, 0.018 mMol) were subsequently added and the mixture was stirred at room temperature until the reaction was complete. The organic matter was washed with saturated NH4Cl, H2O and brine, then dried with MgSO4, filtered and volatiles were removed under reduced pressure. Purification was performed by isolera chromatography (CH2Cl2 / (CH2Cl2+10%MeOH) 98:2 to 30:70 to obtain product 27 (141 mg, 0.16 mMol, 87.7% yield). LCMS 3 min:ES + =1.23min,m / z 870.9[M+H] + .

[0693] f) (10S,10aS)-10-hydroxy-6-methoxy-7-((5-(((S)-6-methoxy-4-oxo-1,2,4,10a-tetrahydroazetidin[1,2-a]benzo[e][1,4]diazepine -7-yl)oxy)pentyl)oxy)-4-oxo-1,2,10,10a-tetrahydroazetidin[1,2-a]benzo[e][1,4]diazepine -9(4H)-carboxylic acid 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-triazatriacontanoylamino)benzyl ester (Ex3)

[0694] The reaction was carried out in a glove box. Under argon and room temperature, compound 27 (70 mg, 0.080 mMol) was dissolved in CH2Cl2 (10 mL) in a flask. 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 the reaction was complete. The organic matter was washed with H2O and brine, then dried with MgSO4, filtered and the volatiles were removed under reduced pressure. Purification was performed by isolera chromatography (CH2Cl2 / (CH2Cl2+10%MeOH) 98:2 to 30:70 to obtain an impure product. Further purification by reverse phase isolera gave pure Ex3 (4 mg, 0.027 mMol, 3.4% yield) plus some unclean fractions (22 mg). LCMS 3min:ES + =1.51min,m / z 1445.6[M+H] + .

[0695] Example 4

[0696] ConjA(Her2-Ex3)

[0697] A 10 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate buffered saline pH 7.4 (PBS) was added (50 molar equivalents / antibody, 7.6 micromoles, 762.7 μL) to trastuzumab (22.9 mg, 153 nanomoles) in a 20.8 mL solution containing 30 mM histidine / histidine HCl, 30 mM arginine pH 6.8, and 1 mM ethylenediaminetetraacetic acid (EDTA) at a final antibody concentration of 1.1 mg / mL. The reduction mixture was reacted at 37° C. for 2 hours (or until complete reduction was observed by UHPLC) in an orbital shaker under gentle (60 rpm) shaking. The reduced antibody solution was buffer exchanged (to remove all excess reducing agent) to a conjugation buffer containing 30 mM histidine / histidine HCl, 30 mM arginine, and 1 mM EDTA by spin filter centrifugation to achieve a final antibody concentration of 1.1 mg / mL. Ex3 was added to 18.6 mL of this reduced antibody solution (20.5 mg, 136 nanomoles) as a DMSO solution (12.5 molar equivalents / antibody, 1.9 micromoles in 2.1 mL DMSO) to achieve a 10% (v / v) final DMSO concentration. The solution was mixed at room temperature for 17 hours, then the conjugation was quenched by adding N-acetyl cysteine ​​(8.5 micromoles, 68 μL, 100 mM), then purified by spin filter centrifugation using a 15 mL Amicon Ultracell 30 KDa MWCO spin filter, sterile filtered and analyzed.

[0698] UHPLC analysis of reduced samples of ConjA at 214 nm and 330 nm (SG3931 specific) using a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluted with a gradient of water and acetonitrile showed that the mixture of unconjugated light chain, light chain linked to a single molecule of SG3931, unconjugated heavy chain, and heavy chain linked to up to three molecules of SG3931 was consistent with a drug / antibody ratio (DAR) of 7.32 molecules of SG3931 / antibody.

[0699] UHPLC analysis of the ConjA sample at 280 nm showed a monomer purity of 94.2% using a Tosoh Bioscience TSKgel SuperSW mAbHTP 4μm 4.6x150mm column (with a 4μm 3.0x20mm guard column) on a Shimadzu Prominence system, eluting with 0.3mL / min sterile filtered SEC buffer containing 200mM potassium phosphate pH 6.95, 250mM potassium chloride, and 10% isopropanol (v / v). UHPLC SEC analysis gave a final ConjA concentration of 1.29mg / mL in 5.8mL, and the mass of ConjA obtained was 7.5mg (37% yield).

[0700] Example 5 - Cytotoxicity Assay

[0701] The potency of the molecules was measured by an in vitro cytotoxicity assay in the cancer cell line NCI-N87.

[0702] Solid material was dissolved in DMSO to a 2 mM stock solution, from which eight serial dilutions were prepared at a 1:10 ratio in DMSO and stored at -20°C until use.

[0703] The attached NCI-N87 cells were washed with D-PBS and detached with trypsin-EDTA, and then counted using an automated cell counter (LUNA-II TM ) Cell density and viability were determined in duplicate by trypan blue exclusion assay. Cell suspensions were incubated in growth medium (with Glutamax + 10% (v / v) HyClone TM Dilute to 1x10 in RPMI 1640 containing fetal bovine serum 5 Cells / ml and vortexed, then 2mL per well was distributed into a sterile 3mL polypropylene plate. The bullet diluent was then distributed into the appropriate wells at 10μl / well and mixed by repeated pipetting. For the control wells, 10μl of DMSO was distributed into the 2mL cell suspension and mixed thoroughly. Then 100μl of each sample was aliquoted into 2 replicate wells of a sterile flat 96-well microplate and incubated in a 37°C CO2-aerated (5%) incubator. At the end of the incubation period (7 days), cell viability was measured by CellTiter96Aqueous One (MTS) assay, which was distributed at 20μl / well and incubated at 37°C, 5% CO2 for 4 hours. Then on the EnVision Multi-label plate reader (Perkin Elmer), the absorbance at 490nm was used to read the plate.

