Antibody drug conjugates comprising toxins having polar groups and uses thereof

Abstract

The present disclosure relates to antibody drug conjugates comprising toxins having polar groups and uses thereof. The present disclosure relates to drugs and toxins functionalized with at least one sugar, sulfate or sulfonate; a drug conjugate comprising the drug or toxin and a cleavable linker; and targeting conjugates comprising the drug or toxin, a cleavable linker, and a targeting moiety. The disclosure also relates to methods of treating cancer, autoimmune diseases, and inflammatory diseases using the compounds and conjugates of the disclosure.

Description

[0001] This application is a divisional application of the PCT international application PCT / IB2022 / 000772 filed on December 20, 2022, which entered the Chinese national phase on June 20, 2024, and whose Chinese patent application number is 202280084624.9 and whose invention name is “Antibody-drug conjugates containing toxins with polar groups and their uses”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 292,101, filed on December 21, 2021. The entirety of this application is incorporated herein by reference. Technical Field

[0004] The present invention relates to an antibody drug conjugate comprising a toxin having a polar group and uses thereof. Background Art

[0005] Cleavable drug conjugates have the potential to combine the binding specificity of antibodies or other targeting groups with the potency of chemotherapeutic agents. Since targeting allows the drug to be accurately delivered to the target cancer cells and released under specific conditions while minimizing collateral damage to healthy cells, this technology improves the efficacy of therapeutic agents and reduces the risk of adverse reactions. However, conventional treatments show non-selective uptake of drugs into normal cells and cancer cells, and their therapeutic effects are not significant. Non-selective uptake is mainly due to the hydrophobicity of the linker-drug, and although research is underway to reduce the hydrophobicity of the linker-drug, limited success has been achieved so far. Summary of the invention

[0006] In certain aspects, provided herein are drug conjugates comprising a compound represented by Formula (VII) or Formula (VIII) and a linker group:

[0007]

[0008] or a pharmaceutically acceptable salt thereof;

[0009] in:

[0010] A is a heterocycle;

[0011] Each R a ' and R b ' is independently halogen, amino, hydroxy, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0012] Two R b'Optionally together to form oxo or =CH 2 ; or two R b 'Together with the intervening atoms, optionally form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group;

[0013] R c 'is a sulfonate, sulfate, hydroxy, amino or thiol;

[0014] R d ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0015] The prerequisite is that at least one R c ' is a sulfonate or sulfate, or at least one R d 'is-L"-Gly;

[0016] R e ' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0017] m is an integer selected from 0-3;

[0018] n is an integer selected from 0 to 8, as long as the valence permits;

[0019] Ring Cy is selected from aryl, heteroaryl, cycloalkyl or heterocycloalkyl;

[0020] is a single bond or a double bond;

[0021] X' is a halogen;

[0022] X” is -NR-, -S-, or -O-;

[0023] R is hydrogen or alkyl;

[0024] Each R a ” and R b " is independently halogen, amino, hydroxy, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or -(L"') r -X"-Gly;

[0025] d is an integer selected from 0-4;

[0026] r is an integer from 0 to 1;

[0027] Each L'' is a bond or a linker,

[0028] R e ” is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0029] p is an integer selected from 0-4;

[0030] DBD is the DNA binding domain;

[0031] L" is a bond or linker; and

[0032] Gly is a monosaccharide, a disaccharide or an oligosaccharide.

[0033] In certain aspects, provided herein are targeted drug conjugates comprising a drug conjugate comprising a compound of the disclosure, a linker group, and a targeting moiety.

[0034] In certain embodiments, the targeted drug conjugate is a compound represented by Formula (XII), Formula (XIII), or Formula (XIV):

[0035]

[0036] or a pharmaceutically acceptable salt thereof;

[0037] Wherein TM is the targeting moiety.

[0038] In certain aspects, provided herein are drug conjugates comprising an active agent and a linking group; wherein the active agent is substituted with a polar group. In some embodiments, the polar group is selected from a sugar, a sulfate, or a sulfonate.

[0039] In some embodiments, the drug conjugate comprising an active agent and a linker group is a drug conjugate of formula (I):

[0040]

[0041] or a pharmaceutically acceptable salt thereof;

[0042] in:

[0043] Z' is a coupling group;

[0044] Ar is aryl, heteroaryl, cycloalkyl or heterocycloalkyl;

[0045] Y' is -(CR b 2 ) y N(R a )-、-(CR b 2 ) y O-or-(CR b 2 ) y S-, which is positioned so that if y is 1, the N, O or S atom is attached to the TG;

[0046] TG is a trigger group that, when activated, generates N, O, or S atoms that can react with SO 2 Reaction by displacement (Q) q -(L') w And form including X-SO 2 and a 5- to 6-membered ring of intervening atoms of Ar;

[0047] X is -O-, -C(R b ) 2 -or-N(R c )-;

[0048] L' is a spacer moiety, if present, which is connected to SO via a heteroatom selected from O, S and N 2 , and is selected so that L' and SO 2 The breaking of the bonds between promotes the release of the active agent;

[0049] Each Q is independently an active agent substituted with a sugar, sulfate or sulfonate;

[0050] q is an integer from 1 to 3;

[0051] w and y are each independently 0 or 1; and

[0052] R a , R b and R c are independently hydrogen or C 1-6 Alkyl; or two R b Together with the atoms to which they are attached, they form a 3- to 5-membered ring;

[0053] The prerequisite is that when w is 0, q is 1.

[0054] In some embodiments, each Q is independently selected from a chemotherapeutic agent substituted with a sugar, sulfate, or sulfonate or a toxin substituted with a sugar, sulfate, or sulfonate.

[0055] In some embodiments, the active agent is a chemotherapeutic agent substituted with a sugar, sulfate, or sulfonate.

[0056] In some embodiments, each Q is independently selected from an immunomodulatory compound substituted with a sugar, sulfate or sulfonate, an anticancer agent substituted with a sugar, sulfate or sulfonate, an antiviral agent substituted with a sugar, sulfate or sulfonate, an antibacterial agent substituted with a sugar, sulfate or sulfonate, an antifungal agent substituted with a sugar, sulfate or sulfonate, or an antiparasitic agent substituted with a sugar, sulfate or sulfonate, and an amanitin substituted with a sugar, sulfate or sulfonate.

[0057] In some embodiments, each Q is independently selected from a benzodiazepine substituted with a sugar, sulfate, or sulfonate, a duocarmycin substituted with a sugar, sulfate, or sulfonate, an auristatin substituted with a sugar, sulfate, or sulfonate, a tubulysin substituted with a sugar, sulfate, or sulfonate, a SN-38 substituted with a sugar, sulfate, or sulfonate, a PNU substituted with a sugar, sulfate, or sulfonate, or an exitecan substituted with a sugar, sulfate, or sulfonate.

[0058] In some embodiments, each Q is independently represented by:

[0059]

[0060] in:

[0061] Z 2 is a linking group;

[0062] Z 3 is a linking group;

[0063] t is an integer from 1 to 5;

[0064] e is an integer from 1 to 5; and

[0065] Each Q' is independently a modified benzodiazepine. In some embodiments, each Q' is independently represented by formula (IIa) or formula (IIb):

[0066]

[0067] or a pharmaceutically acceptable salt thereof;

[0068] in:

[0069] Each A is a heterocycle;

[0070] Each R a ' is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl (preferably lower alkyl), alkenyl, alkynyl, cycloalkyl, aryl or heterocyclic, preferably a five-membered ring or a six-membered ring; optionally, fused to or substituted by one or more aryl or heteroaryl rings;

[0071] Each R b ' is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclic, preferably a five-membered ring or a six-membered ring; optionally, fused to or substituted by one or more aryl or heteroaryl rings;

[0072] Or two R b 'Optionally together to form oxo or =CH 2 ; or two R b' together with the intervening atoms optionally form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group, optionally substituted by one or more R f 'replace

[0073] Each R f ' is independently halogen, hydroxy, -O-Gly, cyano, nitro, alkyl, haloalkyl, cycloalkyl, carboxyl, amino, aminoalkyl (-CH 2 NH 2 、-CH 2 NH(Me) or -CH 2 N(Me) 2 ), aryl or heteroaryl;

[0074] R c 'is a sulfonate, sulfate, hydroxy, amino or thiol;

[0075] R d ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0076] The prerequisite is that at least one R c ' is a sulfonate or sulfate, or at least one R d 'is-L"-Gly;

[0077] L” is a key or joint;

[0078] m is an integer selected from 0-3;

[0079] n is an integer selected from 0 to 8, as long as the valence permits; and

[0080] Gly is a sugar group, preferably a monosaccharide, a disaccharide or an oligosaccharide.

[0081] In certain embodiments, the drug conjugate is a compound of Formula (IIIa) or Formula (IIIb):

[0082]

[0083]

[0084] or a pharmaceutically acceptable salt thereof.

[0085] In a further embodiment, the drug conjugate is a compound represented by formula (V):

[0086]

[0087] or a pharmaceutically acceptable salt thereof.

[0088] In some embodiments, the drug conjugate is represented by Formula (Va) or Formula (Vb):

[0089]

[0090] or a pharmaceutically acceptable salt thereof.

[0091] In some embodiments, DBD-(L'') r The -X"-Gly unit is selected from:

[0092]

[0093] or a pharmaceutically acceptable salt thereof;

[0094] in:

[0095] Y” is C or N;

[0096] Each R b " is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0097] R k is alkyl or hydroxyalkyl, preferably C 1 -C 3 alkyl;

[0098] q is an integer selected from 0-3; and

[0099] Is a single bond or a double bond.

[0100] In a further aspect, provided herein is a targeted drug conjugate of formula (VI), comprising a targeting moiety conjugated to any one of the drug conjugates of the present disclosure:

[0101]

[0102] Wherein TM is the targeting moiety.

[0103] In some embodiments, Z' is selected from

[0104]

[0105]

[0106]

[0107]

[0108] in

[0109] R za is H or methyl;

[0110] R zbis -OH, =O or =NOH;

[0111] Single or double bonds;

[0112] n and m are each independently an integer selected from 1-10;

[0113] x is an integer selected from 1-2;

[0114] a" represents the bond between Z' and the drug conjugate;

[0115] b" represents the bond between Z' and TM; and

[0116] Z" is selected from

[0117]

[0118] In a further aspect, provided herein is a targeted drug conjugate of formula (VIb), comprising a targeting moiety conjugated to a drug conjugate of the present disclosure:

[0119]

[0120] in:

[0121] TM is the targeting moiety;

[0122] R is hydrogen or a hydroxyl protecting group;

[0123] X is -C(O)-, -NH-, -O- or -S-;

[0124] Q is an active agent substituted with a sugar, sulfonate, or sulfate;

[0125] T is

[0126] n is an integer selected from 0 or 1;

[0127] Y is hydrogen, halogenated C 1 -C 8 alkyl, halogen, cyano or nitro; z is an integer selected from 1 to 3, and if z is an integer not less than 2, Y may be the same as or different from each other;

[0128] z1 is an integer selected from 0 or 1;

[0129] W 1 yes

[0130] W 2 yes

[0131] W a1 and W a2are each independently -NH-, -C(=O)- or -CH 2 -;

[0132] W a3 and W a4 are independently -NH-, -C(=O)-, -CH 2 -, -C(=O)NH-, -NHC(=O)- or triazolylene;

[0133] W b1 is an amide bond or a triazole group;

[0134] L is used as a connection W a2 and an amino acid, peptide or amide bond of a linker to Z;

[0135] Z is a single bond, -W a5 -(CH 2 ) a2 -W b2 -(CH 2 ) a3 -W a6 -or-W a7 -(CH 2 ) a4 -CR'R"-X"'-;

[0136] R' is C 1 -C 8 Alkyl or TM-W a8 -Q 3 -W c1 -(CH 2 ) a5 -;

[0137] R" is TM-W a8 -Q 3 -W c1 -(CH 2 ) a5 -;

[0138] Q 1 and Q 3 Each independently is -(CH 2 ) a6 -(X 1 CH 2 CH 2 ) b1 -(CH 2 ) a7 -;

[0139] X 1 and X 3 are independently -O-, -S-, -NH- or -CH 2 -;

[0140] X'' is -NHC(=O)-(CH 2 ) a8 -W a9 - or -C(=O)NH-(CH 2 ) a8 -W a9 -;

[0141] W a5 , W a6 , W a7 , W a8 and W a9 are each independently -NH-, -C(=O)- or -CH 2 -;

[0142] W b2 is an amide bond or a triazole group;

[0143] W c1 is -NHC(=O)- or -C(=O)NH-;

[0144] Q 2 is a linear or branched saturated or unsaturated alkylene group having 1 to 50 carbon atoms, satisfying any one of the following (i) to (iii);

[0145] (i) at least one -CH 2 - is substituted by one or more heteroatoms selected from -NH-, -C(=O), -O- and -S-,

[0146] (ii) at least one arylene group or heteroarylene group is contained in the alkylene group,

[0147] (iii) the alkylene group is further selected from C 1 -C 20 Alkyl, C 6 -C 20 Aryl C 1 -C 8 Alkyl, -(CH 2 ) s1 COOR 3 、-(CH 2 ) s1 COR 3 、-(CH 2 ) s2 CONR 4 R 5 and -(CH 2 ) s2 NR 4 R 5 Replacement by one or more of the group consisting of;

[0148] The arylene or heteroarylene group in (ii) above may be further substituted by a nitro group;

[0149] R 3 , R 4 and R 5 are independently hydrogen or C 1 -C 15 alkyl;

[0150] X 2 is -O-, -S-, -NH- or -CH 2 -;

[0151] U 1 It is bound to B' at the position of the asterisk (*) via a linker selected from the following structures:

[0152]

[0153] R is for C 1 -C 10 Alkyl, C 6 -C 20 Aryl or C 2 -C 20 heteroaryl;

[0154] TM and B' are each independently a ligand or a protein having the property of selectively targeting a drug to a specific organ, tissue or cell, i.e., the property of binding to a receptor;

[0155] a1, a2, a3, a4, a5, a6, a8, b1, p1, p2, p3 and p4 are each independently an integer selected from 1-10;

[0156] a7, y, s1, s2 and s4 are each independently an integer selected from 0-10; and

[0157] R 1 and R 2 Each is independently hydrogen, C 1 -C 8 Alkyl or C 3 -C 8 Cycloalkyl.

[0158] In a further aspect, provided herein is a targeted drug conjugate of formula (VIc), comprising a targeting moiety conjugated to a drug conjugate of the present disclosure:

[0159]

[0160] in:

[0161] TM is the targeting moiety;

[0162] G is a glucuronic acid moiety or a derivative thereof;

[0163] Q is an active agent substituted with a sugar, sulfonate, or sulfate;

[0164] W is an electron withdrawing group;

[0165] Z is hydrogen, C 1 -C 8 Alkyl, halogen, cyano or nitro;

[0166] n is an integer selected from 1 to 3, and when n is an integer of 2 or greater, each Z is the same as or different from each other;

[0167] L is a connector connecting TM and W; and

[0168] R 1 and R 2 Each is independently hydrogen, C 1 -C 8 Alkyl or C 3 -C 8 Cycloalkyl.

[0169] In certain aspects, provided herein is a targeted drug conjugate of formula (VId), comprising a targeting moiety conjugated to a drug conjugate of the present disclosure:

[0170] TM-L 1 -(A a -W w -Y y -Q 1-4 ) p

[0171] (VId);

[0172] Wherein TM is the targeting moiety;

[0173] L 1 is the ligand part;

[0174] Q is an active agent substituted with a sugar, sulfonate, or sulfate;

[0175] -A a -W w -Y y - is the joint part;

[0176] A is an optional extension;

[0177] a is an integer selected from 0-3;

[0178] Each W is independently a glucuronide unit having one of the following formulae:

[0179]

[0180] Su is the sugar part;

[0181] Each R is independently hydrogen, halogen, -CN or -NO 2 ;

[0182] w is an integer selected from 1-2;

[0183] Y is an optional self-immolative spacer moiety;

[0184] y is an integer selected from 0-2; and

[0185] p is an integer selected from 1-20.

[0186] In yet a further aspect, provided herein is a pharmaceutical composition comprising any one of the targeted drug conjugates provided herein.

[0187] In yet a further aspect, provided herein is a compound of formula (VII) or formula (VIII):

[0188]

[0189] or a pharmaceutically acceptable salt thereof;

[0190] in:

[0191] A is a heterocycle;

[0192] Each R a ' and R b ' is independently halogen, amino, hydroxy, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0193] Two R b 'Optionally together to form oxo or =CH 2 ; or two R b 'Together with the intervening atoms, optionally form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group;

[0194] R c 'is a sulfonate, sulfate, hydroxy, amino or thiol;

[0195] R d ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0196] The prerequisite is that at least one R c ' is a sulfonate or sulfate, or at least one R d 'is-L"-Gly;

[0197] R e ' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0198] m is an integer selected from 0-3;

[0199] n is an integer selected from 0 to 8, as long as the valence permits;

[0200] Ring Cy is selected from aryl, heteroaryl, cycloalkyl or heterocycloalkyl;

[0201] is a single bond or a double bond;

[0202] X' is a halogen;

[0203] X” is -NR-, -S-, or -O-;

[0204] Each R a ” and R b " is independently halogen, amino, hydroxy, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or -(L"') r -X"-Gly;

[0205] d is an integer selected from 0-4;

[0206] r is an integer selected from 0-1;

[0207] Each L'' is a bond or a linker,

[0208] R e ” is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0209] p is an integer selected from 0-4;

[0210] DBD is the DNA binding domain;

[0211] L" is a bond or linker; and

[0212] Gly is a monosaccharide, a disaccharide or an oligosaccharide.

[0213] In some embodiments, each L'" is C 10 -C 100 The straight or branched, saturated or unsaturated alkylene moiety optionally contains one or more double and / or triple bonds.

[0214] In some embodiments, each p and each d are independently an integer from 0 to 1. In certain embodiments, the compound is a compound of formula (VII):

[0215]

[0216] or a pharmaceutically acceptable salt thereof.

[0217] In some embodiments, A is a 5- to 6-membered heterocyclic ring.

[0218] In some embodiments, R c ' is hydroxyl. In some embodiments, R c ' is a sulfonate or sulfate.

[0219] In some embodiments, R d ' is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocycloalkyl, C 6-10 aryl or 5- to 10-membered heteroaryl.

[0220] In some embodiments, R d ' is L"-Gly. In some embodiments, R d 'It's hydrogen.

[0221] In some embodiments, each Q' is independently selected from:

[0222]

[0223] or a pharmaceutically acceptable salt thereof;

[0224] in Is a single bond or a double bond.

[0225] In some embodiments, the compound is selected from:

[0226]

[0227] or a pharmaceutically acceptable salt thereof;

[0228] in Is a single bond or a double bond.

[0229] In some embodiments, R a ' is halogen, amino, hydroxy, alkoxy, cyano, nitro, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 4- to 10-membered heterocycloalkyl, C 6-10 aryl or 5- to 10-membered heteroaryl.

[0230] In some embodiments, an R b ' is an alkyl group or two geminal R b 'Together they form an alkenyl group.

[0231] In some embodiments, both R b 'Together with the intervening atoms, form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group; preferably, wherein the aryl or heteroaryl group is optionally substituted by one or more R f 'Substituted 6-membered aryl or heteroaryl.

[0232] In some embodiments, both R b ' together with the intervening atoms form an aryl or heteroaryl group; preferably, wherein the aryl or heteroaryl group is optionally substituted by one or more R f 'Substituted 6-membered aryl or heteroaryl.

[0233] In some embodiments, the two geminal R b 'Together form =CH 2 .

[0234] In some embodiments, both R b ' together with the intervening atoms form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group.

[0235] In some embodiments, both R b ' together with the intervening atoms form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group.

[0236] In some embodiments, both R b ' together with the intervening atoms constitute an aryl or heteroaryl group.

[0237] In some embodiments, both R b ' together with the intervening atoms constitute an aryl group.

[0238] In some embodiments, both R b ' together with the intervening atoms constitute a heteroaryl group.

[0239] In some embodiments, each Q' is independently selected from:

[0240]

[0241] or a pharmaceutically acceptable salt thereof;

[0242] in is a single bond or a double bond; and

[0243] Where g is an integer from 0 to 4.

[0244] In some embodiments, R e ' is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocycloalkyl, C 6-10In some embodiments, R e ' is hydrogen, C 1-6 Alkyl or C 3-10 In some embodiments, R e 'It's hydrogen.

[0245] In some embodiments, the compound is selected from:

[0246]

[0247] or a pharmaceutically acceptable salt thereof. In a further embodiment, the compound is a compound of formula (VIII):

[0248]

[0249] or a pharmaceutically acceptable salt thereof.

[0250] In some embodiments, Cy is phenyl.

[0251] In some embodiments, Cy is pyrrolidine or pyrrole.

[0252] In further embodiments, the compound has the structure of Formula (VIIIa) or Formula (VIIIb):

[0253]

[0254] or a pharmaceutically acceptable salt thereof.

[0255] In some embodiments, DBD-(L'') r The -X"-Gly unit is selected from:

[0256]

[0257] or a pharmaceutically acceptable salt thereof;

[0258] in:

[0259] Y” is C or N;

[0260] X" is selected from -NR-, -S- or -O-;

[0261] R is hydrogen or alkyl;

[0262] r is an integer from 0 to 1;

[0263] Each R b " is independently halogen, amino, hydroxy, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or -(L"') r -X"-Gly;

[0264] R k is an alkyl group, preferably C 1 -C 3 alkyl;

[0265] q is an integer selected from 0-3; and

[0266] is a single bond or a double bond. In some embodiments, the drug conjugate is selected from a group of Formula (Vc), Formula (Vd), Formula (Ve), or Formula (Vf):

[0267]

[0268]

[0269] or a pharmaceutically acceptable salt thereof.

[0270] In some embodiments, the drug conjugate is represented by Formula (Vc) or Formula (Vd):

[0271]

[0272] or a pharmaceutically acceptable salt thereof.

[0273] In some embodiments, each R a " is independently hydrogen, halogen, amino, hydroxy, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L') r -X"-Gly; and

[0274] Each R b " is independently hydrogen, halogen, amino, hydroxy, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L') r -X”-Gly.

[0275] In some embodiments, Q is selected from:

[0276]

[0277]

[0278] or a pharmaceutically acceptable salt thereof.

[0279] In some embodiments, each Q is independently selected from a group of Formula (IVc), Formula (IVd), Formula (IVe), Formula (IVf), Formula (IVg), Formula (IVh), Formula (IVi), or Formula (IVj):

[0280]

[0281]

[0283] or a pharmaceutically acceptable salt thereof.

[0284] In further embodiments, each Q is independently selected from a group of formula (IVc), formula (IVd), formula (IVe), or formula (IVf):

[0285]

[0287] or a pharmaceutically acceptable salt thereof.

[0288] In further embodiments, the compound has the structure of Formula (VIIIc), Formula (VIIId), Formula (VIIIe), or Formula (VIIIf):

[0289]

[0291] or a pharmaceutically acceptable salt thereof.

[0292] In some embodiments, each R a " is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and each R b " is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, C 1-6 Alkyl, heteroalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 4- to 10-membered heterocycloalkyl, C 6-10 aryl or 5- to 10-membered heteroaryl.

[0293] In some embodiments, X' is Cl. In some embodiments, X' is Br. In some embodiments, Y" is C. In some embodiments, Y" is N.

[0294] In some embodiments, R e ' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. In some embodiments, R e " is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocycloalkyl, C 6-10 In some embodiments, R e " is hydrogen, C1-6 Alkyl or C 3-10 In some embodiments, R e "It's hydrogen.

[0295] In a further embodiment, the compound is selected from:

[0296]

[0297]

[0298] or a pharmaceutically acceptable salt thereof.

[0299] In some embodiments, L''' is a bond.

[0300] In some embodiments, L'' is a linker selected from:

[0301]

[0302]

[0303] in:

[0304] R a "' is hydrogen, halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and

[0305] Each R b "' is independently hydrogen, halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and

[0306] h is an integer selected from 0 to 4, as far as the valence permits.

[0307] In some embodiments, L"' is

[0308] In some embodiments, Gly is a monosaccharide. In some embodiments, Gly is a monosaccharide selected from glucose, glucuronic acid, fucose and galactose.

[0309] In some embodiments, Gly is

[0310]

[0311] Optionally, one or more -OH groups are masked by a protecting group.

[0312] In some embodiments, Gly is

[0313]

[0314] In some embodiments, Gly is a disaccharide. In some embodiments, Gly is a disaccharide comprising glucose, glucuronic acid, fucose, galactose, or a combination thereof.

[0315] In some embodiments, Gly is

[0316]

[0317] Optionally, one or more -OH groups are masked by a protecting group.

[0318] In some embodiments, Gly is

[0319]

[0320] In some embodiments, X" or L" is coupled to Gly at the anomeric position.

[0321] In some embodiments, X" is coupled to Gly at the anomeric position. In certain aspects, provided herein are drug conjugates comprising any of the disclosed compounds and a linker group. In certain embodiments, the drug conjugate is a compound of formula (IX), formula (X), or formula (XI):

[0322]

[0323] or a pharmaceutically acceptable salt thereof;

[0324] in:

[0325] Z' is a coupling group;

[0326] Ar is aryl, heteroaryl, cycloalkyl or heterocycloalkyl;

[0327] Y' is -(CR b 2 ) y N(R a )-、-(CR b 2 ) y O-or-(CR b 2 ) y S-, which is positioned so that if y is 1, the N, O or S atom is attached to the TG;

[0328] TG is a trigger group that, when activated, generates N, O, or S atoms that can react with SO 2 Reaction by displacement (Q) q -(L') w And form including X-SO 2and a 5- to 6-membered ring of intervening atoms of Ar;

[0329] X is -O-, -C(R b ) 2 -or-N(R c )-;

[0330] L' is a spacer moiety, if present, which is connected to SO via a heteroatom selected from O, S and N 2 , and is selected so that L' and SO 2 The breaking of the bonds between promotes the release of the active agent;

[0331] w is an integer selected from 0-1;

[0332] r is an integer from 0 to 1;

[0333] Z 2 is a linking group;

[0334] Z 3 is a linking group;

[0335] R a , R b and R c are each independently hydrogen or lower alkyl;

[0336] y is an integer selected from 0-1;

[0337] t is an integer from 1 to 5; and

[0338] e is an integer from 1 to 5.

[0339] In some embodiments, Z 3 Selected from:

[0340]

[0341] in:

[0342] X 5 Is -O- or -NR x -;

[0343] Y 1 Yes CR y or N;

[0344] R x and R y are independently hydrogen or C 1-6 alkyl;

[0345] Each b is independently an integer from 1 to 3; and

[0346] c is an integer from 1 to 5.

[0347] In some embodiments, Z 3 Selected from:

[0348]

[0349] In some embodiments, Z 2 In some embodiments, Z 2 yes

[0350]

[0351] in:

[0352] Y 5 Yes CR Y1 or N, provided there is only one Y 5 is N;

[0353] R Y1 is H, hydroxyl, amino, amide or (CH 2 ) y (R Y1a );

[0354] R Y1a is amino (eg, secondary or tertiary amino), aryl (eg, phenyl), or heteroaryl; and

[0355] y is an integer having a value of 1 to about 10.

[0356] In some embodiments, Z 2 yes

[0357]

[0358] In some embodiments, Z 2 yes

[0359]

[0360] in:

[0361] Y 6 Yes CR Y2 or N;

[0362] R Y2 is H or alkyl, preferably lower alkyl;

[0363] R Z2 Yes (CH 2 ) z R Z2a ;

[0364] R Z2a is amino (preferably tertiary amino), aryl (e.g. phenyl) or heteroaryl; and

[0365] z is an integer having a value from 0 to about 10.

[0366] In some embodiments, Z 2 yes

[0367]

[0368] In some embodiments, Z 2 yes in:

[0369] Y 5 Yes CR Y1 or N, provided there is only one Y 5 is N;

[0370] R Y1 is H, hydroxyl, amino, amide or (CH 2 ) z (R Y1a );

[0371] R Y1a is amino (eg, secondary or tertiary amino), aryl (eg, phenyl), or heteroaryl; and

[0372] z is an integer having a value from 1 to about 10.

[0373] In some embodiments, Z 2 yes

[0374] In yet further embodiments, each Q is independently a group of formula (IV):

[0375]

[0376] or a pharmaceutically acceptable salt thereof;

[0377] in:

[0378] Ring Cy is selected from aryl, heteroaryl, cycloalkyl or heterocycloalkyl;

[0379] is a single bond or a double bond;

[0380] X' is a halogen;

[0381] X" is selected from -NR-, -S- or -O-;

[0382] Each R a ” and R b" are independently halogen, amino, hydroxy, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L'")rX"-Gly;

[0383] p is an integer selected from 0-4;

[0384] d is an integer selected from 0-4;

[0385] r is an integer from 0 to 1;

[0386] DBD is the DNA binding domain;

[0387] Each L'' is a bond or a linker, and

[0388] Gly is a monosaccharide, a disaccharide or an oligosaccharide.

[0389] In some embodiments, each Q is independently a group of formula (IVa) or formula (IVb):

[0390]

[0391] or a pharmaceutically acceptable salt thereof.

[0392] In some embodiments, Ar is aryl. In some embodiments, Ar is C 6-10 In some embodiments, Ar is phenyl. In some embodiments, Ar is heteroaryl. In some embodiments, Ar is a 5- to 10-membered heteroaryl.

[0393] In some embodiments, Y' is -(CR b 2 ) y N(R a )-or-(CR b 2 ) y In some embodiments, Y' is -(CR b 2 ) y O-.

[0394] In some embodiments, y is 0 or 1. In some embodiments, y is 0. In some embodiments, y is 1.

[0395] In some embodiments, X is -O- or -N(R c In some embodiments, X is -O-.

