Peptide linkers containing glycosides for antibody-drug conjugates

Abstract

The present disclosure provides antibody-drug conjugate structures comprising a cleavable linker connecting an antibody to a drug and having a first cleavable module and a second cleavable module that hinders cleavage of the first cleavable module. The disclosure also encompasses methods of production and methods of use of such conjugates.

Description

[0001] This application is a divisional application of an invention application with a filing date of January 22, 2020, Chinese application number 202080013223.5, and invention name “Glycoside-containing peptide linker for antibody-drug conjugates”.

[0002] Cross-reference to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 795,875, filed on January 23, 2019, the disclosure of which is incorporated herein by reference. Technical Field

[0004] The present disclosure relates to antibody-drug conjugates, and in particular to glycoside-containing peptide linkers of antibody-drug conjugates. Background Art

[0005] The field of protein-small molecule therapeutic conjugates has made great progress, providing a variety of clinically beneficial drugs and the promise of providing more in the coming years. Protein conjugate therapeutics can provide several advantages due to, for example, specificity, functional diversity and relatively low off-target activity, resulting in fewer side effects. Chemical modification of proteins can extend these advantages by making them more potent, stable, or multimodal. Summary of the invention

[0006] The present disclosure provides antibody-drug conjugate structures, which include a cleavable linker that connects the antibody to the drug and has a first cleavable moiety and a second cleavable moiety that hinders cleavage of the first cleavable moiety. The present disclosure also encompasses methods of producing such conjugates and methods of use.

[0007] Aspects of the disclosure include a conjugate comprising an antibody, a drug, and a cleavable linker that connects the antibody to the drug and has a first cleavable moiety and a second cleavable moiety that blocks cleavage of the first cleavable moiety.

[0008] In some embodiments, the first cleavable moiety is an enzymatically cleavable moiety and the second cleavable moiety is a chemically cleavable moiety.

[0009] In some embodiments, the first cleavable moiety is a chemically cleavable moiety and the second cleavable moiety is an enzymatically cleavable moiety.

[0010] In some embodiments, the first cleavable moiety is a first enzyme cleavable moiety and the second cleavable moiety is a second enzyme cleavable moiety. For example, the first enzyme cleavable moiety may include a first peptide and the second enzyme cleavable moiety may include a second peptide. In some cases, the first enzyme cleavable moiety includes a peptide and the second enzyme cleavable moiety includes a glycoside.

[0011] In some embodiments, the conjugate is of formula (I):

[0012]

[0013] in

[0014] Z is CR 4 or N;

[0015] R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0016] R 2 and R 3 each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R 2 and R 3 Optionally linked in a ring to form a 5- or 6-membered heterocyclic group;

[0017] Each R 4 independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0018] Each R 5 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl;

[0019] Each R 6 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0020] k is an integer from 1 to 10;

[0021] R 7is selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0022] L 1 It is the first joint;

[0023] L 2 It is the second joint;

[0024] W 1 is a medicine; and

[0025] W 2 It's an antibody.

[0026] Where L 1 , R 6 or R 7 One of the moieties comprises a second cleavable module.

[0027] In some embodiments, k is 2, and the conjugate is of Formula (Ia):

[0028]

[0029] in

[0030] R 6’ or R 6” One of them comprises a second cleavable module, and R 6’ and R 6” The other of which is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0031] In some embodiments, the second cleavable moiety is an enzymatic cleavable moiety comprising a glycoside.

[0032] In some embodiments, the conjugate is of Formula (Ib):

[0033]

[0034] In some embodiments, the conjugate is of formula (Ic):

[0035]

[0036] In some embodiments, k is 2, and the conjugate is of formula (Id):

[0037]

[0038] in

[0039] R 6’ and R 6” are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

[0040] R 7 A second cleavable module is included.

[0041] In some embodiments, the second cleavable moiety is an enzymatic cleavable moiety comprising a glycoside.

[0042] In some embodiments, the conjugate is of Formula (Ie):

[0043]

[0044] In some embodiments, k is 2, and the conjugate is of formula (If):

[0045]

[0046] in

[0047] R 6’ and R 6” are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

[0048] R 8 A second cleavable module is included.

[0049] In some embodiments, the conjugate is of formula (Ig):

[0050]

[0051] In some embodiments, L 1 Include:

[0052] -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d-,

[0053] in

[0054] a, b, c and d are each independently 0 or 1;

[0055] T 1 , T 2 , T 3 and T 4 Each independently selected from a covalent bond, (C1-C 12 ) alkyl, substituted (C1-C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p ,-(CR 13 OH)m-, 4-amino-piperidine (4AP), acetal groups, hydrazines, disulfides, and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;

[0056] V 1 , V 2 , V 3 and V 4 Each is independently selected from the group consisting of: covalent bond, -CO-, -NR 15 -,-NR 15 (CH2) q -,-NR 15 (C6H4)-,-CONR 15 -,-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -,-NR 15 SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6;

[0057] Each R 13 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

[0058] Each R 15 independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0059] In some embodiments, L2 Include:

[0060] -(T 5 -V 5 ) e -(T 6 -V 6 ) f -(T 7 -V 7 ) g -(T 8 -V 8 ) h -,

[0061] in

[0062] e, f, g, and h are each independently 0 or 1;

[0063] T 5 , T 6 , T 7 and T 8 Each independently selected from a covalent bond, (C1-C 12 ) alkyl, substituted (C1-C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p ,-(CR 13 OH)m-, 4-amino-piperidine (4AP), acetal groups, hydrazines, disulfides, and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;

[0064] V 5 , V 6 , V 7 and V 8 Each is independently selected from the group consisting of: covalent bond, -CO-, -NR 15 -,-NR 15 (CH2) q -,-NR 15 (C6H4)-,-CONR 15 -,-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -,-NR 15 SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6;

[0065] Each R 13 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

[0066] Each R 15 independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0067] In some embodiments,

[0068] T 1 Selected from (C1-C 12 ) alkyl and substituted (C1-C 12 )alkyl;

[0069] T 2 , T 3 and T 4 each independently selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, (AA) p ,-(CR 13 OH)m-, 4-amino-piperidin-(4AP), an acetal group, a hydrazine, and an ester; and

[0070] V 1 , V 2 , V 3 and V 4 Each is independently selected from the group consisting of: covalent bond, -CO-, -NR 15 -,-NR 15 (CH2) q -,-NR 15 (C6H4)-,-CONR 15 -,-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -,-NR 15 SO2-, and -P(O)OH-; among which:

[0071] (PEG) n yes wherein n is an integer from 1 to 30;

[0072] EDA is an ethylenediamine module with the following structure: wherein y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine (4AP) is Each R 12 and R 15 are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl and substituted aryl, wherein any two adjacent R 12 The groups may be linked in a ring to form a piperazinyl ring; and

[0073] R 13 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl.

[0074] In some embodiments,

[0075] T 1 Yes (C1-C 12 ) alkyl and V 1 is -CO-;

[0076] T 2 Is 4AP and V 2 It is a covalent bond;

[0077] T 3 Yes (PEG) n And V 3 is -CO-; and

[0078] d is 0.

[0079] In some embodiments,

[0080] T 1 Yes (C1-C 12 ) alkyl and V 1 is -CO-;

[0081] T 2 Is 4AP and V 2 It is a covalent bond;

[0082] T 3 Yes (PEG) n And V 3 is -CONH-; and

[0083] T 4 is aryl or substituted aryl and V 4 It's -CO-.

[0084] In some embodiments,

[0085] T 5 is a covalent bond and V 5 is -CO-; and

[0086] f, g and h are 0.

[0087] In some embodiments, the drug is selected from auristatin, maytansine, and duocarmycin.

[0088] Aspects of the disclosure include a compound comprising a cleavable linker for attaching an antibody to a drug, wherein the cleavable linker comprises a first cleavable moiety and a second cleavable moiety that blocks cleavage of the first cleavable moiety.

[0089] In some embodiments, the first cleavable moiety is an enzymatically cleavable moiety and the second cleavable moiety is a chemically cleavable moiety.

[0090] In some embodiments, the first cleavable moiety is a chemically cleavable moiety and the second cleavable moiety is an enzymatically cleavable moiety.

[0091] In some embodiments, the first cleavable moiety is a first enzyme cleavable moiety and the second cleavable moiety is a second enzyme cleavable moiety. For example, the first enzyme cleavable moiety can be a first peptide and the second enzyme cleavable moiety can be a second peptide. In some cases, the first enzyme cleavable moiety includes a peptide and the second enzyme cleavable moiety includes a glycoside.

[0092] In some embodiments, the compound is of formula (II):

[0093]

[0094] in

[0095] Z is CR 4 or N;

[0096] R 2 and R 3 each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R 2 and R 3 Optionally linked in a ring to form a 5- or 6-membered heterocyclic group;

[0097] Each R 4independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0098] Each R 5 independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0099] Each R 6 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0100] k is an integer from 1 to 10;

[0101] R 7 is selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0102] L 1 It is the first joint;

[0103] L 2 is the second connector; and

[0104] W 1 It's medicine.

[0105] Where L 1 , R 6 or R 7 One of the moieties comprises a second cleavable module.

[0106] In some embodiments, k is 2, and the compound is of formula (IIa):

[0107]

[0108] in

[0109] R 6’ or R 6”One of them comprises a second cleavable module, and R 6’ and R 6” The other of which is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0110] In some embodiments, the second cleavable moiety is an enzymatic cleavable moiety comprising a glycoside.

[0111] In some embodiments, the compound is of formula (IIb):

[0112]

[0113] In some embodiments, the compound is of formula (IIc):

[0114]

[0115] In some embodiments, k is 2, and the compound is of formula (IId):

[0116]

[0117] in

[0118] R 6’ and R 6” are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

[0119] R 7 A second cleavable module is included.

[0120] In some embodiments, the second cleavable moiety is an enzymatic cleavable moiety comprising a glycoside.

[0121] In some embodiments, the compound is of formula (IIe):

[0122]

[0123] In some embodiments, k is 2, and the compound is of formula (IIf):

[0124]

[0125] in

[0126] R 6’ and R 6”are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

[0127] R 8 A second cleavable module is included.

[0128] In some embodiments, the compound is of formula (IIg):

[0129]

[0130] In some embodiments, L 1 Include:

[0131] -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -,

[0132] in

[0133] a, b, c and d are each independently 0 or 1;

[0134] T 1 , T 2 , T 3 and T 4 Each independently selected from a covalent bond, (C1-C 12 ) alkyl, substituted (C1-C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p ,-(CR 13 OH) m -, 4-amino-piperidine (4AP), acetal groups, hydrazines, disulfides, and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;

[0135] V 1 , V 2 , V 3 and V4 Each is independently selected from the group consisting of: covalent bond, -CO-, -NR 15 -,-NR 15 (CH2) q -,-NR 15 (C6H4)-,-CONR 15 -,-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -,-NR 15 SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6;

[0136] Each R 13 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

[0137] Each R 15 independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0138] In some embodiments, L 2 Include:

[0139] -(T 5 -V 5 ) e -(T 6 -V 6 ) f -(T 7 -V 7 ) g -(T 8 -V 8 ) h -,

[0140] in

[0141] e, f, g, and h are each independently 0 or 1;

[0142] T 5 , T 6 , T 7 and T 8 Each independently selected from a covalent bond, (C1-C 12 ) alkyl, substituted (C1-C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA)p ,-(CR 13 OH) m -, 4-amino-piperidine (4AP), acetal groups, hydrazines, disulfides, and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;

[0143] V 5 , V 6 , V 7 and V 8 Each is independently selected from the group consisting of: covalent bond, -CO-, -NR 15 -,-NR 15 (CH2) q -,-NR 15 (C6H4)-,-CONR 15 -,-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -,-NR 15 SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6;

[0144] Each R 13 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

[0145] Each R 15 independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0146] In some embodiments,

[0147] T 1 Selected from (C1-C 12 ) alkyl and substituted (C1-C 12 )alkyl;

[0148] T 2 , T 3 , and T 4 each independently selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (C1-C 12 ) alkyl, substituted (C1-C 12) alkyl, (AA) p ,-(CR 13 OH) m -, 4-amino-piperidine (4AP), an acetal group, a hydrazine, and an ester; and

[0149] V 1 , V 2 , V 3 and V 4 Each is independently selected from the group consisting of: covalent bond, -CO-, -NR 15 -,-NR 15 (CH2) q -,-NR 15 (C6H4)-,-CONR 15 -,-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -,-NR 15 SO2-, and -P(O)OH-;

[0150] in:

[0151] (PEG) n yes wherein n is an integer from 1 to 30;

[0152] EDA is an ethylenediamine module with the following structure: wherein y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine (4AP) is Each R 12 and R 15 are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl and substituted aryl, wherein any two adjacent R 12 The groups may be linked in a ring to form a piperazinyl ring; and

[0153] R 13 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl.

[0154] In some embodiments,

[0155] T 1 Yes (C1-C 12 ) alkyl and V 1 is -CO-;

[0156] T 2 Is 4AP and V 2 It is a covalent bond;

[0157] T 3 Yes (PEG) n And V3 is -CO-; and

[0158] d is 0.

[0159] In some embodiments,

[0160] T 1 Yes (C1-C 12 ) alkyl and V 1 is -CO-;

[0161] T 2 Is 4AP and V 2 It is a covalent bond;

[0162] T 3 Yes (PEG) n And V 3 is -CONH-; and

[0163] T 4 is aryl or substituted aryl and V 4 It's -CO-.

[0164] In some embodiments,

[0165] T 5 is a covalent bond and V 5 is -CO-; and

[0166] f, g and h are 0.

[0167] In some embodiments, the drug is selected from auristatins, maytansinoids, and duocarmycins.

[0168] Aspects of the present disclosure include a pharmaceutical composition comprising a conjugate of the present disclosure and a pharmaceutically acceptable excipient.

[0169] Aspects of the disclosure include a method comprising administering to a subject an effective amount of a conjugate of the disclosure.

[0170] Aspects of the disclosure include a method of treating cancer in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate of the disclosure, wherein the administration is effective to treat the cancer in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0171] Figure 1A A graph showing the serum stability over time of antibody-drug conjugates containing MMAE constructs according to embodiments of the present disclosure.

[0172] Figure 1BA graph showing the serum stability over time of an antibody-drug conjugate containing duocarmycin construct 66, according to an embodiment of the present disclosure.

[0173] Figure 2A A graph showing the in vitro efficacy of antibody-drug conjugates containing MMAE constructs against the NCI-N87 cell line in accordance with embodiments of the present disclosure.

[0174] Figure 2B A graph showing the in vitro efficacy of an antibody-drug conjugate containing duocarmycin construct 66 against the JeKo-1 cell line, in accordance with an embodiment of the present disclosure.

[0175] Figure 3 A graph showing the in vivo efficacy of anti-CD79b 13 antibody-drug conjugate and anti-CD79b 48 antibody-drug conjugate against JeKo-1 xenografts in mice, according to embodiments of the present disclosure. DETAILED DESCRIPTION

[0176] definition

[0177] Unless otherwise indicated, the following terms have the following meanings. Any undefined terms have their art-recognized meanings.

[0178] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, such as 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. For example, this term includes straight and branched hydrocarbon groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), tert-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0179] The term "substituted alkyl" refers to an alkyl group, as defined herein, in which one or more carbon atoms in the alkyl chain (excluding the C1 carbon atom) have been optionally replaced with a heteroatom (such as -O-, -N-, -S-, -S(O)- n-(wherein n is 0 to 2), -NR-(wherein R is hydrogen or alkyl)) replaced and having 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R b , wherein R' and R" may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclyl.

[0180] "Alkylene" refers to a divalent aliphatic hydrocarbon group preferably having 1 to 6 and more preferably 1 to 3 carbon atoms, which is straight chain or branched and optionally interrupted by one or more groups selected from the group consisting of: -O-, -NR 10 -,-NR 10 C(O)-,-C(O)NR 10 -, etc. For example, this term includes methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), and the like.

[0181] "Substituted alkylene" refers to an alkylene group having from 1 to 3 hydrogen atoms replaced with substituents as described with respect to carbon in the definition of "substituted" below.

[0182] The term "alkane" refers to alkyl and alkylene groups as defined herein.

[0183] The terms "alkylaminoalkyl", "alkylaminoalkenyl" and "alkylaminoalkynyl" refer to the group R'NHR"-, wherein R' is alkyl as defined herein and R" is alkylene, alkenylene or alkynylene as defined herein.

[0184] The term "alkaryl" or "aralkyl" refers to the groups -alkylene-aryl and -substituted alkylene-aryl, wherein alkylene, substituted alkylene and aryl are defined herein.

[0185] "Alkoxy" refers to the group -O-alkyl, wherein alkyl is as defined herein. For example, alkoxy includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, wherein alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0186] The term "substituted alkoxy" refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, wherein substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

[0187] The term "alkoxyamino" refers to the group -NH-alkoxy wherein alkoxy is defined herein.

[0188] The term "haloalkoxy" refers to the group alkyl-O- wherein one or more hydrogen atoms on the alkyl group have been replaced with a halo group and includes, by way of example, groups such as trifluoromethoxy.

[0189] The term "haloalkyl" refers to a substituted alkyl as described above, wherein one or more hydrogen atoms on the alkyl have been substituted with a halogen group. Examples of such groups include, but are not limited to, fluoroalkyl groups such as trifluoromethyl, difluoromethyl, trifluoroethyl, and the like.

[0190] The term "alkylalkoxy" refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl, wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

[0191] The term "alkylthioalkoxy" refers to the groups -alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substituted alkyl, wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

[0192] "Alkenyl" refers to a straight or branched chain hydrocarbon radical having 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably 1 to 2 sites of double bond unsaturation. For example, this term includes vinyl, allyl, and but-3-en-1-yl. Included within this term are cis and trans isomers or mixtures of these isomers.

[0193] The term "substituted alkenyl" refers to an alkenyl group as defined herein having from 1 to 5 substituents or from 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0194] "Alkynyl" refers to a straight or branched monovalent hydrocarbon radical having 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include ethynyl (-C≡CH) and propargyl (-CH2C≡CH).

[0195] The term "substituted alkynyl" refers to an alkynyl group as defined herein having from 1 to 5 substituents or from 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0196] "Alkynyloxy" refers to the group -O-alkynyl, wherein alkynyl is as defined herein. For example, alkynyloxy includes ethynyloxy, propynyloxy, and the like.

[0197] "Acyl" refers to the radicals HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-. C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein. For example, acyl includes the "acetyl" group CH3C(O)-.

[0198] "Acylamino" refers to the group -NR 20 C(O)alkyl, -NR 20 C(O) substituted alkyl, -NR 20 C(O)cycloalkyl, -NR 20 C(O)-substituted cycloalkyl, -NR 20 C(O)cycloalkenyl, -NR 20 C(O)-substituted cycloalkenyl, -NR 20 C(O)alkenyl, -NR 20 C(O) substituted alkenyl, -NR 20 C(O)alkynyl, -NR 20 C(O) substituted alkynyl, -NR 20 C(O)aryl, -NR 20 C(O) substituted aryl, -NR 20 C(O)heteroaryl, -NR 20 C(O) substituted heteroaryl, -NR 20 C(O)heterocyclyl, and -NR 20 C(O)-substituted heterocyclic group, wherein R 20 is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

[0199] The term "aminocarbonyl" or "aminoacyl" refers to the group -C(O)NR 21 R 22 , where R 21 and R 22is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl and wherein R 21 and R 22 and optionally linked together with the nitrogen to which it is attached to form a heterocyclyl or substituted heterocyclyl, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

[0200] "Aminocarbonylamino" refers to the group -NR 21 C(O)NR 22 R 23 , where R 21 , R 22 , and R 23 R is independently selected from hydrogen, alkyl, aryl or cycloalkyl, or wherein two R groups are linked to form a heterocyclyl.

[0201] The term "alkoxycarbonylamino" refers to the group -NRC(O)OR, wherein each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl, wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0202] The term "acyloxy" refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclyl-C(O)O-, wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0203] "Aminosulfonyl" refers to the group -SO2NR 21 R 22 , where R 21 and R 22 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, and wherein R 21 and R 22are optionally linked together with the nitrogen to which they are attached to form a heterocyclyl or substituted heterocyclyl, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl are as defined herein.

[0204] "Sulfonylamino" refers to the group -NR 21 S02R 22 , where R 21 and R 22 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl and wherein R 21 and R 22 and optionally joined together with the atoms to which they are attached to form a heterocyclyl or substituted heterocyclyl, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

[0205] "Ar" or "Ar" refers to a monovalent aromatic carbocyclic radical of 6 to 18 carbon atoms having a single ring (such as that found in phenyl) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthracenyl and indanyl) which may or may not be aromatic provided that the point of attachment is through an atom of the aromatic ring. For example, this term includes phenyl and naphthyl. Unless otherwise constrained by the definition of aryl substituents, such aryl radicals may be optionally substituted with 1 to 5 substituents or 1 to 3 substituents selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, Azide, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl and trihalomethyl.

[0206] "Aryloxy" refers to the group -O-aryl wherein aryl is as defined herein and includes, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups also as defined herein.

[0207] "Amino" refers to the group -NH2.

[0208] The term "substituted amino" refers to the group -NRR, wherein each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, with the proviso that at least one R is not hydrogen.

[0209] The term "azido" refers to the group -N3.

[0210] "Carboxy" or "carboxylate" refers to -CO2H or a salt thereof.

[0211] The term "carboxyl ester" or "carboxylalkyl" refers to the radicals -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-alkynyl, -C(O)O-substituted alkynyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cycloalkyl, -C(O)O-substituted cycloalkyl, -C(O)O-cycloalkenyl, -C(O)O-cycloalkyl, -C(O)O-substituted cycloalkyl, -C(O)O-cycloalkylene ... -C(O)O-substituted cycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocyclyl, and -C(O)O-substituted heterocyclyl, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

[0212] "(Carboxy ester)oxy" or "carbonate" refers to the radical -OC(O)O-alkyl, -OC(O)O-substituted alkyl, -OC(O)O-alkenyl, -OC(O)O-substituted alkenyl, -OC(O)O-alkynyl, -OC(O)O-substituted alkynyl, -OC(O)O-aryl, -OC(O)O-substituted aryl, -OC(O)O-cycloalkyl, -OC(O)O-substituted cycloalkyl, -OC(O)O-cycloalkenyl, -OC(O)O-substituted cycloalkenyl, -OC(O)O-heteroaryl, -OC(O)O-substituted heteroaryl, -OC(O)O-heterocyclyl, and -OC(O)O-substituted heterocyclyl, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

[0213] "Cyano" or "nitrile" refers to the group -CN.

[0214] "Cycloalkyl" refers to a cyclic alkyl group of 3 to 10 carbon atoms having a single ring or multiple rings including fused, bridged, and spiro ring systems. For example, examples of suitable cycloalkyl groups include adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. For example, such cycloalkyl groups include single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like; or multiple ring structures such as adamantyl, and the like.

[0215] The term "substituted cycloalkyl" refers to a cycloalkyl group having 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0216] "Cycloalkenyl" refers to a non-aromatic cyclic alkyl group of 3 to 10 carbon atoms having a single ring or multiple rings and having at least one double bond and preferably 1 to 2 double bonds.

[0217] The term "substituted cycloalkenyl" refers to a cycloalkenyl group having 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, keto, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0218] "Cycloalkynyl" refers to a non-aromatic cycloalkyl group of 5 to 10 carbon atoms having a single or multiple rings and having at least one triple bond.

[0219] "Cycloalkoxy" refers to an -O-cycloalkyl group.

[0220] "Cycloalkenyloxy" refers to an -O-cycloalkenyl group.

[0221] "Halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.

[0222] "Hydroxy" refers to the radical -OH.

[0223] "Heteroaryl" refers to an aromatic group having 1 to 15 carbon atoms (such as 1 to 10 carbon atoms) and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur in the ring. Such heteroaryl groups can have a single ring (such as pyridyl, imidazolyl or furanyl) or multiple condensed rings in the ring system (for example, in groups such as indolizinyl, quinolyl, benzofuran, benzimidazolyl or benzothiophenyl), wherein at least one ring in the ring system is aromatic. In order to meet the valence requirements, any heteroatom in such heteroaryl rings may or may not form a bond with H or a substituent group, such as an alkyl group or other substituents described herein. In certain embodiments, the nitrogen and / or sulfur ring atoms of the heteroaryl group are optionally oxidized to provide N-oxide (N→O), sulfinyl, or sulfonyl moieties. For example, this term includes pyridyl, pyrrolyl, indolyl, phenylthio, and furanyl. Unless otherwise constrained by the definition of heteroaryl substituents, such heteroaryl groups may be optionally substituted with 1 to 5 substituents, or 1 to 3 substituents selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, Azide, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl, and trihalomethyl.

[0224] The term "heteroaralkyl" refers to the group -alkylene-heteroaryl, wherein alkylene and heteroaryl are defined herein. For example, this term includes pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

[0225] "Heteroaryloxy" refers to an -O-heteroaryl group.

[0226] "Heterocycle", "heterocycloalkyl", and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple condensed rings (including fused, bridged, and spiro ring systems) and having from 3 to 20 ring atoms, including from 1 to 10 heteroatoms. These ring atoms are selected from nitrogen, sulfur, or oxygen, wherein in a fused ring system, one or more rings may be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclyl are optionally oxidized to provide N-oxides, -S(O)-, or -SO2- moieties. To meet valence requirements, any heteroatom in such heterocyclic rings may or may not be bonded to one or more H or one or more substituent groups, such as alkyl groups or other substituents described herein.

[0227] Examples of heterocyclic and heteroaryl groups include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indoline, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, Phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also known as thiomorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0228] Unless otherwise constrained by the definition of heterocyclic substituents, such heterocyclic radicals may be optionally substituted with 1 to 5 or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxyl, carboxyalkyl, thio Aryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0229] "Heterocyclyloxy" refers to the group -O-heterocyclyl.

[0230] The term "heterocyclylthio" refers to the group heterocyclyl-S-.

[0231] The term "heterocyclylene" refers to a diradical formed from a heterocycle as defined herein.

[0232] The term "hydroxyamino" refers to the group -NHOH.

[0233] "Nitro" refers to the radical -NO2.

[0234] "Oxo" refers to the (=O) atom.

[0235] "Sulfonyl" refers to the group -SO2-alkyl, -SO2-substituted alkyl, -SO2-alkenyl, -SO2-substituted alkenyl, -SO2-cycloalkyl, -SO2-substituted cycloalkyl, -SO2-cycloalkenyl, -SO2-substituted cycloalkenyl, -SO2-aryl, -SO2-substituted aryl, -SO2-heteroaryl, -SO2-substituted heteroaryl, -SO2-heterocyclyl, and -SO2-substituted heterocyclyl, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein. For example, sulfonyl includes methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0236] "Sulfonyloxy" refers to the radical -OSO2-alkyl, -OSO2-substituted alkyl, -OSO2-alkenyl, -OSO2-substituted alkenyl, -OSO2-cycloalkyl, -OSO2-substituted cycloalkyl, -OSO2-cycloalkenyl, -OSO2-substituted cycloalkenyl, -OSO2-aryl, -OSO2-substituted aryl, -OSO2-heteroaryl, -OSO2-substituted heteroaryl, -OSO2-heterocyclyl, and -OSO2-substituted heterocyclyl wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

[0237] "Sulfate" or "sulfate ester" refers to the group -O-SO2-OH, -O-SO2-O-alkyl, -O-SO2-O-substituted alkyl, -O-SO2-O-alkenyl, -O-SO2-O-substituted alkenyl, -O-SO2-O-cycloalkyl, -O-SO2-O-substituted cycloalkyl, -O-SO2-O-cycloalkenyl, -O-SO2-O-substituted cycloalkenyl, -O-SO2-O-aryl, -O-SO2- O-substituted aryl, -O-SO2-O-heteroaryl, -O-SO2-O-substituted heteroaryl, -O-SO2-O-heterocyclyl, and -O-SO2-O-substituted heterocyclyl, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

[0238] The term "aminocarbonyloxy" refers to the group -OC(O)NRR, wherein each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl, wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0239] "Thiol" refers to the group -SH.

