Double-cleavage ester linker for antibody-drug conjugates

Abstract

The present disclosure provides an antibody-drug conjugate structure, the antibody-drug conjugate comprising a cleavable linker comprising an ester group that connects the antibody to the drug. The present disclosure also includes compounds and methods for producing such conjugates. In addition, the present disclosure also includes pharmaceutical compositions and methods using the conjugates.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 138,255, filed on 15 January 2021, the disclosures of which are incorporated herein by reference. [Background technology]

[0002] Antibody-drug conjugates (ADCs) have emerged over the past 20 years as a new class of targeted delivery therapies. A typical ADC (Figure 1) contains an antibody-based targeting element linked to a highly potent drug (payload) via a chemical linker. The molar ratio of the targeting element (e.g., antibody) to the conjugated payload can vary and is referred to as the drug-to-antibody ratio (DAR). [Overview of the project]

[0003] This disclosure provides antibody-drug conjugate structures, the antibody-drug conjugate comprising a cleavable linker containing an ester group that binds the antibody to the drug. This disclosure also includes compounds and methods for producing such conjugates. In addition, this disclosure also includes pharmaceutical compositions and methods for using the conjugates.

[0004] A part of this disclosure is a conjugate of formula (I), [ka] During the ceremony, W 1 However, it is a drug, W 2 However, it is a polypeptide, A is an amino acid residue, and k is 0 or an integer from 1 to 5. L is the linker, G is the conjugated part, X 1 but, [ka] and -(CHR 1 ) j (CHR 2 )- selected from, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, and R 3 selected from, R 1 is optionally substituted with R 3 , j is 0 or an integer from 1 to 5, R 2 is R 3 or R 2 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, and R 2 is substituted with R 3 , X 2 is -NH- or -C(O)-, each R 3 is independently a glycoside or a glycoside derivative, and includes conjugates.

[0005] In some embodiments, X 1 is [Chemical formula] is.

[0006] In some embodiments, X 1 is -(CHR 1 ) j (CHR 2 )-. [[ID=6�]]

[0007] In some embodiments, j is 0.

[0008] In some embodiments, R 2 It is an alkyl group.

[0009] In some embodiments, R 2 That is Ariel.

[0010] In some embodiments, j is 1.

[0011] In some embodiments, R 1 It is hydrogen.

[0012] In some embodiments, R 1 R 3 It will be replaced with.

[0013] Several embodiments, each R 3 Independently, [ka] Selected from.

[0014] In some embodiments, the conjugate is the conjugate of the following formula (II): [ka] In the formula, R 4 However, these are amino acid side chains.

[0015] In some embodiments, the conjugate is [ka] Selected from TIFF0007912016000007.tif25150.

[0016] In some embodiments, the conjugate is the conjugate of the following formula (III): [ka] In the formula, R 4 However, these are amino acid side chains.

[0017] In some embodiments, the conjugate is [ka] Selected from.

[0018] In some embodiments, k is 2.

[0019] In some embodiments, L is -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) f - includes, During the ceremony, a, b, c, d, e, and f are each independently either 0 or 1. T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 However, each is independent, and covalent, (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-, selected from 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), acetals, hydrazines, disulfides, and esters, where EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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. V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 However, each operates independently as 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 Selected from the group consisting of SO2- and -P(O)OH-, where each q is an integer from 1 to 6. Each R 13 However, they are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. Each R 15 However, these are 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.

[0020] In some embodiments, the conjugate includes a linker L. T 1is (C1-C 12 ) alkyl, V 1 is -CO-, T 2 is an amino acid analog, V 2 is -NH-, T 3 is (PEG) n and V 3 is -CO-, d to f are each 0, or T 1 is (C1-C 12 ) alkyl, V 1 is -CO-, T 2 is an amino acid analog, V 2 is -NH-, T 3 is (PEG) n and V 3 is -CONH-, T 4 is (PEG)[[ID=四十八]] n and V 4 is -CO-, e and f are each 0.

[0021] In some embodiments, G is as follows

Chemical formula

[0022] In some embodiments, G is selected from acetals, hydrazones, oximes, sulfides, disulfides, triazoles, esters, and amides.

[0023] Embodiments of this disclosure are compounds of formula (IV), [ka] During the ceremony, W 1 However, it is a drug, A is an amino acid residue, and k is 0 or an integer from 1 to 5. L is the linker, G is the conjugated part, X 1 but, [ka] and -(CHR 1 ) j (CHR 2 )- Selected from, R 1 However, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, and R 3 Selected from, R 1 However, R 3 Replaced by, j is an integer from 0 to 5. R 2 However, R 3 is or R 2 However, 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. 2 However, R 3 Replaced by, X 2 However, it is -NH- or -C(O)-, Each R 3 However, it independently includes a compound of formula (IV) which is a glycoside or a glycoside derivative.

[0024] In some embodiments, X 1 teeth, [ka] That is the case.

[0025] In some embodiments, X 1 is, -(CHR 1 ) j (CHR 2 )-is.

[0026] In some embodiments, j is 0.

[0027] In some embodiments, R 2 It is an alkyl group.

[0028] In some embodiments, R 2 That is Ariel.

[0029] In some embodiments, j is 1.

[0030] In some embodiments, R 1 It is hydrogen.

[0031] In some embodiments, R 1 R 3 It will be replaced with.

[0032] Several embodiments, each R 3 Independently, [ka] Selected from.

[0033] In some embodiments, the compound is a compound of the following formula (V), [ka] In the formula, R 4 However, these are amino acid side chains.

[0034] In some embodiments, the compound is [ka] Selected from.

[0035] In some embodiments, the compound is a compound of formula (VI), [ka] In the formula, R 4 However, these are amino acid side chains.

[0036] In some embodiments, the compound is [ka] Selected from.

[0037] In some embodiments, k is 2.

[0038] In some embodiments, L is -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) f - includes, During the ceremony, a, b, c, d, e, and f are each independently either 0 or 1. T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 However, each is independent, and covalent, (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-, selected from 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), acetals, hydrazines, disulfides, and esters, where EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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. V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 However, each operates independently as 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 Selected from the group consisting of SO2- and -P(O)OH-, where each q is an integer from 1 to 6. Each R 13 However, they are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. Each R 15 However, these are 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.

[0039] In some embodiments, the compound includes linker L. T 1However, (C1~C 12 ) is alkyl, V 1 However, it is -CO-, T 2 However, it is an amino acid analog, V 2 However, it is -NH-, T 3 However, (PEG) n V 3 However, it is -CO-, d~f are each 0, or T 1 However, (C1~C 12 ) is alkyl, V 1 However, it is -CO-, T 2 However, it is an amino acid analog, V 2 However, it is -NH-, T 3 However, (PEG) n V 3 However, it is -CONH-, T 4 However, (PEG) n V 4 However, it is -CO-, e and f are both 0.

[0040] In some embodiments, G is as follows: [ka] During the ceremony, Z, CR 10 or N, R 8 and R 9 However, each is 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 R8 and R 9 However, they are optionally linked in a ring to form a 5-membered or 6-membered heterocycline. Each R 10 However, these 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, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0041] In some embodiments, G is selected from acetals, NHS esters, PFP esters, TFP esters, isocyanates, isothiocyanates, acyl halides, maleimides, iodoacetamides, phosphines, alkynes, azides, hydrazines, alkoxyamines, disulfides, esters, and amides.

[0042] Aspects of this disclosure include pharmaceutical compositions comprising a conjugate described herein and a pharmaceutically acceptable excipient.

[0043] Aspects of this disclosure include methods of administering a conjugate, which include administering a conjugate as described herein. [Brief explanation of the drawing]

[0044] [Figure 1] A schematic diagram of the main components of an antibody-drug conjugate (ADC) according to embodiments of this disclosure is shown. [Figure 2A] Linker strategies for ADCs are shown. (Figure 2, Panel A) Carbamate linkage for amine-containing payloads; (Figure 2, Panel B) Carboxyl ester linker for hydroxyl group-containing payloads. [Figure 2B]Linker strategies for ADCs are shown. (Figure 2, Panel A) Carbamate linkage for amine-containing payloads; (Figure 2, Panel B) Carboxyl ester linker for hydroxyl group-containing payloads. [Figure 3] This embodiment of the disclosure shows a double cleavage design for an ester linker having adjacent monosaccharides. [Figure 4] An example of a structural design for a double-cleavage carboxyl ester linker for ADC according to embodiments of this disclosure is shown. [Figure 5] A schematic diagram of HIPS ligation for ADC synthesis is shown. An antibody containing an aldehyde moiety is reacted with a Hydrazino-iso-Pictet-Spengler (HIPS) linker and payload to generate a site-specifically conjugated ADC with a stable azacarborin bond. [Figure 6] The graph of compound 21 CH1-3 / CT-tagged sacituzumab conjugate is shown, and when determined by hydrophobic interaction column (HIC), a DAR of 3.41 is obtained. [Figure 7] The graph of compound 21 CH1-3 / CT-tagged sacituzumab conjugate shows that 98.1% is monomer, as determined by analytical size exclusion chromatography (SEC). [Figure 8] The graph of compound 21 CH1-3 / CT-tagged polatuzumab conjugate is shown, and when determined by HIC, it yields a DAR of 3.67. [Figure 9] The graph of compound 21 CH1-3 / CT-tagged polatuzumab conjugate shows that, as determined by analysis SEC, 97.4% is monomer. [Figure 10] The graph of compound 52 CT-tagged anti-FITC conjugate is shown, and when determined by HIC, a DAR of 1.61 is obtained. [Figure 11] The graph of compound 52 CT-tagged anti-FITC conjugate shows that, as determined by analysis SEC, 98.1% is monomer. [Figure 12]The graph of compound 52 CT-tagged trastuzumab conjugate is shown, and when determined by HIC, it yields a DAR of 1.56. [Figure 13] The graph of compound 52 CT-tagged trastuzumab conjugate shows that, as determined by analysis SEC, 95.6% is monomer. [Figure 14] The graph of compound 52 CT-tagged sacituzumab conjugate is shown, and when determined by HIC, a DAR of 1.02 is obtained. [Figure 15] The graph of compound 52 CT-tagged sacituzumab conjugate shows that, as determined by analysis SEC, 98.5% is monomer. [Figure 16] The graph of compound 49 CT-tagged anti-FITC conjugate is shown, and when determined by HIC, a DAR of 1.09 is obtained. [Figure 17] The graph of compound 49 CT-tagged anti-FITC conjugate shows that 90.0% is monomer, as determined by analysis SEC. [Figure 18] The graph of compound 49, CT-tagged trastuzumab conjugate, shows a DAR of 1.09 when determined by HIC. [Figure 19] The graph of compound 49 CT-tagged trastuzumab conjugate shows that, as determined by analysis SEC, 90.0% is monomer. [Figure 20] The graph of compound 49 CT-tagged sacituzumab conjugate is shown, and when determined by HIC, a DAR of 0.80 is obtained. [Figure 21] The graph of compound 49 CT-tagged sacituzumab conjugate shows that, as determined by analysis SEC, 93.3% is monomer. [Figure 22] The graph shows the in vitro efficacy of HER2-targeting ADCs, isotype-controlled ADCs, or CL2A-SN38 having (21) compared to (28) against SK-BR-3 cells. [Figure 23]The graph shows the in vitro efficacy of a TROP-2-targeting ADC having (21), or an isotype control ADC, or CL2A-SN38, compared to (28) against MDA-MB-468 cells. [Figure 24] The graph shows the in vitro efficacy of TROP-2-targeting ADCs having (21) or isotype-controlled ADCs compared to (28) or (12) against MDA-MB-468 cells. [Figure 25] The graph shows the in vitro efficacy of TROP-2-targeting ADCs or isotype-controlled ADCs having (49) compared to (41) against MDA-MB-468 cells. [Figure 26] The graph shows the in vitro efficacy of HER2-targeting ADCs or isotype-controlled ADCs having (21) compared to (28) or (12) against SK-BR-3 cells. [Figure 27] The graph shows the in vitro efficacy of HER2-targeting ADCs or isotype-controlled ADCs having (49) compared to (41) against SK-BR-3 cells. [Figure 28] The graph shows the in vitro efficacy of HER2-targeting ADCs or isotype-controlled ADCs having (49) compared to (41) against NCI-N87 cells. [Figure 29] The graph shows the in vitro efficacy of HER2-targeting ADCs or isotype-controlled ADCs having (21) compared to (28) or (12) against NCI-N87 cells. [Figure 30] The graph shows the in vitro efficacy of HER2-targeting ADCs or isotype-controlled ADCs having (52) compared to (41) against SK-BR-3 cells. [Figure 31] The graph shows the in vitro efficacy of TROP-2-targeting ADCs or isotype-controlled ADCs having (52) compared to (41) against MDA-MB-468 cells.

[0045] definition The following terms have the meanings set forth below unless otherwise indicated. Any undefined terms have the meanings recognized in the art.

[0046] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms, for example, 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0047] 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) are optionally -O-, -N-, -S-, -S(O) n - (wherein n is 0 to 2), -NR- (wherein R is hydrogen or alkyl), and are replaced by heteroatoms such as alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R bThe ring has 1 to 5 substituents selected from the group consisting of the following, 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 heterocyclic rings.

[0048] "Alkylene" is a divalent aliphatic hydrocarbyl group having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms, which are either linear or branched, and optionally -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 This term refers to a divalent aliphatic hydrocarbyl group that is interrupted by one or more groups selected from the following. Examples of this term include methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), iso-propylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), etc.

[0049] "Substitutive alkylene" refers to an alkylene group having 1 to 3 hydrogen atoms replaced by substituents, as described in the definition of "substitution" below for carbon.

[0050] The term "alkane" refers to alkyl and alkylene groups as defined herein.

[0051] The terms "alkylaminoalkyl," "alkylaminoalkenyl," and "alkylaminoalkynyl" refer to an R'NHR"-group, where R' is an alkyl group as defined herein and R" is an alkylene, alkenylene, or alkynylene group as defined herein.

[0052] The terms "alkaryl" or "aralkyl" refer to alkylene-aryl and substituted alkylene-aryl groups, and alkylene, substituted alkylene and aryl are defined herein.

[0053] "Alkoxy" refers to an -O-alkyl group, where alkyl is as defined herein. Examples of alkoxys include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and n-pentoxy. The term "alkoxy" also refers to alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O- groups, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0054] The term "substituted alkoxy" refers to the groups of substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0055] The term "alkoxyamino" refers to an -NH-alkoxy group, where alkoxy is defined herein.

[0056] The term "haloalkoxy" refers to an alkyl-O- group in which one or more hydrogen atoms on the alkyl group are substituted with a halo group, and includes groups such as trifluoromethoxy as an example.

[0057] The term "haloalkyl" refers to the substituted alkyl group described above, in which one or more hydrogen atoms on the alkyl group are substituted with a halo group. Examples of such groups include, but are not limited to, fluoroalkyl groups such as trifluoromethyl, difluoromethyl, and trifluoroethyl.

[0058] The term "alkylalkoxy" refers to the groups of alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl, where alkyl, substituted alkyl, alkyl, alkylene, and substituted alkylene are as defined herein.

[0059] The term "alkylthioalkoxy" refers to the groups of alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl, and substituted alkylene-S-substituted alkyl, where alkyl, substituted alkyl, alkyl, and substituted alkyl are as defined herein.

[0060] "Alkenyl" refers to a linear or branched hydrocarbyl group having 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, and at least 1, preferably 1 to 2 double bond unsaturated sites. This term includes, as examples, vivinyl, allyl, and buta-3-en-1-yl. This term includes cis and trans isomers, or mixtures thereof.

[0061] The term "substituted alkenyl" refers to an alkenyl group as defined herein having 1 to 5 substituents 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, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0062] "Alkynyl" refers to a linear or branched monovalent hydrocarbyl group having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and at least 1, preferably 1 to 2 triple bond unsaturated sites. Examples of such alkynyl groups include acetylenyl (-C≡CH) and propargyl (-CH2C≡CH).

[0063] The term "substituted alkynyl" refers to an alkynyl group as defined herein, having 1 to 5 substituents 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, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0064] "Alkynyloxy" refers to an -O-alkynyl group, where alkynyl is as defined herein. Examples of alkynyloxy include ethynyloxy and propynyloxy.

[0065] "Acyl" includes 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)-, heteroaryl-C(O)-, and substituted he The terms refer to teloaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)- groups, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein. For example, acyl contains the "acetyl" group CH3C(O)-.

[0066] "Acylamino" is -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) heterocyclic rings, and -NR 20 R refers to the group of a C(O)-substituted heterocyclic ring. 20The elements are hydrogen or alkyl, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein.

[0067] The terms "aminocarbonyl" or "aminoacyl" are defined as -C(O)NR 21 R 22 It refers to the base, R 21 and R 22 However, 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, heterocyclic ring, and substituted heterocyclic ring, R 21 and R 22 However, optionally, they bond with the nitrogen attached to them to form a heterocyclic ring or a substituted heterocyclic ring group, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein.

[0068] "Aminocarbonylamino" is -NR 21 C(O)NR 22 R 23 It refers to the base, R 21 , R 22 , and R 23 However, independently, the R groups are selected from hydrogen, alkyl, aryl, or cycloalkyl, or two R groups are bonded to form a heterocycline group.

[0069] The term "alkoxycarbonylamino" refers to the -NRC(O)OR group, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl, and alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0070] 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-, where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0071] "Aminosulfonyl" is -SO2NR 21 R 22 It refers to the base, R 21 and R 22 However, 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, heterocyclic ring, substituted heterocyclic ring, R 21 and R 22 However, optionally, they bond with the nitrogen attached to them to form a heterocyclic ring or a substituted heterocyclic ring group, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein.

[0072] "Sulfonylamino" is -NR 21 SO2R 22 It refers to the base, R 21 and R 22However, 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, heterocyclic ring, and substituted heterocyclic ring, R 21 and R 22 However, optionally, they bond with atoms attached to them to form heterocyclic rings or substituted heterocyclic ring groups, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein.

[0073] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a single ring (such as those found in a phenyl group), or a ring system having multiple fused rings (examples of such aromatic ring systems include naphthyl, anthryl, and indanyl), where the fused rings may or may not be aromatic, provided that the bonding sites are mediated by atoms of the aromatic ring. This term includes, by example, phenyl and naphthyl. Unless otherwise restricted by the definition of aryl substituents, such aryl 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, aryloxy, azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, 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.

[0074] "Aryloxy" refers to an -O-aryl group, where aryl includes optionally substituted aryl groups as also defined herein, including, for example, phenoxy, naphthoxy, and the like.

[0075] "Amino" refers to the -NH2 group.

[0076] The term "substituted amino" refers to a -NRR group, where 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, provided that at least one R is not hydrogen.

[0077] The term "azide" refers to the -N3 group.

[0078] "Carboxyl," "carboxy," or "carboxylate" refers to -CO2H or its salts.

[0079] The terms "carboxyl ester" or "carboxyester," or "carboxyalkyl" or "carboxylalkyl" refer to -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, and -C(O) The term refers to the groups of O-substituted cycloalkenyls, -C(O)O-heteroaryls, -C(O)O-substituted heteroaryls, -C(O)O-heterocyclic rings, and -C(O)O-substituted heterocyclic rings, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring are as defined herein.

[0080] "(carboxyl ester) oxy" or "carbonate" refers to -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-heterocyclic ring, and -OC(O)O-substituted heterocyclic ring groups are defined herein as alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring.

[0081] "Cyano" or "nitrile" refers to the -CN group.

[0082] "Cycloalkyl" refers to a cyclic alkyl group of 3 to 10 carbon atoms having one or more cyclic rings, including fused ring systems, crosslinked ring systems, and spiro ring systems. Suitable examples of cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl. Such cycloalkyl groups include, for example, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl, or multi-cyclic structures such as adamantanyl.

[0083] The term "substituted cycloalkyl" refers to a cycloalkyl group having 1 to 5 substituents or 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0084] "Cycloalkenyl" refers to a non-aromatic cyclic alkyl group having 3 to 10 carbon atoms, having one or more rings, at least one double bond, preferably 1 to 2 double bonds.

[0085] The term "substituted cycloalkenyl" refers to a cycloalkenyl group having 1 to 5 substituents 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, azide, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0086] "Cycloalkynyl" refers to a non-aromatic cycloalkyl group consisting of 5 to 10 carbon atoms, having one or more rings and at least one triple bond.

[0087] "Cycloalkoxy" refers to -O-cycloalkyl.

[0088] "Cycloalkenyloxy" refers to -O-cycloalkenyl.

[0089] "Halo" or "halogen" refers to fluoro, chloro, bromo, and iodine.

[0090] "Hydroxy" or "hydroxyl" refers to the -OH group.

[0091] A "heteroaryl" refers to an aromatic group consisting of 1 to 15 carbon atoms, for example, 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur in the ring. Such a heteroaryl group may have a monocyclic ring (such as pyridinyl, imidazolyl, or furyl) or a plurality of fused rings (such as those found in groups such as indolidinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl) in the ring system, and at least one ring in the ring system is aromatic. To satisfy the valence requirement, any heteroatom in such a heteroaryl ring may or may not be bonded to H or a substituent, for example, an alkyl group or other substituent as described herein. In certain embodiments, the nitrogen and / or sulfur ring atoms of the heteroaryl group are optionally oxidized to provide an N-oxide (N→O), sulfinyl, or sulfonyl moiety. This term includes, by example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise restricted 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, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, 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.

[0092] The term "heteroaralkyl" refers to the alkylene-heteroaryl group as defined herein, where alkylene and heteroaryl are also used. Examples of this term include pyridylmethyl, pyridylethyl, and indolylmethyl.

[0093] "Heteroaryloxy" refers to -O-heteroaryl.

[0094] "Heterocyclic," "heterocyclic ring," "heterocycloalkyl," and "heterocyclyl" refer to saturated or unsaturated groups having a monocyclic or multiple fused rings, including fused ring systems, bridging ring systems, and spiro-ring systems, and containing 1 to 10 heteroatoms and 3 to 20 ring atoms. These ring atoms are selected from nitrogen, sulfur, or oxygen, and in fused ring systems, one or more of the rings may be cycloalkyl, aryl, or heteroaryl, provided that the bonding site is via a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclic ring group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety. To satisfy valency requirements, any heteroatom in such a heterocyclic ring may or may not be bonded to one or more H atoms or one or more substituents, such as alkyl groups or other substituents as described herein.

[0095] Examples of heterocyclic and heteroaryl compounds include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indidine, isoindole, indole, dihydroindole, indazole, purine, quinoridine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carborin, phenanthridine, acridine, phenanthroline, isothiazole, and phenazine. Examples include 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, morpholinil, thiomorpholinil (also called thiamorpholinil), 1,1-dioxothiomorpholinil, piperidinil, pyrrolidine, and tetrahydrofuranil.

[0096] Unless otherwise restricted by the definition of heterocyclic ring substituents, such heterocyclic ring groups 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, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and condensed heterocyclic rings.

[0097] "Heterocyclyloxy" refers to the -O-heterocyclyl group.

[0098] The term "heterocyclilthio" refers to a heterocyclic ring-S-group.

[0099] The term "heterocyclene" refers to a diradical group formed from a heterocycle, as defined herein.

[0100] The term "hydroxyamino" refers to the -NHOH group.

[0101] "Nitro" refers to the -NO2 group.

[0102] "Oxo" refers to an atom (=O).

