Beta-glucuronide linker-payloads, protein conjugates thereof, and methods thereof

Abstract

The present invention provides compounds, conjugate products thereof, methods and pharmaceutical compositions for treatment and diagnosis.

Description

Cross-references to related patent applications

[0001] This application claims under 35 U.S.C. §119 and enjoys the benefit of U.S. Provisional Application No. 63 / 355,975, filed on June 27, 2022, the contents of which are hereby incorporated by reference in their entirety. Field of the Invention

[0002] The present invention provides β-glucuronide linker-payload compounds and macromolecular conjugates thereof; pharmaceutical compositions comprising the β-glucuronide linker-payload compounds and / or conjugates; methods for preparing the β-glucuronide linker-payload compounds and / or conjugates; and methods for treating using the β-glucuronide linker-payload compounds, conjugates and compositions. The β-glucuronide linker-payload compounds, conjugates and compositions can all be used, for example, in methods for treating and preventing cell proliferation and cancer, methods for detecting cell proliferation and cancer, and methods for diagnosing cell proliferation and cancer. Background of the Invention

[0003] Biotherapeutics offer a wealth of therapeutic and diagnostic potential for patients worldwide. However, many drugs based on large molecules, such as proteins, peptides, and antibodies, have limitations in their effective use, including limitations in bioavailability, absorption, distribution, metabolism, and excretion (ADME). Some of these limitations affect drug dosing, half-life, side effects, and toxicity. There is still a need to develop strategies to improve the effectiveness of biotherapeutics. Summary of the Invention

[0004] The present invention provides compounds of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), (IIIB), (IV), (IVA), (IVB) and their sub-general formulae, compositions comprising such compounds, methods of preparing such compounds, and methods of using such compounds, conjugates and compositions in treatment and diagnosis. The compounds of the present invention, including compounds of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), (IIIB), (IV), (IVA), (IVB) and their sub-general formulae and embodiments, can be used to regulate the bioavailability and ADME of macromolecular compounds. In certain embodiments, the compounds can be used to prepare prodrug conjugates of macromolecular compounds for use in vivo or elsewhere. In certain embodiments, the compounds and conjugates have the function of being easily enzymatically cleaved to release the payload compound for use in vivo or elsewhere. The compounds can be altered to adjust the physiochemical properties and plasma stability of the conjugates. This provides a platform for regulating the bioavailability and ADME of macromolecules in vivo.

[0005] In some embodiments, the present invention provides a compound represented by formula (I): or a pharmaceutically acceptable salt thereof, wherein L 1 Yes-C 1-6 Alkylene-; Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -[X 1 ] p –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -[X 1 ] p –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -[X 1 ] p -, wherein at least one alkylene, alkenylene or alkynylene group in Y is replaced by one or more selected from R 50 Substituents are substituted, and wherein the alkylene, alkenylene or alkynylene in Y is optionally replaced by one or more selected from R 51 Substituents are substituted; R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, –C 2-6 Alkenylene-X 2 -[C 2-6 alkenylene] m -POLY, or –C 2-6 Alkynylidene-X 2 -[C 2-6 Alkynylidene] m -POLY, where R 50 Each alkylene, alkenylene or alkynylene group is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; R 51 independently selected from halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; X 1 and X 2 are independently selected from -N(R 10 )–, –C(O)–, and –N(R 10 )C(O)–; R 10 At each occurrence, independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; POLY is a water-soluble polymer; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0 and 1; p is an integer selected from 0 and 1; Su is the hexose form of a monosaccharide; D is a drug group moiety; and RL is the reactive linker group residue.

[0006] In some embodiments, the present invention provides a compound represented by formula (I): or a pharmaceutically acceptable salt thereof, wherein L 1 Yes-C 1-6 Alkylene-; Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -X 1 –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -X 1 -, wherein at least one alkylene, alkenylene or alkynylene group in Y is replaced by one or more selected from R 50 Substituents are substituted; R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, –C 2-6 Alkenylene-X 2 -[C 2-6 alkenylene] m -POLY, or –C 2-6 Alkynylidene-X 2 -[C 2-6 Alkynylidene] m -POLY, where R 50Each alkylene, alkenylene or alkynylene group is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; X 1 and X 2 are independently selected from -C(O)-, and -N(R 10 )C(O)–; R 10 At each occurrence, independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; POLY is a water-soluble polymer; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0 and 1; Su is the hexose form of a monosaccharide; D is a drug group moiety; and RL is the active linker group residue.

[0007] In some aspects, the present invention provides a compound having a structure shown in formula (III): or a pharmaceutically acceptable salt thereof, wherein L 1 Yes-C 1-6 Alkylene-; Z is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -X 1 –, or –X 1 -C2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -X 1 -, wherein the alkylene, alkenylene or alkynylene in Z is optionally substituted by one or more substituents selected from the following: halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; X 1 and X 2 are independently selected from -C(O)-, and -N(R 10 )C(O)–; R 10 At each occurrence, independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; POLY is a water-soluble polymer; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0 and 1; Su is the hexose form of a monosaccharide; CYTO is a cytotoxic payload; and RL is the active linker group residue.

[0008] In some embodiments, the present invention provides a compound selected from the group consisting of: or a salt thereof.

[0009] In some embodiments, the present invention provides a compound selected from the group consisting of: or a salt thereof.