[0396] In some embodiments, L' is a spacer moiety and forms an -O-, -OC(O)-, -OC(O)O-, -NHC(O)O-, or -OC(O)NH- linkage including the heteroatom of the active agent. In some embodiments, L' is a spacer moiety and forms an -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH- linkage including the heteroatom of the active agent. In some embodiments, Q-(L') w -Selected from

[0397]

[0398] in:

[0399] X 4 does not exist or forms an -O-, -OC(O)-, -OC(O)O- or -OC(O)NH- linkage, including the heteroatom of Q;

[0400] X 1 Is -O- or -NR a -;

[0401] R a is hydrogen or alkyl;

[0402] X 2 is -O-, -OC(O)-, -OC(O)O- or -OC(O)NH-;

[0403] X 3 is -OC(=O)-;

[0404] w' is an integer having a value from 1 to 5;

[0405] R 9 and R 10 are each independently hydrogen, alkyl, aryl or heteroaryl, wherein alkyl, aryl and heteroaryl are unsubstituted or substituted with one or more radicals selected from, for example, alkyl, -(CH 2 ) u NH 2 、-(CH 2 ) u NR u1 R u2 and -(CH 2 ) u SO 2 R u3 Substituents are substituted;

[0406] R u1 , R u2 and R u3 are each independently hydrogen, alkyl, aryl or heteroaryl; and

[0407] u is an integer having a value of 1-10.

[0408] In some embodiments, Q-(L') w -yes

[0409]

[0410] In some embodiments, TG is a monosaccharide. In some embodiments, TG is a monosaccharide selected from glucose, glucuronic acid, fucose, and galactose.

[0411] In some embodiments, TG is

[0412]

[0413] Optionally, one or more -OH groups are masked by a protecting group.

[0414] In some embodiments, TG is

[0415]

[0416] In some embodiments, the TG is a disaccharide. In some embodiments, the TG is a disaccharide comprising glucose, glucuronic acid, fucose, galactose, or a combination thereof.

[0417] In some embodiments, TG is

[0418]

[0419] Optionally, one or more -OH groups are masked by a protecting group.

[0420] In some embodiments, TG is

[0421]

[0422] In some embodiments, L' is coupled to TG at the anomeric position.

[0423] In some embodiments, Y' is coupled to TG at the anomeric position.

[0424] In some embodiments, Z' is selected from

[0425]

[0426]

[0427]

[0428] in:

[0429] R zais H or methyl;

[0430] R zb is -OH, =O or =NOH;

[0431] m and n are each an integer selected from 1-10;

[0432] x is an integer selected from 1-2;

[0433] single or double bonds; and

[0434] Z" is selected from

[0435]

[0436] In some embodiments, Z' is selected from

[0437]

[0438]

[0439]

[0440]

[0441] in:

[0442] R za is H or methyl;

[0443] R zb is -OH, =O or =NHOH;

[0444] n and m are each independently an integer selected from 1-10;

[0445] x is an integer selected from 1-2;

[0446] represents the bond between Z' and the drug conjugate;

[0447] single or double bonds; and

[0448] Z" is selected from

[0449]

[0450] In some embodiments, the drug conjugate is selected from

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460] or a pharmaceutically acceptable salt thereof.

[0461] In a further aspect, provided herein is a pharmaceutical composition comprising any of the drug conjugates provided herein.

[0462] In a further aspect, provided herein is a targeted drug conjugate comprising any one of the drug conjugates provided herein and a targeting moiety. In certain embodiments, the drug conjugate is a compound of formula (XII), formula (XIII) or formula (XIV):

[0463]

[0464]

[0465] or a pharmaceutically acceptable salt thereof;

[0466] Wherein TM is the targeting moiety.

[0467] In some embodiments, Z' is selected from

[0468]

[0469]

[0470]

[0471]

[0472] in

[0473] R za is H or methyl;

[0474] R zb is -OH, =O or =NOH;

[0475] n and m are each independently an integer selected from 1-10;

[0476] x is an integer selected from 1-2;

[0477] Single or double bonds;

[0478] a" represents the bond between Z' and the drug conjugate;

[0479] b" represents the bond between Z' and TM; and

[0480] Z" is selected from

[0481]

[0482] In some embodiments, R za is H or methyl; R zb is -OH, =O or =NHOH; n and m are each independently an integer selected from 1-10; x is an integer selected from 1-2; single bond or double bond; a" represents the bond between Z' and the drug conjugate; b" represents the bond between Z' and TM; and Z" is selected from

[0483]

[0484] In some embodiments, the TM is a nanoparticle, an immunoglobulin, a nucleic acid, a protein, an oligopeptide, a polypeptide, an antibody, a fragment or a repeat of an antigenic polypeptide.

[0485] In some embodiments, the TM is an antibody selected from the group consisting of an intact polyclonal antibody, an intact monoclonal antibody, an antibody fragment, a single-chain Fv (scFv) mutant, a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a fusion protein comprising an antigenic determinant portion of an antibody, and other modified immunoglobulin molecules comprising an antigen recognition site.

[0486] In some embodiments, the antibody is selected from muromonab-CD3, abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab (Herceptin), etanercept, basiliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, afacept, omalizumab, efalizumab, tositumomab-I 131, cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, rilocept, becelimumab, romiplostim, AMG-531, CNTO-148, CNTO-1275, ABT-874, LEA-29Y, belimumab, TACI-Ig, second generation anti-CD20, ACZ-885, tocilizumab, alizumab, mepolizumab, pertuzumab, HumaxCD20, tesitumumab (CP-675 206), tesitumumab, MDX-010, IDEC-114, ituzumab ozogamicin, HuMax EGFR, aflibercept, HuMax-CD4, Ala-Ala, ChAglyCD3, TRX4, catumaxomab, IGN101, MT-201, prigovinomab, CH-14.18, WX-G250, AMG-162, AAB-001, motuzumab, MEDI-524, ifenguzumab, oloriba, raxicumab, third-generation anti-CD20, LY2469298, and velutuzumab.

[0487] In a further aspect, provided herein is a method for treating cancer, comprising administering to a subject in need thereof any one of the compounds, drug conjugates, targeted drug conjugates, or pharmaceutical compositions provided herein. In certain embodiments, the cancer is selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, urinary bladder cancer, kidney cancer, renal pelvic cancer, oral cancer, pharyngeal cancer, uterine body cancer, or melanoma.

[0488] In yet further aspects, provided herein is a method for treating an autoimmune disease or inflammatory disease, the method comprising administering to a subject in need thereof any one of the compounds, drug conjugates, targeted drug conjugates, or pharmaceutical compositions provided herein. In certain embodiments, the autoimmune disease or inflammatory disease is selected from a B cell-mediated autoimmune disease or inflammatory disease, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom's hypergammaglobulinaemia, Sjogren's syndrome, multiple sclerosis (MS), or lupus nephritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0489] Figure 1A The in vivo efficacy of T-2-AB, T-3-AB, and T-4-AB in the JIMT-1 xenograft model was demonstrated.

[0490] Figure 1B The in vivo efficacy of T-103-AB, T-104-AB, T-116-AB, and T-117-AB in the JIMT-1 xenograft model was demonstrated.

[0491] Figure 2A Plasma stability of the seco-MCBI-HAI duocarmycin payload is shown, where A indicates an unsubstituted toxin-linker conjugate and B indicates a sugar-substituted toxin-linker conjugate.

[0492] Figure 2B Plasma stability of seco-DUBA duocarmycin payloads is shown, where C indicates an unsubstituted toxin-linker conjugate and D indicates a sugar-substituted toxin-linker conjugate. DETAILED DESCRIPTION

[0493] Provided herein are compounds, drug conjugates, and targeted drug conjugates that can be used to treat cancer and / or autoimmune or inflammatory diseases. Compounds, drug conjugates, and targeted drug conjugates can be derived from various toxin payloads that have been modified with sugars, sulfates, or sulfonates. Compounds, drug conjugates, and targeted drug conjugates can significantly reduce the nonspecific uptake of drugs.

[0494] Some components of the technology disclosed herein, including cleavable linker technology and targeting moieties, are further described in WO 2019 / 008441, WO 2019 / 229536, WO 2020 / 141459, WO 2020 / 141460, PCT / IB2021 / 000445, US16,472,983, US14,898,932 and US 11,996,009, each of which is incorporated herein by reference in its entirety.

[0495] definition

[0496] Unless otherwise defined herein, the scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. In general, the terms and techniques used in conjunction with chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are those well-known and commonly used in the art.

[0497] Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and described in various general and more specific references cited and discussed throughout this specification. See, for example, "Principles of Neural Science", McGraw-Hill Medical, New York, NY (2000); Motulsky, "Intuitive Biostatistics", Oxford University Press, Inc. (1995); Lodish et al., "Molecular Cell Biology, 4th Edition", WH Freeman & Co., New York (2000); Griffiths et al., "Introduction to Genetic Analysis, 7th Edition", WH Freeman & Co., NY (1999); and Gilbert et al., "Developmental Biology, 6th Edition", Sinauer Associates, Inc., Sunderland, MA (2000).

[0498] Unless otherwise defined herein, chemical terms used herein are used according to conventional usage in the art as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., ed., McGraw-Hill, San Francisco, CA (1985).

[0499] All of the above, as well as any other publications, patents, and published patent applications mentioned in this application, are expressly incorporated herein by reference. In the event of a conflict, the present specification, including its specific definitions, will control.

[0500] The term "agent" is used herein to refer to a compound (such as an organic or inorganic compound, a mixture of compounds), a biopolymer (such as a nucleic acid, an antibody, including portions thereof and humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portions thereof, such as peptides, lipids, carbohydrates), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents of known structure and agents of unknown structure. The ability of such agents to inhibit AR or promote AR degradation may make them suitable as "therapeutic agents" in the methods and compositions of the present disclosure.

[0501] "Patient", "subject" or "individual" are used interchangeably and refer to human or non-human animals. These terms include mammals, such as humans, primates, livestock animals (including cattle, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats).

[0502] "Treatment" of a disease or patient refers to taking measures to obtain beneficial or desired results, including clinical results. As used herein and as fully understood in the art, "treatment" is a method for obtaining beneficial or desired results (including clinical results). Beneficial or desired clinical results may include, but are not limited to, alleviation or improvement of one or more symptoms or diseases, reduction of disease extent, stabilization of disease state (i.e., no deterioration), prevention of disease spread, delay or slowing of disease progression, improvement or alleviation of disease state, and relief (whether partial relief or total relief), whether detectable or undetectable. "Treatment" may also mean prolonged survival compared to expected survival when not receiving treatment.

[0503] The term "prevention" is art-recognized and well known in the art when used with respect to a condition such as local recurrence (e.g., pain), a disease such as cancer, a symptom such as heart failure, or any other medical condition, and includes the administration of a composition that reduces the frequency of symptoms of the medical condition in a subject, or delays the onset thereof, relative to a subject not receiving the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population relative to an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0504] "Administering / administration" a substance, compound or agent to a subject can be implemented using one of a variety of methods known to those skilled in the art. For example, a compound or agent can be administered in the following ways: intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, through the eye, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinal, intracerebrally, and transdermally (by absorption, such as through a skin tube). A compound or agent can also be suitably introduced by a refillable or biodegradable polymer device or other devices (such as patches and pumps) or preparations that provide extended, slow or controlled release for a compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more extended time periods.

[0505] The appropriate method for administering a substance, compound or agent to a subject will also depend on, for example, the age and / or physical condition of the subject and the chemical and biological properties (e.g., solubility, digestibility, bioavailability, stability and toxicity) of the compound or agent. In some embodiments, the compound or agent is orally administered to a subject, for example, by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or is administered using a device for such slow or extended release.

[0506] As used herein, the phrase "combined administration" refers to any form of administration of two or more different therapeutic agents such that a second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., both agents are effective in the patient simultaneously, which may include a synergistic effect of the two agents). For example, different therapeutic compounds may be administered simultaneously or sequentially in the same formulation or in separate formulations. Thus, an individual receiving such treatment may benefit from the combined effects of the different therapeutic agents.

[0507] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or agent is an amount of the drug or agent that will have the intended therapeutic effect when administered to a subject. The full therapeutic effect does not necessarily occur by administering a single dose, and may only occur after a series of doses are administered. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount required for a subject will depend, for example, on the subject's size, health, and age, and the nature and extent of the condition (such as cancer or MDS) being treated. A skilled person can readily determine the effective amount for a given situation by routine experimentation.

[0508] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, "optionally substituted alkyl" refers to instances where the alkyl group may be substituted as well as instances where the alkyl group is not substituted.

[0509] It is understood that substituents and substitution patterns on the compounds of the present disclosure can be selected by one of ordinary skill in the art to yield chemically stable compounds that can be readily synthesized from readily available starting materials by techniques known in the art and those described below. If a substituent is itself substituted with more than one group, it is understood that the multiple groups can be on the same carbon or on different carbons, as long as a stable structure is obtained.

[0510] As used herein, the term "optionally substituted" refers to the replacement of one to six hydrogen atoms in a given structure with specified substituents, including but not limited to hydroxy, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silicon, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH 2 -O-alkyl, -OP(O)(O-alkyl) 2 or -CH 2 -OP(O)(O-alkyl) 2 Preferably, "optionally substituted" refers to the replacement of one to four hydrogen atoms in a given structure with the substituents mentioned above. More preferably, one to three hydrogen substituents are replaced with the substituents mentioned above. It should be understood that the substituents may be further substituted.

[0511] As used herein, the term "alkyl" refers to a saturated aliphatic group, including but not limited to C 1 -C 10 Straight chain alkyl or C 1 -C 10 Preferably, an "alkyl" group refers to a C 1 -C 6 Straight chain alkyl or C 1 -C 6 Most preferably, an "alkyl" group refers to a C 1 -C 4 Straight chain alkyl or C 1 -C 4 Branched chain alkyl. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neopentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl or 4-octyl, etc. "Alkyl" groups can be optionally substituted.

[0512] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0513] The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group, and may be represented, for example, by the formula hydrocarbyl C(O)NH-.

[0514] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0515] The term "alkoxy" refers to an alkyl group attached to an oxygen. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0516] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group, and may be represented by the general formula alkyl-O-alkyl.

[0517] The term "alkyl" refers to a saturated aliphatic group, including straight chain alkyl, branched chain alkyl, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl and cycloalkyl substituted alkyl. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., for a straight chain alkyl having 30 carbon atoms or less, for a straight chain alkyl having 30 carbon atoms or less, for a branched ... 1-30 , for the branched chain C 3-30 ), and more preferably 20 or less. The term "lower alkyl" refers to an alkyl group having 1 to 6 carbon atoms.

[0518] In addition, the term "alkyl" as used throughout the specification, examples and claims is intended to include unsubstituted and substituted alkyl groups, the latter referring to alkyl moieties having substituents replacing hydrogen on one or more carbon atoms of the hydrocarbon backbone, including halogenated alkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl and the like.

[0519] When used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, the term "C x-y " or "C x -C y " is intended to include groups containing from x to y carbons in the chain. 0 Alkyl indicates hydrogen where the group is in terminal position, or a bond if internal. For example, C 1-6 Alkyl groups contain from one to six carbon atoms in the chain.

[0520] As used herein, the term "alkylamino" refers to an amino group substituted with at least one alkyl group.

[0521] As used herein, the term "alkylthio" refers to a thiol group substituted with an alkyl group, and may be represented by the general formula alkylS-.

[0522] As used herein, the term "amide" refers to a group

[0523]

[0524] Where R 9 and R 10 Each independently represents hydrogen or a hydrocarbon group, or R 9 and R 10 Together with the N atom to which they are attached they form a heterocyclic ring having 4 to 8 atoms in the ring structure.

[0525] The terms "amine" and "amino" are art-recognized and refer to unsubstituted and substituted amines and salts thereof, for example, a moiety represented by the formula

[0526]

[0527] Where R 9 , R 10 and R 10 ' each independently represents hydrogen or a hydrocarbon group, and R 9 and R 10 Together with the N atom to which they are attached they form a heterocyclic ring having 4 to 8 atoms in the ring structure.

[0528] As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group.

[0529] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0530] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups, wherein each atom of the ring is carbon. The ring is preferably a 5-7 ring, more preferably a 6-ring. The term "aryl" also includes a polycyclic system with two or more rings, wherein two or more carbons are common to two adjacent rings, wherein at least one ring is aromatic, for example, other rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclic. Aryl includes benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0531] The term "carbamate" is art-recognized and refers to the group

[0532]

[0533] Where R 9 and R 10 independently represent hydrogen or a hydrocarbon group.

[0534] As used herein, the term "carbocyclylalkyl" refers to an alkyl group substituted with a carbocyclyl group.

[0535] The term "carbocycle" includes 5-7 membered monocycles and 8-12 membered bicyclic rings. Each ring of the bicyclic carbocycle can be selected from saturated, unsaturated and aromatic rings. Carbocycles include bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each ring shares two adjacent atoms with another ring. Each ring of the fused carbocycle can be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring (e.g., phenyl) can be fused with a saturated or unsaturated ring (e.g., cyclohexane, cyclopentane or cyclohexene). When the valence number allows, any combination of saturated, unsaturated and aromatic bicyclic rings is included in the definition of carbocycle. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo [2.2.1] heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo [4.2.0] oct-3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decahydronaphthalene, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocycle" may be substituted at any one or more positions capable of carrying a hydrogen atom.

[0536] As used herein, the term "carbocyclylalkyl" refers to an alkyl group substituted with a carbocyclyl group.

[0537] The term "carbonate" is art-recognized and refers to the group -OCO 2 -.

[0538] As used herein, the term "carboxyl" refers to a group of the formula -CO 2 The group represented by H.

[0539] As used herein, the term "ester" refers to the group -C(O)OR 9 , where R 9 It represents a hydrocarbon group.

[0540] As used herein, the term "ether" refers to a hydrocarbyl group connected to another hydrocarbyl group through an oxygen. Thus, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. An ether may be symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which may be represented by the general formula alkyl-O-alkyl.

[0541] As used herein, the terms "halo" and "halogen" refer to halogen and include chlorine (Cl), fluorine (F), bromine (Br) and iodine (I).

[0542] As used herein, the terms "hetaralkyl" and "heteroaralkyl" refer to an alkyl group substituted with a heteroaryl group.

[0543] The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic monocyclic structures, preferably 5 to 7 rings, more preferably 5 to 6 rings, whose ring structure includes at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic systems with two or more rings, wherein two or more carbons are shared by two adjacent rings, wherein at least one ring is heteroaromatic, for example, other rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclic radicals. Heteroaryl includes, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine etc.

[0544] As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen and sulfur.

[0545] As used herein, the term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl group.

[0546] The terms "heterocyclyl", "heterocycle" and "heterocyclic" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3 to 10-membered rings, more preferably 3 to 7-membered rings, whose ring structures contain at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more rings, wherein two or more carbon atoms are common to two adjacent rings, wherein at least one ring is heterocyclic, for example, the other rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclyl. Heterocyclyl includes, for example, piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam, etc.

[0547] As used herein, the term "hydrocarbyl" refers to a group bonded by a carbon atom that does not have a =O or =S substituent, and typically has at least one carbon-hydrogen bond and a main chain that is mainly carbon, but may optionally contain heteroatoms. Therefore, for the purposes of this application, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl, but substituents such as acetyl (which has a =O substituent on the connecting carbon) and ethoxy (which is connected by oxygen rather than carbon) are not hydrocarbyl. Hydrocarbyl includes, but is not limited to, aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[0548] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0549] When used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy, the term "lower" is meant to include groups having ten or fewer atoms, preferably six or fewer atoms, in the substituent. For example, "lower alkyl" refers to an alkyl group containing ten or fewer, preferably six or fewer carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl or lower alkoxy, whether they occur alone or in combination with other substituents, such as in describing hydroxyalkyl and aralkyl (in this case, for example, when calculating the carbon atoms in the alkyl substituent, the atoms in the aryl are not counted).

[0550] The terms "polycyclic group", "polycyclic" and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclyl) wherein two or more atoms are common to two adjacent rings, e.g., the ring is a "fused ring". Each ring of the polycyclic ring may be substituted or unsubstituted. In certain embodiments, each ring of the polycyclic ring contains 3 to 10 atoms, preferably 5 to 7 atoms, in the ring.

[0551] The term "sulfate" is art-recognized and refers to the group -OSO 3 H or a pharmaceutically acceptable salt thereof.

[0552] The term "sulfonamide" is art-recognized and refers to a group represented by the general formula

[0553]

[0554] Where R 9 and R 10 independently represent hydrogen or a hydrocarbon group.

[0555] The term "sulfoxide" is art-recognized and refers to the group -S(O)-.

[0556] The term "sulfonate" is art-recognized and refers to the group SO 3 H or a pharmaceutically acceptable salt thereof.

[0557] The term "sulfite" is art-recognized and refers to the group -OS(O)OH or a pharmaceutically acceptable salt thereof.

[0558] The term "sulfate" is art-recognized and refers to the group -OSO 3 H or a pharmaceutically acceptable salt thereof.

[0559] The term "sulfone" is art-recognized and refers to the group -S(O) 2 -.

[0560] The term "substituted" refers to the part of the substituent having a replacement hydrogen on one or more carbons of the main chain. It will be understood that "replacement" or "replaced by ... " includes implicit conditions, that is, such substitution is based on the allowed valence of the substituted atom and the substituent, and the substitution produces a stable compound, for example, it will not spontaneously undergo transformations such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad sense, permissible substituents include non-cyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Permissible substituents can be one or more substituents and are the same or different for appropriate organic compounds. For the purposes of this disclosure, heteroatoms (such as nitrogen) can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that meet heteroatom valences. Substituents may include any of the substituents described herein, for example, halogen, hydroxy, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl or aromatic or heteroaromatic moieties. Those skilled in the art will appreciate that, where appropriate, the moieties substituted on the hydrocarbon chain may themselves be substituted.

[0561] As used herein, the term "thioalkyl" refers to an alkyl group substituted with a thiol group.

[0562] As used herein, the term "thioester" refers to the group -C(O)SR 9 or -SC(O)R 9 , where R 9 It represents a hydrocarbon group.

[0563] As used herein, the term "thioether" is equivalent to an ether in which the oxygen is replaced by sulfur.

[0564] The term "urea" is art-recognized and may be represented by the general formula

[0565]

[0566] Where R 9 and R 10 independently represent hydrogen or a hydrocarbon group.

[0567] As used herein, the term "modulate" includes inhibiting or suppressing a function or activity (such as cell proliferation) as well as enhancing a function or activity.

[0568] "Pharmaceutically acceptable salt" or "salt" is used herein to refer to acid addition salts or base addition salts that are suitable for use in or compatible with the treatment of a patient.

[0569] As used herein, the term "pharmaceutically acceptable acid addition salt" means any nontoxic organic or inorganic salt of any base compound represented by Formula I. Exemplary inorganic acids forming suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid and metal salts, such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids forming suitable salts include monocarboxylic acids, dicarboxylic acids and tricarboxylic acids, such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid and salicylic acid and sulfonic acids, such as p-toluenesulfonic acid and methanesulfonic acid. Monobasic or dibasic acid salts can be formed, and such salts can exist in hydrated form, solvated form or substantially anhydrous form. In general, the acid addition salts of Formula I compounds are more soluble in water and various hydrophilic organic solvents, and generally exhibit higher melting points compared to their free base forms. The selection of appropriate salts is known to those skilled in the art. Other non-pharmaceutically acceptable salts, such as oxalates, may be used, for example, in isolating a compound of the disclosure for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0570] As used herein, the term "pharmaceutically acceptable base addition salt" means any non-toxic organic or inorganic base addition salt of any acid compound represented by Formula I or any intermediate thereof. Illustrative inorganic bases that form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide or barium hydroxide. Illustrative organic bases that form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of appropriate salts will be known to those skilled in the art.

[0571] Many compounds that can be used in the methods and compositions of the present disclosure have at least one stereocenter in their structure. This stereocenter can exist in the R or S configuration, and the R and S symbols are used according to the rules described in Pure Appl. Chem. (1976), 45, 11-30. The present disclosure contemplates all stereoisomeric forms, such as enantiomeric and diastereomeric forms of the compound, its salt, prodrug or mixture (including all possible mixtures of stereoisomers). See, for example, WO 01 / 062726.

[0572] In certain embodiments, the compounds of the present disclosure may be racemic. In certain embodiments, the compounds of the present disclosure may be enriched in one enantiomer. For example, the compounds of the present disclosure may have greater than about 30% ee, 40% ee, 50% ee, 60% ee, 70% ee, 80% ee, 90% ee, 95% ee, 96% ee, 97% ee, 98% ee, 99% ee or higher ee.

[0573] As is generally understood in the art, a single bond drawn without stereochemistry does not indicate the stereochemistry of the compound. The compounds of Formula I provide examples of compounds for which no stereochemistry is indicated.

[0574] In certain embodiments, compositions or compounds of the present disclosure can be enriched to mainly provide a kind of enantiomer of compound.Enantiomer-enriched compositions or compounds can include, for example, at least 60 mol % of a kind of enantiomer, or more preferably at least 75 mol %, 90 mol %, 95 mol % or even 99 mol %.In certain embodiments, the compound that is rich in a kind of enantiomer is substantially free of other enantiomers, wherein substantially free of means that compared with the amount of other enantiomers in, for example, compositions or compound mixtures, the material in question accounts for less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%.For example, if compositions or compounds contain 98 grams of the first enantiomer and 2 grams of the second enantiomer, it can be said that it contains 98 mol % of the first enantiomer and only 2 mol % of the second enantiomer.

[0575] Additionally, certain compounds containing alkenyl groups may exist as Z (same side) or E (hetero side) isomers. In each case, the disclosure includes both the mixture and the separate individual isomers.

[0576] Some compounds may also exist in tautomeric forms. Although not explicitly indicated in the formulas described herein, such forms are intended to be included within the scope of the present disclosure.

[0577] "Prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized (e.g., hydrolyzed or oxidized) in a host to form a compound of the present disclosure (e.g., a compound of formula I) after administration. Typical examples of prodrugs include compounds having biologically unstable or cleavable (protective) groups on the functional portion of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminized, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce an active compound. Examples of prodrugs using esters or phosphoramidates as biologically unstable or cleavable (protective) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. Prodrugs of the present disclosure are metabolized to produce compounds of formula I. The present disclosure includes within its scope prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.

[0578] As used herein, the term "logarithm of solubility", "LogS" or "logS" is used in the art to quantify the water solubility of a compound. The water solubility of a compound significantly affects its absorption and distribution characteristics. Low solubility is often associated with poor absorption. The LogS value is the unit exfoliation logarithm (base 10) of the solubility measured in moles / liter.

[0579] As used herein, the term "glycosyl" refers to a monovalent substituent formed by any natural sugar, its metabolite / catabolite, its prodrug, or a combination thereof. This term includes oligosaccharides and polysaccharides in linear and branched forms, as well as α and β configurations or any combination thereof. The preferred chain length of a polysaccharide is one or two (i.e., a monosaccharide or a disaccharide). In certain preferred embodiments, a glycosyl refers to a substituent formed by glucose, fucose, galactose, mannose, xylose, galactosamine, glucuronic acid, galacturonic acid, mannuronic acid, sialic acid, iduronic acid, neuraminic acid, a derivative thereof, or a combination thereof.

[0580] Toxin Payload

[0581] Many toxin payloads are suitable for the conjugates disclosed herein. In certain embodiments, the toxin payload is selected from a chemotherapeutic agent substituted with a sugar, sulfate or sulfonate or a toxin substituted with a sugar, sulfate or sulfonate. In further embodiments, the active agent is a chemotherapeutic agent substituted with a sugar, sulfate or sulfonate. In further embodiments, the toxin payload is independently selected from an immunomodulatory compound substituted with a sugar, sulfate or sulfonate, an anticancer agent substituted with a sugar, sulfate or sulfonate, an antiviral agent substituted with a sugar, sulfate or sulfonate, an antibacterial agent substituted with a sugar, sulfate or sulfonate, an antifungal agent substituted with a sugar, sulfate or sulfonate, or an antiparasitic agent substituted with a sugar, sulfate or sulfonate.

[0582] In yet further embodiments, the toxin payload is independently selected from a benzodiazepine substituted with a sugar, sulfate or sulfonate, a duocarmycin substituted with a sugar, sulfate or sulfonate, an auristatin substituted with a sugar, sulfate or sulfonate, tubulysine substituted with a sugar, sulfate or sulfonate, SN-38 substituted with a sugar, sulfate or sulfonate, PNU substituted with a sugar, sulfate or sulfonate, or exatecan substituted with a sugar, sulfate or sulfonate, amanitin substituted with a sugar, sulfate or sulfonate.

[0583] In yet further embodiments, the toxin payload may be functionalized at one or more functional groups selected from -C(O)-, -O-, -NH-, -S-, and -C(O)O-. In further embodiments, the functional groups are functionalized with sugars, sulfates, or sulfonates. In some embodiments, the toxin payload may comprise a modified portion bound to a sugar via a functional group selected from esters, amides, sulfyls, carbamates, oximes, hydrazones, and the like. In some embodiments, the toxin payload may comprise a modified portion bound to a polar group such as a sulfonate (see, e.g., WO 2006 / 111759 A1), a sulfate, a sulfite, and the like.