[0103] "Sulfonyl" refers to the groups of -SO2-alkyl, -SO2-substituted alkyl, -SO2-alkenyl, -SO2-substituted alkenyl, -SO2-cycloalkyl, -SO2-substituted cycloalkyl, -SO2-cycloalkenyl, -SO2-substituted cycloalkenyl, -SO2-substituted aryl, -SO2-substituted aryl, -SO2-heteroaryl, -SO2-substituted heteroaryl, -SO2-heterocyclic, and -SO2-substituted heterocyclic, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Examples of sulfonyls include methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0104] "Sulfonyloxy" refers to the groups of -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-heterocyclic, and -OSO2-substituted heterocyclic, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0105] "Sulfate" or "sulfate ester" refers to -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-heterocyclic, and -O-SO2-O-substituted heterocyclic groups, while alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0106] The term "aminocarbonyloxy" refers to the -OC(O)NRR group, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic ring, where alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclic rings are as defined herein.

[0107] "Thiol" refers to the -SH group.

[0108] The terms "thioxo" or "thioketo" refer to an atom (=S).

[0109] The terms "alkylthio" or "thioalkoxy" refer to an -S-alkyl group, where alkyl is as defined herein. In certain embodiments, sulfur may be oxidized to -S(O)-. The sulfoxide may exist as one or more stereoisomers.

[0110] The term "substituted thioalkoxy" refers to an -S-substituted alkyl group.

[0111] The term "thioaryloxy" refers to an aryl-S- group, and the aryl group is as defined herein, including optionally substituted aryl groups, also as defined herein.

[0112] The term "thioheteroaryloxy" refers to a heteroaryl-S-group, and the heteroaryl group is as defined herein, including optionally substituted aryl groups, also as defined herein.

[0113] The term "thioheterocyclooxy" refers to a heterocyclyl-S-group, and the heterocyclyl group is as defined herein, including optionally substituted heterocyclyl groups, also as defined herein.

[0114] In addition to the disclosures herein, the term “substituted” may also mean, when used to modify a designated group or radical, that one or more hydrogen atoms of the designated group or radical are replaced independently of each other by the same or different substituents as defined below.

[0115] In addition to the groups disclosed with respect to individual terms herein, substituents for substituting one or more hydrogens on a saturated carbon atom in a particular group or radical (any two hydrogens on a single carbon, =O, =NR) are also available. 70 、=N-OR 70 (Can be replaced with =N2 or =S) Unless otherwise specified, -R 60 , halo, =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 )O - 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 , -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 And R 60 However, selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each R 70 However, independently, hydrogen or R 60 And each R 80 However, R is independent 70 Either or, two R's 80 However, together with the nitrogen atom to which they are bonded, they form 5-membered, 6-membered, or 7-membered heterocycloalkyl groups, which may optionally contain 1 to 4 additional heteroatoms, one or more of the same or different, selected from the group consisting of O, N, and S, where N may have a -H or C1-C3 alkyl substitution, and each M + However, each M is a counterion with a single positive charge. + These are independent, for example, alkaline ions, for example, K + na + Li + , ammonium ions, for example, + N(R 60 )4, or alkaline earth ions, for example, [Ca 2+ ] 0.5 [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5(The subscript 0.5 means that one of the counterions of such divalent alkaline earth ions may be an ionized form of the compound of the present invention, and that other typical counterions such as chlorides, or the two ionized compounds disclosed herein, may function as counterions of such divalent alkaline earth ions, or that the biionized compound of the present invention may function as a counterion of such divalent alkaline earth ions.) For example, -NR 80 R 80 This means that it includes -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methylpiperazin-1-yl, and N-morpholinyl.

[0116] In addition to the disclosures herein, the substituents of hydrogen on unsaturated carbon atoms in "substituted" alkenes, alkynes, aryl and heteroaryl groups are, unless otherwise specified, -R 60 Hello, -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 )O - 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 And in the formula, R 60 , R 70 , R 80 , and M + However, as already defined, except in the case of substituted alkenes or alkynes, the substituent is -O - M + , -OR 70 , -SR 70 , or -S - M + isn't it.

[0117] In addition to the groups disclosed with respect to the individual terms herein, substituents on the nitrogen atom of a "substituted" heteroalkyl and cycloheteralkyl group are, unless otherwise specified, -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR80 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 )O - M + 、 -P(O)(OR 70 )(OR 70 )、 -C(O)R 70 、 -C(S)R 70 、 -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 wherein, R 60 、 R 70 、 R 80 、 and M +However, this is already defined.

[0118] In addition to the disclosures herein, in certain embodiments, the substituted group has one, two, three, or four substituents, one, two, or three substituents, one or two substituents, or one substituent.

[0119] It is understood that polymers obtained by defining substituents that have further substituents themselves (for example, a substituted aryl group having a substituted aryl group as a substituent, being itself substituted with a substituted aryl group, and being further substituted with a substituted aryl group) are not intended to be included herein. In such cases, the maximum number of such substitutions is three. For example, the sequential substitution of substituted aryl groups specifically intended herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0120] Unless otherwise indicated, the nomenclature of substituents not explicitly defined herein is reached by naming the terminal portion of the functional group followed by the adjacent functional group toward the bonding point. For example, the substituent "arylalkyloxycarbonyl" refers to the (aryl)-(alkyl)-OC(O)- group.

[0121] With respect to any of the groups disclosed herein that include one or more substituents, it should be understood that such groups naturally do not include any substitutions or substitution patterns that are sterically unfeasible and / or synthetically unfeasible. In addition, the compounds in question include all stereochemical isomers resulting from the substitutions of these compounds.

[0122] The term “pharmaceutically acceptable salt” means a salt that is acceptable for administration to a patient, e.g., a mammal (a salt having a counterion that has mammalian safety acceptable for a given dosage plan). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable salt of a compound, the salts of which are derived from a variety of organic and inorganic counterions well known in the art, and include, but are not limited to, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and, if the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, and oxalate.

[0123] The term "salt" refers to a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds not intended for administration to a patient. As an example, salts of the compounds of the present invention include those in which the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

[0124] A "solvate" refers to a complex formed by a combination of solvent molecules and solute molecules or ions. The solvent can be an organic compound, an inorganic compound, or a mixture of both. 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.

[0125] "Stereoisomers" refer to compounds that have the same atomic bonding but different atomic arrangements in space. Stereoiomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[0126] "Tautomers" refers to alternative molecular forms that differ only in the electron bonding of atoms and / or in the position of protons, such as enolketo and imine-enamine tautomers, or tautomers of heteroaryl groups containing the -N=C(H)-NH- ring configuration, such as pyrazole, imidazole, benzimidazole, triazole, and tetrazole. Those skilled in the art will recognize that other tautomer ring configurations are possible.

[0127] It should be understood that the term "or its salt, solvate, or stereoisomer" is intended to include all substitutions (permutations) of salts, solvates, and stereoisomers, such as solvates of pharmaceutically acceptable salts of the stereoisomer of the compound in question.

[0128] "Pharmacologically effective dose" and "therapeutic effective dose" refer to the amount of a compound sufficient to treat one or more of a particular disorder or disease, or its symptoms, and / or prevent the onset of the disease or disorder. With respect to tumorigenic proliferative disorders, the pharmacochemically effective dose or therapeutic effective dose includes, in particular, an amount sufficient to shrink the tumor or slow its growth rate.

[0129] The term "patient" refers to both human and non-human subjects, particularly mammals.

[0130] As used herein, the terms “to treat” or “to cure” mean treating or curing a disease or medical condition of a patient, such as a mammal (especially a human), and include (a) preventing the onset of a disease or medical condition, such as by prophylactic treatment of the subject; (b) improving a disease or medical condition, such as eliminating or reversing a disease or medical condition of a patient; (c) suppressing a disease or medical condition, such as by delaying or stopping the onset of a disease or medical condition of a patient; or (d) alleviating the symptoms of a disease or medical condition of a patient.

[0131] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymeric forms of amino acids of any length. Unless otherwise specified, “polypeptide,” “peptide,” and “protein” may include genetically encoded and unencoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone. The terms are not limited to, but include, fusion proteins having heterologous amino acid sequences, fusions having heterologous and homologous leader sequences, fusion proteins containing at least one N-terminal methionine residue (e.g., to facilitate production in recombinant bacterial host cells), immunotagged proteins, and the like.

[0132] "Natural amino acid sequence" or "parent amino acid sequence" is used interchangeably herein and refers to the amino acid sequence of the polypeptide before modification, such as the sequence containing modified amino acid residues.

[0133] The terms “amino acid analog” and “non-natural amino acid” may be used interchangeably and include amino acid-like compounds that are structurally and / or in shape similar 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 backbone. Amino acid analogs also include amino acid analogs that have the same stereochemistry as the naturally occurring D form, as well as the L form of amino acid analogs. In some cases, amino acid analogs share the backbone structure and / or side chain structure of one or more natural amino acids, the difference being one or more modifying groups in the molecule. Such modifications may include, but are not limited to, the substitution of an atom (e.g., N) with a related atom (e.g., S), the addition of a group (e.g., methyl or hydroxyl) or an atom (e.g., Cl or Br), the deletion of a group, the substitution of a covalent bond (e.g., a single bond to a double bond), or combinations thereof. For example, amino acid analogs may include α-hydroxy acids and α-amino acids.

[0134] Terms such as “amino acid side chain” or “side chain of amino acids” may be used to refer to substituents 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 herein in the context of modified amino acids and / or conjugates.

[0135] The term "carbohydrate" 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 and disaccharides. The term "carbohydrate derivative" includes compounds in which one or more functional groups of the carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemical), or absent (e.g., removed or replaced by H). A variety of carbohydrates and carbohydrate derivatives are available and may be suitable for use in the subject compound and conjugate.

[0136] The terms “glycoside” or “glycosyl” refer to a sugar molecule or group linked to a part via a glycosidic bond. For example, the part to which a glycoside is linked may be a cleavable linker as described herein. Glycosidic bonds can link a glycoside to another part 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 may be cleaved from the part to which they are linked by, for example, chemically mediated hydrolysis or enzymatically mediated hydrolysis.

[0137] The term "antibody" is used in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, single-chain antibodies, chimeric antibodies, and antibody fragments (e.g., Fab fragments). Antibodies can bind to target antigens. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). Target antigens may have one or more binding sites, also called epitopes, that are recognized by complementarity-determining regions (CDRs) formed by one or more variable regions of the antibody.

[0138] The term "natural antibody" refers to an antibody in which the heavy and light chains are 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 antibodies produced by cells isolated from a first animal immunized with an antigen are natural antibodies.

[0139] The terms "humanized antibody" or "humanized immunoglobulin" refer to non-human (e.g., mouse or rabbit) antibodies that contain one or more amino acids (e.g., in the framework region, constant region, or CDR) that are substituted with amino acids corresponding to those found in human antibodies. Generally, humanized antibodies produce a reduced immune response in the human host compared to the non-humanized form of the same antibody. Antibodies can be humanized using various techniques known in the art, including, for example, CDR grafting (EP239,400, PCT Publication WO91 / 09967, U.S. Patents 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP592,106, EP519,596, Padlan, Molecular Immunology 28(4 / 5):489-498 (1991), Studnicka et al., Protein Engineering 7(6):805-814 (1994), Roguska et al., PNAS91:969-973 (1994)), and chain shuffling (U.S. Patent 5,565,332). In certain embodiments, framework substitutions are identified by modeling the interaction between CDRs and framework residues, identifying framework residues important for antigen binding and sequence comparison, and identifying abnormal framework residues at specific locations (see, for example, U.S. Patent No. 5,585,089, Riechmann et al., Nature 332:323 (1988)). Additional methods for humanizing the antibodies intended for use in the present invention are described in U.S. Patents 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, as well as PCT Publications WO98 / 45331 and WO98 / 45332. In certain embodiments, the subject rabbit antibody may be humanized according to the methods described in US2004 / 0086979 and US2005 / 0033031. Thus, the antibodies described above may be humanized using methods well known in the art.

[0140] The term "chimeric antibody" refers to an antibody in which the light and heavy chain genes are constructed, typically through genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segment of a gene from a mouse monoclonal antibody can bind to human constant segments such as gamma 1 and gamma 3. An example of a therapeutic chimeric antibody is a hybrid protein composed of a variable domain or antigen-binding domain derived from a mouse antibody and a constant domain or effector domain derived from a human antibody, although domains from other mammalian species may also be used.

[0141] The immunoglobulin light chain or heavy chain variable region of an immunoglobulin polypeptide consists of a framework region (FR) interrupted by three hypervariable regions, also known as the “complementarity-determining region” or “CDR.” The extent of the framework region and CDR is defined (see “Sequences of Proteins of Immunological Interest,” E. Kabat et al., USD Department of Health and Human Services, 1991). The framework region of an antibody, which is a combined framework region of its constituent light and heavy chains, helps to locate and align the CDR. The CDR primarily contributes to the binding of the antigen to the epitope.

[0142] The "parent Ig polypeptide" is a polypeptide comprising an amino acid sequence lacking the aldehyde-tagged constant region described herein. The parent polypeptide may include a natural sequence constant region, or it may include a constant region having existing amino acid sequence modifications (such as additions, deletions, and / or substitutions).

[0143] As used herein, the term “isolated” means describing a compound of interest that is in an environment different from that in which it naturally occurs. “Isolated” means including a compound in a sample in which the compound of interest is substantially concentrated and / or partially or substantially purified.

[0144] As used herein, the term “substantially purified” means a compound that has been removed from its natural environment and contains at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more than 98% of other components associated with its natural origin.

[0145] The term "physiological conditions" means those conditions that are compatible with living cells, such as temperature, pH, salinity, and other primarily aqueous conditions that are compatible with living cells.

[0146] A “reactive partner” means a molecule or molecular part that specifically reacts with another reactive partner to produce a reaction product. An exemplary reactive partner comprises a sulfatase motif of cysteine ​​or serine and a formylglycine-producing enzyme (FGE), which react to form a converted aldehyde tag reaction product containing formylglycine (FGly) instead of cysteine ​​or serine in the motif. Another exemplary reactive partner comprises an “aldehyde-reactive reactive partner” comprising the aldehyde (e.g., a reactive aldehyde group) of the fGly residue of the converted aldehyde tag and the moiety of interest, which react to form a modified aldehyde-tagged polypeptide reaction product having the moiety of interest conjugated to a modified polypeptide via a modified fGly residue.

[0147] The "N-terminus" refers to the terminal amino acid residue of a polypeptide that has a free amine group, and the amine group in the non-N-terminal amino acid residue usually forms part of the covalent backbone of the polypeptide.

[0148] The "C-terminus" refers to the terminal amino acid residue of a polypeptide that has a free carboxyl group, and the carboxyl groups in non-C-terminal amino acid residues usually form part of the covalent backbone of the polypeptide.

[0149] When used in reference to polypeptides or the amino acid sequences of polypeptides, "internal region" refers to the region of the polypeptide that is neither N-terminus nor C-terminus.

[0150] Before further description of the present invention, it should be understood that the present invention is not limited to the specific embodiments described and is, of course, subject to change. It should also be understood that the terms used herein are merely for describing specific embodiments and are not intended to be limiting, as the scope of the present invention is limited solely by the appended claims.

[0151] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, each intervening value up to one-tenth of the lower limit between the upper and lower limits of that range, and any other stated or intervening values ​​within the stated range, are understood to be included within the invention. The upper and lower limits of these smaller ranges may independently be included within those smaller ranges and are also included within the invention, assuming any specifically excluded boundaries within those stated ranges. If a stated range includes one or both of the boundaries, the range excluding one or both of the boundaries that they include is also included within the invention.

[0152] For clarity, it is understood that certain features of the Invention described in the context of separate embodiments may also be combined to be provided in a single embodiment. Conversely, for brevity, various features of the Invention described in the context of a single embodiment may also be provided separately or in any preferred subcombination. All combinations of embodiments relating to the Invention are specifically encompassed by the Invention, and indeed, every conceivable combination is disclosed herein as if individually and explicitly disclosed, to the extent that such combinations encompass subject matter, for example, compounds that are stable compounds (i.e., compounds that can be prepared, isolated, characterized, and tested for biological activity). In addition, all subcombinations of various embodiments and their elements (e.g., elements of chemical groups enumerated in embodiments describing such variables) are also specifically encompassed by the Invention, and indeed, every conceivable subcombination is disclosed herein as if individually and explicitly disclosed.

[0153] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of the present invention, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference to disclose and describe methods and / or materials in relation to those cited herein.

[0154] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context explicitly indicates otherwise. It should also be noted that claims may be written to exclude any optional elements. Therefore, this statement is intended to serve as an antecedent for the use of exclusive terms such as "simply," "only," or "negative" limitations relating to the description of elements of the claims.

[0155] For clarity, it is understood that certain features of the invention described in the context of separate embodiments may also be combined and provided in a single embodiment. Conversely, for brevity, various features of the invention described in the context of a single embodiment may also be provided separately or in any preferred subcombination.

[0156] The publications discussed herein were made available solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as acknowledging that the present invention does not have prior rights to such disclosure by prior art. Furthermore, the dates of the publications provided may differ from the actual publication dates which may need to be independently verified. [Modes for carrying out the invention]

[0157] This disclosure provides an antibody-drug conjugate structure comprising a cleavable linker containing an ester group that binds an antibody to a drug. This disclosure also includes methods for producing such a conjugate and methods for using such a conjugate.

[0158] Most payloads require cleavage from the ADC assembly to exhibit activity. Therefore, the chemical linkers used in ADCs are primarily cleavable under intracellular (lysosomal) conditions. Typically, such linkers are susceptible to enzymatic cleavage by proteases and glycosidases, or are acid-degradable. For example, many amine-containing drugs can readily bind to protease-cleavable dipeptide linkers via self-immolative spacers and carbamate groups (Figure 2, Panel A). In contrast, payloads with only free hydroxyl groups as the bindable portion have proven difficult to use directly in ADCs. Cleavable linkers for such payloads require special design and often necessitate cumbersome synthetic approaches. Binding hydroxyl-containing payloads to antibodies using carboxyl ester groups appears to be the most direct and synthetically manageable approach for producing ADCs with this class of payloads (Figure 2, Panel B). However, esters are highly sensitive to various plasma hydrolases (esterases) and therefore typically lack high stability in circulation (t 1 / 2 This is not considered a useful linker for ADCs requiring (1 week). This disclosure provides an ADC that includes the use of an ester linker for directly conjugating a hydroxyl-containing drug to an antibody for the formation of a plasma-stable conjugate.

[0159] ADCs containing a carboxyl ester linker adjacent to the monosaccharide moiety may exhibit higher plasma stability compared to fully exposed ester groups, and can be efficiently cleaved during ADC internalization and transport, as shown in Figure 3.

[0160] Ester cleavage by extracellular hydrolases before entry into target cells may be negligible due to steric hindrance provided by the adjacent glycoside, which is itself very stable in plasma. Conversely, upon ADC internalization, the monosaccharide group may be efficiently removed by lysosomal glycosidases, leaving an ester group accessible by enzymatic hydrolysis by intracellular esterases. This latter step would lead to the release of the original payload, accompanied by the recovery of the free hydroxyl group used for conjugate (Figure 3). Examples of such double-cleaved ester glycoside linkers are shown in Figure 4. These include salicylic acid ester linkers containing an orthoglycoside moiety (1), and structurally related derivatives of α-arylglycine (2). Applying the same structural design concept to serine, isoserine, and threonine provides glycosidicated ester linkers 3, 4, and 5, respectively. Monosaccharide-containing derivatives of malic acid and tartaric acid (6 and 7, respectively) are also provided according to the same principle (Figure 4).

[0161] Antibody-drug conjugates This disclosure provides conjugates, such as antibody-drug conjugates (ADCs). “Conjugate” means a first part (e.g., an antibody) that stably associates with a second part (e.g., a drug or active agent). For example, an antibody-drug conjugate includes a drug or active agent stably associated with another part (e.g., an antibody). “Stable association” means that one part is bonded to another part or structure under standard conditions. In certain embodiments, the first and second parts are linked to each other via one or more functional groups and covalent bonds. For example, one or more functional groups and covalent bonds may include cleavable linkers as described herein.

[0162] In certain embodiments, the conjugate is a polypeptide conjugate comprising a polypeptide conjugated in a second portion. In certain embodiments, the portion conjugated to the polypeptide may be any of a variety of purposes, but is not limited to a drug, an active agent, a detectable label, a water-soluble polymer, or a portion for immobilizing the polypeptide onto a membrane or surface. In certain embodiments, the conjugate is a drug conjugate, where the polypeptide is an antibody, and therefore provides an antibody-drug conjugate. For example, the conjugate may be a drug conjugate in which the polypeptide is conjugated to a drug or active agent. Various types of drugs or active agents may be used in the conjugate, as described in more detail below.

[0163] A target portion (e.g., a drug or active agent) can be conjugated to a polypeptide (e.g., an antibody) at any desired site of the polypeptide. Therefore, this disclosure provides, for example, modified polypeptides having a conjugated portion at or near the C-terminus of the polypeptide. Other examples include modified polypeptides having a conjugated portion at or near the N-terminus of the polypeptide. Examples also include modified polypeptides having a conjugated portion between the C-terminus and N-terminus of the polypeptide (e.g., an internal site of the polypeptide). The above combinations are also possible when the modified polypeptide is conjugated to two or more portions.

[0164] 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 an amino acid residue. In other words, the conjugates include polypeptides in which the side chains of one or more amino acid residues in the polypeptide are modified to bind to drugs or active agents (e.g., via linkers as described herein). For example, the conjugates include polypeptides in which the α-carbon of one or more amino acid residues in the polypeptide is modified to bind to drugs or active agents (e.g., via linkers as described herein).

[0165] Embodiments of the present disclosure include conjugates in which a polypeptide is conjugated to one or more parts, for example, two, three, four, five, six, seven, eight, nine, or ten or more parts. A part can be conjugated to one or more sites in the polypeptide. For example, one or more parts can be conjugated to a single amino acid residue of the polypeptide. In some cases, one part is conjugated to an amino acid residue of the polypeptide. In other embodiments, two parts can be conjugated to the same amino acid residue of the polypeptide. In other embodiments, a first part is conjugated to a first amino acid residue of the polypeptide, and a second part is conjugated to a second amino acid residue of the polypeptide. For example, the above combination is also possible when the polypeptide is conjugated to a first part at a first amino acid residue and to two other parts at a second amino acid residue. Other combinations are also possible, but are not limited to these, such as polypeptides in which the first amino acid residue is conjugated to the first and second parts, and the second amino acid residue is conjugated to the third and fourth parts.

[0166] One or more amino acid residues of a polypeptide that are conjugated to one or more parts may be naturally occurring amino acids, unnatural amino acids, or a combination thereof. For example, a conjugate may include a part conjugated to a naturally occurring amino acid residue of the polypeptide. In other examples, a conjugate may include a part conjugated to an unnatural amino acid residue of the polypeptide. One or more parts may be conjugated to a polypeptide with a single natural or unnatural amino acid residue, as described above. One or more natural or unnatural amino acid residues in a polypeptide may be conjugated to one or more parts, as described herein. For example, two (or more) amino acid residues (e.g., natural or unnatural amino acid residues) in a polypeptide may each be conjugated to one or two parts, such that multiple sites in the polypeptide are modified.