[0704] The percentage of cell survival (%) was calculated from the average absorbance of two replicate wells for each sample and compared with the average absorbance in two control wells treated with DMSO alone (100%). IC was determined by fitting each data set to a dose-response curve with a variable slope using a nonlinear curve fitting algorithm on GraphPad Prism software (San Diego, CA). 50 .

[0705] All experiments reported in this report were performed and tested in three independent experiments. Data are reported as the average of three independent replicates.

[0706] <![CDATA[IC 50 (nM)]]> Ex2A 70.11 Ex1 2.202 Ex2B 4.035

[0707] Example 6 - ADC Cytotoxicity Assay

[0708] The concentration and viability of cells from subconfluent (80-90% confluent) T75 flasks were measured by trypan blue staining and analyzed using LUNA-II TM Count the cells using an automated cell counter. Dilute the cells to 2x10 5 / ml, dispense (50 μl / well) into 96-well flat-bottom plates.

[0709] The stock solution (1 ml) of the test antibody drug conjugate (ADC) (20 μg / ml) was prepared by diluting the filter-sterilized ADC into the cell culture medium. A set of 8x10 dilutions of the reserve ADC was performed in a 24-well plate by continuously transferring 100 μl to 900 μl of cell culture medium. The ADC dilutions were distributed (50 μl / well) to 4 replicate wells of a 96-well plate containing 50 μl of cell suspension previously inoculated. The control wells received 50 μl of cell culture medium. The 96-well plates containing cells and ADC were incubated at 37°C in a CO2-aerated incubator for a continuous exposure time.

[0710] At the end of the incubation period, cell viability was measured by MTS assay. MTS (Promega) was dispensed (20 μl per well) into each well and incubated for 4 hours at 37° C. in a CO2-filled incubator. The well absorbance at 490 nm was measured. The cell survival percentage was calculated by the average absorbance in the 4 ADC-treated wells compared to the average absorbance in the 4 control untreated wells (100%). IC was determined from the dose-response data using GraphPad Prism using a nonlinear curve fitting algorithm. 50 : Sigmod dose-response curve with variable slope.

[0711] ADC incubation time was 4 days for MDA-MB-468 and 7 days for NCI-N87. TM MDA-MB-468 and NCI-N87 were cultured in RPMI 1640 medium supplemented with fetal bovine serum.

[0712] EC was determined by fitting the data to a sigmoidal dose-response curve with a variable slope using GraphPad Prism software v6.05 (GraphPad, San Diego, CA). 50 value.

[0713]

[0714] Example 7 - Xenograft Testing

[0715] NCI-N87 xenograft mice

[0716] On day 1 of the study, female severe combined immunodeficient mice (Fox Chase CB-17 / Icr-Prkdcscid, Charles River) were eight weeks old with a body weight (BW) range of 16.5 g to 21.6 g. Animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and NIH 31 Modified and Irradiated Laboratory Feed consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. Mice were housed in static microisolators on irradiated Enricho'cobs at 20-22°C (68-72°F) and 40-60% humidity using a 12-h light cycle. TM Laboratory Animal Bedding. CR Discovery Services specifically follows the recommendations of the Guide for Care and Use of Laboratory Animals regarding restraint, housing, surgical procedures, feed and fluid regulations, and veterinary care. The animal care and use procedures at CR Discovery Services are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which ensures compliance with generally accepted standards for the care and use of laboratory animals.

[0717] Tumor cell culture

[0718] Human NCI-N87 gastric lymphoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units / mL penicillin G sodium, 100 μg / mL streptomycin sulfate, and 25 μg / mL gentamicin. The cells were grown in tissue culture flasks in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air.

[0719] In vivo transplantation and tumor growth

[0720] NCI-N87 cells for transplantation were harvested during the logarithmic growth phase and resuspended in 50% Matrigel TM (BD Biosciences) in phosphate buffered saline (PBS). On the day of tumor implantation, each test mouse was injected subcutaneously in the right abdomen with 1×10 7 cells (0.1 mL of cell suspension) and when the average size is close to 100 to 150 mm 3 Fourteen days later, designated as study day 1, mice were sorted into groups based on the calculated tumor size, with each group consisting of mice with tumors ranging from 108 to 144 mm 3 The group consisted of ten animals with individual tumor volumes ranging from 1.1 to 1.5 mm and the group mean tumor volume was 115 mm 3 .

[0721] Tumors were measured in two dimensions using calipers, and volumes were calculated using the following formula:

[0722]

[0723] Where w = width of the tumor and l = length of the tumor in mm. Assuming 1 mg equals 1 mm3 of tumor volume, tumor weight can be estimated.

[0724] treat

[0725] On day 1, in patients with established subcutaneous NCI-N87 tumors (108–144 mm 3 Treatment was started in groups of 10 mice (n=10) with 10 mg / kg of ConjA. ConjA (4 mg / kg) was administered intravenously once on day 1 (qd x 1). The vehicle-treated group was used as the control group for efficacy analysis. Tumors were measured twice a week until the end of the study on day 79. Each mouse was given a 4-day follow-up of 10 mice at a time when its tumor reached 800 mm. 3 The mice were euthanized when the volume reached the endpoint or on the last day (whichever came first). The time to endpoint (TTE) was calculated for each mouse.