[0584] In yet a further aspect, provided herein is a compound of formula (VII) or formula (VIII):

[0585]

[0586]

[0587] or a pharmaceutically acceptable salt thereof;

[0588] in:

[0589] A is a heterocycle;

[0590] Each R a ' and R b ' is independently halogen, amino, hydroxy, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0591] Two R b 'Optionally together to form oxo or =CH 2 ; or two R b 'Together with the intervening atoms, optionally form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group;

[0592] R c 'is a sulfonate, sulfate, hydroxy, amino or thiol;

[0593] R d ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0594] The prerequisite is that at least one R c ' is a sulfonate or sulfate, or at least one R d 'is-L"-Gly;

[0595] R e ' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0596] m is an integer selected from 0-3;

[0597] n is an integer selected from 0 to 8, as long as the valence permits;

[0598] Ring Cy is selected from aryl, heteroaryl, cycloalkyl or heterocycloalkyl;

[0599] is a single bond or a double bond;

[0600] X' is a halogen;

[0601] X” is -NR-, -S-, or -O-;

[0602] Each R a ” and R b " is independently halogen, amino, hydroxy, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or -(L"') r -X"-Gly;

[0603] d is an integer selected from 0-4;

[0604] r is an integer selected from 0-1;

[0605] Each L'' is a bond or a linker,

[0606] R e ” is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0607] p is an integer selected from 0-4;

[0608] DBD is the DNA binding domain;

[0609] L" is a bond or linker; and

[0610] Gly is a monosaccharide, a disaccharide or an oligosaccharide.

[0611] In certain embodiments, each L'" is C 10 -C 100 A linear or branched, saturated or unsaturated alkylene moiety optionally contains one or more double bonds and / or triple bonds. In a further embodiment, each p and each d is independently an integer from 0 to 1.

[0612] In yet further embodiments, the compound is represented by formula (VII):

[0613]

[0614] or a pharmaceutically acceptable salt thereof.

[0615] In yet further embodiments, A is a 5- to 6-membered heterocyclic ring. c In a further embodiment, R d ' is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocycloalkyl, C 6-10 In a further embodiment, R d ' is L"-Gly. In yet further embodiments, the compound is selected from:

[0616]

[0617] or a pharmaceutically acceptable salt thereof;

[0618] in Is a single bond or a double bond.

[0619] In certain embodiments, R a ' is halogen, amino, hydroxy, alkoxy, cyano, nitro, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10Cycloalkyl, 4- to 10-membered heterocycloalkyl, C 6-10 In a further embodiment, the two geminal R b 'Together form =CH 2 In a further embodiment, the two R b ' together with the intervening atoms form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl. In yet further embodiments, the two R b ' together with the intervening atoms form an aryl or heteroaryl group. In certain embodiments, two R b ' together with the intervening atoms constitute an aryl group.

[0620] In a further embodiment, R e ' is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocycloalkyl, C 6-10 In a further embodiment, R e ' is hydrogen, C 1-6 Alkyl or C 3-10 In yet further embodiments, R e 'It's hydrogen.

[0621] In certain embodiments, the compound is selected from:

[0622]

[0623] or a pharmaceutically acceptable salt thereof.

[0624] In a further embodiment, the compound is represented by formula (VIII):

[0625]

[0626] or a pharmaceutically acceptable salt thereof.

[0627] In further embodiments, Cy is phenyl. In yet further embodiments, Cy is pyrrolidine or pyrrole. In certain embodiments, the compound is represented by formula (VIIIa) or formula (VIIIb):

[0628]

[0629]

[0630] or a pharmaceutically acceptable salt thereof.

[0631] In a further embodiment, DBD-(L"') r The -X"-Gly unit is selected from:

[0632]

[0633] or a pharmaceutically acceptable salt thereof;

[0634] in:

[0635] Y” is C or N;

[0636] X" is selected from -NR-, -S- or -O-;

[0637] R is hydrogen or alkyl;

[0638] r is an integer selected from 0-1;

[0639] Each R b " is independently halogen, amino, hydroxy, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or -(L"') r -X"-Gly;

[0640] R k is an alkyl group, preferably C 1 -C 3 alkyl;

[0641] q is an integer selected from 0-3; and

[0642] Is a single bond or a double bond.

[0643] In still further embodiments, the compound is represented by Formula (VIIIc), Formula (VIIId), Formula (VIIIe), or Formula (VIIIf):

[0644]

[0646] or a pharmaceutically acceptable salt thereof.

[0647] In yet further embodiments, each R a " is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and each R b " is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, C 1-6 Alkyl, heteroalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 4- to 10-membered heterocycloalkyl, C 6-10 aryl or 5- to 10-membered heteroaryl.

[0648] In certain embodiments, X' is -Cl. In further embodiments, X' is Br. In still further embodiments, Y" is C. In certain embodiments, Y" is N. In further embodiments, R e ' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. In a further embodiment, R e " is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocycloalkyl, C 6-10 In yet further embodiments, R e ' is hydrogen, C 1-6 Alkyl or C 3-10 In certain embodiments, R e "It's hydrogen.

[0649] In certain embodiments, the compound is selected from:

[0650]

[0651]

[0652] or a pharmaceutically acceptable salt thereof.

[0653] In further embodiments, L'' is a bond. In still further embodiments, L'' is a linker selected from:

[0654]

[0655] in:

[0656] R a "' is hydrogen, halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and

[0657] Each R b "' is independently hydrogen, halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and

[0658] h is an integer selected from 0 to 4, as far as the valence permits.

[0659] In yet further embodiments, L"' is

[0660]

[0661] In certain embodiments, Gly is a monosaccharide. In further embodiments, Gly is a monosaccharide selected from glucose, glucuronic acid, fucose, and galactose. In still further embodiments, Gly is

[0662]

[0663] Optionally, one or more -OH groups are masked by a protecting group.

[0664] In yet further embodiments, Gly is

[0665]

[0666] In certain embodiments, Gly is a disaccharide. In further embodiments, Gly is a disaccharide comprising glucose, glucuronic acid, fucose, galactose, or a combination thereof. In still further embodiments, Gly is

[0667]

[0668] Optionally, one or more -OH groups are masked by a protecting group.

[0669] In certain embodiments, Gly is

[0670]

[0671] In certain embodiments, X" is coupled to Gly at the anomeric position.

[0672] Drug conjugates

[0673] In certain aspects, provided herein are drug conjugates comprising a compound represented by Formula (VII) or Formula (VIII) and a linker group:

[0674]

[0676] or a pharmaceutically acceptable salt thereof;

[0677] in:

[0678] A is a heterocycle;

[0679] Each R a ' and R b ' is independently halogen, amino, hydroxy, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0680] Two R b'Optionally together to form oxo or =CH 2 ; or two R b 'Together with the intervening atoms, optionally form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group;

[0681] R c 'is a sulfonate, sulfate, hydroxy, amino or thiol;

[0682] R d ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0683] The prerequisite is that at least one R c ' is a sulfonate or sulfate, or at least one R d 'is-L"-Gly;

[0684] R e ' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0685] m is an integer selected from 0-3;

[0686] n is an integer selected from 0 to 8, as long as the valence permits;

[0687] Ring Cy is selected from aryl, heteroaryl, cycloalkyl or heterocycloalkyl;

[0688] is a single bond or a double bond;

[0689] X' is a halogen;

[0690] X” is -NR-, -S-, or -O-;

[0691] R is hydrogen or alkyl;

[0692] Each R a ” and R b " is independently halogen, amino, hydroxy, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or -(L"') r -X"-Gly;

[0693] d is an integer selected from 0-4;

[0694] r is an integer from 0 to 1;

[0695] Each L'' is a bond or a linker,

[0696] R e ” is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0697] p is an integer selected from 0-4;

[0698] DBD is the DNA binding domain;

[0699] L" is a bond or linker; and

[0700] Gly is a monosaccharide, a disaccharide or an oligosaccharide.

[0701] In certain embodiments, each L'" is C 10 -C 100 A linear or branched, saturated or unsaturated alkylene moiety optionally contains one or more double bonds and / or triple bonds. In a further embodiment, each p and each d is independently an integer from 0 to 1.

[0702] In still further embodiments, the drug conjugate comprises a compound of formula (VII):

[0703]

[0704] or a pharmaceutically acceptable salt thereof.

[0705] In yet further embodiments, A is a 5- to 6-membered heterocyclic ring. c In a further embodiment, R d ' is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocycloalkyl, C 6-10 In a further embodiment, R d ' is L"-Gly. In yet further embodiments, the compound is selected from:

[0706]

[0707] or a pharmaceutically acceptable salt thereof;

[0708] in Is a single bond or a double bond.

[0709] In certain embodiments, R a ' is halogen, amino, hydroxy, alkoxy, cyano, nitro, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 4- to 10-membered heterocycloalkyl, C 6-10 In a further embodiment, the two geminal R b 'Together form =CH 2 In a further embodiment, the two R b' together with the intervening atoms form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl. In yet further embodiments, the two R b ' together with the intervening atoms form an aryl or heteroaryl group. In certain embodiments, two R b ' together with the intervening atoms constitute an aryl group.

[0710] In a further embodiment, R e ' is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocycloalkyl, C 6-10 In a further embodiment, R e ' is hydrogen, C 1-6 Alkyl or C 3-10 In yet further embodiments, R e 'It's hydrogen.

[0711] In certain embodiments, the compound is selected from:

[0712]

[0713] or a pharmaceutically acceptable salt thereof.

[0714] In a further embodiment, the compound is represented by formula (VIII):

[0715]

[0716] or a pharmaceutically acceptable salt thereof.

[0717] In further embodiments, Cy is phenyl. In yet further embodiments, Cy is pyrrolidine or pyrrole. In certain embodiments, the compound is represented by formula (VIIIa) or formula (VIIIb):

[0718]

[0719]

[0720] or a pharmaceutically acceptable salt thereof.

[0721] In a further embodiment, DBD-(L"') r The -X"-Gly unit is selected from:

[0722]

[0723] or a pharmaceutically acceptable salt thereof;

[0724] in:

[0725] Y” is C or N;

[0726] X" is selected from -NR-, -S- or -O-;

[0727] R is hydrogen or alkyl;

[0728] r is an integer selected from 0-1;

[0729] Each R b " is independently halogen, amino, hydroxy, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or -(L"') r -X"-Gly;

[0730] R k is an alkyl group, preferably C 1 -C 3 alkyl;

[0731] q is an integer selected from 0-3; and

[0732] Is a single bond or a double bond.

[0733] In still further embodiments, the compound is represented by Formula (VIIIc), Formula (VIIId), Formula (VIIIe), or Formula (VIIIf):

[0734]

[0736] or a pharmaceutically acceptable salt thereof.

[0737] In yet further embodiments, each R a " is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and each R b " is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, C 1-6 Alkyl, heteroalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 4- to 10-membered heterocycloalkyl, C 6-10 In certain preferred embodiments, at least one R a " is alkoxy, such as methoxy, ethoxy or propoxy. In certain embodiments, X' is -Cl. In further embodiments, X' is Br. In yet further embodiments, Y" is C. In certain embodiments, Y" is N. In further embodiments, R e' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. In a further embodiment, R e " is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocycloalkyl, C 6-10 In yet further embodiments, R e ' is hydrogen, C 1-6 Alkyl or C 3-10 In certain embodiments, R e "It's hydrogen.

[0738] In certain embodiments, at least one R a " at position 8. In a further embodiment, p is 1, d is 0, and R a ” at position 8, that is, the compound is represented by formula (VIIIg) or formula (VIIIh):

[0739]

[0741] or a pharmaceutically acceptable salt thereof. In certain embodiments of Formula (VIIIg) or Formula (VIIIh), R a " is an alkoxy group, such as methoxy, ethoxy or propoxy, preferably methoxy. In certain such embodiments, the compound is selected from:

[0742]

[0743] or a pharmaceutically acceptable salt thereof.

[0744] In further embodiments, L'' is a bond. In still further embodiments, L'' is a linker selected from:

[0745]

[0746] in:

[0747] R a "' is hydrogen, halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and

[0748] Each R b "' is independently hydrogen, halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and

[0749] h is an integer selected from 0 to 4, as far as the valence permits.

[0750] In yet further embodiments, L"' is

[0751]

[0752] In certain embodiments, Gly is a monosaccharide. In further embodiments, Gly is a monosaccharide selected from glucose, glucuronic acid, fucose, and galactose. In still further embodiments, Gly is

[0753]

[0754] Optionally, one or more -OH groups are masked by a protecting group.

[0755] In yet further embodiments, Gly is

[0756]

[0757] In certain embodiments, Gly is a disaccharide. In further embodiments, Gly is a disaccharide comprising glucose, glucuronic acid, fucose, galactose, or a combination thereof. In still further embodiments, Gly is

[0758]

[0759] Optionally, one or more -OH groups are masked by a protecting group.

[0760] In certain embodiments, Gly is

[0761]

[0762] In certain embodiments, X" is coupled to Gly at the anomeric position.

[0763] In certain aspects, provided herein are drug conjugates comprising any of the disclosed compounds and a linker group. In certain embodiments, the drug conjugate is a compound of formula (IX), formula (X) or formula (XI):

[0764]

[0765] or a pharmaceutically acceptable salt thereof;

[0766] in:

[0767] Z' is a coupling group;

[0768] Ar is aryl, heteroaryl, cycloalkyl or heterocycloalkyl;

[0769] Y' is -(CR b 2 ) yN(R a )-、-(CR b 2 ) y O-or-(CR b 2 ) y S-, which is positioned so that if y is 1, the N, O or S atom is attached to the TG;

[0770] TG is a trigger group that, when activated, generates N, O, or S atoms that can react with SO 2 Reaction by displacement (Q) q -(L') w And form including X-SO 2 and a 5- to 6-membered ring of intervening atoms of Ar;

[0771] X is -O-, -C(R b ) 2 -or-N(R c )-;

[0772] L' is a spacer moiety, if present, which is connected to SO via a heteroatom selected from O, S and N 2 , and is selected so that L' and SO 2 The breaking of the bonds between promotes the release of the active agent;

[0773] w is an integer selected from 0-1;

[0774] r is an integer from 0 to 1;

[0775] Z 2 is a linking group;

[0776] Z 3 is a linking group;

[0777] R a , R b and R c are each independently hydrogen or lower alkyl;

[0778] y is an integer selected from 0-1;

[0779] t is an integer from 1 to 5; and

[0780] e is an integer from 1 to 5.

[0781] In certain embodiments, Z 3 Selected from:

[0782]

[0783] in:

[0784] X5 Is -O- or -NR x -;

[0785] Y 1 Yes CR y or N;

[0786] R x and R y are independently hydrogen or C 1-6 alkyl;

[0787] Each b is independently an integer from 1 to 3; and

[0788] c is an integer from 1 to 5.

[0789] In a further embodiment, Z 3 Selected from:

[0790]

[0791] In a further embodiment, Z 2 In yet further embodiments, Z 2 yes

[0792]

[0793] in:

[0794] Y 5 Yes CR Y1 or N, provided there is only one Y 5 is N;

[0795] R Y1 is H, hydroxyl, amino, amide or (CH 2 ) y (R Y1a );

[0796] R Y1a is amino (eg, secondary or tertiary amino), aryl (eg, phenyl), or heteroaryl; and

[0797] y is an integer having a value of 1 to about 10.

[0798] In certain embodiments, Z 2 yes

[0799]

[0800] In a further embodiment, Z 2 yes:

[0801]

[0802] in:

[0803] Y 6 Yes CR Y2 or N;

[0804] R Y2 is H or alkyl, preferably lower alkyl;

[0805] R Z2 Yes (CH 2 ) z R Z2a ;

[0806] R Z2a is amino (preferably tertiary amino), aryl (e.g. phenyl) or heteroaryl; and

[0807] z is an integer having a value from 0 to about 10.

[0808] In a further embodiment, Z 2 yes:

[0809]

[0810] In a further embodiment, Ar is aryl. In an even further embodiment, Ar is C 6-10 In yet further embodiments, Ar is phenyl. In certain embodiments, Ar is heteroaryl. In further embodiments, Ar is a 5- to 10-membered heteroaryl. In yet further embodiments, Y' is -(CR b 2 ) y N(R a )-or-(CR b 2 ) y In yet further embodiments, Y' is -(CR b 2 ) y In certain embodiments, y is 0. In further embodiments, y is 1. In still further embodiments, X is -O-, C(R b )(R c )-or-N(R c )-. In yet further embodiments, X is -O-. In certain embodiments, L' is a spacer moiety and forms an -O-, -OC(O)-, -OC(O)O-, -NHC(O)O-, or -OC(O)NH- linkage including the heteroatom of the active agent.

[0811] In certain embodiments, L' is selected from

[0812]

[0813] in:

[0814] X 4 does not exist or forms an -O-, -OC(O)-, -OC(O)O- or -OC(O)NH- linkage, including the heteroatom of Q;

[0815] X 1 Is -O- or -NR a -;

[0816] X 2 is -O-, -OC(O)-, -OC(O)O- or -OC(O)NH-;

[0817] X 3 is -OC(=O)-;

[0818] w' is an integer having a value of 1, 2, 3, 4 or 5;

[0819] R 9 and R 10 are each independently hydrogen, alkyl, aryl or heteroaryl, wherein alkyl, aryl and heteroaryl are unsubstituted or substituted with one or more radicals selected from, for example, alkyl, -(CH 2 ) u NH 2 、-(CH 2 ) u NR u1 R u2 and -(CH 2 ) u SO 2 R u3 Substituents are substituted;

[0820] R u1 , R u2 and R u3 are each independently hydrogen, alkyl, aryl or heteroaryl; and

[0821] u is an integer having a value from 1 to about 10.

[0822] In a further embodiment, L' is

[0823]

[0824] In certain aspects, provided herein is a drug conjugate comprising any one of the toxin payload compounds disclosed herein (active agent) and a linker; wherein the active agent is substituted with a polar group. In some embodiments, the polar group is selected from a sugar, a sulfate, or a sulfonate.

[0825] In some embodiments, the drug conjugate comprising an active agent and a linker group is a drug conjugate of formula (I):

[0826]

[0827] or a pharmaceutically acceptable salt thereof;

[0828] in:

[0829] Z' is a coupling group;

[0830] Ar is aryl, heteroaryl, cycloalkyl or heterocycloalkyl;

[0831] Y' is -(CR b 2 ) y N(R a )-、-(CR b 2 ) y O-or-(CR b 2 ) y S-, which is positioned so that if y is 1, the N, O or S atom is attached to the TG;

[0832] TG is a trigger group that, when activated, generates N, O, or S atoms that can react with SO 2 Reaction by displacement (Q) q -(L') w And form including X-SO 2 and a 5- to 6-membered ring of intervening atoms of Ar;

[0833] X is -O-, -C(R b ) 2 -or-N(R c )-;

[0834] L' is a spacer moiety, if present, which is connected to SO via a heteroatom selected from O, S and N 2 , and is selected so that L' and SO 2 The breaking of the bonds between promotes the release of the active agent;

[0835] Each Q is independently an active agent substituted with a sugar, sulfate or sulfonate;

[0836] q is an integer from 1 to 3;

[0837] w and y are each independently 0 or 1; and

[0838] R a , R b and R c are independently hydrogen or C1-6 Alkyl; or two R b Together with the atoms to which they are attached, they form a 3- to 5-membered ring;

[0839] The prerequisite is that when w is 0, q is 1.

[0840] In certain embodiments, each Q is independently selected from a chemotherapeutic agent substituted with a sugar, sulfate or sulfonate or a toxin substituted with a sugar, sulfate or sulfonate. In further embodiments, the active agent is a chemotherapeutic agent substituted with a sugar, sulfate or sulfonate. In still further embodiments, each Q is independently selected from an immunomodulatory compound substituted with a sugar, sulfate or sulfonate, an anticancer agent substituted with a sugar, sulfate or sulfonate, an antiviral agent substituted with a sugar, sulfate or sulfonate, an antibacterial agent substituted with a sugar, sulfate or sulfonate, an antifungal agent substituted with a sugar, sulfate or sulfonate, or an antiparasitic agent substituted with a sugar, sulfate or sulfonate.

[0841] In yet further embodiments, each Q is independently selected from a benzodiazepine substituted with a sugar, sulfate or sulfonate, a duocarmycin substituted with a sugar, sulfate or sulfonate, an auristatin substituted with a sugar, sulfate or sulfonate, a tubulysin substituted with a sugar, sulfate or sulfonate, SN-38 substituted with a sugar, sulfate or sulfonate, PNU substituted with a sugar, sulfate or sulfonate, or an exitecan substituted with a sugar, sulfate or sulfonate, an amanitin substituted with a sugar, sulfate or sulfonate.

[0842] In certain embodiments, Q may be a modified moiety bonded to the sugar via a linking group. In further embodiments, Q may be a modified moiety bonded to the sugar via a functional group selected from -C(O)-, -OH, -NH-, -SH, -COH, -COOH, etc. In yet further embodiments, Q may be a modified moiety bonded to the sugar via a functional group selected from esters, amides, thiols, carbamates, oximes, hydrazones, etc.

[0843] In certain embodiments, Q may comprise a modified portion bonded to a sugar via a functional group selected from esters, amides, thiols, carbamates, oximes, hydrazones, etc. In further embodiments, Q may comprise a modified portion bonded to a polar group such as sulfonate, sulfate, sulfite, etc.

[0844] In certain embodiments, each Q is independently represented by:

[0845]

[0846] in:

[0847] Z 2 is a linking group;

[0848] Z 3 is a linking group;

[0849] t is an integer from 1 to 5;

[0850] e is an integer from 1 to 5;

[0851] And each Q' is independently a modified benzodiazepine.

[0852] In further embodiments, each Q' is independently represented by Formula (IIa) or Formula (IIb):

[0853]

[0854]

[0855] or a pharmaceutically acceptable salt thereof;

[0856] in:

[0857] Each A is a heterocycle;

[0858] Each R a ' is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl (preferably lower alkyl), alkenyl, alkynyl, cycloalkyl, aryl or heterocyclic, preferably a five-membered ring or a six-membered ring; optionally, fused to or substituted by one or more aryl or heteroaryl rings;

[0859] Each R b ' is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclic, preferably a five-membered ring or a six-membered ring; optionally, fused to or substituted by one or more aryl or heteroaryl rings;

[0860] Or two R b 'Optionally together to form oxo or =CH 2 ; or two R b ' together with the intervening atoms optionally form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group, optionally substituted by one or more R f 'replace

[0861] Each R f ' is independently halogen, hydroxy, -O-Gly, cyano, nitro, alkyl, haloalkyl, cycloalkyl, carboxyl, amino, aminoalkyl (-CH 2 NH 2 、-CH 2 NH(Me) or -CH 2 N(Me) 2 ), aryl or heteroaryl;

[0862] Rc 'is a sulfonate, sulfate, hydroxy, amino or thiol;

[0863] R d ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0864] The prerequisite is that at least one R c ' is a sulfonate or sulfate, or at least one R d 'is-L"-Gly;

[0865] m is an integer selected from 0-3;

[0866] n is an integer selected from 0 to 8, as long as the valence permits; and

[0867] Gly is a saccharide group, preferably, wherein Gly is a monosaccharide, a disaccharide or an oligosaccharide.

[0868] As will be appreciated, in formula (IIb), R b '(or two co-position R as described above b ' together) will serve as the point of attachment to the rest of the conjugate (e.g., where Q is ). According to the definitions provided below, the remainder of the conjugate may be understood as R b '. This type of R b Examples are thus selected from the substituents listed above, which may be divalent, such as amino, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclic, preferably five-membered or six-membered, optionally fused to or substituted by one or more aryl or heteroaryl rings. b When they form a point of attachment together, they are also selected from potentially divalent substituents, such as =CH 2 or they are taken together with the intervening atoms to form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group, optionally substituted by one or more R as further described herein f 'replace.

[0869] In certain embodiments, the drug conjugate is represented by Formula (IIIa) or Formula (IIIb):

[0870]

[0872] or a pharmaceutically acceptable salt thereof.

[0873] In some embodiments herein, the drug conjugate is represented by Formula (IIIa) or Formula (IIIb), wherein:

[0874] Each A is a heterocycle;

[0875] Each R a ' is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl (preferably lower alkyl), alkenyl, alkynyl, cycloalkyl, aryl or heterocyclic, preferably a five-membered ring or a six-membered ring; optionally, fused to or substituted by one or more aryl or heteroaryl rings;

[0876] Each R b ' is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclic, preferably a five-membered ring or a six-membered ring; optionally, fused to or substituted by one or more aryl or heteroaryl rings;

[0877] Or two R b 'Optionally together to form oxo or =CH 2 ; or two R b ' together with the intervening atoms optionally form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group, optionally substituted by one or more R f 'replace

[0878] Each R f ' is independently halogen, hydroxy, -O-Gly, cyano, nitro, alkyl, haloalkyl, cycloalkyl, carboxyl, amino, aminoalkyl (-CH 2 NH 2 、-CH 2 NH(Me) or -CH 2 N(Me) 2 ), aryl or heteroaryl;

[0879] R c 'is a sulfonate, sulfate, hydroxy, amino or thiol;

[0880] R d ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0881] The prerequisite is that at least one R c ' is a sulfonate or sulfate, or at least one R d 'is-L"-Gly;

[0882] m is an integer selected from 0-3;

[0883] n is an integer selected from 0 to 8, as long as the valence permits; and

[0884] Gly is a saccharide group, preferably, wherein Gly is a monosaccharide, a disaccharide or an oligosaccharide.

[0885] In some embodiments herein, Formula (IIIa) and Formula (IIIb) are referred to as Formula (IX) and Formula (X), respectively, as will be apparent from their context.

[0886] In certain embodiments, A is a 5- to 6-membered heterocyclic ring. In further embodiments, R c In a further embodiment, R c ' is a sulfonate or sulfate. In yet further embodiments, R d ' is -L"-Gly. In certain embodiments, R d 'It's hydrogen.

[0887] In certain embodiments, each Q' is independently selected from:

[0888]

[0889] or a pharmaceutically acceptable salt thereof;

[0890] in Is a single bond or a double bond.

[0891] In a further embodiment, R a ' is halogen, amino, hydroxy, alkoxy, cyano, nitro, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 4- to 10-membered heterocycloalkyl, C 6-10 In a further embodiment, one R b ' is an alkyl group, or two geminal R b In yet a further embodiment, two R b 'Together with the intervening atoms, form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group; preferably, wherein the aryl or heteroaryl group is optionally substituted by one or more R f 'Substituted 6-membered aryl or heteroaryl.

[0892] In a further embodiment, both R b ' together with the intervening atoms form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group; preferably, wherein the aryl or heteroaryl group is optionally substituted by one or more R f In a further embodiment, two R b ' together with the intervening atoms form an aryl group. In a further embodiment, the two R b ' together with the intervening atoms constitute a heteroaryl group.

[0893] In certain embodiments, each Q' is independently selected from:

[0894]

[0895] or a pharmaceutically acceptable salt thereof;

[0896] in is a single bond or a double bond; and

[0897] Where g is an integer from 0 to 4.

[0898] In certain embodiments, Z 3 Selected from:

[0899]

[0900] in:

[0901] X 5 Is -O- or -NR x -;

[0902] Y 1 Yes CR y or N;

[0903] R y is hydrogen or C 1-6 alkyl;

[0904] Each b is independently an integer from 1 to 3; and

[0905] c is an integer from 1 to 5.

[0906] In a further embodiment, Z 3 Selected from:

[0907]

[0908] In a further embodiment, Z 2 In yet further embodiments, Z 2 yes

[0909]

[0910] in:

[0911] Y 5 Yes CR Y1 or N, provided there is only one Y 5 is N;

[0912] R Y1 is H, hydroxyl, amino, amide or (CH 2 ) y (R Y1a );

[0913] RY1a is amino (eg, secondary or tertiary amino), aryl (eg, phenyl), or heteroaryl; and

[0914] y is an integer having a value of 1 to about 10.

[0915] In certain embodiments, Z 2 yes

[0916]

[0917] In a further embodiment, Z 2 yes:

[0918]

[0919] in:

[0920] Y 6 Yes CR Y2 or N;

[0921] R Y2 is H or alkyl, preferably lower alkyl;

[0922] R Z2 Yes (CH 2 ) z R Z2a ;

[0923] R Z2a is amino (preferably tertiary amino), aryl (e.g. phenyl) or heteroaryl; and

[0924] z is an integer having a value from 0 to about 10.

[0925] In a further embodiment, Z 2 yes:

[0926]

[0927] In yet further embodiments, each Q is independently a group of formula (IV):

[0928]

[0929] or a pharmaceutically acceptable salt thereof;

[0930] in:

[0931] Ring Cy is selected from aryl, heteroaryl, cycloalkyl or heterocycloalkyl;

[0932] is a single bond or a double bond;

[0933] X' is a halogen;

[0934] X" is selected from -NR-, -S- or -O-;

[0935] Each R a ” and R b" are independently halogen, amino, hydroxy, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L'")rX"-Gly;

[0936] p is an integer selected from 0-4;

[0937] d is an integer selected from 0-4;

[0938] r is an integer from 0 to 1;

[0939] DBD is the DNA binding domain;

[0940] Each L'' is a bond or a linker, and

[0941] Gly is a monosaccharide, a disaccharide or an oligosaccharide.

[0942] In a further embodiment, each L'" is C 10 -C 100 A linear or branched, saturated or unsaturated alkylene moiety, optionally comprising one or more double bonds and / or triple bonds. In yet further embodiments, each p and each d are independently an integer from 0 to 1. In certain embodiments, Cy is phenyl. In further embodiments, Cy is pyrrolidine or pyrrole.

[0943] In certain embodiments, each Q is independently a group of formula (IVa) or formula (IVb):

[0944]

[0945] or a pharmaceutically acceptable salt thereof.

[0946] In a further embodiment, DBD-(L"') r The -X"-Gly unit is selected from:

[0947]

[0948]

[0949] or a pharmaceutically acceptable salt thereof;

[0950] in:

[0951] Y” is C or N;

[0952] X" is selected from -NR-, -S- or -O-;

[0953] Each R b " is independently halogen, amino, hydroxy, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or -(L"') r -X"-Gly;

[0954] R k is alkyl or hydroxyalkyl, preferably C 1 -C 3 alkyl;

[0955] q is an integer selected from 0-3; and

[0956] Is a single bond or a double bond.