[0167] As described herein, polypeptides can be conjugated to one or more parts. In certain embodiments, the part of interest is a chemical entity such as a drug, an active agent, or a detectable label. For example, a drug (or active agent) may be conjugated to a polypeptide, or in other embodiments, a detectable label may be conjugated to a polypeptide. Thus, embodiments of the present disclosure, for example, include, but are not limited to,: a polypeptide and a drug conjugate, a polypeptide and an active agent conjugate, a polypeptide and a detectable label conjugate, a conjugate of two or more drugs and polypeptides, a conjugate of two or more detectable labels and polypeptides, and so on.

[0168] In certain embodiments, a polypeptide (e.g., an antibody) and a moiety of interest (e.g., a drug or active agent) are conjugated via a conjugation moiety. For example, the polypeptide and the moiety of interest may each be bound (e.g., covalently) to the conjugation moiety, and thus indirectly linked together via the conjugation moiety. In some cases, the conjugation moiety includes a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound, or a derivative of a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound. For example, a general scheme for coupling a moiety of interest to a polypeptide via a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety is shown in the general reaction scheme below. The hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moieties are also referred to herein as the hydrazino-iso-Pictet-Spengler (HIPS) conjugated moiety and the aza-hydrazino-iso-Pictet-Spengler (aza-HIPS) conjugated moiety, respectively. [ka]

[0169] In the above reaction scheme, R comprises a moiety of interest (e.g., a drug or active agent) to be 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) is reacted with a drug or active agent modified to include a conjugation moiety (e.g., a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety) to produce a polypeptide conjugate bound to the conjugation moiety, thereby conjugating the drug or active agent to the polypeptide via the conjugation moiety.

[0170] As described herein, this part may be any of the following: a variety of parts, for example, a chemical entity, for example, a detectable label, or a drug or active agent. R' and R'' may each be independently any desired substituent, for example, 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 is CR 21 , NR 22 It may be N, O, or S, in the formula, R 21 and R 22 Each of these substituents is independently selected from among the substituents described above for R' and R''.

[0171] Other hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugate moieties are also possible, as shown in the conjugates and compounds described herein. For example, hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugate moieties may be modified to be bonded (e.g., covalently bonded) to a linker. Thus, embodiments of the present disclosure include hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugate moieties that are bonded to a drug or active agent via a linker. Various embodiments of linkers capable of coupling hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugate moieties to a drug or active agent are described in detail herein. For example, in some cases, the linker is a cleavable linker, e.g., a cleavable linker as described herein.

[0172] In certain embodiments, the polypeptide may be conjugated to a moiety of interest, and the polypeptide is modified before conjugation to the moiety of interest. Modification of the polypeptide may produce a modified polypeptide containing one or more reactive groups suitable for conjugation to the moiety of interest. In some cases, the polypeptide may be modified with one or more amino acid residues to provide one or more reactive groups suitable for conjugation to a moiety of interest (e.g., a moiety containing a conjugation moiety such as the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety described above). For example, the polypeptide may be modified to contain a reactive aldehyde group (e.g., reactive aldehyde). The reactive aldehyde may be contained in an "aldehyde tag" or "ald tag," which, as used herein, refer to an amino acid sequence derived from a sulfatase motif (e.g., L(C / S)TPSR) that has been converted to contain a 2-formylglycine residue (referred to herein as "FGly") by the action of formylglycine-producing enzyme (FGE). The FGly residue produced by FGE may also be referred to as “formylglycine.” In other words, the term “aldehyde tag” is used herein to refer to an amino acid sequence containing 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, e.g., L(FGly)TPSR). The converted sulfatase motif may derive from an amino acid sequence containing 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 being converted, e.g., an unconverted sulfatase motif having the sequence L(C / S)TPSR). When used in the context of the action of formylglycinase (FGE) on a sulfatase motif, “conversion” refers to the biochemical modification of a cysteine ​​or serine residue in the sulfatase motif to a formylglycine (FGly) residue (e.g., Cys to FGly, or Ser to FGly).Further embodiments of aldehyde tags in site-directed protein modification and their use are described in U.S. Patents No. 7,985,783 and No. 8,729,232, the respective disclosures of which are incorporated herein by reference.

[0173] In some cases, a modified polypeptide containing an FGly residue may be conjugated to a desired moiety by a reaction between FGly and a compound (e.g., a compound containing the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety described above). For example, an FGly-containing polypeptide may be contacted with a drug containing a reactive partner under conditions suitable for providing drug conjugation to the polypeptide. In some cases, the drug containing the reactive partner may contain the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety described above. For example, a drug or active agent may be modified to contain a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety. In some cases, the drug or active agent may bind to hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl and covalently bond to it via, for example, a linker (e.g., a cleavable linker as described in detail herein).

[0174] In certain embodiments, the conjugate of the Disclosure comprises a polypeptide (e.g., an antibody) having at least one modified amino acid residue. The modified amino acid residue of the polypeptide can be coupled to a drug or active agent containing the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugate moiety described above. In certain embodiments, the modified amino acid residue of the polypeptide (e.g., an antibody) may be derived from a cysteine ​​or serine residue converted to an FGly residue as described above. In certain embodiments, the FGly residue is conjugated to a drug or active agent containing the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugate moiety described above, providing the conjugate of the Disclosure in which the drug is conjugated to the polypeptide via the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugate moiety. As used herein, the term 'FGly' refers to a modified amino acid residue of a polypeptide (e.g., an antibody) coupled to the moiety of interest (e.g., a drug or active agent).

[0175] In certain embodiments, the conjugate comprises at least one modified amino acid residue as described herein, the modified amino acid residue being bound to a linker (cleavable linker) as described herein, and the linker (cleavable linker) being bound to a drug or active agent. For example, the conjugate may comprise the above-described at least one modified amino acid residue (FGly').

[0176] In some embodiments, the conjugate is a conjugate of the following formula (I): [ka] During the ceremony, W 1 However, it is a drug, W 2 However, it is a polypeptide, A is an amino acid residue, and k is 0 or an integer from 1 to 5. L is the linker, G is the conjugated part, X 1 but, [ka] and -(CHR 1 ) j (CHR 2 )- Selected from, R 1 However, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, and R 3 Selected from, R 1 However, R 3 Replaced by, j is an integer from 0 to 5. R 2 However, R 3 is or R 2 However, 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. 2 However, R 3 Replaced by, X 2 However, it is -NH- or -C(O)-, Each R 3 However, they are independently glycosides or glycoside derivatives.

[0177] The substituents associated with the conjugate of formula (I) are described in more detail below.

[0178] In a particular embodiment, X 1 teeth, [ka] and -(CHR 1 ) j (CHR 2 )- Selected from.

[0179] In some cases, X 1 teeth, [ka] That is the case.

[0180] In a particular embodiment, X 1 teeth, [ka] That is the case.

[0181] In some cases, X 1 is, -(CHR 1 ) j (CHR 2 )-. In certain embodiments, j is 0 or an integer from 1 to 5. In some cases, j is 0, and therefore X 1 It does not exist. In some cases, j is 1. In some cases, j is 2. In some cases, j is 3. In some cases, j is 4. In some cases, j is 5.

[0182] In a particular embodiment, R 1 This includes hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, and R 3 Selected from, R 1 However, R 3 It is replaced by R. In certain embodiments, 1 is hydrogen. In a particular embodiment, R 1 is an alkyl or substituted alkyl, for example, C 1~6 Alkyl or C 1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is a substituted alkyl. In certain embodiments, R 1This refers to an alkenyl or a substituted alkenyl, for example, C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is a substituted alkenyl. In certain embodiments, R 1 This refers to alkynyl or substituted alkynyl, for example, C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is a substituted alkenyl. In certain embodiments, R 1 is an aryl or substituted aryl, for example, C 5~8 Aryl or C 5~8 Substituting aryls, for example, C5 aryls or C5-substituted aryls, or C6 aryls or C6-substituted aryls. In certain embodiments, R 1 R is phenyl. In certain embodiments, R 1 This refers to heteroaryl or substituted heteroaryl, for example, C 5~8 Heteroaryl or C 5~8 Substituting heteroaryls, for example, C5 heteroaryls or C5-substituted heteroaryls, or C6 heteroaryls or C6-substituted heteroaryls. In certain embodiments, R 1 This refers to cycloalkyl or substituted cycloalkyl, for example, C 3~8 Cycloalkyl or C 3~8 Substitutive cycloalkyls, for example, C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 It is a substituted cycloalkyl. In certain embodiments, R 1 This refers to heterocyclines or substituted heterocyclines, for example, C 3~8 Heterocycline or C 3~8 Substituting heterocyclyls, e.g., C3~6 Heterocycline or C 3~6 Substituted heterocyclyl, or C 3~5 Heterocycline or C 3~5 It is a substituted heterocyclyl. In certain embodiments, R 1 R 3 That is the case.

[0183] In a particular embodiment, R 1 R is, at will, 3 It is replaced by R. For example, in some cases, 1 R 3 It is not replaced by R. In other embodiments, 1 R 3 It will be replaced with.

[0184] In a particular embodiment, R 2 R 3 That is the case.

[0185] In a particular embodiment, R 2 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. 2 However, R 3 It is replaced by R. In certain embodiments, 2 R 3 It is replaced with R. 2 R can be 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 2 is an alkyl or substituted alkyl, for example, C 1~6 Alkyl or C 1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3It is a substituted alkyl. In certain embodiments, R 2 is methyl. In certain embodiments, R 2 is ethyl. In certain embodiments, R 2 This refers to an alkenyl or a substituted alkenyl, for example, C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is a substituted alkenyl. In certain embodiments, R 2 This refers to an alkenyl or a substituted alkenyl, for example, C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is a substituted alkenyl. In certain embodiments, R 2 is an aryl or substituted aryl, for example, C 5~8 Aryl or C 5~8 Substituting aryls, for example, C5 aryls or C5-substituted aryls, or C6 aryls or C6-substituted aryls. In certain embodiments, R 2 R is phenyl. In certain embodiments, R 2 This refers to heteroaryl or substituted heteroaryl, for example, C 5~8 Heteroaryl or C 5~8 Substituting heteroaryls, for example, C5 heteroaryls or C5-substituted heteroaryls, or C6 heteroaryls or C6-substituted heteroaryls. In certain embodiments, R 2 This refers to cycloalkyl or substituted cycloalkyl, for example, C 3~8 Cycloalkyl or C 3~8 Substitutive cycloalkyls, for example, C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 It is a substituted cycloalkyl. In certain embodiments, R2 This refers to heterocyclines or substituted heterocyclines, for example, C 3~8 Heterocycline or C 3~8 Substituting heterocyclyls, e.g., C 3~6 Heterocycline or C 3~6 Substituted heterocyclyl, or C 3~5 Heterocycline or C 3~5 It is a substituted heterocyclyl.

[0186] In a particular embodiment, X 2 It is -NH- or -C(O)-. In some cases, X 2 It is -NH-. In some cases, X 2 It is -C(O)-.

[0187] In a specific method of operation, each R 3 These are independently glycosides or glycoside derivatives. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronides, galactosides, glucosides, mannosides, fucosides, O-GlcNAc, and O-GalNAc.

[0188] For example, in some embodiments, the glycoside or glycoside derivative has the following structure: [ka] It can be selected from the following.

[0189] In a particular embodiment, W 1 is a drug (or active agent). Examples of drugs and active agents that may be used in the conjugates of this disclosure are described in more detail below.

[0190] In a particular embodiment, W 2 This is a polypeptide (e.g., an antibody). Examples of polypeptides and antibodies that may be used in the conjugates of this disclosure are described in more detail below.

[0191] In certain embodiments, A is an amino acid residue. In certain embodiments, k is 0 or an integer from 1 to 5. In some cases, k is 0, and therefore A is absent. In some cases, k is 1. In some cases, k is 2. In some cases, k is 3. In some cases, k is 4. In some cases, k is 5.

[0192] Amino acid residues include, but are not limited to, amino acids 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). In some cases, amino acid residues may be amino acid analogs or unnatural amino acids.

[0193] The terms "amino acid analog" and "non-natural amino acid" may be used interchangeably and include amino acid-like compounds that are structurally and / or overall similar in shape to one or more amino acids commonly found in naturally occurring proteins. Amino acid analogs also include natural amino acids that have modified side chains or skeletons. Amino acid analogs also include amino acid analogs that have the same stereochemistry as the naturally occurring D-form, as well as the L-form of amino acid analogs. In some cases, amino acid analogs share the skeletal structure and / or side-chain structure of one or more natural amino acids, the difference being one or more modifying groups in the molecule. Such modifications may include, but are not limited to, substitution of an atom (e.g., N) with a related atom (e.g., S), addition of a group (e.g., methyl or hydroxyl) or an atom (e.g., Cl or Br), deletion of a group, substitution of a covalent bond (e.g., a single bond to a double bond), or combinations thereof. For example, amino acid analogs may include α-hydroxy acids and α-amino acids.

[0194] In certain embodiments, L is a linker. In some cases, the linker is a detachable linker. Examples of linkers that may be used in the conjugates of this disclosure are described in more detail below.

[0195] In certain embodiments, G is a conjugated portion. As described above, in the ADC of the present disclosure, a polypeptide (e.g., an antibody) and a drug or active agent can be conjugated to each other via a conjugated portion. For example, the polypeptide (antibody) and the drug may each be bound (e.g., covalently) to the conjugated portion, and thus indirectly bound together via the conjugated portion. Any convenient conjugated portion can be used as the conjugate of the present disclosure, and the conjugated portion is sufficient to provide stable binding of the polypeptide (e.g., an antibody) to the drug or active agent in the ADC.

[0196] In certain embodiments, the conjugated portion includes a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound, or a derivative of a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compound. In certain embodiments, the conjugated portion G is as follows: [ka] During the ceremony, Z, CR 10 or N, R 7 However, selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, R 8 and R 9However, each is 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 8 and R 9 However, they are optionally linked in a ring to form a 5-membered or 6-membered heterocycline. Each R 10 However, these 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, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0197] In certain embodiments, Z is CR 10 Or N. In certain embodiments, Z is CR 10 In certain embodiments, Z is N.

[0198] In a particular embodiment, R 7 R is selected from 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 7 is hydrogen. In a particular embodiment, R 7 is an alkyl or substituted alkyl, for example, C 1~6 Alkyl or C 1~6Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is a substituted alkyl. In certain embodiments, R 7 is methyl. In certain embodiments, R 7 This refers to an alkenyl or a substituted alkenyl, for example, C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is a substituted alkenyl. In certain embodiments, R 7 This refers to alkynyl or substituted alkynyl, for example, C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is a substituted alkenyl. In certain embodiments, R 7 is an aryl or substituted aryl, for example, C 5~8 Aryl or C 5~8 Substituting aryls, for example, C5 aryls or C5-substituted aryls, or C6 aryls or C6-substituted aryls. In certain embodiments, R 7 This refers to heteroaryl or substituted heteroaryl, for example, C 5~8 Heteroaryl or C 5~8 Substituting heteroaryls, for example, C5 heteroaryls or C5-substituted heteroaryls, or C6 heteroaryls or C6-substituted heteroaryls. In certain embodiments, R 7 This refers to cycloalkyl or substituted cycloalkyl, for example, C 3~8 Cycloalkyl or C 3~8 Substitutive cycloalkyls, for example, C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5It is a substituted cycloalkyl. In certain embodiments, R 7 This refers to heterocyclines or substituted heterocyclines, for example, C 3~8 Heterocycline or C 3~8 Substituting heterocyclyls, e.g., C 3~6 Heterocycline or C 3~6 Substituted heterocyclyl, or C 3~5 Heterocycline or C 3~5 It is a substituted heterocyclyl.

[0199] In a particular embodiment, R 8 and R 9 Each is 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 8 and R 9 However, they can be selectively linked together in a ring to form a 5-membered or 6-membered heterocycline.

[0200] In a specific method of operation, each R 8 is 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. In certain embodiments, R 8 is hydrogen. In a particular embodiment, R 8 is an alkyl or substituted alkyl, for example, C 1~6 Alkyl or C1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is a substituted alkyl. In certain embodiments, R 8 is methyl. In certain embodiments, R 8 This refers to an alkenyl or a substituted alkenyl, for example, C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is a substituted alkenyl. In certain embodiments, R 8 is an alkynyl or substituted alkynyl. In certain embodiments, R 8 is an alkoxy or substituted alkoxy. In certain embodiments, R 8 is an amino acid or a substituted amino acid. In certain embodiments, R 8 is a carboxyl or carboxyl ester. In certain embodiments, R 8 is an acyl or acyloxy. In certain embodiments, R 8 is acylamino or aminoacyl. In certain embodiments, R 8 is an alkylamide or a substituted alkylamide. In certain embodiments, R 8 is a sulfonyl. In certain embodiments, R 8 is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 8 is an aryl or substituted aryl, for example, C 5~8 Aryl or C 5~8 Substituting aryls, for example, C5 aryls or C5-substituted aryls, or C6 aryls or C6-substituted aryls. In certain embodiments, R 8 This refers to heteroaryl or substituted heteroaryl, for example, C 5~8 Heteroaryl or C 5~8Substituting heteroaryls, for example, C5 heteroaryls or C5-substituted heteroaryls, or C6 heteroaryls or C6-substituted heteroaryls. In certain embodiments, R 8 This refers to cycloalkyl or substituted cycloalkyl, for example, C 3~8 Cycloalkyl or C 3~8 Substitutive cycloalkyls, for example, C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 It is a substituted cycloalkyl. In certain embodiments, R 8 This refers to heterocyclines or substituted heterocyclines, for example, C 3~6 Heterocycline or C 3~6 Substituted heterocyclyl, or C 3~5 Heterocycline or C 3~5 It is a substituted heterocyclyl.

[0201] In a specific method of operation, each R 9 is 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. In certain embodiments, R 9 is hydrogen. In a particular embodiment, R 9 is an alkyl or substituted alkyl, for example, C 1~6 Alkyl or C 1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is a substituted alkyl. In certain embodiments, R 9 is methyl. In certain embodiments, R 9This refers to an alkenyl or a substituted alkenyl, for example, C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is a substituted alkenyl. In certain embodiments, R 9 is an alkynyl or substituted alkynyl. In certain embodiments, R 9 is an alkoxy or substituted alkoxy. In certain embodiments, R 9 is an amino acid or a substituted amino acid. In certain embodiments, R 9 is a carboxyl or carboxyl ester. In certain embodiments, R 9 is an acyl or acyloxy. In certain embodiments, R 9 is acylamino or aminoacyl. In certain embodiments, R 9 is an alkylamide or a substituted alkylamide. In certain embodiments, R 9 is a sulfonyl. In certain embodiments, R 9 is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 9 is an aryl or substituted aryl, for example, C 5~8 Aryl or C 5~8 Substituting aryls, for example, C5 aryls or C5-substituted aryls, or C6 aryls or C6-substituted aryls. In certain embodiments, R 9 This refers to heteroaryl or substituted heteroaryl, for example, C 5~8 Heteroaryl or C 5~8 Substituting heteroaryls, for example, C5 heteroaryls or C5-substituted heteroaryls, or C6 heteroaryls or C6-substituted heteroaryls. In certain embodiments, R 9 This refers to cycloalkyl or substituted cycloalkyl, for example, C 3~8 Cycloalkyl or C 3~8 Substitutive cycloalkyls, for example, C 3~6 Cycloalkyl or C 3~6Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 It is a substituted cycloalkyl. In certain embodiments, R 9 This refers to heterocyclines or substituted heterocyclines, for example, C 3~8 Heterocycline or C 3~8 Substituting heterocyclyls, e.g., C 3~6 Heterocycline or C 3~6 Substituted heterocyclyl, or C 3~5 Heterocycline or C 3~5 It is a substituted heterocyclyl.

[0202] In a particular embodiment, R 8 and R 9 These are optionally linked in a ring to form a 5-membered or 6-membered heterocycline. In certain embodiments, R 8 and R 9 These are linked in a ring to form a 5-membered or 6-membered heterocycline. In certain embodiments, R 8 and R 9 These are linked in a ring to form a 5-membered heterocycline. In certain embodiments, R 8 and R 9 These are linked together in a ring to form a six-membered heterocycline.

[0203] In a specific method of operation, each R 10 These 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, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0204] Each R 10 Various possibilities regarding this are described in more detail below. In certain embodiments, R 10is hydrogen. In a particular embodiment, each R 10 is hydrogen. In a particular embodiment, R 10 is a halogen such as F, Cl, Br, or I. In certain embodiments, R 10 is F. In a particular embodiment, R 10 is Cl. In certain embodiments, R 10 is Br. In certain embodiments, R 10 is I. In a particular embodiment, R 10 is an alkyl or substituted alkyl, for example, C 1~6 Alkyl or C 1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is a substituted alkyl. In certain embodiments, R 10 is methyl. In certain embodiments, R 10 This refers to an alkenyl or a substituted alkenyl, for example, C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is a substituted alkenyl. In certain embodiments, R 10 is an alkynyl or substituted alkynyl. In certain embodiments, R 10 is an alkoxy or substituted alkoxy. In certain embodiments, R 10 is an amino acid or a substituted amino acid. In certain embodiments, R 10 is a carboxyl or carboxyl ester. In certain embodiments, R 10 is an acyl or acyloxy. In certain embodiments, R 10 is acylamino or aminoacyl. In certain embodiments, R 10 is an alkylamide or a substituted alkylamide. In certain embodiments, R 10 is a sulfonyl. In certain embodiments, R 10is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 10 is an aryl or substituted aryl, for example, C 5~8 Aryl or C 5~8 Substituting aryls, for example, C5 aryls or C5-substituted aryls, or C6 aryls or C6-substituted aryls (for example, phenyl or substituted phenyl). In certain embodiments, R 10 This refers to heteroaryl or substituted heteroaryl, for example, C 5~8 Heteroaryl or C 5~8 Substituting heteroaryls, for example, C5 heteroaryls or C5-substituted heteroaryls, or C6 heteroaryls or C6-substituted heteroaryls. In certain embodiments, R 10 This refers to cycloalkyl or substituted cycloalkyl, for example, C 3~8 Cycloalkyl or C 3~8 Substitutive cycloalkyls, for example, C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 It is a substituted cycloalkyl. In certain embodiments, R 10 This refers to heterocyclines or substituted heterocyclines, for example, C 3~8 Heterocycline or C 3~8 Substituting heterocyclyls, e.g., C 3~6 Heterocycline or C 3~6 Substituted heterocyclyl, or C 3~5 Heterocycline or C 3~5 It is a substituted heterocyclyl.

[0205] In certain embodiments, the conjugated portion G is polypeptide (antibody) W 2It contains a functional group that conjugates to linker L. In some cases, G is selected from acetals, hydrazones, oximes, sulfides, disulfides, triazoles, esters, and amides. In some cases, the conjugated moiety G is an acetal. In some cases, the conjugated moiety G is a hydrazone. In some cases, the conjugated moiety G is an oxime. In some cases, the conjugated moiety G is a sulfide. In some cases, the conjugated moiety G is a disulfide. In some cases, the conjugated moiety G is a triazole. In some cases, the conjugated moiety G is an ester. In some cases, the conjugated moiety G is an amide.