[0726] The results are Figure 1 Shown in , which shows the changes in normalized tumor growth (■ - Control; ◆ - ConjA).

[0727] Endpoint and Tumor Growth Delay (TGD) Analysis

[0728] Tumors were measured twice weekly using calipers and when they reached 800 mm 3 Each animal was euthanized when the tumor volume reached the endpoint or at the end of the study (day 79), whichever came first. Animals that withdrew from the study due to reaching the tumor volume endpoint were recorded as euthanized due to tumor progression (TP), and the date of euthanasia was recorded. The endpoint time (TTE) for each mouse for analysis was calculated by the following formula:

[0729]

[0730] Where TTE is expressed in days and endpoint volume is expressed in mm 3 Denotes, b is the intercept, and m is the slope of the line obtained by linear regression of the logarithmic transformed tumor growth data set. The data set consists of the first observation that exceeds the endpoint volume used in the analysis and the three consecutive observations before reaching this endpoint volume. The calculated TTE is usually less than the TP date, that is, the day when the animal is euthanized due to tumor size. Animals whose tumors have not reached the endpoint volume are assigned a TTE value equal to the last day of the study (the 79th day). In the case of the logarithmic transformed calculated TTE less than the day before reaching the endpoint or exceeding the day when the tumor volume endpoint is reached, linear interpolation is performed to estimate TTE. Any animal classified as dying from NTR (non-treatment related) reasons due to accidents (NTRa) or due to unknown reasons (NTRu) is excluded from TTE calculations (and all further analyses). Animals classified as TR (treatment related) deaths or NTRm (non-treatment related deaths caused by metastasis) are assigned TTE values ​​equal to the day of death. Treatment outcomes are assessed by tumor growth delay (TGD), and TGD is defined as the increase in the median endpoint time (TTE) of the treatment group compared to the control group:

[0731] TGD = T – C,

[0732] Expressed in days or as a percentage of the median TTE of the control group:

[0733]

[0734] in:

[0735] T = median TTE for the treatment group, and

[0736] C = median TTE of the indicated control group.

[0737] Tumor growth inhibition

[0738] Tumor growth inhibition (TGI) analysis evaluates the difference in median tumor volume (MTV) between treated and control mice. For this study, the endpoint for determining TGI was day 19, which was the last day that all evaluable control mice remained in the study. The MTV (n) for each group was determined, i.e., the median tumor volume of n animals on the day of the TGI analysis. The percentage of tumor growth inhibition (% TGI) is defined as the difference between the MTV of a given control group and the MTV of a drug-treated group, expressed as a percentage of the MTV of the control group.

[0739]

[0740] The data set used for TGI analysis included all animals in the group except those that died from treatment-related (TR) or non-treatment-related (NTR) causes prior to the day of TGI analysis.

[0741] MTV and criteria for extinction response

[0742] The efficacy of treatment can be determined by the tumor volume of the animals remaining in the study on the last day. The MTV (n) is defined as the median tumor volume on the last day of the study for the remaining number of animals (n) whose tumors have not reached the endpoint volume. The efficacy of treatment can also be determined by the incidence and magnitude of regression responses observed during the study. Treatment can result in partial regression (PR) or complete regression (CR) of the animal's tumor. In a PR response, the tumor volume is 50% or less of its day 1 volume for three consecutive measurements during the course of the study, and one or more of these three measurements is equal to or greater than 13.5 mm 3 In a CR response, the tumor volume was less than 13.5 mm for three consecutive measurements during the course of the study. 3 Animals were scored only once for PR or CR events during the study, and only scored as CR if both PR and CR criteria were met. Animals with CR responses at study termination were additionally classified as tumor-free survivors (TFS). Animals were monitored for regression responses.

[0743] toxicity

[0744] Animals were weighed daily on days 1-5, and then twice weekly until the study was completed. Mice were frequently observed for obvious signs of any adverse treatment-related (TR) side effects, and clinical signs were recorded when observed. Individual body weights were monitored according to the protocol, and any animal with a weight loss of more than 30% or more than 25% in three consecutive measurements was euthanized as a TR death. Group average weight loss was also monitored according to the CR Discovery Services protocol. Acceptable toxicity was defined as a group average body weight (BW) loss of less than 20% and no more than 10% TR deaths during the study. Dosing was suspended when the average weight loss in any group exceeded the acceptable limit. If the group average body weight returned to an acceptable level, dosing was modified to a lower level and / or a reduced frequency and then restarted. If clinical signs and / or necropsy demonstrated that death was attributed to treatment side effects, it was classified as TR. Death due to unknown causes during dosing or within 14 days of the last dose was also assigned a TR classification. If there was no evidence that the death was related to treatment side effects, the death was classified as non-treatment related (NTR). NTR deaths are also classified as follows: NTRa describes deaths caused by accidents or human error; deaths believed to be caused by tumor spread (by invasion) and / or metastasis (based on autopsy results) are designated as NTRm; NTRu describes deaths of unknown cause, lacking available evidence that the death is related to metastasis, tumor progression, accidents or human error. It should be noted that treatment side effects cannot be excluded in deaths classified as NTRu.