[0010] In some embodiments, the present invention provides a conjugate comprising a residue of a compound such as Formula (I), (IA), (IB), (III), (IIIA), (IIIB) or a pharmaceutically acceptable salt thereof, linked to a second compound.

[0011] In some embodiments, the present invention provides a conjugate having the following chemical structure: and a pharmaceutically acceptable salt of any one thereof, in is the residue of the second compound.

[0012] In some embodiments, the present invention provides a conjugate having the following chemical structure: and a pharmaceutically acceptable salt of any one thereof, in is the residue of the second compound.

[0013] In certain embodiments, the present invention provides a pharmaceutical composition comprising a compound of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), (IIIB), (IV), (IVA) and (IVB) and a pharmaceutically acceptable excipient, carrier or diluent. Any suitable pharmaceutical composition may be used.

[0014] In certain embodiments, the compounds shown in formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), (IIIB), (IV), (IVA) and (IVB) or their pharmaceutical compositions can be used for treatment. In certain embodiments, the compounds shown in formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), (IIIB), (IV), (IVA) and (IVB) or their pharmaceutical compositions can be used for the treatment of cancer. In certain embodiments, the compounds shown in formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), (IIIB), (IV), (IVA) and (IVB) or their pharmaceutical compositions can be used for medicine.

[0015] In certain embodiments, the present invention provides a method of inhibiting tubulin polymerization in a subject in need thereof, comprising administering to the subject an effective amount of a compound shown in Formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), (IIIB), (IV), (IVA) and (IVB) or a pharmaceutical composition thereof. In certain embodiments, the present invention provides a method of reducing cell proliferation in a subject in need thereof, comprising administering to the subject an effective amount of a compound shown in Formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), (IIIB), (IV), (IVA) and (IVB) or a pharmaceutical composition thereof. In certain embodiments, the present invention provides a method of treating cell proliferation or cancer in a subject in need thereof, comprising administering to the subject an effective amount of a compound shown in Formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), (IIIB), (IV), (IVA) and (IVB) or a pharmaceutical composition thereof.

[0016] In certain embodiments, the present invention provides a method for preparing a conjugate, comprising contacting a compound of formula (I), (IA), (IB), (III), (IIIA) or (IIIB) with a second compound under conditions suitable for coupling the compound of formula (I), (IA), (IB), (III), (IIIA) or (IIIB) with the second compound; wherein the second compound comprises an alkyne, a cyclooctyne, a strained olefin, a tetrazine, a methylcyclopropene, a thiol, a maleimide, a carbonyl, an amine, an oxyamine, or an azide. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 MALDI-ToF data of drug to antibody ratio (DAR) after conjugation to the antibody are shown.

[0018] FIG. 2A to FIG. 2F The cell killing activity of certain β-Glu linker-payloads coupled to ADCs with DAR=8, as well as certain free payloads, is shown.

[0019] FIG. 3A to FIG. 3D Shown is the susceptibility of the β-glucuronide linker-payload to enzymatic cleavage following treatment with β-glucuronidase. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention describes compounds of formula (I), (IA), (IB), (III), (IIIA) and (IIIB), and conjugates of formula (II), (IIA), (IIB), (IV), (IVA) and (IVB), which can be used to regulate the bioavailability and ADME of such macromolecular conjugate compounds. In some embodiments, the compounds described herein can be used to prepare macromolecular conjugates for in vivo use, such as prodrugs. definition

[0021] Unless otherwise defined, all technical terms, symbol descriptions, and other scientific terms used in the present invention are intended to have the meanings commonly understood by those having ordinary knowledge in the art to which the present invention belongs. In many cases, terms with commonly understood meanings are defined in the present invention for clarity and / or ease of reference. The techniques and procedures described or referred to in the present invention are generally widely understood by those having ordinary knowledge in the art and are frequently employed using conventional methods, such as, for example, the widely used Green & Sambrook, Molecular Cloning: A Laboratory Manual 4 th Molecular cloning methods described in ed. (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons. Where appropriate, methods involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and conditions / parameters unless otherwise specified.

[0022] As used herein, the singular forms "a", "an", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0023] The term "about" means and encompasses the indicated value and the range above and below this value. In certain embodiments, the term "about" means a specified value ± 10%, ± 5%, or ± 1%. In certain embodiments, the term "about" means a specified value ± one standard deviation of this value. In certain embodiments, for example, for a logarithmic scale (e.g., pH), the term "about" means a specified value ± 0.3, ± 0.2, or ± 0.1.

[0024] When referring to the compounds provided by the present invention, unless otherwise stated, the following terms have the following meanings. Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those of ordinary skill in the art are generally understood. If a term of the present invention has multiple definitions, unless otherwise stated, the definitions in this section shall prevail.

[0025] "Alkoxy" and "alkoxy group" refer to the group -OR", where R" is alkyl or cycloalkyl. In certain embodiments, alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

[0026] The term "alkoxyamine" as used herein refers to the group -alkylene-O-NH2, wherein alkylene is as defined herein. In some embodiments, the alkoxyamine group can react with an aldehyde to form an oxime residue. Examples of alkoxyamine groups include -CH2CH2-O-NH2, -CH2-O-NH2, and -O-NH2.