[0206] In certain embodiments, the conjugated moiety G may be described in relation to an intermediate or precursor functional group that reacts with a corresponding reactive group on the polypeptide (antibody) to conjugate the polypeptide (antibody) to linker L. In these examples, the conjugated moiety G may be selected from acetals, NHS esters, PFP esters, TFP esters, isocyanates, isothiocyanates, acyl halides, maleimides, iodoacetamides, phosphines, alkynes, azides, hydrazines, alkoxyamines, disulfides, esters, and amides. In some cases, the conjugated moiety G is an acetal. In some cases, the conjugated moiety G is an NHS ester. In some cases, the conjugated moiety G is a PFP ester. In some cases, the conjugated moiety G is a TFP ester. In some cases, the conjugated moiety G is an isocyanate. In some cases, the conjugated moiety G is an isothiocyanate. In some cases, the conjugated moiety G is an acyl halide. In some cases, the conjugated moiety G is maleimide. In some cases, the conjugated moiety G is iodoacetamide. In some cases, the conjugated moiety G is phosphine. In some cases, the conjugated moiety G is alkyne. In some cases, the conjugated moiety G is azide. In some cases, the conjugated moiety G is hydrazine. In some cases, the conjugated moiety G is alkoxyamine. In some cases, the conjugated moiety G is disulfide. In some cases, the conjugated moiety G is ester. In some cases, the conjugated moiety G is amide.

[0207] In certain embodiments, the conjugate of formula (I) comprises a linker L. The linker can be used to conjugate one or more moieties of interest (e.g., a drug or active agent) to one or more polypeptides via the conjugated moiety. The linker can be conjugated to the conjugated moiety G at any convenient position (e.g., as described herein) (e.g., covalently). For example, the linker can conjugate a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety to a drug (e.g., camptothecin or a camptothecin derivative). The linker (and therefore the drug) can be conjugated to a polypeptide, such as an antibody, using the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety.

[0208] For example, as shown in equation (I) above, L is W via the conjugated part G. 2 It joins to W, and therefore W 2 It is indirectly coupled to linker L via the conjugate portion. As described above, W 2 L is a polypeptide (e.g., an antibody), and therefore L binds to the polypeptide (e.g., an antibody) via a conjugated moiety, and linker L, for example, binds indirectly to the polypeptide (e.g., an antibody) via a conjugated moiety (via the hydrazinyl-indol or hydrazinyl-pyrrolo-pyridinyl conjugated moiety as described herein).

[0209] Any suitable linker may be used for linker L in the conjugate and compound of this subject. In certain embodiments, linker L may include a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, linker L may include alkyl or substituted alkyl groups. In certain embodiments, linker L may include alkenyl or substituted alkenyl groups. In certain embodiments, linker L may include alkynyl or substituted alkynyl groups. In certain embodiments, linker L may include alkoxy or substituted alkoxy groups. In certain embodiments, linker L may include amino or substituted amino groups. In certain embodiments, linker L may include carboxyl or carboxyl ester groups. In certain embodiments, linker L may contain an acylamino group. In certain embodiments, linker L may contain an alkylamide or substituted alkylamide group. In certain embodiments, linker L may contain an aryl or substituted aryl group. In certain embodiments, linker L may contain a heteroaryl or substituted heteroaryl group. In certain embodiments, linker L may contain a cycloalkyl or substituted cycloalkyl group. In certain embodiments, linker L may contain a heterocyclyl or substituted heterocyclyl group.

[0210] In certain embodiments, linker L may include a polymer. For example, the polymer may include polyalkylene glycols and their derivatives, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymer, polypropylene glycol homopolymer, copolymer of ethylene glycol and propylene glycol (e.g., the homopolymer and copolymer are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinylpyrrolidone, and combinations thereof. 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.

[0211] In some embodiments, L is given by the following formula: -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f - is a linker described by, In the formula, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 Each of them is an independent linker subunit, and a, b, c, d, e, and f are each independently 0 or 1, and the sum of a, b, c, d, e, and f is between 1 and 6.

[0212] In certain embodiments, the sum of a, b, c, d, e, and f is 1. In certain embodiments, the sum of a, b, c, d, e, and f is 2. In certain embodiments, the sum of a, b, c, d, e, and f is 3. In certain embodiments, the sum of a, b, c, d, e, and f is 4. In certain embodiments, the sum of a, b, c, d, e, and f is 5. In certain embodiments, the sum of a, b, c, d, e, and f is 6. In certain embodiments, a, b, c, d, e, and f are each 1. In certain embodiments, a, b, c, d, and e are each 1, and f is 0. In certain embodiments, a, b, c, and d are each 1, and e and f are each 0. In certain embodiments, a, b, and c are each 1, and d, e, and f are each 0. In certain embodiments, a and b are each 1, and c, d, e, and f are each 0.

[0213] In a particular embodiment, linker subunit L 1 It is coupled to the conjugated portion (for example, as shown in equation (I) above). In a particular embodiment, the linker subunit L 2 If present, the drug or active agent W 1 It joins to L. 2 -A in the structure of equation (I) k -X 2 -X 1 -C(O)O- moiety via drug or active agent W 1 It can be indirectly coupled to the linker subunit L. In a particular embodiment, the linker subunit L 3 If present, it binds to the drug or active agent. For example, L 3 -A in the structure of equation (I) k -X 2 -X 1 -C(O)O- moiety via drug or active agent W 1 It can be indirectly coupled to the linker subunit L. In a particular embodiment, the linker subunit L 4 If present, it binds to the drug or active agent. For example, L 4-A in the structure of equation (I) k -X 2 -X 1 -C(O)O- moiety via drug or active agent W 1 It can be indirectly coupled to the linker subunit L. In a particular embodiment, the linker subunit L 5 If present, it binds to the drug or active agent. For example, L 5 -A in the structure of equation (I) k -X 2 -X 1 -C(O)O- moiety via drug or active agent W 1 It can be indirectly coupled to the linker subunit L. In a particular embodiment, the linker subunit L 6 If present, it binds to the drug or active agent. For example, L 6 -A in the structure of equation (I) k -X 2 -X 1 -C(O)O- moiety via drug or active agent W 1 It can be indirectly linked to it.

[0214] Any suitable linker subunit can be used for linker L. The linker subunit of interest may include, but are not limited to, polyethylene glycol, polyethylene and polyacrylates, amino acid residues, carbohydrate polymers or their carbohydrate residues and derivatives, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, and other polymer units, combinations thereof, as well as substituted versions thereof. In some embodiments, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 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 including alkylenediamines).

[0215] In some embodiments, L 1(If present) includes polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 1 It contains polyethylene glycol. In some embodiments, L 1 It contains modified polyethylene glycol. In some embodiments, L 1 L contains an amino acid residue. In some embodiments, L 1 This includes an alkyl group or a substituted alkyl group. In some embodiments, L 1 This includes an aryl group or a substituted aryl group. In some embodiments, L 1 It contains a diamine (for example, a linking group including alkylenediamine).

[0216] In some embodiments, L 2 (If present) includes polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 2 It contains polyethylene glycol. In some embodiments, L 2 It contains modified polyethylene glycol. In some embodiments, L 2 L contains an amino acid residue. In some embodiments, L 2 This includes an alkyl group or a substituted alkyl group. In some embodiments, L 2 This includes an aryl group or a substituted aryl group. In some embodiments, L 2 It contains a diamine (for example, a linking group including alkylenediamine).

[0217] In some embodiments, L 3 (If present) includes polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 3contains polyethylene glycol. In some embodiments, L 3 contains modified polyethylene glycol. In some embodiments, L 3 contains amino acid residues. In some embodiments, L 3 contains an alkyl group or a substituted alkyl. In some embodiments, L 3 contains an aryl group or a substituted aryl group. In some embodiments, L 3 contains a diamine (e.g., a linking group containing an alkylenediamine).

[0218] In some embodiments, L 4 (if present) contains polyethylene glycol, modified polyethylene glycol, amino acid residues, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 4 contains polyethylene glycol. In some embodiments, L 4 contains modified polyethylene glycol. In some embodiments, L 4 contains amino acid residues. In some embodiments, L 4 contains an alkyl group or a substituted alkyl. In some embodiments, L 4 contains an aryl group or a substituted aryl group. In some embodiments, L 4 contains a diamine (e.g., a linking group containing an alkylenediamine).

[0219] In some embodiments, L 5 (if present) contains polyethylene glycol, modified polyethylene glycol, amino acid residues, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L 5 contains polyethylene glycol. In some embodiments, L 5 contains modified polyethylene glycol. In some embodiments, L 5 contains amino acid residues. In some embodiments, L 5This includes an alkyl group or a substituted alkyl group. In some embodiments, L 5 This includes an aryl group or a substituted aryl group. In some embodiments, L 5 It contains a diamine (for example, a linking group including alkylenediamine).

[0220] In some embodiments, L 6 (If present) includes polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 6 It contains polyethylene glycol. In some embodiments, L 6 It contains modified polyethylene glycol. In some embodiments, L 6 L contains an amino acid residue. In some embodiments, L 6 This includes an alkyl group or a substituted alkyl group. In some embodiments, L 6 This includes an aryl group or a substituted aryl group. In some embodiments, L 6 It contains a diamine (for example, a linking group including alkylenediamine).

[0221] In some embodiments, L is -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f - is a linker that includes, in the formula, -(L 1 ) a - is, - (T 1 -V 1 ) a -and, -(L 2 ) b - is, - (T 2 -V 2 ) b -and, -(L 3 ) c - is, -(T 3 -V 3 ) c - and -(L 4 ) d - is, -(T 4 -V 4 ) d - and -(L 5 ) e - is, -(T 5 -V 5 ) e - and -(L 6 ) f - is, -(T 6 -V 6 ) f - and T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are, when present, a tether group, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 are, when present, a covalent bond or a linking functional group, a, b, c, d, e, and f are each independently 0 or 1, and the sum of a, b, c, d, e, and f is 1 - 6.

[0222] As described above, in certain embodiments, L 1 is attached to the conjugating moiety G (e.g., as shown in formula (I) above). Thus, in certain embodiments, T 1 is attached to the conjugating moiety G (e.g., as shown in formula (I) above). In certain embodiments, V 1 is (indirectly) attached to a drug or bioactive agent (e.g., the -A k -X 2 -X 1Bonded (via the -C(O)O- moiety). In certain embodiments, L 2 If present, it binds to the drug or active agent. Therefore, in certain embodiments, T 2 If present, it may (indirectly) bind to the drug or active agent, or V 2 If present, it (indirectly) contributes to the drug or active agent (for example, the structure of formula (I) -A k -X 2 -X 1 Bonded (via the -C(O)O- moiety). In certain embodiments, L 3 If present, it binds to the drug or active agent. Therefore, in certain embodiments, T 3 If present, it may (indirectly) bind to the drug or active agent, or V 3 If present, it (indirectly) contributes to the drug or active agent (for example, the structure of formula (I) -A k -X 2 -X 1 Bonded (via the -C(O)O- moiety). In certain embodiments, L 4 If present, it is bound to camptothecin or a camptothecin derivative. Therefore, in certain embodiments, T 4 If present, it may (indirectly) bind to the drug or active agent, or V 4 If present, it (indirectly) contributes to the drug or active agent (for example, the structure of formula (I) -A k -X 2 -X 1 Bonded (via the -C(O)O- moiety). In certain embodiments, L 5 If present, it is bound to camptothecin or a camptothecin derivative. Therefore, in certain embodiments, T 5 If present, it may (indirectly) bind to the drug or active agent, or V 5 If present, it (indirectly) contributes to the drug or active agent (for example, the structure of formula (I) -A k -X 2 -X 1 Bonded (via the -C(O)O- moiety). In certain embodiments, L 6If present, it is bound to camptothecin or a camptothecin derivative. Therefore, in certain embodiments, T 6 If present, it may (indirectly) bind to the drug or active agent, or V 6 If present, it (indirectly) contributes to the drug or active agent (for example, the structure of formula (I) -A k -X 2 -X 1 They bond (via the -C(O)O- moiety).

[0223] Tether group, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 In relation to this, any convenient tether group can be used in the linker of this subject. In some embodiments, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each is 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 - comprises one or more groups independently selected from 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxyphenyl (PHP), acetal groups, hydrazines, disulfides, and esters, 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.

[0224] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) is (C1~C 12 ) Alkyl or substituted (C1~C 12 ) Contains alkyl. In certain embodiments, (C1~C 12 Alkyl is a linear or branched alkyl group containing 1 to 12 carbon atoms, for example, 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, (C1-C 12 ) Alkyl refers to alkyl or substituted alkyl, for example, C1~C 12 Alkyl, or C1-C 10 It may be alkyl, or C1-C6 alkyl, or C1-C3 alkyl. In some cases, (C1-C 12 ) Alkyl is C2-alkyl. For example, (C1~C 12 ) Alkyl refers to alkylene or substituted alkylene, for example, C1-C 12 Alkylene, or C1-C 10 Alkylene, or C1-C6 alkylene, or C1-C3 alkylene may also be used. In some cases, (C1-C 12 Alkyl is a C2-alkylene (e.g., CH2CH2).

[0225] In certain embodiments, substitution (C1~C 12 Alkyl is a linear or branched substituted alkyl group containing 1 to 12 carbon atoms, for example, 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 ) Alkyl refers to substituted alkyl, for example, substituted C1~C 12 Alkyl or substituted C1-C 10Alkyl, or substituted C1-C6 alkyl, or substituted C1-C3 alkyl may also be used. In some cases, substituted (C1-C 12 )alkyl is a substituted C2-alkyl. For example, substituted (C1~C 12 ) Alkyl refers to substituted alkylenes, for example, substituted C1-C 12 Alkylene or substituted C1-C 10 Alkylene, or substituted C1-C6 alkylene, or substituted C1-C3 alkylene may also be used. In some cases, substitution (C1-C 12 Alkyl is a substituted C2-alkylene.

[0226] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 ) includes aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some cases, a tether group (e.g., T) may be included. 1 , T 2 , T 3 , T 4 , T 5 , and T 6 ) includes aryl or substituted aryl. For example, aryl can be phenyl. In some cases, substituted aryl is substituted phenyl. Substituted phenyl is (C1~C 12 ) alkyl, substituted (C1~C 12 The substituents may be substituted with one or more substituents selected from alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In some cases, the substituted aryl is a substituted phenyl, and the substituents include cleavable moieties as described herein (e.g., enzymatically cleavable moieties, e.g., glycosides or glycoside derivatives).

[0227] In some cases, a tether group (for example, T 1 , T 2 , T 3 , T4 , T 5 , and T 6 ) includes heteroaryl or substituted heteroaryl groups. In some cases, a tether group (e.g., T) may be present. 1 , T 2 , T 3 , T 4 , T 5 , and T 6 ) includes cycloalkyl or substituted cycloalkyl groups. In some cases, a tether group (e.g., T) may be present. 1 , T 2 , T 3 , T 4 , T 5 , and T 6 ) comprises a heterocyclyl or a substituted heterocyclyl. In some cases, a substituted heteroaryl, substituted cycloalkyl, or substituent on a substituted heterocyclyl comprises a cleavable moiety as described herein (e.g., an enzymatically cleavable moiety, e.g., a glycoside or glycoside derivative).

[0228] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and is T 6 ) comprises an ethylenediamine (EDA) moiety, for example, an EDA-containing tether group. In certain embodiments, (EDA) w The formula includes one or more EDA moieties, for example, where w is an integer from 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, e.g., 1, 2, 3, 4, 5, or 6). The linked ethylenediamine (EDA) moieties may be optionally substituted at one or more favorable positions with any favorable substituent, e.g., alkyl, substituted alkyl, acyl, substituted acyl, aryl, or substituted aryl. In a particular embodiment, the EDA moiety has the following structure: [ka] Described by, In the formula, y is an integer from 1 to 6, or 0 or 1, and each R 12However, 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. In certain embodiments, y is 1, 2, 3, 4, 5, or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R 12 These are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In certain embodiments, any two adjacent R of EDA 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 group is an alkyl group, which is linked cyclically to form a piperazinyl ring. In certain embodiments, y is 1, and adjacent R 12 The group is selected from hydrogen, alkyl (e.g., methyl), and substituted alkyl (e.g., lower alkyl-OH, e.g., ethyl-OH, or propyl-OH).

[0229] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and is T 6) comprises a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidine-4-amino, P4A). The 4AP moiety may be optionally substituted at one or more favorable positions with any preferred substituent, such as alkyl, substituted alkyl, polyethylene glycol moiety, acyl, substituted acyl, aryl, or substituted aryl. In certain embodiments, the 4AP moiety is described by the following structure: [ka] In the formula, R 12 R is selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety (e.g., polyethylene glycol or 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 This is the polyethylene glycol portion. In certain embodiments, R 12 This is carboxy-modified polyethylene glycol.

[0230] In a particular embodiment, R 12 is the formula (PEG) k It contains a polyethylene glycol portion as described by, which is represented by the following structure: [ka] In the formula, k is an integer between 1 and 20, for example, 1 to 18, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1 or 2, for example, 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 a particular embodiment, R17 R is selected from OH, COOH, or COOR, where 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 COOH is.

[0231] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 ) is (PEG) n Includes, in the formula, (PEG) n However, it is a polyethylene glycol or modified polyethylene glycol linked unit. In a particular embodiment, (PEG) n It is described by the following structure: [ka] In the formula, n is an integer between 1 and 50, for example, 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, for example, 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.

[0232] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 ) is (AA) pThe formula includes, where AA is an amino acid residue. Any suitable amino acid may be used. The target amino acid may include, but is not limited to, L-amino acids and D-amino acids, naturally occurring amino acids, e.g., any of the 20 main alpha-amino acids and beta-alanine, or amino acids that do not exist naturally (e.g., amino acid analogs), e.g., alpha-amino acids or beta-amino acids that do not exist naturally. In certain embodiments, p is an integer from 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, e.g., 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.

[0233] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 ) is formula - (CR 13 OH) m -Includes the portion described by, where m is 0 or n is an integer between 1 and 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, R 13 is 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. In certain embodiments, R 13 is hydrogen. In a particular embodiment, R 13is an alkyl or substituted alkyl, for example, C 1~6 Alkyl or C 1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is a substituted alkyl. In certain embodiments, R 13 This refers to an alkenyl or a substituted alkenyl, for example, C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is a substituted alkenyl. In certain embodiments, R 13 is an alkynyl or substituted alkynyl. In certain embodiments, R 13 is an alkoxy or substituted alkoxy. In certain embodiments, R 13 is an amino acid or a substituted amino acid. In certain embodiments, R 13 is a carboxyl or carboxyl ester. In certain embodiments, R 13 is an acyl or acyloxy. In certain embodiments, R 13 is acylamino or aminoacyl. In certain embodiments, R 13 is an alkylamide or a substituted alkylamide. In certain embodiments, R 13 is a sulfonyl. In certain embodiments, R 13 is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 13 is an aryl or substituted aryl, for example, C 5~8 Aryl or C 5~8 Substituting aryls, for example, C5 aryls or C5-substituted aryls, or C6 aryls or C6-substituted aryls. In certain embodiments, R 13 This refers to heteroaryl or substituted heteroaryl, for example, C 5~8 Heteroaryl or C 5~8Substituting heteroaryls, for example, C5 heteroaryls or C5-substituted heteroaryls, or C6 heteroaryls or C6-substituted heteroaryls. In certain embodiments, R 13 This refers to cycloalkyl or substituted cycloalkyl, for example, C 3~8 Cycloalkyl or C 3~8 Substitutive cycloalkyls, for example, C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 It is a substituted cycloalkyl. In certain embodiments, R 13 This refers to heterocyclines or substituted heterocyclines, for example, C 3~8 Heterocycline or C 3~8 Substituting heterocyclyls, e.g., C 3~6 Heterocycline or C 3~6 Substituted heterocyclyl, or C 3~5 Heterocycline or C 3~5 It is a substituted heterocyclyl.

[0234] In a particular embodiment, R 13 R is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are R 13 The details are as described above.

[0235] In certain embodiments, a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 This includes meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), or para-hydroxyphenyl (PHP).

[0236] In some embodiments, the tether has the following structure [ka] It contains the MABO group described by [the relevant authority].

[0237] In some embodiments, the tether has the following structure [ka] It contains the MABC group described by [the relevant authority].

[0238] In some embodiments, the tether has the following structure [ka] Contains the PABO group as described by [author's name].

[0239] In some embodiments, the tether has the following structure [ka] It contains the PABC group described by [the relevant authority].

[0240] In some embodiments, the tether has the following structure [ka] Contains the PAB group described by [the relevant authority].

[0241] In some embodiments, the tether has the following structure [ka] Contains PABA groups as described by [author's name].

[0242] In some embodiments, the tether has the following structure [ka] Contains the PAP group described by [translate].

[0243] In some embodiments, the tether has the following structure [ka] Includes the PHP base described by [the author / source].

[0244] In a specific method of operation, each R 14 These 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, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0245] In a particular embodiment, R 14 is hydrogen. In a particular embodiment, each R 14 is hydrogen. In a particular embodiment, R 14 is an alkyl or substituted alkyl, for example, C 1~6 Alkyl or C 1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is a substituted alkyl. In certain embodiments, R 14 This refers to an alkenyl or a substituted alkenyl, for example, C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is a substituted alkenyl. In certain embodiments, R 14 is an alkynyl or substituted alkynyl. In certain embodiments, R 14is an alkoxy or substituted alkoxy. In certain embodiments, R 14 is an amino acid or a substituted amino acid. In certain embodiments, R 14 is a carboxyl or carboxyl ester. In certain embodiments, R 14 is an acyl or acyloxy. In certain embodiments, R 14 is acylamino or aminoacyl. In certain embodiments, R 14 is an alkylamide or a substituted alkylamide. In certain embodiments, R 14 is a sulfonyl. In certain embodiments, R 14 is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 14 is an aryl or substituted aryl, for example, C 5~8 Aryl or C 5~8 Substituting aryls, for example, C5 aryls or C5-substituted aryls, or C6 aryls or C6-substituted aryls. In certain embodiments, R 14 This refers to heteroaryl or substituted heteroaryl, for example, C 5~8 Heteroaryl or C 5~8 Substituting heteroaryls, for example, C5 heteroaryls or C5-substituted heteroaryls, or C6 heteroaryls or C6-substituted heteroaryls. In certain embodiments, R 14 This refers to cycloalkyl or substituted cycloalkyl, for example, C 3~8 Cycloalkyl or C 3~8 Substitutive cycloalkyls, for example, C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 It is a substituted cycloalkyl. In certain embodiments, R 14 This refers to heterocyclines or substituted heterocyclines, for example, C 3~8 Heterocycline or C 3~8 Substituting heterocyclyls, e.g., C 3~6 Heterocycline or C 3~6 Substituted heterocyclyl, or C 3~5Heterocycline or C 3~5 It is a substituted heterocyclyl.

[0246] In some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring may be substituted with one or more additional groups selected from halogens, alkyls, substituted alkyls, alkenyls, substituted alkenyls, alkynyls, substituted alkynyls, alkoxys, substituted alkoxys, aminos, substituted aminos, carboxyls, carboxyl esters, acyls, acyloxys, acylaminos, aminoacyls, alkylamides, substituted alkylamides, sulfonyls, thioalkoxys, substituted thioalkoxys, aryls, substituted aryls, heteroaryls, substituted heteroaryls, cycloalkyls, substituted cycloalkyls, heterocyclyls, and substituted heterocyclyls.

[0247] In a particular embodiment of the linker L, the tether group T 1 , T 2 , T 3 , T 4 , T 5 , or T 6 One or more of these are optionally substituted with a glycoside or glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0248] In certain embodiments, the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above may be substituted with one or more additional groups selected from glycosides and glycoside derivatives. For example, in some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring may be substituted with one or more additional groups selected from glycosides and glycoside derivatives. In certain embodiments, the glycoside or glycoside derivative may be selected from glucuronides, galactosides, glucosides, mannosides, fucosides, O-GlcNAc, and O-GalNAc.