[0745] Statistical and graphical analysis

[0746] All statistical analyses and graphical presentations were performed using GraphPad Prism 8.0 for Windows. Study groups that experienced toxicity beyond acceptable limits (>20% group mean weight loss or >10% treatment-related deaths) or had fewer than five evaluable observations were excluded from the statistical analyses. The log-rank test was used to assess the significance of differences between the survival experiences of the two groups. The log-rank test analyzed the individual TTEs of all animals in the group, excluding those that withdrew from the study due to NTR death. The Mann-Whitney U test was used to complete the statistical analysis of the differences between the median tumor volume (MTV) on day 19 of the control group and the treatment group. For statistical analyses, a two-tailed test was performed at a significance level of P = 0.05. Prism summarized the test results as not significant (ns), P > 0.05; significant (sign denoted as “*”), 0.01 < P ≤ 0.05; very significant (“**”), 0.001 < P ≤ 0.01; and extremely significant (“***”), P ≤ 0.001. Since tests of statistical significance do not provide an estimate of the magnitude of the difference between groups, all significance levels are described as significant or not significant in the context reported herein.

[0747] n Median TTE TC %TGD MTV(n), Day 79 vehicle - 10 24.8 -- - 466(10) Conj 4mg / kg 10 79.0 54.2 219 32(9)

[0748] PR CR TFS BW Nadir TR NTR NTR vehicle 0 0 0 -2.0(2) 0 0 0 Conj 6 4 0 -1.9(2) 0 0 0

[0749] The MTV (10) on day 19 of the animals treated with ConjA was 32 mm 3 , or a significant 93% TGI (P < 0.001, Mann-Whitney). Nine animals survived the study and the median TTE allotted was 79.0 days; this represents a maximum possible significant 219% TGD (P < 0.001, log-rank). The MTV (9) on day 79 was 320 mm 3 and there were six PRs and four CRs.

[0750] All of the documents and other references mentioned above are incorporated herein by reference.

[0751] Statement of Invention

[0752] 1. A compound of formula IV:

[0753]

[0754] and its salts and solvates, wherein:

[0755] R 2 and R 2’ are H;

[0756] R 6 and R9 is independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halogen;

[0757] wherein R and R' are independently selected from optionally substituted C 1-12 Alkyl, C 3-20 Heterocyclic and C 5-20 Aryl;

[0758] and

[0759] (a)R 7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halogen;

[0760] R 7’ is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halogen; or

[0761] (b)R 7 and R 7 'Together form a group which is: (i) -O-(CH2) n -O-, wherein n is 7 to 16; or (ii) -O-(CH2CH2O) m -, wherein m is 2 to 5;

[0762] R″ is C 3-12 Alkylene, the chain may be separated by one or more heteroatoms, such as O, S, NR N2 (where R N2 H or C 1-4 alkyl), and / or aromatic rings, such as benzene or pyridine;

[0763] Y and Y' are selected from O, S or NH;

[0764] R 6’ and R 9’ Selected from R 6 and R 9 The same group;

[0765] and

[0766] (ia)R 10 and R 11 together to form a double bond between the N atom and the C atom to which they are bound; or

[0767] (ib)R 10 H and R 11 Selected from OH and OR A , where R A C 1-4Alkyl; or (ic) R 10 and R 11 All are H;

[0768] and

[0769] (ii-a)R 20 and R 21 together to form a double bond between the N atom and the C atom to which they are bound; or

[0770] (ii-b)R 20 H and R 21 Selected from OH and OR B , where R B C 1-4 Alkyl; or (ii-c) R 20 and R 21 Both are H.

[0771] 2. A compound according to statement 1, wherein Y and Y' are both O.

[0772] 3. A compound according to Statement 1 or Statement 2, wherein R" is C 3-7 Alkylene.

[0773] 4. A compound according to Statement 1 or Statement 2, wherein R" is a group of the formula:

[0774]

[0775] Where r is 1 or 2.

[0776] 5. A compound according to any one of statements 1 to 4, wherein R 9 For H.

[0777] 6. A compound according to any one of Statements 1 to 5, wherein R 6 For H.

[0778] 7. A compound according to any one of Statements 1 to 6, wherein R 7 is selected from H, OH or OR and R 7’ Selected from H, OH and OR.

[0779] 8. A compound according to statement 7, wherein R 7 C 1-4 Alkoxy and R 7’ C 1-4 Alkoxy.

[0780] 9. A compound according to any one of Statements 1 to 8, wherein R 2’ is with R 2 The same group, R 6 ' is with R6 The same group, R 7 ' is with R 7 The same group, R 9’ is with R 9 is the same group as Y and Y' is the same group as Y.

[0781] 10. A compound according to any one of statements 1 to 9, wherein R 10 and R 11 Together they form a double bond between the N atom and the C atom to which they are bound.

[0782] 11. A compound according to any one of statements 1 to 9, wherein R 10 H and R 11 Selected from OH and OR A .

[0783] 12. A compound according to statement 11, wherein R A It is methyl.

[0784] 13. A compound according to any one of Statements 1 to 9, wherein R 10 and R 11 Both are H.

[0785] 14. A compound according to any one of Statements 1 to 13, wherein R 20 and R 21 Together they form a double bond between the N atom and the C atom to which they are bound.

[0786] 15. A compound according to any one of Statements 1 to 13, wherein R 20 H and R 21 Selected from OH and OR B .

[0787] 16. A compound according to statement 14, wherein R B It is methyl.