[0240] "Thio" or the term "thioketo" refers to the atom (=S).

[0241] "Alkylthio" or the term "thioalkoxy" refers to the group -S-alkyl, wherein alkyl is as defined herein. In certain embodiments, the sulfur can be oxidized to -S(O)-. The sulfoxide can exist as one or more stereoisomers.

[0242] The term "substituted thioalkoxy" refers to the group -S-substituted alkyl.

[0243] The term "thioaryloxy" refers to the group aryl-S- wherein aryl is as defined herein including optionally substituted aryl groups as also defined herein.

[0244] The term "thioheteroaryloxy" refers to the group heteroaryl-S- wherein heteroaryl is as defined herein including optionally substituted aryl groups also as defined herein.

[0245] The term "thioheterocyclyl" refers to the group heterocyclyl-S- wherein heterocyclyl is as defined herein including optionally substituted heterocyclyl groups also as defined herein.

[0246] Outside the disclosure herein, the term "substituted" when used to modify a specified group or radical may also mean that one or more hydrogen atoms of the specified group or radical are each independently replaced with the same or different substituents as defined below.

[0247] In addition to the groups disclosed with respect to individual terms herein, unless otherwise specified, the group used to replace one or more hydrogens on a saturated carbon atom in the specified group or radical (any two hydrogens on a single carbon can be replaced with =O, =NR 70 , =N-OR 70 , =N2 or =S) is replaced by -R 60 , halogenated, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 , -SO2O - M + , -SO2OR 70 ,-OSO2R 70 , -OSO2O - M + , -OSO2OR 70 , -P(O)(O - )2(M + )2,-P(O)(OR 70 ) - M + , -P(O)(OR 70 )2,-C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)O - M + , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)O - M + , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and-NR 70 C(NR 70 )NR 80 R 80 , where R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each R 70 Independently hydrogen or R 60 ; Each R 80 Independence is R 70 , or, two R 80’ Together with the nitrogen atom to which it is bound, it forms a 5-, 6- or 7-membered heterocycloalkyl group, which may optionally include 1 to 4 identical or different additional heteroatoms selected from the group consisting of O, N and S, wherein N may be substituted with -H or C1-C3 alkyl; and each M + is a counterion with a net single positive charge. For example, each M + Can be independently an alkali metal ion, such as K + , Na + , Li + ; ammonium ions, such as + N(R 60 )4; or alkaline earth metal ions, such as [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5 (The subscript 0.5 means that one counterion of such divalent alkaline earth metal ions can be the ionized form of the compound of the present invention and the other is a typical counterion such as chloride, or two ionized compounds disclosed herein can serve as counterions of such divalent alkaline earth metal ions, or the doubly ionized compounds of the present invention can serve as counterions of such divalent alkaline earth metal ions.) As a specific example, -NR 80 R 80Intended to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl and N-morpholinyl.

[0248] Outside the disclosure herein, unless otherwise specified, a substituent for a hydrogen on an unsaturated carbon atom in a "substituted" alkene, alkynyl, aryl and heteroaryl group is -R 60 , halogenated, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO3 - M + , -SO3R 70 ,-OSO2R 70 , -OSO3 - M + ,-OSO3R 70 ,-PO3 -2 (M + )2,-P(O)(OR 70 ) - M + , -P(O)(OR 70 )2,-C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -CO2 - M + , -CO2R 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OCO2 - M + , -OCO2R 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2- M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and-NR 70 C(NR 70 )NR 80 R 80 , where R 60 , R 70 , R 80 and M + As previously defined, provided that in the case of a substituted alkene or alkyne, the substituent is not -O - M + , -OR 70 , -SR 70 , or -S - M + .

[0249] In addition to the groups disclosed with respect to individual terms herein, unless otherwise specified, a substituent for a hydrogen on a nitrogen atom in a "substituted" heteroalkyl or cycloheteroalkyl group is -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 , -S(O)2O - M + , -S(O)2OR 70 , -OS(O)2R 70 , -OS(O)2O - M + , -OS(O)2OR 70 , -P(O)(O - )2(M + )2,-P(O)(OR 70 ) - M + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70 , -C(S)R70 , -C(NR 70 )R 70 , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and-NR 70 C(NR 70 )NR 80 R 80 , where R 60 , R 70 , R 80 and M + As defined previously.

[0250] In addition to the disclosure herein, in certain embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0251] It is understood that, in all substituted groups defined above, polymers obtained by defining a substituent that itself has further substituents (e.g., a substituted aryl has a substituted aryl as a substituent, which itself is substituted with a substituted aryl, which is further substituted with a substituted aryl, etc.) are not intended to be included herein. In such cases, the maximum number of such substitutions is three. For example, the serial substitution of substituted aryls specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0252] Unless otherwise indicated, substituents not expressly defined herein are named by calling out the terminal portion of the functionality followed by the adjacent functionality proximal to the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0253] With respect to any group containing one or more substituents disclosed herein, it is to be understood that, of course, such groups do not contain any substitution or substitution pattern that is not sterically feasible and / or synthetically feasible. In addition, the subject compounds include all stereochemical isomers produced by the substitution of these compounds.

[0254] The term "pharmaceutically acceptable salt" means a salt that is acceptable for administration to a patient, such as a mammal (a salt with a counterion that has acceptable mammalian safety for a given dosage regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which are derived from various organic and inorganic counterions known in the art, and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functional group, include salts of organic or inorganic acids, such as hydrochloric acid or hydrochloride, hydrobromide, formates, tartrates, benzenesulfonates, methanesulfonates, acetates, maleates, oxalates, and the like.

[0255] The term "salt thereof" means a compound formed when a proton of an acid is replaced with a cation such as a metal cation or an organic cation, etc. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not necessary for salts of intermediate compounds that are not intended for administration to a patient. For example, salts of the compounds of the invention include those in which the compound is protonated by an inorganic or organic acid to form a cation, wherein the conjugate base of the inorganic or organic acid serves as the anionic component of the salt.

[0256] "Solvate" refers to a complex formed by the combination of solvent molecules with molecules or ions of a solute. The solvent can be an organic compound, an inorganic compound, or a mixture of the two. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

[0257] "Stereoisomers" refer to compounds that have the same atomic connectivity but different arrangements of their atoms in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[0258] "Tautomers" refer to alternative forms of molecules that differ only in the electronic bonding of atoms and / or the position of protons, such as enol-keto and imine-enamine tautomers; or tautomeric forms of heteroaryl groups containing an -N=C(H)-NH- ring atom arrangement, such as pyrazole, imidazole, benzimidazole, triazole, and tetrazole. One of ordinary skill in the art will recognize that other tautomeric ring atom arrangements are possible.

[0259] It should be understood that the term "or a salt or solvate or stereoisomer thereof" is intended to include all arrangements of salts, solvates and stereoisomers, such as solvates of pharmaceutically acceptable salts of stereoisomers of the subject compound.

[0260] "Pharmaceutically effective amount" and "therapeutically effective amount" refer to an amount of a compound sufficient to treat a specified condition or disease or one or more symptoms thereof and / or to prevent the occurrence of a disease or condition. When referring to a neoplastic proliferative disorder, a pharmaceutically or therapeutically effective amount includes an amount sufficient to cause tumor shrinkage or reduce the rate of tumor growth, etc.

[0261] "Patient" refers to human and non-human subjects, particularly mammalian subjects.

[0262] As used herein, the terms "treatment" or "treating" mean the treatment or management of a disease or medical condition in a patient, such as a mammal (particularly a human), including: (a) preventing the disease or medical condition from occurring, such as prophylactic treatment of a subject; (b) ameliorating the disease or medical condition, such as eliminating the disease or medical condition in a patient or causing regression of the disease or medical condition in a patient; (c) inhibiting the disease or medical condition, for example by slowing or arresting the occurrence of the disease or medical condition in a patient; or (d) alleviating the symptoms of the disease or medical condition in a patient.

[0263] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length. Unless otherwise specifically indicated, "polypeptide", "peptide", and "protein" may include genetically encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. This term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, proteins containing at least one N-terminal methionine residue (e.g., to promote production in recombinant host cells); proteins with immunological tags; and the like.

[0264] "Native amino acid sequence" or "parent amino acid sequence" are used interchangeably herein to refer to the amino acid sequence of a polypeptide prior to being modified to include modified amino acid residues.

[0265] The terms "amino acid analogs," "non-natural amino acids," and the like are used interchangeably and include amino acid-like compounds that are similar in structure and / or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include natural amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs that have the same stereochemistry as the naturally occurring D-form, as well as amino acid analogs of the L-form. In some cases, amino acid analogs share the backbone structure and / or side chain structure of one or more natural amino acids, but differ in one or more modified groups in the molecule. Such modifications may include, but are not limited to, substitution of atoms (such as N) for related atoms (such as S), addition of groups (such as methyl or hydroxyl, etc.) or atoms (such as Cl or Br, etc.), deletion of groups, substitution of covalent bonds (single bonds for double bonds, etc.), or combinations thereof. For example, amino acid analogs may include α-hydroxy acids and α-amino acids, etc.

[0266] The term "amino acid side chain" or "amino acid side chain" and the like may be used to refer to a substituent attached to the α-carbon of an amino acid residue (including natural amino acids, non-natural amino acids, and amino acid analogs). Amino acid side chains may also include amino acid side chains described in the context of modified amino acids and / or conjugates described herein.

[0267] The term "carbohydrate" and the like may be used to refer to monomeric units and / or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term sugar may be used to refer to smaller carbohydrates, such as monosaccharides, disaccharides. The term "carbohydrate derivative" includes compounds in which one or more functional groups of the carbohydrate of interest are substituted (replaced with any convenient substituent), modified (converted to another group using any convenient chemical conversion), or missing (e.g., eliminated or replaced with H). A variety of carbohydrates and carbohydrate derivatives are available and may be adapted for use in the subject compounds and conjugates.

[0268] The term "glycoside" or "glycosyl" refers to a sugar molecule or group that is bound to a module via a glycosidic bond. For example, the module to which the glycoside is bound can be a cleavable linker as described herein. Glycosidic bonds can connect glycosides to other modules via various types of bonds, such as, but not limited to, O-glycosidic bonds (O-glycosides), N-glycosidic bonds (glycosylamines), S-glycosidic bonds (thioglycosides), or C-glycosidic bonds (C-glycosides or C-glycosyls). In some cases, glycosides can be cleaved from the module to which they are attached, such as by chemically mediated hydrolysis or enzyme-mediated hydrolysis.

[0269] The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (such as bispecific antibodies), humanized antibodies, single-chain antibodies, chimeric antibodies, antibody fragments (such as Fab fragments), and the like. Antibodies are capable of binding to target antigens (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5 th Ed., Garland Publishing, New York). A target antigen may have one or more binding sites, also called epitopes, which are recognized by a complementarity determining region (CDR) formed by one or more variable regions of an antibody.

[0270] The term "natural antibody" refers to an antibody in which the heavy and light chains of the antibody have been produced and paired by the immune system of a multicellular organism. The spleen, lymph nodes, bone marrow, and serum are examples of tissues that produce natural antibodies. For example, antibodies produced by antibody-producing cells isolated from a first animal immunized with an antigen are natural antibodies.

[0271] The term "humanized antibody" or "humanized immunoglobulin" refers to a non-human (e.g., mouse or rabbit) antibody in which one or more amino acids (e.g., in a framework region, a constant region, or a CDR) have been replaced with an amino acid from a human antibody at the corresponding position. In general, a humanized antibody produces a reduced immune response in a human host compared to a non-humanized version of the same antibody. Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; PCT Publication WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). In certain embodiments, framework replacements are identified by modeling the interactions of the CDR and framework residues to identify framework residues that are important for antigen binding and by sequence comparison to identify unusual framework residues at specific positions (see, e.g., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)). Additional methods for humanizing antibodies that can be used in the present invention are described in U.S. Pat. Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417; 5,693,493; 5,558,864; 4,935,496; and 4,816,567, and PCT Publications WO 98 / 45331 and WO 98 / 45332. In certain embodiments, the subject rabbit antibodies may be humanized according to the methods outlined in US20040086979 and US20050033031. Thus, the antibodies described above may be humanized using methods well known in the art.

[0272] The term "chimeric antibody" refers to an antibody in which the light and heavy chain genes have been constructed, usually by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segments of the genes from a mouse monoclonal antibody can be linked to human constant segments, such as gamma 1 and gamma 3. An example of a therapeutic chimeric antibody is a hybrid protein consisting of a variable domain or antigen binding domain from a mouse antibody and a constant domain or effector domain from a human antibody, although domains from other mammalian species can be used.

[0273] The immunoglobulin light chain or heavy chain variable region of an immunoglobulin polypeptide is composed of a framework region (FR) interspersed with three hypervariable regions (also called "complementarity determining regions" or "CDRs"). The boundaries of the framework region and CDR have been defined (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., US Department of Health and Human Services, 1991). The framework region of an antibody, which is the combined framework region that constitutes the light chain and the heavy chain, is used to position and align the CDR. The CDR is primarily responsible for binding to the epitope of the antigen.

[0274] A "parent Ig polypeptide" is a polypeptide comprising an amino acid sequence that lacks a constant region with an aldehyde tag as described herein. The parent polypeptide may comprise a native sequence constant region, or may comprise a constant region with pre-existing amino acid sequence modifications (such as additions, deletions and / or substitutions).

[0275] As used herein, the term "isolated" is intended to describe a compound of interest in an environment different from that in which the compound naturally occurs."Isolated" is intended to include a compound in a sample that is substantially enriched for the compound of interest and / or partially or substantially purified.

[0276] As used herein, the term "substantially purified" refers to a compound that is removed from its native environment and is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or more than 98% free from other components with which it is naturally associated.

[0277] The term "physiological conditions" is intended to encompass those conditions that are compatible with living cells, such as temperature, pH, salinity, etc., primarily aqueous conditions that are compatible with living cells.

[0278] " reactive spouse " means with another reactive spouse specific reaction to produce the molecule or molecular module of reaction product.Exemplary reactive spouse comprises cysteine ​​or serine and formylglycine generating enzyme (FGE) of sulfatase motif, and they react to form the reaction product of the conversion aldehyde label containing cysteine ​​or serine in formylglycine (FGly) alternative motif.Other exemplary reactive spouse comprises aldehyde and " aldehyde reactive reactive spouse " of the fGly residue of conversion aldehyde label (such as reactive aldehyde group), it comprises aldehyde reactive group and part of interest, and reacts to form the polypeptide with the aldehyde label of modification of reaction product, wherein module of interest is conjugated to the polypeptide of modification via the fGly residue of modification.

[0279] "N-terminus" refers to the terminal amino acid residue in a polypeptide having a free amine group. The amine group in the non-N-terminal amino acid residue generally forms part of the covalent backbone of the polypeptide.

[0280] "C-terminus" refers to the terminal amino acid residue in a polypeptide having a free carboxyl group. The carboxyl group in non-C-terminal amino acid residues generally forms part of the covalent backbone of the polypeptide.

[0281] As used in reference to a polypeptide or an amino acid sequence of a polypeptide, "internal site" means a region of the polypeptide that is not at the N-terminus or the C-terminus.

[0282] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, which may of course vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be restrictive, as the scope of the present invention will only be limited by the appended claims.

[0283] Where a range of values ​​is provided, it is to be understood that, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of the range separated by one-tenth of the unit of the lower limit and any other specified value or intervening value within the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present invention, subject to any specifically excluded limits within the specified range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0284] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for simplicity may also be provided separately or in any suitable sub-combination. The present invention specifically encompasses and discloses herein all combinations of embodiments belonging to the present invention, just as each and every combination is disclosed individually and explicitly, to the extent that the combination covers the subject matter, for example, compounds as stable compounds (i.e., compounds that can be generated, separated, characterized, and tested for biological activity). In addition, the present invention also specifically encompasses and discloses herein all sub-combinations of various embodiments and elements thereof (e.g., elements of chemical groups listed in embodiments describing such variables), just as each and every such sub-combination is disclosed individually and explicitly herein.

[0285] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to the methods and materials described herein may also be used to implement or test the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and record methods and / or materials related to the cited publications.

[0286] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional elements. As such, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "only," "only" and the like in connection with reciting claim elements or using a "negative" limitation.

[0287] It will be appreciated that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination.

[0288] The publications discussed herein are provided solely because they were disclosed prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Furthermore, the dates of publications provided may differ from the actual publication dates, which may need to be independently confirmed.

[0289] Details

[0290] The present disclosure provides antibody-drug conjugate structures, which include a cleavable linker connecting an antibody to a drug. The cleavable linker includes a first cleavable module and a second cleavable module that hinders cleavage of the first cleavable module. The present disclosure also covers methods for producing such conjugates and methods of use.

[0291] Antibody-drug conjugates

[0292] The present disclosure provides conjugates, such as antibody-drug conjugates (ADCs). "Conjugate" means that a first module (such as an antibody) is stably associated with a second module (such as a drug or an active agent). For example, an antibody-drug conjugate includes a drug (such as a maytansinoid, auristatin or a duocarmycin active agent module) that is stably associated with another module (such as an antibody). "Stably associated" means that a module is bound to another module or structure under standard conditions. In certain embodiments, the first and second modules are bound to each other via one or more functional groups and covalent bonds. For example, one or more functional groups and covalent bonds may include a cleavable linker as described herein.

[0293] In certain embodiments, the conjugate is a polypeptide conjugate, which includes a polypeptide (such as an antibody) conjugated to a second module. In certain embodiments, the module conjugated to the polypeptide can be any of a variety of modules of interest, such as but not limited to detectable markers, drugs, water-soluble polymers, or for immobilizing the polypeptide to a film or a surface module. In certain embodiments, the conjugate is a drug conjugate, wherein the polypeptide is an antibody, so providing an antibody-drug conjugate. For example, the conjugate can be a drug conjugate, wherein the polypeptide is conjugated to a drug or an active agent module. For example, a drug or an active agent can be maytansinoid. "Maytansinoid", "maytansinoid module", "maytansinoid active agent module" and "maytansinoid alkaloid" refer to maytansinoid and its analogs and derivatives, and pharmaceutically active maytansinoid modules and / or portions thereof. The maytansinoid conjugated to the polypeptide can be any of a variety of maytansinoid alkaloid modules, such as but not limited to maytansinoid and its analogs and derivatives, as described herein (e.g., deacetyl maytansinoid). In other cases, the drug or active agent can be auristatin, or an analog or derivative thereof, or a pharmaceutically active auristatin module and / or a portion thereof. The auristatin conjugated to the polypeptide can be any of a variety of auristatin modules, such as but not limited to auristatin and its analogs and derivatives, as described herein. In other cases, the drug or active agent can be a duocarmycin, or an analog or derivative thereof, or a pharmaceutically active duocarmycin module and / or a portion thereof. The duocarmycin conjugated to the polypeptide can be any of a variety of duocarmycin modules, such as but not limited to duocarmycin and its analogs and derivatives, as described herein.

[0294] In certain embodiments, the conjugate is an antibody-drug conjugate, wherein the antibody and the drug are connected together by a joint. In some cases, the joint is a cleavable joint. A cleavable joint is a joint including one or more cleavable modules, wherein the cleavable module includes one or more keys that can dissociate under certain conditions, so that the cleavable joint is separated into two or more separable parts. For example, the cleavable module can include one or more covalent bonds, which can dissociate or break under certain conditions to separate the cleavable joint into two or more parts. As such, a cleavable joint can be included in an antibody-drug conjugate so that under suitable conditions, the cleavable joint is cut at the desired target of action of the drug to separate or release the drug from the antibody.

[0295] In some cases, the cleavable linker includes two cleavable modules, such as a first cleavable module and a second cleavable module. The cleavable module can be configured so that in order to separate or release the drug from the antibody at the desired target of action of the drug, the cleavage of the two cleavable modules is required. For example, the cleavage of the cleavable linker can be achieved by first cutting one of the two cleavable modules and then cutting the other of the two cleavable modules. In certain embodiments, the cleavable linker includes a first cleavable module and a second cleavable module that hinders the cleavage of the first cleavable module. "Hindering cleavage" means that the presence of the second cleavable module that has not been cut reduces the possibility of cutting the first cleavable module or substantially inhibits the cutting of the first cleavable module, so that the amount of cleavage of the cleavable linker is substantially reduced or the cleavage of the cleavable linker is prevented. For example, the presence of the second cleavable module that has not been cut can hinder the enzyme and / or chemical cleavage of the first cleavable module. The presence of the second cleavable module hinders the cleavage of the first cleavable module and then substantially reduces the amount of the drug released from the antibody or prevents the drug from being released from the antibody. For example, premature release of the drug from the antibody can be substantially reduced or prevented until the antibody-drug conjugate is at or near the desired target of the drug.

[0296] In some cases, because the second cleavable module hinders the cleavage of the first cleavable module, the cleavage of the cleavable linker can be achieved by first cleaving the second cleavable module and then cleaving the first cleavable module. The cleavage of the second cleavable module can reduce or eliminate the obstruction of the cleavage of the first cleavable module, thus allowing the cleavage of the first cleavable module. As described above, the cleavage of the first cleavable module can cause the cleavable linker to dissociate or separate into two or more parts to release the drug from the antibody-drug conjugate. In some cases, the cleavage of the first cleavable module does not substantially occur in the presence of the second cleavable module that has not been cleaved. Substantial means that about 10% or less of cleavage of the first cleavable moiety occurs in the presence of the uncleaved second cleavable moiety, such as about 9% or less, or about 8% or less, or about 7% or less, or about 6% or less, or about 5% or less, or about 4% or less, or about 3% or less, or about 2% or less, or about 1% or less, or about 0.5% or less, or about 0.1% or less of cleavage of the first cleavable moiety occurs in the presence of the uncleaved second cleavable moiety.

[0297] In other words, the second cleavable module can protect the first cleavable module from cleavage. For example, the presence of the second cleavable module that has not been cleaved can protect the first cleavable module from cleavage, thus substantially reducing or preventing the premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target of the drug. In this way, the cutting of the second cleavable module exposes the first cleavable module (e.g., deprotecting the first cleavable module), so that the first cleavable module is allowed to be cut, resulting in the cutting of the cleavable linker, which is then separated from or released from the antibody at the desired target of the drug, as described above. In some cases, the cutting of the second cleavable module exposes the first cleavable module to subsequent cutting, but the cutting of the second cleavable module itself does not lead to the cutting of the cleavable linker (i.e., the first cleavable module still needs to be cut in order to cut the cleavable linker).

[0298] The cleavable modules included in the cleavable linker can each be a chemical cleavable module or an enzyme cleavable module. For example, the first cleavable module can be a chemical cleavable module and the second cleavable module can be a chemical cleavable module. In other cases, the first cleavable module can be an enzyme cleavable module and the second cleavable module can be a chemical cleavable module, or the first cleavable module can be a chemical cleavable module and the second cleavable module can be an enzyme cleavable module. In some embodiments, the first cleavable module is a first enzyme cleavable module and the second cleavable module is a second enzyme cleavable module.

[0299] For example, the cleavable module can be a chemical cleavable module. The chemical cleavable module includes a cleavable module that can be cleaved in the presence of certain chemical conditions. In some cases, the chemical cleavable module includes one or more bonds that can dissociate in the presence of certain chemical conditions, so that the cleavable module is separated into two or more separable parts. For example, the chemical cleavable module can be cleaved in the presence of chemical conditions such as acidic conditions or alkaline conditions, which can cause the hydrolysis of the chemical cleavable module. In some cases, the chemical conditions under which the chemical cleavable module is cleaved can be present at the desired target of action, such as the desired target of action of the drug to be released from the antibody-drug conjugate. In some cases, the chemical conditions found at the desired cleavage site of the chemical cleavable module are not present in significant amounts in other regions, such as in whole blood, plasma or serum. As such, the cutting of the chemical cleavable module can be controlled so that substantial cutting occurs at the desired site of action, and cutting does not occur significantly in other regions or before the antibody-drug conjugate reaches the desired site of action.

[0300] In some cases, the cleavable module can be an enzyme cleavable module. As described above, the enzyme cleavable module is a cleavable module that can be separated into two or more parts via the enzymatic action of an enzyme. The enzyme cleavable module can be any cleavable module that can be cut via the enzymatic action of an enzyme, such as but not limited to peptides, glycosides, etc. In some cases, the enzyme that cuts the enzyme cleavable module is present at the desired target of action, such as the desired target of action of the drug to be released from the antibody-drug conjugate. In some cases, the enzyme that cuts the enzyme cleavable module is not present in significant amounts in other regions, such as in whole blood, plasma or serum. As such, the cutting of the enzyme cleavable module can be controlled so that substantial cutting occurs at the desired site of action, and cutting does not occur significantly in other regions or before the antibody-drug conjugate reaches the desired site of action.

[0301] For example, as described herein, the antibody-drug conjugates of the present disclosure can be used to treat cancer, such as for delivering cancer therapeutic drugs to the desired site of action where cancer cells are present. In some cases, enzymes (such as protease cathepsin B) can be biomarkers of cancers overexpressed in cancer cells. Overexpression of certain enzymes in cancer and thus positioning to specifically release drugs at the desired site of action (i.e., sites of cancer (and overexpressed enzymes)) can be used in the context of enzyme cleavable modules included in the cleavable linkers of the antibody-drug conjugates of the present disclosure. Thus, in some embodiments, the enzyme cleavable module is a cleavable module (e.g., peptide) that can be cleaved by an enzyme overexpressed in cancer cells. For example, the enzyme can be a protease cathepsin B. As such, in some cases, the enzyme cleavable module is a cleavable module (e.g., peptide) that can be cleaved by a protease, such as cathepsin B.

[0302] In certain embodiments, the enzyme cleavable module is a peptide. The peptide can be any peptide suitable for use in a cleavable linker and can be cleaved via the enzymatic action of an enzyme. Non-limiting examples of peptides that can be used as enzyme cleavable modules include, for example, Val-Ala, Phe-Lys, and the like. For example, the first cleavable module described above (i.e., a cleavable module protected from premature cleavage by a second cleavable module) may include a peptide. The presence of the uncut second cleavable module can protect the first cleavable module (peptide) from cleavage by a protease (e.g., cathepsin B), thereby substantially reducing or preventing the drug from being released prematurely from the antibody until the antibody-drug conjugate is located at or near the desired target of action of the drug. In some cases, one of the amino acid residues of the peptide constituting the first cleavable module includes a substituent, wherein the substituent includes a second cleavable module. In some cases, the second cleavable module includes a glycoside. In other embodiments, the second cleavable module may include a peptide. In other embodiments, the first cleavable module includes a peptide and the second cleavable module includes a peptide.

[0303] In some embodiments, the enzyme cleavable module is a sugar module, such as a glycoside (or glycosyl). In some cases, compared with a cleavable linker that does not include a glycoside, a glycoside can promote the increase in the hydrophilicity of the cleavable linker. A glycoside can be any glycoside suitable for use in a cleavable linker and can be cleaved via the enzymatic action of an enzyme. For example, a second cleavable module (i.e., a cleavable module that protects the first cleavable module from premature cleavage) can be a glycoside. In other embodiments, the first cleavable module may include a glycoside. In other embodiments, the first cleavable module includes a glycoside and the second cleavable module includes a glycoside. In other embodiments, the first cleavable module includes a peptide and the second cleavable module includes a glycoside. In other embodiments, the first cleavable module includes a glycoside and the second cleavable module includes a peptide.