[0249] For example, in some embodiments, the glycoside or glycoside derivative has the following structure: [ka] It can be selected from the following.

[0250] Linking functional group, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 Regarding this, any convenient linking functional group can be used in linker L. Examples of desired linking functional groups, but not limited to, include amino, carbonyl, amide, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, and thiophosphoride. In some embodiments, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 These are, independently, covalent, -CO-, and -NR bonds. 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 Selected from SO2- and -P(O)OH-, 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. In certain embodiments, q is 3. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6.

[0251] In some embodiments, each R 15 These 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, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0252] In a particular embodiment, R 15 is hydrogen. In a particular embodiment, each R 15 is hydrogen. In a particular embodiment, R 15 is an alkyl or substituted alkyl, for example, C 1~6 Alkyl or C 1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is a substituted alkyl. In certain embodiments, R 15 This refers to an alkenyl or a substituted alkenyl, for example, C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C2~3 Alkenil or C 2~3 It is a substituted alkenyl. In certain embodiments, R 15 is an alkynyl or substituted alkynyl. In certain embodiments, R 15 is an alkoxy or substituted alkoxy. In certain embodiments, R 15 is an amino acid or a substituted amino acid. In certain embodiments, R 15 is a carboxyl or carboxyl ester. In certain embodiments, R 15 is an acyl or acyloxy. In certain embodiments, R 15 is acylamino or aminoacyl. In certain embodiments, R 15 is an alkylamide or a substituted alkylamide. In certain embodiments, R 15 is a sulfonyl. In certain embodiments, R 15 is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 15 is an aryl or substituted aryl, for example, C 5~8 Aryl or C 5~8 Substituting aryls, for example, C5 aryls or C5-substituted aryls, or C6 aryls or C6-substituted aryls. In certain embodiments, R 15 This refers to heteroaryl or substituted heteroaryl, for example, C 5~8 Heteroaryl or C 5~8 Substituting heteroaryls, for example, C5 heteroaryls or C5-substituted heteroaryls, or C6 heteroaryls or C6-substituted heteroaryls. In certain embodiments, R 15 This refers to cycloalkyl or substituted cycloalkyl, for example, C 3~8 Cycloalkyl or C 3~8 Substitutive cycloalkyls, for example, C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 It is a substituted cycloalkyl. In certain embodiments, R 15 This refers to heterocyclines or substituted heterocyclines, for example, C 3~8Heterocycline or C 3~8 Substituting heterocyclyls, e.g., C 3~6 Heterocycline or C 3~6 Substituted heterocyclyl, or C 3~5 Heterocycline or C 3~5 It is a substituted heterocyclyl.

[0253] In a specific method of operation, each R 15 The substituents are 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, the substituents are R 15 The details are as described above.

[0254] In certain embodiments, the tether group includes an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group includes an acetal group. In some embodiments, the tether group includes a hydrazine. In some embodiments, the tether group includes a disulfide. In some embodiments, the tether group includes an ester.

[0255] As described above, in some embodiments, L is -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 )e -(T 6 -V 6 ) f - is a linker containing a, b, c, d, e, and f, where a, b, c, d, e, and f are each independently 0 or 1, and the sum of a, b, c, d, e, and f is between 1 and 6.

[0256] In some embodiments, in the linker L, T 1 (C1~C 12 ) Alkyl and substituted (C1~C 12 ) Selected from alkyl groups, T 2 , T 3 , T 4 , T 5 , and T 6 However, each operates independently, (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 - Selected from 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal group, disulfide, hydrazine, and ester, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 However, each operates independently as 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 15Selected from SO2- and -P(O)OH-, where q is an integer from 1 to 6. (PEG) n but, [ka] In the formula, n is an integer between 1 and 30. The EDA has the following structure: [ka] The ethylenediamine moiety has the following characteristics, where y is an integer from 1 to 6, and r is 0 or 1. 4-amino-piperidine (4AP) [ka] And, AA is an amino acid residue, and p is an integer from 1 to 20. Each R 12 However, independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, any two adjacent R 12 The groups may be linked in a ring to form a piperazinyl ring. Each R 13 However, they are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. Each R 15 However, these are 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.

[0257] In some embodiments, L is a linker, T 1 However, (C1~C 12 ) is alkyl, V 1 However, it is -CO-, T 2 However, it is an amino acid analog, V2 However, it is -NH-, T 3 However, (PEG) n V 3 However, it is -CO-, d~f are each 0, or T 1 However, (C1~C 12 ) is alkyl, V 1 However, it is -CO-, T 2 However, it is an amino acid analog, V 2 However, it is -NH-, T 3 However, (PEG) n V 3 However, it is -CONH-, T 4 However, (PEG) n V 4 However, it is -CO-, e and f are both 0.

[0258] In certain embodiments, the left side of the upper linker structure (for example, T 1 ) is coupled to the conjugated portion G and to the right side of the upper linker structure (e.g., V 3 or V 4 ) is (for example, the structure of equation (I) -A k -X 2 -X 1 Binds to a drug or active agent (via the -C(O)O- moiety).

[0259] In certain embodiments, the conjugate is an antibody-drug conjugate in which an antibody and a drug are linked together by a linker, as described above. In some cases, the linker is a cleavable linker. A cleavable linker is a linker comprising one or more cleavable moieties, each comprising one or more bonds that can dissociate under certain conditions, and thus separate the cleavable linker into two or more separable parts. For example, the cleavable moieties may comprise one or more covalent bonds, each of which can dissociate or separate under certain conditions to separate the cleavable linker into two or more parts. Such a linker contained in an antibody-drug conjugate may be a cleavable linker, and as a result, under appropriate conditions, the cleavable linker is cleaved, separating or releasing the drug from the antibody at a desired target site of action for that drug.

[0260] In some cases, a cleavable linker includes two cleavable portions, for example, a first cleavable portion and a second cleavable portion. The cleavable portions may be configured such that cleavage of both cleavable portions is required to separate or release the drug from the antibody at a desired target site of action for the drug. For example, cleavage of a cleavable linker can be achieved by first cleaving one of the two cleavable portions and then cleaving the other of the two cleavable portions. In certain embodiments, a cleavable linker includes a first cleavable portion and a second cleavable portion that interferes with the cleavage of the first cleavable portion. "Interfering with cleavage" means that the presence of the uncleaved second cleavable portion reduces the possibility of cleavage of the first cleavable portion or substantially inhibits cleavage, and therefore substantially reduces or prevents cleavage of the cleavable linker. For example, the presence of an uncleaved second cleavable portion can interfere with the cleavage of the first cleavable portion. The presence of a second cleavable portion interferes with the cleavage of the first cleavable portion, thereby substantially reducing or preventing the release of the drug 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 site of action for the drug.

[0261] In some cases, the second cleavable portion interferes with the cleavage of the first cleavable portion, so cleavage of the cleavable linker can be achieved by first cleaving the second cleavable portion and then the first cleavable portion. Cleavage of the second cleavable portion can reduce or eliminate the interference with cleavage of the first cleavable portion, and thus enable cleavage of the first cleavable portion. Cleavage of the first cleavable portion allows the cleavable linker to dissociate or separate into two or more portions as described above, thereby releasing the drug from the antibody-drug conjugate. In some cases, cleavage of the first cleavable portion does not substantially occur in the presence of the uncleaved second cleavable portion. "Substantially" means that, in the presence of a second, uncuttable portion, the cutting of the first cleavable portion is less than approximately 10%; for example, in the presence of a second, uncuttable portion, the cutting of the first cleavable portion is less than approximately 9%, or less than approximately 8%, or less than approximately 7%, or less than approximately 6%, or less than approximately 5%, or less than approximately 4%, or less than approximately 3%, or less than approximately 2%, or less than approximately 1%, or less than approximately 0.5%, or less than approximately 0.1%.

[0262] In other words, the second cleavable portion can protect the first cleavable portion from being cleaved. For example, the presence of an uncleaved second cleavable portion can protect the first cleavable portion from being cleaved, and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action for the drug. Thus, cleavage of the second cleavable portion exposes the first cleavable portion (e.g., deprotects the first cleavable portion), and thus enables cleavage of the first cleavable portion, resulting in cleavage of the cleavable linker, and then separation or release of the drug from the antibody at the desired target site of action for the drug. In certain cases, cleavage of the second cleavable portion exposes the first cleavable portion to subsequent cleavage, but the second cleavable portion is not in the cleavable linker and does not cause cleavage of the cleavable linker itself (i.e., cleavage of the first cleavable portion is still required to cleave the cleavable linker).

[0263] Each cleavable portion within a cleavable linker may be an enzymatically cleavable portion. For example, the first cleavable portion may be the first enzymatically cleavable portion, and the second cleavable portion may be the second enzymatically cleavable portion. An enzymatically cleavable portion is a cleavable portion that can be separated into two or more parts as described above by the enzymatic action of an enzyme. An enzymatically cleavable portion may be any cleavable portion that can be cleaved by the enzymatic action of an enzyme, such as, but not limited to, esters, peptides, glycosides, etc. In some cases, the enzyme that cleaves the enzymatically cleavable portion is present at the desired target site of action, such as the desired target site of action of the drug released from the antibody-drug conjugate. In other cases, the enzyme that cleaves the enzymatically cleavable portion is not present in significant amounts in other regions such as whole blood, plasma, or serum. Therefore, cleavage of the enzymatically cleavable portion can be controlled so that substantial cleavage occurs at the desired site of action, while significant cleavage does not occur in other regions or before the antibody-drug conjugate reaches the desired site of action.

[0264] For example, as described herein, the antibody-drug conjugates of this disclosure can be used for the treatment of cancer, for example, for the delivery of cancer-therapeutic drugs to desired sites of action where cancer cells are present. In some cases, an enzyme, such as an esterase that cleaves ester bonds, may be a biomarker for cancer that is overexpressed in cancer cells. Overexpression, and therefore localization, of a specific enzyme in cancer can be used in the context of an enzymatically cleavable moiety contained in the cleavable linker of the antibody-drug conjugate of this disclosure to specifically release the drug at a desired site of action (i.e., the site of cancer (and the overexpressed enzyme)). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester) that can be cleaved by an enzyme overexpressed in cancer cells. For example, the enzyme may be an esterase. Thus, in some cases, the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester) that can be cleaved by an esterase enzyme.

[0265] In certain embodiments, the enzymatically cleavable portion is an ester bond. For example, the first cleavable portion described above (i.e., the cleavable portion protected from premature cleavage by the second cleavable portion) may contain an ester. The presence of the uncleaved second cleavable portion can protect the first cleavable portion (ester) from being cleaved by the esterase enzyme, and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action for the drug. In some cases, the portion of linker L adjacent to the first cleavable portion is linked to or contains a substituent, the substituent containing the second cleavable portion. In some cases, the second cleavable portion contains a glycoside.

[0266] In some embodiments, the enzymatically cleavable portion is a sugar portion, such as a glycoside (or glycosyl) or a glycoside derivative. In some cases, the glycoside or glycoside derivative can promote increased hydrophilicity of the cleavable linker compared to a cleavable linker that does not contain a glycoside or glycoside derivative. The glycoside or glycoside derivative may be any glycoside or glycoside derivative that is suitable for use in a cleavable linker and can be cleaved by the enzymatic action of an enzyme. For example, the second cleavable portion (i.e., a cleavable portion that protects the first cleavable portion from premature cleavage) may be a glycoside or glycoside derivative. For example, in some embodiments, the first cleavable portion includes an ester, and the second cleavable portion includes a glycoside or glycoside derivative. In certain embodiments, the second cleavable portion is a glycoside or glycoside derivative selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. In some cases, the second cleavable portion is a glucuronide. In some cases, the second cleavable portion is a galactoside. In some cases, the second cleavable portion is a glucoside. In some cases, the second cleavable portion is a mannoside. In some cases, the second cleavable portion is a fucoside. In some cases, the second cleavable portion is O-GlcNAc. In some cases, the second cleavable portion is O-GalNAc.

[0267] Glycosides or glycoside derivatives can be covalently bonded to cleavable linkers via glycosidic bonds. Glycosidic bonds can link glycosides or glycoside derivatives to cleavable linkers via various types of bonds, including, but not limited to, O-glycosidic bonds (O-glycosids), N-glycosidic bonds (glycosylamines), S-glycosidic bonds (thioglycosids), or C-glycosidic bonds (C-glycosids or C-glycosyls). In some cases, the glycosidic bond is an O-glycosidic bond (O-glycosid). In other cases, glycosides or glycoside derivatives can be cleaved from cleavable linkers by enzymes (e.g., via enzyme-mediated hydrolysis of the glycosidic bond). A glycoside or glycoside derivative can be removed from or cleaved from a cleavable linker by any convenient enzyme capable of performing cleavage (hydrolysis) of the glycosidic bond that binds the glycoside or glycoside derivative to the cleavable linker. Examples of enzymes that may be used to mediate the cleavage (hydrolysis) of the glycosidic bond that binds the glycoside or glycoside derivative to the cleavable linker include glycosidases, such as glucuronidase, galactosidase, glucosidase, mannosidase, and fucosidase. Other suitable enzymes may also be used to mediate the cleavage (hydrolysis) of the glycosidic bond that binds the glycoside or glycoside derivative to the cleavable linker. In some cases, the enzyme used to mediate the cleavage (hydrolysis) of the glycosidic bond that binds the glycoside or glycoside derivative to the cleavable linker is found at or near the desired site of action for the drug in the antibody-drug conjugate. For example, the enzyme may be a lysosomal enzyme, such as a lysosomal glycosidase, found at or near the desired site of action for the drug in the antibody-drug conjugate. In some cases, the enzyme is found at or near the target site where the enzyme mediating the cleavage of the first cleavable portion is found.

[0268] In a particular embodiment, the conjugate is the conjugate of formula (II), [ka] In the formula, R 4 However, these are amino acid side chains.

[0269] In a particular embodiment, R 4 R represents the side chain of an amino acid. For example, R 4 R may represent substituents bonded to the α-carbon of an amino acid residue, including natural amino acids, unnatural amino acids, and amino acid analogs. In some cases, R 4 is a side chain of an amino acid 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). In certain embodiments, R 4 This represents the side chain of valine (Val), i.e., R 4 However, it is isopropyl. In certain embodiments, R 4 This represents the side chain of alanine (Ala), i.e., R 4 However, it is methyl. In a particular embodiment, R 4 This represents the side chain of phenylalanine (Phe), i.e., R 4 However, it is benzyl. In a particular embodiment, R 4 represents the side chain of lysine (Lys), i.e., R 4 However, it is 4-aminobutyl.

[0270] In a particular embodiment of formula (II), the conjugate is [ka] Selected from TIFF0007912016000046.tif51149.

[0271] In certain embodiments, the conjugate is the conjugate of formula (III), [ka] In the formula, R 4 However, these are amino acid side chains.

[0272] In a particular embodiment of formula (III), the conjugate is [ka] Selected from.

[0273] In formulas (II) and (III), substituent W 1 , W 2 , k, L, G, and R 3 As stated above regarding equation (I).

[0274] Any of the chemical entities, linkers, and conjugated portions shown in the above structure may be suitable for use in the compounds and conjugates of this subject.

[0275] Further disclosures regarding hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl compounds and methods for producing conjugates can be found in U.S. Patents 9,310,374 and 9,493,413, the respective disclosures of which are incorporated herein by reference.

[0276] Compounds useful for generating conjugates This disclosure provides compounds useful for generating the conjugates described herein. In certain embodiments, the compounds may be conjugated to a drug or active agent and may contain a conjugation moiety useful for conjugating the drug or active agent to a polypeptide (e.g., an antibody). For example, the conjugation moiety in the compound may be conjugated to a polypeptide (e.g., an antibody), thereby indirectly linking the drug or active agent and the polypeptide (antibody) together.

[0277] In certain embodiments, the compound is a compound of formula (IV), [ka] During the ceremony, W 1 However, it is a drug, A is an amino acid residue, and k is 0 or an integer from 1 to 5. L is the linker, G is the conjugated part, X 1 but, [ka] and -(CHR 1 ) j (CHR 2 )- Selected from, R 1 However, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, and R 3 Selected from, R 1 However, R 3 Replaced by, j is an integer from 0 to 5. R 2 However, R 3 is or R 2 However, 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. 2 However, R 3 Replaced by, X 2 However, it is -NH- or -C(O)-, Each R 3 However, they are independently glycosides or glycoside derivatives.

[0278] Regarding the compound of formula (IV), substituent W 1 A, k, L, G, X 1 , X 2 , R 1, j, R 2 , and R 3 The conjugate of equations (I), (II), and (III) is as described above.

[0279] In certain embodiments, the compound is a compound of formula (V), [ka] In the formula, R 4 However, these are amino acid side chains.

[0280] In a particular embodiment of formula (V), the compound is [ka] Selected from.

[0281] In certain embodiments, the compound is a compound of formula (VI), [ka] In the formula, R 4 However, these are amino acid side chains.

[0282] In a particular embodiment of formula (VI), the compound is [ka] Selected from.

[0283] In formulas (V) and (VI), substituent W 1 , k, L, G, R 3 , and R 4 The same applies to equations (I), (II), and (III) as described above.

[0284] Polypeptides and antibodies As shown above, the subject conjugate is substituent W 2The polypeptide may also include a polypeptide (e.g., an antibody). The polypeptide (antibody) may be 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 one-letter abbreviations, for example, 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.

[0285] In certain embodiments, the amino acid sequence of a polypeptide or antibody is modified to include a sulfatase motif containing a serine or cysteine ​​residue that can be converted (oxidized) to a 2-formylglycine (FGly) residue by the action of formylglycine-producing enzyme (FGE), either in vivo (e.g., during translation of an aldehyde tag-containing protein in cells) or in vitro (e.g., by contacting an aldehyde tag-containing protein with FGE in a cell-free system). Such a sulfatase motif may also be referred to herein as an FGE modification site.

[0286] Sulfatase motif The minimum sulfatase motif of an aldehyde tag is typically the length of 5 or 6 amino acid residues, and is typically the length of 6 or fewer amino acid residues. The sulfatase motif provided in an Ig polypeptide is at least 5 or 6 amino acid residues and can be 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, for example, to define sulfatase motifs with amino acid residue lengths of 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or less than 6 amino acid residues.

[0287] In certain embodiments, the target polypeptide includes one or more amino acid residues, for example, two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or ten or more, or eleven or more, or twelve or more, or thirteen or more, or fourteen or more, or fifteen or more, or sixteen or more, or seventeen or more, or eighteen or more, or nineteen or more, or twenty or more, that have been inserted, deleted or substituted (replaced) from the native amino acid sequence to provide a sequence of sulfatase motifs in the polypeptide. In certain embodiments, the polypeptide includes modifications (insertions, additions, deletions, and / or substitutions) of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or fewer than two amino acid residues in the amino acid sequence compared to the native amino acid sequence of the polypeptide. If 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 site-specific modifications (insertion, addition, deletion, substitution / replacement) of amino acid residues adjacent to native amino acid residues that provide the sequence of the desired sulfatase motif. In certain embodiments, the degree of modification of the native amino acid sequence of the target antibody is minimized so as to minimize the number of amino acid residues inserted, deleted, substituted (replaced), and / or added (e.g., relative to the N-terminus or C-terminus). By minimizing the degree of modification of the amino acid sequence of the target antibody, the potential impact of such modifications on the antibody's function and / or structure can be minimized.

[0288] While aldehyde tags for specific purposes include at least one minimal sulfatase motif (also referred to as the “consensus sulfatase motif”), it should be noted that longer aldehyde tags are intended and included by this disclosure and may be used in the compositions and methods of this disclosure. Accordingly, aldehyde tags may include a minimal sulfatase motif of five or six residues, or longer minimal sulfatase motifs that may be flanked by additional amino acid residues on the N-terminus and / or C-terminus of the motif. For example, aldehyde tags of five or six amino acid residues, as well as longer amino acid sequences of five, six, seven, eight, nine, ten, eleven, twelfth, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more amino acid residues, are intended.

[0289] Aldehyde tags can be located at or near the C-terminus of the Ig heavy chain. For example, aldehyde tags may be located within the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10th amino acids at the C-terminus of the natural wild-type Ig heavy chain. Aldehyde tags may be located within the CH1 domain of the Ig heavy chain. Aldehyde tags may be located within the CH2 domain of the Ig heavy chain. Aldehyde tags may be located within the CH3 domain of the Ig heavy chain. Aldehyde tags may be located within the constant region of the Ig light chain, for example, the kappa light chain constant region or the lambda light chain constant region.

[0290] In certain embodiments, the sulfatase motif used may be described by the following formula: X 1 Z 10 X 2 Z 20 X 3 Z 30 (I') During the ceremony, Z 10 However, it is cysteine ​​or serine (which can also be represented by (C / S)), Z 20 However, it is either a proline or alanine residue (which can also be represented by (P / A)), Z 30However, basic amino acids (for example, arginine (R), lysine (K) or histidine (H), for example, lysine), or aliphatic amino acids (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), for example, A, G, L, V, or I, X 1 However, whether present or absent, if present, any amino acid, e.g., aliphatic amino acid, sulfur-containing amino acid, or polar uncharged amino acid (i.e., anything other than aromatic amino acids or charged amino acids), e.g., L, M, V, S, or T, e.g., L, M, S, or V, provided that the sulfatase motif is at the N-terminus of the target polypeptide, X 1 There exists, X 2 and X 3 However, independently, any amino acid may be used, but usually aliphatic amino acids, polar uncharged amino acids, or sulfur-containing amino acids (i.e., those other than aromatic amino acids or charged amino acids), for example, S, T, A, V, G, or C, for example, S, T, A, V, or G.

[0291] The amino acid sequence of the antibody heavy chain and / or light chain is given by formula X 1 Z 10 X 2 Z 20 X 3 Z 30 It may be modified to provide a sequence of at least 5 amino acids, in the formula, Z 10 However, it is cysteine ​​or serine, Z 20 However, it is a proline or alanine residue, Z 30 However, they are aliphatic amino acids or basic amino acids. X 1 However, it may or may not exist, and if it exists, it is any amino acid, provided that a heterologous sulfatase motif is at the N-terminus of the polypeptide, X 1 There exists, X 2 and X 3However, each of them is an arbitrary amino acid, independently of the others.

[0292] The sulfatase motif is generally selected so that it can be converted by a selected FGE, for example, an FGE present in a host cell expressing an aldehyde-tagged polypeptide, or an FGE that is contacted with the aldehyde-tagged polypeptide in a cell-free in vitro method.

[0293] For example, if FGE is a eukaryotic FGE (e.g., mammalian FGE including human FGE), the sulfatase motif is given by the following formula: X 1 CX 2 PX 3 Z 30 It may also be the sulfatase motif (I”) During the ceremony, X 1 However, it may or may not be present, and if present, it may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., something other than an aromatic amino acid or a charged amino acid), such as L, M, S, or V, provided that the sulfatase motif is at the N-terminus of the target polypeptide, X 1 There exists, X 2 and X 3 However, independently, any amino acid, for example, an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., something other than an aromatic amino acid or a charged amino acid), for example, S, T, A, V, G, or C, for example, S, T, A, V, or G. Z 30 However, it may be a basic amino acid (for example, arginine (R), lysine (K), or histidine (H), for example, lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), for example, A, G, L, V, or I).