[0788] 17. A compound according to any one of Statements 1 to 13, wherein R 20 and R 21 Both are H.

[0789] 18. A compound according to Statement 1, which has the formula IVa, IVb or IVc:

[0790]

[0791] Where R 1a Selected from methyl and benzyl.

[0792] 19. A compound of formula I:

[0793]

[0794] and salts and solvates thereof, wherein:

[0795] Y, Y', R", R 2 , R 2’ , R 6 , R 6’ , R 7 , R 7’ , R 9 and R 9’ As defined in any one of statements 1 to 18;

[0796] R 11b Selected from OH, OR A , where R A C 1-4 Alkyl; and

[0797] R L is a linker for connecting to a cell binding agent selected from:

[0798] (iiia):

[0799]

[0800] in

[0801] Q is:

[0802] Where Q X is such that Q is an amino acid residue, a dipeptide residue or a tripeptide residue;

[0803] X is:

[0804]

[0805] Where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5;

[0806] G L is a linker for attachment to a Ligand unit; and

[0807] (iiib):

[0808]

[0809] Where R L1 and R L2 are independently selected from H and methyl, or together with the carbon atoms to which they are attached form cyclopropylene or cyclobutylene; and e is 0 or 1;

[0810] and:

[0811] (a)R30 and R 31 together to form a double bond between the N atom and the C atom to which they are bound; or

[0812] (b)R 30 H and R 31 Selected from OH and OR B , where R B C 1-4 Alkyl; or

[0813] (c)R 30 and R 31 All are H; or

[0814] (d) R 31 OH or OR B , where R B C 1-4 Alkyl and R 30 Selected from:

[0815] (i)

[0816] (ii)

[0817] (iii) Where R Z Selected from:

[0818] (zi)

[0819] (z-ii) OC(=O)CH3;

[0820] (z-iii) NO2;

[0821] (z-iv)OMe;

[0822] (zv) glucuronide;

[0823] (z-vi)NH-C(=O)-X1-NHC(=O)X2-NH-C(=O)-R ZC , wherein -C(=O)-X1-NH- and -C(=O)-X2-NH- represent natural amino acid residues and R ZC Selected from Me, OMe, CH2CH2OMe and (CH2CH2O)2Me.

[0824] 20. A compound according to statement 19, wherein R 30 and R 31 Together they form a double bond between the N atom and the C atom to which they are bound.

[0825] 21. A compound according to statement 19, wherein R30 H and R 31 Selected from OH and OR B , where R B C 1-4 alkyl.

[0826] 22. A compound according to statement 19, wherein R 30 and R 31 Both are H.

[0827] 23. A compound according to statement 19, wherein R 31 OH or OR A And R 30 Selected from:

[0828]

[0829]

[0830] 24. A compound according to statement 23, wherein -C(=O)-X1-NHC(=O)X2-NH- is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys- and -Val-Cit-.

[0831] 25. A compound according to Statement 23, wherein -C(=O)-X1-NHC(=O)X2-NH- is selected from: -Phe-Lys- and -Val-Ala-.

[0832] 26. A compound according to any one of Statements 23 to 25, wherein R ZC Selected from CH2CH2OMe and (CH2CH2O)2Me.

[0833] 27. A compound according to statement 26, wherein R ZC It is (CH2CH2O)2Me.

[0834] 28. A compound according to Statement 19, which has Formula Ia, Ib or Ic:

[0835]

[0836]

[0837] Where R 1a Selected from methyl and benzyl.

[0838] 29. A compound according to any one of Statements 19 to 28, wherein R 11b For OH.

[0839] 30. A compound according to any one of Statements 19 to 29, wherein R 11b OR A , where R A C 1-4 alkyl.

[0840] 31. A compound according to statement 30, wherein R A It is methyl.

[0841] 32. A compound according to any one of Statements 19 to 31, wherein R L It has formula IIIa, and Q is an amino acid residue selected from the group consisting of Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg and Trp.

[0842] 33. A compound according to any one of Statements 19 to 31, wherein R L has the formula IIIa, and Q is a dipeptide residue selected from:

[0843] CO -Phe-Lys- NH ,

[0844] CO -Val-Ala- NH ,

[0845] CO -Val-Lys- NH ,

[0846] CO -Ala-Lys- NH ,

[0847] CO -Val-Cit- NH ,

[0848] CO -Phe-Cit- NH ,

[0849] CO -Leu-Cit- NH ,

[0850] CO -Ile-Cit- NH ,

[0851] CO -Phe-Arg- NH ,as well as

[0852] CO -Trp-Cit- NH .

[0853] 34. A compound according to Statement 33, wherein Q is selected from CO -Phe-Lys- NH , CO -Val-Cit- NH and CO -Val-Ala- NH .

[0854] 35. A compound according to any one of Statements 19 to 31, wherein R L has the formula IIIa, and Q is a tripeptide residue selected from:

[0855] CO -Glu-Val-Ala- NH ,

[0856] CO -Glu-Val-Cit- NH ,

[0857] CO -αGlu-Val-Ala- NH ,as well as

[0858] CO -αGlu-Val-Cit- NH .

[0859] 36. A compound according to any one of Statements 19 to 35, wherein R L It has the formula IIIa and a is 0 to 3.

[0860] 37. A compound according to statement 36, wherein a is 0.

[0861] 38. A compound according to any one of Statements 19 to 37, wherein R L has the formula IIIa and b is 0 to 12.

[0862] 39. A compound according to Statement 38, wherein b is 0 to 8.