[0304] Glycosides can be attached (covalently bonded) to cleavable linkers via glycosidic bonds. Glycosidic bonds can connect glycosides to cleavable linkers via various types of bonds, such as, but not limited to, O-glycosidic bonds (O-glycosides), N-glycosidic bonds (glycosylamines), S-glycosidic bonds (thioglycosides), or C-glycosidic bonds (C-glycosides or C-glycosyls). In some cases, the glycosidic bond is an O-glycosidic bond (O-glycoside). In some cases, the glycoside can be cut from the cleavable linker to which the glycoside is attached by an enzyme (e.g., via enzyme-mediated hydrolysis of the glycosidic bond). Glycosides can be removed or cut from the cleavable linker by any convenient enzyme that is capable of cutting (hydrolyzing) the glycosidic bond of the attached glycoside to the cleavable linker. An example of an enzyme that can be used to mediate the cutting (hydrolysis) of the glycosidic bond of the attached glycoside to the cleavable linker is β-glucuronidase. Other suitable enzymes can also be used to mediate the cutting (hydrolysis) of the glycosidic bond of the attached glycoside to the cleavable linker. In some cases, an enzyme is found at or near the desired site of action of the drug of the antibody-drug conjugate for mediating the cleavage (hydrolysis) of the glycosidic bond of the attached glycoside to the cleavable linker. For example, the enzyme can be a lysosomal enzyme found in a cell located at or near the desired site of action of the drug of the antibody-drug conjugate. In some cases, the enzyme is an enzyme found at or near the target site where the enzyme that mediates the cleavage of the first cleavable module is found.

[0305] In certain embodiments, the first cleavable moiety is an enzymatic cleavable moiety.

[0306] The module of interest (e.g., a drug or an active agent) can be conjugated to a polypeptide at any desired site of a polypeptide (e.g., an antibody). Thus, for example, the present disclosure provides a modified polypeptide having a module conjugated at a site near the C-terminus or C-terminus of a polypeptide. Other examples include a modified polypeptide having a module conjugated at a position near the N-terminus or N-terminus of a polypeptide. Examples also include a modified polypeptide having a module conjugated at a position between the C-terminus and the N-terminus of a polypeptide (e.g., at an internal site of a polypeptide). In the case where a modified polypeptide is conjugated to two or more modules, the above combination is also possible.

[0307] In certain embodiments, the conjugates of the present disclosure include drugs or active agents conjugated to amino acid residues of a polypeptide at the α-carbon of the amino acid residue. In other words, the conjugate includes a polypeptide in which the side chains of one or more amino acid residues in the polypeptide have been modified to attach to a drug or active agent (e.g., attached to a drug or active agent via a linker as described herein). For example, the conjugate includes a polypeptide in which the α-carbon of one or more amino acid residues in the polypeptide has been modified to attach to maytansin (e.g., attached to maytansin via a linker as described herein). In other cases, the conjugate includes a polypeptide in which the α-carbon of one or more amino acid residues in the polypeptide has been modified to attach to auristatin (e.g., attached to auristatin via a linker as described herein). In other cases, the conjugate includes a polypeptide in which the α-carbon of one or more amino acid residues in the polypeptide has been modified to attach to duocarmycin (e.g., attached to duocarmycin via a linker as described herein).

[0308] Embodiments of the present disclosure include conjugates in which a polypeptide is conjugated to one or more modules, such as 2 modules, 3 modules, 4 modules, 5 modules, 6 modules, 7 modules, 8 modules, 9 modules, or 10 or more modules. A module may be conjugated to a polypeptide at one or more sites in a polypeptide. For example, one or more modules may be conjugated to a single amino acid residue of a polypeptide. In some cases, a module is conjugated to an amino acid residue of a polypeptide. In other embodiments, two modules may be conjugated to the same amino acid residue of a polypeptide. In other embodiments, a first module is conjugated to a first amino acid residue of a polypeptide and a second module is conjugated to a second amino acid residue of a polypeptide. The above combination is also possible, for example, wherein a polypeptide is conjugated to a first module at a first amino acid residue and to two other modules at a second amino acid residue. Other combinations are also possible, such as, but not limited to, a polypeptide conjugated to a first and a second module at a first amino acid residue and to a third and a fourth module at a second amino acid residue, etc.

[0309] One or more amino acid residues conjugated to one or more modules in a polypeptide can be naturally occurring amino acids, non-natural amino acids, or combinations thereof. For example, a conjugate can include a module conjugated to a naturally occurring amino acid residue of a polypeptide. In other cases, a conjugate can include a module conjugated to a non-natural amino acid residue of a polypeptide. As described above, one or more modules can be conjugated to a polypeptide at a single natural or non-natural amino acid residue. As described herein, one or more natural or non-natural amino acid residues in a polypeptide can be conjugated to one or more modules. For example, two (or more) amino acid residues (e.g., natural or non-natural amino acid residues) in a polypeptide can each be conjugated to one or two modules so that multiple sites in the polypeptide are modified.

[0310] As described herein, a polypeptide may be conjugated to one or more modules. In certain embodiments, the module of interest is a chemical entity, such as a drug or a detectable marker. For example, a drug (e.g., maytansinoids, auristatins, or duocarmycins) may be conjugated to a polypeptide, or in other embodiments, a detectable marker may be conjugated to a polypeptide. Thus, for example, embodiments of the present disclosure include, but are not limited to, the following: a conjugate of a polypeptide and a drug; a conjugate of a polypeptide and a detectable marker; a conjugate of two or more drugs and a polypeptide; a conjugate of two or more detectable markers and a polypeptide; and the like.

[0311] In certain embodiments, polypeptide and module of interest are conjugated via coupling module.For example, polypeptide and module of interest can each be combined (for example covalently bonded) to coupling module, so polypeptide and module of interest (for example medicine, such as maytansinoid) are indirectly combined via coupling module.In some cases, coupling module includes hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compound, or derivative of hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compound.For example, a general scheme for coupling module of interest (for example maytansinoid) to polypeptide via hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling module is shown in general reaction scheme below.Hydrazino-indolyl and hydrazino-pyrrolo-pyridinyl coupling module are also referred to as hydrazino-iso-Pictet-Spengler (HIPS) coupling module and aza-hydrazino-iso-Pictet-Spengler (azaHIPS) coupling module respectively in this article.

[0312]

[0313] In the above reaction scheme, R comprises a moiety of interest (e.g., a drug or an active agent) conjugated to a polypeptide (e.g., conjugated to a polypeptide via a cleavable linker as described herein). As shown in the above reaction scheme, a polypeptide comprising a 2-formylglycine residue (fGly) reacts with a drug or an active agent that has been modified to comprise a coupling moiety (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety) to generate a polypeptide conjugate attached to the coupling moiety, thereby attaching the drug or active agent to the polypeptide via the coupling moiety.

[0314] As described herein, the moiety can be any of a variety of moieties, such as, but not limited to, a chemical entity, such as a detectable label or a drug. R' and R" can each independently be any desired substituent, such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z can be CR 21 , NR 22 , N, O or S, where R 21 and R 22 Each is independently selected from any of the substituents described above for R' and R".

[0315] Other hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling modules are also possible, as shown in the conjugates and compounds described herein. For example, the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling module can be modified to attach (e.g., covalently attach) to the joint. As such, embodiments of the present disclosure include hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling modules attached to a drug or an active agent via a joint. Various embodiments of the joints in which the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling modules can be coupled to a drug or an active agent are described in detail herein. For example, in some cases, the joint is a cleavable joint, such as the cleavable joint described herein.

[0316] In certain embodiments, polypeptide can be conjugated to module of interest, wherein polypeptide is modified before being conjugated to module of interest.Modified polypeptide can produce modified polypeptide containing one or more reactive groups suitable for being conjugated to module of interest.In some cases, polypeptide can be modified at one or more amino acid residues to provide one or more reactive groups suitable for being conjugated to module of interest (for example, including coupling module as described above, such as the module of hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling module).For example, polypeptide can be modified to include reactive aldehyde group (for example reactive aldehyde).Reactive aldehyde can be included in " aldehyde tag " or " ald tag ", as used in this article, " aldehyde tag " or " ald tag " refers to the amino acid sequence derived from the sulfatase motif (for example L (C / S) TPSR) containing 2-formylglycine residues (referred to as " FGly " in this article) that has been transformed into by the action of formylglycine generating enzyme (FGE).The FGly residue generated by FGE can also be referred to as " formylglycine ". In other words, the term "aldehyde tag" is used herein to refer to an amino acid sequence comprising a "converted" sulfatase motif (i.e., a sulfatase motif in which a cysteine ​​or serine residue has been converted to FGly by the action of FGE, such as L(FGly)TPSR). The converted sulfatase motif can be derived from an amino acid sequence comprising an "unconverted" sulfatase motif (i.e., a sulfatase motif in which a cysteine ​​or serine residue has not been converted to FGly by FGE, but is capable of conversion, such as an unconverted sulfatase motif having the sequence L(C / S)TPSR). As used in the context of a formylglycine generating enzyme (FGE) acting on a sulfatase motif, "conversion" refers to a biochemical modification in which a cysteine ​​or serine residue in a sulfatase motif is converted to a formylglycine (FGly) residue (e.g., Cys is converted to FGly, or Ser is converted to FGly). Additional aspects of aldehyde tags and their use in site-specific protein modification are described in US Pat. No. 7,985,783 and US Pat. No. 8,729,232, the disclosures of each of which are incorporated herein by reference.

[0317] In some cases, the modified polypeptide containing FGly residues can be conjugated to the module of interest by the reaction of FGly and a compound (e.g., a compound containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling module as described above). For example, a polypeptide containing FGly can be contacted with a drug containing a reactive spouse under conditions suitable for providing a drug conjugated to a polypeptide. In some cases, a drug containing a reactive spouse may include a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling module as described above. For example, a drug or an active agent may be modified to include a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling module. In some cases, a drug or an active agent is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl, such as via a joint, such as a cleavable joint as described in detail herein covalently attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl.

[0318] In certain embodiments, the conjugate of the present disclosure includes a polypeptide (such as an antibody) with at least one modified amino acid residue. The modified amino acid residue of the polypeptide can be coupled to a drug or an activating agent containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling module as described above. In certain embodiments, the modified amino acid residue of a polypeptide (such as an antibody) can be derived from a cysteine ​​or serine residue that has been transformed into a FGly residue as described above. In certain embodiments, the FGly residue is conjugated to a drug or an activating agent containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling module as described above to provide a conjugate of the present disclosure, wherein the drug or activating agent is conjugated to a polypeptide via a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling module. As used herein, the term FGly' refers to a modified amino acid residue coupled to a module of interest (such as a drug or an activating agent) in a polypeptide (such as an antibody).

[0319] In certain embodiments, the conjugate includes at least one modified amino acid residue as described herein, wherein the modified amino acid residue is attached to a linker as described herein (cleavable linker), which in turn is attached to a drug or active agent. For example, the conjugate may include at least one modified amino acid residue (FGly') as described above. In some embodiments, the conjugate is of formula (I):

[0320] in

[0321] Z is CR 4 or N;

[0322] R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0323] R 2 and R 3 each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R 2 and R 3 Optionally linked in a ring to form a 5- or 6-membered heterocyclic group;

[0324] Each R 4 independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0325] Each R 5 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl;

[0326] Each R 6 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0327] k is an integer from 1 to 10;

[0328] R 7 is selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0329] L 1 It is the first joint;

[0330] L 2 It is the second joint;

[0331] W 1 is a medicine; and

[0332] W 2 It's an antibody.

[0333] Where L 1 , R 6 or R 7 One of the moieties comprises a second cleavable module.

[0334] In certain embodiments of Formula (I), n is 2, and the conjugate is of Formula (Ia):

[0335]

[0336] in

[0337] R 6’ or R 6” One of them comprises a second cleavable module, and R 6’ and R 6” The other of which is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0338] For example, the conjugate may be a conjugate of formula (Ib):

[0339]

[0340] In other cases, the conjugate may be a conjugate of formula (Ic):

[0341]

[0342] In certain embodiments of Formula (I), k is 2, and the conjugate is of Formula (Id):

[0343]

[0344] in

[0345] R 6’ and R 6” are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

[0346] R 7 A second cleavable module is included.

[0347] For example, the conjugate may be a conjugate of formula (Ie):

[0348]

[0349] In certain embodiments of Formula (I), k is 2, and the conjugate is of Formula (If):

[0350]

[0351] in

[0352] R 6’ and R 6” are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

[0353] R 8 A second cleavable module is included.

[0354] For example, the conjugate may be a conjugate of formula (Ig):

[0355]

[0356] Substituents relating to conjugates of formula (I) are described in more detail below. Reference to formula (I) is intended to also encompass formulas (Ia), (Ib), (Ic),

[0357] (Id), (Ie), (If) and (Ig).

[0358] In certain embodiments, Z is CR 4 or N. In certain embodiments, Z is CR 4 In certain embodiments, Z is N.

[0359] In certain embodiments, R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 1 is hydrogen. In certain embodiments, R 1 is an alkyl or substituted alkyl group, such as C 1-6 Alkyl or substituted C 1-6 Alkyl, or C 1-4 Alkyl or substituted C 1-4 Alkyl, or C 1-3 Alkyl or substituted C 1-3 In certain embodiments, R 1 In certain embodiments, R 1 is alkenyl or substituted alkenyl, such as C 2-6 Alkenyl or substituted C 2-6 Alkenyl, or C 2-4 Alkenyl or substituted C2-4 Alkenyl, or C 2-3 Alkenyl or substituted C 2-3 In certain embodiments, R 1 is alkynyl or substituted alkynyl, such as C 2-6 Alkynyl or substituted C 2-6 Alkynyl, or C 2-4 Alkynyl or substituted C 2-4 Alkynyl, or C 2-3 Alkynyl or substituted C 2-3 In certain embodiments, R 1 is aryl or substituted aryl, such as C 5-8 Aryl or substituted C 5-8 In certain embodiments, R 1 is a heteroaryl or substituted heteroaryl, such as C 5-8 Heteroaryl or substituted C 5-8 In certain embodiments, R 1 is a cycloalkyl or substituted cycloalkyl group, such as C 3-8 Cycloalkyl or substituted C 3-8 Cycloalkyl, such as C 3-6 Cycloalkyl or substituted C 3-6 Cycloalkyl, or C 3-5 Cycloalkyl or substituted C 3-5 In certain embodiments, R 1 is a heterocyclic group or a substituted heterocyclic group, such as C 3-8 Heterocyclic or substituted C 3-8 Heterocyclic groups, such as C 3-6 Heterocyclic or substituted C 3-6 Heterocyclic group, or C 3-5 Heterocyclic or substituted C 3-5 Heterocyclic group.

[0360] In certain embodiments, R 2 and R 3 each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl,

[0361] substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R 2 and R 3The rings are optionally linked to form a 5- or 6-membered heterocyclic group.

[0362] In certain embodiments, R 2 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,

[0363] In certain embodiments, R 2 is hydrogen. In certain embodiments, R 2 is an alkyl or substituted alkyl group, such as C 1-6 Alkyl or substituted C 1-6 Alkyl, or C 1-4 Alkyl or substituted C 1-4 Alkyl, or C 1-3 Alkyl or substituted C 1-3 In certain embodiments, R 2 In certain embodiments, R 2 is alkenyl or substituted alkenyl, such as C 2-6 Alkenyl or substituted C 2-6 Alkenyl, or C 2-4 Alkenyl or substituted C 2-4 Alkenyl, or C 2-3 Alkenyl or substituted C 2-3 In certain embodiments, R 2 is alkynyl or substituted alkynyl. In certain embodiments, R 2 is alkoxy or substituted alkoxy. 2 is amino or substituted amino. In certain embodiments, R 2 is carboxyl or carboxyl ester. In certain embodiments, R 2 is acyl or acyloxy. In certain embodiments, R 2 is acylamino or aminoacyl. In certain embodiments, R 2 is an alkylamide or a substituted alkylamide. 2 In certain embodiments, R 2 is thioalkoxy or substituted thioalkoxy. 2 is aryl or substituted aryl, such as C 5-8 Aryl or substituted C 5-8In certain embodiments, R 2 is a heteroaryl or substituted heteroaryl, such as C 5-8 Heteroaryl or substituted C 5-8 In certain embodiments, R 2 is a cycloalkyl or substituted cycloalkyl group, such as C 3-8 Cycloalkyl or substituted C 3-8 Cycloalkyl, such as C 3-6 Cycloalkyl or substituted C 3-6 Cycloalkyl, or C 3-5 Cycloalkyl or substituted C 3-5 In certain embodiments, R 2 is a heterocyclic group or a substituted heterocyclic group, such as C 3-6 Heterocyclic or substituted C 3-6 Heterocyclic group, or C 3-5 Heterocyclic or substituted C 3-5 Heterocyclic group.

[0364] In certain embodiments, R 3 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl,

[0365] In certain embodiments, R 3 is hydrogen. In certain embodiments, R 3 is an alkyl or substituted alkyl group, such as C 1-6 Alkyl or substituted C 1-6 Alkyl, or C 1-4 Alkyl or substituted C 1-4 Alkyl, or C 1-3 Alkyl or substituted C 1-3 In certain embodiments, R 3 In certain embodiments, R 3 is alkenyl or substituted alkenyl, such as C 2-6 Alkenyl or substituted C 2-6 Alkenyl, or C 2-4 Alkenyl or substituted C 2-4 Alkenyl, or C 2-3 Alkenyl or substituted C 2-3 In certain embodiments, R3 is alkynyl or substituted alkynyl. In certain embodiments, R 3 is alkoxy or substituted alkoxy. 3 is amino or substituted amino. In certain embodiments, R 3 is carboxyl or carboxyl ester. In certain embodiments, R 3 is acyl or acyloxy. In certain embodiments, R 3 is acylamino or aminoacyl. In certain embodiments, R 3 is an alkylamide or a substituted alkylamide. 3 In certain embodiments, R 3 is thioalkoxy or substituted thioalkoxy. 3 is aryl or substituted aryl, such as C 5-8 Aryl or substituted C 5-8 In certain embodiments, R 3 is a heteroaryl or substituted heteroaryl, such as C 5-8 Heteroaryl or substituted C 5-8 In certain embodiments, R 3 is a cycloalkyl or substituted cycloalkyl group, such as C 3-8 Cycloalkyl or substituted C 3-8 Cycloalkyl, such as C 3-6 Cycloalkyl or substituted C 3-6 Cycloalkyl, or C 3-5 Cycloalkyl or substituted C 3-5 In certain embodiments, R 3 is a heterocyclic or substituted heterocyclic group, such as C 3-8 Heterocyclic or substituted C 3-8 Heterocyclic groups, such as C 3-6 Heterocyclic or substituted C 3-6 Heterocyclic group, or C 3-5 Heterocyclic or substituted C 3-5 Heterocyclic group.

[0366] In certain embodiments, R 2 and R 3 Optionally, the rings are linked to form a 5- or 6-membered heterocyclic group. 2 and R 3 In some embodiments, R 2 and R 3In some embodiments, R 2 and R 3 The rings are connected to form a 6-membered heterocyclic group.

[0367] In certain embodiments, each R 4 are independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy,

[0368] acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0369] Each R 4 The various possibilities are described in more detail below. In certain embodiments, R 4 is hydrogen. In certain embodiments, each R 4 is hydrogen. In certain embodiments, R 4 is a halogen, such as F, Cl, Br or I. In certain embodiments, R 4 is F. In certain embodiments, R 4 is Cl. In certain embodiments, R 4 is Br. In certain embodiments, R 4 is 1. In certain embodiments, R 4 is an alkyl or substituted alkyl group, such as C 1-6 Alkyl or substituted C 1-6 Alkyl, or C 1-4 Alkyl or substituted C 1-4 Alkyl, or C 1-3 Alkyl or substituted C 1-3 In certain embodiments, R 4 In certain embodiments, R 4 is alkenyl or substituted alkenyl, such as C 2-6 Alkenyl or substituted C 2-6 Alkenyl, or C 2-4 Alkenyl or substituted C 2-4 Alkenyl, or C 2-3 Alkenyl or substituted C 2-3 In certain embodiments, R 4 is alkynyl or substituted alkynyl. In certain embodiments, R 4 is alkoxy or substituted alkoxy. 4 is amino or substituted amino. In certain embodiments, R 4is carboxyl or carboxyl ester. In certain embodiments, R 4 is acyl or acyloxy. In certain embodiments, R 4 is acylamino or aminoacyl. In certain embodiments, R 4 is an alkylamide or a substituted alkylamide. 4 In certain embodiments, R 4 is thioalkoxy or substituted thioalkoxy. 4 is aryl or substituted aryl, such as C 5-8 Aryl or substituted C 5-8 In certain embodiments, R 4 is a heteroaryl or substituted heteroaryl, such as C 5-8 Heteroaryl or substituted C 5-8 In certain embodiments, R 4 is a cycloalkyl or substituted cycloalkyl group, such as C 3-8 Cycloalkyl or substituted C 3-8 Cycloalkyl, such as C 3-6 Cycloalkyl or substituted C 3-6 Cycloalkyl, or C 3-5 Cycloalkyl or substituted C 3-5 In certain embodiments, R 4 is a heterocyclic or substituted heterocyclic group, such as C 3-8 Heterocyclic or substituted C 3-8 Heterocyclic groups, such as C 3-6 Heterocyclic or substituted C 3-6 Heterocyclic group, or C 3-5 Heterocyclic or substituted C 3-5 Heterocyclic group.

[0370] In certain embodiments, each R 5 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. In certain embodiments, R 5 is hydrogen. In certain embodiments, R 5 is an alkyl or substituted alkyl group, such as C 1-6 Alkyl or substituted C 1-6 Alkyl, or C 1-4 Alkyl or substituted C 1-4 Alkyl, or C 1-3 Alkyl or substituted C 1-3 In certain embodiments, R 5In certain embodiments, R 5 In certain embodiments, R 5 is propyl (eg, n-propyl or isopropyl). In certain embodiments, R 5 is butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). In certain embodiments, R 5 is pentyl (eg, n-pentyl or neopentyl, etc.). In certain embodiments, R 5 In certain embodiments, R 5 is alkenyl or substituted alkenyl, such as C 2-6 Alkenyl or substituted C 2-6 Alkenyl, or C 2-4 Alkenyl or substituted C 2-4 Alkenyl, or C 2-3 Alkenyl or substituted C 2-3 In certain embodiments, R 5 It is an alkynyl group or a substituted alkynyl group.

[0371] In certain embodiments, each R 6 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 6 is hydrogen. In certain embodiments, R 6 is an alkyl or substituted alkyl group, such as C 1-6 Alkyl or substituted C 1-6 Alkyl, or C 1-4 Alkyl or substituted C 1-4 Alkyl, or C 1-3 Alkyl or substituted C 1-3 In certain embodiments, R 6 In certain embodiments, R 6 In certain embodiments, R 6 is propyl (eg, n-propyl or isopropyl). In certain embodiments, R 6 is butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). In certain embodiments, R 6 is pentyl (eg, n-pentyl or neopentyl, etc.). In certain embodiments, R 6 In certain embodiments, R 6 is alkenyl or substituted alkenyl, such as C 2-6 Alkenyl or substituted C 2-6 Alkenyl, or C 2-4 Alkenyl or substituted C 2-4 Alkenyl, or C 2-3Alkenyl or substituted C 2-3 In certain embodiments, R 6 It is an alkynyl group or a substituted alkynyl group.

[0372] In certain embodiments, R 6 is aryl or substituted aryl, such as C 5-8 Aryl or substituted C 5-8 In certain embodiments, R 6 is a heteroaryl or substituted heteroaryl, such as C 5-8 Heteroaryl or substituted C 5-8 In certain embodiments, R 6 is a cycloalkyl or substituted cycloalkyl group, such as C 3-8 Cycloalkyl or substituted C 3-8 Cycloalkyl, such as C 3-6 Cycloalkyl or substituted C 3-6 Cycloalkyl, or C 3-5 Cycloalkyl or substituted C 3-5 In certain embodiments, R 6 is a heterocyclic or substituted heterocyclic group, such as C 3-8 Heterocyclic or substituted C 3-8 Heterocyclic groups, such as C 3-6 Heterocyclic or substituted C 3-6 Heterocyclic group, or C 3-5 Heterocyclic or substituted C 3-5 Heterocyclic group.

[0373] In certain embodiments, R 6 represents the side chain of an amino acid. For example, R 6 Can represent a substituent attached to the α-carbon of an amino acid residue (including natural amino acids, unnatural amino acids, and amino acid analogs). 6 represents the side chain of an amino acid found in a naturally occurring protein (e.g., the side chain of Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). In certain embodiments, R 6 represents the side chain of valine (Val); that is, R 6 In certain embodiments, R 6represents the side chain of alanine (Ala); that is, R 6 In certain embodiments, R 6 represents the side chain of phenylalanine (Phe); that is, R 6 is benzyl. In certain embodiments, R 6 represents the side chain of lysine (Lys); that is, R 6 It is 4-amino-butyl.

[0374] In certain embodiments, k is an integer from 1 to 10. In certain embodiments, k is 1. In certain embodiments, k is 2. In certain embodiments, k is 3. In certain embodiments, k is 4. In certain embodiments, k is 5. In certain embodiments, k is 6. In certain embodiments, k is 7. In certain embodiments, k is 8. In certain embodiments, k is 9. In certain embodiments, k is 10.

[0375] In certain embodiments, R 7 is selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino,

[0376] aminoacyl, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0377] In certain embodiments, R 7 is hydrogen. In certain embodiments, R 7 is a halogen, such as F, Cl, Br or I. In certain embodiments, R 7 is an alkyl or substituted alkyl group, such as C 1-6 Alkyl or substituted C 1-6 Alkyl, or C 1-4 Alkyl or substituted C 1-4 Alkyl, or C 1-3 Alkyl or substituted C 1-3 In certain embodiments, R 7 In certain embodiments, R 7 In certain embodiments, R 7 is propyl (eg, n-propyl or isopropyl). In certain embodiments, R 7 is butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). In certain embodiments, R 7 is pentyl (eg, n-pentyl or neopentyl, etc.). In certain embodiments, R 7 In certain embodiments, R7 is alkenyl or substituted alkenyl, such as C 2-6 Alkenyl or substituted C 2-6 Alkenyl, or C 2-4 Alkenyl or substituted C 2-4 Alkenyl, or C 2-3 Alkenyl or substituted C 2-3 In certain embodiments, R 7 It is an alkynyl group or a substituted alkynyl group.

[0378] In certain embodiments, R 7 is alkoxy or substituted alkoxy. 7 is amino or substituted amino. In certain embodiments, R 7 is carboxyl or carboxyl ester. In certain embodiments, R 7 In certain embodiments, R 7 In certain embodiments, R 7 In certain embodiments, R 7 In certain embodiments, R 7 It is an alkylamide or a substituted alkylamide.

[0379] In certain embodiments, R 7 is aryl or substituted aryl, such as C 5-8 Aryl or substituted C 5-8 In certain embodiments, R 7 is a heteroaryl or substituted heteroaryl, such as C 5-8 Heteroaryl or substituted C 5-8 In certain embodiments, R 7 is a cycloalkyl or substituted cycloalkyl group, such as C 3-8 Cycloalkyl or substituted C 3-8 Cycloalkyl, such as C 3-6 Cycloalkyl or substituted C 3-6 Cycloalkyl, or C 3-5 Cycloalkyl or substituted C 3-5 In certain embodiments, R 7 is a heterocyclic or substituted heterocyclic group, such as C 3-8 Heterocyclic or substituted C 3-8 Heterocyclic groups, such as C 3-6 Heterocyclic or substituted C 3-6 Heterocyclic group, or C 3-5 Heterocyclic or substituted C 3-5Heterocyclic group.

[0380] In certain embodiments, L 1 is the first connector. The following describes in more detail the 1 of the connector.

[0381] In certain embodiments, L 2 is the second connector. The following describes in more detail the 2 of the connector.

[0382] In certain embodiments, W 1 is a drug. For example, W 1 Can be maytansinoid. Other descriptions of maytansinoid are disclosed herein. In some cases, W 1 is auristatin. Auristatin is further described in the disclosure herein. In some cases, W 1 It is duocarmycin. Additional description of duocarmycin is provided in the disclosure herein.