[0294] Specific examples of sulfatase motifs include LCTPSR (sequence number / / ), MCTPSR (sequence number / / ), VCTPSR (sequence number / / ), LCSPSR (sequence number / / ), LCAPSR (sequence number / / ), LCVPSR (sequence number / / ), LCGPSR (sequence number / / ), ICTPAR (sequence number / / ), LCTPSK (sequence number / / ), MCTPSK (sequence number / / ), VCTPSK (sequence number / / ), LCSPSK (sequence number / / ), LCAPSK (sequence number / / ), LCVPSK (sequence number / / ), LCGPSK (sequence number / / ), LCTPSA (sequence number / / ), ICTPAA (sequence number / / ), MCTPSA (sequence number / / ), VCTPSA (sequence number / / ), LCSPSA (sequence number / / ), LCAPSA (sequence number / / ), LCVPSA ​​(sequence number / / ), and LCGPSA (sequence number / / ).

[0295] FGly-containing sequence When FGE acts on the heavy and / or light chains of a modified antibody, serine or cysteine ​​in the sulfatase motif is modified to FGly. Therefore, the FGly-containing sulfatase motif has the following formula: X 1 (FGly)X 2 Z 20 X 3 Z 30( It may also be the FGly-containing sulfatase motif of I'''). During the ceremony, FGly is a formylglycine residue, Z 20 However, it is either a proline or alanine residue (which can also be represented by (P / A)), Z 30 However, basic amino acids (e.g., arginine (R), lysine (K), or histidine (H), usually lysine), or aliphatic amino acids (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I) X 1However, it may or may not be present, and if present, any amino acid, for example, an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., one other than an aromatic amino acid or a charged amino acid), for example, L, M, V, S, or T, for example, L, M, or V, provided that the sulfatase motif is at the N-terminus of the target polypeptide, X 1 There exists, X 2 and X 3 However, independently, any amino acid may be used, for example, an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., something other than an aromatic amino acid or a charged amino acid), for example, S, T, A, V, G, or C, for example, S, T, A, V, or G.

[0296] As described above, a modified polypeptide containing an FGly residue may be conjugated to a drug (e.g., a mytansinoid) by reaction of FGly with a drug (e.g., a drug containing a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugated moiety as described above) to produce an FGly'-containing sulfatase motif. As used herein, the term FGly' refers to the modified amino acid residue of the sulfatase motif coupled to a drug such as mytansin or auristatin. Thus, the FGly'-containing sulfatase motif has the following formula: X 1 (FGly')X 2 Z 20 X 3 Z 30 (II) It may also be an FGly'-containing sulfatase motif, During the ceremony, FGly' is the modified amino acid residue in formula (I), Z 20 However, it is either a proline or alanine residue (which can also be represented by (P / A)), Z 30However, basic amino acids (e.g., arginine (R), lysine (K), or histidine (H), usually lysine), or aliphatic amino acids (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I) X 1 However, it may or may not be present, and if present, any amino acid, for example, an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., one other than an aromatic amino acid or a charged amino acid), for example, L, M, V, S, or T, for example, L, M, or V, provided that the sulfatase motif is at the N-terminus of the target polypeptide, X 1 There exists, X 2 and X 3 However, independently, any amino acid may be used, for example, an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., something other than an aromatic amino acid or a charged amino acid), for example, S, T, A, V, G, or C, for example, S, T, A, V, or G.

[0297] Modification site As shown 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) to an FGly residue by the action of FGE, either in vivo (e.g., during translation of an aldehyde tag-containing protein in a cell) or in vitro (e.g., by contacting an aldehyde tag-containing protein with FGE in a cell-free system). The antibody used to generate the conjugate of this disclosure includes at least an Ig constant region, e.g., an Ig heavy chain constant region (e.g., at least a CH1 domain; at least CH1 and CH2 domains; CH1, CH2, and CH3 domains; or CH1, CH2, CH3, and CH4 domains), or an Ig light chain constant region. Such an Ig polypeptide is referred to herein as a “targeted Ig polypeptide” or “targeted antibody”.

[0298] The site in the antibody to which the sulfatase motif is introduced can be any convenient site. As shown above, in some cases the degree of modification of the native amino acid sequence of the target polypeptide is kept to a minimum so as to minimize the number of amino acid residues inserted, deleted, substituted, and / or added (e.g., to the N-terminus or C-terminus). By minimizing the degree of modification of the amino acid sequence of the target antibody, the potential impact of such modifications on the antibody's function and / or structure can be minimized.

[0299] The antibody heavy chain constant region may include the Ig constant region of any heavy chain isotype, or an Ig heavy chain constant region that does not exist naturally (including the consensus Ig heavy chain constant region). The Ig constant region may be modified to include an aldehyde tag, which is located in or adjacent to the solvent-accessible loop region of the Ig constant region. The Ig constant region may be modified by insertions and / or substitutions 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 the amino acid sequence of the sulfatase motif as described above.

[0300] In some cases, the aldehyde-tagged antibody comprises an aldehyde-tagged Ig heavy chain constant region (e.g., at least a CH1 domain; at least CH1 and CH2 domains; CH1, CH2, and CH3 domains; or CH1, CH2, CH3, and CH4 domains). The aldehyde-tagged Ig heavy chain constant region may comprise an IgA, IgM, IgD, IgE, IgG1, IgG2, IgG3, or IgG4 isotype heavy chain or any allotype 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 modified by FGE to include at least one sulfatase motif capable of producing an FGly-modified Ig polypeptide. Allotype variants of Ig heavy chains are known in the art. See, for example, Jefferis and Lefranc (2009) MAbs1:4.

[0301] In some cases, the aldehyde-tagged antibody includes an aldehyde-tagged Ig light chain constant region. The aldehyde-tagged Ig light chain constant region includes a kappa light chain, a lambda light chain constant region sequence, e.g., a human kappa or lambda light chain constant region, a hybrid light chain constant region, a synthetic light chain constant region, or a consensus light chain constant region sequence, and is modified to include at least one sulfatase motif that can be modified by FGE to produce an FGly-modified antibody. An exemplary constant region includes the human gamma 1 and gamma 3 regions. Apart from the sulfatase motif, the modified constant region may have a wild-type amino acid sequence, or may 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.

[0302] In some embodiments, the sulfatase motif is located at a position other than the C-terminus of the Ig polypeptide heavy chain, or at a position added to it. As shown above, the isolated aldehyde-tagged antibody may include a heavy chain constant region modified to include the sulfatase motif described above, where the sulfatase motif is located within or adjacent to a surface-accessible loop region of the antibody heavy chain constant region.

[0303] The sulfatase motif may be provided within or adjacent to one or more of these amino acid sequences at the modification site of the Ig heavy chain. For example, an Ig heavy chain polypeptide can be modified with one or more of these amino acid sequences (e.g., the modification involves the insertion, deletion, and / or substitution of one or more amino acid residues) and provide a sulfatase motif adjacent to these modification sites at the N-terminus and / or adjacent to the C-terminus. Alternatively or in addition, an Ig heavy chain polypeptide can be modified with one or more of these amino acid sequences (e.g., the modification involves the insertion, deletion, and / or substitution of one or more amino acid residues) and provide a sulfatase motif between any two residues of the Ig heavy chain modification site. In some embodiments, the Ig heavy chain polypeptide may be modified to include two motifs that are adjacent to each other or separated by one, two, three, four or more (e.g., about 1 to about 25, about 25 to about 50, or about 50 to about 100, or more than that number of amino acids). Alternatively or in addition thereto, if the native amino acid sequence provides one or more amino acid residues of a sulfatase motif sequence, selected amino acid residues of the modification site of the Ig heavy chain polypeptide amino acid sequence may be modified to provide a sulfatase motif to the modification site (e.g., if the modification involves the insertion, deletion, and / or substitution of one or more amino acid residues).

[0304] The antibodies used in the antibody-drug conjugates of this disclosure are, but are 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), and antigens present on disease cells, and may have any of the following antigen-binding specificities. For example, the antibody conjugate may bind to an antigen, and the antigen may be present on the surface of a cell. The antibody conjugates of this disclosure have a suitable binding affinity, for example, 5 × 10⁻⁶ -6 M~10 -7 M, 10 -7 M~5×10 -7 M, 5×10 -7 M~10 -8 M, 10 -8M~5×10 -8 M, 5×10 -8 M~10 -9 M, or 10 -9 It can bind to antigens with a binding affinity greater than M.

[0305] As a non-limiting example, the antibody conjugate of the subject may bind to an antigen present on cancer cells (e.g., tumor-specific antigen, antigen overexpressed on cancer cells, etc.), and the conjugated portion may be a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.). For example, the antibody conjugate of the subject may be specific to an antigen on cancer cells, and the conjugated portion may be a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).

[0306] As a further non-limiting example, the antibody conjugate of the subject may bind to an antigen present on a virus-infected cell (for example, if the antigen is encoded by a virus, or if the antigen is expressed on a cell type that is infected by the virus), and the conjugated portion may be a drug, such as a viral fusion inhibitor.

[0307] Drugs for conjugation into polypeptides As shown above, the conjugate or compound of the present disclosure is substituent W 1 This may include drugs or active agents. Any of several drugs may be suitable for use as a reactive partner to conjugate to an antibody, or may be modified to be suitable for use. Examples of drugs include small molecule drugs and peptide drugs.

[0308] As used herein, “small molecule drug” refers to a compound, such as an organic compound, which exhibits the desired pharmaceutically active properties and has a molecular weight of generally about 800 Da or less, or 2000 Da or less, but may include molecules up to 5 kDa and be as large as 10 kDa. Small inorganic molecules refer to molecules that do not contain carbon atoms, while small organic molecules refer to compounds that contain at least one carbon atom.

[0309] For example, the drug or active agent may be camptothecin, or its analogues or derivatives, or a pharmaceutically active camptothecin moiety and / or part thereof. The camptothecin conjugated to the polypeptide may be any of the various camptothecin moieties, including, but not limited to, camptothecin and its analogues and derivatives as described herein. Examples of drugs found to be used in the conjugates and compounds described herein include, but are not limited to, SN-38, berotecan, exatecan, 9-aminocamptothecin (9-AC), and their derivatives, as well as other camptothecin or camptothecin derivatives.

[0310] In other embodiments, the drug or active agent may be mytansine. "Mytansine," "mytansine moiety," "mytansine active agent moiety," and "mytansinoid" refer to mytansine, as well as its analogs and derivatives, and pharmaceutically active mytansine moieties and / or moieties thereof. Mytansine conjugated to a polypeptide may be any of a variety of mytansinoid moieties, including, but not limited to, mytansine and its analogs and derivatives as described herein (e.g., deacylmytansine).

[0311] In other examples, the drug or active agent may be auristatin, or its analogues or derivatives, or a pharmaceutically active auristatin moiety and / or its moiety. The auristatin conjugated to the polypeptide may be any of the various auristatin moieties, including, but not limited to, auristatin and its analogues and derivatives as described herein. Examples of drugs found to be used with the conjugates and compounds described herein include, but are not limited to, auristatin or auristatin derivatives such as monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and their derivatives.

[0312] In other cases, the drug or active agent may be duocalmycin, or its analogs or derivatives, or a pharmaceutically active duocalmycin moiety and / or its moiety. Duocalmycin conjugated to a polypeptide may be any of the various duocalmycin moieties, including, but not limited to, duocalmycin and its analogs and derivatives as described herein. Examples of drugs found to be used with the conjugates and compounds described herein include, but are not limited to, duocalmycin A, duocalmycin B1, duocalmycin B2, duocalmycin C1, duocalmycin C2, duocalmycin D, duocalmycin SA, and CC-1065, and their derivatives, as well as duocalmycin or duocalmycin derivatives. In some embodiments, duocalmycin is a duocalmycin analog, such as adzeresin, bizeresin, or karzeresin, but is not limited to these.

[0313] In certain embodiments, the drug is selected from cytotoxins, kinase inhibitors, immunostimulants, Toll-like receptor (TLR) agonists, oligonucleotides, aptamers, cytokines, steroids, and peptides.

[0314] For example, cytotoxins may include any compound that causes cell death (e.g., necrosis or apoptosis) or a decrease in cell viability.

[0315] Kinase inhibitors include, but are not limited to, adavocertib, afatinib, axitinib, bosutinib, cetuximab, cobimetinib, crizotinib, cabozantinib, dacomitinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mbritinib, nilotinib, pazopanib, pegaptanib, ruxolitinib, sorafenib, sunitinib, tucatinib, vandetanib, and vemurafenib.

[0316] Examples of immunostimulants, though not limited to these, include vaccines (e.g., bacterial or viral vaccines), colony-stimulating factors, interferons, and interleukins. Examples of TLR agonists, though not limited to these, include imiquimod and reximod.

[0317] Examples of oligonucleotide drugs, though not limited to these, include fomivirsen, pegaptanib, mipomersen, eteplirsen, defibrotide, nusinersen, golozyrsen, viltolarsen, boranesolsen, inotercene, tofersen, and tominersen.

[0318] Examples of aptamer drugs, though not limited to them, include pegaptanib, AS1411, REG1, ARC1779, NU172, ARC1905, E10030, NOX-A12, and NOX-E36.

[0319] Cytokines include, but are not limited to, Albuinterferon alpha-2B, Aldesleukin, ALT-801, Anakinra, Ancestim, Avotermin, Valgrastim, Benpegaldesleukin, Vinetraquin, Syntredekin Besdotox, CTCE-0214, Darbepoetin alfa, Denileukin Difutox, Duranermin, Edodekin alfa, Emfilamine, Epoetin Delta, Erythropoietin, Human Interleukin 2, Interferon α, Interferon α-2c, Interferon α-n1, Interferon α-n3, Interferon α-con-1, Interferon β-1a, Interferon β-1b, and Interferon γ-1 b. Examples include interferon κ, interleukin-1α, interleukin-10, interleukin-7, lenograstim, religistim, ripegfilgrastim, lorcafusp alpha, Maxy-G34, methoxypolyethylene glycol epoetin beta, moglamostim, muprestim, nagrestipen, oprelbequin, pegfilgrastim, pegrodecakine, pegyinterferon α-2a, pegyinterferon α-2b, pegyinterferon β-1a, pegyinterferon λ-1a, recombinant CD40 ligand, reglamostim, romiplostim, salglamostim, thrombopoietin, tucozotsumab cermoloukin, and viral macrophage inflammatory proteins.

[0320] Examples of steroid drugs, though not limited to these, include prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, and deflazacort.

[0321] As used herein, “peptide drugs” refers to amino acids including polymer compounds and encompasses native and unnatural peptides, oligopeptides, cyclic peptides, and proteins, as well as peptide mimes. Peptide drugs can be obtained by chemical synthesis or produced from genetically encoded sources (e.g., recombinant sources). Peptide drugs may have a molecular weight range of 200 Da to 10 kDa or greater. Suitable peptides include, but are not limited to, cytotoxic peptides, angiogenic peptides, anti-angiogenic peptides, B cell activating peptides, T cell activating peptides, antiviral peptides, peptides that inhibit viral fusion, peptides that increase the production 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-nociceptive peptides, vasodilator peptides, platelet aggregation inhibitors, and analgesics.

[0322] Additional examples of drugs found to be used with the conjugates and compounds described herein include, but are not limited to, tubulisin M, calicheamycin, STAT3 inhibitors, α-amanitin, aurora kinase inhibitors, berotecan, and anthracyclines.

[0323] Other examples of drugs include small molecule drugs such as cancer chemotherapeutic agents. For example, if the polypeptide is an antibody (or fragment thereof) having specificity for tumor cells, the antibody may be modified to include modified amino acids, as described herein, and then conjugate to a cancer chemotherapeutic agent. Cancer chemotherapeutic agents include non-peptidic (i.e., non-proteinic) compounds that reduce the proliferation of cancer cells and encompass cytotoxic agents and cell proliferation inhibitors. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, or steroid hormones. Peptidic compounds may also be used.

[0324] Suitable cancer chemotherapeutic agents include drastatin 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, WO96 / 33212, WO96 / 14856, and US6,323,315. For example, drastatin 10 or auristatin PE may be included in the antibody-drug conjugate of this disclosure. Suitable cancer chemotherapeutic agents also include mytansinoids and their active analogues and derivatives (see, for example, EP1391213 and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623) and duocalmycin and its active analogues and derivatives (including, for example, synthetic analogues, KW-2189 and CB1-TM1).

[0325] Drugs that act to reduce cell proliferation are known and widely used in the art. Such drugs include, but are not limited to, mechloretamine, cyclophosphamide (Cytoxane®), melphalan (L-sarcolicin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozosin, chlorozotosin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobromane, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide, as well as alkylating agents such as nitrogen mustard, nitrosourea, ethyleneimine derivatives, alkyl sulfonates, and triazeneses.

[0326] Examples of antimetabolites include, but are not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), phloxuridine (FudR), 6-thioguanine, 6-mercaptopurine (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, as well as folate analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors.

[0327] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinal; alkaloids, e.g., vincristine, vinblastine, vinorelbine, vindesine; podophyllotoxins, e.g., etoposide, teniposide; antibiotics, e.g., anthracyclines, daunorubicin salts Examples include, but are not limited to, salts (daunomycin, rubidomycin, serubicin), idarubicin, doxorubicin, epirubicin, and morpholino derivatives; phenoxyzombine cyclopeptides, e.g., cutinomycin; basic glycopeptides, e.g., bleomycin; anthraquinone glycosides, e.g., plicamycin (mitramycin); anthracendions, e.g., mitoxantrone; azilinopyrroindolediones, e.g., mitomycin; macrocyclic immunosuppressants, e.g., cyclosporine, FK-506 (tacrolimus, Prograf), rapamycin, etc.

[0328] Other antiproliferative cytotoxic agents include navelbine, CPT-11, anastrozole, letrozole, capecitabine, raloxafene, cyclophosphamide, ifosfamide, and droloxifene.

[0329] Microtubule agonists having antiproliferative activity are also suitable for use, but are not limited to, allocolchicine (NSC406042), halichondrin B (NSC609395), colchicine (NSC757), colchicine derivatives (e.g., NSC33410), dorastatin 10 (NSC376128), meitansine (NSC153858), rhizoxin (NSC332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC361792), tritylcysteine, vinblastine sulfate, vincristine sulfate, and, but are not limited to, epotilon A, epotilon B, natural and synthetic epotilons including discodermolide; estramustine, nocodazole, etc.

[0330] Suitable hormone regulators and steroids (including synthetic analogs) for use include, but are not limited to, corticosteroids such as prednisone and dexamethasone; estrogens and pregestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, and tamoxifen; and corticosteroids such as aminoglutethimide, 17α-ethinylestradiol, and diethyl steroids. Examples include rubestrol, testosterone, fluoxymesterone, dromostanolone propionate, testactone, methylprednisolone, methyl-testosterone, prednisone, triamcinolone, chlorotrianicene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), toremifene (Fareston), and Zoladex®. Estrogen stimulates proliferation and differentiation, and therefore compounds that bind to estrogen receptors are used to inhibit this activity. Adrenocortical steroids can inhibit T cell proliferation.

[0331] Other suitable chemotherapeutic agents include metal complexes, such as cisplatin (cis-DDP) and carboplatin; urea, such as hydroxyurea; and hydrazine, such as N-methylhydrazine; epipophyllotoxin; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; and tegafur. Other antiproliferative agents of interest include immunosuppressants, such as mycophenolic acid, thalidomide, desoxysperguarin, azasporin, leflunomide, mizoribine, azaspiran (SKF105685); and Iressa (registered trademark) (ZD1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline).

[0332] Taxanes are suitable for use. "Taxane" includes paclitaxel, as well as any active taxane derivatives or prodrugs. "Paclitaxel" (which should be understood herein to include analogs, formulations, and derivatives such as, for example, docetaxel, Taxol®, Taxotere®, 10-desacetyl analogs of paclitaxel, and 3'N-desbenzoyl-3'Nt-butoxycarbonyl analogs of paclitaxel) can be readily prepared using techniques known to those skilled in the art (WO94 / 07882, WO94 / 07882, WO94 / 07882) It can be obtained from various commercial sources, including, for example, Sigma Chemical Co., St. Louis, Mo (T7402 from Taxus brevifolia, or T-1912 from Taxus yannanensis).

[0333] It should be understood that paclitaxel refers not only to the commonly available chemical forms of paclitaxel, but also to its analogues and derivatives (e.g., Taxotere® docetaxel as described above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

[0334] Various known derivatives, including both hydrophilic and hydrophobic derivatives, are also included within the term “taxane.” Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. 99 / 18113; piperazino and other derivatives described in WO99 / 14209; taxane derivatives described in WO99 / 09021, WO98 / 22451, and U.S. Patent No. 5,869,680; 6-thio derivatives described in WO98 / 282886; sulfenamide derivatives described in U.S. Patent No. 5,821,263; and taxol derivatives described in U.S. Patent No. 5,415,869. It also further includes prodrugs of paclitaxel, including, but are not limited to, those described in WO98 / 58927, WO98 / 13059, and U.S. Patent No. 5,824,701.

[0335] Suitable biological reaction 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 factors; and (9) inhibitors of angiogenesis.

[0336] Examples of drugs include small molecule drugs such as cancer chemotherapy agents. For example, if the polypeptide is an antibody (or fragment thereof) having specificity for tumor cells, the antibody may be modified to include modified amino acids, as described herein, and then conjugate to cancer chemotherapy agents such as microtubule agonists. In certain embodiments, the drug is a microtubule agonist having antiproliferative activity, such as a mytansinoid.

[0337] Embodiments of the present disclosure include a conjugate in which an antibody is conjugated to one or more drug moieties, e.g., two, three, four, five, six, seven, eight, nine, or ten or more drug moieties. The drug moieties may 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, e.g., 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, e.g., 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 5. In certain embodiments, the conjugate has an average DAR of 1 to 4. In certain embodiments, the conjugate has an average DAR of 1 to 3. In certain embodiments, the conjugate has an average DAR of 1 to 2. "Average" refers to the arithmetic mean.

[0338] Drugs conjugated to polypeptides can be modified to incorporate a reactive partner for reaction with the polypeptide. If the drug is a peptide drug, the reactive moiety (e.g., aminooxy or hydrazide) can be located in the N-terminal region, N-terminus, C-terminal region, C-terminus, or internally relative to the peptide. For example, one method involves synthesizing a peptide drug having an aminooxy group. In this example, the peptide is synthesized from a Boc-protected precursor. The amino group of the peptide can react with compounds containing a carboxylic acid group and an oxy-N-Boc group. For example, the amino group of the peptide reacts with 3-(2,5-dioxopyrrolidine-1-yloxy)propanoic acid. Other modifications on compounds containing a carboxylic acid group and an oxy-N-protecting group may involve alkylene linkers and substituents on the alkylene linker with varying numbers of carbon atoms. Reactions between the amino group of the peptide and compounds containing a carboxylic acid group and an oxy-N-protecting group occur through standard peptide coupling chemistry.Examples of peptide-binding reagents that can be used include, but are not limited to, DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluylcarbodiimide, BDP (1-benzotriazolediethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC (1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuryl fluoride, cyanuryl chloride, and TFFH (tetramethylamine). (O-benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), DPPA (diphenylphosphorazidate), BOP (benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), HBTU (O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate), TBTU (O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate) ), TSTU (O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), HATU (N-[(dimethylamino)-1-H-1,2,3-triazolo[4,5,6]-pyridine-1-ylmethylene]--N-methylmethaneaminium hexafluorophosphate N-oxide), BOP-Cl (bis(2-oxo-3-oxazolidinyl)phosphinate chloride), PyBOP ((1-H-1,2,3-benzotriazole- Examples include 1-yloxy)-tris(pyrrolidino)phosphonium tetrafluorophosphate, BrOP (bromotris(dimethylamino)phosphonium hexafluorophosphate), and DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)-one)PyBrOP (bromotris(pyrrolidino)phosphonium hexafluorophosphate). Non-limiting examples include HOBt and DIC, which can be used as peptide linking reagents.