[0027] The term "alkyl" as used herein, unless otherwise specified, refers to a saturated straight or branched chain hydrocarbon. In certain embodiments, the alkyl group is a primary, secondary or tertiary hydrocarbon. In certain embodiments, the alkyl group comprises 1-10 carbon atoms (i.e., C1-C 10 In certain embodiments, the alkyl group is a lower alkyl group, such as C 1-6 Alkyl etc. In certain embodiments, the alkyl group is selected from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl and 2,3-dimethylbutyl. In certain embodiments, "substituted alkyl" refers to alkyl being substituted by, for example, 1, 2 or 3 groups independently selected from halogen (e.g., fluorine (F), chlorine (Cl), bromine (Br) or iodine (I)), alkyl, -CN, -NO2, amide, -C (O) -, -C (S) -, ester, carbamate, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, dialkylamino, haloalkyl, hydroxyl, amino, alkylamino and alkoxy groups. In some embodiments, alkyl is unsubstituted.

[0028] As used herein, the term "alkylene", unless otherwise indicated, refers to a divalent alkyl group, the alkyl being as defined herein. "Substituted alkylene" refers to an alkylene group substituted as described herein for alkyl. In some embodiments, the alkylene is unsubstituted.

[0029] "Alkenyl" refers to an ethylenically unsaturated hydrocarbon radical, in certain embodiments, having up to about 11 carbon atoms or 2 to 6 carbon atoms (e.g., "lower alkenyl"), which may be straight chain or branched, and having at least 1 or 1-2 sites of ethylenic unsaturation. "Substituted alkenyl" refers to an alkenyl group substituted as described herein for alkyl.

[0030] "Alkenylene" refers to a divalent alkenyl group as defined herein. Lower alkenylene is, for example, C2-C6-alkenylene.

[0031] "Alkynyl" refers to an acetylenically unsaturated hydrocarbon radical, in certain embodiments, having up to about 11 carbon atoms or 2-6 carbon atoms (e.g., "lower alkynyl"), which may be straight or branched, and having at least 1 or 1-2 sites of acetylenic unsaturation. Non-limiting examples of alkynyl groups include ethynyl (-C≡CH), propargyl (-CH2C≡CH), and the like. "Substituted alkynyl" refers to an alkynyl group substituted as described herein for alkyl.

[0032] "Alkyne" refers to a divalent alkynyl group as defined herein. Lower alkynyl is, for example, C2-C6-alkynyl.

[0033] "Amino" refers to -NH2.

[0034] The term "alkylamino" as used herein, unless otherwise indicated, refers to the group -NHR", wherein R" is, for example, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, C 1-10 In certain embodiments, alkylamino is C 1-6 Alkylamino.

[0035] The term "dialkylamino" as used herein, unless otherwise specified, refers to the group -NR"R", wherein each R" is independently C 1-10 In certain embodiments, dialkylamino is, for example, di-C 1-6 Alkylamino, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, C 1-10 Haloalkyl, etc.

[0036] The term "aryl" used in the present invention refers to phenyl, biphenyl or naphthyl, unless otherwise indicated. The term includes substituted and unsubstituted radicals. The aryl group can be substituted by any described radical, including but not limited to one or more radicals selected from the group consisting of the following (for example, in some embodiments, 1, 2 or 3 radicals): halogen (fluorine (F), chlorine (Cl), bromine (Br) or iodine (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate (phosphate) and phosphonate (phosphonate), wherein each radical is independently unprotected, or protected as required, as known to those skilled in the art (see, for example, Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991); and wherein the aryl in the arylamino and aryloxy substituents is not further substituted.

[0037] The term "arylamino", as used herein, and unless otherwise indicated, refers to a -NR'R" group, wherein R' is H or C1-C6-alkyl; and R" is aryl, as defined herein.

[0038] The term "arylene" as used herein, unless otherwise indicated, refers to a divalent aryl group, wherein aryl is as defined herein.

[0039] The term "aryloxy" as used herein, unless otherwise indicated, refers to a -OR group, wherein R is an aryl group, as defined herein.

[0040] "Alkarylene" refers to an arylene group, as defined herein, wherein the aryl ring is substituted with 1 or 2 alkyl groups. "Substituted alkarylene" refers to an alkylarylene group, as defined herein, wherein the arylene group is further substituted as defined herein for aryl.

[0041] "Aralkylene" refers to a -CH2-arylene-, -arylene-CH2-, or -CH2-arylene-CH2- group, wherein arylene is as defined herein. "Substituted aralkylene" refers to arylenealkyl, as defined herein, wherein the aralkylene group is substituted as defined herein for aryl.

[0042] "Carboxyl" or "carboxyl group" refers to -C(O)OH or -COOH.

[0043] The term "cycloalkyl" as used herein, unless otherwise indicated, refers to a saturated cyclic hydrocarbon. In certain embodiments, the cycloalkyl group may be a saturated, and / or bridged, and / or non-bridged, and / or fused bicyclic group. In certain embodiments, the cycloalkyl group contains 3-10 carbon atoms (i.e., C3-C 10 In some embodiments, the cycloalkyl group has 3 to 15 carbon atoms (C 3-15 ), 3-10 carbon atoms (C 3-10 ), 3-7 carbon atoms (C 3-7 ), or 3-6 carbon atoms (C3-C6) (i.e., "lower cycloalkyl"). In certain embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, cycloheptyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, decalinyl, or adamantyl.