[0383] In certain embodiments, W 2 is an antibody. Additional description of antibodies that can be used in the subject conjugates is found in the disclosure herein.

[0384] In certain embodiments, the compound of formula (I) includes one or more joints. The coupling module can be bound to one or more modules of interest and / or one or more polypeptides using a joint. In some embodiments, the coupling module is bound to any polypeptide or chemical entity (such as a drug) by a joint. The joint can be bound (e.g., covalently bonded) to a coupling module (e.g., as described herein) at any convenient position. For example, a joint can attach a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling module to a drug (e.g., maytansinoids or auristatins). A hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling module can be used to conjugate a joint (and thus a drug) to a polypeptide, such as an antibody. For example, a coupling module can be used to conjugate a joint (and thus a drug) to a modified amino acid residue of a polypeptide, such as an antibody's FGly residue.

[0385] In certain embodiments, the linker includes one or more linkers, such as a first linker L 1 and the second connector L 2 In addition, the linker can include one or more cleavable modules (e.g., a first cleavable module and a second cleavable module), as described herein. In some cases, the linker includes one or more linkers, such as a first linker L 1 and the second connector L 2For example, the linker may include a first linker (L) connecting a first cleavable moiety to a coupling moiety (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described herein). 1 ) and a second linker (L) connecting the first cleavable moiety to a chemical entity, such as a drug as described herein. 2 As such, the linker may comprise a first linker (L) that connects the first cleavable moiety to the antibody (e.g., via a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described herein). 1 ) and a second linker (L) connecting the first cleavable moiety to a chemical entity, such as a drug as described herein. 2 ).

[0386] For example, as shown in Formula (I) above, L 1 Attached to W via coupling module 2 , and thus W 2 Indirectly bonded to the first connector L via a coupling module 1 As described above, W 2 is an antibody, and thus L 1 Attached to the antibody via a coupling moiety, e.g., a first linker L 1 Indirectly bonded to the antibody via a coupling moiety (e.g., via a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described herein). In addition, as shown in Formula (I) above, L 1 (Indirectly) attached to L 2 , and L 2 Attached to W 1 As described above, W 1 is the drug, and thus the second connector L 2 Via the first connector L 1 The drug is attached to the antibody by using a coupling moiety (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described herein). 2 .

[0387] The first linker (L) in the subject conjugates and compounds may be 1 ) and the second connector (L 2 ) using any convenient linker. In certain embodiments, the first linker (L 1 ) and the second connector (L 2 ) each independently may include a member selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino,

[0388] In certain embodiments, the first linker (L) is selected from the group consisting of alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 1 ) and the second connector (L 2 ) each independently may comprise an alkyl or substituted alkyl group. In certain embodiments, the first linker (L 1 ) and the second connector (L 2 ) each independently may include an alkenyl or substituted alkenyl group. In certain embodiments, the first linker (L 1 ) and the second connector (L 2 ) each independently may include an alkynyl or substituted alkynyl group. In certain embodiments, the first linker (L 1 ) and the second connector (L 2 ) each independently may include an alkoxy or substituted alkoxy group. In certain embodiments, the first linker (L 1 ) and the second connector (L 2 ) each independently may include an amino group or a substituted amino group. In certain embodiments, the first linker (L 1 ) and the second connector (L 2 ) each independently may include a carboxyl group or a carboxyl ester group. In certain embodiments, the first linker (L 1 ) and the second connector (L 2 ) each may independently include an acylamino group. In certain embodiments, the first linker (L 1 ) and the second connector (L 2 ) each independently may comprise an alkylamide or substituted alkylamide group. In certain embodiments, the first linker (L 1 ) and the second connector (L 2 ) each independently may include an aryl or substituted aryl group. In certain embodiments, the first linker (L 1 ) and the second connector (L 2 ) each independently may include a heteroaryl or substituted heteroaryl group. In certain embodiments, the first linker (L 1 ) and the second connector (L 2 ) each independently may include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the first linker (L 1 ) and the second connector (L 2 ) each independently may include a heterocyclyl or substituted heterocyclyl group.

[0389] In certain embodiments, the first linker (L 1 ) and the second connector (L 2) Each can independently include a polymer. For example, the polymer can include polyalkylene glycol and its derivatives, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymer, polypropylene glycol homopolymer, copolymers of ethylene glycol and propylene glycol (e.g., wherein the homopolymer and copolymer are unsubstituted or substituted with an alkyl group at one end), polyvinyl alcohol, polyvinyl ethyl ether, polyvinyl pyrrolidone, combinations thereof, and the like. In certain embodiments, the polymer is polyalkylene glycol. In certain embodiments, the polymer is polyethylene glycol. Other linkers are also possible, as shown in the conjugates and compounds described in more detail below.

[0390] In some embodiments, L 1 is through the formula -(L 11 ) a -(L 12 ) b -(L 13 ) c -(L 14 ) d - to describe the first joint, where L 11 , L 12 , L 13 and L 14 Each is independently a first linker subunit, and a, b, c and d are each independently 0 or 1, wherein the sum of a, b, c and d is 1-4.

[0391] In certain embodiments, the sum of a, b, c, and d is 1. In certain embodiments, the sum of a, b, c, and d is 2. In certain embodiments, the sum of a, b, c, and d is 3. In certain embodiments, the sum of a, b, c, and d is 4. In certain embodiments, a, b, c, and d are each 1. In certain embodiments, a, b, and c are each 1, and d is 0. In certain embodiments, a and b are each 1, and c and d are each 0. In certain embodiments, a is 1, and b, c, and d are each 0.

[0392] In certain embodiments, L 11 is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety (e.g., as shown in Formula (I) above). In certain embodiments, L 12 , if present, is attached to the first cleavable moiety. In certain embodiments,

[0393] L 13 , if present, is attached to the first cleavable moiety. In certain embodiments, L 14 , if present, attached to the first cleavable module.

[0394] At the first joint L 1Any convenient linker subunit is utilized in the invention. Linker subunits of interest include, but are not limited to, units of polymers such as polyethylene glycol, polyethylene and polyacrylates, amino acid residues, carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclyl groups, combinations thereof, and substitution patterns thereof. In some embodiments, L 11 , L 12 , L 13 and L 14 Each of (if present) comprises one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., linking groups comprising alkylenediamines).

[0395] In some embodiments, L 11 (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 11 In some embodiments, L 11 In some embodiments, L 11 In some embodiments, L 11 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 11 In some embodiments, L 11 A diamine-containing linking group (eg, an alkylene diamine-containing linking group) is provided.

[0396] In some embodiments, L 12 (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 12 In some embodiments, L 12 In some embodiments, L 12 In some embodiments, L 12 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 12 In some embodiments, L 12 A diamine-containing linking group (eg, an alkylene diamine-containing linking group) is provided.

[0397] In some embodiments, L 13(if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 13 In some embodiments, L 13 In some embodiments, L 13 In some embodiments, L 13 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 13 In some embodiments, L 13 A diamine-containing linking group (eg, an alkylene diamine-containing linking group) is provided.

[0398] In some embodiments, L 14 (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 14 In some embodiments, L 14 In some embodiments, L 14 In some embodiments, L 14 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 14 In some embodiments, L 14 A diamine-containing linking group (eg, an alkylene diamine-containing linking group) is provided.

[0399] In some embodiments, L 1 Is included-(L 11 ) a -(L 12 ) b -(L 13 ) c -(L 14 ) d - a connector, wherein:

[0400] -(L 11 ) a -Yes-(T 1 -V 1 ) a -;

[0401] -(L 12 ) b -Yes-(T 2 -V 2 ) b -;

[0402] -(L 13 ) c -Yes-(T 3 -V 3 ) c -;and

[0403] -(L 14 ) d -Yes-(T 4 -V 4 ) d -,

[0404] Where T 1 , T 2 , T 3 and T 4 , if present, is the tethering group;

[0405] V 1 , V 2 , V 3 and V 4 , if present, is a covalent bond or a linking functional group; and

[0406] a, b, c and d are each independently 0 or 1, wherein the sum of a, b, c and d is 1 to 4.

[0407] As described above, in certain embodiments, L 11 is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety (e.g., as shown in Formula (I) above). As such, in certain embodiments, T 1 is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety (e.g., as shown in Formula (I) above). In certain embodiments, V 1 Attached to the first cleavable module. In certain embodiments, L 12 , if present, is attached to the first cleavable moiety. As such, in certain embodiments, T 2 , if present, attached to the first cleavable module, or V 2 , if present, is attached to the first cleavable moiety. In certain embodiments, L 13 , if present, is attached to the first cleavable moiety. As such, in certain embodiments, T 3 , if present, attached to the first cleavable module, or V 3 , if present, is attached to the first cleavable moiety. In certain embodiments, L 14 , if present, is attached to the first cleavable moiety. As such, in certain embodiments, T 4 , if present, attached to the first cleavable module, or V 4 , if present, attached to the first cleavable module.

[0408] About the tether group T 1 , T 2 , T 3 and T 4 Any convenient tethering group may be utilized in the subject linker. In some embodiments, T 1 , T 2 , T 3 and T 4 Each comprises one or more independently selected from (C1-C 12 ) alkyl, substituted (C1-C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w ,

[0409] (PEG) n , (AA) p ,-(CR 13 OH) h -, 4-amino-piperidine (4AP), acetal group, disulfide, hydrazine, and ester group, wherein w is an integer from 1 to 20, n is an integer from 1 to 30, p is an integer from 1 to 20, and h is an integer from 1 to 12.

[0410] In certain embodiments, the tethering group (eg, T 1 , T 2 , T 3 and / or T 4 ) includes (C1-C 12 ) alkyl or substituted (C1-C 12 In certain embodiments, (C1-C 12 )alkyl is a straight or branched chain alkyl group comprising 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. 12 ) The alkyl group may be an alkyl group or a substituted alkyl group, such as C1-C 12 Alkyl, or C1-C 10 alkyl, or C1-C6 alkyl, or C1-C3 alkyl. 12 )alkyl is a C2 alkyl. For example, (C1-C 12 ) The alkyl group may be an alkylene group or a substituted alkylene group, such as C1-C 12 Alkylene, or C1-C 10 In some cases, (C1-C 12)Alkyl is C2 alkylene (eg CH2CH2).

[0411] In certain embodiments, the substituted (C1-C 12 )alkyl is a linear or branched substituted alkyl group comprising 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. 12 The alkyl group may be a substituted alkyl group, such as a substituted C1-C 12 Alkyl, or substituted C1-C 10 In some cases, the substituted (C1-C6) alkyl group is 12 )alkyl is a substituted C2 alkyl. For example, a substituted (C1-C 12 ) The alkyl group may be a substituted alkylene group, such as a substituted C1-C 12 Alkylene, or substituted C1-C 10 alkylene, or substituted C1-C6 alkylene, or substituted C1-C3 alkylene. In some cases, substituted (C1-C 12 )Alkyl is a substituted C2 alkylene.

[0412] In certain embodiments, the tethering group (eg, T 1 , T 2 , T 3 and / or T 4 ) includes aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some cases, the tethering group (e.g., T 1 ,

[0413] T 2 , T 3 and / or T 4 ) includes aryl or substituted aryl. For example, aryl can be phenyl. In some cases, substituted aryl is substituted phenyl. Substituted phenyl can be substituted with one or more selected from (C1-C 12 ) alkyl, substituted (C1-C 12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl substituents. In some cases, the substituted aryl is substituted phenyl, wherein the substituent includes a second cleavable moiety as described herein (e.g., an enzyme cleavable moiety, such as a glycoside).

[0414] In some cases, the tethering group (e.g., T 1 , T 2 , T3 and / or T 4 ) includes heteroaryl or substituted heteroaryl. In some cases, the tethering group (e.g., T 1 , T 2 , T 3 and / or T 4 ) includes cycloalkyl or substituted cycloalkyl. In some cases, the tethering group (e.g., T 1 , T 2 , T 3 and / or T 4 ) include heterocyclyl or substituted heterocyclyl. In some cases, the substituents on the substituted heteroaryl, substituted cycloalkyl or substituted heterocyclyl include a second cleavable moiety as described herein (e.g., an enzyme cleavable moiety, such as a glycoside).

[0415] In certain embodiments, the tethering group (eg, T 1 , T 2 , T 3 and / or T 4 ) includes an ethylenediamine (EDA) moiety, such as an EDA tether. In certain embodiments, (EDA) w One or more EDA modules are included, such as wherein w is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12 or 1 to 6, such as 1, 2, 3, 4, 5 or 6. The connected ethylenediamine (EDA) module can be optionally substituted with any convenient substituent at one or more convenient positions, for example, with alkyl, substituted alkyl, acyl, substituted acyl, aryl or substituted aryl. In certain embodiments, the EDA module is described by the following structure:

[0416]

[0417] wherein y is an integer from 1 to 6, r is 0 or 1, and each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy,

[0418] In some embodiments, y is 1, 2, 3, 4, 5 or 6. In some embodiments, y is 1 and r is 0. In some embodiments, y is 1 and r is 1. In some embodiments, y is 2 and r is 0. In some embodiments, y is 2 and r is 1. In some embodiments, each R 12are independently selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl. In certain embodiments, any two adjacent R 12 The groups may be linked in a ring, for example to form a piperazinyl ring. In certain embodiments, y is 1 and two adjacent R 12 The groups are alkyl groups, cyclically linked to form a piperazinyl ring. In certain embodiments, y is 1 and the adjacent R 12 The radical is selected from hydrogen, alkyl (eg methyl) and substituted alkyl (eg lower alkyl-OH such as ethyl-OH or propyl-OH).

[0419] In certain embodiments, the tethering group (eg, T 1 , T 2 , T 3 and / or T 4 ) includes a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidine-4-amino, P4A). The 4AP moiety may optionally be substituted at one or more convenient positions with any convenient substituent, such as an alkyl, substituted alkyl, a polyethylene glycol moiety, an acyl, a substituted acyl, an aryl, or a substituted aryl. In certain embodiments, the 4AP moiety is described by the following structure:

[0420]

[0421] Where R 12 is selected from hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety (e.g., polyethylene glycol or a modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 12 is a polyethylene glycol moiety. 12 It is a carboxyl-modified polyethylene glycol.

[0422] In certain embodiments, R 12 Including through formula (PEG) k To describe the polyethylene glycol module, it can be represented by the following structure:

[0423]

[0424] wherein k is an integer from 1 to 20, such as from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 6, or from 1 to 4, or from 1 to 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some cases, k is 2. In certain embodiments, R 17 is selected from OH, COOH, or COOR, wherein R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 17 It's COOH.

[0425] In certain embodiments, the tethering group (eg, T 1 , T 2 , T 3 and / or T 4 ) including (PEG) n , where (PEG) n is polyethylene glycol or a modified polyethylene glycol linking unit. In certain embodiments, (PEG) n It is described by the following structure:

[0426]

[0427] wherein n is an integer from 1 to 50, such as from 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, n is 2. In some cases, n is 3. In some cases, n is 6. In some cases, n is 12.

[0428] In certain embodiments, the tethering group (eg, T 1 , T 2 , T 3 and / or T 4 ) includes (AA) p, wherein AA is an amino acid residue. Any convenient amino acid can be used. Amino acids of interest include, but are not limited to, L- and D-amino acids, naturally occurring amino acids, such as any of the 20 main α-amino acids and β-alanine, non-naturally occurring amino acids (e.g., amino acid analogs), such as non-naturally occurring α-amino acids or non-naturally occurring β-amino acids, etc. In certain embodiments, p is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12 or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In certain embodiments, p is 1. In certain embodiments, p is 2.

[0429] In certain embodiments, the tethering group (eg, T 1 , T 2 , T 3 and / or T 4 ) including the formula -(CR 13 OH) h - described module, wherein h is 0 or n is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12 or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In certain embodiments, h is 1. In certain embodiments, h is 2. In certain embodiments, R 13 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,

[0430] In certain embodiments, R 13 is hydrogen. In certain embodiments, R 13 is an alkyl or substituted alkyl group, such as C 1-6 Alkyl or substituted C 1-6 Alkyl, or C 1-4 Alkyl or substituted C 1-4 Alkyl, or C 1-3 Alkyl or substituted C 1-3 In certain embodiments, R 13 is alkenyl or substituted alkenyl, such as C 2-6 Alkenyl or substituted C 2-6 Alkenyl, or C 2-4 Alkenyl or substituted C 2-4 Alkenyl, or C 2-3 Alkenyl or substituted C2-3 In certain embodiments, R 13 is alkynyl or substituted alkynyl. In certain embodiments, R 13 is alkoxy or substituted alkoxy. 13 is amino or substituted amino. In certain embodiments, R 13 is carboxyl or carboxyl ester. In certain embodiments, R 13 is acyl or acyloxy. In certain embodiments, R 13 is acylamino or aminoacyl. In certain embodiments, R 13 is an alkylamide or a substituted alkylamide. 13 In certain embodiments, R 13 is thioalkoxy or substituted thioalkoxy. 13 is aryl or substituted aryl, such as C 5-8 Aryl or substituted C 5-8 In certain embodiments, R 13 is a heteroaryl or substituted heteroaryl, such as C 5-8 Heteroaryl or substituted C 5-8 In certain embodiments, R 13 is a cycloalkyl or substituted cycloalkyl group, such as C 3-8 Cycloalkyl or substituted C 3-8 Cycloalkyl, such as C 3-6 Cycloalkyl or substituted C 3-6 Cycloalkyl, or C 3-5 Cycloalkyl or substituted C 3-5 In certain embodiments, R 13 is a heterocyclic or substituted heterocyclic group, such as C 3-8 Heterocyclic or substituted C 3-8 Heterocyclic groups, such as C 3-6 Heterocyclic or substituted C 3-6 Heterocyclic group, or C 3-5 Heterocyclic or substituted C 3-5 Heterocyclic group.

[0431] In certain embodiments, R 13 is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R 13 Described.

[0432] About the Linking Functional Group V1 , V 2 , V 3 and V 4 , can be at the first joint L 1 Any convenient linking functional group is utilized in the invention. Linking functional groups of interest include, but are not limited to, amino, carbonyl, (acyl)amide, oxycarbonyl, carboxyl, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phosphate, phosphoramidate, thiophosphoramidate, and the like. In some embodiments, V 1 ,

[0433] V 2 , V 3 and V 4 Each is independently selected from a covalent bond, -CO-, -NR 15 -,-NR 15 (CH2) q -,-NR 15 (C6H4)-,-CONR 15 -,-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -,-NR 15 SO2- and -P(O)OH-, wherein q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In certain embodiments, q is 1. In certain embodiments, q is 2.

[0434] In some embodiments, each R 15 independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0435] Each R 15 The various possibilities are described in more detail below. In certain embodiments, R 15 is hydrogen. In certain embodiments, each R 15 is hydrogen. In certain embodiments, R 15 is an alkyl or substituted alkyl group, such as C 1-6 Alkyl or substituted C 1-6 Alkyl, or C 1-4 Alkyl or substituted C 1-4 Alkyl, or C 1-3Alkyl or substituted C 1-3 In certain embodiments, R 15 is alkenyl or substituted alkenyl, such as C 2-6 Alkenyl or substituted C 2-6 Alkenyl, or C 2-4 Alkenyl or substituted C 2-4 Alkenyl, or C 2-3 Alkenyl or substituted C 2-3 In certain embodiments, R 15 is alkynyl or substituted alkynyl. In certain embodiments, R 15 is alkoxy or substituted alkoxy. 15 is amino or substituted amino. In certain embodiments, R 15 is carboxyl or carboxyl ester. In certain embodiments, R 15 is acyl or acyloxy. In certain embodiments, R 15 is acylamino or aminoacyl. In certain embodiments,

[0436] R 15 is an alkylamide or a substituted alkylamide. 15 In certain embodiments, R 15 is thioalkoxy or substituted thioalkoxy. 15 is aryl or substituted aryl, such as C 5-8 Aryl or substituted C 5-8 In certain embodiments, R 15 is a heteroaryl or substituted heteroaryl, such as C 5-8 Heteroaryl or substituted C 5-8 In certain embodiments, R 15 is a cycloalkyl or substituted cycloalkyl group, such as C 3-8 Cycloalkyl or substituted C 3-8 Cycloalkyl, such as C 3-6 Cycloalkyl or substituted C 3-6 Cycloalkyl, or C 3-5 Cycloalkyl or substituted C 3-5 In certain embodiments, R 15 is a heterocyclic or substituted heterocyclic group, such as C 3-8 Heterocyclic or substituted C 3-8 Heterocyclic groups, such as C 3-6 Heterocyclic or substituted C 3-6 Heterocyclic group, or C 3-5Heterocyclic or substituted C 3-5 Heterocyclic group.

[0437] In certain embodiments, each R 15 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,

[0438] alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,

[0439] Substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl substituents are as described above for R 15 Descriptive.

[0440] In certain embodiments, the tethered group comprises an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tethered group comprises an acetal group. In some embodiments, the tethered group comprises a disulfide. In some embodiments, the tethered group comprises a hydrazine. In some embodiments, the tethered group comprises an ester.

[0441] As described above, in some embodiments, L 1 is included-(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -a first linker, wherein a, b, c and d are each independently 0 or 1, wherein the sum of a, b, c and d is 1 to 4.

[0442] In some embodiments, at the first linker L 1 middle:

[0443] T 1 Selected from (C1-C 12 ) alkyl and substituted (C1-C 12 )alkyl;

[0444] T 2 , T 3 and T 4 Each independently selected from (C1-C 12 ) alkyl, substituted (C1-C12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p ,-(CR 13 OH) h -, 4-amino-piperidine (4AP), acetal groups, disulfides, hydrazines, and esters; and

[0445] V 1 , V 2 , V 3 and V 4 Each is independently selected from a covalent bond, -CO-, -NR 15 -,-NR 15 (CH2) q -,-NR 15 (C6H4)-,-CONR 15 -,-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -,-NR 15 SO2- and -P(O)OH-, wherein q is an integer from 1 to 6;

[0446] in:

[0447] (PEG) n yes wherein n is an integer from 1 to 30;

[0448] EDA is an ethylenediamine module with the following structure: wherein y is an integer from 1 to 6 and r is 0 or 1;

[0449] 4-Amino-piperidine (4AP) is

[0450] AA is an amino acid residue, wherein p is an integer from 1 to 20;

[0451] Each R 15 and R 12 are independently selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl, wherein any two adjacent R 12 The groups may be linked in a ring to form a piperazinyl ring; and

[0452] R 13 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl.

[0453] In certain embodiments, T 1 , T2 , T 3 and T 4 and V 1 , V 2 , V 3 and V 4 Select from the following:

[0454] T 1 Yes (C1-C 12 ) alkyl and V 1 is -CO-;

[0455] T 2 Is 4AP and V 2 It is a covalent bond;

[0456] T 3 Yes (PEG) n And V 3 is -CO-; and

[0457] T 4 and V 4 Does not exist.

[0458] For example, T 1 , T 2 , T 3 and T 4 and V 1 , V 2 , V 3 and V 4 You can choose from the following:

[0459] T 1 is CH2CH2 and V 1 is -CO-;

[0460] T 2 Is 4AP and V 2 It is a covalent bond;

[0461] T 3 is (PEG)2 and V 3 is -CO-; and

[0462] T 4 and V 4 Does not exist.

[0463] In certain embodiments, T 1 , T 2 , T 3 and T 4 and V 1 , V 2 , V 3 and V 4 Select from the following:

[0464] T1 Yes (C1-C 12 ) alkyl and V 1 is -CO-;

[0465] T 2 Is 4AP and V 2 It is a covalent bond;

[0466] T 3 Yes (PEG) n And V 3 is -CONH-; and

[0467] T 4 is aryl or substituted aryl and V 4 It's -CO-.

[0468] For example, T 1 , T 2 , T 3 and T 4 and V 1 , V 2 , V 3 and V 4 You can choose from the following:

[0469] T 1 is CH2CH2 and V 1 is -CO-;

[0470] T 2 Is 4AP and V 2 It is a covalent bond;

[0471] T 3 is (PEG)2 and V 3 is -CONH-; and

[0472] T 4 is phenyl or substituted phenyl and V 4 It's -CO-.

[0473] In certain embodiments, the first linker L 1 It is described by the following structure:

[0474]

[0475] In certain embodiments, the first linker L 1 It is described by the following structure:

[0476]

[0477] In certain embodiments, the first linker L 1 It is described by the following structure:

[0478]

[0479] In certain embodiments, the left-hand side of the linker structure is attached to the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety, and the right-hand side of the linker structure is attached to the first cleavable moiety. For example, where the first cleavable moiety comprises a peptide, the right-hand side of the linker structure can be attached to an amino acid of the peptide comprising the first cleavable moiety. In some cases, the carbonyl group on the right-hand side of the linker structure can form an amide bond with an amino acid of the peptide comprising the first cleavable moiety.

[0480] In some embodiments, L 2 is through the formula -(L 21 ) e -(L 22 ) f -(L 23 ) g -(L 24 ) h - to describe the second linker, where L 21 , L 22 , L 23 and L 24 Each is independently a second linker subunit, and e, f, g and h are each independently 0 or 1, wherein the sum of e, f, g and h is 0-4.

[0481] In certain embodiments, the sum of e, f, g and h is 0. In these cases, the second linker L 2 In other words, when the sum of e, f, g and h is 0, the second joint L 2 is a covalent bond. In certain embodiments, the sum of e, f, g, and h is 1. In certain embodiments, the sum of e, f, g, and h is 2. In certain embodiments, the sum of e, f, g, and h is 3. In certain embodiments, the sum of e, f, g, and h is 4. In certain embodiments, e, f, g, and h are each 1. In certain embodiments, e, f, and g are each 1 and h is 0. In certain embodiments, e and f are each 1 and g and h are each 0. In certain embodiments, e is 1 and f, g, and h are each 0.

[0482] In certain embodiments, L 21 Attached to the drug (e.g., W as shown in Formula (I) above) 1 ). In certain embodiments, L 22 , if present, is attached to the drug. In certain embodiments, L 23 , if present, is attached to the drug. In certain embodiments, L 24 , if present, attached to the drug.

[0483] The second connector L 2 Any convenient linker subunit is utilized in the invention. Linker subunits of interest include, but are not limited to, units of polymers such as polyethylene glycol, polyethylene and polyacrylates, amino acid residues, carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclyl groups, combinations thereof, and substitution patterns thereof. In some embodiments, L 21 , L 22 , L 23 and L 24 Each of (if present) comprises one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., linking groups comprising alkylenediamines).

[0484] In some embodiments, L 21 (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 21 In some embodiments, L 21 In some embodiments, L 21 In some embodiments, L 21 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 21 In some embodiments, L 21 A diamine-containing linking group (eg, an alkylene diamine-containing linking group) is provided.

[0485] In some embodiments, L 22 (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 22 In some embodiments, L 22 In some embodiments, L 22 In some embodiments, L 22 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 22 In some embodiments, L 22 A diamine-containing linking group (eg, an alkylene diamine-containing linking group) is provided.

[0486] In some embodiments, L 23(if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 23 In some embodiments, L 23 In some embodiments, L 23 In some embodiments, L 23 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 23 In some embodiments, L 23 A diamine-containing linking group (eg, an alkylene diamine-containing linking group) is provided.

[0487] In some embodiments, L 24 (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 24 In some embodiments, L 24 In some embodiments, L 24 In some embodiments, L 24 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 24 In some embodiments, L 24 A diamine-containing linking group (eg, an alkylene diamine-containing linking group) is provided.

[0488] In some embodiments, L 2 Is included-(L 21 ) e -(L 22 ) f -(L 23 ) g -(L 24 ) h - a second connector, wherein:

[0489] -(L 21 ) e -Yes-(T 5 -V 5 ) e -;

[0490] -(L 22 ) f -Yes-(T 6 -V 6 ) f -;

[0491] -(L 23 ) g -Yes-(T 7 -V 7 ) g -;and

[0492] -(L 24 ) h -Yes-(T 8 -V 8 ) h -,

[0493] Where T 5 , T 6 , T 7 and T 8 , if present, is the tethering group;

[0494] V 5 , V 6 , V 7 and V 8 , if present, is a covalent bond or a linking functional group; and

[0495] e, f, g and h are each independently 0 or 1, wherein the sum of e, f, g and h is 0 to 4.