[0863] 40. A compound according to any one of Statements 19 to 39, wherein R L has the formula IIIa and d is 0 to 3.

[0864] 41. A compound according to Statement 38, wherein d is 2.

[0865] 42. A compound according to any one of Statements 19 to 35, wherein R L having formula IIIa, and a is 0, c is 1 and d is 2, and b is 0-8.

[0866] 43. A compound according to statement 42, wherein b is 0, 4 or 8.

[0867] 44. A compound according to any one of Statements 19 to 43, wherein R L having the formula IIIa and G L Selected from:

[0868]

[0869]

[0870] Where Ar represents C 5-6 Arylene.

[0871] 45. A compound according to Statement 44, wherein Ar is phenylene.

[0872] 46. ​​A compound according to Statement 44 or Statement 45, wherein G L Selected from G L1-1 and G L1-2 .

[0873] 47. A compound according to statement 46, wherein G L G L1-1 .

[0874] 48. A compound according to any one of Statements 19 to 31, wherein R L has the formula IIIb, and R L1 and R L2 Both are H.

[0875] 49. A compound according to any one of Statements 19 to 31, wherein R L Having the formula IIIb, R L1 H and R L2 It is methyl.

[0876] 50. A compound according to any one of Statements 19 to 31, wherein R L has the formula IIIb, and R L1 and R L2 All are methyl.

[0877] 51. A compound according to any one of Statements 19 to 31, wherein R L has the formula IIIb, and R L1 and R L2 Together with the carbon atom to which they are attached they form a cyclopropylene group.

[0878] 52. A compound according to any one of Statements 19 to 31, wherein R L has the formula IIIb, and R L1and R L2 Together with the carbon atom to which they are attached they form a cyclobutylene group.

[0879] 53. A compound according to any one of Statements 19 to 31 and 48 to 52, wherein R L It has the formula IIIb, and e is 0.

[0880] 54. A compound according to any one of Statements 19 to 31 and 48 to 52, wherein R L It has the formula IIIb, and e is 1.

[0881] 55. A compound according to statement 54, wherein the nitro group is in the para position.

[0882] 56. A compound according to Statement 19, wherein the compound has Formula Id:

[0883]

[0884] Where Q is selected from:

[0885] (a) -CH2-;

[0886] (b) -C3H6-; and

[0887] (c)

[0888] 57. A conjugate of formula II:

[0889] L-(D L ) p (II)

[0890] Wherein L is a ligand unit (i.e., a targeting agent), D L A drug linker unit of formula I':

[0891]

[0892] in:

[0893] Y, Y', R", R 2 , R 2’ , R 6 , R 6’ , R 7 , R 7’ , R 9 and R 9’ As defined in any one of statements 1 to 18;

[0894] R 11b , R 30 and R 31 As defined in any of statements 19 to 27 and 29 to 31;

[0895] R LL is a linker for connecting to a cell binding agent selected from:

[0896] (iiia):

[0897]

[0898] wherein Q and X are as defined in any of Statements 19 and 32 to 43, and G LL is a linker attached to the Ligand unit; and

[0899] (iiib):

[0900]

[0901] Where R L1 and R L2 As defined in any of statements 19 and 48 to 52;

[0902] wherein p is an integer from 1 to 20.

[0903] 58. The conjugate according to statement 57, wherein G LL Selected from:

[0904]

[0905]

[0906] Where Ar represents C 5-6 Arylene.

[0907] 59. The conjugate according to statement 58, wherein Ar is phenylene.

[0908] 60. The conjugate according to Statement 58 or Statement 59, wherein G LL Selected from G LL1-1 and G LL1-2 .

[0909] 61. The conjugate according to statement 60, wherein G LL G LL1-1 .

[0910] 62. The conjugate according to statement 57, wherein D L Having the formula (Id'):

[0911]

[0912] Where Q is selected from:

[0913] (a) -CH2-;

[0914] (b) -C3H6-; and

[0915] (c)#imgpt111#

[0916] 63. A conjugate according to any one of Statements 57 to 62, wherein the Ligand unit is an antibody or an active fragment thereof.

[0917] 64. A conjugate according to statement 63, wherein the antibody or antibody fragment is an antibody or antibody fragment to a tumor-associated antigen.

[0918] 65. The conjugate of statement 64, wherein the antibody or antibody fragment is an antibody that binds to one or more tumor-associated antigens or cell surface receptors selected from (1)-(88):

[0919] (1) BMPR1B;

[0920] (2) E16;

[0921] (3) STEAP1;

[0922] (4)0772P;

[0923] (5)MPF;

[0924] (6) Napi3b;

[0925] (7) Sema 5b;

[0926] (8)PSCA hlg;

[0927] (9) ETBR;

[0928] (10)MSG783;

[0929] (11) STEAP2;

[0930] (12)TrpM4;

[0931] (13) CRIPTO;

[0932] (14)CD21;

[0933] (15) CD79b;

[0934] (16) FcRH2;

[0935] (17) HER2;

[0936] (18) NCA;

[0937] (19) MDP;

[0938] (20) IL20R-α;

[0939] (21) Brevican;

[0940] (22) EphB2R;

[0941] (23)ASLG659;

[0942] (24) PSCA;

[0943] (25)GEDA;

[0944] (26) BAFF-R;

[0945] (27) CD22;

[0946] (28)CD79a;

[0947] (29) CXCR5;

[0948] (30) HLA-DOB;