[0044] The term "carbocycle" used in the present invention, unless otherwise indicated, refers to a saturated, unsaturated or aromatic ring in which each atom on the ring is a C atom. In certain embodiments, the carbocyclic group can be a saturated, and / or bridged, and / or non-bridged, and / or fused bicyclic group, and / or a spirocyclic bicyclic group. In some embodiments, the carbocyclic ring comprises a monocyclic ring composed of 3 to 10 atoms, a bicyclic ring composed of 6 to 12 atoms, and a bridged ring composed of 6 to 12 atoms. In some embodiments, each ring of the bicyclic carbocyclic ring can be selected from saturated rings, unsaturated rings, and aromatic rings. In some embodiments, an aromatic ring (such as phenyl) can be fused with a saturated or unsaturated ring (for example, cyclohexane, cyclopentane, or cyclohexene). Bicyclic carbocyclic rings include any combination of saturated bicyclic, unsaturated bicyclic, and aromatic bicyclic, as long as valence allows. Bicyclic carbocycles include any combination of ring sizes, such as 4-5 fused ring systems, 5-5 fused ring systems, 5-6 fused ring systems, 6-6 fused ring systems, 5-7 fused ring systems, 6-7 fused ring systems, 5-8 fused ring systems, and 6-8 fused ring systems. In certain embodiments, the carbocyclic group contains 3-10 carbon atoms (i.e., C3-C 10 In certain embodiments, the carbocyclic group contains 3-12 carbon atoms (i.e., C3-C 12 In some embodiments, the carbocyclic ring has 3-15 carbon atoms (C 3-15 ), 3-12 carbon atoms (C 3-12 ), 3-10 carbon atoms (C 3-10 ), 3-7 carbon atoms (C 3-7), or 3-6 carbon atoms (C3-C6). In certain embodiments, the carbocyclic group is cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cyclohexylmethyl, cycloheptyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, decalinyl, phenyl, indanyl, naphthyl or adamantyl.

[0045] The term "cycloalkylene" as used herein refers to a divalent cycloalkyl group, wherein the cycloalkyl group is as defined herein. In certain embodiments, the cycloalkylene group is cyclopropylene. Cyclobutylene Cyclopentylene Cyclohexylene Cycloheptylene etc. Lower cycloalkylene refers to C3-C6-cycloalkylene.

[0046] The term "cycloalkylalkyl" as used herein, unless otherwise indicated, refers to an alkyl group as defined herein substituted with 1 or 2 cycloalkyl groups as defined herein.

[0047] The term "ester" as used herein refers to -C(O)OR or -COOR, wherein R is an alkyl group as defined herein.

[0048] The term "fluorene" used in the present invention refers to Any one or more C atoms carrying one or more H atoms may be replaced by the chemical functional groups described in the present invention.

[0049] The term "haloalkyl" refers to an alkyl group, as defined herein, substituted with one or more independently selected halogen atoms (eg, in some embodiments 1, 2, 3, 4, or 5).

[0050] The term "heteroalkyl" refers to an alkyl group defined in the present invention, wherein one or more carbon atoms are replaced by heteroatoms. "Heteroalkenyl" used in the present invention refers to an alkenyl group defined in the present invention, wherein one or more carbon atoms are replaced by heteroatoms. "Heteroalkynyl" used in the present invention refers to an alkynyl group defined in the present invention, wherein one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen (N), oxygen (O) and sulfur (S) atoms. Heteroalkyl, heteroalkenyl and heteroalkynyl groups may all be optionally substituted. Examples of heteroalkyl group moieties include, but are not limited to, aminoalkyl, sulfonylalkyl and sulfinylalkyl. Examples of heteroalkyl group moieties also include, but are not limited to, methylamino, methylsulfonyl and methylsulfinyl. "Substituted heteroalkyl" refers to heteroalkyl substituted by 1, 2 or 3 groups independently selected from halogen (e.g., fluorine (F), chlorine (Cl), bromine (Br) or iodine (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino and alkoxy groups. In some embodiments, the heteroalkyl group may contain 1, 2, 3 or 4 heteroatoms. Those skilled in the art will recognize that heteroalkyl groups consisting of 4 atoms may typically contain 1 or 2 heteroatoms, heteroalkyl groups consisting of 5 or 6 atoms may typically contain 1, 2 or 3 heteroatoms, and heteroalkyl groups consisting of 7 to 10 atoms may typically contain 1, 2, 3 or 4 heteroatoms.

[0051] As used herein, the term "heteroalkylene" refers to a divalent heteroalkyl group, which is as defined herein. "Substituted heteroalkylene" refers to a divalent heteroalkyl group, as defined herein, which is substituted as described herein for heteroalkyl groups.