[0496] In certain embodiments, L 21 Attached to the first cleavable module. As such, in certain embodiments, T 5 Attached to the first cleavable module. In certain embodiments, V 5 Attached to the drug (e.g., W as shown in Formula (I) above) 1 ). In certain embodiments,

[0497] L 22 , if present, is attached to the drug. As such, in certain embodiments, T 6 , if present, attached to the drug, or V 6 , if present, is attached to the drug. In certain embodiments, L 23 , if present, is attached to the drug. As such, in certain embodiments, T 7 , if present, attached to the drug, or V 7 , if present, is attached to the drug. In certain embodiments,

[0498] L 24 , if present, is attached to the drug. As such, in certain embodiments, T 8 , if present, attached to the drug, or V 8 , if present, attached to the drug.

[0499] About the tether group T 5 , T 6 , T 7 and T 8 Any convenient tethering group may be utilized in the subject linker. In some embodiments, T 5 , T 6 , T 7 and T 8 Each comprises one or more independently selected from (C1-C 12 ) alkyl, substituted (C1-C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w ,

[0500] (PEG) n , (AA) p ,-(CR 13 OH) h -, 4-amino-piperidine (4AP), acetal group, disulfide, hydrazine, and ester group, wherein w is an integer from 1 to 20, n is an integer from 1 to 30, p is an integer from 1 to 20, and h is an integer from 1 to 12.

[0501] In certain embodiments, the tethering group (eg, T 5 , T 6 , T 7 and / or T 8 ) includes (C1-C 12 ) alkyl or substituted (C1-C 12 In certain embodiments, (C1-C 12 )alkyl is a straight or branched chain alkyl group comprising 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. 12 ) The alkyl group may be an alkyl group or a substituted alkyl group, such as C1-C 12 Alkyl, or C1-C 10 alkyl, or C1-C6 alkyl, or C1-C3 alkyl. 12 )alkyl is a C2 alkyl. For example, (C1-C 12 ) The alkyl group may be an alkylene group or a substituted alkylene group, such as C1-C 12 Alkylene, or C1-C 10 In some cases, (C1-C 12)alkyl is C2 alkylene (e.g., CH2CH2). In some cases, (C1-C 12 )Alkyl is C1 alkylene (eg CH2).

[0502] In certain embodiments, the substituted (C1-C 12 )alkyl is a substituted straight or branched chain alkyl group comprising 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, substituted (C1-C 12 The alkyl group may be a substituted alkyl group, such as a substituted C1-C 12 Alkyl, or substituted C1-C 10 In some cases, the substituted (C1-C6) alkyl group is 12 )alkyl is a substituted C2 alkyl. For example, a substituted (C1-C 12 ) The alkyl group may be a substituted alkylene group, such as a substituted C1-C 12 Alkylene, or substituted C1-C 10 alkylene, or substituted C1-C6 alkylene, or substituted C1-C3 alkylene. In some cases, substituted (C1-C 12 In some cases, the substituted (C1-C 12 )Alkyl is a substituted C1 alkylene.

[0503] In certain embodiments, the tethering group (eg, T 5 , T 6 , T 7 and / or T 8 ) includes aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some cases, the tethering group includes aryl or substituted aryl. For example, the aryl group can be phenyl or substituted phenyl. In some cases, the tethering group (e.g., T 5 , T 6 , T 7 and / or T 8 ) includes heteroaryl or substituted heteroaryl. In some cases, the tethering group (e.g., T 5 , T 6 , T 7 and / or T 8 ) includes cycloalkyl or substituted cycloalkyl. In some cases, the tethering group (e.g., T 5 , T 6 , T 7 and / or T 8) includes a heterocyclic group or a substituted heterocyclic group.

[0504] In certain embodiments, the tethering group (eg, T 5 , T 6 , T 7 and / or T 8 ) includes an ethylenediamine (EDA) module, for example, an EDA module as described above, such as an EDA module described by the following structure:

[0505]

[0506] wherein y is an integer from 1 to 6, r is 0 or 1, and each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy,

[0507] In some embodiments, y is 1, 2, 3, 4, 5 or 6. In some embodiments, y is 1 and r is 0. In some embodiments, y is 1 and r is 1. In some embodiments, y is 2 and r is 0. In some embodiments, y is 2 and r is 1. In some embodiments, each R 12 are independently selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl. In certain embodiments, any two adjacent R 12 The groups may be linked in a ring, for example to form a piperazinyl ring. In certain embodiments, y is 1 and two adjacent R 12 The groups are alkyl groups, cyclically linked to form a piperazinyl ring. In certain embodiments, y is 1 and the adjacent R 12 The radical is selected from hydrogen, alkyl (eg methyl) and substituted alkyl (eg lower alkyl-OH such as ethyl-OH or propyl-OH).

[0508] In certain embodiments, the tethering group (eg, T 5 , T 6 , T 7 and / or T 8 ) includes a 4-amino-piperidine (4AP) module as described above, such as the 4AP module described by the following structure:

[0509]

[0510] Where R 12is selected from hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety (e.g., polyethylene glycol or a modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 12 is a polyethylene glycol moiety. 12 It is a carboxyl-modified polyethylene glycol.

[0511] In certain embodiments, R 12 Including through formula (PEG) k To describe the polyethylene glycol module, it can be represented by the following structure:

[0512]

[0513] wherein k is an integer from 1 to 20, such as from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 6, or from 1 to 4, or from 1 to 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some cases, k is 2. In certain embodiments, R 17 is selected from OH, COOH, or COOR, wherein R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 17 It is COOH.

[0514] In certain embodiments, the tethering group (eg, T 5 , T 6 , T 7 and / or T 8 ) comprises a polyethylene glycol moiety (PEG) as described above n , such as that described by the following structure (PEG) n Modules:

[0515]

[0516] wherein n is an integer from 1 to 50, such as from 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, n is 2. In some cases, n is 3. In some cases, n is 6. In some cases, n is 12.

[0517] In certain embodiments, the tethering group (eg, T 5 , T 6 , T 7 and / or T 8 ) includes (AA) p , wherein AA is an amino acid residue. Any convenient amino acid can be used. Amino acids of interest include, but are not limited to, L- and D-amino acids, naturally occurring amino acids, such as any of the 20 main α-amino acids and β-alanine, non-naturally occurring amino acids (e.g., amino acid analogs), such as non-naturally occurring α-amino acids or non-naturally occurring β-amino acids, etc. In certain embodiments, p is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12 or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In certain embodiments, p is 1. In certain embodiments, p is 2.

[0518] In certain embodiments, the tethering group (eg, T 5 , T 6 , T 7 and / or T 8 ) including the formula -(CR 13 OH) h - described module, wherein h is 0 or n is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12 or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In certain embodiments, h is 1. In certain embodiments, h is 2. In certain embodiments, R 13 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,

[0519] In certain embodiments, R 13 is hydrogen. In certain embodiments, R 13is an alkyl or substituted alkyl group, such as C 1-6 Alkyl or substituted C 1-6 Alkyl, or C 1-4 Alkyl or substituted C 1-4 Alkyl, or C 1-3 Alkyl or substituted C 1-3 In certain embodiments, R 13 is alkenyl or substituted alkenyl, such as C 2-6 Alkenyl or substituted C 2-6 Alkenyl, or C 2-4 Alkenyl or substituted C 2-4 Alkenyl, or C 2-3 Alkenyl or substituted C 2-3 In certain embodiments, R 13 is alkynyl or substituted alkynyl. In certain embodiments, R 13 is alkoxy or substituted alkoxy. 13 is amino or substituted amino. In certain embodiments, R 13 is carboxyl or carboxyl ester. In certain embodiments, R 13 is acyl or acyloxy. In certain embodiments, R 13 is acylamino or aminoacyl. In certain embodiments, R 13 is an alkylamide or a substituted alkylamide. 13 In certain embodiments, R 13 is thioalkoxy or substituted thioalkoxy. 13 is aryl or substituted aryl, such as C 5-8 Aryl or substituted C 5-8 In certain embodiments, R 13 is a heteroaryl or substituted heteroaryl, such as C 5-8 Heteroaryl or substituted C 5-8 In certain embodiments, R 13 is a cycloalkyl or substituted cycloalkyl group, such as C 3-8 Cycloalkyl or substituted C 3-8 Cycloalkyl, such as C 3-6 Cycloalkyl or substituted C 3-6 Cycloalkyl, or C 3-5 Cycloalkyl or substituted C 3-5 In certain embodiments, R 13 is a heterocyclic or substituted heterocyclic group, such as C 3-8 Heterocyclic or substituted C3-8 Heterocyclic groups, such as C 3-6 Heterocyclic or substituted C 3-6 Heterocyclic group, or C 3-5 Heterocyclic or substituted C 3-5 Heterocyclic group.

[0520] In certain embodiments, R 13 is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R 13 Described.

[0521] About the Linking Functional Group V 5 , V 6 , V 7 and V 8 , you can use the second connector L 2 Any convenient linking functional group is utilized in the invention. Linking functional groups of interest include, but are not limited to, amino, carbonyl, (acyl)amide, oxycarbonyl, carboxyl, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phosphate, phosphoramidate, thiophosphoramidate, and the like. In some embodiments, V 5 ,

[0522] V 6 , V 7 and V 8 Each is independently selected from a covalent bond, -CO-, -NR 15 -,-NR 15 (CH2) q -,-NR 15 (C6H4)-,-CONR 15 -,-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -,-NR 15 SO2- and -P(O)OH-, wherein q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In certain embodiments, q is 1. In certain embodiments, q is 2.

[0523] In some embodiments, each R 15independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0524] Each R 15 The various possibilities are described in more detail below. In certain embodiments, R 15 is hydrogen. In certain embodiments, each R 15 is hydrogen. In certain embodiments, R 15 is an alkyl or substituted alkyl group, such as C 1-6 Alkyl or substituted C 1-6 Alkyl, or C 1-4 Alkyl or substituted C 1-4 Alkyl, or C 1-3 Alkyl or substituted C 1-3 In certain embodiments, R 15 is alkenyl or substituted alkenyl, such as C 2-6 Alkenyl or substituted C 2-6 Alkenyl, or C 2-4 Alkenyl or substituted C 2-4 Alkenyl, or C 2-3 Alkenyl or substituted C 2-3 In certain embodiments, R 15 is alkynyl or substituted alkynyl. In certain embodiments, R 15 is alkoxy or substituted alkoxy. 15 is amino or substituted amino. In certain embodiments, R 15 is carboxyl or carboxyl ester. In certain embodiments, R 15 is acyl or acyloxy. In certain embodiments, R 15 is acylamino or aminoacyl. In certain embodiments,

[0525] R 15 is an alkylamide or a substituted alkylamide. 15 In certain embodiments, R 15 is thioalkoxy or substituted thioalkoxy. 15 is aryl or substituted aryl, such as C 5-8 Aryl or substituted C 5-8 In certain embodiments, R15 is a heteroaryl or substituted heteroaryl, such as C 5-8 Heteroaryl or substituted C 5-8 In certain embodiments, R 15 is a cycloalkyl or substituted cycloalkyl group, such as C 3-8 Cycloalkyl or substituted C 3-8 Cycloalkyl, such as C 3-6 Cycloalkyl or substituted C 3-6 Cycloalkyl, or C 3-5 Cycloalkyl or substituted C 3-5 In certain embodiments, R 15 is a heterocyclic or substituted heterocyclic group, such as C 3-8 Heterocyclic or substituted C 3-8 Heterocyclic groups, such as C 3-6 Heterocyclic or substituted C 3-6 Heterocyclic group, or C 3-5 Heterocyclic or substituted C 3-5 Heterocyclic group.

[0526] In certain embodiments, each R 15 R is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl substituents are as described above for R 15 Described.

[0527] In certain embodiments, the tethering group comprises an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tethering group comprises an acetal group. In some embodiments, the tethering group comprises a disulfide. In some embodiments, the tethering group comprises a hydrazine. In some embodiments, the tethering group comprises an ester.

[0528] As described above, in some embodiments, L 2 is included-(T 5 -V 5 ) e -(T 6 -V 6 ) f -(T 7 -V 7 ) g -(T8 -V 8 ) h -a second linker, wherein e, f, g and h are each independently 0 or 1, wherein the sum of e, f, g and h is 0 to 4.

[0529] In some embodiments, in the second linker L 2 middle:

[0530] T 5 , T 6 , T 7 and T 8 Each independently selected from (C1-C 12 ) alkyl, substituted (C1-C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p ,-(CR 13 OH) h -, 4-amino-piperidine (4AP), acetal groups, disulfides, hydrazines, and esters; and

[0531] V 5 , V 6 , V 7 and V 8 Each is independently selected from a covalent bond, -CO-, -NR 15 -,-NR 15 (CH2) q -,-NR 15 (C6H4)-,-CONR 15 -,-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -,-NR 15 SO2- and -P(O)OH-, wherein q is an integer from 1 to 6;

[0532] in:

[0533] (PEG) n yes wherein n is an integer from 1 to 30;

[0534] EDA is an ethylenediamine module having the following structure:

[0535] wherein y is an integer from 1 to 6 and r is 0 or 1;

[0536] 4-Amino-piperidine (4AP) is

[0537] AA is an amino acid residue, wherein p is an integer from 1 to 20;

[0538] Each R 13 are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

[0539] Each R 15 independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0540] In certain embodiments, T 5 , T 6 , T 7 and T 8 and V 5 , V 6 , V 7 and V 8 Select from the following:

[0541] T 5 is a covalent bond and V 5 is -OC(O)-;

[0542] T 6 and V 6 does not exist;

[0543] T 7 and V 7 does not exist; and

[0544] T 8 and V 8 Does not exist.

[0545] In certain embodiments, T 5 , T 6 , T 7 and T 8 and V 5 , V 6 , V 7 and V 8 does not exist (i.e., the sum of e, f, g, and h is 0).

[0546] In certain embodiments, the left hand side of the above linker structure is attached to the first cleavable moiety and the right hand side of the above linker structure is attached to the drug.

[0547] Any of the chemical entities, linkers, and coupling moieties listed in the structures above may be adapted for use in the subject compounds and conjugates.

[0548] Additional disclosure relating to hydrazino-indolyl and hydrazino-pyrrolo-pyridinyl compounds and methods for forming conjugates is found in U.S. Pat. No. 9,310,374 and U.S. Pat. No. 9,493,413, the disclosures of each of which are incorporated herein by reference.

[0549] Compounds useful for forming conjugates

[0550] The present disclosure provides hydrazino-indolyl and hydrazino-pyrrolo-pyridinyl compounds useful for generating the conjugates described herein. In certain embodiments, the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compounds can be useful coupling modules for conjugation of antibodies and drugs. For example, the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compounds can bind to the antibody and also bind to the drug, thus indirectly binding the antibody and drug together.

[0551] In certain embodiments, the compound is of formula (II):

[0552]

[0553] in

[0554] Z is CR 4 or N;

[0555] R 2 and R 3 each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R 2 and R 3 Optionally linked in a ring to form a 5- or 6-membered heterocyclic group;

[0556] Each R 4 independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0557] Each R 5independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0558] Each R 6 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0559] k is an integer from 1 to 10;

[0560] R 7 is selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0561] L 1 It is the first joint;

[0562] L 2 is the second connector; and

[0563] W 1 It's medicine.

[0564] Where L 1 , R 6 or R 7 One of the moieties comprises a second cleavable module.

[0565] In some cases, k is 2, and the compound is of formula (IIa):

[0566]

[0567] in

[0568] R 6’ or R 6” One of them comprises a second cleavable module, and R 6’ and R 6” The other of which is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0569] For example, the compound may be a compound of formula (IIb):

[0570]

[0571] In some cases, the compound may be a compound of formula (IIc):

[0572]

[0573] In certain embodiments, k is 2, and the compound is of formula (IId):

[0574]

[0575] in

[0576] R 6’ and R 6” are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

[0577] R 7 A second cleavable module is included.

[0578] For example, the compound may be a compound of formula (IIe):

[0579]

[0580] In certain embodiments, k is 2, and the compound is of Formula (IIf):

[0581]

[0582] in

[0583] R 6’ and R 6” are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

[0584] R 8 A second cleavable module is included.

[0585] For example, the compound may be a compound of formula (IIg):

[0586]

[0587] Substituents are described herein with respect to the conjugates of formula (II). Reference to formula (II) is intended to also encompass formulas (IIa), (IIb), (IIc), (IId), (IIe), (IIf) and (IIg).

[0588] Regarding the compound of formula (II), the substituents Z, R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , and W 1 As described above with respect to the conjugate of formula (I). Similarly, with respect to the first linker L of formula (II) 1 and the second connector L 2 , T 1 , T 2 , T 3 , T 4 , V 1 , V 2 , V 3 , and V 4 , and T 5 , T 6 , T 7 , T 8 , V 5 , V 6 , V 7 and V 8 The substituents are as described above in relation to the conjugates of formula (I).

[0589] In certain embodiments, the compound is of the structure:

[0590]

[0591] For example, a compound may have the following structure:

[0592]

[0593] In certain embodiments, the compound is of the structure:

[0594]

[0595] For example, a compound may have the following structure:

[0596]

[0597] In certain embodiments, the compound is of the structure:

[0598]

[0599] For example, a compound may have the following structure:

[0600]

[0601] In certain embodiments, the compound is of the structure:

[0602]

[0603] For example, a compound may have the following structure:

[0604]

[0605] In certain embodiments, the compound is of the structure:

[0606]

[0607] For example, a compound may have the following structure:

[0608]

[0609] Antibody

[0610] As described above, the subject conjugates may comprise an antibody as a substituent. 2 , wherein the antibody is modified to include a 2-formylglycine (FGly) residue. As used herein, amino acids may be referred to by their standard names, their standard three letter abbreviations, and / or their standard single letter abbreviations, such as: Alanine or Ala or A; Cysteine ​​or Cys or C; Aspartic acid or Asp or D; Glutamic acid or Glu or E; Phenylalanine or Phe or F; Glycine or Gly or G; Histidine or His or H; Isoleucine or Ile or I; Lysine or Lys or K; Leucine or Leu or L; Methionine or Met or M; Asparagine or Asn or N; Proline or Pro or P; Glutamine or Gln or Q; Arginine or Arg or R; Serine or Ser or S; Threonine or Thr or T; Valine or Val or V; Tryptophan or Trp or W; and Tyrosine or Tyr or Y.

[0611] In certain embodiments, the amino acid sequence of the antibody is modified to include a sulfatase motif containing a serine or cysteine ​​residue that can be converted (oxidized) into a 2-formylglycine (FGly) residue by the action of a formylglycine generating enzyme (FGE) in vivo (e.g., when translating a protein containing an aldehyde tag in a cell) or in vitro (e.g., by contacting a protein containing an aldehyde tag with FGE in a cell-free system). Such sulfatase motifs may also be referred to herein as FGE modification sites.

[0612] Sulfatase motif

[0613] The minimal sulfatase motif of an aldehyde tag is typically 5 or 6 amino acid residues in length, and typically is no more than 6 amino acid residues in length. The sulfatase motif provided in an Ig polypeptide is at least 5 or 6 amino acid residues in length, and can be, for example, 5-16, 6-16, 5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10, 5-9, 6-9, 5-8, or 6-8 amino acid residues, thereby defining a sulfatase motif of less than 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acid residues in length.

[0614] In certain embodiments, polypeptides of interest include those in which one or more amino acid residues, such as 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20 or more amino acid residues have been inserted, deleted, substituted (replaced) relative to the native amino acid sequence to provide a sequence of a sulfatase motif in the polypeptide. In certain embodiments, the polypeptide includes modifications (insertions, additions, deletions, and / or substitutions / replacements) of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues of the amino acid sequence relative to the native amino acid sequence of the polypeptide. In the case where the native amino acid sequence of a polypeptide (e.g., an antibody) contains one or more residues of a desired sulfatase motif, the total number of residue modifications can be reduced, for example, by providing a sequence of the desired sulfatase motif through site-specific modifications (insertions, additions, deletions, substitutions / replacements) of the amino acid residues flanking the native amino acid residues. In certain embodiments, the degree of modification of the native amino acid sequence of the target antibody is minimized to minimize the number of amino acid residues that are inserted, deleted, substituted (replaced), or added (e.g., to the N or C termini). Minimizing the degree of modification of the amino acid sequence of the target antibody can minimize the impact that such modifications may have on antibody function and / or structure.

[0615] It should be noted that while aldehyde tags of particular interest are those that contain at least a minimal sulfatase motif (also referred to as a "consensus sulfatase motif"), it will be readily appreciated that the present disclosure relates to and encompasses longer aldehyde tags that can be used in the compositions and methods of the present disclosure. The aldehyde tag may thus contain a minimal sulfatase motif of 5 or 6 residues, or may be longer, containing a minimal sulfatase motif and the N- and / or C-terminal sides of the motif may be flanked by additional amino acid residues. For example, aldehyde tags of 5 or 6 amino acid residues are encompassed, as well as longer amino acid sequences of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues.

[0616] The aldehyde tag may be present at or near the C-terminus of the Ig heavy chain; for example, the aldehyde tag may be present within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C-terminus of a native, wild-type Ig heavy chain. The aldehyde tag may be present in the CH1 domain of the Ig heavy chain. The aldehyde tag may be present in the CH2 domain of the Ig heavy chain. The aldehyde tag may be present in the CH3 domain of the Ig heavy chain. The aldehyde tag may be present in the constant region of an Ig light chain, such as a kappa light chain constant region or a lambda light chain constant region.

[0617] In certain embodiments, the sulfatase motif used can be described by the following formula:

[0618] X 1 Z 10 X 2 Z 20 X 3 Z 30 (I'),

[0619] in

[0620] Z 10 is cysteine ​​or serine (it can also be represented by (C / S));

[0621] Z 20 is either a proline or an alanine residue (it can also be represented by (P / A));

[0622] Z 30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), such as lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), such as A, G, L, V, or I);

[0623] X 1is present or absent, and, when present, may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), such as L, M, V, S or T, such as L, M, S or V, provided that, when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists; and

[0624] X 2 and X 3 It can independently be any amino acid, although typically it is an aliphatic amino acid, a polar, uncharged amino acid, or a sulfur-containing amino acid (i.e. other than an aromatic amino acid or a charged amino acid), such as S, T, A, V, G or C, such as S, T, A, V or G.

[0625] The amino acid sequence of the antibody heavy and / or light chain may be modified to provide a 1 Z 10 X 2 Z 20 X 3 Z 30 A sequence of at least 5 amino acids, wherein

[0626] Z 10 is cysteine ​​or serine;

[0627] Z 20 is a proline or alanine residue;

[0628] Z 30 is an aliphatic amino acid or a basic amino acid;

[0629] X 1 is present or absent, and, when present, is any amino acid, provided that, when the heterologous sulfatase motif is at the N-terminus of the polypeptide, X 1 exist;

[0630] X 2 and X 3 Each independently is any amino acid.

[0631] The sulfatase motif is typically selected to be capable of being converted by a selected FGE, such as an FGE present in a host cell in which the aldehyde-tagged polypeptide is expressed or an FGE to be contacted with the aldehyde-tagged polypeptide in a cell-free in vitro method.

[0632] For example, where the FGE is eukaryotic FGE (e.g., mammalian FGE, including human FGE), the sulfatase motif may be of the following formula:

[0633] X 1 CX 2 PX 3 Z30 (I”),

[0634] in

[0635] X 1 may be present or absent, and, when present, may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), such as L, M, S or V, provided that, when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exist;

[0636] X 2 and X 3 can independently be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), such as S, T, A, V, G, or C, such as S, T, A, V or G; and Z 30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), such as lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), such as A, G, L, V, or I).

[0637] Specific examples of sulfatase motifs include LCTPSR (SEQ ID NO: / / ), MCTPSR (SEQ ID NO: / / ), VCTPSR (SEQ ID NO: / / ), LCSPSR (SEQ ID NO: / / ), LCAPSR (SEQ ID NO: / / ), LCVPSR (SEQ ID NO: / / ), LCGPSR (SEQ ID NO: / / ), ICTPAR (SEQ ID NO: / / ), LCTPSK (SEQ ID NO: / / ), MCTPSK (SEQ ID NO: / / ) NO: / / ), VCTPSK(SEQ ID NO: / / ), LCSPSK(SEQ ID NO: / / ), LCAPSK(SEQ IDNO: / / ), LCVPSK(SEQ ID NO: / / ), LCGPSK(SEQ ID NO: / / ), LCTPSA(SEQ ID NO: / / ), ICTPAA(SEQ ID NO: / / ), MCTPSA(SEQ ID NO: / / ), VCTPSA(SEQ ID NO: / / ), LCSPSA(SEQ ID NO: / / ), LCAPSA (SEQ ID NO: / / ), LCVPSA ​​(SEQ ID NO: / / ), and LCGPSA (SEQ ID NO: / / ).

[0638] Containing FGly sequence

[0639] Once FGE acts on the modified antibody heavy and / or light chain, the serine or cysteine ​​in the sulfatase motif is modified to FGly. Thus, the FGly-containing sulfatase motif can be of the following formula:

[0640] X 1 (FGly)X 2 Z 20 X 3 Z 30 (I''),

[0641] in

[0642] FGly is a formylglycine residue;

[0643] Z 20 is either a proline or an alanine residue (also represented by (P / A));

[0644] Z 30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), typically lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I);

[0645] X 1 may be present or absent, and, when present, may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), such as L, M, V, S or T, such as L, M or V, provided that, when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists; and

[0646] X 2 and X 3 It can independently be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid), such as S, T, A, V, G or C, such as S, T, A, V or G.

[0647] As described above, by the reaction of FGly and a drug (e.g., a drug containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling module as described above) to generate a FGly' sulfatase motif, a modified polypeptide containing a FGly residue can be conjugated to a drug (e.g., maytansinoid alkaloid). As used herein, the term FGly' refers to a modified amino acid residue in a sulfatase motif that is coupled to a drug, such as maytansinoid or auristatin. Thus, a FGly' sulfatase motif can be of the following formula:

[0648] X 1 (FGly')X 2 Z 20 X 3 Z 30 (II)

[0649] in

[0650] FGly' is a modified amino acid residue of formula (I);

[0651] Z 20 is either a proline or an alanine residue (also represented by (P / A));

[0652] Z 30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), typically lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I);

[0653] X 1 may be present or absent, and, when present, may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid (i.e., other than an aromatic amino acid or a charged amino acid), such as L, M, V, S or T, such as L, M or V, provided that, when the sulfatase motif is at the N-terminus of the target polypeptide, X 1 exists; and

[0654] X 2 and X 3 It can independently be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid (i.e. other than an aromatic amino acid or a charged amino acid), such as S, T, A, V, G or C, such as S, T, A, V or G.

[0655] Modification site

[0656] As described above, the amino acid sequence of the antibody is modified to include a sulfatase motif containing a serine or cysteine ​​residue that can be converted (oxidized) into a FGly residue by the action of FGE in vivo (e.g., when translating a protein containing an aldehyde tag in a cell) or in vitro (e.g., by contacting a protein containing an aldehyde tag with FGE in a cell-free system). The antibody used to generate the conjugate of the present disclosure includes at least one Ig constant region, such as an Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and CH2 domain; a CH1, CH2, and CH3 domain; or a CH1, CH2, CH3, and CH4 domain), or an Ig light chain constant region. Such Ig polypeptides are referred to herein as "target Ig polypeptides" or "target antibodies."