[0957] In still further embodiments, each Q is independently selected from a group of Formula (IVc), Formula (IVd), Formula (IVe), Formula (IVf), Formula (IVg), Formula (IVh), Formula (IVi), or Formula (IVj).

[0958]

[0959]

[0960]

[0961] or a pharmaceutically acceptable salt thereof.

[0962] In preferred embodiments, each Q is independently selected from a group of formula (IVc), formula (IVd), formula (IVe) or formula (IVf):

[0963]

[0964]

[0965] or a pharmaceutically acceptable salt thereof.

[0966] In certain embodiments, the drug conjugate is represented by Formula (V):

[0967]

[0968] or a pharmaceutically acceptable salt thereof;

[0969] in:

[0970] Z' is a coupling group;

[0971] Ar is aryl, heteroaryl, cycloalkyl or heterocycloalkyl;

[0972] Y' is -(CRb 2 ) y N(R a )-、-(CR b 2 ) y O-or-(CR b 2 ) y S-, which is positioned so that if y is 1, the N, O or S atom is attached to the TG;

[0973] TG is a trigger group that, when activated, generates N, O, or S atoms that can react with SO 2 Reaction by displacement (Q) q -(L') w And form including X-SO 2 and a 5- to 6-membered ring of intervening atoms of Ar;

[0974] X is -O-, -C(R b ) 2 -or-N(R c )-;

[0975] L' is a spacer moiety, if present, which is connected to SO via a heteroatom selected from O, S and N 2 , and is selected so that L' and SO 2 Breaking of the bonds between promotes release of the active agent; and

[0976] r is an integer from 0 to 1.

[0977] In some embodiments herein, Formula (V) is referred to as Formula (XI), as will be apparent from the context.

[0978] In a further embodiment, Cy is phenyl. In an even further embodiment, Cy is selected from pyrrolidine or pyrrole. In yet a further embodiment, the drug conjugate is represented by formula (Va) or formula (Vb):

[0979]

[0980] or a pharmaceutically acceptable salt thereof.

[0981] In certain embodiments, DBD-(L"') r The -X"-Gly unit is selected from:

[0982]

[0983] or a pharmaceutically acceptable salt thereof;

[0984] in:

[0985] Y” is C or N;

[0986] Each R b " is independently halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[0987] R k is alkyl or hydroxyalkyl, preferably C 1 -C 3 alkyl;

[0988] q is an integer selected from 0-3; and

[0989] Is a single bond or a double bond.

[0990] In certain embodiments, the drug conjugate is selected from a group of Formula (Vc), Formula (Vd), Formula (Ve), or Formula (Vf):

[0991]

[0992]

[0993] or a pharmaceutically acceptable salt thereof.

[0994] In a preferred embodiment, the drug conjugate is represented by Formula (Vc) or Formula (Vd):

[0995]

[0996] or a pharmaceutically acceptable salt thereof.

[0997] In certain embodiments, each R a " is independently hydrogen, halogen, amino, hydroxy, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L') r -X"-Gly; and each R b " is independently hydrogen, halogen, amino, hydroxy, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L') r -X”-Gly.

[0998] In further embodiments, X' is Cl. In still further embodiments, X' is Br. In still further embodiments, Y" is C. In certain embodiments, Y" is N.

[0999] In certain embodiments, Q is selected from:

[1000]

[1001] or a pharmaceutically acceptable salt thereof.

[1002] In further embodiments, L'' is a bond. In still further embodiments, L'' is a linker selected from:

[1003]

[1004] in:

[1005] Each R a "' is independently hydrogen, halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and

[1006] Each R b ” ' is independently hydrogen, halogen, amino, hydroxy, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

[1007] h is an integer selected from 0-4, as far as the valence permits; and

[1008] It is the point of connection with the adjacent functional group.

[1009] In yet further embodiments, L"' is

[1010]

[1011] In certain embodiments, Gly is a monosaccharide. In further embodiments, Gly is a monosaccharide selected from glucose, glucuronic acid, fucose, and galactose. In still further embodiments, Gly is

[1012]

[1013] Optionally, one or more -OH groups are masked by a protecting group.

[1014] In yet further embodiments, Gly is

[1015]

[1016] In certain embodiments, Gly is a disaccharide. In further embodiments, Gly is a disaccharide comprising glucose, glucuronic acid, fucose, galactose, or a combination thereof. In still further embodiments, Gly is

[1017]

[1018] Optionally, one or more -OH groups are masked by a protecting group.

[1019] In yet further embodiments, Gly is

[1020]

[1021] In certain embodiments, X" or L" is coupled to Gly at the anomeric position. In further embodiments, Ar is aryl. In still further embodiments, Ar is C 6-10 In yet further embodiments, Ar is phenyl. In certain embodiments, Ar is heteroaryl. In further embodiments, Ar is a 5- to 10-membered heteroaryl. In yet further embodiments, Y' is -(CR b 2 ) y N(R a )-or-(CR b 2 ) y In yet further embodiments, Y' is -(CR b 2 ) y In certain embodiments, y is 0 or 1. In further embodiments, y is 1. In still further embodiments, X is -O-, C(R b ) 2 -or-N(R c )-. In yet further embodiments, X is -O-. In certain embodiments, L' is a spacer moiety and forms an -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH- linkage including the heteroatom of the active agent.

[1022] In certain embodiments, Q-(L') w -Selected from:

[1023]

[1024] in:

[1025] X 4 does not exist or forms an -O-, -OC(O)-, -OC(O)O- or -OC(O)NH- linkage, including the heteroatom of Q;

[1026] X 1 Is -O- or -NR a -;

[1027] X 2 is -O-, -OC(O)-, -OC(O)O- or -OC(O)NH-;

[1028] X 3 is -OC(=O)-;

[1029] w' is an integer having a value of 1, 2, 3, 4 or 5;

[1030] R 9 and R 10 are each independently hydrogen, alkyl, aryl or heteroaryl, wherein alkyl, aryl and heteroaryl are unsubstituted or substituted with one or more radicals selected from, for example, alkyl, -(CH 2 ) u NH 2 、-(CH 2 ) u NR u1 R u2 and -(CH 2 ) u SO 2 R u3 Substituents are substituted;

[1031] R u1 , R u2 and R u3 are each independently hydrogen, alkyl, aryl or heteroaryl; and

[1032] u is an integer having a value from 1 to about 10.

[1033] In a further embodiment, Q-(L') w -Selected from:

[1034]

[1035] In certain embodiments, the drug conjugate is selected from:

[1036]

[1037]

[1038]

[1039]

[1040]

[1041]

[1042]

[1043]

[1044]

[1045] or a pharmaceutically acceptable salt thereof.

[1046] In certain embodiments, the drug conjugate is not selected from:

[1047]

[1048]

[1049]

[1050]

[1051]

[1052] In certain embodiments, the drug conjugate is not a compound disclosed in US2022 / 0047717.

[1053] Targeted drug conjugates

[1054] In certain aspects, provided herein are targeted drug conjugates comprising a drug conjugate comprising a compound of the disclosure, a linker group, and a targeting moiety.

[1055] In certain embodiments, the targeted drug conjugate is a compound represented by Formula (XII), Formula (XIII), or Formula (XIV):

[1056]

[1057] or a pharmaceutically acceptable salt thereof;

[1058] Wherein TM is the targeting moiety.

[1059] In certain aspects, the drug conjugates of the present disclosure further comprise a targeting moiety. In a further aspect, provided herein is a targeted drug conjugate of formula (VI), comprising a targeting moiety conjugated to any one of the drug conjugates of the present disclosure:

[1060]

[1061] Wherein TM is the targeting moiety.

[1062] In certain embodiments, the targeted drug conjugate is a compound of formula (XII), formula (XIII), or formula (XIV):

[1063]

[1064]

[1065] or a pharmaceutically acceptable salt thereof;

[1066] Wherein TM is the targeting moiety.

[1067] In a further aspect, provided herein is a targeted drug conjugate of formula (VIb), comprising a targeting moiety conjugated to a drug conjugate of the present disclosure:

[1068]

[1069] in:

[1070] TM is the targeting moiety;

[1071] R is hydrogen or a hydroxyl protecting group;

[1072] X is -C(O)-, -NH-, -O- or -S-;

[1073] Q is an active agent substituted with a sugar, sulfonate, or sulfate;

[1074] T is

[1075] n is an integer selected from 0 or 1;

[1076] Y is hydrogen, halogenated C 1 -C 8 alkyl, halogen, cyano or nitro; z is an integer selected from 1 to 3, and if z is an integer not less than 2, Y may be the same as or different from each other;

[1077] z1 is an integer selected from 0 or 1;

[1078] W 1 yes

[1079] W 2 yes

[1080] W a1 and W a2 are each independently -NH-, -C(=O)- or -CH 2 -;

[1081] W a3 and W a4 are independently -NH-, -C(=O)-, -CH 2 -, -C(=O)NH-, -NHC(=O)- or triazolylene;

[1082] W b1 is an amide bond or a triazole group;

[1083] L is used as a connection Wa2 and an amino acid, peptide or amide bond of a linker to Z;

[1084] Z is a single bond, -W a5 -(CH 2 ) a2 -W b2 -(CH 2 ) a3 -W a6 -or-W a7 -(CH 2 ) a4 -CR'R"-X"'-;

[1085] R' is C 1 -C 8 Alkyl or TM-W a8 -Q 3 -W c1 -(CH 2 ) a5 -;

[1086] R" is TM-W a8 -Q 3 -W c1 -(CH 2 ) a5 -;

[1087] Q 1 and Q 3 Each independently is -(CH 2 ) a6 -(X 1 CH 2 CH 2 ) b1 -(CH 2 ) a7 -;

[1088] X 1 and X 3 are independently -O-, -S-, -NH- or -CH 2 -;

[1089] X'' is -NHC(=O)-(CH 2 ) a8 -W a9 - or -C(=O)NH-(CH 2 ) a8 -W a9 -;

[1090] W a5 , W a6 , W a7 , W a8 and W a9are each independently -NH-, -C(=O)- or -CH 2 -;

[1091] W b2 is an amide bond or a triazole group;

[1092] W c1 is -NHC(=O)- or -C(=O)NH-;

[1093] Q 2 is a linear or branched saturated or unsaturated alkylene group having 1 to 50 carbon atoms, satisfying any one of the following (i) to (iii);

[1094] (i) at least one -CH 2 - is substituted by one or more heteroatoms selected from -NH-, -C(=O), -O- and -S-,

[1095] (ii) at least one arylene group or heteroarylene group is contained in the alkylene group,

[1096] (iii) the alkylene group is further selected from C 1 -C 20 Alkyl, C 6 -C 20 Aryl C 1 -C 8 Alkyl, -(CH 2 ) s1 COOR 3 、-(CH 2 ) s1 COR 3 、-(CH 2 ) s2 CONR 4 R 5 and -(CH 2 ) s2 NR 4 R 5 Replacement by one or more of the group consisting of;

[1097] The arylene or heteroarylene group in (ii) above may be further substituted by a nitro group;

[1098] R 3 , R 4 and R 5 are independently hydrogen or C 1 -C 15 alkyl;

[1099] X 2 is -O-, -S-, -NH- or -CH 2 -;

[1100] U 1 It is bound to B' at the position of the asterisk (*) via a linker selected from the following structures:

[1101]

[1102] R is for C 1 -C 10 Alkyl, C 6 -C 20 Aryl or C 2 -C 20 heteroaryl;

[1103] TM and B' are each independently a ligand or a protein having the property of selectively targeting a drug to a specific organ, tissue or cell, i.e., the property of binding to a receptor;

[1104] a1, a2, a3, a4, a5, a6, a8, b1, p1, p2, p3 and p4 are each independently an integer selected from 1-10;

[1105] a7, y, s1, s2 and s4 are each independently an integer selected from 0-10; and

[1106] R 1 and R 2 Each is independently hydrogen, C 1 -C 8 Alkyl or C 3 -C 8 Cycloalkyl.

[1107] In a further aspect, provided herein is a targeted drug conjugate of formula (VIc), comprising a targeting moiety conjugated to a drug conjugate of the present disclosure:

[1108]

[1109] in:

[1110] TM is the targeting moiety;

[1111] G is a glucuronic acid moiety or a derivative thereof;

[1112] Q is an active agent substituted with a sugar, sulfonate, or sulfate;

[1113] W is an electron withdrawing group;

[1114] Z is hydrogen, C 1 -C 8 Alkyl, halogen, cyano or nitro;

[1115] n is an integer selected from 1 to 3, and when n is an integer of 2 or greater, each Z is the same as or different from each other;

[1116] L is a connector connecting TM and W; and

[1117] R 1 and R 2 Each is independently hydrogen, C 1 -C 8 Alkyl or C 3 -C 8 Cycloalkyl.

[1118] In certain aspects, provided herein is a targeted drug conjugate of formula (VId), comprising a targeting moiety conjugated to a drug conjugate of the present disclosure:

[1119] TM-L 1 -(A a -W w -Y y -Q 1-4 ) p

[1120] (VId);

[1121] Wherein TM is the targeting moiety;

[1122] L 1 is the ligand part;

[1123] Q is an active agent substituted with a sugar, sulfonate, or sulfate;

[1124] -A a -W w -Y y - is the joint part;

[1125] A is an optional extension;

[1126] a is an integer selected from 0-3;

[1127] Each W is independently a glucuronide unit having one of the following formulae:

[1128]

[1129] Su is the sugar part;

[1130] Each R is independently hydrogen, halogen, -CN or -NO 2 ;

[1131] w is an integer selected from 1-2;

[1132] Y is an optional self-immolative spacer moiety;

[1133] y is an integer selected from 0-2; and

[1134] p is an integer selected from 1-20.

[1135] Release of active agent

[1136] As described above, in certain embodiments, the compounds and conjugates disclosed herein are capable of dissociating one or more active agents by intramolecular cyclization reaction after activating a chemical reaction of a triggering group. In certain embodiments, the chemical reaction is a physicochemical reaction and / or a biochemical reaction.

[1137] In some embodiments, the compounds and conjugates disclosed herein comprise a nucleophilic functional group (Y or Y') introduced at an atom adjacent to X (e.g., O) on Ar. Typically, the nucleophilic functional group is masked by a trigger group (TG), as described in further detail below. Upon activation, the trigger group releases the nucleophilic functional group to bind to a nearby SO 2 Part of the intramolecular cyclization reaction, ultimately releasing one or more compounds of Formula (II), Formula (IIa) or Formula (IIb). In some such embodiments, one or more active agents are released by intramolecular cyclization after a chemical reaction, a physicochemical reaction and / or a biochemical reaction (see, e.g., Reaction Scheme 1), or the active agent is released by 1,6-elimination or 1,4-elimination after an intramolecular cyclization reaction (see, e.g., Reaction Scheme 2).

[1138] As an example, when Y is -Y'-TG and Q is directly conjugated to SO 2 When the active agent of the group is released, the active agent can be released by the mechanism shown in Reaction Scheme 1:

[1139] Reaction Scheme 1

[1140]

[1141] When Q is When 1 Release can be achieved via the mechanism shown in Reaction Scheme 2:

[1142] Reaction Scheme 2

[1143]

[1144] In some embodiments, Q 1 When released, the active agent comprises at least one functional group selected from -C(O)-, -OH, -NH-, -SH, -COH and -COOH. According to these embodiments, as further described herein, Q 1Conjugation to compounds as described herein is via -C(O)-, -OH, -NH-, -SH, -COH and -COOH, for example via a functional group selected from esters, amides, thioesters, carbamates, ureas, oximes, hydrazones, etc. In some such embodiments, Q is used 2 Replace Q 1 , and Q 2 is a drug containing an amine group. In other embodiments, Q 2 is an active agent capable of binding to an ammonium unit. In other embodiments, Q 2 Can be in Q 2 After release, it dissociates in its original form with amine groups, wherein the active agent can be a drug, a toxin, an affinity ligand, a probe for detection, or a combination thereof.

[1145] In some embodiments, the compounds and conjugates disclosed herein are chemically and physiologically stable. In some such embodiments, the compounds and conjugates disclosed herein reach the desired target cells in a state where the active agent is minimally dissociated in the blood, thereby selectively releasing the drug.

[1146] Trigger Group (TG)

[1147] In some embodiments, the conjugates of the present disclosure include a trigger group (TG). TG is a group that can be cleaved, preferably selectively cleaved, by a chemical reaction, such as a biological reaction. In general, the trigger group is used to mask the nucleophilic nature of the Y' group, thereby providing stability to the compounds and conjugates disclosed herein (e.g., by preventing self-immolation or intramolecular cyclization before the conjugate reaches the target position or undergoes a predetermined trigger condition). As described above, after activation, the trigger group releases the nucleophilic Y group and allows self-immolation or intramolecular cyclization to occur.

[1148] In some embodiments, TG comprises a sequence (such as a peptide sequence) or a portion that can be recognized by TEV, trypsin, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase 1, matrix metalloproteinase (MMP), etc., which can be hydrolyzed by an enzyme (e.g., oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, etc.), and / or can include a portion selected from sulfate, phosphodiester, phospholipid, ester, β-galactose, β-glucose, fucose, oligosaccharide, etc.

[1149] In some embodiments, the TG comprises a reactive chemical moiety or functional group that can be cleaved under nucleophilic conditions (e.g., silyl ether, 2-N-acylnitrobenzenesulfonamide, unsaturated vinyl thioether, activated sulfonamide, malondialdehyde-indole derivative, levulinyl ester, hydrazone or acylhydrazone).

[1150] In some embodiments, the TG may comprise a reactive chemical moiety or functional group that is cleavable under alkaline reagent conditions (eg, 2-cyanoethyl ester, ethylene glycol disuccinate, 2-sulfonylethyl ester, alkylthioester, or phenylthioester).

[1151] In some embodiments, the TG may comprise a reactive chemical moiety or functional group that can be cleaved by photoirradiation (e.g., 2-nitrobenzyl derivatives, benzoylmethyl esters, 8-quinolylbenzenesulfonate, coumarins, phosphotriesters, bisarylhydrazones, or bimane bisthiopropionic acid derivatives).

[1152] In some embodiments, a TG may comprise a reactive chemical moiety or functional group (eg, hydroxylamine, disulfide, levulinate, nitro, or 4-nitrobenzyl derivative) that may be cleaved by reducing agent conditions.

[1153] In some embodiments, the TG may comprise a reactive chemical moiety or functional group that can be cleaved using acidic conditions (e.g., sugars, tert-butylcarbamate analogs, dialkyl or diaryldialkoxysilanes, orthoesters, acetals, aconityl, hydrazones, β-thiopropionates, phosphoramidates, imines, trityl, vinyl ethers, polyketals, and alkyl 2-(diphenylphosphino)benzoate derivatives; alkyl esters, 8-hydroxyquinoline esters, and picolinates).

[1154] In some embodiments, the TG may comprise a reactive chemical moiety or functional group that can be cleaved under oxidative conditions (eg, a boronate ester, a vicinal diol, a p-methoxybenzyl derivative, or a selenium compound).

[1155] In certain preferred embodiments, the TG comprises a sugar that can be cleaved under acidic or enzymatic conditions. In certain preferred embodiments, the trigger group is -NO 2 , which can be cleaved under reducing conditions. In certain preferred embodiments, the trigger group is a borate ester, which can be cleaved under oxidative conditions. In certain preferred embodiments, the trigger group is an ester, which can be cleaved under acidic, alkaline or enzymatic conditions. In certain preferred embodiments, the trigger group is a hydrazone, which can be cleaved under nucleophilic conditions or under acidic conditions. In certain preferred embodiments, the trigger group is a hydroxylamine, which can be cleaved under reducing conditions.

[1156] Sugar trigger group

[1157] In some embodiments, the compounds and conjugates disclosed herein comprise a sugar trigger group, such as a trigger group selected from:

[1158]

[1159] Each R 21 are independently hydrogen or are selected such that OR 21 is a hydroxy protecting group (eg, acetyl); and R 22 is hydrogen or a lower alkyl group (e.g. C 1 -C 6 In certain embodiments, hydroxy protecting groups can be used in organic synthesis, including but not limited to: methyl ether, methoxymethyl ether, methylthiomethyl ether, 2-methoxyethoxymethyl ether, bis(2-chloroethoxy)methyl ether, tetrahydropyranyl ether, tetrahydrothiopyranyl ether, 4-methoxytetrahydropyranyl ether, 4-methoxytetrahydrothiopyranyl ether, tetrahydrofuranyl ether, 1-ethoxyethyl ether, 1-methyl-1-methoxyethyl ether, 2-(phenylseleno)ethyl ether, tert-butyl ether, allyl ether, benzyl ether, o-nitrobenzyl ether, triphenylmethyl ether, α-naphthyl diphenylmethyl ether, p-methoxyphenyl diphenylmethyl ether, 9-(9-phenyl- 10-oxo)anthryl ether, trimethylsilyl ether, isopropyldimethylsilyl ether, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether, tritylsilyl ether, triisopropylsilyl ether, formates, acetates, trichloroacetates, phenoxyacetates, isobutyrates, pivalates, adamantates, benzoates, 2,4,6-trimethylbenzoate, methyl carbonate, 2,2,2-trichloroethyl carbonate, allyl carbonate, p-nitrophenyl carbonate, benzyl carbonate, p-nitrobenzyl carbonate, S-benzylthiocarbonate, N-phenylcarbamate, nitrates, 2,4-dinitrophenylsulfenate, etc., but are not limited thereto.

[1160] In certain embodiments, TG is a monosaccharide. In further embodiments, TG is a monosaccharide selected from glucose, glucuronic acid, fucose, and galactose. In still further embodiments, TG is

[1161]

[1162] Optionally, one or more -OH groups are masked by a protecting group.

[1163] In yet a further embodiment, TG is

[1164]

[1165] In certain embodiments, TG is a disaccharide. In further embodiments, TG is a disaccharide comprising glucose, glucuronic acid, fucose, galactose, or a combination thereof. In still further embodiments, TG is

[1166]

[1167] Optionally, one or more -OH groups are masked by a protecting group.

[1168] In yet a further embodiment, TG is

[1169]

[1170] In certain embodiments, Y' or L' is coupled to TG at the anomeric position.

[1171] Protecting groups as triggering groups

[1172] In some embodiments, TG is a group that can be cleaved by chemical reaction, physicochemical reaction and / or biological reaction. In certain embodiments, TG is a protecting group. In some such embodiments, the protecting group is an amine protecting group, an alcohol protecting group or a thiol protecting group.

[1173] Amine protecting group

[1174] In certain embodiments, the amine protecting group is a general protecting group that can be used in organic synthesis, including but not limited to: m-nitrophenylcarbamate, 3,5-dimethoxybenzylcarbamate, o-nitrobenzylcarbamate, phenyl(o-nitrophenyl)methylcarbamate, alkylcarbamate, 9-fluorenylmethylcarbamate, 2,2,2-trichloroethylcarbamate, 2-trimethylsilylethylcarbamate (Teoc), tert-butylcarbamate (Boc), vinylcarbamate (Voc), allylcarbamate (Alloc), 1-isopropylallylcarbamate (Ipaoc), 8-quinolylcarbamate, N-hydroxypiperidinylcarbamate, benzylcarbamate, p-methoxybenzylcarbamate, p-nitrobenzylcarbamate, diphenylmethylcarbamate, acetamide, chloroacetamide, trichloroacetamide, phenylacetamide, benzamide, N-phthalimide, N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, benzenesulfonamide, o-nitrobenzenesulfonamide, triphenylmethylsulfonamide, p-toluenesulfonamide, methanesulfonamide, etc., but are not limited thereto.

[1175] Alcohol protecting group

[1176] In certain embodiments, the alcohol protecting group is a general protecting group that can be used in organic synthesis, including but not limited to: methyl ether, methoxymethyl ether (MOM ether), benzyloxymethyl ether (BOM ether), 2-(trimethylsilyl)ethoxymethyl ether (SEM ether), phenylthiomethyl ether (PTM ether), 2,2-dichloro-1,1-difluoroethyl ether, p-bromophenacyl methyl ether, chloropropyl methyl ether, isopropyl ether, cyclohexyl ether, 4-methoxybenzyl, 2,6-dichlorobenzyl ether, 4-( dimethylaminocarbonyl)benzyl ether, 9-anthryl methyl ether, 4-picolyl methyl ether, methylthiomethyl ether (MTM ether), 2-methoxyethoxymethyl ether (MEM ether), bis(2-chloroethoxy)methyl ether, tetrahydropyranyl ether (THP ether), tetrahydrothiopyranyl ether, 4-methoxytetrahydropyranyl ether, 4-methoxytetrahydrothiopyranyl ether, tetrahydrofuranyl ether, 1-ethoxyethyl ether, 1-methyl-1-methoxyethyl ether, 2-(phenylseleno)ethyl ether, tert-butyl ether, allyl ether, benzyl ether, o-nitrobenzyl ether, triphenyl methyl ether, α-naphthyl diphenyl methyl ether, p-methoxyphenyl diphenyl methyl ether, 9-(9-phenyl-10-oxo) anthracenyl ether, trimethylsilyl ether (TMS ether), isopropyl dimethylsilyl ether, tert-butyl dimethylsilyl ether (TBDMS ether), tert-butyl diphenylsilyl ether, trityl silyl ether, triisopropyl silyl ether, formates, acetates, trichloroacetates, phenoxy acetates, isobutyrates, pivalates, adamantates, benzoates, 2 , 4,6-trimethylbenzoate (Mesitoate), methyl carbonate, 2,2,2-trichloroethyl carbonate, allyl carbonate, p-nitrophenyl carbonate, benzyl carbonate, p-nitrobenzyl carbonate, S-benzylthiocarbonate, N-phenylcarbamate, nitrate, 2,4-dinitrophenylsulfonate, dimethyl phosphinate (DMP ester), dimethyl phosphinate thioester (MPT ester), aryl methanesulfonate, aryl toluenesulfonate, etc., but not limited to these.

[1177] Thiol Protecting Group

[1178] In certain embodiments, thiol protecting groups can be used in organic synthesis, including but not limited to: S-benzyl sulfide, S-p-methoxybenzyl sulfide, S-o-hydroxy or p-hydroxy or acetoxybenzyl sulfide, S-p-nitrobenzyl sulfide, S-4-picolyl sulfide, S-2-picolyl N-oxide sulfide, S-9-anthryl methyl sulfide, S-9-fluorenyl methyl sulfide, S-methoxymethyl monothioacetal, A-acetyl derivatives, S-benzoyl derivatives, S-(N-ethylcarbamate), S-(N-methoxymethylcarbamate), etc., but are not limited thereto.

[1179] Coupling group (linker) from drug conjugate to targeting moiety

[1180] In some embodiments, the compounds and conjugates disclosed herein comprise a linker group that connects each TM and Ar via a covalent bond. Typical linkers are stable, non-hydrolyzable moieties such as C 10 -C 100 Straight or branched, saturated or unsaturated alkylene. In certain embodiments, the linking unit meets at least two of the following four criteria, and more preferably at least three:

[1181] (i) at least one -CH 2 - is replaced by one or more heteroatoms selected from -NH-, -C(=O), -O-, -S- and -P-;

[1182] (ii) at least one heteroarylene group is contained in the alkylene portion;

[1183] (iii) at least one amino acid moiety, sugar bond, peptide bond or amide bond is contained in the alkylene moiety; and

[1184] (iv) The alkylene group may also be replaced by one or more selected from C 1 -C 20 Alkyl, C 6 -C 20 Aryl C 1 -C 8 Alkyl, -(CH 2 ) s COOH and -(CH 2 ) p NH 2 The substituents consisting of the group substituted wherein s is an integer having a value from 0 to 10, and p is an integer having a value from 1 to about 10.

[1185] In certain embodiments, the linking unit comprises at least two, and more preferably at least three, of the following:

[1186] (i) at least one heteroatom selected from -NH-, -C(=O), -O-, -S- and -P-;

[1187] (ii) at least one heteroarylene group;

[1188] (iii) at least one amino acid moiety, sugar bond, peptide bond or amide bond; and

[1189] (iv) The alkylene group may also be replaced by one or more selected from C 1 -C 20 Alkyl, C 6 -C 20 Aryl C 1 -C 8 Alkyl, -(CH 2 )s COOH and -(CH 2 ) p NH 2 The substituents consisting of the group substituted wherein s is an integer having a value from 0 to 10, and p is an integer having a value from 1 to about 10.

[1190] In other embodiments, the linking group connecting each TM and Ar comprises a functional group generated by a click chemistry reaction.

[1191] In alternative embodiments, the linking unit comprises a reactive functional group capable of participating in a click chemistry reaction.

[1192] Click chemistry is a reaction that can be performed under mild conditions and has extremely high selectivity for uncommon functional groups in biomolecules (e.g., azido, acetylene, etc.). Therefore, this reaction can be performed in the presence of complex trigger groups, targeting moieties, etc. In addition, click chemistry has a high degree of reaction specificity. For example, the click chemistry reaction between an azide and an acetylene group is selectively performed without interference from other functional groups present in the molecule. For example, azide-acetylene click chemistry can obtain a triazole moiety in high yield.

[1193] Thus, in some embodiments, the linking group connecting each TM and Ar comprises V can be a single bond, -O-, -S-, -NR 21 -、-C(O)NR 22 -、-NR 23 C(O)-、-NR 24 SO 2 -or-SO 2 NR 25 -, R 21 To R 25 may each independently be hydrogen, (C 1 -C 6 ) alkyl, (C 1 -C 6 )alkyl(C 6 -C 20 ) aryl or (C 1 -C 6 )alkyl(C 3 -C 20 ) heteroaryl, r may be an integer having a value from 1 to about 10, p may be an integer having a value from 0 to about 10, q may be an integer having a value from 1 to about 10, and L" may be a single bond.