[0052] The term "heterocycloalkyl" refers to a monovalent monocyclic or polycyclic non-aromatic ring system, wherein one or more ring atoms are independently selected from oxygen (O), sulfur (S) and nitrogen (N) heteroatoms (e.g., wherein the N or S atom may be optionally oxidized, and the N atom may be optionally quaternized), and the remaining ring atoms of the non-aromatic ring are all carbon atoms. In certain embodiments, heterocycloalkyl is a monovalent monocyclic or polycyclic fully saturated ring system. In certain embodiments, the heterocycloalkyl group has 3 to 20, 3 to 15, 3 to 10, 3 to 8, 4 to 7, 4 to 11, or 5 to 6 ring atoms. The heterocycloalkyl may be connected to a parent core structure at any heteroatom or carbon atom thereof, thereby forming a stable compound. In certain embodiments, the heterocycloalkyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include a fused or bridged ring system, and wherein the N or S atom may be optionally oxidized, and / or the N atom may be optionally quaternized. In some embodiments, heterocycloalkyl groups include, but are not limited to, 2,5-diazabicyclo[2.2.2]octyl, decahydroisoquinolyl, dihydrobenzisoxazinyl, dihydrofuranyl, dihydroisoindolyl, dihydropyranyl, dihydropyrazolyl, dihydropyrazinyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl, 1,4-dithianyl, furanonyl, imidazolidinyl, imidazolinyl, indolyl, , isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinone, oxazolidinyl, oxiranyl, piperazinyl, piperidinyl, 4-piperidone, pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydrothienyl, thiamorpholinyl, thiazolidinyl, tetrahydroquinolinyl, and 1,3,5-trithiacyclohexane. In certain embodiments, heterocycloalkyl may also be optionally substituted as described herein. In certain embodiments, heterocycloalkyl is substituted by 1, 2 or 3 groups independently selected from halogen (e.g., fluorine (F), chlorine (Cl), bromine (Br) or iodine (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy. In some embodiments, the heterocycloalkyl group may contain 1, 2, 3 or 4 heteroatoms. Those skilled in the art will recognize that heterocycloalkyl groups consisting of 4 atoms may typically contain 1 or 2 heteroatoms, heterocycloalkyl groups consisting of 5 or 6 atoms may typically contain 1, 2 or 3 heteroatoms, and heterocycloalkyl groups consisting of 7 to 10 atoms may typically contain 1, 2, 3 or 4 heteroatoms.

[0053] The term "heterocycle" refers to a saturated, unsaturated or aromatic ring containing one or more heteroatoms. Exemplary heteroatoms include nitrogen (N), oxygen (O), silicon (Si), phosphorus (P), boron (B) and sulfur (S) atoms, wherein N or S atoms may be optionally oxidized, and N atoms may be optionally quaternized, and the remaining ring atoms of the non-aromatic ring are carbon atoms. Heterocycles include monocycles composed of 3 to 10 atoms, monocycles composed of 3 to 12 atoms, bicycles composed of 6 to 12 atoms, and bridged rings composed of 6 to 12 atoms. In certain embodiments, heterocycles are monocyclic or polycyclic fully saturated ring systems of monovalence. Bicyclic heterocycles include any combination of saturated, unsaturated and aromatic bicyclics, as long as valence allows. In some embodiments, aromatic rings (e.g., pyridyl) may be fused with saturated or unsaturated rings (e.g., cyclohexane, cyclopentane, morpholine, piperidine or cyclohexene). Bicyclic heterocycles include any combination of ring sizes, such as 4-5 fused ring systems, 5-5 fused ring systems, 5-6 fused ring systems, 6-6 fused ring systems, 5-7 fused ring systems, 6-7 fused ring systems, 5-8 fused ring systems, and 6-8 fused ring systems. In certain embodiments, the "heterocycloalkyl" or "heterocycle" group can be unsaturated, and / or bridged, and / or non-bridged, and / or fused bicyclic groups, and / or spirocyclic bicyclic groups. The term "unsaturated heterocycle" refers to a heterocycle with at least one degree of unsaturation, but does not include aromatic heterocycles. Examples of unsaturated heterocycles include dihydropyrroles, dihydrofurans, oxazoline, pyrazoline, and dihydropyridine.

[0054] "Heterocycloalkylene" refers to a divalent heterocycloalkyl group, wherein the heterocycloalkyl group is as defined herein.

[0055] The term "heteroaryl" refers to a monovalent monocyclic aromatic group and / or a polycyclic aromatic group, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S and N in the ring. Each ring of the heteroaryl group may contain 1 or 2 O atoms, 1 or 2 S atoms and / or 1-4 N atoms, provided that the total number of heteroatoms in each ring is 4 or less, and each ring contains at least one carbon atom. In certain embodiments, the heteroaryl group has 5-20, 5-15 or 5-10 ring atoms. The heteroaryl group may be connected to the rest of the molecule by a nitrogen or carbon atom. In some embodiments, the monocyclic heteroaryl group includes, but is not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, triazolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzimidazolyl, benzisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothiophenyl, benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothiophenyl, isoindolyl, isoquinolyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrolopyridinyl, quinolyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidinyl, and thienopyridinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthroline, phenanthridinyl, phenanthroline, phenanthroline, phenanthroline, phenanthroline and xanthenyl. In certain embodiments, heteroaryl can also be optionally substituted as described herein. "Substituted heteroaryl" is a heteroaryl substituted as defined by the present invention for aryl.

[0056] The term "heteroarylene" refers to a divalent heteroaryl radical, wherein heteroaryl is as defined herein. "Substituted heteroarylene" is a heteroarylene that is substituted as defined herein for aryl.

[0057] The term "protecting group" used in the present invention, unless otherwise specified, refers to a group added to an oxygen, nitrogen or phosphorus atom to prevent (protected) oxygen, nitrogen or phosphorus from further reaction or for other purposes. Various oxygen protecting groups and nitrogen protecting groups are known to those skilled in the art of organic synthesis (see, for example, Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Fourth Edition, 2006, which is incorporated herein by reference in its entirety).