[0657] The site at which the sulfatase motif is introduced into the antibody may be any convenient site. As described above, in some cases, the degree of modification of the native amino acid sequence of the target polypeptide is minimized to minimize the number of amino acid residues that are inserted, deleted, substituted (replaced), and / or added (e.g., to the N or C termini). Minimizing the degree of modification of the amino acid sequence of the target antibody can minimize the effects that such modifications may have on antibody function and / or structure.

[0658] The antibody heavy chain constant region may include an Ig constant region of any heavy chain isotype, a non-naturally occurring Ig heavy chain constant region (including a consensus Ig heavy chain constant region). The Ig constant region may be modified to include an aldehyde tag, wherein the aldehyde tag is present in or adjacent to a solvent accessible loop region of the Ig constant region. The Ig constant region may be modified by insertion and / or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids, or more than 16 amino acids, to provide an amino acid sequence of a sulfatase motif as described above.

[0659] In some cases, the antibody with an aldehyde tag comprises an Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and CH2 domain; a CH1, CH2, and CH3 domain; or a CH1, CH2, CH3, and CH4 domain) with an aldehyde tag. The Ig heavy chain constant region with an aldehyde tag may include a heavy chain constant region sequence of an IgA, IgM, IgD, IgE, IgG1, IgG2, IgG3, or IgG4 isotype heavy chain or any allotypic variant thereof, such as a human heavy chain constant region sequence or a mouse heavy chain constant region sequence, a hybrid heavy chain constant region, a synthetic heavy chain constant region, or a consensus heavy chain constant region sequence, etc., which is modified to include at least one sulfatase motif, which may be modified by FGE to generate an FGly-modified Ig polypeptide. Allotypic variants of Ig heavy chains are known in the art. See, e.g., Jefferis and Lefranc (2009) MAbs 1:4.

[0660] In some cases, the antibody with aldehyde tag comprises the Ig light chain constant region with aldehyde tag. The Ig light chain constant region with aldehyde tag can include kappa light chain, the constant region sequence of lambda light chain, such as human kappa or lambda light chain constant region, hybrid light chain constant region, synthetic light chain constant region, or a common light chain constant region sequence, etc., which is modified to include at least one sulfatase motif, which can be modified by FGE to generate FGly modified antibodies. Exemplary constant regions include human γ1 and γ3 regions. In addition to the sulfatase motif, the modified constant region can have a wild-type amino acid sequence, or it can have an amino acid sequence that is at least 70% identical (e.g., at least 80%, at least 90% or at least 95% identical) to the wild-type amino acid sequence.

[0661] In some embodiments, the sulfatase motif is located at a position other than or at the C-terminus of the Ig polypeptide heavy chain. As described above, an isolated aldehyde-tagged antibody may comprise a heavy chain constant region modified to include a sulfatase motif as described above, wherein the sulfatase motif is located within or adjacent to a surface accessible loop region of the antibody heavy chain constant region.

[0662] A sulfatase motif may be provided within or adjacent to one or more of these amino acid sequences of such modification sites of the Ig heavy chain. For example, an Ig heavy chain polypeptide may be modified (e.g., wherein the modification comprises one or more amino acid residue insertions, deletions, and / or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif at the N-terminus and adjacent to these modification sites and / or at the C-terminus and adjacent to these modification sites. Alternatively or additionally, an Ig heavy chain polypeptide may be modified (e.g., wherein the modification comprises one or more amino acid residue insertions, deletions, and / or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif between any two residues of the Ig heavy chain modification site. In some embodiments, an Ig heavy chain polypeptide may be modified to include two motifs, which may be adjacent to each other, or may be separated by 1, 2, 3, 4 or more (e.g., about 1 to about 25, about 25 to about 50, or about 50 to about 100, or more) amino acids. Alternatively, or additionally, where the native amino acid sequence provides one or more amino acid residues of a sulfatase motif sequence, selected amino acid residues at a modification site of the Ig heavy chain polypeptide amino acid sequence may be modified (e.g., where the modification comprises one or more amino acid residue insertions, deletions, and / or substitutions) to provide a sulfatase motif at the modification site.

[0663] The antibodies used in the antibody-drug conjugates of the present disclosure may have any of a variety of antigen binding specificities, including, but not limited to, antigens present on cancer cells; antigens present on autoimmune cells; antigens present on pathogenic microorganisms; antigens present on virus-infected cells (e.g., human immunodeficiency virus-infected cells); antigens present on diseased cells; and the like. For example, the antibody conjugates may bind to an antigen, wherein the antigen is present on the surface of a cell. The antibody conjugates of the present disclosure may bind to an antigen with a suitable binding affinity, e.g., 5 x 10 -6 M to 10 -7 M, 10 -7 M to 5x 10 -7 M, 5x10 -7 M to 10 -8 M, 10 -8 M to 5x 10 -8 M, 5x10 -8 M to 10 -9 M, or greater than 10 -9 The binding affinity of M.

[0664] As a non-limiting example, a subject antibody conjugate can bind to an antigen present on a cancer cell (e.g., a tumor-specific antigen; an antigen overexpressed on a cancer cell; etc.), and the conjugated moiety can be a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.). For example, a subject antibody conjugate can be specific for an antigen on a cancer cell, wherein the conjugated moiety is a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).

[0665] As another non-limiting example, a subject antibody conjugate can bind to an antigen present on a cell infected with a virus (e.g., where the antigen is encoded by a virus; where the antigen is expressed on a cell type infected by the virus; etc.), and the conjugated moiety can be a drug, such as a viral fusion inhibitor. For example, a subject antibody conjugate can bind to an antigen present on a cell infected with a virus, and the conjugated moiety can be a drug, such as a viral fusion inhibitor.

[0666] Drugs for conjugation to peptides

[0667] The present disclosure provides drug-polypeptide conjugates (eg, antibody-drug conjugates). Any of a variety of drugs are suitable for use, or can be modified to make them suitable for use, as a reactive partner conjugated to an antibody. Examples of drugs include small molecule drugs and peptide drugs.

[0668] As used herein, "small molecule drug" refers to a compound, such as an organic compound, that exhibits a pharmaceutical activity of interest and generally has a molecular weight of 800Da or less, or 2000Da or less, but may encompass molecules up to 5kDa and may be as large as 10kDa. An inorganic small molecule refers to a molecule that does not contain a carbon atom, while an organic small molecule refers to a compound that contains at least one carbon atom.

[0669] As used herein, "peptide drugs" refer to polymeric compounds containing amino acids, and are intended to cover naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides, and proteins, as well as peptide mimetics. Peptide drugs can be obtained by chemical synthesis, or can be generated from a genetically encoded source (e.g., a recombinant source). Peptide drugs can have a range of molecular weights, and the molecular weight can be 200Da to 10kDa or greater. Suitable peptides include, but are not limited to, cytotoxic peptides; angiogenic peptides; anti-angiogenic peptides; peptides that activate B cells; peptides that activate T cells; antiviral peptides; peptides that inhibit viral fusion; peptides that increase the generation of one or more lymphocyte populations; antimicrobial peptides; growth factors; growth hormone releasing factors; vasoactive peptides; anti-inflammatory peptides; peptides that regulate glucose metabolism; antithrombotic peptides; anti-injury (nociceptive) peptides; vasodilating peptides; platelet aggregation inhibitors; analgesics; and the like.

[0670] Examples of drugs include small molecule drugs, such as cancer chemotherapeutics. For example, in the case where the polypeptide is an antibody (or fragment thereof) that is specific for tumor cells, the antibody can be modified as described herein to include modified amino acids, which can then be conjugated to cancer chemotherapeutics. Cancer chemotherapeutics include non-peptide (i.e., non-protein) compounds that reduce cancer cell proliferation, and also encompass cytotoxic agents and cytostatics. Non-limiting examples of chemotherapeutics include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptide compounds can also be used.

[0671] Suitable cancer chemotherapeutic agents include dolastatin and its active analogs and derivatives; and auristatin and its active analogs and derivatives (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). See, for example, WO 96 / 33212, WO 96 / 14856, and US 6,323,315. For example, dolastatin 10 or auristatin PE can be included in the antibody-drug conjugates of the present disclosure. Suitable cancer chemotherapeutic agents also include maytansinoids and their active analogs and derivatives (see, e.g., EP 1391213; and Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and their active analogs and derivatives (e.g., including the synthetic analogs KW-2189 and CB 1-TM1); and benzodiazepines and their active analogs and derivatives (e.g., pyrrolobenzodiazepines (PBD)).

[0672] Agents that act to reduce cell proliferation are known in the art and are widely used. Such agents include alkylating agents such as nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkyl sulfonates, and triazenes, including but not limited to mechlorethamine, cyclophosphamide (Cytoxan TM ), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0673] Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including but not limited to cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (Mercaptopurine), and cytosine arabinoside. ine)(6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolate (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0674] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include, but are not limited to, Ara-C, paclitaxel Docetaxel deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids such as vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins such as etoposide, teniposide teniposide, etc.; antibiotics, such as anthracyclines, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxazine bicyclic peptides (phenoxizone biscyclopeptide, such as dactinomycin; basic glycopeptide, such as bleomycin; anthraquinone glycoside, such as plicamycin (mithramycin); anthracenedione, such as mitoxantrone; azirinopyrrolo indoledione, such as mitomycin; macrocyclic immunosuppressants, such as cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc.; and the like.

[0675] Other antiproliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosfamide, and droloxafine.

[0676] Microtubule-affecting agents with antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (NSC 33619), and sirolimus (NSC 33617). Derivatives, docetaxel Thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones (including but not limited to epothilone A, epothilone B), discodermolide; estramustine, nocodazole, and the like.

[0677] Hormone modulators and steroids (including synthetic analogs) suitable for use include, but are not limited to, adrenocortical steroids, such as prednisone, dexamethasone, etc.; estrogens and progestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, etc.; and adrenocortical suppressants, such as aminoglutethimide; 17α-ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, 17α-ethinylestradiol ... propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Estrogen stimulates proliferation and differentiation; therefore, compounds that bind to estrogen receptors are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0678] Other suitable chemotherapeutic agents include metal complexes, such as cis-DDP, carboplatin, etc.; ureas, such as hydroxyurea; and hydrazines, such as N-methylhydrazine; epipodophyllotoxin; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Other antiproliferative agents of interest include immunosuppressants, such as mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline); etc.

[0679] Taxanes are suitable for use. "Taxane" includes paclitaxel, and any active taxane derivative or prodrug. "Paclitaxel" (which should be understood herein to include analogs, formulations, and derivatives, such as docetaxel, TAXOL TM ,TAXOTERE TM (a formulation of docetaxel), a 10-deacetyl analog of paclitaxel and a 3'N-debenzoyl-3'N-tert-butyloxycarbonyl analog of paclitaxel) can be readily prepared using techniques known to those skilled in the art (see also WO 94 / 07882, WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO 93 / 10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtained from a variety of commercial sources, including, for example, Sigma Chemical Co., St. Louis, Mo. (T7402, from Taxus brevifolia; or T-1912, from Taxus yannanensis).

[0680] Paclitaxel should be understood to refer not only to the common chemically available form of paclitaxel, but also to analogs and derivatives (e.g., Taxotere TMdocetaxel) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextraside, or paclitaxel-xylose).

[0681] Also included within the term "taxane" are various known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99 / 18113; piperazino and other derivatives described in WO 99 / 14209; taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98 / 28288; sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and taxol derivatives described in U.S. Pat. No. 5,415,869. It further includes prodrugs of paclitaxel, including, but not limited to, those described in WO 98 / 58927; WO 98 / 13059; and U.S. Pat. No. 5,824,701.

[0682] Suitable biological response modifiers for use include, but are not limited to: (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine / threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) colony stimulating factor; and (9) inhibitors of angiogenesis.

[0683] Examples of drugs include small molecule drugs, such as cancer chemotherapeutics. For example, in the case where the polypeptide is an antibody (or fragment thereof) specific to tumor cells, the antibody can be modified as described herein to include modified amino acids, which can then be conjugated to cancer chemotherapeutics, such as agents that affect microtubules. In certain embodiments, the drug is an agent that affects microtubules with antiproliferative activity, such as maytansinoids. In certain embodiments, the drug is a maytansinoid of the following structure:

[0684]

[0685] in Indications for the conjugates and compounds described herein are midaminoid alkaloids and second linkers L 2 The attachment point between The symbol indicates the N and the second linker L of the maytansinoid in the conjugates and compounds described herein. 2 For example, in formula (I), W 1It may be a maytansinoid, such as a maytansinoid of the structure above, wherein Indication of maytansinoid and second linker L 2 In some cases, the maytansinoid of the above structure may be referred to as deacetyl maytansinoid.

[0686] In certain embodiments, the drug is an antimitotic agent, such as auristatin or an active auristatin analog or derivative thereof (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). In certain embodiments, the drug is MMAE, which has the following structure:

[0687]

[0688] For example, an MMAE active agent can be included in an antibody-drug conjugate as follows:

[0689]

[0690] in Indications of the auristatin and second linker L in the conjugates and compounds described herein 2 For example, The symbol indicates N and the second linker L of auristatin 2 For example, in formula (I), W 1 It can be an auristatin, such as MMAE, wherein Indicates MMAE and the second linker L 2 The attachment points between them.

[0691] In certain embodiments, the drug is a DNA alkylating agent, such as a duocarmycin. Examples of duocarmycins include, but are not limited to, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065. In some embodiments, the duocarmycin is a duocarmycin analog, such as, but not limited to, adozelesin, bizelesin, or carzelesin.

[0692] In some cases, the duocarmycin is a compound having the following structure:

[0693]

[0694] For example, a duocarmycin active agent can be included in an antibody-drug conjugate as follows:

[0695]

[0696] in Indications for the duocarmycin and second linker L in the conjugates and compounds described herein 2 For example, Symbols indicate duocarmycin and the second linker L 2 The bonds between are as shown in formula (I). For example, in formula (I), W 1 It may be a duocarmycin, such as the duocarmycin of the structure above, wherein Indicator Duocarmycin and second linker L 2 The attachment points between them.

[0697] As described above, in certain embodiments, L 2 It is through the formula -(L 21 ) e -(L 22 ) f -(L 23 ) g -(L 24 ) h - to describe the second linker, wherein L 21 , L 22 , L 23 and L 24 Each independently is a second linker subunit. In certain embodiments, L 21 Attached to the first cleavable module. In certain embodiments, L 21 , if present, also attached to W 1 (Drug). In certain embodiments, L 22 , if present, attached to W 1 (Drug). In certain embodiments, L 23 , if present, attached to W 1 (Drug). In certain embodiments, L 24 , if present, attached to W 1 (drug).

[0698] As described above, in certain embodiments, the second linker -(L 21 ) e -(L 22 ) f -(L 23 ) g -(L 24 ) h -Through-(T 5 -V 5 ) e -(T 6 -V 6 )f -(T 7 -V 7 ) g -(T 8 -V 8 ) h -, wherein e, f, g and h are each independently 0 or 1, wherein the sum of e, f, g and h is 0 to 4. In certain embodiments, as described above, L 21 Attached to the first cleavable module. As such, in certain embodiments, T 5 Attached to the first cleavable module. In certain embodiments, V 5 Attached to W 1 (Drug). In certain embodiments, as described above, L 22 , if present, attached to W 1 (Drug). Accordingly, in certain embodiments, T 6 , if present, attached to W 1 (drug), or V 6 , if present, attached to W 1 (Drug). In certain embodiments, as described above, L 23 , if present, attached to W 1 (Drug). Accordingly, in certain embodiments, T 7 , if present, attached to W 1 (drug), or V 7 , if present, attached to W 1 (Drug). In certain embodiments, as described above, L 24 , if present, attached to W 1 (Drug). Accordingly, in certain embodiments, T 8 , if present, attached to W 1 (drug), or V 8 , if present, attached to W 1 (drug).

[0699] Embodiments of the present disclosure include conjugates in which antibodies are conjugated to one or more drug moieties, such as 2 drug moieties, 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drug moieties, or 10 or more drug moieties. The drug moiety can be conjugated to the antibody at one or more sites in the antibody, as described herein. In certain embodiments, the conjugate has an average drug to antibody ratio (DAR) (molar ratio) in the range of 0.1 to 10, or 0.5 to 10, or 1 to 10, such as 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In certain embodiments, the conjugate has an average DAR of 1 to 2, such as 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2. In certain embodiments, the conjugate has an average DAR of 1 to 1.5. In certain embodiments, the conjugates have an average DAR of 1.5 to 2. Average means the arithmetic mean.

[0700] The drug to be conjugated to the polypeptide can be modified to incorporate a reactive partner for reacting with the polypeptide. In the case where the drug is a peptide drug, a reactive module (such as an aminooxy group or a hydrazide) can be located at the N-terminal region, N-terminal, C-terminal region, C-terminal, or internal position of the peptide. For example, an example of a method relates to synthesizing a peptide drug with an aminooxy group. In this example, a peptide is synthesized from a precursor protected by Boc. The amino group of the peptide can react with a compound comprising a carboxylic acid group and an oxy-N-Boc group. As an example, the amino group of the peptide reacts with 3-(2,5-dioxopyrrolidine-1-yloxy) propionic acid. Other changes on the compound comprising a carboxylic acid group and an oxy-N-protecting group can include a substituent on the different number of carbons in the alkylene joint and the alkylene joint. The reaction between the amino group of the peptide and the compound comprising a carboxylic acid group and an oxy-N-protecting group occurs via standard peptide coupling chemistry. Examples of peptide coupling agents that can be used include, but are not limited to, DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1-benzotriazole diethylphospho-1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide), EDC (1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride (cyanuric acid chloride), TFFH (tetramethylfluoroformamide hexafluorophosphate), DPPA (diphenylphosphoryl azide), BOP (benzotriazol-1-yloxytris(dimethylamino)phosphine hexafluorophosphate), HBTU (O-benzotriazol-1-yl-N,N,N',N'-tetramethylurea hexafluorophosphate), TBTU (O-benzotriazol-1-yl-N,N,N',N'-tetramethylurea tetrafluoroborate), TSTU (O-(N-succinimidyl)-N,N,N',N'-tetramethylurea tetrafluoroborate), HATU (N-[(dimethylamino)-1-H-1,2,3-triazolo[2- ... [4,5,6]-pyridin-1-ylmethylene]--N-methylmethanamine N-oxide), BOP-Cl (bis(2-oxo-3-oxazolidinyl)phosphinyl chloride), PyBOP ((1-H-1,2,3-benzotriazol-1-yloxy)-tris(pyrrolidinyl)phosphine tetrafluorophosphate), BrOP (bromotris(dimethylamino)phosphine hexafluorophosphate), DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)-one), PyBrOP (bromotris(pyrrolidinyl)phosphine hexafluorophosphate). As a non-limiting example, HOBt and DIC can be used as peptide coupling reagents.

[0701] Deprotection is performed on peptides containing N-protecting groups to expose aminooxy functionality. For example, deprotection of N-oxysuccinimide groups occurs according to standard deprotection conditions for cyclic amide groups. Deprotection conditions can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons NY and Harrison et al. Certain deprotection conditions include hydrazine reagents, amino reagents, or sodium borohydride. Deprotection of Boc protecting groups can occur with TFA. Other reagents used for deprotection include, but are not limited to, hydrazine, methylhydrazine, phenylhydrazine, sodium borohydride, and methylamine. Products and intermediates can be purified by conventional means, such as HPLC purification.

[0702] It will be appreciated by those of ordinary skill that factors such as pH and steric hindrance (i.e., the accessibility of the amino acid residue to the reactive partner of interest) are important. Modifying the reaction conditions to provide optimal conjugation conditions is within the skill of those of ordinary skill and is routine in the art. In the case of conjugation with a polypeptide present in or on a living cell, conditions are selected to make it physiologically compatible. For example, pH can temporarily decline for a period of time, enough to allow the reaction to occur but within a period of time (e.g., about 30 minutes to 1 hour) tolerated by the cell. Physiological conditions for carrying out polypeptide modification on the cell surface can be similar to those used in the ketone-azide reaction of cells carrying cell surface azides (see, e.g., US 6,570,040).

[0703] Small molecule compounds containing or modified to contain α-nucleophilic groups that serve as reactive partners of the compounds or conjugates disclosed herein also encompass use as drugs in the polypeptide-drug conjugates of the present disclosure. General methods for chemical synthesis schemes and conditions useful for synthesizing compounds of interest are known in the art (see, e.g., Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).

[0704] Formulations

[0705] The conjugates of the present disclosure may be formulated in a variety of different ways. Generally, where the conjugate is an antibody-drug conjugate, the conjugate is formulated in a manner that is compatible with the drug, the antibody, the condition to be treated, and the route of administration to be used.

[0706] In some embodiments, provided is a pharmaceutical composition comprising any conjugate of the present disclosure and a pharmaceutically acceptable excipient.

[0707] The conjugate (e.g., antibody-drug conjugate) may be provided in any suitable form (e.g., in the form of a pharmaceutically acceptable salt) and may be formulated for any suitable route of administration, such as oral, topical or parenteral administration. In the case where the conjugate is provided as a liquid injectable (such as in those embodiments where they are administered intravenously or directly into a tissue), the conjugate may be provided as a ready-to-use dosage form, or as a reconstituted storage-stable powder or liquid consisting of a pharmaceutically acceptable carrier and excipients.

[0708] Methods for formulating the conjugate can be adapted from those readily available. For example, the conjugate can be provided in a pharmaceutical composition comprising a therapeutically effective amount of the conjugate and a pharmaceutically acceptable carrier (e.g., saline). The pharmaceutical composition may optionally include other additives (e.g., buffers, stabilizers, preservatives, etc.). In some embodiments, the formulation is suitable for administration to mammals, such as those suitable for administration to humans.

[0709] Treatment

[0710] The antibody-drug conjugates of the disclosure can be used to treat a condition or disease in a subject that is amenable to treatment by administration of the parent drug (ie, the drug prior to conjugation to the antibody).

[0711] In some embodiments, provided are methods comprising administering to a subject an effective amount (eg, a therapeutically effective amount) of any conjugate of the disclosure.

[0712] In certain aspects, provided is a method of delivering a drug to a target site in a subject, the method comprising administering to a subject a pharmaceutical composition comprising any conjugate of the present disclosure, wherein the administration effectively releases a therapeutically effective amount of the drug from the conjugate at the target site in the subject. For example, as described herein, the antibody-drug conjugate of the present disclosure may include a cleavable linker, such as an enzyme cleavable linker or a chemical cleavable linker, comprising a first cleavable module and a second cleavable module, wherein the second cleavable module hinders the cleavage of the first cleavable module. In some cases, the cleavable linker may be cleaved under suitable conditions to separate or release the drug from the antibody at the desired target site of the drug. For example, the second cleavable linker that protects the first cleavable linker from cleavage may be cleaved to allow the first cleavable module to be cleaved, resulting in the cleavage of the cleavable linker into two or more parts, so that the drug is released from the antibody-drug conjugate at the desired site of action.

[0713] In certain embodiments, the first cleavable module can be an enzyme cleavable module. In some cases, the enzyme that promotes the cleavage of the first cleavable module is an enzyme applied to the subject to be treated (i.e., exogenous to the subject to be treated). For example, the first enzyme can be applied before the antibody-drug conjugate described herein is applied, in parallel with the antibody-drug conjugate described herein, or after the antibody-drug conjugate described herein is applied.

[0714] In certain embodiments, the second cleavable module can be an enzyme cleavable module. In some cases, the enzyme that promotes the cleavage of the second cleavable module is an enzyme applied to the subject to be treated (that is, exogenous to the subject to be treated). For example, before applying the antibody-drug conjugate described herein, in parallel with the antibody-drug conjugate described herein, or after the antibody-drug conjugate described herein, the second enzyme is applied. In certain embodiments, the first enzyme and the second enzyme are different enzymes.

[0715] In other cases, the first enzyme that promotes the cutting of the first cleavable module is an enzyme present in the subject to be treated (that is, endogenous to the subject to be treated). For example, the first enzyme may be present at the desired action site of the drug of the antibody-drug conjugate. The antibody-drug conjugate may be targeted to the desired action site (for example, the antigen present at the desired action site may be specifically bound), wherein the desired action site also includes the presence of the first enzyme. In some cases, compared with other regions in the body of the subject to be treated, the first enzyme is present in excess at the desired action site. For example, compared with other regions in the body of the subject to be treated, the first enzyme may be overexpressed at the desired action site. In some cases, due to the positioning of the first enzyme in a specific region or position, the first enzyme is present in excess at the desired action site. For example, the first enzyme may be associated with a certain structure in the desired action site, such as a lysosome. In some cases, compared with other regions in the body of the subject, the first enzyme is present in excess in a lysosome. In some embodiments, a lysosome comprising a first enzyme is found at the desired site of action of the drug of the antibody-drug conjugate, such as the site of a cancer or tumor to be treated with the drug. In certain embodiments, the first enzyme is a protease, such as a human protease (e.g., cathepsin B).

[0716] In other cases, the second enzyme that promotes the cutting of the second cleavable module is an enzyme present in the subject to be treated (that is, endogenous to the subject to be treated). For example, the second enzyme may be present at the desired action site of the drug of the antibody-drug conjugate. The antibody-drug conjugate may be targeted to the desired action site (for example, the antigen present at the desired action site may be specifically bound), wherein the desired action site also includes the presence of the second enzyme. In some cases, compared with other regions in the body of the subject to be treated, the second enzyme is present in excess at the desired action site. For example, compared with other regions in the body of the subject to be treated, the second enzyme may be overexpressed at the desired action site. In some cases, due to the positioning of the second enzyme in a specific region or position, the second enzyme is present in excess at the desired action site. For example, the second enzyme may be associated with a certain structure in the desired action site, such as a lysosome. In some cases, compared with other regions in the body of the subject, the second enzyme is present in excess in a lysosome. In some embodiments, the lysosome comprising the second enzyme is found at the desired site of action of the drug of the antibody-drug conjugate, such as the site of a cancer or tumor to be treated with the drug. In certain embodiments, the second enzyme is a glucuronidase, such as human glucuronidase (e.g., lysosomal β-glucuronidase).

[0717] Any suitable enzyme can be used to cleave the first cleavable moiety and the second cleavable moiety of the antibody-drug conjugate described herein. Other enzymes may also be suitable for cleaving the first cleavable moiety and the second cleavable moiety of the antibody-drug conjugate described herein, such as but not limited to enzymes from other vertebrates (e.g., primates, mice, rats, cats, pigs, quail, goats, dogs, etc.).

[0718] In certain embodiments, the antibody-drug conjugate is substantially stable under standard conditions. Substantial stability means that the cleavable joint of the antibody-drug conjugate does not undergo a significant amount of cleavage under the absence of the first enzyme and the second enzyme as described above. For example, as described above, the second cleavable module can protect the first cleavable module from being cleaved, and as such, the cleavable joint of the antibody-drug conjugate does not undergo a significant amount of cleavage under the absence of the second enzyme as described above. For example, the cleavable joint of the antibody-drug conjugate can be substantially stable, so that 25% or less of the antibody-drug conjugate is cleaved under the absence of the first enzyme and / or the second enzyme, such as 20% or less, or 15% or less, or 10% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less. In some cases, the antibody-drug conjugate is substantially stable, so that the cleavable joint of the antibody-drug conjugate does not undergo a significant amount of cleavage under the absence of the first enzyme and / or the second enzyme, but can be cleaved when the first enzyme and the second enzyme are present. For example, antibody-drug conjugates can be substantially stable after being applied to a subject. In some cases, antibody-drug conjugates are substantially stable after being applied to a subject, and then, when the antibody-drug conjugate is in the presence of a second enzyme at the desired site of action, the second cleavable module can be cut from a cleavable linker, so exposing the first cleavable module to the subsequent cutting of the first enzyme, followed by release of the drug at the desired site of action. In certain embodiments, after being applied to a subject, antibody-drug conjugates are stable for a period of time extended under the absence of the first enzyme and / or the second enzyme, such as 1 hour or more, or 2 hours or more, or 3 hours or more, or 4 hours or more, or 5 hours or more, or 6 hours or more, or 7 hours or more, or 8 hours or more, or 9 hours or more, or 10 hours or more, or 15 hours or more, or 20 hours or more, or 24 hours (1 day) or more, or 2 days or more, or 3 days or more, or 4 days or more, or 5 days or more, or 6 days or more, or 7 days (1 week) or more. In certain embodiments, the antibody-drug conjugate is stable in the absence of the first enzyme and / or the second enzyme for an extended period of time over a range of pH values, such as a pH in the range of 2 to 10, or 3 to 9, or 4 to 8, or 5 to 7, or 6 to 7.