[0339] Deprotection for exposure to aminooxy functionality is carried out on peptides containing an N-protecting group. Deprotection of the N-oxysuccinimide group occurs, for example, according to standard deprotection conditions for cyclic amino 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. Specific deprotection conditions involve hydrazine reagents, amino reagents, or sodium borohydride. Deprotection of the Boc protecting group occurs with TFA. Other reagents 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.

[0340] Those skilled in the art will understand that factors such as pH and steric hindrance (i.e., the possibility of contact between amino acid residues for reaction with the desired reactive partner) are important, and modification reaction conditions to provide optimal conjugate conditions are well-established within the art and are customary in the art. When conjugate is performed on polypeptides present in or on living cells, the conditions are selected to be physiologically compatible. For example, pH may be temporarily reduced for a sufficient time to allow the reaction to occur, but not within a period tolerable by the cells (e.g., about 30 minutes to 1 hour). Physiological conditions for modifying polypeptides on a cell surface may be similar to those used in ketone-azide reactions during modification of cells possessing cell surface azides (see, e.g., US6,570,040).

[0341] Small molecule compounds containing or modified to contain an α-nucleophilic group that serve as a reactive partner with the compounds or conjugates disclosed herein are also intended for use as drugs in the polypeptide-drug conjugates of this disclosure. General methods are known in the art for useful chemical synthesis schemes and conditions for synthesizing the compounds of interest (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).

[0342] formulation The conjugates of this disclosure may be formulated in a variety of different ways. Generally, if the conjugate is a gradient-drug conjugate, the conjugate is formulated in a manner that is appropriate to the drug, antibody, condition being treated, and route of administration used.

[0343] In some embodiments, a pharmaceutical composition is provided comprising one of the conjugates of the present disclosure and a pharmaceutically acceptable excipient.

[0344] The conjugate (e.g., antibody-drug conjugate) may be provided in any preferred form, for example, in the form of a pharmaceutically acceptable salt, and may be formulated for any preferred route of administration, e.g., oral, topical, or parenteral administration. Where the conjugate is provided as an injectable liquid (such as in those embodiments where they are administered intravenously or directly to tissue), the conjugate may be provided as a ready-to-use dosage form, or as a liquid consisting of a reconstituted, storage-stable powder or a pharmaceutically acceptable carrier and excipient.

[0345] Methods for formulating the conjugate can be adapted from those readily available. For example, the conjugate may be provided as a pharmaceutical composition comprising a therapeutically effective amount of the conjugate and a pharmaceutically acceptable carrier (e.g., physiological saline). The pharmaceutical composition may optionally contain other additives (e.g., buffers, stabilizers, preservatives, etc.). In some embodiments, the formulation is suitable for administration to mammals, for example, to humans.

[0346] Treatment method The antibody-drug conjugates of this disclosure are useful for treating conditions or diseases in subjects who are able to receive treatment by administering the parent drug (i.e., the drug before conjugation to the antibody).

[0347] In some embodiments, methods are provided that include administering an effective amount (e.g., a therapeutically effective amount) of any of the conjugates of this disclosure to a target.

[0348] In certain embodiments, a method is provided for delivering a drug to a target site of a subject, the method comprising administering to the subject a pharmaceutical composition comprising one of the conjugates of the Disclosure, wherein the administration is effective in releasing a therapeutically effective amount of the drug from the conjugate at the target site of the subject. For example, as described herein, the antibody-drug conjugate of the Disclosure may comprise an enzymatically cleavable linker, e.g., an enzymatically cleavable linker comprising a first enzymatically cleavable portion and a second enzymatically cleavable portion. In some cases, the cleavable linker may be cleaved under appropriate conditions to separate or release the drug from the antibody at a desired target site of action for the drug. For example, a second cleavable linker protecting the first cleavable linker from cleavage may be cleaved to enable cleavage of the first cleavable portion, thereby cleaving the cleavable linker into two or more portions, and thus releasing the drug from the antibody-drug conjugate at a desired site of action.

[0349] In certain embodiments, the first cleavable portion may be an enzymatically cleavable portion. In some cases, the enzyme that facilitates the cleavage of the first cleavable portion is an enzyme administered to the subject being treated (i.e., an exogenous enzyme to the subject being treated). For example, the first enzyme may be administered before, concurrently with, or after the administration of the antibody-drug conjugate described herein.

[0350] In certain embodiments, the second cleavable portion may be an enzymatically cleavable portion. In some cases, the enzyme that facilitates the cleavage of the second cleavable portion is an enzyme administered to the subject being treated (i.e., an exogenous enzyme to the subject being treated). For example, the second enzyme may be administered before, concurrently with, or after the administration of the antibody-drug conjugate described herein. In certain embodiments, the first enzyme and the second enzyme are different enzymes.

[0351] In other examples, the first enzyme that facilitates the cleavage of the first cleavable portion is an enzyme present in the target being treated (i.e., endogenous to the target being treated). For example, the first enzyme may be present at the desired site of action for the drug in an antibody-drug conjugate. The antibody in the antibody-drug conjugate may be specifically targeted to the desired site of action (e.g., specifically bound to an antigen present at the desired site of action), and the desired site of action also includes the presence of the first enzyme. In some cases, the first enzyme is present in excess at the desired site of action compared to other areas of the body of the target being treated. For example, the first enzyme may be overexpressed at the desired site of action compared to other areas of the body of the target being treated. In some cases, the first enzyme is present in excess at the desired site of action due to the localization of the first enzyme in a particular region or location. For example, the first enzyme may be associated with a particular structure within the desired site of action, such as a lysosome. In some cases, the first enzyme is present in excess in lysosomes compared to other areas of the body of the target. In some embodiments, lysosomes containing the first enzyme are located at the desired site of action for the drug in the antibody-drug conjugate, for example, at the site of cancer or tumor to be treated with the drug. In certain embodiments, the first enzyme is an esterase.

[0352] In certain embodiments, the second enzyme that facilitates the cleavage of the second cleavable portion is an enzyme present in the target being treated (i.e., endogenous to the target being treated). For example, the second enzyme may be present at the desired site of action for the drug in an antibody-drug conjugate. The antibody in the antibody-drug conjugate may be specifically targeted to the desired site of action (e.g., specifically bound to an antigen present at the desired site of action), and the desired site of action also includes the presence of the second enzyme. In some cases, the second enzyme is present in excess at the desired site of action compared to other areas of the body of the target being treated. For example, the second enzyme may be overexpressed at the desired site of action compared to other areas of the body of the target being treated. In some cases, the second enzyme is present in excess at the desired site of action due to localization of the second enzyme in a particular region or location. For example, the second enzyme may be associated with a particular structure within the desired site of action, such as a lysosome. In some cases, the second enzyme is present in excess in lysosomes compared to other areas of the body of the target. In some embodiments, lysosomes containing the second enzyme are located at the desired site of action for the drug in the antibody-drug conjugate, for example, at the site of cancer or tumor to be treated with the drug. In certain embodiments, the second enzyme is glucuronidase, galactosidase, glucosidase, mannosidase, fucosidase, etc.

[0353] Any suitable enzyme can be used to cleave the first and second cleavable portions of the antibody-drug conjugate described herein. Other enzymes, for example, but not limited to, those derived from other vertebrates (e.g., primates, mice, rats, cats, pigs, quail, goats, dogs, etc.), may also be suitable for cleaving the first and second cleavable portions of the antibody-drug conjugate described herein.

[0354] In certain embodiments, the antibody-drug conjugate is substantially stable under standard conditions. Substantially stable means that the cleavable linker of the antibody-drug conjugate does not undergo significant cleavage in the absence of the first and second enzymes described above. For example, as described above, the second cleavable portion can be protected from cleavage of the first cleavable portion, and therefore the cleavable linker of the antibody-drug conjugate does not undergo significant cleavage in the absence of the second enzyme described above. For example, the cleavable linker of the antibody-drug conjugate may be substantially stable such that 25% or less of the antibody-drug conjugate, e.g., 20%, or 15%, or 10%, or 5%, or 4%, or 3%, or 2%, or 1%, is cleaved in the absence of the first and / or second enzymes. In some cases, an antibody-drug conjugate is substantially stable such that its cleavable linker does not undergo significant cleavage in the absence of a first enzyme and / or a second enzyme, but can be cleaved in the presence of both the first and second enzymes. For example, an antibody-drug conjugate may be substantially stable after administration to a subject. In some cases, an antibody-drug conjugate is substantially stable after administration to a subject, and then, when the antibody-drug conjugate is in the presence of a second enzyme at a desired site of action, a second cleavable portion is cleaved from the cleavable linker, thus exposing the first cleavable portion to subsequent cleavage by the first enzyme, and subsequently releasing the drug at its desired site of action. In certain embodiments, after administration to a subject, the antibody-drug conjugate remains stable for a long period of time, for example, 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 the absence of the first enzyme and / or the second enzyme.In certain embodiments, the antibody-drug conjugate is stable for extended periods in a certain pH range, for example, in the range of 2-10, or 3-9, or 4-8, or 5-8, or 6-8, or 7-8, in the absence of the first and / or second enzymes.

[0355] As stated above, the antibody-drug conjugates of this disclosure find use in the treatment of a condition or disease in subjects suitable for treatment by administration of the parent drug. "Treatment" means that at least improvement of the symptoms associated with a condition that afflicts the host is achieved, and improvement is used broadly to refer to the magnitude of a parameter, e.g., at least reduction of the symptoms associated with the condition being treated. Thus, treatment includes situations in which a pathological condition, or at least symptoms associated therewith, is completely inhibited so that the host no longer suffers the condition, or at least symptoms that characterize the condition, e.g., prevention of onset or cessation, e.g., termination. Thus, treatment includes (i) prevention, i.e., reducing the risk of developing clinical symptoms, including preventing the development of clinical symptoms, such as preventing disease progression to an adverse condition; (ii) inhibition, i.e., preventing the development or further development of clinical symptoms, such as mitigating or completely inhibiting active disease; and / or (iii) mitigation, i.e., reversing clinical symptoms.

[0356] The subjects to be treated may be subjects in need of therapy, and the subjects to be treated may be subjects suitable for treatment with the parent drug. Thus, a variety of subjects may be suitable for treatment with the antibody-drug conjugates disclosed herein. Generally, such subjects are “mammals,” including humans of interest. Other subjects may include domestic pets (e.g., dogs and cats), livestock (e.g., cattle, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, e.g., animal models of diseases), and non-human primates (e.g., chimpanzees and monkeys).

[0357] The amount of antibody-drug conjugate administered may be initially determined based on the dose of the parent drug and / or the dosage regimen. Generally, the administered antibody-drug conjugate may provide targeted delivery of the conjugated drug and / or an enhanced serum half-life, thus providing at least one of a reduced dose or reduced administration of the dosage regimen. Therefore, an antibody-drug conjugate may provide a reduced dose and / or reduced administration of the dosage regimen relative to the parent drug before being conjugated into the antibody-drug conjugate of this disclosure.

[0358] Furthermore, as described above, since antibody-drug conjugates can provide a controlled stoichiometry of drug delivery, the dosage of an antibody-drug conjugate can be calculated based on the number of drug molecules delivered per antibody-drug conjugate principal component.

[0359] In some embodiments, multiple doses of the antibody-drug conjugate may be administered. The frequency of administration of the antibody-drug conjugate may vary depending on any of several factors, such as the severity of symptoms or the condition of the subject. For example, in some embodiments, the antibody-drug conjugate may be 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, daily (qd / od), twice a day (bds / bid), or three times a day (tds / tid). [Examples]

[0360] The following examples are provided to those skilled in the art to provide a complete disclosure and description of the methods of preparation and use of the present invention and are not intended to limit the scope of what the inventors consider to be the invention, nor are they intended to represent that the following experiments are all or only experiments that can be performed. Efforts have been made to ensure accuracy to the figures used (e.g., quantity, temperature, etc.), but some degree of experimental error and deviation should be taken into consideration. Unless otherwise indicated, parts are by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure. "Average" means arithmetic mean. Standard abbreviations may be used, for example, bp, base pair, kb, kilobase, pl, picoliters, s or sec, seconds, min, minutes, h or hr, hours, aa, amino acid, kb, kilobase, bp, base pair, nt, nucleotide, im, intramuscular, ip, intraperitoneal, sc, subcutaneous, etc.

[0361] General synthesis procedure Many common references are available that provide generally 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).

[0362] The compounds described herein may be purified by any purification protocol known in the art, including chromatography such as HPLC, preparative thin-layer chromatography, flash column chromatography, and ion-exchange chromatography. Any suitable stationary phase, including normal-phase and reverse-phase chromatography, and ionic resins, may be used. 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. LRSnyder and JJ Kirkland, John Wiley and Sons, 1979, and Thin Layer Chromatography, edited by E. Stahl, Springer-Verlag, New York, 1969.

[0363] During any of the processes for preparing the target compound, it may be necessary and / or desirable to protect any sensitive or reactive groups of the molecule in question. This can be seen, for example, in JFW McOmie, “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 der organischen Chemie”, Houben-Weyl, 4th edition, Vol. 15 / l, 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 This can be achieved by using conventional protecting groups, as described in standard studies such as "Kohlenhydrate: Monosaccharide and Derivate," Georg Thieme Verlag, Stuttgart 1974. The protecting groups can be removed at a convenient subsequent step using methods known in the art.

[0364] 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. Various examples of synthetic routes that may be used to synthesize the compounds disclosed herein are described in the following scheme.

[0365] Example 1 Synthesis of double-cleavage ester linkers Materials and methods General information Synthetic reagents were purchased from Sigma-Aldrich, Acros, AK Scientific, or other commercial sources and used without purification. Anhydrous solvents were obtained from commercial sources in sealed bottles. Cytotoxin SN-38(12) was purchased from MedChemExpress and used as is. Cytotoxins 28 and 41 were purchased from commercial sources and used as is. Compound 9 was synthesized using previously reported procedures. Synthetic intermediates 16, 19, 20, 22, 25, and 48 were commercially obtained from Shanghai Medicilon and used without purification. All other starting materials were purchased from commercial sources or synthesized using previously reported procedures. In all cases, the solvent was removed under reduced pressure using a Buchi Rotovapor R-114 fitted with a Buchi V-700 vacuum pump. Column chromatography was performed using a Biotage chromatography purification system. Preparative HPLC purification was performed using a Waters preparative HPLC unit fitted with a Phenomenex Kinetex 5μm EVO C18 150×21.2mm column. At 30°C, HPLC analysis was performed using an Agilent 1100 series analytical HPLC system fitted with a Model G1322A degasser, Model G1311A quaternary 4 pump, Model G1329A autosampler, Model G1314 tunable wavelength detector, and an Agilent Poroshell 120 SB C18, 4.6mm×50mm column, using a 10-100% gradient of water and acetonitrile containing 0.05% trifluoroacetic acid. The HPLC was monitored at 254nm or 205nm. Low-resolution mass spectra (LRMS) were acquired at 30°C using a 10–100% gradient of water and acetonitrile containing 0.1% formic acid on an Agilent Technology 6120 quadrupole LC / MS system equipped with an Agilent 1260 Infinity HPLC system, a G1314 tunable wavelength detector, and an Agilent Poroshell 120 SB C18, 4.6 mm × 50 mm column.

[0366] [ka] Preparation of (2S,3R,4S,5S,6S)-2-(2-(tert-butoxycarbonyl)-5-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (10) A mixture of tert-butyl 2-hydroxy-4-nitrobenzoic acid (9, 160 mg, 0.67 mmol) and methyl acetobromo-D-glucuronate (8, 797 mg, 2.0 mmol) in 20 mL of anhydrous acetonitrile was mixed with silver(I) oxide (463 mg, 2.0 mmol). The resulting mixture was vigorously stirred in the dark for 24 hours. The reaction mixture was filtered through a silica gel pad and eluted with ethyl acetate. The combined filtrate was concentrated to dryness, and the residue was purified on silica gel (ethyl acetate-hexane, 0-30% v / v gradient) to obtain 210 mg of product 10 (0.38 mmol, yield 56%) as a white solid. LRMS (ESI): m / z 578.1 [M+Na] + , C 24 H 29 NO 14 The calculated value for m / z is 578.2.

[0367] Preparation of 4-nitro-2-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzoic acid (11) 180 mg (0.32 mmol) of tert-butyl ester 10 was dissolved in 4 mL of a TFA-DCM mixture (1:1) at room temperature. The resulting solution was allowed to stand for 30 minutes, then the solvent was removed under reduced pressure. The residue was purified on silica gel (methanol-DCM, 0-5% v / v gradient) to obtain 155 mg of product 11 (0.31 mmol, 97% yield) as a pink foamy solid. LRMS (ESI): m / z 522.1 [M+Na] + , C 20 H 21 NO 14 The calculated value for m / z is 522.1.

[0368] [ka] Preparation of (2S,3R,4S,5S,6S)-2-(2-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-9-yl)oxy)carbonyl)-5-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (53) To a mixture of SN-38 (12, 20 mg, 51 μmole) and carboxylic acid 11 (26 mg, 51 μmole) in 2 mL of anhydrous THF, 10.5 mg of DCC (51 μmole) was added, followed by the addition of DMAP (6 mg, 51 μmole). The reaction mixture was stirred for 24 hours, then the solid was removed by filtration. The resulting filtrate was concentrated under vacuum and purified on silica gel (MeOH-DCM 0-10% v / v gradient) to obtain 40 mg of product 53 (46 μmole, 90% yield) as an off-white solid. LRMS (ESI): m / z 874.2 [M+H] + , C 42 H 39 N3O 18 The calculated value for m / z is 874.2.

[0369] Preparation of (2S,3R,4S,5S,6S)-2-(5-amino-2-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-9-yl)oxy)carbonyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (54) To a stirred solution of compound 53 (40 mg, 46 μmol) in 5 mL of ethyl acetate, 5 mg of 10 wt% palladium-carbon (4.6 μmol) and 50 μL of triethylamine (0.36 mmol) were added. The reaction flask was washed with argon, sealed with a rubber diaphragm, and fitted with a hydrogen balloon. The reaction mixture was stirred overnight at room temperature, then filtered through a Celite pad, concentrated under vacuum, and purified on silica gel (MeOH-DCM 0-10% v / v gradient) to obtain 28 mg of compound 54 (34 μmole, 73% yield) as a yellowish solid. LRMS (ESI): m / z 844.3 [M+H] + , C 42 H 41 N3O 16 The calculated value for m / z is 844.3.

[0370] Preparation of (2S,3R,4S,5S,6S)-2-(5-((S)-2-((tert-butoxycarbonyl)amino)propanamide)-2-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-9-yl)oxy)carbonyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (56) To a mixture of compound 54 (28 mg, 34 μmol) and Boc-L-Ala-OH (7 mg, 34 μmol) in 1 mL of DCM, EEDQ (10 mg, 40 μmol) was added at room temperature. The resulting mixture was stirred in the dark for 24 hours and then purified directly on silica gel (MeOH-DCM 0-10% v / v gradient) to obtain 24 mg of product 56 (24 μmole, 70% yield) as a yellowish-brown solid. LRMS (ESI): m / z 1015.4 [M+H] + , C 50 H 54 N4O 19 The calculated value for m / z is 1015.3.

[0371] Preparation of (2S,3S,4S,5R,6S)-6-(5-((S)-2-((tert-butoxycarbonyl)amino)propanamide)-2-((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-9-yl)oxy)carbonyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (57) To a solution of compound 55 (19 mg, 19 μmole) in 2 mL of methanol-water mixture (4:1), scandium(III) triflate (19 mg, 38 μmole) was added at room temperature. The resulting mixture was stirred at 30°C for 4 days, then the solvent was removed under vacuum, and the residue was purified by reverse-phase HPLC (C18 column, CH3CN-H2O, 0.05% TFA, 0-50% v / v gradient) to obtain 3.5 mg of a mixture of the desired product 57 and the corresponding partially hydrolyzed methyl ester 58 in approximately a 1:1 ratio (mixture yield 21%). The mixture was used in the next step without further purification. LRMS(ESI) m / z 875.4[M+H] + , C 43 H 46 N4O 16 Calculated value for (57) 875.3; LRMS(ESI)m / z 889.4[M+H] + , C 44 H 48 N4O 16 The calculated value for (58) is 889.3.

[0372] [ka] Preparation of (S)-tert-butyl(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-9-yl) carbonate (13) To a solution of SN-38 12 (158 mg, 0.40 mmol) and Boc2O (114 mg, 1.3 mmol) in dichloromethane (16 mL), pyridine (0.98 mL, 12.2 mmol) was added at 0°C. After 1 hour, the solution was warmed to room temperature and stirred for 2 hours. The reaction mixture was then concentrated under vacuum, and the residue was purified using silica gel (hexane-siRNA, 100:0~0:100 v / v) to obtain 13 (160 mg, 0.33 mmol, 83%) as an off-white solid. LRMS(ESI): m / z 493.2[M+H] + , C 27 H 29 The calculated value for N2O7 (m / z) is 493.2.

[0373] Preparation of (2S,3R,4S,5S,6S)-2-(2-((((S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)oxy)carbonyl)-5-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (14) To a solution of carboxylic acid 11 (18 mg, 36 μmol) in dichloromethane (1 mL) and DMF (0.5 mL), Boc-protected SN-38 13 (14 mg, 28 μmol), followed by DCC (6 mg, 29 μmol) and DMAP (3 mg, 25 μmol), were added at 0°C. After 1 hour, the reaction mixture was heated to room temperature and stirred for 2 hours. The reaction mixture was purified by reverse-phase chromatography using a C18 column (H2O / CH3CN containing 0.05% TFA, 100:0~0:100 v / v) to obtain compound 14 (25 mg, 26 μmol, 93% yield) as a yellow solid. LRMS (ESI): m / z 974.3 [M+H] + , C 47 H 47 N3O 20 The calculated value for m / z is 974.3.

[0374] Preparation of (2S,3R,4S,5S,6S)-2-(5-amino-2-((((S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)oxy)carbonyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (15) To a solution of compound 14 (35 mg, 36 μmol) in à (0.5 mL), Pd / C (10 wt%, 2 mg) and triethylamine (2 μL, 22 μmol) were added. The flask was then evacuated and filled with hydrogen gas from a balloon in three repeated cycles. The reaction mixture was vigorously stirred at room temperature for 48 hours with the H2 balloon attached. The solid was removed by filtration through a Celite pad, the filtrate was concentrated, and dried under high pressure to obtain 35 mg of crude compound 15, which was used in the next step without purification. LRMS(ESI): m / z 944.3[M+H] + , C 47 H 49 N3O 18 The calculated value for m / z is 944.3.