[1194] A variety of linking groups are suitable for use with the drug conjugates disclosed herein. When a targeting moiety is not present (i.e., the drug conjugate is not a targeted drug conjugate), the linking group comprises a terminal reactive functional moiety that can react with the targeting moiety. In some embodiments, Z' is selected from

[1195]

[1196]

[1197]

[1198]

[1199] in:

[1200] R za is H or methyl;

[1201] R zb is -OH, =O or =NHOH;

[1202] n and m are each independently an integer selected from 1-10;

[1203] x is an integer selected from 1-2;

[1204] represents the bond between Z' and the drug conjugate;

[1205] Single or double bonds;

[1206] Z" is selected from

[1207]

[1208] In further embodiments, when a targeting moiety is present (ie, the drug conjugate is a targeted drug conjugate), the linking group links the conjugate to the targeting moiety. In some embodiments, Z' is selected from

[1209]

[1210]

[1211] in

[1212] R za is H or methyl;

[1213] R zb is -OH, =O or =NHOH;

[1214] Single or double bonds;

[1215] n and m are each independently an integer selected from 1-10;

[1216] x is an integer selected from 1-2;

[1217] a" represents the bond between Z' and the drug conjugate;

[1218] b" represents the bond between Z' and TM; and

[1219] Z" is selected from

[1220]

[1221] Targeting moiety

[1222] The compounds and conjugates of the present disclosure may also include one or more ligands or targeting moieties TM. In some embodiments, the ligand or targeting moiety is any molecular recognition element that can specifically interact with at least one other molecule by, for example, non-covalent bonding, such as hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, π-π interactions, halogen bonding, electrostatic and / or electromagnetic effects. In certain embodiments, the TM is selected from nanoparticles, immunoglobulins, nucleic acids, proteins, oligopeptides, polypeptides, antibodies, fragments of antigenic polypeptides, repebodies, etc.

[1223] Compounds and conjugates of the present disclosure may include one or more targeting moieties. In certain embodiments, the targeting moiety is a fragment or repeat of a nanoparticle, an immunoglobulin, a nucleic acid, a protein, an oligopeptide, a polypeptide, an antibody, an antigenic polypeptide. In further embodiments, the targeting moiety is an antibody selected from the group consisting of a complete polyclonal antibody, a complete monoclonal antibody, an antibody fragment, a single-chain Fv (scFv) mutant, a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a fusion protein comprising an antigenic determinant portion of an antibody, and other modified immunoglobulin molecules comprising an antigen recognition site.

[1224] In a further embodiment, the antibody is selected from Muromonab-CD3, Abciximab, Rituximab, Daclizumab, Palivizumab, Infliximab, Trastuzumab (herceptin), Etanercept, Basiliximab, Gemtuzumab Ozogamicin, Alemtuzumab, Ibritumomabtiuxetan, Adalimumab, Alefacept, Omalizumab, Efalizumab, Tositumomab-I 131、Cetuximab、Bevacizumab、Natalizumab、Ranibizumab、Panitumumab、Eculizu mab、Rilonacept、Certolizumab pegol、Romiplostim、AMG-531、CNTO-148、CNTO-1275、ABT-874、LEA-29Y、Belimumab、TACI-Ig、Second-generation anti-CD20、ACZ-885、Tocilizumab、Atlizumab、Mepolizumab、Pertuzumab、Humax CD20, Tremelimumab (CP-675 206), Ticilimumab, MDX-010, IDEC-114, Inotuzumab abozogamycin, HuMax EGFR, Aflibercept, HuMax-CD4, Ala-Ala, ChAglyCD3, TRX4, Catumaxomab, IGN101, MT-201, Pregovomab, CH-14.18, WX-G250, AMG-162, AAB-001, Motavizumab, MEDI-524, Efumgumab, Aurograb, Raxibacumab, third-generation anti-CD20, LY2469298, and Veltuzumab.

[1225] In some embodiments, the TM comprises two or more independently selected natural or unnatural amino acids conjugated by covalent bonds (e.g., peptide bonds), and the peptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more natural or unnatural amino acids conjugated by peptide bonds. In some embodiments, the ligand comprises shorter amino acid sequences (e.g., fragments of natural proteins or synthetic polypeptide fragments) as well as full-length proteins (e.g., pre-engineered proteins).

[1226] In some embodiments, TM is selected from antibodies, hormones, drugs, antibody analogs (e.g., non-IgG), proteins, oligopeptides, polypeptides, etc. that bind to receptors. In certain embodiments, TM selectively targets drugs to specific organs, tissues, or cells. In other embodiments, TM specifically binds to receptors that are overexpressed in cancer cells compared to normal cells, and can be divided into monoclonal antibodies (mAbs) or antibody fragments and low molecular weight non-antibodies. Preferably, TM is selected from peptides, tumor cell-specific peptides, tumor cell-specific aptamers, tumor cell-specific carbohydrates, tumor cell-specific monoclonal antibodies, polyclonal antibodies, and antibody fragments identified in library screening.

[1227] Exemplary ligands or targeting moieties include, but are not limited to, carnitine, inositol, lipoic acid, pyridoxal, ascorbic acid, niacin, pantothenic acid, folic acid, riboflavin, thiamine, biotin, vitamin B 12, other water-soluble vitamins (vitamin B), fat-soluble vitamins (vitamin A, D, E, K), RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), transferrin, VIP (vasoactive intestinal peptide) receptor, APRPG (Ala-Pro-Arg-Pro-Gly) peptide, TRX-20 (thioredoxin-20), integrin, nucleolin, aminopeptidase N (CD13), endoglin, vascular epithelial growth factor receptor, low-density lipoprotein receptors, transferrin receptors, somatostatin receptors, bombesin, neuropeptide Y, luteinizing hormone-releasing hormone receptors, folate receptors, epidermal growth factor receptors, transforming growth factor, fibroblast growth factor receptors, asialoglycoprotein receptors, galectin 3 receptors, E-selectin receptors, hyaluronic acid receptors, prostate-specific membrane antigen (PSMA), cholecystokinin A receptor, cholecystokinin B receptor, discoidin domain receptors, mucin receptors, opioid receptors, plasminogen receptors, bradykinin receptors, pancreatic Insulin receptor, insulin-like growth factor receptor, angiotensin AT1 receptor, angiotensin AT2 receptor, granulocyte macrophage colony stimulating factor receptor (GM-CSF receptor), galactosamine receptor, σ2 receptor, delta-like 3 (DLL-3), aminopeptidase P, melanocyte transferrin, leptin, tetanus toxin Tet1, tetanus toxin G23, RVG (rabies virus glycoprotein) peptide, HER2 (human epidermal growth factor receptor 2), GPNMB (glycoprotein non-transferase b), Ley, CA6, CanAng, S LC44A4 (solute carrier family 44 member 4), CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5), Nectin-4, carbonic anhydrase 9, TNNB2, 5T4, CD30, CD37, CD74, CD70, PMEL17, EphA2 (ephrin A2 receptor), Trop-2, SC-16, tissue factor, ENPP-3 (AGS-16), SLITRK6 (SLIT and NTRK-like family member 6), CD27, LewisY antigen, LIV1, GPR161 (G protein-coupled receptor 161), PBR (peripheral benzodiazepine receptor), MERTK (Mer receptor tyrosine kinase) receptor, CD71, LLT1 (lectin-like transcript 1 or CLED2D), interleukin 22 receptor, σ1 receptor, peroxisome proliferator-activated receptor, DLL3, C4.4a, cKIT, ephrin A, CTLA4 (cytotoxic T lymphocyte-associated protein 4), FGFR2b (fibroblast cell growth factor receptor 2b), N-acetylcholine receptor, gonadotropin-releasing hormone receptor, gastrin-releasing peptide receptor, bone morphogenetic protein receptor type 1B (BMPR1B), E16 (LAT1, SLC7A5), STEAP1 (six-transmembrane epithelial antigen of the prostate), 0772P (CA125, MUC16), MPF (MSLN, mesothelin), Napi3b (SLC34A2), Sema5b (semaphorin 5b5b)), ETBR (endothelin type B receptor), MSG783 (RNF124), STEAP2 (six transmembrane epithelial antigen of the prostate 2), TrpM4 (transient receptor potential cation 5 channel, M subfamily, member 4), CRIPTO (teratocarcinoma-derived growth factor), CD21, CD79b, FcRH2 (IFGP4), HER2 (ErbB2), NCA (CEACM6), MDP (DPEP1), IL20R-α (IN20Ra), Brevican (BCAN), EphB2R, ASLG659 (B7h), CD276, PS CA (prostate stem cell antigen precursor), GEDA, BAFF-R (BR3), CD22 (BL-CAM), CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, SSTR2, SSTR5, SSTR1, SSTR3, SSTR4, ITGAV (integrin α5), ITGB6 (integrin β6), MET, MUC1, EGFRvIII, CD33, CD19, IL2RA (interleukin 2 receptor α), AXL, BCMA, CTA (cancer testis antigen), CD174, CLEC 14A, GPR78, CD25, CD32, LGR5 (GPR49), CD133 (Prominin), ASG5, ENPP3 (Exonucleotide pyrophosphatase / phosphodiesterase 3), PRR4 (Proline-rich protein 4), GCC (Guanylate cyclase 2C), Liv-1 (SLC39A6), CD56, CanAg, TIM-1, RG-1, B7-H4, PTK7, CD138, Claudins, Her3 (ErbB3), RON (MST1R), CD20, TNC (Tenasin C), FAP, DKK- 1. CD52, CS1 (SLAMF7), annexin A1, V-CAM, gp100, MART-1, MAGE-1 (melanoma antigen encoding gene 1), MAGE-3 (melanoma associated antigen 3), BAGE, GAGE-1, MUM-1 (multiple myeloma oncogene 1), CDK4, TRP-1 (gp75), TAG-72 (tumor-associated glycoprotein 72), ganglioside GD2, GD3, GM2, GM3, VEP8, VEP9, My1, VIM-D5, D156-22, OX40, RNAK, PD-L1, TNFR1, TNFR2, etc.

[1228] Target

[1229] In some embodiments, one or more targets of molecular recognition elements are specifically related to one or more specific cells or tissue types. In some embodiments, the target is specifically related to one or more specific disease states. In some embodiments, the target is specifically related to one or more specific developmental stages. For example, the expression level of cell type specific markers in the cell type is usually higher than at least 2 times in the reference cell population. In some embodiments, cell type specific markers exist at a level that is at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 50 times, at least 100 times or at least 1,000 times higher than its average expression in the reference population. The detection or measurement of cell type specific markers is likely to distinguish one or more cell types of interest from many, most or all other types of cells. In some embodiments, the target can include proteins, carbohydrates, lipids and / or nucleic acids, as described herein.

[1230] In some embodiments, a substance is considered "targeted" if it specifically binds to a targeting moiety, such as a nucleic acid targeting moiety. In some embodiments, a targeting moiety, such as a nucleic acid targeting moiety, specifically binds to a target under stringent conditions.

[1231] In certain embodiments, conjugates and compounds described herein include a targeting portion specifically bound to one or more targets (e.g., antigens) associated with organs, tissues, cells, extracellular matrix components, and / or intracellular compartments. In some embodiments, conjugates and compounds described herein include a targeting portion specifically bound to a target associated with a particular organ or organ system. In some embodiments, conjugates and compounds described herein include a targeting portion specifically bound to one or more intracellular targets (e.g., organelles, intracellular proteins). In some embodiments, conjugates and compounds described herein include a targeting portion specifically bound to a target associated with a diseased organ, tissue, cell, extracellular matrix component, and / or intracellular compartment. In some embodiments, conjugates and compounds described herein include a targeting portion specifically bound to a target associated with a particular cell type (e.g., endothelial cells, cancer cells, malignant cells, prostate cancer cells, etc.).

[1232] In some embodiments, the conjugates and compounds described herein include a targeting moiety that is bound to a target that is specific to one or more specific tissue types (e.g., liver tissue vs. prostate tissue). In some embodiments, the conjugates and compounds described herein include a targeting moiety that is bound to a target that is specific to one or more specific cell types (e.g., T cells vs. B cells). In some embodiments, the conjugates and compounds described herein include a targeting moiety that is bound to a target that is specific to one or more specific disease states (e.g., tumor cells vs. healthy cells). In some embodiments, the conjugates and compounds described herein include a targeting moiety that is bound to a target that is specific to one or more specific developmental stages (e.g., stem cells vs. differentiated cells).

[1233] In some embodiments, target can be exclusively or mainly with one or several cell types, with one or several diseases and / or with one or several developmental stages related markers.The expression level of cell type specific markers in the cell type is usually higher than at least 2 times in reference cell colony, and the reference cell colony can be composed of, for example, a mixture of cells from multiple (for example 5-10 or more) different tissues or organs containing approximately equal amounts.In some embodiments, cell type specific markers exist at a level higher than at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 50 times, at least 100 times or at least 1000 times of its average expression in reference colony.Detection or measurement of cell type specific markers are likely to distinguish one or more cell types of interest from many, most or all other types of cells.

[1234] In some embodiments, the target comprises a protein, a carbohydrate, a lipid, and / or a nucleic acid. In some embodiments, the target comprises a protein and / or a characteristic portion thereof, such as a tumor marker, an integrin, a cell surface receptor, a transmembrane protein, an intercellular protein, an ion channel, a membrane transporter, an enzyme, an antibody, a chimeric protein, a glycoprotein, etc. In some embodiments, the target comprises a carbohydrate and / or a characteristic portion thereof, such as a glycoprotein, a sugar (e.g., a monosaccharide, a disaccharide, a polysaccharide), a glycocalyx (i.e., a carbohydrate-rich peripheral region on the outer surface of most eukaryotic cells), etc. In some embodiments, the target comprises a lipid and / or a characteristic portion thereof, such as an oil, a fatty acid, a glyceride, a hormone, a steroid (e.g., cholesterol, bile acid), a vitamin (e.g., vitamin E), a phospholipid, a sphingolipid, a lipoprotein, etc. In some embodiments, the target comprises a nucleic acid and / or a characteristic portion thereof, such as a DNA nucleic acid; an RNA nucleic acid; a modified DNA nucleic acid; a modified RNA nucleic acid; a nucleic acid comprising any combination of DNA, RNA, modified DNA, and modified RNA.

[1235] Many markers are known in the art. Typical markers include cell surface proteins, such as receptors. Exemplary receptors include, but are not limited to, transferrin receptors; LDL receptors; growth factor receptors, such as epidermal growth factor receptor family members (e.g., EGFR, Her2, Her3, Her4) or vascular endothelial growth factor receptors, cytokine receptors, cell adhesion molecules, integrins, selectins, and CD molecules. Markers can be molecules that exist exclusively or in higher amounts in malignant cells, such as tumor antigens.

[1236] Antibody-drug conjugates (ADCs)

[1237] In some embodiments, TM is an antibody and Q is a drug. Thus, the compounds and conjugates disclosed herein can be used to conjugate an antibody to a drug moiety to form a targeted drug conjugate, which is an antibody-drug conjugate (ADC). Antibody-drug conjugates (ADCs), like other targeted drug conjugates, can improve therapeutic efficacy in treating diseases (e.g., cancer) because ADCs are able to selectively deliver one or more drug moieties to target tissues, such as tumor-associated antigens. Thus, in certain embodiments, the present disclosure provides ADCs for therapeutic uses, such as treating cancer.

[1238] The ADC of the present disclosure comprises an antibody connected to one or more drug moieties. The specificity of the ADC is defined by the specificity of the antibody. In one embodiment, the antibody is connected to one or more cytotoxic drugs that are delivered internally to cancer cells.

[1239] Examples of drugs that can be used in the ADCs of the present disclosure are provided below. The terms "drug," "agent," and "drug moiety" are used interchangeably herein. The terms "linked" and "conjugated" are also used interchangeably herein and indicate that the antibody and moiety are covalently linked.

[1240] In certain aspects, the present disclosure relates to ADC, compositions comprising ADC, methods of treatment, and methods of preparing ADC compositions. ADC comprises an antibody or antibody fragment conjugated to a cytotoxic compound. In some embodiments, the cytotoxic compound is conjugated to the antibody via a linker. In other embodiments, the cytotoxic compound is directly attached to the antibody. The types of antibodies, linkers, and cytotoxic compounds encompassed by the present disclosure are described below.

[1241] Antibody

[1242] The antibody of ADC can be any antibody that is usually but not necessarily specifically bound to an antigen expressed on the surface of the target cell of interest. Antigen is not required, but in some embodiments, the ADC bound thereto can be internalized into the cell. The target cell of interest may include cells that need to induce apoptosis. The target antigen can be any protein, glycoprotein, polysaccharide, lipoprotein, etc. expressed on the target cell of interest, but it is usually a protein that is uniquely expressed on the target cell and not expressed on normal or healthy cells, or a protein that is overexpressed on the target cell compared to normal or healthy cells, so that the ADC selectively targets the specific cells of interest, such as tumor cells. As the technician will understand, the specific antigen selected and the antibody therefore will depend on the identity of the desired target cell of interest. In a specific embodiment, the antibody of ADC is an antibody suitable for administration to humans.

[1243] Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural features. Although antibodies show binding specificity to specific targets, immunoglobulins include antibodies and other antibody-like molecules that lack target specificity. Primary antibodies and immunoglobulins are typically heterotetrameric glycoproteins of about 150,000 daltons, which are composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end.

[1244] References to "VH" refer to the variable region of an immunoglobulin heavy chain (including the heavy chain of an Fv, scFv, or Fab) of an antibody. References to "VL" refer to the variable region of an immunoglobulin light chain (including the light chain of an Fv, scFv, dsFv, or Fab).

[1245] The term "antibody" herein is used in the broadest sense and refers to an immunoglobulin molecule that specifically binds to or immunoreacts with a specific antigen, and includes polyclonal, monoclonal, genetically engineered and other modified forms of antibodies, including but not limited to murine antibodies, chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies and tetrabodies), and antigen-binding fragments of antibodies, including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments. The term "scFv" refers to a single-chain Fv antibody in which the variable domains of the heavy and light chains from traditional antibodies have been linked to form one chain.

[1246] Antibodies can be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th edition, Garland Publishing, New York). Target antigens generally have many binding sites, also referred to as epitopes, recognized by CDRs on a variety of antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen can have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules or portions thereof that contain antigen binding sites that immunospecifically bind to an antigen of a target of interest, such targets include but are not limited to cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. The immunoglobulins can be derived from any species. However, in one aspect, the immunoglobulins are of human, murine, or rabbit origin.

[1247] The term "antibody fragment" refers to a portion of a full-length antibody, generally a target binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments. The "Fv" fragment is the smallest antibody fragment containing a complete target recognition and binding site. This region consists of a dimer (VH-VL dimer) of a heavy chain variable domain and a light chain variable domain in tight non-covalent association. It is this configuration of interaction of three CDRs of each variable domain that defines the target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer target binding specificity to antibodies. However, in some cases, even a single variable domain (or half an Fv containing only three CDRs specific to a target) can also have the ability to recognize and bind to a target. A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody in a single polypeptide chain. In general, the Fv polypeptide also comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the structure required for target binding. A "single domain antibody" consists of a single VH or VL domain that exhibits sufficient affinity for the target. In a specific embodiment, the single domain antibody is a camelized antibody (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231: 25-38).

[1248] The Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that it has several residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. The F(ab') fragment is produced by cleavage of the disulfide bond at the hinge cysteine ​​of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.

[1249] The light chain and heavy chain variable domains all have complementary determining regions (CDRs), also referred to as hypervariable regions. The more highly conserved parts of the variable domains are referred to as frameworks (FRs). As known in the art, the amino acid positions / boundaries depicting the hypervariable regions of antibodies can vary, depending on the context and various definitions known in the art. Some positions within the variable domains can be considered as mixed hypervariable positions, because these positions can be considered to be within the hypervariable regions under one set of criteria, and are considered to be outside the hypervariable regions under another set of criteria. One or more of these positions can also be found in the extended hypervariable regions. The CDRs in each chain are tightly bound together by the FR region, and together with the CDRs from another chain, contribute to the formation of the target binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health), Bethesda, Md. 1987). Unless otherwise indicated, as used herein, the numbering of immunoglobulin amino acid residues is carried out according to the immunoglobulin amino acid residue numbering system of Kabat et al.

[1250] In certain embodiments, the antibody of the ADC of the present disclosure is a monoclonal antibody. The term "monoclonal antibody" (mAb) refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic or phage clone, rather than a method for producing it. Preferably, the monoclonal antibodies of the present disclosure are present in a homogeneous or substantially homogeneous population. Monoclonal antibodies include complete molecules, as well as antibody fragments (such as Fab and F(ab')2 fragments) that can specifically bind to proteins. Fab and F(ab')2 fragments lack the Fc fragment of a complete antibody, are cleared faster from animal circulation, and may have less nonspecific tissue binding than complete antibodies (Wahl et al., 1983, J.Nucl.Med24:316). The monoclonal antibodies that can be used in the present disclosure can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant and phage display technologies or combinations thereof. The antibodies of the present disclosure include chimeric antibodies, primatized antibodies, humanized antibodies or human antibodies.

[1251] Although in most cases antibodies consist only of genetically encoded amino acids, in some embodiments, non-coding amino acids may be incorporated at specific positions. Examples of non-coding amino acids that can be incorporated into antibodies for controlling stoichiometry and attachment positions and methods for preparing such modified antibodies are discussed in Tian et al., 2014, Proc Nat'l Acad Sci USA 111 (5): 1766-1771 and Axup et al., 2012, Proc Nat'l Acad Sci USA 109 (40): 16101-16106, the entire contents of which are incorporated herein by reference.

[1252] In certain embodiments, the antibody of the ADC described herein is a chimeric antibody. As used herein, the term "chimeric" antibody refers to an antibody having a variable sequence derived from a non-human immunoglobulin (such as a rat or mouse antibody) and a human immunoglobulin constant region generally selected from a human immunoglobulin template. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229 (4719): 1202-7; Oi et al., 1986, BioTechniques 4: 214-221; Gillies et al., 1985, J. Immunol. Methods 125: 191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety.

[1253] In certain embodiments, the antibody of ADC described herein is a humanized antibody. The "humanized" form of non-human (e.g., murine) antibodies is a chimeric immunoglobulin, an immunoglobulin chain, or a fragment thereof (such as Fv, Fab, Fab', F(ab')2 or other target binding subdomains of an antibody), which contains a minimal sequence derived from a non-human immunoglobulin. In general, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins and all or substantially all of the FR regions are those of human immunoglobulin sequences. Humanized antibodies may also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a constant region of a human immunoglobulin consensus sequence. The method of antibody humanization is known in the art. See, e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and U.S. Pat. No. 6,180,370 to Queen et al.; EP 239400; PCT Publication WO 91 / 09967; U.S. Pat. No. 5,225,539; EP 592106; EP 519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad Sci. USA 91:969-973; and U.S. Patent No. 5,565,332, all of which are hereby incorporated by reference in their entirety.

[1254] In certain embodiments, the antibody of ADC described herein is a human antibody. Completely "human" antibodies may be ideal for therapeutic treatment of human patients. As used herein, "human antibodies" include antibodies with the amino acid sequence of human immunoglobulins, and include antibodies isolated from human immunoglobulin libraries or from one or more human immunoglobulin transgenics and animals that do not express endogenous immunoglobulins. Human antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887, 4,716,111, 6,114,598, 6,207,418, 6,235,883, 7,227,002, 8,809,151 and U.S. Publication Application No. 2013 / 189218, the contents of which are incorporated herein by reference in their entirety. Human antibodies can also be produced using transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. See, e.g., U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 7,723,270; 8,809,051 and U.S. Published Application No. 2013 / 117871, which are incorporated herein by reference in their entirety. In addition, companies such as Medarex (Princeton, NJ), Astellas Pharma (Deerfield, Ill.), and Regeneron (Tarrytown, NY) may also be collaborated to provide human antibodies against selected antigens using techniques similar to those described above. A technique known as "guided selection" may be used to generate fully human antibodies that recognize selected epitopes. In this approach, a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (Jespers et al., 1988, Biotechnology 12:899-903).

[1255] In certain embodiments, the antibodies of the ADCs described herein are primatized antibodies. The term "primatized antibody" refers to an antibody comprising a monkey variable region and a human constant region. Methods for producing primatized antibodies are known in the art. See, e.g., U.S. Pat. Nos. 5,658,570; 5,681,722; and 5,693,780, which are incorporated herein by reference in their entirety.

[1256] In certain embodiments, the antibody of the ADC described herein is a bispecific antibody or a dual variable domain antibody (DVD). Bispecific and DVD antibodies are monoclonal antibodies, typically human or humanized antibodies, that have binding specificity to at least two different antigens. DVD is described in, for example, U.S. Patent No. 7,612,181, the disclosure of which is incorporated herein by reference.

[1257] In certain embodiments, the antibody of the ADC described herein is a derivatized antibody. For example, but not limited to, the derivatized antibody is usually modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting groups / blocking groups, proteolytic cleavage, connection with cell ligands or other proteins, etc. Any of a variety of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, tunicamycin metabolic synthesis, etc. In addition, the derivative can contain one or more non-natural amino acids, for example, using ambrx technology (see, for example, Wolfson, 2006, Chem.Biol.13(10):1011-2).

[1258] In certain embodiments, the antibodies of the ADCs described herein have sequences that have been modified relative to the corresponding wild-type sequence to alter at least one constant region-mediated biological effector function. For example, in some embodiments, the antibody can be modified to reduce at least one constant region-mediated biological effector function relative to the unmodified antibody, such as reducing binding to Fc receptors (FcRs). FcR binding can be reduced by mutating the immunoglobulin constant region segments of the antibody at specific regions necessary for FcR interaction (see, e.g., Canfield and Morrison, 1991, J. Exp. Med 173: 1483-1491; and Lund et al., 1991, J. Immunol. 147: 2657-2662).

[1259] In certain embodiments, the antibodies of the ADCs described herein are modified to obtain or improve at least one constant region-mediated biological effector function relative to an unmodified antibody, for example, to enhance FcγR interactions (see, e.g., US2006 / 0134709). For example, antibodies having a constant region that binds to FcγRIIA, FcγRIIB, and / or FcγRIIIA with greater affinity than the corresponding wild-type constant region can be produced according to the methods described herein.

[1260] In certain specific embodiments, the antibody of ADC described herein is an antibody that binds tumor cells, such as an antibody for a cell surface receptor or a tumor-associated antigen (TAA). In an attempt to find an effective cell target for cancer diagnosis and therapy, researchers attempt to identify transmembrane or other tumor-associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells compared to expression on one or more normal non-cancerous cells. Typically, the expression of such tumor-associated polypeptides on the surface of cancer cells is more abundant than expression on the surface of non-cancerous cells. Such cell surface receptors and tumor-associated antigens are known in the art and can be prepared for use in the art to generate antibodies using methods and information well known in the art.

[1261] Exemplary Cell Surface Receptors and TAAs

[1262] Examples of cell surface receptors and TAAs that the antibodies of the ADCs described herein can target include, but are not limited to, the various receptors and TAAs listed in Table 1 below. For convenience, information related to these antigens (all known in the art) is listed below and includes names, alternative names, Genbank accession numbers, and one or more primary references, following the National Center for Biotechnology Information (NCBI) nucleic acid and protein sequence identification conventions. Nucleic acid and protein sequences corresponding to the listed cell surface receptors and TAAs are available in public databases such as GenBank.

[1263] Table I.