[0949] (31)P2X5;

[0950] (32) CD72;

[0951] (33) LY64;

[0952] (34) FcRH1;

[0953] (35) IRTA2;

[0954] (36)TENB2;

[0955] (37) PSMA–FOLH1;

[0956] (38) SST;

[0957] (38.1)SSTR2;

[0958] (38.2)SSTR5;

[0959] (38.3)SSTR1;

[0960] (38.4)SSTR3;

[0961] (38.5)SSTR4;

[0962] (39)ITGAV;

[0963] (40)ITGB6;

[0964] (41) CEACAM5;

[0965] (42) MET;

[0966] (43)MUC1;

[0967] (44) CA9;

[0968] (45) EGFRvIII;

[0969] (46)CD33;

[0970] (47)CD19;

[0971] (48)IL2RA;

[0972] (49) AXL;

[0973] (50)CD30-TNFRSF8;

[0974] (51) BCMA-TNFRSF17;

[0975] (52) CT Ags–CTA;

[0976] (53) CD174 (Lewis Y)-FUT3;

[0977] (54)CLEC14A;

[0978] (55) GRP78–HSPA5;

[0979] (56) CD70;

[0980] (57) Stem cell specific antigens;

[0981] (58)ASG-5;

[0982] (59)ENPP3;

[0983] (60)PRR4;

[0984] (61)GCC–GUCY2C;

[0985] (62) Liv-1–SLC39A6;

[0986] (63)5T4;

[0987] (64)CD56–NCMA1;

[0988] (65) CanAg;

[0989] (66)FOLR1;

[0990] (67)GPNMB;

[0991] (68)TIM-1–HAVCR1;

[0992] (69) RG-1 / prostate tumor target Mindin – Mindin / RG-1;

[0993] (70) B7-H4–VTCN1;

[0994] (71)PTK7;

[0995] (72)CD37;

[0996] (73)CD138–SDC1;

[0997] (74) CD74;

[0998] (75) tight junction protein – CL;

[0999] (76) EGFR;

[1000] (77) Her3;

[1001] (78)RON-MST1R;

[1002] (79)EPHA2;

[1003] (80)CD20–MS4A1;

[1004] (81) tenascin C–TNC;

[1005] (82) FAP;

[1006] (83)DKK-1;

[1007] (84)CD52;

[1008] (85)CS1-SLAMF7;

[1009] (86) Endoglin – ENG;

[1010] (87) Annexin A1–ANXA1;

[1011] (88) V-CAM (CD106)-VCAM1;

[1012] (89)ASCT2(SLC1A5).

[1013] 66. A conjugate according to any one of statements 63 to 65, wherein the antibody or antibody fragment is a cysteine ​​engineered antibody.

[1014] 67. The conjugate according to any one of Statements 57 to 66, wherein p is an integer from 1 to 8.

[1015] 68. The conjugate according to statement 67, wherein p is 1, 2, 3 or 4.

[1016] 69. A composition comprising a mixture of conjugates according to any one of Statements 57 to 68, wherein in the mixture of conjugated compounds, the average p is from about 1 to about 8.

[1017] 70. A conjugate according to any one of statements 57 to 68 for use in therapy.

[1018] 71. A pharmaceutical composition comprising the conjugate of any one of Statements 57 to 68, and a pharmaceutically acceptable diluent, carrier or excipient.

[1019] 72. A conjugate according to any one of Statements 57 to 68 or a pharmaceutical composition according to Statement 71 for use in treating a proliferative disease in a subject.

[1020] 73. The conjugate for use according to statement 72, wherein the disease to be treated is cancer.

[1021] 74. Use of a conjugate according to any one of statements 57 to 68 or a pharmaceutical composition according to statement 71 in a method of medical treatment.

[1022] 75. A method of medical treatment comprising administering to a patient a pharmaceutical composition as described in statement 71.

[1023] 76. The method of statement 75, wherein the medical treatment method is for treating cancer.

[1024] 77. The method of statement 76, wherein a chemotherapeutic agent is administered to the patient in combination with the conjugate.

[1025] 78. Use of a conjugate according to any one of Statements 57 to 68 in a method for the manufacture of a medicament for the treatment of a proliferative disease.

[1026] 79. A method of treating a mammal having a proliferative disease comprising administering an effective amount of a conjugate according to any one of Statements 57 to 68 or a pharmaceutical composition according to Statement 71.

Claims

1. A compound of formula IV: and salts thereof, wherein: R 2 and R 2’ is H; R 6 and R 9 is independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halogen; wherein R and R' are independently selected from optionally substituted C 1-12 Alkyl, C 3-20 Heterocyclic group, C 6-10 Carboaryl and C 5-14 heteroaryl; and (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-(CH2) n -O-, wherein n is 7 to 16; or (ii) -O-(CH2CH2O) m -, wherein m is 2 to 5; R″ is C 3-12 Alkylene, the chain of which may be interrupted by one or more heteroatoms and / or by aromatic rings, the heteroatoms being selected from O, S or NR N2 , where R N2 H or C 1-4 Alkyl, the aromatic ring is selected from benzene or pyridine; Y and Y' are selected from O, S or NH; R 6’ and R 9’ Selected from R 6 and R 9 The same group; and (ia)R 10 and R 11 together to form a double bond between the N atom and the C atom to which they are bound; or (ib)R 10 H and R 11 Selected from OH and OR A , where R A C 1-4 Alkyl; or (ic)R 10 and R 11 All are H; and (ii-a)R 20 and R 21 together to form a double bond between the N atom and the C atom to which they are bound; or (ii-b)R 20 H and R 21 Selected from OH and OR B , where R B C 1-4 Alkyl; or (ii-c)R 20 and R 21 Both are H.