[0058] "Pharmaceutically acceptable salt" refers to any salt of a compound provided herein that retains the biological properties of the compound and is non-toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from various organic and inorganic counterions known in the art. Such salts include, but are not limited to: (1) with organic or inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, glutaric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, picric acid , cinnamic acid, mandelic acid, phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphoric acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthyl or (2) when an acidic proton is present in the parent compound, it is (a) replaced by a metal ion, such as an alkali metal ion, an alkaline earth metal ion or an aluminum ion, or an alkali metal or alkaline earth metal hydroxide, such as sodium, potassium, calcium, magnesium, aluminum, lithium, zinc and barium, or ammonia, or (b) coordinated with an organic base, such as an aliphatic, alicyclic or aromatic organic amine, including but not limited to ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, procaine, N-benzylphenethylamine, N-methylglucamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and the like to form a base addition salt.

[0059] By way of example only and not limitation, pharmaceutically acceptable salts further include sodium salts, potassium salts, calcium salts, magnesium salts, ammonium salts, and tetraalkylammonium salts and the like; when the compound contains a basic functional group, it is a salt of a non-toxic organic or inorganic acid, such as a hydrohalide such as hydrochloride and hydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate, tartrate, citrate, benzoate, 3-(4-hydroxybenzoyl) Benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate), ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (benzenesulfonate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylaminosulfonate, quinate, muconate, etc.

[0060] In terms of composition, the term "substantially free of" or "substantially absent" refers to a composition comprising at least 85% by weight or 90% by weight, in certain embodiments, comprising 95%, 98%, 99% by weight or 100% by weight, or in certain embodiments, comprising 95%, 98%, 99% or 100% of the specified enantiomer or diastereomer of the compound. In certain embodiments, in the methods and compounds provided by the invention, the compound is substantially free of one of two enantiomers. In certain embodiments, in the methods and compounds provided by the invention, the compound is substantially free of one of two diastereomers. In certain embodiments, in the methods and compounds provided by the invention, the compound is substantially free of enantiomers (that is, the compound is not a racemic compound or a mixture of 50:50 compounds).

[0061] Similarly, with respect to a composition, the term "isolated" means that the composition contains at least 85%, 90%, 95%, 98%, 99% to 100% by weight of the compound, with the remainder comprising other chemical species, enantiomers, or diastereomers.

[0062] "Solvate" means that the compound or salt thereof provided by the present invention further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0063] "Isotopic composition" refers to the amount of each isotope present for a given atom, and "natural isotopic composition" refers to the naturally occurring isotopic composition or abundance of a given atom. Atoms having their natural isotopic composition may also be referred to as "non-enriched" atoms in the present invention. Unless otherwise indicated, atoms of the compounds described herein are intended to represent any stable isotopes of the atom. For example, unless otherwise indicated, when a position is specifically designated as "hydrogen" (H), the position is understood to be hydrogen having its natural isotopic composition.

[0064] "Isotopic enrichment" refers to the percentage of incorporation of a specific isotope at a given atom in a molecule in place of the natural isotopic abundance of the atom. For example, a 1% deuterium (D) enrichment at a given position means that 1% of the molecules in a given sample contain deuterium at the specified position. Since the natural distribution of deuterium is about 0.0156%, the deuterium enrichment at any position in a compound synthesized using a non-enriched starting material is about 0.0156%. The isotopic enrichment of the compounds provided by the present invention can be measured using conventional analytical methods known to those of ordinary skill in the art, including mass spectrometry and nuclear magnetic resonance spectroscopy.

[0065] "Isotopically enriched" refers to atoms having an isotopic composition other than the natural isotopic composition of said atoms. "Isotopically enriched" may also refer to a compound containing at least one atom having an isotopic composition other than the natural isotopic composition of said atoms.

[0066] As used herein, “alkyl,” “alkylene,” “alkylamino,” “dialkylamino,” “cycloalkyl,” “aryl,” “arylene,” “alkoxy,” “amino,” “carboxy,” “heterocycloalkyl,” “heteroaryl,” “heteroarylene,” “carboxy,” and “amino acid” groups optionally contain deuterium (D) at one or more positions where there is a hydrogen atom (H), and wherein the deuterium composition of said one or more atoms is not the natural isotopic composition.

[0067] Likewise, the "alkyl", "alkylene", "alkylamino", "dialkylamino", "cycloalkyl", "aryl", "arylene", "alkoxy", "amino", "carboxyl", "heterocycloalkyl", "heteroaryl", "heteroarylene", "carboxyl" and "amino acid" groups used herein optionally contain C-13 ( 13 C).

[0068] The term "macromolecule" or "macromolecule moiety" refers to a protein, peptide, antibody, nucleic acid, carbohydrate, or other macromolecule composed of polymerized monomers. It includes peptides of two or more residues or ten or more residues. In certain embodiments, the mass of the macromolecule is at least 1000 Da. In certain embodiments, the macromolecule has at least 1000 atoms. In certain embodiments, the macromolecule can be modified. For example, the protein, peptide or antibody can be modified with one or more carbohydrates and / or small molecule therapeutic compounds.

[0069] The term "immunoglobulin" refers to a class of structurally related proteins that generally comprise two pairs of polypeptide chains: a pair of light (L) chains and a pair of heavy (H) chains. In a "complete immunoglobulin," all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been characterized in detail. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. In brief, each heavy chain generally comprises a heavy chain variable region (V H or VH) and the heavy chain constant region (C H The heavy chain constant region generally contains three domains, abbreviated as C H1 (or CH1), C H2 (or CH2) and C H3 (or CH3). Each light chain generally comprises a light chain variable region (V L or VL) and a light chain constant region. The light chain constant region generally comprises a domain, abbreviated as C L or CL.