[0375] Preparation of (2S,3R,4S,5S,6S)-2-(5-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)propanamide)-2-((((S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)oxy)carbonyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (17) A mixture of crude amine 15 (35 mg) and Fmoc-Val-Ala-OH16 (60 mg, 0.15 mmol) in DMF (0.5 mL) was mixed with HATU (56 mg, 0.15 mmol) and DIPEA (51 μL, 0.30 mmol) at room temperature. The reaction mixture was stirred overnight and purified by reverse-phase chromatography on a C18 column (H2O / CH3CN containing 0.05% TFA, 100:0~0:100 v / v) to obtain compound 17 (46 mg, 34 μmol, 94% yield in 2 steps) as a yellow solid. LRMS (ESI): m / z 1336.5 [M+H] + , C 70 H 73 N5O 22 The calculated value for m / z is 1336.5.

[0376] Preparation of (2S,3S,4S,5R,6S)-6-(5-((S)-2-((S)-2-amino-3-methylbutanamide)propanamide)-2-((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)oxy)carbonyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (18) Compound 17 (20 mg, 15 μmol) was dissolved in a MeOH-H2O mixture (4:1 v / v, 1 mL) to which Sc(OTf)3 (180 mg, 0.36 mmol) was added at room temperature. The resulting mixture was stirred for 2 days and concentrated under vacuum. The residue was reconstituted in a DMF-piperidine mixture (10:1 v / v, 1.1 mL) and stirred at room temperature for 1 hour. The reaction mixture was purified by reverse-phase chromatography on a C18 column (H2O / CH3CN containing 0.05% TFA, 90:10~35:65 v / v) to obtain compound 18 (8 mg, 9 μmol, 60% yield). LRMS (ESI): m / z 874.3 [M+H] + , C 43 H 47 N5O 15 The calculated value for m / z is 874.3.

[0377] Preparation of (R)-2-(3-(2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridine-1-yl)propanamide)-3,31-dioxo-31-(perfluorophenoxy)-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontane-1-sulfonic acid (20) [ka] A mixture of carboxylic acid 19 (180 mg, 0.17 mmol) and pentafluorophenol (125 mg, 0.68 mmol) in 4 mL of anhydrous THF was mixed with DCC (68 mg, 0.33 mmol) at room temperature. The resulting mixture was stirred overnight, filtered through a Celite pad, concentrated under vacuum, and purified by reverse-phase chromatography (C18, 0-80% acetonitrile water / 0.05% TFA) to obtain 100 mg of PFP ester 20 as a colorless oil (0.08 mmol, yield 47%). LRMS (ESI): m / z 1225.4 [M+H] + , C 56 H 69 F5N6O 17 The calculated value for S is m / z 1225.4.

[0378] (2S,3S,4S,5R,6S)-6-(2-((((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolidino[1,2-b]quinoline-4-yl)oxy)carbonyl)-5-((2S,5S,36R)-40-(2-((1,2-dimethylhydrazinyl)methyl)-1H Preparation of -pyrrolo[2,3-b]pyridine-1-yl)-5-isopropyl-2-methyl-4,7,35,38-tetraoxo-36-(sulfomethyl)-10,13,16,19,22,25,28,31-octaoxa-3,6,34,37-tetraazatetracontanamid)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (21) To a mixture of amine 18 (8 mg, 9 μmol) and PFP ester 20 (12 mg, 10 μmol) in DMF (0.5 mL), HOAt (1.2 mg, 9 μmol) and DIPEA (5 μL, 27 μmol) were added at ambient temperature. The reaction mixture was stirred for 1 hour, then DMF (0.5 mL) was added to the mixture, followed by piperidine (50 μL). After stirring at room temperature for 15 minutes, the reaction mixture was directly purified by reverse-phase HPLC using a C18 column (H2O / CH3CN containing 0.05% TFA, 90:10~45:55 v / v). The fraction containing the desired compound was pooled and lyophilized to obtain compound 21 as a yellow solid (3.5 mg, 2 μmol, yield 22%). LRMS (ESI): m / z 1692.7 [M+H] + , C 78 H 105 N 11 O 29 The calculated value for S is m / z 1692.7.

[0379] [ka] Preparation of ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-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)carbamate (42) A mixture of MMAE41 (100 mg, 0.14 mmol) and Boc2O (56 mg, 0.26 mmol) in CH3CN (2 mL) was mixed with DIPEA (44 μL, 0.26 mmol) at room temperature. After stirring overnight, the reaction mixture was purified using silica gel column chromatography (DCM / MeOH, 100:0~90:10 v / v) to obtain compound 42 (100 mg, 0.12 mmol, 86%) as an off-white solid. LRMS (ESI): m / z 818.6 [M+H] + , C44 H 75 The calculated value for N5O9 (m / z) is 818.6.

[0380] Preparation of (2S,3R,4S,5S,6S)-2-(5-amino-2-(((1S,2R)-2-((2R,3R)-3-((S)-1-((6S,9S,12S,13R)-12-((S)-sec-butyl)-6,9-diisopropyl-13-methoxy-2,2,5,11-tetramethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadecane-15-oil)pyrrolidine-2-yl)-3-methoxy-2-methylpropanamide)-1-phenylpropoxy)carbonyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (44) To a solution of carboxylic acid 11 (160 mg, 320 μmol) in dichloromethane (1 mL) and DMF (0.5 mL), Boc-MMAE42 (100 mg, 120 μmol) was added, followed by the addition of DCC (54 mg, 210 μmol) and DMAP (32 mg, 210 μmol) at 0°C. After 1 hour, the reaction mixture was heated to room temperature and stirred overnight. The mixture was purified by silica gel flash chromatography (0-6% MeOH in DCM) to obtain semi-pure compound 43, which was dissolved in 2 mL of ethyl acetate. To this solution, Pd / C (10% by weight, 40 mg) and triethylamine (44 μL, 440 μmol) were added. The flask was then evacuated and filled with hydrogen gas from a balloon in three repeated cycles. The reaction mixture was vigorously stirred at room temperature for 48 hours with the H2 balloon attached. After removing the catalyst by filtration through a Celite pad, the filtrate was concentrated under vacuum and purified by silica gel flash chromatography (0-5% MeOH in DCM) to obtain compound 44 (120 mg, 95 μmol, 79% yield) as a yellow solid. LRMS (ESI): m / z 1269.7 [M+H] + , C 64 H 96 N6O 20 The calculated value for m / z is 1269.7.

[0381] 2S,3R,4S,5S,6S)-2-(5-((S)-2-((S)-2-((((9H-Fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutanamide)propanamide)-2-(((1S,2R)-2-((2R,3R)-3-((S)-1-((6S,9S,12S,13R)-12-((S)-sec-butyl)-6,9-diisopropyl-13-methyl Preparation of Toxy-2,2,5,11-tetramethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadecane-15-oil)pyrrolidine-2-yl)-3-methoxy-2-methylpropanamide)-1-phenylpropoxy)carbonyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (46) A mixture of amine 44 (120 mg, 94 μmol) and Fmoc-Ala-Cl32 (63 mg, 180 μmol) in anhydrous DMF (1 mL) was mixed with DIPEA (32 μL, 180 μmol) at room temperature. After 30 minutes, the reaction mixture was briefly purified by silica gel flash chromatography (0-5% MeOH in DCM). Next, piperidine (80 μL) was added to a solution of compound 45 in CH3CN (5 mL). After 1 hour, the reaction mixture was concentrated and reconstituted in CH3CN (1 mL). Fmoc-Val-OPfp34 (94 mg, 180 μmol) and DIPEA (22 μL, 120 μmol) were added to this solution. After 20 minutes, the reaction mixture was purified by silica gel chromatography (0-5% v / v MeOH in DCM) to obtain compound 46 (120 mg, 72 μmol, yield 77%) as a yellow solid. LRMS(ESI): m / z 1661.9[M+H] + , C 87 H 120 N8O 24 The calculated value for m / z is 1661.8.

[0382] Preparation of (2S,3S,4S,5R,6S)-6-(5-((S)-2-((S)-2-amino-3-methylbutanamide)propanamide)-2-(((1S,2R)-2-((2R,3R)-3-((S)-1-((6S,9S,12S,13R)-12-((S)-sec-butyl)-6,9-diisopropyl-13-methoxy-2,2,5,11-tetramethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadecane-15-oil)pyrrolidine-2-yl)-3-methoxy-2-methylpropanamide)-1-phenylproxy)carbonyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (47) To a solution of compound 46 (15 mg, 9 μmol) in a 4:1 (v / v) mixture of MeOH / H2O (0.3 mL), ScOTf3 (100 mg, 0.20 mmol) was added at room temperature with stirring. After 2 days, the reaction mixture was concentrated under vacuum and reconstituted in a DMF-piperidine mixture (10:1 v / v, 1.1 mL), and stirred at room temperature for 1 hour. The reaction mixture was then purified by reverse-phase chromatography on a C18 column (H2O / CH3CN containing 0.05% TFA, 90:10~20:80 v / v). The fraction containing the desired compound was pooled and concentrated under vacuum to obtain compound 47 (1.5 mg, yield 13%). LRMS (ESI): m / z 1299.7 [M+H] + , C 65 H 102 N8O 19 The calculated value for m / z is 1299.7.

[0383] (2S,3S,4S,5R,6S)-6-(2-(((1S,2R)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropanamide)-1-phenoxypropoxy)carbonyl)-5 Preparation of -((2S,5S,18R)-22-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-5-isopropyl-2-methyl-4,7,17,20-tetraoxo-18-(sulfomethyl)-10,13-dioxa-3,6,16,19-tetrazadocosaneamide)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (49) A mixture of carboxylic acid 48 (1 mg, 1.3 μmol) and HATU (0.4 mg, 1.1 μmol) in DMF (0.5 mL) was mixed with DIPEA (1.0 μL, 6.1 μmol). The reaction mixture was stirred at room temperature for 30 minutes, and then amine 47 (1.5 mg, 1.2 μmol) was added to the mixture. After 1 hour, the reaction mixture was concentrated and then reconstituted in formic acid (1 mL). After 30 minutes, the solvent was removed under vacuum. The residue was dissolved in DMF (1 mL) and treated with piperidine (50 μL) at room temperature. After stirring at room temperature for 15 minutes, the reaction mixture was directly purified by reverse-phase HPLC using a C18 column (H2O / CH3CN containing 0.05% TFA, 90:10~35:65 v / v). The fraction containing the desired compound was pooled and lyophilized to obtain compound 49 (0.7 mg, 35% yield). LRMS(ESI): m / z 1753.9[M+H] + , C 84 H 129 N 13 O 25 The calculated value for S is m / z 1753.9.

[0384] [ka] (2S,3S,4S,5R,6S)-6-(5-(1-(9H-Fluorene-9-yl)-3-Oxo-2,7,10,13,16,19,22,25,28-Nonaoxa-4-Azahentricontane-31-amide)-2-(((1S,2R)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-Dimethicone Preparation of 2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamide)-1-phenylpropoxy)carbonyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (50) Compound 44 (32 mg, 25 μmol) and a mixture of carboxylic acid 37 (52 mg, 77 μmol) and MeOH (50 μL) in DCM (0.5 mL) were mixed with EEDQ (25 mg, 100 μmol) at room temperature. The resulting mixture was stirred overnight, concentrated under vacuum, and purified by silica gel chromatography (0-10% MeOH / DCM). The resulting intermediate was dissolved in 300 μL of MeOH and 75 μL of water. This solution was treated with scandium(III) triflate (310 mg), stirred at room temperature for 2 days, and then directly purified by reverse-phase chromatography (C18, 0-80% acetonitrile-water, 0.05% TFA) to obtain 7 mg of compound 50 (4 μmol, yield 16%) as a colorless solid. LRMS (ESI): m / z 1674.9 [M+H] + , C 86 H 127 N7O 26 The calculated value for m / z is 1674.9.

[0385] Preparation of (2S,3S,4S,5R,6S)-6-(5-(1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide)-2-(((1S,2R)-2-((2R,3R)-3-((S)-1-(6S,9S,12S,13R)-12-((S)-sec-butyl)-6,9-diisopropyl-13-methoxy-2,2,5,11-tetramethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadecane-15-oil)pyrrolidine-2-yl)-3-methoxy-2-methylpropanamide)-1-phenylpropoxy)carbonyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (51) A solution of compound 50 (7 mg, 4 μmol) in 1.5 mL of anhydrous acetonitrile was treated at room temperature with Boc2O (10 mg, 40 μmol) and DIPEA (7.3 μL, 40 μmol). The reaction mixture was stirred for 4 hours and then directly treated with piperidine (100 μL). After 30 minutes, the reaction mixture was purified by reverse-phase chromatography (C18, 0-70% acetonitrile-water / 0.05% TFA) to obtain 3.6 mg of compound 51 (2 μmol, 50% yield) as a colorless solid. LRMS (ESI): m / z 1553.8 [M+H] + , C 76 H 125 N7O 26 The calculated value for m / z is 1553.9.

[0386] (2S,3S,4S,5R,6S)-6-(2-(((1S,2R)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropanamide)-1-phenylpropoxy)carbonyl)-5-( Preparation of (R)-44-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-29,39,42-trioxo-40-(sulfomethyl)-4,7,10,13,16,19,22,25,32,35-decaxa-28,38,41-triazatetratetracontanamid)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (52) A solution of carboxylic acid 48 (3.6 mg, 4.5 μmol) in 0.3 mL of anhydrous DMF was treated with HATU (1.5 mg, 4 μmol) and DIPEA (3 μL) at room temperature. The resulting mixture was stirred for 30 minutes and then combined with compound 51 (3.5 mg, 2 μmol). After stirring at room temperature for 1 hour, the reaction mixture was concentrated under vacuum and reconstituted in 1 mL of formic acid. After 1 hour at room temperature, the formic acid was removed under vacuum, the residue was dissolved in 1 mL of DMF, and treated with piperidine (100 μL) at room temperature. After 30 minutes, the reaction mixture was purified by reverse-phase preparative HPLC (0-70% acetonitrile-water / 0.05% TFA). The pure fractions were combined and lyophilized to obtain 6 mg of compound 52 (1.8 μmol, 90% yield) as a white solid. LRMS (ESI): m / z 1004.6 [M+2H] 2+ , C 94 H 151 N 13 O 32 The calculated value for m / z is 1004.5.

[0387] Example 2 Preparation of Conjugates Cytotoxin constructs 21, 49, and 52 containing double-cleaved ester linkers were conjugated to a set of aldehyde-tagged monoclonal antibodies using HIPS ligation (Figure 5). The analytical characterization of the resulting conjugates is shown in Figures 6-21.

[0388] Example 3 Bioconjugate, purification, and HPLC analysis Aldehyde-tagged antibody (15 mg / mL) was conjugated to a linker-payload (8–28 mol equivalents of drug:antibody) in sodium citrate / NaCl buffer (pH 5.3–5.5) containing 0.85% DMA at 37°C for 72 hours. In some cases, 3% sucrose, 0.05% Tween-20, and / or up to 10% vol / vol of DMA were added to improve linker-payload solubility. After conjugation, the free drug was removed by diluting it multiple times in the conjugation buffer and concentrating it using an Amicon 0.5 mL 30 kD MWCO centrifuge filter (Millipore Sigma #UFC5030BK) or a Zeba desalting column (Fisher #PI87766). ADC was performed by analytical HIC or PLRP to determine the DAR of the final product. HIC columns (Tosoh #14947) were tested with mobile phase A: 1.5 M ammonium sulfate, 25 mM sodium phosphate (pH 7.0), and mobile phase B: 25% isopropanol, 18.75 mM sodium phosphate (pH 7.0). PLRP columns (Agilent #PL1912-1802) were tested with mobile phase A: 0.1% trifluoroacetic acid in H2O, and mobile phase B: 0.1% trifluoroacetic acid in CH3CN, with the columns heated to 80°C. To measure aggregation, samples were analyzed using analytical size exclusion chromatography (SEC, Tosoh #08541) with mobile phases of 300 mM NaCl, 25 mM sodium phosphate (pH 6.8), and 5% isopropanol.

[0389] Maleimide conjugation of untagged (wild-type) antibodies The antibody (5 mg / mL) was reduced in PBS (pH 8.0) and 1 mM DTPA at 37°C for 90 minutes using 2.5–4 mol equivalents of TCEP. After removing the TCEP, the protein was replaced with PBS (pH 7.4) and 1 mM DTPA. The reduced antibody (3 mg / mL) was conjugated with 10 mol equivalents of CL2A-SN-38 on ice for 60 minutes. After removing the free drug, the final ADC was replaced with PBS (pH 7.4).

[0390] In vitro cytotoxicity assay -On day 1, the cell line was plated into a 96-well plate (Corning #3603) at a density of 4 × 10³ cells / well in 100 μL of growth medium. On day 0, serial dilutions of the test sample were performed in 6 × final concentration growth medium, and 20 μL was added to the cells. After incubation at 5% CO2 and 37°C for 5 days, viability was measured using Promega CellTiter Glo® as recommended by the manufacturer. GI50 curves were calculated using GraphPad Prism normalized to payload concentration. Graphs of the cytotoxicity assay (viability (%) relative to drug concentration (nM)) are shown in Figures 22-31.

[0391] Although the present invention is described with reference to its specific embodiments, various modifications may be made, and it should be understood by those skilled in the art that equivalents can be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt specific circumstances, materials, composition of substances, processes, or one or more process steps to the spirit and scope of the invention. All such modifications are intended to be within the scope of the claims appended herein.

Claims

1. The conjugate of equation (I), 【Chemistry 1】 During the ceremony, W １ However, it is a drug, W ２ However, it is a polypeptide, A is an amino acid residue, and k is 0 or an integer from 1 to 5. L is the linker, G is a conjugated moiety containing hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl. X １ but, 【Chemistry 2】 And, X ２ However, it is -NH- or -C(O)-, R ３ but, 【Transformation 3】 A conjugate, chosen from among them.

2. The aforementioned conjugate is the conjugate of equation (II), 【Chemistry 4】 In the formula, R ４ The conjugate according to claim 1, wherein the side chain is an amino acid.

3. The aforementioned conjugate, 【Transformation 5】 A conjugate according to claim 2, selected from the following.

4. The aforementioned conjugate is the conjugate of equation (III), 【Transformation 6】 In the formula, R ４ The conjugate according to claim 1, wherein the side chain is an amino acid.

5. The aforementioned conjugate, 【Transformation 7】 A conjugate according to claim 4, selected from the following.

6. The conjugate according to any one of claims 2 to 5, wherein k is 2.

7. L is, -(T １ -V １ ) ａ -(T ２ -V ２ ) ｂ -(T ３ -V ３ ) ｃ -(T ４ -V ４ ) ｄ -(T ５ -V ５ ) ｅ -(T ６ -V ６ ) ｆ - and includes, where a, b, c, d, e, and f are each independently 0 or 1. T １ , T ２ , T ３ , T ４ , T ５ , and T ６ However, each is independent, and covalently bonded, (C １ ~C １２ ) alkyl, substituted (C １ ~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), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), acetal, hydrazine, disulfide, and ester, wherein EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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. V １ , V ２ , V ３ , V ４ , V ５ , and V ６ However, each is independent of the covalent bond, -CO-, -NR １５ -, -NR １５ (CH ２ ) ｑ -, -NR １５ (C ６ H ４ )-,-CONR １５ -, -NR １５ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ２ -, -SO ２ NR １５ -, -NR １５ SO ２ Selected from the group consisting of - and -P(O)OH-, where each q is an integer from 1 to 6, Each R １３ However, they are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. Each R １５ The conjugate according to any one of claims 1 to 6, wherein the conjugate 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.

8. T １ However, (C １ ~C １２ ) is alkyl, V １ However, it is -CO-, T ２ However, it is an amino acid analog, V ２ However, it is -NH-, T ３ However, (PEG) ｎ V ３ However, it is -CO-, d to f are each 0, or T １ However, (C １ ~C １２ ) is alkyl, V １ However, it is -CO-, T ２ However, it is an amino acid analog, V ２ However, it is -NH-, T ３ However, (PEG) ｎ V ３ However, it is -CONH-, T ４ However, (PEG) ｎ V ４ However, it is -CO-, The conjugate according to claim 7, wherein e and f are each 0.

9. W2-G is as follows: 【Transformation 8】 During the ceremony, Z is CR １０ or N, R ７ However, selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, R ８ and R ９ However, each is 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 ８ and R ９ However, they can be optionally linked in a ring to form a 5-membered or 6-membered heterocycline. Each R １０ is 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, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, the conjugate according to any one of claims 1 to 8.

10. A compound of formula (IV), 【Chemistry 9】 During the ceremony, W １ However, it is a drug, A is an amino acid residue, and k is 0 or an integer from 1 to 5. L is the linker, G is a conjugated moiety containing hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl. X １ but, 【Chemistry 10】 And, X ２ However, it is -NH- or -C(O)-, R ３ but, 【change】 A compound selected from among them.

11. The aforementioned compound is a compound of formula (V), 【Chemistry 11】 In the formula, R ４ The compound according to claim 10, wherein the side chain is an amino acid.

12. The aforementioned compound, 【Chemistry 12】 A compound according to claim 11, selected from the above.

13. The aforementioned compound is a compound of formula (VI), 【Chemistry 13】 In the formula, R ４ The compound according to claim 10, wherein the side chain is an amino acid.

14. The aforementioned compound, 【Chemistry 14】 A compound according to claim 13, selected from the above.

15. The compound according to any one of claims 10 to 14, wherein k is 2.

16. L is, -(T １ -V １ ) ａ -(T ２ -V ２ ) ｂ -(T ３ -V ３ ) ｃ -(T ４ -V ４ ) ｄ -(T ５ -V ５ ) ｅ -(T ６ -V<000--0113>) ｆ including - During the ceremony, a, b, c, d, e, and f are each independently 0 or 1. T １ , T ２ , T ３ , T ４ , T ５ , and T ６ However, each is independent, and covalently bonded, (C １ ~C １２ ) alkyl, substituted (C １ ~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), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), acetal, hydrazine, disulfide, and ester, wherein EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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. V １ , V ２ , V ３ , V ４ , V ５ , and V ６ However, each is independent of the covalent bond, -CO-, -NR １５ -, -NR １５ (CH ２ ) ｑ -, -NR １５ (C ６ H ４ )-,-CONR １５ -, -NR １５ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ２ -, -SO ２ NR １５ -, -NR １５ SO ２ Selected from the group consisting of - and -P(O)OH-, where each q is an integer from 1 to 6, Each R １３ However, they are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. Each R １５ The compound according to any one of claims 10 to 15, wherein the compound 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.

17. T １ However, (C １ -C １２ ) is alkyl, V １ However, it is -CO-, T ２ However, it is an amino acid analog, V ２ However, it is -NH-, T ３ However, (PEG) ｎ V ３ However, it is -CO-, d to f are each 0, or T １ However, (C １ -C １２ ) is alkyl, V １ However, it is -CO-, T ２ However, it is an amino acid analog, V ２ However, it is -NH-, T ３ However, (PEG) ｎ V ３ However, it is -CONH-, T ４ However, (PEG) ｎ V ４ However, it is -CO-, The compound according to claim 16, wherein e and f are each 0.

18. G is as follows: 【Chemistry 15】 During the ceremony, Z is CR １０ or N, R ８ and R ９ However, each is 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 ８ and R ９ However, they can be optionally linked in a ring to form a 5-membered or 6-membered heterocycline. Each R １０ The compound according to any one of claims 10 to 17, which is 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, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

19. A pharmaceutical composition, A conjugate according to any one of claims 1 to 9, A pharmaceutical composition comprising a pharmaceutically acceptable excipient.

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