[0719] As described above, the antibody-drug conjugates of the present disclosure can be used to treat a condition or disease in a subject that is suitable for treatment by administering the parent drug. "Treatment" means at least achieving an improvement in the symptoms associated with the condition that afflicts the host, wherein improvement is used in a broad sense to refer to at least a reduction in the degree of a parameter (e.g., symptom) associated with the condition being treated. As such, treatment also includes situations in which a pathological condition or at least a symptom associated therewith is completely suppressed (e.g., prevented from occurring), or stopped (e.g., terminated), such that the host no longer suffers from the condition or at least the symptoms that characterize the condition. Thus, treatment includes: (i) prevention, i.e., reducing the risk of clinical symptoms occurring, including causing clinical symptoms not to occur, such as preventing the disease from progressing to a harmful state; (ii) inhibition, i.e., blocking the occurrence or further development of clinical symptoms, such as alleviating or completely inhibiting active disease; and / or (iii) relief, i.e., causing clinical symptoms to subside.

[0720] The subject to be treated can be a subject in need of treatment, wherein the subject to be treated is a subject suitable for treatment with the parent drug. Thus, a variety of subjects may be suitable for treatment with the antibody-drug conjugate disclosed herein. In general, such subjects are "mammals", of which humans are of interest. Other subjects may include domesticated pets (e.g., dogs and cats), livestock (e.g., cattle, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, e.g., in animal models of disease), and non-human primates (e.g., chimpanzees and monkeys).

[0721] The amount of the antibody-drug conjugate administered can initially be determined based on the dosage of the parent drug and / or the guidance of the dosage regimen. In general, the antibody-drug conjugate can provide targeted delivery and / or extended serum half-life to the bound drug, thus providing at least one of reduced dosage or reduced administration in the dosage regimen. Thus, relative to the parent drug before conjugation in the antibody-drug conjugate of the present disclosure, the antibody-drug conjugate can provide reduced dosage and / or reduced administration in the dosage regimen.

[0722] Furthermore, as described above, because antibody-drug conjugates can provide controlled stoichiometry of drug delivery, the dosage of the antibody-drug conjugate can be calculated based on the number of drug molecules provided on a per antibody-drug conjugate basis.

[0723] In some embodiments, multiple doses of the antibody-drug conjugate are administered. The frequency of administration of the antibody-drug conjugate may vary with any of a variety of factors, such as the severity of the symptoms, the condition of the subject, etc. For example, in some embodiments, the antibody-drug conjugate is administered once a month, twice a month, three times a month, every other week, once a week (qwk), twice a week, three times a week, four times a week, five times a week, six times a week, every other day, every day (qd / od), twice a day (bds / bid), or three times a day (tds / tid), etc.

[0724] Methods for treating cancer

[0725] The present disclosure provides methods comprising delivering a conjugate of the present disclosure to an individual with cancer. The methods are useful for treating a wide variety of cancers, including carcinomas, sarcomas, leukemias, and lymphomas. In the context of cancer, the term "treating" includes one or more (e.g., each) of the following: reducing the growth of solid tumors, inhibiting the replication of cancer cells, reducing overall tumor burden, and ameliorating one or more symptoms associated with cancer.

[0726] Cancers that can be treated using the subject methods include, but are not limited to, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (of various tissues), bladder cancer (including transitional cell carcinoma (a malignant neoplasm of the bladder)), bronchogenic carcinoma, colon cancer, colorectal cancer, gastric cancer, lung cancer (including small cell carcinoma and non-small cell carcinoma of the lung), adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, osteogenic cancer, epithelial cancer, and nasopharyngeal carcinoma, among others.

[0727] Sarcomas that can be treated using the subject methods include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0728] Other solid tumors that can be treated using the subject methods include, but are not limited to, gliomas, astrocytomas, medulloblastomas,

[0729] Craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma,

[0730] neuroblastoma, and retinoblastoma.

[0731] Leukemias that can be treated using the subject methods include, but are not limited to: a) chronic myeloproliferative syndrome (a neoplastic disorder of a multipotent hematopoietic stem cell); b) acute myeloid leukemia (a neoplastic transformation of a multipotent hematopoietic stem cell or a hematopoietic cell of restricted lineage potential); c) chronic lymphocytic leukemia (CLL; a clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell CLL, T-cell CLL, prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemia (characterized by the accumulation of lymphoblasts). Lymphomas that can be treated using the subject methods include, but are not limited to, B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; non-Hodgkin's B cell lymphoma; and the like.

[0732] In certain aspects, provided are methods of treating cancer in a subject, such methods comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising any of the conjugates of the disclosure, wherein the administration is effective to treat the cancer in the subject.

[0733] Example

[0734] The following examples are presented to provide one of ordinary skill in the art with a comprehensive disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to claim that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numerical values ​​used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric pressure. "Average" means the arithmetic mean. Standard abbreviations may be used, for example, bp, base pairs; kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; kb, kilobases; bp, base pairs; nt, nucleotides; im, intramuscular; ip, intraperitoneal; sc, subcutaneous; and the like.

[0735] General synthetic protocol

[0736] Many general references are available that provide commonly known chemical synthesis schemes and conditions useful for synthesizing the disclosed compounds (see, for example, Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).

[0737] Compounds as described herein can be purified by any purification scheme known in the art, including chromatography, such as HPLC, preparative thin layer chromatography, flash column chromatography, and ion exchange chromatography. Any suitable stationary phase can be used, including normal phase and reverse phase and ionic resins. In certain embodiments, the disclosed compounds are purified via silica gel and / or alumina chromatography. See, for example, Introduction to Modern Liquid Chromatography, 2nd Edition, ed. LR Snyder and JJ Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed. E. Stahl, Springer-Verlag, New York, 1969.

[0738] During any process for preparing the subject compounds, it may be necessary and / or desirable to protect sensitive or reactive groups on any molecule of interest. This may be accomplished with the aid of conventional protecting groups as described in standard texts such as JFW McOmie,

[0739] "Protective Groups in Organic Chemistry", Plenum Press, London and New York 1973; TW Greene and PGMWuts, "Protective Groups in Organic Synthesis", Third edition, Wiley, New York 1999; "The Peptides"; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981; "Methoden derorganischen Chemie", Houben-Weyl, 4 th edition, Vol. 15 / 1, Georg Thieme Verlag, Stuttgart 1974; H.-D. Jakubke and H. Jescheit, "Aminosauren, Peptide, Protein", Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982; and / or Jochen Lehmann, "Chemie der Kohlenhydrate: Monosaccharide and Derivate", Georg Thieme Verlag, Stuttgart 1974. The protecting groups can be removed at a convenient subsequent stage using methods known in the art.

[0740] The subject compounds can be synthesized via a variety of different synthetic routes using commercially available starting materials and / or starting materials prepared by conventional synthetic methods. Several examples of synthetic routes that can be used to synthesize the compounds disclosed herein are described in the schemes below.

[0741] Synthetic reagents were purchased from Sigma-Aldrich, Acros, or other commercial sources and used without purification. Anhydrous solvents were obtained from commercial sources in sealed bottles. Acetyl bromide-α-D-glucuronic acid methyl ester 1, MMAE 8, 17, 46, 57, and 62 were purchased from commercial sources. Unless otherwise noted, all reactions were carried out in flame-dried glassware under N2. In all cases, the solvent was removed by decompression using a Buchi Rotovapor R-114 equipped with a Buchi V-700 vacuum pump. Column chromatography was performed using a Biotage Isolera Prime chromatograph. Preparative HPLC purification was performed using a Waters preparative HPLC unit equipped with a Phenomenex Kinetex 5μmEVO C18 150x 21.2mm column. HPLC analysis was performed on an Agilent 1100 series analytical HPLC equipped with a G1322A degasser, a G1311A quadrupole pump, a G1329A autosampler, a G1314 variable wavelength detector, an Agilent Poroshell 120SB C18, 4.6 mm x 50 mm column at room temperature using a 10-100% gradient of water and acetonitrile containing 0.1% formic acid. HPLC was monitored at 254 nm.

[0742] Example 1

[0743] Synthesis of MMAE construct 13

[0744] The monomethyl auristatin E (MMAE) construct was synthesized according to Scheme 1 shown below.

[0745]

[0746]

[0747]

[0748] Preparation of (2S,3R,4S,5S,6S)-2-(5-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2)

[0749] To a solution of methyl acetyl bromide-α-D-pyranoglucopyranose ester 1 (6.3 g, 15.9 mmol) and 3-hydroxy-4-nitrobenzaldehyde (0.9 g, 5.4 mmol) in acetonitrile (100 mL) was added AgO (10.0 g, 43.1 mmol). The reaction mixture was stirred in the dark at room temperature for 60 hours. The reaction mixture was then concentrated under vacuum, and the residue was purified using column chromatography (hexane / EtOAc, 9:1 to 1:9 v / v) to produce compound 2 (2.1 g, 81%) as an off-white solid.

[0750] Calculation: [M+Na] + (C 20 H 21 NNaO 13 )m / z=506.1; found ESI: [M+Na] + (C 20 H 21 NNaO 13 )m / z=505.7.

[0752] Preparation of (2S,3R,4S,5S,6S)-2-(2-amino-5-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (3)

[0753] Pd / C (10wt%, 20mg) and triethylamine (20 μL, 0.14mmol) were added to a solution of compound 2 (460mg, 0.95mmol) in EtOAc (15mL). The flask was then emptied and hydrogen was injected from a balloon, and three cycles were repeated. The reaction was stirred vigorously at room temperature for 24 hours with a H2 balloon connected. After removing the catalyst by filtration through a diatomaceous earth pad, the filtrate was concentrated under vacuum. The residue was dried under high vacuum for 1 hour to obtain a white solid (425mg, 99%). It was used directly in the next step without further purification.

[0754] Calculation: [M+H] + (C 20 H 26 NO 11 )m / z=456.2; found ESI: [M+H] + (C 20 H 26 NO 11 )m / z=455.6.

[0755] Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propionamido)-5-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (4)

[0756] To a mixture of Fmoc-Ala-OH (400 mg, 1.29 mmol) and crude intermediate 3 (425 mg, 0.93 mmol) in DCM (0.9 mL) and MeOH (0.1 mL) was added N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 390 mg, 1.58 mmol). The reaction was stirred at room temperature for 1 hour. After removing the solvent under vacuum, the residue was purified using column chromatography (hexane / EtOAc, 9:1 to 1:9 v / v) to produce compound 4 (0.64 g, 90% for two steps) as a white solid.

[0757] Calculation: [M+H] + (C 38 H 41 N2O 14 )m / z=749.3; found ESI: [M+H] + (C 38 H 41 N2O 14 )m / z=748.7.

[0758] Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-aminopropionamido)-5-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (5)

[0759] Pd / C (10wt%, 40mg) was added to a mixture of compound 4 (640mg, 0.86mmol) in MeOH (15mL). The flask was then evacuated and H2 gas was injected from a balloon, and three cycles were repeated. The reaction was vigorously stirred at room temperature for 3 days while a H2 balloon was connected. After removing the catalyst by filtration through a diatomaceous earth pad, the filtrate was concentrated under vacuum. The residue was purified by reverse phase chromatography using acetonitrile-water (0.05% TFA, 5-70%) as eluent. The fractions containing the desired compound were combined and concentrated under vacuum to produce compound 5 (341mg, 76%) as a white solid.

[0760] Calculation: [M+H] + (C 23 H 31 N2O 12 )m / z=527.2; found ESI: [M+H] +(C 23 H 31 N2O 12 )m / z=526.8.

[0761] Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)propionamido)-5-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (6)

[0762] To the solution of amine 5 (255mg, 0.49mmol) and Fmoc-valine-OH (170mg, 0.50mmol) in DMF (2mL), DIPEA (0.15mL, 0.86mmol) and PyAOP (260mg, 0.50mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. The mixture was diluted with DCM (70mL) and washed with NH4Cl saturated aqueous solution (50mL), water (50mL), and NaCl saturated aqueous solution (10mL). The organic layer was dried with MgSO4, filtered, and evaporated. The residue was purified by column chromatography (hexane / EtOAc, 1:9 to 9:1 v / v) to produce compound 6 (334mg, 81%) as a white solid.

[0763] Calculation: [M+H] + (C 43 H 50 N3O 15 )m / z=848.3; found ESI: [M+H] + (C 43 H 50 N3O 15 )m / z=848.3.

[0764] Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)propionamido)-5-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (7)

[0765] To a solution of benzyl alcohol 6 (334 mg, 0.39 mmol) and bis(4-nitrophenyl) carbonate (240 mg, 0.79 mmol) in THF (3 mL) was added DIPEA (0.1 mL, 0.57 mmol). The mixture was stirred at room temperature for 24 hours. After removing the solvent under vacuum, the residue was purified by column chromatography (hexane / EtOAc, 1:9 to 9:1 v / v) to produce compound 7 (328 mg, 82%) as a white solid.

[0766] Calculation: [M+H] + (C 50 H 53 N4O 19 )m / z=1013.3; found ESI: [M+H] + (C 50 H 53 N4O 19 )m / z=1013.3. Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)propionamido)-5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R) (1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (9)

[0767] To the solution of PNP carbonate 7 (112mg, 0.11mmol) and MMAE 8 (130mg, 0.16mmol) in DMF (1.5mL), DIPEA (50uL, 0.29mmol) and HOAt (15mg, 0.11mmol) were added. The reaction was stirred at room temperature for 20 hours. EtOAc (100mL) was added to the mixture. The organic layer was washed with NH4Cl saturated aqueous solution, water, and NaCl saturated aqueous solution. The solution was then dried with MgSO4, filtered, and evaporated. The residue was purified by column chromatography (hexane / EtOAc, 1:9 to 10:0 v / v) to produce compound 9 (81mg, 46%), which was a white solid.

[0768] Calculation: [M+H] + (C 83 H 115 N8O 23)m / z=1591.8; found ESI: [M+H] + (C 83 H 115 N8O 23 )m / z=1591.7. Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)-5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropanoyl)propionamido) (10)

[0769] To a solution of MMAE construct 9 (81 mg, 51 μmol) in DMF (3 mL) was slowly added piperidine (0.1 mL, 1.0 mmol). The reaction was stirred at room temperature for 30 minutes. The residue was purified by reverse phase chromatography using acetonitrile-water as eluent (0.1% formic acid, 5-100%). The fractions containing the desired compound were combined and concentrated under vacuum to produce compound 10 (62 mg, 89%) as a white solid.

[0770] Calculation: [M+H] + (C 68 H 105 N8O 21 )m / z=1369.7; found ESI: [M+H] + (C 68 H 105 N8O 21 )m / z=1369.6. Preparation of (2S,3R,4S,5S,6S)-2-(2-((15S,18S)-4-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)-1-(9H-fluoren-9-yl)-15-isopropyl-18-methyl-3,13,16-trioxo-2,7,10-trioxa-4,14,17-triazanonadecacarboxamido)-5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-

[0771] (((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (12)

[0772] To the solution of MMAE-amine 10 (32 mg, 24 μmol) and Fmoc-azaHIPS-PNH acid 11 (27 mg, 29 μmol) in DMF (1 mL) was added DIPEA (10 μL, 57 μmol) and HATU (11 mg, 29 μmol). The reaction mixture was stirred at room temperature for 1 hour. The mixture was diluted with DCM (70 mL) and washed with NH4Cl saturated aqueous solution (50 mL), water (50 mL), and NaCl saturated aqueous solution (10 mL). The organic layer was dried with MgSO4, filtered, and evaporated. The residue was purified by column chromatography (hexane / EtOAc, 1:9 to 9:1 v / v) to produce compound 12 (33 mg, 61%) as a white solid.

[0773] Calculation: [M+2H] 2+ (C 123 H 164 N 14 O 29 )m / z=1150.6; found ESI: [M+2H] 2+ (C 123 H 164 N 14 O 29 )m / z=1151.0.

[0774] Preparation of (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2, 13-dioxa-4,7,10-triazatetradecyl)-2-((2S,5S)-15-((1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)amino)-5-isopropyl-2-methyl-4,7-dioxa-10,13-dioxa-3,6-diazapentadecamidyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (13)

[0775] A solution of LiOH (3 mg) in water (0.25 mL) was added to a solution of MMAE compound 12 (33 mg, 14 μmol) in THF (1 mL) at 0 ° C. The mixture was stirred at room temperature for 1 hour. The reaction was quenched by adding acetic acid (3 μL, 53 μmol). After removing the organic solvent, the residue was purified by reverse phase chromatography using acetonitrile-water as eluent (0.05% TFA, 5-80%). The fractions containing the desired compound were combined and concentrated under vacuum to produce compound 13 (13 mg, 52%) as a white solid.

[0776] Calculation: [M+H] + (C 86 H 135 N 14 O 22 )m / z=1716.0;Found ESI:[M+H] + (C 86 H 135 N 14 O 22 )m / z=1715.9.

[0777] Synthesis of PNP intermediate 16

[0778] The PNP intermediate was synthesized according to Scheme 2 shown below.

[0779]

[0780] Preparation of (2S,3R,4S,5S,6S)-2-(4-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (14)

[0781] To a mixture of acetyl bromide-α-D-pyranoglucopyranose methyl ester 1 (1.0 g, 2.5 mmol) and 4-hydroxybenzaldehyde (0.15 g, 1.2 mmol) in acetonitrile (20 mL) was added AgO (1.4 g, 6.1 mmol). The reaction mixture was stirred in the dark at room temperature for 20 hours. The reaction mixture was then concentrated under vacuum, and the residue was purified using column chromatography (hexane / EtOAc, 1:9 to 9:1 v / v) to produce compound 14 (0.52 g, 97%) as a white solid.

[0782] Calculation: [M+Na] + (C 20 H 22 NaO 11 )m / z=461.1; found ESI: [M+Na] + (C 20 H 22 NaO 11 )m / z=460.8. Preparation of (2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (15)

[0783] Silica gel (0.041 g, 0.40 mmol) was added to a solution of aldehyde 14 (0.175 g, 0.40 mmol) in chloroform (1.45 mL) and iPr-OH (0.36 mL) at 0 ° C. The resulting mixture was warmed to room temperature and stirred for 2 hours. Acetic acid (0.05 mL, 0.87 mmol) was added to quench the reaction. The reaction mixture was then concentrated under vacuum, and the residue was purified using column chromatography (DCM / MeOH, 0.1: 9 to 1: 9 v / v) to produce compound 15 (0.113 g, 70%) as a white solid.

[0784] Calculation: [M+Na] + (C 20 H 24 NaO 11 )m / z=463.1; found ESI: [M+Na] + (C 20 H 24 NaO 11 )m / z=462.8. Preparation of (2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (16)

[0785] Under argon, a solution of alcohol 15 (0.05 g, 0.11 mmol) in anhydrous THF (2.6 mL) was added with bis (4-nitrophenyl) carbonate (0.076 g, 0.25 mmol), followed by DIPEA (0.06 mL, 0.35 mmol). The reaction was allowed to stir at room temperature for 18 hours. The reaction mixture was not complete by LCMS. Additional bis (4-nitrophenyl) carbonate (0.02 g, 0.07 mmol) and DIPEA (0.005 mL, 0.03 mmol) were added and the reaction was allowed to stir for another 2 hours. The reaction mixture was then concentrated under vacuum, and the residue was purified using column chromatography (hexane / EtOAc, 1:9 to 1:1 v / v) to produce compound 16 (0.064 g, 93%), which was a white solid.

[0786] Calculation: [M+Na] + (C 27 H 27 NNaO 15 )m / z=628.1; found ESI: [M+Na] + (C 27 H 27 NNaO 15 )m / z=627.7.

[0787] Synthesis of MMAE construct 23

[0788] MMAE constructs were synthesized according to Scheme 3 shown below.

[0789]

[0790]

[0791] Preparation of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropionamido)-6-(tritylamino)hexanamido)benzyl ((S)-1-(((S)-1-((((3R,4S,5S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (18)

[0792] To a mixture of PNP 17 (0.048 g, 0.047 mmol) and MMAE 8 (0.05 g, 0.070 mmol) in DMF (1.5 mL) under argon was added DIPEA (0.032 mL, 0.18 mmol) and HOAt (0.11 mmol). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was then taken and purified directly using reverse phase column chromatography (MeCN / H2O, 1:9 to 10:0 v / v) to produce compound 18 (0.061 g, 82%) as a white solid.

[0793] Calculation: [M+H] + (C 96 H 120 N9O 13 )m / z=1606.9; found ESI: [M+H] + (C 96 H 120 N9O 13 )m / z=1606.7. Preparation of 4-((S)-2-(((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamic acid ester (19)

[0794] Trifluoroacetic acid (0.30 mL, 3.9 mmol) was added to a solution of MMAE derivative 18 (0.061 g, 0.038 mmol) in DCM (3.0 mL). The reaction was allowed to stir at room temperature for 6 hours. The reaction mixture was then concentrated under vacuum and the residue was purified using reverse phase column chromatography (MeCN / H2O, 0.1% formic acid in the eluent, 1:9 to 10:0 v / v) to produce compound 19 (0.028 g, 54%) as a white solid.

[0795] Calculation: [M+H] + (C 77 H 106 N9O 13 )m / z=1364.8; found ESI: [M+H] + (C 77 H 106 N9O 13)m / z=1363.8. Preparation of (2S,3R,4S,5S,6S)-2-(4-((5S,8S)-5-benzyl-8-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2 (20)

[0796] To a solution of amine 19 (0.028 g, 0.020 mmol) and PNP sugar 16 (0.018 g, 0.030 mmol) in DMF (1 mL) under argon was added HOAt (8.3 mg, 0.061 mmol) and DIPEA (0.014 mL, 0.080 mmol). The reaction mixture was stirred at room temperature for 16 h and used directly in the next step without further purification.

[0797] Preparation of (2S,3R,4S,5S,6S)-2-(4-(((((S)-5-((S)-2-amino-3-phenylpropionamido)-6-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1 (21)

[0798] To a solution of 20 in DMF was added piperidine (0.014 mL, 0.14 mmol). The reaction mixture was stirred at room temperature for 1.5 hours. The reaction mixture was then taken directly for purification using reverse phase column chromatography (MeCN / H2O, 0.1% trifluoroacetic acid in the eluent, 1:9 to 10:0 v / v) to produce compound 21 (0.021 g, 65%) as a white solid.

[0799] Calculation: [M+H] + (C 83 H 118 N9O 23 )m / z=1608.8; found ESI: [M+H] + (C 83 H 118 N9O 23 )m / z=1607.6. Preparation of (2S,3R,4S,5S,6S)-2-(4-((15S,18S)-4-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)-15-benzyl-18-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane- 2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)carbamoyl)-1-(9H-fluoren-9-yl)-3,13,16,24-tetraoxo-2,7,10,25-tetraoxa-4,14,17,23-tetraazahexacosane-26-yl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (22)

[0800] To a solution of amine 21 (0.020 g, 0.012 mmol) and acid 11 (0.014 g, 0.015 mmol) in DMF (1 mL) was added HATU (0.007 g, 0.018 mmol) and DIPEA (0.009 mL, 0.052 mmol) under argon. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was then taken directly for purification using preparative HPLC (MeCN / H2O, 0.05% trifluoroacetic acid in the eluent, 1:9 to 9.5:0.5 v / v) to yield compound 22 (0.011 g, 35%) as a white solid.

[0801] Calculation: [M+2H] 2+ (C 138 H 177 N 15 O 31 )m / z=1270.1; found ESI: [M+2H] 2+ (C 138 H 177 N 15 O31 )m / z=1269.7.

[0802] Preparation of (2S,3S,4S,5R,6S)-6-(4-((9S,12S)-12-benzyl-9-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl 2-((1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)amino)-3,11,14-trioxo-2,17,20-trioxa-4,10,13-triazadocosyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (23)

[0803] On ice, 1M LiOH (0.035mL, 0.035mmol) was added to a solution of compound 22 (0.011g, 0.004mmol) in THF (0.5mL). The reaction mixture was stirred on ice for 2 hours. Additional 1M LiOH (0.035mL, 0.035mmol) was added and continued to stir on ice for 2 hours. The reaction mixture was then concentrated under vacuum, and preparative HPLC (MeCN / H2O, 0.05% trifluoroacetic acid in the eluent, 0:10 to 7.5:2.5v / v) was used to purify the residue to produce compound 23 (0.005g, 58%), which was a white solid.

[0804] Calculation: [M+2H] 2+ (C 101 H 149 N 15 O 24 )m / z=978.1; found ESI: [M+2H] 2+ (C 101 H 149 N 15 O 24 )m / z=977.8.

[0805] Synthetic intermediate 28

[0806] The intermediates were synthesized according to Scheme 4 shown below.

[0807]

[0808] Preparation of tert-butyl 4-oxopiperidine-1-carboxylate (25)

[0809] To a solution of piperidone 24 (400 mg, 2.6 mmol, 1.0 equiv), DIPEA (540 μL, 3.1 mmol, 1.2 equiv), and Boc2O (850 mg, 3.9 mmol, 1.5 equiv) in CH3CN (12 mL) was added DMAP (32 mg, 0.26 mmol, 0.10 equiv). After stirring for 2 hours, the solution was concentrated and reconstituted in DCM. The solution was washed with 0.1 M HCl and the organics were dried over Na2SO4, filtered and concentrated. The product 25 was obtained as a light yellow solid and used without further purification (515 mg, 99%).

[0810] Calculation: [M+H] + (C 10 H 17 NNaO3) m / z = 222.1; found ESI: [M+H] + C 10 H 17 NNaO3) m / z = 223.0. Preparation of tert-butyl 4-((2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)amino)piperidine-1-carboxylate (26)

[0811] To a solution of compound 25 (515 mg, 2.7 mmol, 1.0 equiv) and amino-PEG2-tert-butyl ester (740 mg, 3.2 mmol, 1.2 equiv) in DCE (12 mL) was added STAB (1.1 g, 5.4 mmol, 2.0 equiv). The mixture was stirred overnight. After concentrating the solution, the residue was used without further purification.

[0812] Calculation: [M+H] + (C 21 H 41 N2O6) m / z = 417.3; found ESI: [M+H] + (C 21 H 41 N2O6)m / z=417.0.

[0813] Preparation of tert-butyl 4-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-(3-(tert-butoxy)-3-oxopropoxy)ethoxy)ethyl)amino)piperidine-1-carboxylate (27)

[0814] To a crude mixture of compound 26 (1.1 g, 2.7 mmol, 1.0 equiv) and DIPEA (920 μL, 5.4 mmol, 2.0 equiv) in DCM (10 mL) was added FmocCl (1.4 g, 2.0 equiv) at 0°C. The mixture was allowed to warm to room temperature and stirred for 2 hours. Afterwards, the mixture was washed with saturated NaHCO3. The organics were dried over Na2SO4, filtered, and concentrated. The crude product was purified by flash chromatography (hexanes / EtOAc, 1:9 to 1:1 v / v) to give compound 27 as a clear oil (1.6 g, 98% for three steps).

[0815] Calculation: [M+Na] + (C 36 H 50 N2NaO8) m / z = 661.3; found ESI: [M+Na] + (C 36 H 50 N2NaO8)m / z=

[0816] 660.9.