[0070] The term "antibody" is used in the broadest sense in the present invention. Antibodies include complete antibodies (e.g., complete immunoglobulins) and antibody fragments (e.g., antigen-binding fragments or antigen-binding fragments of antibodies). Antibodies contain at least one antigen-binding domain. An example of an antigen-binding domain is a V H -V L Dimerized antigen-binding domain.

[0071] The term "amino acid" refers to the 20 common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V), as well as the less common pyrrolysine and selenocysteine. Natural amino acids also include citrulline. Naturally encoded amino acids include post-translational variants of 22 naturally occurring amino acids, such as prenylated amino acids, isoprenylated amino acids, myristoylated amino acids, palmitoylated amino acids, N-linked glycosylated amino acids, O-linked glycosylated amino acids, phosphorylated amino acids, and acylated amino acids. The term "amino acid" also includes non-natural (or unnatural) or synthetic α-, β-, γ-, or δ-amino acids, and includes, but is not limited to, amino acids found in proteins, i.e., glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine. In certain embodiments, the amino acid is in the L configuration. In certain embodiments, the amino acid is in the D configuration. Alternatively, the amino acid may be a derivative of alanyl, valyl, leucyl, isoleucyl, prolyl, phenylalanyl, tryptophanyl, methionyl, glycyl, seryl, threonyl, cysteinyl, tyrosyl, asparaginyl, glutaminyl, aspartyl, glutaryl, lysyl, arginyl, histidyl, β-alanyl, β-valyl, β-leucyl, β-isoleucyl, β-prolyl, β-phenylalanyl, β-tryptophanyl, β-methionyl, β-glycyl, β-seryl, β-threonyl, β-cysteinyl, β-tyrosyl, β-asparaginyl, β-glutaminyl, β-aspartyl, β-glutaryl, β-lysyl, β-arginyl, or β-histidyl. Unnatural amino acids are not proteinogenic amino acids, nor are they post-translationally modified variants thereof. Specifically, the term unnatural amino acid refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine, nor a post-translationally modified variant thereof.

[0072] The term "conjugate" refers to a compound or drug moiety of the present invention connected to one or more macromolecular moieties. The macromolecular moiety is as defined herein, or is any macromolecule deemed suitable by a person skilled in the art. The compound or drug moiety can be any compound or drug moiety described herein. The compound or drug moiety can be directly connected to the macromolecular moiety via a covalent bond, or the compound or drug moiety can be indirectly connected to the macromolecular moiety via a linker. Typically, the linker is covalently linked to the macromolecular moiety and is also covalently linked to the compound or drug moiety.

[0073] "pAMF," "pAMF residue," or "pAMF mutation" refers to a variant phenylalanine residue (ie, p-azidomethyl-L-phenylalanine) that is added to or substituted into a polypeptide.

[0074] The term "connector" refers to a molecular group portion that can form at least two covalent bonds. Typically, a connector can form at least one covalent bond with a macromolecular group portion, and form at least another covalent bond with a compound or drug group portion. In certain embodiments, a connector can form more than one covalent bond with a macromolecular group portion. In certain embodiments, a connector can form more than one covalent bond with a compound or drug group portion, or can form multiple covalent bonds with more than one compound or drug group portion. After a connector forms a bond with a macromolecular group portion, or a compound or drug group portion, or both, the remaining structure (i.e., the residue of the connector after forming one or more covalent bonds ("connector residue")) can still be referred to as a "connector" in the present invention. The term "connector precursor" refers to a connector having one or more reactive groups that can form covalent bonds with a macromolecule, or a compound or drug group portion, or both. A person of ordinary skill in the art, after understanding the context in which the term "linker" is used, will understand whether "linker" refers to a linker precursor having one active group, a linker precursor having more than one active group, a linker residue covalently linked to a macromolecule, a linker residue covalently linked to a compound or drug group portion, and / or a linker residue covalently linked to a macromolecule and covalently linked to a compound or drug group portion. In some embodiments, the linker is a cleavable linker. For example, a cleavable linker can be a linker that is released by biological instability or enzymatic function, which may or may not be engineered. In some embodiments, the linker is a non-cleavable linker. For example, a non-cleavable linker can be a linker that is released after the macromolecular group portion is degraded.

[0075] The term "EC 50” refers to the dose, concentration or amount of a specific test compound that elicits a dose-dependent response at 50% of the maximal expression of a specific response induced, elicited or potentiated by the specific test compound.

[0076] Unless otherwise specified, the term "IC 50 ” refers to the amount, concentration or dose of a particular test compound that achieves a 50% inhibition of the maximal response in an assay measuring such response.

[0077] The terms "subject" and "patient" used herein are used interchangeably. The term "subject" refers to an animal, such as a mammal including non-primates (e.g., cattle, pigs, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys such as cynomolgus monkeys, chimpanzees, and humans), and in certain embodiments is a human. In certain embodiments, the subject is a farm animal (e.g., horse, cattle, pig, etc.) or a pet (e.g., dog or cat). In certain embodiments, the subject is a human.