[1264]

[1265]

[1266]

[1267]

[1268] Exemplary Antibodies

[1269] Exemplary antibodies to be used in the ADCs of the present disclosure include, but are not limited to, 3F8 (GD2), Abagovomab (CA-125 (generic)), Adecatumumab (EpCA M), Afutuzumab (CD20), Alacizumab pego l (VEGFR2), ALD518 (IL-6), Alemtuzumab (CD52), Atumomab Pentetate (Altumomab pentetate)(CEA), Amatuximab(mesothelin), Anatumomnabmafenatox(TAG-72), Apolizumab(HLA-DR), Arcitumomab(CEA), Bavituximab(phosphatidylserine), Bectumomab(CD22), Belimumab(BAFF), Besilesomab(CEA-related antigen), Bevacizumab(VEGF-A), Bivatuzumabmertansine(CD44 v6), Blinatumomab(CD19), Brentuximab vedotin((CD30(TNFRSF8)), Cantuzumab mertansine)(Mucin CanAg), Cantuzumab ravt ansine(MUC1), Capromab pendetide(Prostate cancer cells), Carlumab(MCP-1), Catumaxomab(EpCAM, CD3), CC49(Tag-72), cBR96-DOX ADC(Lewis-Y antigen), Cetuximab(EGFR), Citatuzumab ravt ansine(EpCAM), Cixutumumab(IGF-1 receptor), Clivatuzumab(Clivatuzumab)Tetraxetan (MUC1), Conatumumab (TRAIL-E2), Dacetuzumab (CD40), Dalotuzumab (Insulin-like growth factor 1 receptor), Deratumumab ((CD38 (cyclic ADP ribose hydrolase)), Demcizumab (DLL4), Denosumab (RANKL), Detumomab (B lymphoma cells), Drozitumab (DR5), Dusigitumab (ILGF2), Ecromeximab (D3 ganglioside), Eculizumab (C5), Edrecolomab (EpCAM), Elotuzumab (SLAMF7), Elsilimomab (IL-6), Enavatuzumab (TWEAK receptor), Enoticumab (DLL4), Ensituximab (5AC), Sipilimumab (Ep itumomabcituxetan (epithelial sialoprotein), Epratuzumab (CD22), Ertumaxomab (HER2 / neu, CD3), Etancizumab (integrin αvβ3), Farletuzumab (folate receptor 1), FBTA05 (CD20), Ficlatuzumab (HGF), Figitumumab (IGF-1 receptor) , Flanvotumab ((TYRP1 (glycoprotein 75)), Fresolimumab (TGF-1), Galiximab (CD80), Ganitumab (IGF-I), Gemtuzumab ozogamicin (CD33), Girentuximab ((Carbonic anhydrase 9 (CA-IX)), Glembatumumab vedotin (GPNMB), Ibritumomab tiuxetantiuxetan (CD20), Icrucumab (VEGFR-1), Igovomab (CA-125), IMAB362 (CLDN18.2), Imgatuzumab (EGFR), Indatuximab (SDC1), Intetumumab ( umab)(CD51), Inotuzumab ozogamicin(CD22), Ipilimumab(CD152), Iratumumab((CD30(TNFRSF8)), Labetuzumab(CEA), Lambrolizumab(PDCD1), Lexatumumab(TRAIL-R2), Lintuzumab(CD33), Lorvotuzumabmertansine(CD56), Lucatumumab(CD40), Lumiliximab((CD23(IgE receptor)), Mapatumumab( umumab (TRAIL-R1), Margetuximab (ch4DS), Matuzumab (EGFR), Milatuzumab (CD74), Mitumo mab)(GD3 ganglioside), Mogamulizumab(CCR4), Moxetumomabpasudotox(CD22), Nacolomabtafenatox(C2-42 antigen), Naptumomabestafenatox(5T4), Narnatumab(RON), Natalizumab(Integrin α4), Necitumumab(EGFR), Nesvacumab(Angiopoietin 2), Nimotuzumab(EGFR), Nivolumab(IgG4), Ocaratuzumab(CD20), Ofatumumab(CD20), Olaratumab)(PDGF-Rα), Onartuzumab(Human scatter factor receptor kinase), Ontuxizumab(TEM1), Oportuzumabmonato(EpCA M), Oregovomab(CA-125), Otlertuzumab(CD37), Panitumumab(EGFR), Pankomab(Tumor-specific glycosylation of MUC1), Parsatuzumab(EGFL7), Patritumab(HER3), Pemtumomab(MUC1), Pertuzumab(HER2 / neu), Pidilizumab(PD-1), Vidin-Pinatuzumab( uzumabvedotin)(CD22), Pritumumab(vimentin), Racotumomab(N-glycolylneuraminic acid), Radretumab(fibronectin extra domain B), Ramucirumab(VEGFR2), Rilotumumab(HGF), Rituximab(CD20), Robatumumab(IGF-1 receptor), Samalizumab(CD200), Satumomabpendetide(TAG-72), Seribantumab(ERBB3), Silobizumab(Si brotuzumab (FAP), SGN-CD19A (CD19), SGN-CD33A (CD33), Siltuximab (IL-6), Solitomab (EpCAM), Sonepcizumab (Sphingosine-1-phosphate), Tabalumb (BAFF), Tacatuzumab tetraxetan (AFP), Tapitumomab paptox (CD19), Tenatumomab (Tenasin C), Tepr otumumab (CD221), TGN1412 (CD28), Ticilimumab (CTLA-4), Tigatuzumab (TRAIL-R2), TNX-650 (IL-13), Tovetumab (CD40a), Trastuzumab (HER2 / neu), TRBS07 (GD2), Tisitumab (CTLA-4), Tucotuzumab celmoleukin (EpCAM), Ublituximab (MS4A), Urelumab (4-1BB), Vandetanib (VEGF), Vantictumab (Frizzled receptor), Volociximab (integrin α5β1), Vorsetuzumab mafodotin (CD70), Votumumab (tumor antigen CTAA16.88), Zalutumumab (tumor antigen CTAA16.88), mab) (EGFR), Zanolimumab (CD4), and Zatuximab (HER1).

[1270] Antibody Preparation Method

[1271] The antibody of ADC can be prepared by recombinant expression of immunoglobulin light chain and heavy chain genes in host cells. For example, in order to recombinantly express antibodies, host cells are transfected with recombinant expression vectors of DNA fragments of one or more immunoglobulin light chains and heavy chains encoding antibodies, so that light chains and heavy chains are expressed in host cells, and optionally secreted into the culture medium of the host cells, antibodies can be recovered from the culture medium. Standard recombinant DNA methods are used to obtain antibody heavy and light chain genes, these genes are incorporated into recombinant expression vectors and the vectors are introduced into host cells, such as those described in the following documents: Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (ed.), Cold Spring Harbor, NY, 1989), Current Protocols in Molecular Biology (Ausubel, FM et al., Greene Publishing Associates, 1989), and U.S. Patent No. 4,816,397.

[1272] In one embodiment, an Fc variant antibody is similar to its wild-type equivalent, but its Fc domain is changed. To generate nucleic acids encoding such Fc variant antibodies, a DNA fragment encoding the Fc domain of a wild-type antibody or a portion of the Fc domain (referred to as a "wild-type Fc domain") can be synthesized and used as a mutagenesis template for generating antibodies as described herein using conventional mutagenesis techniques; alternatively, a DNA fragment encoding an antibody can be directly synthesized.

[1273] Once the DNA fragments encoding wild-type Fc domains are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert constant region genes into full-length antibody chain genes. In these operations, the DNA fragment encoding CH is operably connected to another DNA fragment encoding another protein, such as an antibody variable region or a flexible linker. As used in this context, the term "operably connected" is intended to mean that the two DNA fragments are connected so that the amino acid sequence encoded by the two DNA fragments remains in frame.

[1274] In order to express Fc variant antibodies, the DNA encoding part or full-length light chain and heavy chain obtained as described above is inserted into an expression vector so that the gene is operably connected to transcription and translation control sequences. In this context, the term "operably connected" is intended to mean that the antibody gene is joined to a vector so that the transcription and translation control sequences in the vector play the expected function of its regulation of the transcription and translation of the antibody gene. Expression vectors and expression control sequences compatible with the expression host cell used are selected. Variant antibody light chain genes and antibody heavy chain genes can be inserted into a separate vector, or more typically, two genes are inserted into the same expression vector.

[1275] The antibody gene is inserted into the expression vector by standard methods (e.g., joining the complementary restriction sites on the antibody gene fragment and the vector, or flat-end joining if there is no restriction site). Before inserting the variant Fc domain sequence, the expression vector may have carried the antibody variable region sequence. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that promotes secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into a vector so that the signal peptide frame is connected to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[1276] In addition to the antibody chain genes, the recombinant expression vector also carries regulatory sequences for controlling the expression of the antibody chain genes in host cells. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif., 1990). It will be appreciated by those skilled in the art that the design of expression vectors, including the selection of regulatory sequences, may depend on factors such as the selection of host cells to be transformed, the expression level of the desired protein, etc. Regulatory sequences suitable for mammalian host cell expression include viral elements that instruct high levels of protein expression in mammalian cells, such as promoters and / or enhancers (such as CMV promoters / enhancers) derived from cytomegalovirus (CMV), simian virus 40 (SV40) (such as SV40 promoters / enhancers), adenovirus (e.g., adenovirus major late promoter (AdMLP)) and polyomavirus. For further description of viral regulatory elements and sequences thereof, see, e.g., U.S. Pat. No. 5,168,062 to Stinski, U.S. Pat. No. 4,510,245 to Bell et al., and U.S. Pat. No. 4,968,615 to Schaffner.

[1277] In addition to antibody chain genes and regulatory sequences, recombinant expression vectors can also carry additional sequences, such as sequences (e.g., replication origins) and selective marker genes that regulate vector replication in host cells. Selective marker genes help select host cells that have been introduced into vectors (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017, all belonging to Axel et al.). For example, selective marker genes generally confer resistance to drugs such as G418, puromycin, blasticidin, hygromycin, or methotrexate to host cells that have been introduced into vectors. Suitable selective marker genes include dihydrofolate reductase (DHFR) genes (for DHFR-host cells with methotrexate selection / amplification) and neo genes (for G418 selection). In order to express light and heavy chains, one or more expression vectors encoding heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, and the like.

[1278] It is possible to express the antibody in prokaryotic or eukaryotic host cells. In certain embodiments, the expression of the antibody is carried out in eukaryotic cells, such as mammalian host cells, to optimally secrete correctly folded and immunocompetent antibodies. Exemplary mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO cells) (including DHFR-CHO cells, described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77: 4216-4220, used together with DHFR selective markers, for example, as described in Kaufman and Sharp, 1982, Mol. Biol. 159: 601-621), NS0 myeloma cells, COS cells, 293 cells and SP2 / 0 cells. When the recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing a period of host cells that is sufficient to allow the antibody to be expressed in the host cell or the antibody is secreted into the culture medium of the host cell growth. Standard protein purification methods can be used to reclaim the antibody from the culture medium. The host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules.

[1279] In some embodiments, the antibody of the ADC can be a bifunctional antibody. Such antibodies in which one heavy chain and one light chain are specific for one antigen and the other heavy chain and light chain are specific for a second antigen can be produced by cross-linking the antibody with the second antibody by standard chemical cross-linking methods. Bifunctional antibodies can also be prepared by expressing nucleic acids engineered to encode bifunctional antibodies.

[1280] In certain embodiments, dual-specific antibodies, i.e. antibodies that use the same binding site to bind one antigen and a second unrelated antigen, can be produced by mutating amino acid residues in the light chain and / or heavy chain CDRs. Exemplary second antigens include proinflammatory cytokines (such as lymphotoxin, interferon gamma, or interleukin 1). Dual-specific antibodies can be produced, for example, by mutating amino acid residues around the antigen binding site (see, e.g., Bostrom et al., 2009, Science 323: 1610-1614). Dual-functional antibodies can be prepared by expressing nucleic acids engineered to encode dual-specific antibodies.

[1281] Antibodies can also be produced by chemical synthesis (e.g., by methods described in Solid Phase Peptide Synthesis, 2nd Edition, 1984 The Pierce Chemical Co., Rockford, 111.) Antibodies can also be produced using a cell-free platform (see, e.g., Chu et al., Biochemia 2nd, 2001 (Roche Molecular Biologicals)).

[1282] Methods for recombinant expression of Fc fusion proteins are described in Flanagan et al., Methods in Molecular Biology, Vol. 378: Monoclonal Antibodies: Methods and Protocols.

[1283] Once the antibodies have been produced by recombinant expression, they can be purified by any method known in the art for purifying immunoglobulin molecules, for example by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly by affinity chromatography for the antigen after selection with protein A or protein G, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins.

[1284] Once isolated, the antibody can be further purified if necessary, for example, by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology (Work and Burdon, eds., Elsevier, 1980)), or by Superdex TM Gel filtration chromatography on a TALEN 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

[1285] General methods for preparing antibodies

[1286] Various procedures known in the art can be used to generate polyclonal or monoclonal antibodies to a given target (such as B7-H3, tumor-associated antigens or other targets) or to derivatives, fragments, analogs, homologs or orthologs thereof (see, e.g., Antibodies: A Laboratory Manual, Harlow E and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[1287] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which primarily provides the IgG fraction of immune serum. Subsequently or alternatively, a specific antigen or epitope thereof that is the target of the immunoglobulin being sought can be fixed on a column to purify the immunospecific antibodies by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[1288] In some embodiments, the antibodies that can be used in the embodiments disclosed herein are monoclonal antibodies. For example, monoclonal antibodies are generated by using the procedures set forth in the embodiments provided herein. Antibodies can also be generated, for example, by immunizing BALB / c mice with a combination of cell transfectants that express a high level of a given target on its surface. The hybridomas produced by myeloma / B cell fusions are then screened for reactivity to the selected target.

[1289] For example, monoclonal antibodies are prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters or other appropriate host animals are usually immunized with an immunizing agent to induce lymphocytes to produce or be able to produce antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[1290] Immunizing agents will generally include protein antigens, fragments thereof, or fusion proteins thereof. In general, if cells of human origin are required, peripheral blood lymphocytes are used, or if non-human mammalian sources are required, spleen cells or lymph node cells are used. Lymphocytes are then fused to immortalized cell lines using a suitable fusion agent (such as polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) 59-103 pages). Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, cattle, and human origin. Typically, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium, and the culture medium preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas will typically contain hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances will prevent the growth of HGPRT-deficient cells.

[1291] Preferred immortalized cell lines are those that can effectively fuse, support the selected antibody-producing cells to stably express antibodies at high levels, and are sensitive to culture media (such as HAT culture media). Preferred immortalized cell lines are murine myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center (Salk Institute Cell Distribution Center, San Diego, California) and the American Type Culture Collection (American Type Culture Collection, Manassas, Virginia). Human myeloma and mouse-human heteromyeloma cell lines have also been described for producing monoclonal antibodies. (See Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

[1292] The presence of monoclonal antibodies against antigens in the culture medium in which hybridoma cells are cultured can then be determined. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by Munson and Pollard, Anal. Biochem., 107: Scatchard analysis of 220 (1980). In addition, in the therapeutic application of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity to the target antigen.

[1293] After the desired hybridoma cells are identified, clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies:Principles and Practice , Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo as ascites of a mammal.

[1294] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[1295] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding monoclonal antibodies can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. Once separated, DNA can be placed in an expression vector, which is then transfected into a host cell, such as a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise generate immunoglobulins, to synthesize monoclonal antibodies in recombinant host cells. It can also be, for example, by replacing homologous murine sequences with coding sequences of human heavy and light chain constant domains (see U.S. Patent No. 4,816,567; Morrison, Nature 368,812-13 (1994)) or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence to modify DNA. Such non-immunoglobulin polypeptides can replace the constant domains of an antibody, or can replace the variable domains of one antigen combining site of an antibody to create a chimeric bivalent antibody.

[1296] Monoclonal antibodies that can be used in the embodiments disclosed herein include humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without eliciting an immune response in humans against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')) consisting primarily of sequences of human immunoglobulins. 2Or other antigen binding subsequences of antibodies), and contain minimal sequences derived from non-human immunoglobulins. For example, by following the method of Winter and colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), humanization is performed by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. (See also U.S. Patent No. 5,225,539). In some cases, the Fv framework residues of a human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies also include, for example, residues that are not found in either the receptor antibody or the imported CDR or framework sequence. In general, humanized antibodies include substantially all of at least one and usually two variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. Humanized antibodies also optimally include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of an immunoglobulin constant region (Fc) of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[1297] A fully human antibody is an antibody molecule in which the entire sequence of the light chain and the heavy chain (including CDR) is derived from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies". Monoclonal antibodies can be prepared using tri-hybridoma technology; human B cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4: 72); and EBV hybridoma technology (see Cole et al., 1985 Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) for producing monoclonal antibodies. Monoclonal antibodies are available and can be produced by using human hybridomas (see Cote et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein Barr Virus (see Cole et al., 1985 Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[1298] In addition, additional techniques can also be used to generate human antibodies, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991)). Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals, such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. After challenge, the production of human antibodies is observed, which is very similar to the antibodies observed in humans in all aspects including gene rearrangement, assembly and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; and in Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368, 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

[1299] In addition, transgenic non-human animals can be used to produce human antibodies, which are modified to produce fully human antibodies rather than endogenous antibodies produced by animals in response to antigenic attack. (See PCT Publication WO94 / 02602). The endogenous genes encoding heavy and light chain immunoglobulins in the non-human host have been disabled, and the active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. For example, yeast artificial chromosomes containing the necessary human DNA segments are used to incorporate human genes. Then, animals that provide all the desired modifications are obtained as offspring by hybridizing intermediate transgenic animals containing less than the complete complement of modifications. An example of such a non-human animal is called a Xenomouse. TMMice, such as disclosed in PCT publications WO 96 / 33735 and WO 96 / 34096. Such animals produce B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from animals after immunization with the immunogen of interest, for example as preparations of polyclonal antibodies, or from immortalized B cells derived from animals, such as hybridomas that produce monoclonal antibodies. In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly, or can be further modified to obtain analogs of the antibodies, such as single-chain Fv (scFv) molecules.

[1300] An example of a method for producing a non-human host (using mice as an example) lacking endogenous immunoglobulin heavy chain expression is disclosed in U.S. Pat. No. 5,939,598. A non-human host can be obtained by a method comprising deleting a J segment gene from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent the formation of transcripts of the rearranged immunoglobulin heavy chain locus, wherein the deletion is achieved by a targeting vector containing a gene encoding a selective marker; and generating a transgenic mouse from embryonic stem cells, wherein the somatic cells and germ cells of the mouse contain genes encoding selective markers.

[1301] A method for producing an antibody of interest (such as a human antibody) is disclosed in U.S. Pat. No. 5,916,771. This method comprises introducing an expression vector containing a nucleotide sequence encoding a heavy chain into a cultured mammalian host cell, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing a heavy chain and a light chain.

[1302] In a further refinement of this procedure, methods for identifying clinically relevant epitopes on immunogens and related methods for selecting antibodies that specifically bind to the relevant epitopes with high affinity are disclosed in US Publication No. US2003 / 009212.

[1303] The antibodies can be expressed by vectors containing DNA segments encoding the single-chain antibodies described above.

[1304] These may include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates such as those described in WO 93 / 64701, which have a targeting portion (e.g., a ligand for a cell surface receptor) and a nucleic acid binding portion (e.g., polylysine); viral vectors (e.g., DNA or RNA viral vectors); fusion proteins such as those described in U.S. Pat. No. 7,186,697, which are fusion proteins containing a targeting portion (e.g., an antibody specific for a target cell) and a nucleic acid binding portion (e.g., protamine); plasmids; phages, etc. Vectors may be chromosomal, non-chromosomal, or synthetic.

[1305] Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA viral vectors are preferred. These vectors include pox vectors, such as orthopox or fowlpox vectors; herpes virus vectors, such as herpes simplex virus type I (HSV) vectors (see Geller, AI et al., J. Neurochem, 64: 487 (1995); Lim, F. et al., DNA Cloning: Mammalian Systems, D. Glover ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA 90: 7603 (1993); Geller, AI et al., Proc Natl. Acad. Sci USA 87: 1149 (1990); adenovirus vectors (see LeGal LaSalle et al., Science, 259: 988 (1993); Davidson et al., Nat. Genet. 3:219 (1993); Yang et al., J. Virol. 69:2004 (1995); and adeno-associated virus vectors (see Kaplitt, MG et al., Nat. Genet. 8:148 (1994).

[1306] Poxvirus vectors introduce genes into the cytoplasm of cells. Fowlpox virus vectors only facilitate short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acids into neural cells. The expression time facilitated by adenovirus vectors (about 2 months) is shorter than that of adeno-associated virus (about 4 months), which in turn is shorter than that of HSV vectors. The specific vector selected will depend on the target cell and the condition being treated. Introduction can be carried out by standard techniques, such as infection, transfection, transduction, or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO 4 Precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection and viral vectors.

[1307] The vector can be used to target essentially any desired target cell. For example, a vector (e.g., adenovirus, HSV) can be directed to a desired location using stereotaxic injection. In addition, particles can be delivered by intracerebroventricular (icv) infusion using a micropump infusion system (such as the SynchroMed infusion system). A bulk flow-based method (called convection) has also been shown to effectively deliver macromolecules to extended areas of the brain and can be used to deliver vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91: 2076-2080 (1994); Morrison et al., Am. J. Physiol. 266: 292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal and subcutaneous injections, as well as oral or other suitable routes of administration.

[1308] Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for a target such as B7-H3 or any fragment thereof. The second binding target is any other antigen, and advantageously is a cell surface protein or receptor or receptor subunit.

[1309] Many methods for preparing bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain / light chain pairs, wherein the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually accomplished by an affinity chromatography step. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991), published on May 13, 1993.

[1310] Bispecific and / or monovalent antibodies that can be used in the embodiments disclosed herein can be prepared using any of a variety of techniques recognized in the art, including those disclosed in application WO 2012 / 023053 filed on August 16, 2011, the contents of which are hereby incorporated by reference in their entirety. The method described in WO 2012 / 023053 generates bispecific antibodies that are structurally identical to human immunoglobulins. This type of molecule consists of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant κ domain, and a second light chain variable region fused to a constant λ domain. Each combination site exhibits a different antigenic specificity, to which both the heavy chain and the light chain contribute. The light chain variable region can belong to the λ or κ family, and is preferably fused to the λ and κ constant domains, respectively. This is preferred to avoid the generation of non-natural polypeptide connections. However, it is also possible to obtain bispecific antibodies that can be used in the embodiments disclosed herein by fusing the κ light chain variable domain to the constant λ domain to obtain a first specificity and fusing the λ light chain variable domain to the constant κ domain to obtain a second specificity. The bispecific antibodies described in WO 2012 / 023053 are referred to as IgGκλ antibodies or "κλ bodies", a new fully human bispecific IgG format. This κλ body format allows affinity purification of bispecific antibodies that are indistinguishable from standard IgG molecules with characteristics that are indistinguishable from standard monoclonal antibodies and is therefore advantageous compared to previous formats.

[1311] A necessary step of the method is to identify two antibody Fv regions (each consisting of a variable light chain and a variable heavy chain domain) with different antigen specificities and sharing the same heavy chain variable domain. A variety of methods for generating monoclonal antibodies and fragments thereof have been described. (See, for example, Antibodies: A Laboratory Manual, Harlow E and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Fully human antibodies are antibody molecules in which the sequences of light and heavy chains (including CDR 1 and 2) are derived from human genes. The CDR3 region can be of human origin or designed by synthetic means. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies". Human monoclonal antibodies can be prepared by using trioma technology, human B cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4: 72), and EBV hybridoma technology for producing human monoclonal antibodies (see Cole et al., 1985 Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be used and can be produced by using human hybridomas (see Cote et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B cells with Epstein Barr virus in vitro (see Cole et al., 1985 Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[1312] Monoclonal antibodies are generated, for example, by immunizing an animal with a target antigen or an immunogenic fragment, derivative, or variant thereof. Alternatively, an animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target antigen, such that the target antigen is expressed and associated with the surface of the transfected cells. A variety of techniques suitable for producing xenogeneic non-human animals are well known in the art. See, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which are hereby incorporated by reference in their entirety.

[1313] Alternatively, antibodies are obtained by screening libraries containing antibody or antigen binding domain sequences that bind to the target antigen. Such libraries are prepared, for example, in phage as protein or peptide fusions with phage coat proteins that are expressed on the surface of assembled phage particles and the encoding DNA sequence is contained within the phage particles (i.e., "phage display libraries").

[1314] The hybridomas produced by the myeloma / B cell fusion are then screened for reactivity to the target antigen. For example, monoclonal antibodies are prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters or other appropriate host animals are usually immunized with an immunizing agent to induce lymphocytes to produce or be able to produce antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[1315] Although not completely impossible, it is extremely unlikely to accidentally identify different antibodies with the same heavy chain variable domain but against different antigens. In fact, in most cases, the heavy chain contributes greatly to the antigen binding surface and has the greatest variation in sequence. In particular, CDR3 on the heavy chain is the most diverse CDR in sequence, length and structure. Therefore, two antibodies that are specific for different antigens almost always carry different heavy chain variable domains.

[1316] The method disclosed in the application U.S. Patent No. 9,926,382 overcomes this limitation and greatly facilitates the separation of antibodies with the same heavy chain variable domain by using an antibody library, wherein the heavy chain variable domain is the same for all library members, and therefore diversity is limited to the light chain variable domain. Such libraries are described in, for example, U.S. Patent No. 8,921,281 and application WO 2011 / 084255, each of which is hereby incorporated by reference as a whole. However, since the light chain variable domain is co-expressed with the heavy chain variable domain, both domains contribute to antigen binding. In order to further promote this process, an antibody library containing the same heavy chain variable domain and a diversified λ variable light chain or κ variable light chain can be used in parallel to select antibodies against different antigens in vitro. This method is able to identify two antibodies with a common heavy chain, but one carries a λ light chain variable domain and the other carries a κ light chain variable domain, and these two antibodies can be used as building blocks for generating bispecific antibodies in the form of complete immunoglobulins. The bispecific antibodies that can be used in the embodiments disclosed herein can have different isotypes, and their Fc portions can be modified to change the binding properties to different Fc receptors, and in this way change the effector function of the antibody and its pharmacokinetic properties. A variety of methods for modifying the Fc portion have been described, and these methods are applicable to antibodies that can be used in the embodiments disclosed herein. (See, e.g., Strohl, WR Curr Opin Biotechnol 2009 (6): 685-91; U.S. Patent No. 6,528,624; PCT / US2009 / 0191199 filed on January 9, 2009).

[1317] A common heavy chain and two different light chains are co-expressed in a single cell to allow assembly of bispecific antibodies that can be used in the embodiments disclosed herein. If all polypeptides are expressed at the same level and assemble equally well to form immunoglobulin molecules, the ratio of monospecific (same light chain) to bispecific (two different light chains) should be 50%. However, different light chains may be expressed at different levels and / or not assembled with the same efficiency. Therefore, means for regulating the relative expression of different polypeptides are used to compensate for their intrinsic expression characteristics or different tendencies to assemble with a common heavy chain. This regulation can be achieved via promoter strength, the use of internal ribosome entry sites (IRES) with different efficiencies, or other types of regulatory elements that can act at the transcriptional or translational level and act on mRNA stability. Different promoters of different strengths can include CMV (immediate early cytomegalovirus promoter); EF1-1α (human elongation factor 1α subunit promoter); Ubc (human ubiquitin C promoter); SV40 (simian virus 40 promoter). Different IRES of mammalian and viral origin have also been described. (See, e.g., Hellen CU and Sarnow P. Genes Dev 2001 15: 1593-612). These IRESs may vary greatly in length and ribosome recruitment efficiency. In addition, it is possible to further regulate activity by introducing multiple copies of the IRES (Stephen et al. 2000 Proc Natl Acad Sci USA 97: 1536-1541). Regulation of expression can also be achieved by multiple continuous transfections of cells to increase the copy number of a single gene expressing one or the other light chain and thus alter their relative expression. The examples provided herein demonstrate that controlling the relative expression of different chains is essential for maximizing the assembly and total yield of bispecific antibodies.

[1318] The co-expression of heavy chain and two light chains generates a mixture of three different antibodies in the cell culture supernatant: two monospecific bivalent antibodies and a bispecific bivalent antibody. The latter must be purified from the mixture to obtain the molecule of interest. The method described herein greatly facilitates this purification process by using affinity chromatography media that specifically interact with κ or λ light chain constant domains, such as CaptureSelect Fabκ and CaptureSelect Fabλ affinity matrices (BAC BV, Holland). This multi-step affinity chromatography purification method is effective and generally applicable to antibodies that can be used in the embodiments disclosed herein. This is in sharp contrast to the specific purification method that must be developed and optimized for each bispecific antibody derived from a four-source hybridoma or other cell line expressing an antibody mixture. In fact, if the biochemical characteristics of different antibodies in the mixture are similar, it may be challenging or impossible to separate them using standard chromatographic techniques (such as ion exchange chromatography).

[1319] Other suitable purification methods include those disclosed in US 2013 / 0317200, the contents of which are hereby incorporated by reference in their entirety.

[1320] In other embodiments of producing bispecific antibodies, antibody variable domains (antibody-antigen combination sites) with desired binding specificity can be fused with immunoglobulin constant domain sequences. Preferably, it is fused with immunoglobulin heavy chain constant domains, which include at least a portion of hinge region, CH2 and CH3 regions. Preferably, in at least one fusion, there is a first heavy chain constant region (CH1) containing the site necessary for light chain binding. DNA encoding immunoglobulin heavy chain fusions and (if necessary) immunoglobulin light chains are inserted into separate expression vectors and cotransfected into suitable host organisms. For further details of generating bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

[1321] According to the another method described in WO 96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of the heterodimer recovered from recombinant cell culture.Preferred interface includes at least a portion of the CH3 region of antibody constant domain.In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (for example tyrosine or tryptophan).By replacing large amino acid side chains with smaller amino acid side chains (for example alanine or threonine), compensation "cavity" of the same or similar size as one or more large side chains is produced at the interface of the second antibody molecule.This provides a mechanism to improve the output of heterodimer relative to other unwanted final products (such as homodimers).

[1322] The technology of generating bispecific antibodies from antibody fragments has been described in the literature. For example, chemical linkage can be used to prepare bispecific antibodies. The generated bispecific antibodies can be used as agents for selective immobilization of enzymes.

[1323] Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then oxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides an alternative mechanism for preparing bispecific antibody fragments. The fragment comprises a light chain variable domain (V L )'s heavy chain variable domain (V H ), the linker is too short to allow pairing between the two domains on the same chain. H and V L The domain is forced to align with the complementary V L and V H The domains pair to form two antigen binding sites. Another strategy for making bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994).

[1324] Antibodies with more than two valency states are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[1325] Exemplary bispecific antibodies can be bound to two different epitopes, at least one of which is derived from a protein antigen that can be used in the embodiments disclosed herein. Alternatively, the anti-antigen arm of the immunoglobulin molecule can be combined with an arm that binds to a trigger molecule on a leukocyte to focus the cell defense mechanism on cells expressing specific antigens, such as T cell receptor molecules (e.g., CD2, CD3, CD28, or B7) or IgG Fc receptors (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). Bispecific antibodies can also be used to guide cytotoxic agents to cells expressing specific antigens. These antibodies have an antigen binding arm and an arm that binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds to a protein antigen described herein and further binds to a tissue factor (TF).

[1326] Heteroconjugate antibodies are also within the scope of the present disclosure. Heteroconjugate antibodies consist of two covalently linked antibodies. For example, such antibodies have been proposed to target immune system cells to unwanted cells (see U.S. Patent No. 4,676,980), and for treating HIV infection (see WO 91 / 00360; WO 92 / 200373; EP 03089). It is expected that antibodies can be prepared in vitro using synthetic protein chemistry (including those involving cross-linking agents). For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiol esters and methyl-4-mercaptobutyrylimidate, and those disclosed in, for example, U.S. Patent No. 4,676,980.