2. The compound according to claim 1, wherein Y and Y' are both O.

3. The compound according to claim 1 or claim 2, wherein R" is C 3-7 Alkylene.

4. A compound according to claim 1 or claim 2, wherein R" is a group of the formula: Where r is 1 or 2.

5. The compound according to claim 1 or 2, wherein R 9 For H, R 6 For H, R 7 and R 7’ Independently for C 1-4 Alkoxy.

6. The compound according to claim 1 or 2, wherein R 6’ is with R 6 The same group, R 7 ' is with R 7 The same group, R 9 ' is with R 9 is the same group as Y and Y' is the same group as Y.

7. The compound according to claim 1, which has the formula IVa, IVb or IVc: Where R 1a Selected from methyl and benzyl.

8. A compound of formula I: and salts thereof, wherein: Y, Y', R", R 2 , R 2’ , R 6 , R 6’ , R 7 , R 7’ , R 9 and R 9’ As defined in any one of claims 1 to 7; R 11b Selected from OH, OR A , where R A C 1-4 Alkyl; and R L is a linker for connecting to a cell binding agent selected from: (iiia): in Q is: Where Q X is such that Q is an amino acid residue, a dipeptide residue or a tripeptide residue; X is: Where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5; G L is a linker for attachment to the Ligand unit; as well as (iiib): Where R L1 and R L2 are independently selected from H and methyl, or together with the carbon atoms to which they are attached form cyclopropylene or cyclobutylene; and e is 0 or 1; and: (a)R 30 and R 31 together to form a double bond between the N atom and the C atom to which they are bound; or (b)R 30 H and R 31 Selected from OH and OR B , where R B C 1-4 alkyl; (c)R 30 and R 31 All are H; or (d) R 31 OH or OR B , where R B C 1-4 Alkyl and R 30 Selected from: (i) (ii) (iii) Where R Z Selected from: (z-i) (z-ii) OC(=O)CH3; (z-iii) NO2; (z-iv)OMe; (zv) glucuronide; (z-vi)NH-C(=O)-X1-NHC(=O)X2-NH-C(=O)-R ZC , wherein -C(=O)-X1-NH- and -C(=O)-X2-NH- represent natural amino acid residues and R ZC Selected from Me, OMe, CH2CH2OMe and (CH2CH2O)2Me.

9. The compound according to claim 8, wherein R 30 and R 31 Together they form a double bond between the N atom and the C atom to which they are bound.

10. The compound according to claim 8, which has formula Ia, Ib or Ic: Where R 1a Selected from methyl and benzyl.

11. A compound according to any one of claims 8 to 10, wherein R L has the formula IIIa, and Q is a dipeptide residue selected from: CO -Phe-Lys- NH 、 CO -With-Ala- NH 、 CO -Fall-List- NH 、 CO -Lower Lys- NH 、 CO -Val-Cit- NH 、 CO -Phe-Cit- NH , CO -Leo-Cit- NH 、 CO -Ile-Cit- NH 、 CO -Phe-Arg- NH ,as well as CO -Trp-Cit- NH 。 12. A compound according to any one of claims 8 to 10, wherein R L having formula IIIa, and a is 0, c is 1 and d is 2, and b is 0-8.

13. The compound according to claim 12, wherein b is 0, 4 or 8.

14. A compound according to any one of claims 8 to 10, wherein R L having formula IIIa and G L Selected from: wherein Ar represents a phenylene group.

15. The compound according to claim 14, wherein G L G L1-1 .

16. The compound according to claim 8, wherein the compound has Formula Id: Where Q is selected from: (a) -CH2-; (b) -C3H6-; and (c) 17. A conjugate of formula II: L-(D L ) p (II) Wherein L is a ligand unit, the ligand unit is a targeting agent, and D L A drug linker unit of formula I': in: Y, Y', R", R 2 , R 2’ , R 6 , R 6’ , R 7 , R 7’ , R 9 and R 9’ As defined in any one of claims 1 to 6; R 11b , R 30 and R 31 As defined in any one of claims 8 and 9; R LL is a linker for connecting to a cell binding agent selected from: (iiia): wherein Q and X are as defined in any one of claims 8 and 11 to 13, and G LL is a linker attached to the Ligand unit; as well as (iiib): Where R L1 and R L2 As defined in claim 8; wherein p is an integer from 1 to 20.

18. The conjugate according to claim 17, wherein G LL Selected from: wherein Ar represents a phenylene group, and CBA represents the ligand unit.

19. The conjugate according to claim 17, wherein D L Having the formula (Id'): Where Q is selected from: (a) -CH2-; (b) -C3H6-; and (c) 20. A composition comprising a mixture of conjugates according to any one of claims 17 to 19, wherein in the mixture of conjugates, the average p is 1 to 8.

21. Use of the conjugate according to any one of claims 17 to 19 in the preparation of a medicament for treating gastric cancer or breast cancer.

22. A pharmaceutical composition comprising the conjugate according to any one of claims 17 to 19, and a pharmaceutically acceptable diluent, carrier or excipient.

23. Use of the pharmaceutical composition according to claim 22 in the preparation of a medicament for treating gastric cancer or breast cancer in a subject.

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