[0817] Preparation of 1-(9H-fluoren-9-yl)-3-oxo-4-(piperidin-4-yl)-2,7,10-trioxa-4-azatridecane-13-oic acid tert-butyl ester (28)

[0818] To a solution of compound 27 (1.6 g, 2.7 mmol) and TIPS (600 μL) in DCM (6 mL) was added TFA (6 mL). After stirring for 1 hour, the solution was concentrated and purified by reverse phase chromatography using acetonitrile-water (0.05% TFA, 10-70%) as eluent. The product 28 was obtained as a white solid (800 mg, 68%).

[0819] Calculation: [M+H] + (C 27 H 35 N2O6) m / z = 483.2; found ESI: [M+H] + (C 27 H 35 N2O6)m / z=482.9.

[0820] Synthetic intermediate 30

[0821] The intermediates were synthesized according to Scheme 5 shown below.

[0822]

[0823] Preparation of 3-{[(2S)-1-{[(1S,2R,5R,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl]oxy}-1-oxopropan-2-yl](methyl)carbamoyl}propanoic acid (29)

[0824] To a solution of maytansinoid (180 mg, 0.28 mmol, 1.0 equiv) and succinic anhydride (33 mg, 0.33 mmol, 1.2 equiv) in DCM (8 mL) was added DIPEA (96 μL, 0.55 mmol, 2.0 equiv). After stirring overnight, the solution was concentrated and purified by flash chromatography (DCM / MeOH, 100:0 to 10:1 v / v). The product 29 was obtained as a white solid (191 mg, 92%).

[0825] Calculation: [M+H] + (C 36 H 49 ClN3O 12 )m / z=750.3; found ESI: [M+H] + (C 36 H 49 ClN3O 12 )m / z=749.8. Preparation of 2,3,4,5,6-pentafluorophenyl 3-{[(2S)-1-{[(1S,2R,5R,6S,16E,18E,20R,21S)-11-chloro-2-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl]oxy}-1-oxopropan-2-yl](methyl)carbamoyl}propanoate (30)

[0826] To a solution of compound 29 (160 mg, 0.21 mmol, 1.5 equiv) and perfluorophenol (60 mg, 0.33 mmol, 1.3 equiv) in DCM (3 mL) was added DIC (35 mg, 0.28 mmol, 1.3 equiv). After stirring overnight, the solution was filtered, concentrated and purified by flash chromatography (DCM / MeOH, 100:0 to 24:1 v / v). The product 30 was obtained as a white solid (190 mg, 97%).

[0827] Calculation: [M+H] + (C 42 H 48 ClF5N3O 12 )m / z=916.3; found ESI: [M+H] + (C 42 H 48 ClF5N3O 12 )m / z=915.7.

[0828] Synthetic maytansinoid construct 39

[0829] Maytansinoid constructs were synthesized according to Scheme 6 shown below.

[0830]

[0831]

[0832]

[0833] Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutyramido)propionamido)-5-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (31)

[0834] To a solution of amine 5 (341 mg, 0.65 mmol) and Boc-valine-OH (169 mg, 0.78 mmol) in DMF (2.0 mL) was added DIPEA (0.35 mL, 2.0 mmol) and HATU (380 mg, 1.0 mmol). The residue was purified by reverse phase chromatography using acetonitrile-water (0.05% TFA, 5-90%) as eluent. The fractions containing the desired compound were combined and lyophilized to yield compound 31 (386 mg, 82%) as a white solid.

[0835] Calculation: [M+H] + (C 33 H 48 N3O 15 )m / z=726.3; found ESI: [M+H] + (C 33 H 48 N3O 15 )m / z=726.1.

[0836] Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutyramido)propionamido)-5-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (32)

[0837] To a solution of compound 31 (81 mg, 0.11 mmol, 1.0 equiv) and bis-p-nitrophenol carbonate (140 mg, 0.67 mmol, 6.0 equiv) in THF (1.5 mL) was added DIPEA (120 μL, 0.67 mmol, 6.0 equiv). After stirring overnight, the solution was concentrated and purified by flash chromatography (DCM / MeOH, 100:0 to 93:7 v / v). The product 32 was obtained as a white solid (101 mg, quantitative).

[0838] Calculation: [M+Na] + (C 40 H 50 N4NaO 19 )m / z=913.3; found ESI: [M+Na] + (C 40 H 50 N4NaO 19 )m / z=912.7.

[0839] Preparation of 4-(1-(((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutyramido)propionamido)-3-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)piperidin-4-yl)-1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecane-13-oic acid (33)

[0840] To a solution of compound 32 (140 mg, 0.16 mmol, 1.0 equiv) and 28 (110 mg, 0.17 mmol, 1.5 equiv) in DCM (5 mL) was added DIPEA (100 μL, 0.59 mmol, 3.8 equiv). After stirring overnight, the solution was concentrated and purified by reverse phase chromatography using acetonitrile-water as eluent (0.05% TFA, 50-80%). The product 33 was obtained as a white solid (110 mg, 53%).

[0841] Calculation: [M+Na] + (C 61 H79 N5NaO 22 )m / z=1256.5; found ESI: [M+Na] + (C 61 H 79 N5NaO 22 )m / z=1256.7.

[0842] Preparation of (2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutyramido)propionamido)-5-(((4-((2-(2-(2-carboxyethoxy)ethoxy)ethyl)amino)piperidine-1-carbonyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (34)

[0843] To a solution of compound 33 (110 mg, 0.089 mmol) in THF (2 mL) was added 1 M LiOH (1.4 mL) at 0°C. The mixture was allowed to warm to room temperature and stirred for 2 hours. Afterwards, the mixture was concentrated to remove THF and purified by reverse phase chromatography using acetonitrile-water as eluent (0.05% TFA, 0-45%) to give compound 34 as a white solid (68 mg, 87%).

[0844] Calculation: [M+H] + (C 39 H 62 N5O 17 )m / z=872.4; found ESI: [M+H] + (C 39 H 62 N5O 17 )m / z=871.8.

[0845] Preparation of (2S,3S,4S,5R,6S)-6-{2-[(2S)-2-[(2S)-2-{[(tert-butoxy)carbonyl]amino}-3-methylbutyramido]propionamido]-5-{[4-(N-{2-[2-(2-carboxyethoxy)ethoxy]ethyl}-3-{[(2S)-1-{[(1S,2R,5R,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20- Dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosyl-10,12,14(26),16,18-pentaen-6-yl]oxy}-1-oxopropan-2-yl](methyl)carbamoyl}propionamido)piperidine-1-carbonyloxy]methyl}phenoxy}-3,4,5-trihydroxyoxo-2-carboxylic acid (35)

[0846] To a solution of compound 34 (18 mg, 0.021 mmol, 1.0 equiv) and 30 (29 mg, 0.033 mmol, 1.5 equiv) in DMF (0.6 mL) was added DIPEA (30 μL, 0.18 mmol, 8.6 equiv). After stirring overnight, the solution was concentrated and purified by reverse phase chromatography using acetonitrile-water as eluent (0.05% TFA, 10-95%). The product 35 was obtained as a white solid as a mixture with compound 29 (3:2 compound 29 to compound 35, 20 mg, 36% yield).

[0847] Calculation: [M+Na] + (C 75 H 107 ClN5NaO 28 )m / z=1625.7; found ESI: [M+Na] + (C 75 H 107 ClN5NaO 28 )m / z=1624.6.

[0848] Preparation of (2S,3S,4S,5R,6S)-6-{2-[(2S)-2-[(2S)-2-amino-3-methylbutyramido]propionamido]-5-{[4-(N-{2-[2-(2-carboxyethoxy)ethoxy]ethyl}-3-{[(2S)-1-{[(1S,2R,5R,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2 ,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosyl-10,12,14(26),16,18-pentaen-6-yl]oxy}-1-oxopropan-2-yl](methyl)carbamoyl}propionamido)piperidine-1-carbonyloxy]methyl}phenoxy}-3,4,5-trihydroxyoxo-2-carboxylic acid(36)

[0849] To a solution of compound 35 (12 mg, 7.5 μmol, 1.0 equiv) in DCM (2 mL) was added 1 M SnCl4 in DCM (100 μL, 13 equiv). After stirring for 20 min, the solution was quenched with CH3CN and H2O, concentrated to remove DCM, and purified by reverse phase chromatography using acetonitrile-water as eluent (0.05% TFA, 2-60%). The product 36 was obtained as a white solid (5 mg, 44% yield).

[0850] Calculation: [M+2H] 2+ (C 70 H 101 ClN8O 26 )m / z=752.3; found ESI: [M+2H] 2+ C 70 H 101 ClN8O 26 )m / z=752.0.

[0851] Preparation of (2S,3S,4S,5R,6S)-6-(5-{[4-(N-{2-[2-(2-carboxyethoxy)ethoxy]ethyl}-3-{[(2S)-1-{[(1S,2R,5R,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl]oxy}-1-oxopropane alkyl-2-yl](methyl)carbamoyl}propionamido)piperidin-1-carbonyloxy]methyl}-2-[(2S)-2-[(2S)-2-(3-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}({1-[3-(2-{[({[(9H-fluoren-9-yl)methoxy]carbonyl}(methyl)amino)(methyl)amino]methyl}-1H-pyrrolo[2,3-b]pyridin-1-yl)propionyl]piperidin-4-yl})amino)ethoxy]ethoxy}propionamido)-3-methylbutanamido]propionamido]phenoxy)-3,4,5-trihydroxyoxo-2-carboxylic acid (38)

[0852] To a solution of compound 36 (5 mg, 3.3 μmol, 1.0 equiv) and 37 (10 mg, 9.0 μmol, 2.7 equiv) in DMF (0.15 mL) was added DIPEA (1.5 μL, 8.7 μmol, 2.6 equiv). After stirring overnight, the solution was purified by reverse phase chromatography using acetonitrile-water as eluent (0.05% TFA, 10-90%). The white solid product 38 was semi-pure with hydrolyzed compound 37 (6 mg, 81% yield).

[0853] Calculation: [M+2H] 2+ (C 125 H 159 C1N 14 O 34 )m / z=1217.5; found ESI: [M+2H] 2+ (C 125 H 159 C1N 14 O 34 )m / z=1217.3.

[0854] Preparation of (2S,3S,4S,5R,6S)-6-(5-{[4-(N-{2-[2-(2-carboxyethoxy)ethoxy]ethyl}-3-{[(2S)-1-{[(1S,2R,5R,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosyl-10,12,14-(26), 16,18-pentaen-6-yl]oxy}-1-oxopropan-2-yl](methyl)carbamoyl}propionamido)piperidine-1-carbonyloxy]methyl}-2-[(2S)-2-[(2S)-2-{3-[2-(2-{[1-(3-{2-[(1,2-dimethylhydrazine-1-yl)methyl]-1H-pyrrolo[2,3-b]pyridin-1-yl}propionyl)piperidin-4-yl]amino}ethoxy)ethoxy]propionamido}-3-methylbutanamido]propionamido]phenoxy)-3,4,5-trihydroxyoxo-2-carboxylic acid (39)

[0855] To a solution of compound 38 (6 mg, 2.7 μmol) in DMA (600 μL) was added DBU (6 μL, 40 μmol). After stirring for 15 minutes, the solution was purified by reverse phase chromatography using acetonitrile-water as eluent (0.05% TFA, 10-90%). The product 39 was obtained as a white solid (3 mg, 61% yield).

[0856] Calculation: [M+2H] 2+ (C 95 H 159 C1N 14 O 30 )m / z=995.5; found ESI: [M+2H] 2+ (C 95 H 159 C1N 14 O 30 )m / z=995.0.

[0857] Synthesis of MMAE construct 48

[0858] MMAE constructs were synthesized according to Scheme 7 shown below.

[0859]

[0860]

[0861] Preparation of tert-butyl 2-hydroxy-4-nitrobenzoate (41)

[0862] To a solution of benzoic acid 40 (2.07 g, 11.3 mmol) in THF (10 mL), tert-butyl alcohol (10 mL, 105.4 mmol), DMAP (1.2 g, 9.8 mmol), and DCC (2.5 g, 12.1 mmol) were added. The resulting mixture was stirred at room temperature for 20 hours. The mixture was diluted with EtOAc (200 mL) and washed with NH4Cl saturated aqueous solution (100 mL), water (100 mL), NaHCO3 saturated aqueous solution (100 mL), water (100 mL), and NaCl saturated aqueous solution (20 mL). The organic layer was dried with MgSO4, filtered, and evaporated. The residue was purified by column chromatography (hexane / EtOAc, 1:9 to 9:1 v / v) to produce compound 41 (2.35 g, 87%), which was a yellow oil.

[0863] Calculation: [M+H] + (C 11 H 14 NO5) m / z = 240.1; found ESI: [M+H] + (C 11 H 14 NO5)m / z=240.5.

[0864] Preparation of (2S,3R,4S,5S,6S)-2-(2-(tert-butoxycarbonyl)-5-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (42)

[0865] To a mixture of methyl bromide-α-D-pyranoglucopyranose ester 1 (1.32 g, 3.3 mmol) and benzol 41 (434 mg, 1.8 mmol) in acetonitrile (10 mL) was added AgO (1.0 g, 4.3 mmol). The reaction mixture was stirred in the dark at room temperature for 2 days. The reaction mixture was then concentrated under vacuum, and the residue was purified using column chromatography (hexane / EtOAc, 1:9 to 9:1 v / v) to produce compound 42 (495 mg, 49%) as an off-white solid.

[0866] Calculation: [M+H] + (C 24 H 30 NO 14 )m / z=556.2; found ESI: [M+H] + (C 24 H 30 NO 14 )m / z=556.2.

[0867] Preparation of (2S,3R,4S,5S,6S)-2-(5-amino-2-(tert-butoxycarbonyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (43)

[0868] Pd / C (10 wt %, 200 mg) was added to a solution of nitro compound 42 (495 mg, 0.89 mmol) in MeOH (5 mL). The flask was then evacuated and H2 gas was injected from a balloon, and two cycles were repeated. The reaction was vigorously stirred at room temperature for 3 days with a H2 balloon connected. After removing the catalyst by filtration through a celite pad, the filtrate was concentrated under vacuum to produce compound 43 (420 mg, 96%) as an oil. The crude product was used directly in the next step without further purification.

[0869] Calculation: [M+H] + (C 24 H 32 NO 12 )m / z=526.2; found ESI: [M+H] + (C 24 H 32 NO 12 )m / z=526.7.

[0870] Preparation of (2S,3R,4S,5S,6S)-2-(5-(4-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)-1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecanoyl)-2-(tert-butoxycarbonyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (44)

[0871] Compound 11 (57 mg, 60.2 μmol), DIPEA (32 μL, 0.18 mmol), and HATU (23 mg, 60.5 μmol) were added to a solution of aniline 43 (35 mg, 66.7 μmol) in DMF (1 mL). The resulting mixture was stirred at room temperature for 1 hour. The crude product was purified by reverse phase chromatography using acetonitrile-water (0.05% TFA, 5-85%) as eluent. The fractions containing the desired compound were combined and concentrated under vacuum to produce compound 44 (49 mg, 56%) as a white solid.

[0872] Calculation: [M+H] + (C 79 H 90 N7O 20)m / z=1456.6; found ESI: [M+H] + (C 79 H 90 N7O 20 )m / z=1457.3. Preparation of 4-(4-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)-1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecanoyl)-2-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzoic acid (45)

[0873] At 0°C, a solution of compound 44 (49 mg, 33.7 μmol) in DCM (0.5 mL) was added with SnCl4 (1 M, 0.34 mL, 0.34 mmol) in DCM. After stirring for 30 minutes, the reaction was quenched by adding H2O (0.1 mL) and CH3CN (0.2 mL). The mixture was then concentrated under vacuum and the residue was purified by reverse phase chromatography using acetonitrile-water as eluent (0.05% TFA, 5-75%). The fractions containing the desired compound were combined and concentrated under vacuum to produce compound 45 (40 mg, 85%) as an oil.

[0874] Calculation: [M+H] + (C 75 H 82 N7O 20 )m / z=1400.6; found ESI: [M+H] + (C 75 H 82 N7O 20)m / z=1400.4. Preparation of (2S,3R,4S,5S,6S)-2-(5-(4-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)-1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecanoyl)-2-(((S)-1-(((S)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2 -((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (47)

[0875] To a solution of carboxylic acid 45 (15 mg, 10.7 μmol) in DMF (75 μL) was added MMAE compound 46 (12 mg, 10.7 μmol), DIPEA (6 μL, 34.4 μmol) and HATU (5 mg, 13.2 μmol) in DMA (50 μL). The reaction mixture was stirred at room temperature for 1 hour. The crude product was purified by preparative HPLC using acetonitrile-water as mobile phase (0.05% TFA, 5-90%). The fractions containing the desired compound were combined and lyophilized to produce compound 47 (5.2 mg, 19%) as a white solid.

[0876] Calculation: [M+2H] 2+ (C 133 H 175 N 17 O 31 )m / z=1253.1; found ESI: [M+2H] 2+ (C 133 H 175 N 17 O 31 )m / z=1253.6.

[0877] Preparation of (2S,3S,4S,5R,6S)-6-(2-(((S)-1-(((S)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo -2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)-5-(3-(2-(2-((1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)amino)ethoxy)ethoxy)propionamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (48)

[0878] To a solution of compound 47 (5.2 mg, 2.1 μmol) in THF (0.2 mL) was slowly added LiOH solution (1.0 mg in 0.1 mL H2O) at 0°C. The resulting mixture was stirred at room temperature for 2 hours. The crude product was purified by preparative HPLC using acetonitrile-water as mobile phase (0.05% TFA, 5-75%). The fractions containing the desired compound were combined and lyophilized to yield MMAE construct 48 (2.5 mg, 63%) as a white solid.

[0879] Calculation: [M+2H] 2+ (C 96 H 147 N 17 O 24 )m / z=961.1; found ESI: [M+H] + (C 96 H 147 N 17 O 24 )m / z=961.1.

[0880] Synthesis of MMAE construct 53

[0881] MMAE constructs were synthesized according to Scheme 8 shown below.

[0882]

[0883]

[0884] Preparation of tert-butyl 4-amino-2-hydroxybenzoate (49)

[0885] Pd / C (10 wt %, 200 mg) was added to a solution of nitro compound 41 (485 mg, 2.03 mol) in EtOAc (4 mL) and EtOH (4 mL). The flask was then evacuated and H2 gas was injected from a balloon, and two cycles were repeated. The reaction was vigorously stirred at room temperature for 2 days with a H2 balloon connected. After removing the catalyst by filtration through a celite pad, the filtrate was concentrated under vacuum to produce compound 49 (418 mg, 98%) as an oil. The crude product was used directly in the next step without further purification.

[0886] Calculation: [M+H] + (C 11 H 16 NO3) m / z = 210.1; found ESI: [M+H] + (C 11 H 16 NO3)m / z=210.1.

[0887] Preparation of 2-((1-(3-(4-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-(3-((4-(tert-butoxycarbonyl)-3-hydroxyphenyl)amino)-3-oxopropoxy)ethoxy)ethyl)amino)piperidin-1-yl)-3-oxopropyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)-1,2-dimethylhydrazinecarboxylic acid (9H-fluoren-9-yl)methyl ester (50)

[0888] To a solution of aniline 49 (15.7 mg, 75.1 μmol) and 11 (47 mg, 49.6 μmol) in THF (0.5 mL) was added N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 25 mg, 101.2 μmol). The resulting mixture was stirred at room temperature for 1 hour. The crude product was purified by reverse phase chromatography using acetonitrile-water as eluent (0.05% TFA, 5-85%). The fractions containing the desired compound were combined and concentrated under vacuum to yield compound 50 (17.4 mg, 31%) as a white solid.

[0889] Calculation: [M+H] + (C 66 H 74 N7O 11 )m / z=1140.5; found ESI: [M+H] + (C 66 H 74 N7O 11)m / z=1140.4. Preparation of 4-(4-(1-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propanoyl)piperidin-4-yl)-1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecanoyl)-2-hydroxybenzoic acid (51)

[0890] To a solution of compound 50 (17.4 mg, 15.3 μmol) in dioxane (0.1 mL) was added HCl in dioxane (4 M, 0.1 mL, 0.4 mmol) at 0°C. The reaction mixture was stirred at room temperature for 1 hour. The crude product was purified by reverse phase chromatography using acetonitrile-water as eluent (0.05% TFA, 5-85%). The fractions containing the desired compound were combined and concentrated under vacuum to yield compound 51 (8.5 mg, 51%) as a yellow oil.

[0891] Calculation: [M+H] + (C 62 H 66 N7O 11 )m / z=1084.5; found ESI: [M+H] + (C 62 H 66 N7O 11 )m / z=1084.4. Preparation of 2-((1-(3-(4-((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-(3-((4-(((S)-1-(((S)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8 (52)

[0892] To a solution of carboxylic acid 51 (11.6 mg, 10.7 μmol) in DMF (50 μL) was added MMAE compound 46 (12 mg, 10.7 μmol), DIPEA (6 μL, 34.4 μmol) and HATU (5 mg, 13.2 μmol). The reaction mixture was stirred at room temperature for 1 hour. The crude product was purified by preparative HPLC using acetonitrile-water as mobile phase (0.05% TFA, 5-90%). The fractions containing the desired compound were combined and lyophilized to produce compound 52 (2.8 mg, 12%) as a white solid.

[0893] Calculation: [M+2H] 2+ (C 120 H 159 N 17 O 22 )m / z=1095.1; found ESI: [M+2H] 2+ (C 120 H 159 N 17 O 22 )m / z=1095.6.

[0894] Preparation of ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl 4-((S)-2-((S)-2-(4-(3-(2-(2-((1-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propionyl)piperidin-4-yl)amino)ethoxy)ethoxy)propionamido)-2-hydroxybenzamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl-1-oxobutan-2-yl)(methyl)carbamate (53)

[0895] To a solution of compound 52 (2.8 mg, 1.3 μmol) in DMA (0.2 mL) was slowly added piperidine (1.0 μL, 10.1 μmol) at 0°C. The resulting mixture was stirred at room temperature for 30 minutes. The crude product was purified by preparative HPLC using acetonitrile-water as the mobile phase (0.05% TFA, 5-70%). The fractions containing the desired compound were combined and lyophilized to yield compound 53 (1.0 mg, 45%) as a white solid.

[0896] Calculation: [M+H] + (C 90H 138 N 17 O 18 )m / z=1745.0;Found ESI:[M+H] + (C 90 H 138 N 17 O 18 )m / z=1745.6.

[0897] Synthetic duocarmycin construct 66

[0898] The duocarmycin constructs were synthesized according to Scheme 9 shown below.

[0899]

[0900]

[0901] Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-((tert-butoxycarbonyl)amino)propionamido)-5-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (55)

[0902] To a mixture of Boc-Ala-OH 54 (44 mg, 0.23 mmol) and aniline 3 (53 mg, 0.12 mmol) in DCM (2.0 mL) and MeOH (0.2 mL) was added N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 1.25 g, 0.23 mmol). The reaction was stirred at room temperature for 1 hour. After removing the solvent under vacuum, the residue was purified using column chromatography (hexane / EtOAc, 1:9 to 9:1 v / v) to produce compound 55 (41 mg, 55%) as a white solid.

[0903] Calculation: [M+H] + (C 28 H 39 N2O 14 )m / z=627.2; found ESI: [M+H] + (C 28 H 39 N2O 14 )m / z=627.1.

[0904] Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-((tert-butoxycarbonyl)amino)propionamido)-5-(chloromethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (56)

[0905] To a solution of benzyl alcohol 55 (108 mg, 0.17 mmol) in THF (1.5 mL) was added SOCl2 (25 μL, 0.34 mmol) at 0°C. The mixture was stirred at room temperature for 5 hours. After removing the solvent under vacuum, the residue was purified using column chromatography (hexane / EtOAc, 1:9 to 9:1 v / v) to give compound 56 (107.7 mg, 97%) as an oil.

[0906] Calculation: [M+H] + (C 28 H 38 ClN2O 13 )m / z=645.2; found ESI: [M+H] + (C 28 H 38 ClN2O 13 )m / z=645.2. Preparation of (2S,3R,4S,5S,6S)-2-(5-((((S)-3-(tert-butoxycarbonyl)-1-(hydroxymethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)oxy)methyl)-2-((S)-2-((tert-butoxycarbonyl)amino)propionamido)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (58)

[0907] To a solution of phenyl 57 (242 mg, 0.77 mmol) in DMF (3 mL) was added CsCO (235 mg, 0.72 mmol). After the mixture was stirred at room temperature for 20 minutes, chloride 56 (521 mg, 0.81 mmol) in DMF (2 mL) was added. The resulting mixture was stirred at room temperature for 20 hours. The crude product was purified by reverse phase chromatography using acetonitrile-water (0.05% TFA, 5-100%) as eluent. The fractions containing the desired compound were combined and concentrated under vacuum to produce compound 58 (326 mg, 46%) as a light yellow oil.

[0908] Calculation: [M+H] + (C 46 H 58 N3O 17 )m / z=924.4; found ESI: [M+H] + (C 46 H 58 N3O 17 )m / z=924.3.

[0909] Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-aminopropionamido)-5-((((S)-3-(tert-butoxycarbonyl)-1-(hydroxymethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (59)

[0910] To a solution of compound 58 (163 mg, 0.18 mmol) in THF (1 mL) was added HCl in dioxane (4 M, 1 mL, 1 mmol) at 0°C. The reaction mixture was stirred at room temperature for 1 hour. After the solvent was concentrated under vacuum, the residual dioxane was removed by azeotropic distillation. The residue was used directly in the next step without further purification.

[0911] Calculation: [M+H] + (C 36 H 42 N3O 13 )m / z=724.3; found ESI: [M+H] + (C 36 H 42 N3O 13 )m / z=724.3.

[0912] Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)propionamido)-5-((((S)-1-(hydroxymethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (61)

[0913] To a solution of compound 59 (5.8 mg, 8.0 μmol) in DMF (0.1 mL) was added Fmoc-valine pentafluorophenyl ester (4.1 mg, 8.1 μmol) and DIPEA (4.5 μL, 25.8 μmol). After the mixture was stirred at room temperature for 24 hours, the crude product was purified by reverse phase chromatography using acetonitrile-water (0.1% formic acid, 5-100%) as eluent. The fractions containing the desired compound were combined and concentrated under vacuum to yield compound 61 (5.3 mg, 63%) as a light yellow oil.

[0914] Calculation: [M+H] + (C 56 H 61 N4O 16 )m / z=1045.4; found ESI: [M+H] +(C 56 H 61 N4O 16 )m / z=1045.3. Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propionamido)-5-((((S)-1-(chloromethyl)-3-(5-(2-(dimethylamino)ethoxy)-1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (63)

[0915] To a solution of compound 61 (5.3 mg, 5.1 μmol) and 62 (2.6 mg, 10.5 μmol) in DMA (0.1 mL) was added EDC (5 mg, 32.3 μmol). After stirring for 1 day, the residue was purified by preparative HPLC using acetonitrile-water as mobile phase (0.05% TFA, 5-80%). The fractions containing the desired compound were combined and concentrated under vacuum to yield compound 63 (2.9 mg, 42%) as an oil.

[0916] Calculation: [M+H] + (C 69 H 74 ClN6O 17 )m / z=1293.5; found ESI: [M+H] + (C 69 H 74 ClN6O 17 )m / z=1293.4.

[0918] Preparation of (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)-5-((((S)-1-(chloromethyl)-3-(5-(2-(dimethylamino)ethoxy)-1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (64) [09...

Claims

1. A conjugate comprising: Antibody; medicines; and A cleavable linker connects the antibody to the drug and comprises a first cleavable moiety and a second cleavable moiety that blocks cleavage of the first cleavable moiety.

2. A compound comprising: A cleavable linker for linking an antibody to a drug, wherein the cleavable linker comprises a first cleavable moiety and a second cleavable moiety that blocks cleavage of the first cleavable moiety.

3. A pharmaceutical composition comprising: The conjugate of claim 1; and Pharmaceutically acceptable excipients.

4. A method comprising: An effective amount of the conjugate of claim 1 is administered to a subject.

5. A method of treating cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the conjugate of claim 1, wherein the administration is effective to treat cancer in the subject.

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