[1327] It may be desirable to modify the antibodies that can be used in the embodiments disclosed herein in terms of effector function to enhance, for example, the effectiveness of the antibodies in treating cancer and / or other diseases and conditions associated with abnormal B7-H3 expression and / or activity. For example, one or more cysteine ​​residues may be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The homodimeric antibodies thus generated may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, the antibodies may be engineered to have dual Fc regions and thus may have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).

[1328] Conjugated Antibodies

[1329] The present disclosure also relates to conjugated antibodies, also referred to herein as immunoconjugates, comprising an antibody or antigen-binding fragment thereof conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof), or a radioactive isotope (i.e., a radioconjugate).

[1330] In some embodiments, the toxin is a microtubule inhibitor or a derivative thereof. In some embodiments, the toxin is dolastatin or a derivative thereof. In some embodiments, the toxin is auristatin E, auristatin F, AFP, MMAF, MMAE, MMAD, DMAF or DMAE. In some embodiments, the toxin is a maytansinoid or a maytansinoid derivative. In some embodiments, the toxin is DM1 or DM4. In some embodiments, the toxin is a nucleic acid damaging toxin. In some embodiments, the toxin is a duocarmycin or a derivative thereof. In some embodiments, the toxin is a calicheamicin or a derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine or a derivative thereof. In some embodiments, the agent is exatecane or a derivative thereof. In some embodiments, the agent is amanitin or a derivative thereof.

[1331] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, saponin inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and tricothecene. A variety of radionuclides can be used to produce radioconjugated antibodies. Examples include 212 Bi, 131 I. 131 In, 90 Y and 186 Re.

[1332] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimide hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies (see WO94 / 11026).

[1333] One of ordinary skill in the art will recognize that a variety of possible moieties can be coupled to the resulting antibodies that can be used in the embodiments disclosed herein (see, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds.), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[1334] As long as the antibody and the other part retain their respective activities, coupling can be achieved by any chemical reaction that binds the two molecules. This connection can include many chemical mechanisms, such as covalent binding, affinity binding, embedding, coordination binding and complexing. However, the preferred combination is covalent binding. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent linking agents can be used to couple protein molecules (such as antibodies of the present disclosure) to other molecules. For example, representative coupling agents can include organic compounds, such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene and hexamethylenediamine. This list is not intended to exhaustively list the various types of coupling agents known in the art, but rather examples of more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133: 1335-2549 (1984); Jansen et al., Immunological Reviews 62: 185-216 (1982); and Vitetta et al., Science 238: 1098 (1987).

[1335] Suitable linkers are described in the literature. (See, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), describing the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester)). See also U.S. Pat. No. 5,030,719, describing the use of halogenated acetylhydrazide derivatives coupled to antibodies via oligopeptide linkers. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog number (21558G); (iii) SPDP (succinimidyl-6-[3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., catalog number 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6-[3-(2-pyridyldithio)-propionamido] hexanoate (Pierce Chem. Co., catalog number 2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., catalog number 2165-G) conjugated to EDC. Chem. Co., catalog number 24510).

[1336] The joints described above contain components with different properties, so that the conjugates have different physicochemical properties. For example, the sulfo-NHS esters of alkyl carboxylates are more stable than the sulfo-NHS esters of aromatic carboxylates. The solubility of joints containing NHS esters is worse than that of sulfo-NHS esters. In addition, the joint SMPT contains a sterically hindered disulfide bond and can form a conjugate with enhanced stability. Disulfide bonds are generally more unstable than other bonds because disulfide bonds are cleaved in vitro, resulting in less available conjugates. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. When carbodiimide coupling (such as EDC) is used in combination with sulfo-NHS, the ester formed is more resistant to hydrolysis than a separate carbodiimide coupling reaction.

[1337] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies can be prepared by any suitable method, such as Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and described in U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[1338] Particularly useful liposomes can be generated by a reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of defined pore size to obtain liposomes having a desired diameter. Fab' fragments of antibodies of the present disclosure can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982).

[1339] Uses of anti-B7-H3 antibodies

[1340] It will be appreciated that administration of therapeutic entities according to the present disclosure will be administered with suitable carriers, excipients and other agents incorporated into formulations to provide improved transfer, delivery, tolerability, etc. A large number of suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, PA (1975)), especially Chapter 87 of Blaug, Seymour therein. These formulations include, for example, powders, pastes, ointments, pectins, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as Lipofectin TM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. Any of the foregoing mixtures may be suitable for treatment and therapy according to the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerated with the route of administration. See also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts." J Pharm Sci. 89(8):967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311 (1998) and references therein for additional information related to formulations, excipients, and carriers that are familiar to pharmaceutical chemists.

[1341] The therapeutic formulations of the present disclosure (including the conjugates of the present disclosure) are used to treat or alleviate symptoms associated with cancer, such as, by way of non-limiting example, leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung and bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, urinary bladder cancer, kidney cancer and renal pelvic cancer, oral cancer and pharyngeal cancer, uterine body cancer and / or melanoma. The present disclosure also provides methods for treating or alleviating symptoms associated with cancer. The treatment regimen may include identifying a subject, e.g., identifying a human patient suffering from (or at risk of developing) cancer using, e.g., standard methods.

[1342] The therapeutic formulations of the present disclosure (including conjugates of the present disclosure that recognize B7-H3 and, optionally, a second target) can be used to treat or alleviate symptoms associated with autoimmune and / or inflammatory diseases, such as B cell-mediated autoimmune and / or inflammatory diseases, including, by way of non-limiting example, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom's hypergammaglobulinemia, Sjögren's syndrome, multiple sclerosis (MS), and / or lupus nephritis.

[1343] The effectiveness of the treatment can be determined in association with any suitable method for diagnosing or treating a particular immune-related disorder.Amelioration of one or more symptoms of the immune-related disorder indicates that the conjugate has a clinical benefit.

[1344] Conjugates directed against targets such as B7-H3, tumor-associated antigens or other antigens can be used in methods related to the localization and / or quantification of these targets, such as for measuring the levels of these targets in appropriate physiological samples, for diagnostic methods, for protein imaging, etc. For example, conjugates containing antibody-derived antigen-binding domains specific for any of these targets or derivatives, fragments, analogs or homologs thereof can be used as pharmacologically active compounds (hereinafter referred to as "therapeutic agents").

[1345] The conjugates disclosed herein can be used to isolate specific targets using standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. The conjugates disclosed herein can be used diagnostically to monitor protein levels in tissues as part of a clinical testing program, for example, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically connecting) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125 I. 131 I. 35 S or 3 H.

[1346] The conjugates disclosed herein can be used as therapeutic agents. Such agents will generally be used to treat or prevent diseases or conditions associated with abnormal expression or activation of a given target in a subject. A conjugate formulation, preferably one with high specificity and high affinity for its target antigen, is administered to a subject and will generally have an effect due to its binding to the target. Administration of the conjugate can eliminate or inhibit or interfere with the signaling function of the target. Administration of the conjugate can eliminate or inhibit or interfere with the binding of the target to its naturally bound endogenous ligand.

[1347] The therapeutically effective amount of the conjugate of the present disclosure generally relates to the amount required to achieve the therapeutic goal. As noted above, this may be a binding interaction between the antibody and its target antigen, which in some cases interferes with the function of the target and / or the effect of the active agent conjugated to the antibody. In addition, the amount to be administered will depend on the binding affinity of the antibody to its specific antigen and / or the efficacy of the active agent, and will also depend on the rate at which the administered antibody is depleted from the free volume of other subjects to which it is administered. As a non-limiting example, the commonly used range of the therapeutically effective dose of the conjugate of the present disclosure can be from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Common dosing frequencies can be in the range of, for example, twice a day to once a week.

[1348] The conjugates of the present disclosure can be administered in the form of pharmaceutical compositions to treat a variety of diseases and disorders. The principles and considerations involved in preparing such compositions and guidance for component selection are provided in, for example, the following documents: Remington: The Science And Practice Of Pharmacy 19th Edition (Alfonso R. Gennaro et al., ed.) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[1349] The formulation may also contain more than one active compound necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may include an agent that enhances its function, such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are suitably present in combination in an amount effective to achieve the intended purpose.

[1350] The active ingredients can also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions.

[1351] Preparations to be used for in vivo administration are preferably sterile. This is readily achieved by filtration through sterile filtration membranes.

[1352] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT TM ) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins for shorter periods of time.

[1353] The conjugates according to the present disclosure can be used as agents for detecting the presence of a given target (or a protein fragment thereof) in a sample. In some embodiments, the conjugate contains a detectable label. The antibody can be polyclonal, or more preferably monoclonal. The whole antibody or its fragment (e.g., F ab , scFv or F (ab)2). The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present in a subject. Therefore, the use of the term "biological sample" includes blood and fractions or components of blood, including serum, plasma or lymph. That is, the detection methods of the present disclosure can be used to detect analyte mRNA, protein or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detecting analyte mRNA include northern hybridization and in situ hybridization. In vitro techniques for detecting analyte proteins include enzyme-linked immunosorbent assay (ELISA), western blotting, immunoprecipitation and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include southern hybridization. Procedures for performing immunoassays are described, for example, in the following literature: "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, JR Crowther (ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. In addition, in vivo techniques for detecting analyte proteins include introducing labeled anti-analyte conjugates into the subject. For example, antibodies can be labeled with radioactive markers, the presence and location of which in the subject can be detected by standard imaging techniques.

[1354] Pharmaceutical composition

[1355] In certain aspects, provided herein are pharmaceutical compositions comprising a compound, drug conjugate, or targeted drug conjugate of the present disclosure.

[1356] The antibody-drug conjugates can be used to transfer active agents to target cells of a subject to treat the subject using any suitable method of preparing the composition.In some aspects, the disclosure relates to compositions (eg, pharmaceutical compositions) comprising an antibody-drug conjugate as described herein.

[1357] The compositions and methods disclosed herein can be used to treat individuals in need. In certain embodiments, the individual is a mammal, such as a human or non-human mammal. When applied to an animal, such as a human, the composition or compound is preferably applied in the form of a pharmaceutical composition, which comprises, for example, a compound of the present disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art, and include, for example, aqueous solutions, such as water or physiologically buffered saline or other solvents or vehicles, such as glycols, glycerol, oils (such as olive oil) or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are applied to humans, particularly for invasive routes of administration (i.e., such as injections or implants that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to achieve delayed release of an agent or selective targeting of one or more cells, tissues or organs. Pharmaceutical compositions can be in dosage unit form, such as lyophilized agents, powders, solutions, injections, etc. for recovery.

[1358] A pharmaceutically acceptable carrier may contain a physiologically acceptable agent, which, for example, acts to stabilize a compound (such as a compound of the present disclosure), increase its solubility, or increase its absorption. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier (including a physiologically acceptable agent) depends, for example, on the route of administration of the composition. The formulation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (formulation) may also be a liposome or other polymer matrix, into which, for example, a compound of the present disclosure may be incorporated. For example, liposomes comprising phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively simple to prepare and administer.

[1359] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[1360] Pharmaceutical compositions (preparations) can be administered to a subject by any of a variety of routes of administration. For example, the compound can be simply dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found in, for example, U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896 and the patents cited therein.

[1361] The preparation can be conveniently present in unit dosage form and can be prepared by any suitable method in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated, the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect. In general, in one hundred percent, this amount will be in the range of about 1% to about 99% active ingredient, preferably about 5% to about 70%, most preferably about 10% to about 30%.

[1362] The methods for preparing these formulations or compositions include the step of bringing into association the active compound, such as a compound of the present disclosure, with a carrier and optionally one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[1363] As used herein, the phrases "parenteral administration" and "parenteral administration" mean modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise a combination of one or more active compounds with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions prior to use, which combination may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[1364] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). For example, proper fluidity can be maintained by the use of coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.

[1365] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. By including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc., it is possible to ensure that the effects of microorganisms are prevented. It is also possible to expect that isotonic agents, such as sugar, sodium chloride, etc., are included in the compositions. In addition, the extended absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[1366] In some cases, in order to prolong the effect of the drug, it is desirable to slow down the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material with poor water solubility. The absorption rate of the drug depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form can be accomplished by dissolving or suspending the drug in an oil vehicle.

[1367] Injectable depot forms are prepared by forming a microencapsulated matrix of the subject compound in a biodegradable polymer, such as polylactide-polyglycolide. Depending on the ratio of the drug to the polymer and the properties of the specific polymer used, the drug release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by embedding the drug in a liposome or microemulsion compatible with body tissues.

[1368] For use in the methods of the present disclosure, the active compound may be provided per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably 0.5% to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[1369] The method of introduction can also be provided by a refillable or biodegradable device. Regarding the controlled delivery of drugs (including protein biopharmaceuticals), various sustained release polymer devices have been developed and tested in vivo in recent years. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form implants to continuously release compounds at specific target sites.

[1370] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[1371] The selected dosage level will depend upon a variety of factors, including the activity of the specific compound or combination of compounds employed, or the esters, salts or amides thereof; the route of administration; the time of administration; the rate of excretion of the specific compound or compounds employed; the duration of the treatment; other drugs, compounds and / or materials used in combination with the specific compound or compounds employed; the age, sex, weight, prior medical history of illness, general health of the patient being treated; and like factors well known in the medical arts.

[1372] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe a therapeutically effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start the pharmaceutical composition or compound dosage at a level lower than the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. "Therapeutically effective amount" means the concentration of the compound sufficient to cause the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the subject's weight, sex, age and medical history. Other factors affecting the effective amount may include, but are not limited to, the severity of the patient's illness, the condition being treated, the stability of the compound, and (if necessary) another type of therapeutic agent administered with the compound of the present disclosure. A larger total dose can be delivered by multiple administrations of the agent. Many methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13th edition, 1814-1882, incorporated herein by reference).

[1373] In general, a suitable daily dose of the active compound used in the compositions and methods of the present disclosure will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above.

[1374] The patient receiving such treatment may be any animal in need thereof, including primates, particularly humans; and other mammals, such as horses, cattle, pigs, sheep, cats, and dogs; poultry; and pets in general.

[1375] In certain embodiments, the compounds of the present disclosure may be used alone or in combination with another type of therapeutic agent.

[1376] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[1377] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[1378] The composition can be prepared as a liquid solution or suspension in an injectable form. Solid forms suitable for injection can also be prepared as, for example, emulsions, or prepared with antibody-drug conjugates encapsulated in liposomes. Antibody-drug conjugates can be combined with a pharmaceutically acceptable carrier, including any carrier that does not induce the production of antibodies that are harmful to the subject receiving the carrier. Suitable carriers generally include large macromolecules that are slowly metabolized, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates, etc.

[1379] The composition can also contain diluents, such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc. can also be present therein. The composition can be administered parenterally by injection, wherein such injection can be subcutaneous injection or intramuscular injection. In some embodiments, the composition can be administered to a tumor. The composition can be inserted (e.g., injected) into a tumor. Additional preparations are suitable for other forms of administration, such as suppositories or oral administration. Oral compositions can be administered as solutions, suspensions, tablets, pills, capsules or sustained release formulations.

[1380] The composition can be administered in a manner compatible with the dosage and formulation. The composition preferably comprises a therapeutically effective amount of an antibody-drug conjugate. The dosage may vary depending on the subject to be treated, the subject's health and physical condition, the desired degree of protection, and other relevant factors. The exact amount of the active ingredient (e.g., antibody-drug conjugate) may depend on the doctor's judgment. For example, a therapeutically effective amount of an antibody-drug conjugate or a composition containing it may be administered to a patient suffering from cancer or a tumor to treat the cancer or tumor.

[1381] According to the antibody-drug conjugate of the present disclosure or a composition containing it can be administered in the form of a pharmaceutically acceptable salt thereof. In some embodiments, the antibody-drug conjugate according to the present invention or a composition containing it can be administered together with a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable additive. The effective amount and type of pharmaceutically acceptable salts, excipients and additives can be measured using standard methods (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition, 1990).

[1382] In some embodiments, the disclosure relates to a method of treating cancer in a subject, the method comprising administering to the subject a pharmaceutical composition comprising an antibody-drug conjugate as described herein. In preferred embodiments, the subject is a mammal. For example, the subject can be selected from a rodent, a lagomorph, a cat, a dog, a pig, a sheep, a cow, a horse, and a primate. In certain preferred embodiments, the subject is a human.

[1383] The conjugates of the present disclosure (also referred to herein as "active compounds") and their derivatives, fragments, analogs and homologues can be incorporated into pharmaceutical compositions suitable for administration. Such compositions generally comprise a conjugate and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. that are compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in the art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous vehicles (such as fixed oils) can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, it is expected to be used in the composition. Supplementary active compounds can also be incorporated into the composition.

[1384] The pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal and subcutaneous administration. Solutions or suspensions for parenteral, intradermal or subcutaneous application may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates or phosphates; and agents for adjusting tension, such as sodium chloride or dextrose. pH can be adjusted with an acid or a base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes or multidose vials made of glass or plastic.

[1385] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid that is easy to inject. The composition must be stable under preparation and storage conditions and must be preserved for the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium, and the solvent or dispersion medium contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and their suitable mixtures. For example, it can be by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant to maintain appropriate fluidity. The effect of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal (thimerosal), etc. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols (such as mannitol, sorbitol), sodium chloride. The absorption of the injectable composition can be extended by including a delayed absorption agent in the composition, such as aluminum monostearate and gelatin.

[1386] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound into an appropriate solvent with a composition or combination of compositions listed above, followed by filtration sterilization as required. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other components required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze drying, which obtains a powder of the active ingredient and any additional desired components from its previously sterile filtered solution.

[1387] In certain embodiments, the active compound is prepared together with a carrier that will prevent the compound from being quickly eliminated from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The method for preparing such preparations will be apparent to those skilled in the art. These materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to a suitable method, for example, as described in U.S. Patent number 4,522,811.

[1388] It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and the inherent limitations in the art of compounding such active compounds for treating individuals.

[1389] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[1390] Treatment

[1391] The compounds and conjugates disclosed herein can be used in methods of inducing apoptosis.

[1392] Apoptosis dysregulation is associated with a variety of diseases, including, for example, autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, graft-versus-host disease, myasthenia gravis, or Sjögren's syndrome), chronic inflammatory disorders (e.g., psoriasis, asthma, or Crohn's disease), hyperproliferative disorders (e.g., breast cancer, lung cancer), viral infections (e.g., herpes, papilloma, or HIV), and other disorders, such as osteoarthritis and atherosclerosis. The compounds, conjugates, and compositions described herein can be used to treat or ameliorate any of these diseases. Such treatment generally involves administering to a subject suffering from the disease an amount of a compound, conjugate, or composition described herein sufficient to provide a therapeutic benefit. The nature of the antibody to the compound, conjugate, or composition administered will depend on the disease being treated - thus the antibody should bind to a cell surface antigen expressed in a cell type whose inhibition would be beneficial. The therapeutic benefit achieved will also depend on the specific disease being treated. In some cases, when administered as a monotherapy, the compounds and compositions disclosed herein can treat or ameliorate the disease itself or a symptom of the disease. In other cases, the compounds and compositions disclosed herein can be part of an overall treatment regimen that includes other agents that treat or improve the disease or symptoms of the disease being treated together with the inhibitor or the compounds and compositions disclosed herein. It will be apparent to those skilled in the art that agents useful for treating or improving specific diseases can be used in conjunction with or in conjunction with the compounds and compositions disclosed herein.

[1393] Although an absolute cure is always desired in any treatment regimen, achieving a cure does not necessarily provide a therapeutic benefit. A therapeutic benefit may include stopping or slowing the progression of a disease, causing the disease to regress but not cure, and / or improving or slowing the progression of disease symptoms. Prolonged survival and / or improved quality of life compared to a statistical mean may also be considered a therapeutic benefit.

[1394] A class of specific diseases involving apoptosis disorders and causing a significant health burden worldwide is cancer. In a specific embodiment, the compounds and compositions disclosed herein can be used to treat cancer. Cancer can be, for example, a solid tumor or a blood tumor. Cancers that can be treated with the compounds and compositions disclosed herein include, but are not limited to, bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T cell or B cell origin, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, myeloma, prostate cancer, small cell lung cancer and spleen cancer. The compounds and compositions disclosed herein may be particularly beneficial in treating cancer, because antibodies can be used to specifically target tumor cells, thereby potentially avoiding or improving undesirable side effects and / or toxicity that may be associated with systemic administration of unconjugated inhibitors. One embodiment relates to a method of treating a disease involving intrinsic apoptosis dysregulation, the method comprising administering to a subject suffering from a disease involving apoptosis dysregulation an amount of the compounds and compositions disclosed herein effective to provide a therapeutic benefit, wherein the ligand of the compounds and compositions disclosed herein binds to a cell surface receptor on a cell with intrinsic apoptosis dysregulation. One embodiment relates to a method of treating cancer, the method comprising administering to a subject suffering from cancer an amount of the compounds and compositions disclosed herein effective to provide a therapeutic benefit, wherein the ligand is capable of binding to a cell surface receptor or a tumor-associated antigen expressed on the surface of a cancer cell.

[1395] In the context of tumorigenic cancers, therapeutic benefit may specifically include, in addition to the effects discussed above, halting or slowing the progression of tumor growth, causing tumor growth regression, eradicating one or more tumors, and / or increasing patient survival compared to the statistical average for the type and stage of cancer being treated. In one embodiment, the cancer being treated is a tumorigenic cancer.

[1396] The compounds and conjugates disclosed herein can be administered as monotherapy to provide therapeutic benefit, or can be administered in adjunct to or with other chemotherapeutic agents and / or radiotherapy. Chemotherapeutic agents that can utilize the compounds and compositions disclosed herein as adjunct therapy can be targeted (e.g., ADC, protein kinase inhibitors, etc.) or non-targeted (e.g., non-specific cytotoxic agents, such as radionucleotides, alkylating agents, and intercalating agents). Non-targeted chemotherapeutic agents that can be adjunctively administered with the compounds and compositions disclosed herein include, but are not limited to, methotrexate, paclitaxel, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustard, Cytoxan, etoposide, 5-fluorouracil, BCN U, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin D, dactinomycin B, dactinomycin C, dactinomycin D ... mycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, calicheamicin, and docetaxel.

[1397] The compounds and conjugates disclosed herein that may not be effective as monotherapy for the treatment of cancer can be administered in adjunct to or in conjunction with other chemotherapeutic agents or radiotherapy to provide therapeutic benefit. One embodiment relates to a method in which the compounds or compositions disclosed herein are administered in an amount effective to sensitize tumor cells to standard chemotherapy and / or radiotherapy. Thus, in the context of treating cancer, "therapeutic benefit" includes administering the compounds and compositions disclosed herein as an adjunct to or in conjunction with chemotherapeutic agents and / or radiotherapy to patients who have not yet started such therapy or to patients who have developed but have not yet shown signs of resistance, or to patients who have begun to show signs of resistance, as a means of sensitizing tumors to chemotherapy and / or radiotherapy.

[1398] In some aspects, the present disclosure provides pharmaceutical compositions comprising an antibody drug conjugate as described herein, optionally further comprising a therapeutically effective amount of a chemotherapeutic agent.

[1399] In certain aspects, provided herein are methods of treating cancer comprising administering to a subject in need thereof one or more of the compounds, drug conjugates, targeted drug conjugates, or pharmaceutical compositions of the present disclosure.

[1400] In certain embodiments, the cancer is selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, urinary bladder cancer, kidney cancer, renal pelvis cancer, oral cancer, pharyngeal cancer, uterine corpus cancer, or melanoma.

[1401] In a further aspect, provided herein are methods of treating an autoimmune disease or an inflammatory disease, the methods comprising administering to a subject in need thereof one or more of the compounds, drug conjugates, targeted drug conjugates, or pharmaceutical compositions of the present disclosure.

[1402] In certain embodiments, the autoimmune or inflammatory disease is selected from a B cell-mediated autoimmune or inflammatory disease, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom's hypergammaglobulinemia, Sjögren's syndrome, multiple sclerosis (MS), or lupus nephritis.

[1403] Hereinafter, the configuration of the present disclosure will be described in detail by examples, but the following examples are only used to help understand the present disclosure. The scope of the present disclosure is not limited thereto. In addition, unless otherwise specifically described, the reagents, solvents and starting materials described in this specification can be easily obtained from commercial suppliers.

[1404] Example

[1405] Example 1: Preparation of Compound L-1

[1406]

[1407] Preparation of compound L-1-1

[1408] at -78°C in N 2 To a solution of dimethyl 5-hydroxyisophthalate (5 g, 23.79 mmol) in anhydrous THF (300 mL) was added LAH (3.6 g, 95.15 mmol) dropwise under anhydrous THF atmosphere. The reaction mixture was stirred at room temperature for 17 hours. After the reaction was complete, 15% NaOH solution (4 mL), H 2O (8 mL) and EA (100 mL), and then the reaction mixture was stirred for 1 hour. The mixture was filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-1-1 (3.02 g, 82%).

[1409] 1 H NMR (400MHz, DMSO-d6) δ9.21 (s, 1H), 6.66 (s, 1H), 6.58 (s, 2H), 5.07 (t, J = 6.0Hz, 2H), 4.38 (d, J = 4.6Hz, 4H)

[1410] Preparation of compound L-1-2

[1411] In N 2 Compound L-1-1 (2 g, 12.97 mmol) was dissolved in HBr (5.0 mL, 33% in AcOH) under atmosphere. After stirring at 60 °C for 18 hours, NaHCO 3 The reaction was quenched with a solution (pH about 8). Distilled water (50 mL) and EA (100 mL×2) were then added to the reaction mixture. 2 SO 4 The organic layer was dried, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-1-2 (2.9 g, 80%).

[1412] 1 H NMR (400 MHz, CDCl 3 )δ6.99(s,1H),6.81(s,2H),4.85(s,1H),4.41(s,2H).

[1413] Preparation of compound L-1-3

[1414] At room temperature in N 2 To a solution of compound L-1-2 (1.0 g, 3.57 mmol) in DCM (35 mL) was added TEA (0.45 mL, 3.21 mmol) under atmospheric pressure. SO was introduced via a balloon. 2 F 2 Gas, and stir the mixture at room temperature for 1 hour.Then wash the mixture with DCM (50mL) and add water (30mL).Wash the organic layer with NaHCO3 aqueous solution, dry over anhydrous Na2SO4, filter, and concentrate under reduced pressure.Purify the residue by column chromatography to obtain compound L-5 (941.7mg, 73%).

[1415] Preparation of compound L-1

[1416] At room temperature in N 2To a solution of compound L-1-2 (100 mg, 0.36 mmol) in anhydrous DCM (3 mL) was added imidazole (27 mg, 0.39 mmol) and TBDMS-Cl (59 mg, 0.39 mmol) under atmosphere. After stirring for 16 hours, distilled water (50 mL) and EA (100 mL) were added to the reaction mixture. 2 SO 4 The organic layer was dried, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-1 (110 mg, 79%).

[1417] 1 H NMR (400 MHz, CDCl 3 )δ7.00(s,1H),6.80(s,2H),4.41(s,4H),0.99(s,9H),0.21

[1418] Example 2: Preparation of Compound L-2

[1419]

[1420] Preparation of compound L-2-1

[1421] At room temperature in N 2 NaHCO 3 (3.8 g, 45.8 mmol, 2.0 equiv) and BOC 2 11-azido-3,6,9-trioxaundecane-1-amine (Aldrich, CAS 134179-38-7, 5.0 g, 22.9 mmol) in 1,4-dioxane (100 mL) and H 2 O (6.0 g, 27.5 mmol, 1.2 equiv) was treated with 1,4-dioxane (100 mL) and H 2 O (6.0 g, 27.5 mmol, 1.2 equiv). 2 O (25 mL) and then stirred for 6 hours. The reaction was quenched with water (50 mL) and extracted with DCM (100 mL×3). 2 SO 4 The organic layer was dried, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (1% to 3% MeOH / DCM) to obtain compound L-2-1 (7.2 g, 99%) as a colorless oil.

[1422] 1 H NMR (400 MHz, CDCl 3 )δ5.03(brs,1H),3.72-3.60(m,10H),3.98-3.52(m,1H),3.43-3.36(m,1H),3.35-3.24(m,1H),1.26(s,9H).

[1423] EI-MS m / z:319(M + +1).

[1424] Preparation of compound L-2-2

[1425] At 0°C in N 2 To a solution of compound L-8 (2 g, 6.282 mmol) in DMF (25 mL) was added sodium hydride (301 mg, 12.56 mmol, 60%) under N atmosphere. After 10 minutes, the mixture was stirred at the same temperature under N 2 Iodomethane (3.9 mL, 62.82 mmol) was added under N atmosphere. 2 The reaction was stirred for 3 hours under atmosphere. After completion of the reaction, the reaction mixture was quenched with 2N HCl (10 mL) and extracted with EA (500 mL×3). 2 SO 4 The organic layer was dried, filtered and concentrated under reduced pressure to yield compound L-2-2 (3.3 g, quantitative) as a yellow oil, which was used without further purification.

[1426] 1 H NMR (400 MHz, CDCl 3 )δ3.70-3.62(m,12H),3.4(t,J=5.2Hz,4H),2.91(s,3H),1.46(s,9H).

[1427] Preparation of compound L-2

[1428] At 0°C in N 2 Compound L-2-2 (3.3 g, 6.282 mmol) was added to CH 2 Cl 2 To the solution in 4% HCl (70 mL) was added 4N HCl in dioxane (25 mL). 2 The reactant was stirred under atmosphere for 1 hour. After the reaction was completed, the reaction mixture was concentrated under reduced pressure. Compound L-2 (1.8 g, 100%) was obtained as a yellow oil, which was used without further purification.

[1429] 1 H NMR (400 MHz, CDCl 3 )δ3.92(t,J=4.8Hz,2H),3.73-3.69(m,10H),3.45(t,J=5.2Hz,2H),3.22-3.16(m,2H),2.77(t,J=5.6Hz,3H),2.35(brs,1H).

[1430] Example 3: Preparation of Compound L-3

[1431]

[1432] Preparation of compound L-3-1

[1433] At 0°C in N 2 A solution of hexaethylene glycol (5.0 g, 18.0 mmol) in anhydrous THF (20 mL) was treated with 1M t-BuOK (9.4 ml, 4.9 mmol) and propargyl bromide (1.0 mL, 9.4 mmol) under atmosphere. The reaction mixture was allowed to warm to room temperature and stirred overnight. EA (50 mL×2), H 2 The mixture was extracted with 4% paraformaldehyde (20 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by chromatography to obtain compound L-3-1 (2.4 g, 80%).

[1434] 1 H NMR (400 Hz, CDCl 3 )δ4.21(s,2H),3.7-3.6(m,24H),3.05(brs,1H),2.43(s,1H).

[1435] Preparation of compound L-3-2

[1436] At room temperature in N 2 A clear solution of compound L-3-1 (4.23 g, 13.2 mmol) in anhydrous DCM (45 mL) was treated with TEA (4.78 mL, 34.32 mmol, 2.6 eq.) and pTs-Cl (5.03 g, 26.40 mmol, 2.0 eq.) under atmosphere and stirred overnight. The reaction was diluted with water (50 mL) and extracted with DCM (100 mL×2). 2 SO 4 The obtained organic layer was dried, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-3-2 (6.09 g, 97%) as a colorless oil.

[1437] 1 H NMR (400 Hz, CDCl 3 )δ7.80(d,J=8.4Hz,2H),7.35(d,J=8.8Hz,2H),4.22-4.20(m,J=,2H),4.16(t,J=4.8Hz,2H),3.93-3.58(m,22H),2.45(s,3H); EI-MS m / z: 475(M + +1).

[1438] Preparation of compound L-3-3

[1439] At room temperature in N 2 NaN 3 A clear solution of compound L-3-2 (6.09 g, 12.83 mmol) in DMF (45 mL) was treated with 4% paraformaldehyde (1.25 mg, 19.25 mmol, 1.5 equiv) and stirred overnight. The reaction was diluted with water (50 mL) and extracted with DCM (100 mL×3). 2 SO 4 The obtained organic layer was dried, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (HeX:EA=1:4) to obtain compound L-3-3 (3.52 g, 79%) as a slightly yellow oil.

[1440] 1 H NMR (400 Hz, CDCl 3 )δ4.23-4.22(m 2H),3.80-3.62(m,22H),3.44-3.38(m,2H),2.46-2.42(m,1H)

[1441] Preparation of compound L-3

[1442] At 0°C in N 2 A clear solution of compound L-3-3 (3.52 g, 10.19 mmol) in EA (24 mL), ether (24 mL) was treated with 5% HCl solution (48 mL), triphenylphosphine (3.47 g, 13.25 mmol) under atmospheric pressure and stirred overnight. 2 The mixture was diluted with 1% O (30 mL). The aqueous layer was extracted with DCM (100 mL×3). The aqueous phase was concentrated under high vacuum to obtain compound L-3 (2.73 g, 75%); EI-MS m / z: 320 (M + +1).

[1443] Example 4: Preparation of Compound L-4

[1444]

[1445] Preparation of compound L-4-1

[1446] At room temperature in N 2 KI (294 mg, 1.77 mmol), Ag 2A solution of hexaethylene glycol (5.0 g, 17.71 mmol) in anhydrous DCM (178 mL) was treated with 1H-D-O (4.92 g, 19.48 mmol), p-TsCl (3.7 g, 19.48 mmol) and stirred overnight. The reaction mixture was filtered and washed with DCM (100 mL) The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-4-1 (5.98 g, 73%).

[1447] 1 H NMR (400 Hz, CDCl 3 )δ7.80(d,J=8.4Hz,2H),7.35(d,J=8.4Hz,2H),4.16(t,J=4.8Hz,2H),3.71-3.58(m,22H),2.88(br,1H),2.45(s,3H).

[1448] Preparation of compound L-4-2

[1449] At room temperature in N 2 NaN 3 A solution of compound L-4-1 (5.98 g, 13.7 mmol) in DMF (30 mL) was treated with (1.34 g, 20.55 mmol) and stirred at 110° C. for 1 hour. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-4-2 (4.1 g, 97%).

[1450] 1 H NMR (400 Hz, CDCl 3 )δ3.72-3.60(m,22H),3.39(t,J=4.8Hz,2H),2.78(br,1H).

[1451] Preparation of compound L-4-3

[1452] At room temperature in N 2 A solution of compound L-4-2 (1.9 g, 6.18 mmol) in DCM (20 mL) was treated with triethylamine (2.0 mL, 14.22 mmol) and p-TsCl (2.4 g, 12.36 mmol) under atmosphere and stirred overnight. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-4-3 (2.58 g, 91%).

[1453] 1 H NMR (400 Hz, CDCl 3)δ7.80(d,J=8.4Hz,2H),7.35(d,J=8.4Hz,2H),4.16(t,J=4.8Hz,2H),3.70-3.61(m,16H),3.56(s,1H),3.39(t,J=4.8Hz,2H),2.45(s,3H).

[1454] EI-MS m / z:462(M + +1).

[1455] Preparation of compound L-4-4

[1456] At room temperature in H 2 A solution of compound L-4-2 (1.0 g, 3.25 mmol) in EtOH (5 mL) was treated with 5% Pd / C (1.04 g, 0.49 mmol) under atmospheric pressure and stirred for 4 hours. The mixture was filtered to remove Pd / C and concentrated under reduced pressure. The residue was dissolved in DCM (25 mL). BOC was added 2 O (852.1 mg, 3.9 mmol) and the resulting mixture was stirred at room temperature for 3 hours. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-4-4 (330 mg, 28%).

[1457] 1 H NMR (400 Hz, CDCl 3 )δ5.19(brs,1H),3.73(t,J=4.8Hz,2H),3.67(s,12H),3.63-3.60(m,6H),3.54(t,J=5.2Hz,2H),3.34-3.27(m,1H),1.44(s,9H).

[1458] EI-MS m / z:382(M + +1).

[1459] Preparation of compound L-4-5

[1460] At 0°C in N 2 Under atmosphere, a solution of compound L-4-4 (450 mg, 1.18 mmol) in anhydrous THF (10 mL) was treated with NaH (60% dispersion in mineral oil, 47.2 mg, 1.18 mmol) and stirred for 20 minutes. A solution of compound L-4-4 (544.5 mg, 1.18 mmol) was added thereto. The reactant was warmed to room temperature and stirred overnight. The reactant was cooled, quenched with MeOH (5 mL) and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-4-5 (582.9 mg, 74%).

[1461] Preparation of compound L-4

[1462] Compound L-4 was synthesized in a similar manner to the preparation method of compound L-3 in Example 3 (quantitative, colorless oil).

[1463] EI-MS m / z:645(M + +1).

[1464] Example 5: Preparation of Compounds Int-1 and Int-2

[1465]

[1466] Preparation of compound Int-1-1

[1467] At 0°C in N 2 To a solution of vanillic acid (50.0 g, 0.30 mol) in MeOH (700 mL) was added SOCl dropwise under atmosphere. 2 (207 mL, 2.85 mol). After stirring at room temperature for 15 h, saturated NaHCO 3 The reaction was adjusted to pH 7 to 8 with aqueous solution and then diluted with distilled water (100 mL) and EA (400 mL). 2 SO 4 The organic layer was dried, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-1-1 (54.2 g, quantitative).

[1468] 1 H NMR (400 MHz, CDCl 3 )δ7.64(dd,J=6.4,1.6Hz,1H),7.55(s,1H),6.94(d,J=8.4Hz,1H),6.05(s,1H),3.95(s,3H),3.89(s,3H).

[1469] Preparation of compound Int-1-2

[1470] At room temperature, K 2 CO 3 A solution of compound Int-1-1 (54.2 g, 0.30 mol) in DMF (200 mL) was treated with 1,4-dihydro-1,4-dihydro-2-nitropropene (61.6 g, 0.45 mol), benzyl bromide (39.0 mL, 0.33 mol) and stirred at 100 °C for 6 hours. The reaction mixture was cooled to room temperature and diluted with distilled water (100 mL) and EA (400 mL). 2 SO 4The organic layer was dried, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-1-2 (79.8 g, 98%).

[1471] 1 H NMR (400 MHz, CDCl 3 )δ7.60(dd,J=6.4,2.0Hz,1H),7.56(d,J=2.0Hz,1H),7.44-7.31(m,5H),6.89(d,J=8.4Hz,1H),5.22(s,2H),3.94(s,3H),3.88(s,3H).

[1472] Preparation of compound Int-1-3

[1473] At 0°C in N 2 To a solution of compound Int-1-2 (79.8 g, 0.29 mol) in acetic anhydride (550 mL) was added copper (II) nitrate hemi (pentahydrate) (75.0 g, 0.32 mol) in a solution of acetic anhydride (550 mL) under atmosphere and stirred at 0 ° C for 6 hours. The reaction mixture was quenched with ice water (800 mL). The solid was filtered and washed with distilled water (100 mL) and hexane (400 mL) to obtain compound Int-1-3 (85.5 g, 92%).

[1474] 1 H NMR (400 MHz, CDCl 3 )δ7.52(s,1H),7.45-7.35(m,5H),7.08(s,1H),5.22(s,2H),3.98(s,3H),3.91(s,3H).

[1475] Preparation of compound Int-1-4

[1476] To a solution of compound Int-1-3 (85.5 g, 0.27 mol) in THF (800 mL) and MeOH (300 mL) was added 2N NaOH (404 mL, 0.81 mol). After stirring at 65 ° C for 5 hours, the reactant was cooled to room temperature and adjusted to pH 2 by adding 2N HCl solution, and then extracted with distilled water (100 mL) and EA (300 mL×2). After anhydrous Na 2 SO 4 The organic layer was dried, filtered and concentrated under reduced pressure. The residual solid was collected and washed with hexane to obtain compound Int-1-4 (79.2 g, 97%).

[1477] 1H NMR (400MHz, DMSO-d6) δ7.69(s,1H),7.47-7.35(m,5H),7.03(s,1H),5.24(s,2H),3.91(s,3H).

[1478] Preparation of compound Int-1

[1479] At 0°C in N 2 To a solution of compound Int-1-4 (100 mg, 0.33 mmol) in anhydrous THF (500 μL) and anhydrous DCM (1.5 mL) was slowly added dropwise oxalyl chloride (42.4 μL) and 1 drop of DMF under atmosphere. After stirring for 30 minutes, the reaction mixture was concentrated under reduced pressure. Compound Int-1 was used directly in the next step without further purification.

[1480] Preparation of compound Int-1-5

[1481] At 0°C in N 2 To a solution of compound Int-1-3 (5.0 g, 15.8 mmol) in DCM (300 mL) was slowly added dropwise a solution of methanesulfonic acid (50 mL) in DCM (100 mL) under atmospheric pressure and stirred for 2 hours. 3 The reaction mixture was quenched with H 2 O (100 mL) was extracted. 2 SO 4 The organic layer was dried, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-1-5 (2.54 g, 71%)

[1482] 1 H NMR (400 MHz, CDCl 3 )δ7.48(s,1H),7.14(s,1H),6.05(s,1H),4.02(s,3H),3.89(s,3H).

[1483] Preparation of compound Int-1-6

[1484] A solution of compound Int-1-5 (2.0 g, 8.8 mmol) in 1,4-dioxane (28 ml) was treated with 6N NaOH solution (4.4 ml, 26.4 mmol) under N2 atmosphere and stirred at 40 ° C for 4 hours. The reaction mixture was cooled to 0 ° C and acidified with 2N HCl. The mixture was extracted with EA / H2O. The organic layer was dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure and dried in vacuo to obtain a white solid Int-1-6 (2.0 g, quantitative).

[1485] 1H NMR(400MHz,DMSO-d6)δ10.60(s,1H),7.305(s,1H),7.24(s,1H),3.89(s,3H)

[1486] Preparation of compound Int-2

[1487] In N 2 A solution of compound Int-1-6 (1.87 g, 8.77 mmol) in acetic anhydride (1.0 ml, 10.5 mmol) was treated with TEA (1.8 ml, 13.1 mmol) and DMAP (0.2 g, 1.75 mmol) under atmospheric pressure and stirred at room temperature for 3.5 hours. 2 The reaction mixture was extracted with anhydrous Na 2 SO 4 The organic layer was dried, filtered, concentrated under reduced pressure and dried in vacuo to obtain Int-2 as a white solid (2.2 g brown solid, 49%).

[1488] 1H NMR (400MHz, DMSO-d6): δ7.981(s,1H),7.451(s,1H),3.933(s,3H),2.294(s,3H).

[1489] Example 6: Preparation of Compound Int-TG1

[1490]

[1491] Preparation of compound Int-TG1

[1492] At 0°C in N 2 β-D-galactose pentaacetate (Alfa, CAS 4163-60-4, 5.0 g, 12.81 mmol) was dissolved in AcOH (20 mL) containing 33% HBr under an atmosphere. The mixture was warmed to room temperature. After stirring at room temperature for 4 hours, the mixture was concentrated under reduced pressure, and then EA (1000 mL) and saturated aqueous sodium bicarbonate solution (1000 mL) were added. The mixture was washed with anhydrous Na 2 SO 4 The organic layer was dried, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG1 (5.2 g, 99%).

[1493] 1 H NMR (400 Hz, CDCl 3)δ6.70(d,J=4.0Hz,1H),5.52(d,J=2.4Hz,1H),5.41(dd,J=7.6,2.8Hz,1H),5.05(d d, J=6.4, 4.0Hz, 1H), 4.49 (t, J=6.4Hz, 1H), 4.22-4.09 (m, 2H), 2.16-2.01 (m, 12H).

[1494] Example 7: Preparation of Compound Int-TG2

[1495]

[1496] Preparation of compound Int-TG2

[1497] Compound Int-TG2 was synthesized via a method similar to that described in Example 6.

[1498] Yield 80%

[1499] 1 H NMR (400 MHz, CDCl 3 )δ6.654(d,J=4.0Hz,1H),5.627(t,J=10.0Hz,1H),5.252(dd,J=10.4Hz,9.6Hz,1H),4.865(dd,J=1 0.0Hz,4.0Hz,1H),4.593(d,J=10.4Hz,1H),3.777(s,3H),2.113(s,3H),2.071(s,3H),2.065(s,3H)

[1500] Example 8: Preparation of Compound Int-TG3

[1501]

[1502] Preparation of compound Int-TG3-1

[1503] At room temperature in N 2 Ag in atmosphere 2 A solution of compound Int-TG1 (18.5 g, 45.0 mmol), 4-hydroxybenzaldehyde (5.0 g, 40.9 mmol), molecular sieves (10.0 g) in ACN (150 mL) was treated with 1% 4-nitropropane (38.0 g, 0.164 mol) and stirred for 3 hours. The reaction mixture was filtered through a celite pad and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG3-1 (16.0 g, 86%)

[1504] 1 H NMR (400 MHz, CDCl3 )δ9.93(s,1H),7.86(d,J=6.8Hz,2H),7.11(d,J=6.8Hz,2H),5.52-5.47(m,2H) ,5.18-5.14(m,2H),4.24-4.11(m,3H),2.19(s,3H),2.07(s,6H),2.02(s,3H).

[1505] Preparation of compound Int-TG3-2

[1506] At 0°C in N 2 NaBH 4 A solution of compound Int-TG3-1 (540 mg, 1.19 mmol) in anhydrous THF (15 mL) was treated with 4% paraformaldehyde (113 mg, 2.98 mmol) and stirred at 0°C for 10 minutes. After stirring at room temperature for 4 hours, the mixture was added with H 2 O and EA to dilute the reactants. 2 SO 4 The organic layer was dried, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (EA:HEX=1:1) to obtain compound Int-TG3-2 (430 mg, 79%).

[1507] 1 H NMR (400 MHz, CDCl 3 )δ7.30(d,J=8.8Hz,2H),6.99(d,J=8.8Hz,2H),5.51-5.54(m,2H),5.11(dd,J=10.8Hz,1H),5.03(d,J=8.0Hz,1H),4.65(d,J=5.6 2H)4.25-4.04(m,3H),2.19(s,3H),2.07(s,3H),2.06(s,3H),2.01(s,3H).

[1508] Preparation of compound Int-TG3

[1509] At room temperature in N 2 A solution of compound Int-TG3-2 (1.0 g, 2.2 mmol) in anhydrous DMF (6.0 ml) was treated with bis(pentafluorophenyl carbonate) (1.3 g, 3.3 mmol) under atmospheric pressure and stirred for 3 hours. EA (20 mL×2), H 2 The reaction mixture was extracted with anhydrous Na 2 SO 4 The organic layer was dried, filtered, and concentrated under reduced pressure. The reaction mixture was purified by column chromatography to obtain compound Int-TG3 (1.4 g, 98%).1 H NMR (400 MHz, CDCl 3 ): δ7.384(d,J=8.8Hz,2H),7.039(d,J=8.4Hz,2H),5.529-5.465(m,2H),5.280(s,2H),5.141 -5.068(m,2H),4.262-4.070(m,4H),2.195(s,3H),2.078(s,3H),2.073(s,3H),2.025(s,3H).

[1510] Example 9: Preparation of compounds Int-TG4 and Int-TG4a

[1511]

[1512] Compound Int-TG4-1 was synthesized via a similar method as described in Example 8 (yield 72%).

[1513] 1 H NMR (400 MHz, CDCl 3 )δ9.93(s,1H),7.86(d,J=6.8Hz,2H),7.11(d,J=6.8Hz,2H),5.52-5.47(m,2H) ,5.18-5.14(m,2H),4.24-4.11(m,3H),2.19(s,3H),2.07(s,6H),2.02(s,3H).

[1514] Preparation of compound Int-TG4-2

[1515] To a solution of compound Int-TG4-1 (2.06 g, 4.70 mmol) in DCM (50 mL) was added NaBH dropwise at 0 °C under N2 atmosphere. 4 (191 mg, 5.04 mmol) in methanol (50 mL) and stirred for 30 minutes. The reaction mixture was diluted with saturated aqueous ammonium chloride (263 mL), and the aqueous layer was extracted with dichloromethane (88 mL × 3) and ethyl acetate (88 mL × 3) in sequence. 2 SO 4 The organic layer was dried, filtered and concentrated under reduced pressure to give a white foam (2.02 g, 98%). The product was used in the next step without further purification to obtain compound Int-TG4-2.

[1516] 1 H NMR (400 MHz, CDCl 3)δ7.31(d,J=8.8Hz,2H),6.99(d,J=8.8Hz,2H),5.35-5.28(m,3H),5.13(d,J=7.2 Hz, 1H), 4.64 (d, J = 5.2Hz, 2H), 4.18-4.16 (m, 1H), 3.73 (s, 3H), 2.12-2.04 (m, 9H).

[1517] Preparation of compound Int-TG4

[1518] A solution of compound Int-TG4-2 (500 mg, 1.14 mmol) in DMF (7 mL) was treated with bis(PNP) (517 mg, 1.70 mmol), DIPEA (0.395 mL, 2.27 mmol) at room temperature under nitrogen atmosphere and stirred for 2 hours. 2 The reaction mixture was extracted with DCM. 2 SO 4 The organic layer was dried, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG4 (645 mg, 95%) as a white foam.

[1519] 1 H NMR (400 MHz, CDCl 3 )δ8.27(d,J=9.2Hz,2H),7.38-7.36(m,4H),7.03(d,J=8.0Hz,2H),5.36-5.28(m,3H), 5.24(s,2H),5.17(d,J=7.6Hz,1H),4.20-4.18(m,1H),3.73(s,3H),2.06-2.04(m,9H).

[1520] Preparation of compound Int-TG4a

[1521] Yield 98%

[1522] 1 H NMR (400 MHz, CDCl 3 )δ7.54(dd,J=8.8,4.8Hz,2H),7.03(dd,J=4.4,8.8Hz,2H),5.37-5.26(m,5H ), 5.18 (d, J = 6.8 Hz, 2H), 4.21-4.18 (m, 2H), 3.73 (s, 3H), 2.09-2.06 (m, 9H).

[1523] Example 10: Preparation of Compound Int-TG5

[1524]

[1525] Compound Int-TG5 was synthesized via a method similar to that described in Example 9.

[1526] Compound Int-TG5-1

[1527] Yield quantification

[1528] 1 H NMR (400 MHz, CDCl 3 )δ9.98(s,1H),8.31(d,J=2.0Hz,1H),8.07(dd,J=2.0Hz,8.8Hz,1H),7.48(d,J=8.8Hz,1H),5.59(dd,J=7.6Hz,10.4Hz,1H),5.49(d,J=3 .2Hz,1H),5.20(d,J=7.6Hz,1H),5.13(dd,J=3.6Hz,10.4Hz,1H),4.28-4.09(m,3H),2.20(s,3H),2.13(s,3H),2.10(s,3H),2.04(s,3H).

[1529] Compound Int-TG5-2

[1530] Yield 96%

[1531] 1 H NMR (400 MHz, CDCl 3 )δ7.81(d,J=2.0Hz,1H),7.52(dd,J=2.0Hz,8.8Hz,1H),7.35(d,J=8.8Hz,1H),5.54(dd,J=8.0Hz,10.4Hz,1H),5.47(d,J=3.2Hz,1H),5.10(d d,J=3.6Hz,10.4Hz,1H),5.05(d,J=8.0Hz,1H),4.73(d,J=6.0Hz,2H),4.28-4.04(m,3H),2.19(s,3H),2.13(s,3H),2.10(s,3H),2.02(s,3H).

[1532] Compound Int-TG5

[1533] Yield 84%

[1534] 11 H NMR (400 MHz, CDCl 3)δ7.89(d,J=2.4Hz,1H),7.60(dd,J=2.0Hz,8.4Hz,1H),7.41(d,J=8.4Hz,1H),5.56(dd,J=8.0Hz,10.4Hz,1H),5.48(d ,J=2.4Hz,1H),5.32(s,2H),5.13-5.09(m,2H),4.28-4.07(m,3H),2.19(s,3H),2.13(s,3H),2.04(s,3H),2.02(s,3H).

[1535] Example 11: Preparation of compounds Int-TG6 and Int-TG7

[1536]

[1537] Preparation of compound Int-TG6-1

[1538] At room temperature in N 2 Benzyl bromide (5.1 mL, 43.06 mmol) and NaHCO 3 A solution of 3-formyl-4-hydroxybenzoic acid (5 g, 43.06 mmol) in DMF (100 mL) was treated with 1% ethyl acetate (2.53 g, 43.06 mmol) and stirred overnight. The reaction mixture was extracted with EA (200 mL×2) and distilled water (100 mL). 2 SO 4 The obtained organic layer was dried, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG6-1 (2.56 g, 39%).

[1539] 1 H NMR (400 Hz, CDCl 3 )δ11.41(s,1H),9.95(s,1H),8.34(d,J=2.0Hz,1H),8.23(dd,J=6.4Hz,2.4Hz,1H),7.46-7.35(m,5H),7.04(d,J=9.2Hz,1H),5.37(s,2H).

[1540] Preparation of compound Int-TG6-2

[1541] At room temperature in N 2 Molecular sieves (8 g), Ag 2A solution of compound Int-TG6-1 (1.0 g, 3.90 mmol) and compound Int-TG1 (1.6 g, 3.90 mmol) in anhydrous ACN (30 mL) was treated with 1% CO (3.62 g, 15.61 mmol) and stirred for 1 hour. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG6-2 (2.1 g, 92%).

[1542] 1 H NMR (400 Hz, CDCl 3 )δ10.34(s,1H),8.55(d,J=2.0Hz,1H),8.26(dd,J=6.8,2.0Hz,1H),7.45-7.35(m,5H),7.17(d,J=8.8Hz,1H),5.63-5.60(m,1H),5.50 (d,J=3.6Hz,1H),5.37(s,2H),5.23(d,J=8.0Hz,1H),5.16(dd,J=7.2,3.6Hz,1H),4.24-4.10(m,4H),2.20(s,3H),2.10-2.03(m,9H).

[1543] Preparation of compound Int-TG6-3

[1544] At 0°C in N 2 A solution of compound Int-TG6-2 (2.1 g, 3.58 mmol) in DCM (30 mL) was treated with m-CPBA (2.65 g, 10.74 mmol) under atmosphere and stirred for 7 hours. The reaction mixture was quenched by adding saturated sodium bicarbonate (40 mL×2). The mixture was separated and the organic layer was washed with brine and then purified by Na 2 SO 4 Dry, filter, and concentrate under reduced pressure. 2 The residue was dissolved in chloroform (5 mL) under atmosphere and treated with hydrazine hydrate (261 μL, 5.37 mmol). After stirring for 1 hour, the reaction mixture was extracted with EA (30 mL×2) and 1 M aqueous HCl (10 mL) was added. 2 SO 4 The obtained organic layer was dried, filtered and concentrated under reduced pressure to obtain compound Int-TG6-3 (1.1 g, 55%).

[1545] EI-MS m / z:574(M + +Na)

[1546] Preparation of compound Int-TG6-4

[1547] At 0°C in N 2 The mixture was stirred at room temperature with TBDMS-OTf (224 μL, 0.97 mmol), Et 3 A solution of compound Int-TG6-3 (280 mg, 0.49 mmol) in DCM (5 mL) was treated with N (207 μL, 1.46 mmol) and stirred for 1.5 hours. The reaction mixture was quenched by adding citric acid (20 ml). The organic layer was washed with brine (20 mL) and washed with Na 2 SO 4 Dry, filter, and concentrate under reduced pressure.The residue was purified by column chromatography to obtain compound Int-TG6-4 (246.3 mg, 68%).

[1548] 1 H NMR (400 Hz, CDCl 3 )δ7.67(d,J=8.4Hz,1H),7.57(s,1H),7.44-7.34(m,5H),7.02(d,J=8.4 Hz,1H),5.49-5.44(m,2H),5.30(s,2H),5.19(d,J=7.6Hz,1H),5.10(dd, J=6.8,3.2Hz,1H)4.20-4.11(m,2H),4.05(t,J=6.8Hz,2H),2.19(s,3H), 2.04(s,3H),2.01(d,J=6.0Hz,6H),1.02(s,9H),0.20(d,J=15.6Hz,6H).

[1549] Preparation of compound Int-TG6-5

[1550] At room temperature in H 2 To a solution of compound Int-TG6-4 (283.2 mg, 0.41 mmol) in EA (5 mL) was added Pd / C (5%, 87.5 mg, 0.04 mmol). The mixture was stirred for 1 hour and Filtered and then concentrated under reduced pressure. Compound Int-TG6-5 was used directly in the next step without further purification (246 mg, quantitative).

[1551] 1 H NMR (400 Hz, CDCl 3)δ7.67(d,J=8.8Hz,1H),7.57(s,1H),7.05(d,J=8.4Hz,1H),5.49-5.45(m,2H),5.22(d,J=7.6Hz,1H),5.12(dd,J=7. 2,3.6Hz,1H)4.20-4.06(m,4H),2.19(s,3H),2.05(s,3H),2.02(d,J=7.6Hz,6H),1.01(s,9H),0.21(d,J=15.2Hz,6H).

[1552] Preparation of compound Int-TG6

[1553] A...

Claims

1. A drug conjugate comprising a compound represented by formula (VII) or formula (VIII) and a linker group: or a pharmaceutically acceptable salt thereof; in: A is a heterocycle; Each R a ' and R b ' is independently halogen, amino, hydroxy, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; Two R b 'optionally together form oxo or =CH2; or two R b 'Together with the intervening atoms, optionally form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group; R c 'is a sulfonate, sulfate, hydroxy, amino or thiol; R d ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; The prerequisite is that at least one R c ' is a sulfonate or sulfate, or at least one R d 'is-L"-Gly; R e ' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; m is an integer selected from 0-3; n is an integer selected from 0 to 8, as long as the valence permits; Ring Cy is selected from aryl, heteroaryl, cycloalkyl or heterocycloalkyl; is a single bond or a double bond; X' is a halogen; X” is -NR-, -S-, or -O-; R is hydrogen or alkyl; Each R a ” and R b " is independently halogen, amino, hydroxy, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or -(L"') r -X"-Gly; d is an integer selected from 0-4; r is an integer from 0 to 1; Each L'' is a bond or a linker, R e ” is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; p is an integer selected from 0-4; DBD is the DNA binding domain; L" is a bond or linker; and Gly is a monosaccharide, a disaccharide or an oligosaccharide.

2. The drug conjugate of claim 1, wherein each L'" is C 10 -C 100 The straight or branched, saturated or unsaturated alkylene moiety optionally contains one or more double and / or triple bonds.

3. The drug conjugate of claim 1, wherein each p and each d are independently an integer from 0 to 1.

4. The drug conjugate according to claim 1, wherein the compound is represented by formula (VII): or a pharmaceutically acceptable salt thereof.

5. The drug conjugate of any one of claims 1 to 4, wherein A is a 5- to 6-membered heterocycle.

6. The drug conjugate according to any one of claims 1 to 5, wherein R c ' is hydroxyl.

7. The drug conjugate according to any one of claims 1 to 6, wherein R d ' is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocycloalkyl, C 6-10 aryl or 5- to 10-membered heteroaryl.

8. The drug conjugate of claim 7, wherein R d 'It's L"-Gly.

9. The drug conjugate of claim 4, wherein the compound is selected from: or a pharmaceutically acceptable salt thereof; in Is a single bond or a double bond.

10. The drug conjugate according to any one of claims 1 to 9, wherein R a ' is halogen, amino, hydroxy, alkoxy, cyano, nitro, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 4- to 10-membered heterocycloalkyl, C 6-10 aryl or 5- to 10-membered heteroaryl.

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