[0078] As used herein, the terms "therapeutic agent" and "therapeutic drug / agent" refer to any drug / agent or payload that can be used to treat or prevent a disease / disorder or one or more symptoms thereof. In certain embodiments, the term "therapeutic agent" includes a compound or conjugate provided herein. In certain embodiments, a therapeutic agent is a drug / agent that is known to be useful or has been used or is currently being used to treat or prevent a disease / disorder or one or more symptoms thereof.

[0079] "Therapeutically effective amount" refers to the amount of a compound or composition that is sufficient to achieve treatment of the disease or condition when administered to a subject. A "therapeutically effective amount" may vary depending on, inter alia, the compound, the disease or condition and its severity, and the age, weight, etc., of the subject to be treated.

[0080] In certain embodiments, "treating" or "managing" any disease or condition refers to improving the disease or condition present in a subject. In another embodiment, "treating" or "managing" includes improving at least one physical parameter, which may not be perceived by the subject. In yet another embodiment, "treating" or "managing" includes regulating the disease or condition, whether physically (e.g., stabilizing perceptible symptoms) or physiologically (e.g., stabilizing physiological parameters) or both. In another embodiment, "treating" or "managing" includes postponing or preventing the onset of the disease or condition, or postponing or preventing the recurrence of the disease or condition. In yet another embodiment, "treating" or "managing" includes reducing or eliminating the disease or condition, or delaying the progression of the disease or condition or one or more symptoms of the disease or condition, or reducing the severity of the disease or condition or one or more symptoms of the disease or condition.

[0081] As used herein, the term "inhibit growth" (e.g., in reference to cells, such as tumor cells) is intended to include any measurable reduction in cell growth (e.g., tumor cell growth) when contacted with a compound, drug moiety, or conjugate of the invention, compared to the growth of the same cells not contacted with the compound, drug moiety, or conjugate of the invention. In some embodiments, growth may be inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Reduction in cell growth may occur by a variety of mechanisms, including, but not limited to, conjugate, compound, or drug moiety internalization, apoptosis, necrosis, and / or effector function-mediated activity.

[0082] The term "prophylactic agent" as used herein and the term "prophylactic drug / agent" as used herein refer to any drug / agent or payload that can be used to prevent a disease / disorder or one or more symptoms thereof. In certain embodiments, the term "prophylactic agent" includes a compound, drug group portion, or conjugate provided herein. In certain other embodiments, the term "prophylactic agent" does not refer to a compound, drug group portion, or conjugate provided herein. For example, a prophylactic agent is a drug / agent that is known to be useful or has been used or is being used to prevent or stop the onset, development, progression, and / or severity of a disease / disorder.

[0083] The phrase "prophylactically effective amount" as used herein refers to an amount of a treatment / therapy (e.g., a prophylactic agent or a payload) sufficient to result in the prevention or reduction of the development, recurrence, or onset of one or more symptoms associated with a disease / disorder, or to enhance or improve the prophylactic effect of another treatment / therapy (e.g., another prophylactic agent).

[0084] In some chemical structures shown in the present invention, certain substituents, chemical groups and atoms are represented by curved / wavy / bent lines (e.g., ) depicts a curve / wavy line / curved line intersecting one or more bonds to represent the atoms through which the substituents, chemical groups, and atoms are connected. For example, in certain structures, such as but not limited to The curved / wavy / curved lines represent atoms in the backbone of the conjugate, compound or drug group portion of the structure to which the chemical entity is attached. In some structures, for example but not limited to The curved / wavy / curved lines represent atoms in a macromolecule to which the indicated chemical entity is attached and atoms in the backbone of a conjugate, compound or drug group moiety structure to which the indicated chemical entity is attached.

[0085] As used herein, illustrations showing substituents attached to cyclic groups (e.g., aromatic, heteroaromatic, fused rings, and saturated or unsaturated cycloalkyl or heterocycloalkyl) through bonds between ring atoms are intended to indicate that, unless otherwise indicated, the cyclic group may be substituted with the substituent at any ring position in the cyclic group or on any ring in a fused ring group, according to the techniques described herein or known in the art to which the invention pertains. For example, the group wherein the subscript q is an integer from 0 to 4, and wherein the substituent R 1 The positions of the substituents R are described in general terms, i.e., not directly connected to any vertex of the bond line structure, i.e., a specific ring carbon atom, including the following: 1 Non-limiting examples of groups attached to specific ring carbon atoms:

[0086] The term "site-specific" refers to the modification of a polypeptide at a predetermined sequence position in the polypeptide. The modification is at a single predictable residue of the polypeptide with little or no change. In certain embodiments, the modified amino acid is introduced at the sequence position, for example, by recombination or synthesis. Similarly, a group moiety can be "site-specifically" attached to a residue at a specific sequence position in a polypeptide. In certain embodiments, a polypeptide may contain more than one site-specific modification. Compounds represented by formula (I), (IA), (IB), (III), (IIIA) and (IIIB)

[0087] The present invention provides compounds that can be used to modulate one or more properties of macromolecules. The compounds can be formed according to the present invention and used to form conjugates with one or more macromolecules. The conjugates can be used for treatment or diagnosis. In certain embodiments, the treatment is the treatment of cancer or inflammatory diseases or conditions.

[0088] The embodiments of the present invention include the compounds and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, and / or mixtures thereof.

[0089] The present invention provides a compound having a structure shown in formula (I): or a pharmaceutically acceptable salt thereof, wherein, or a pharmaceutically acceptable salt thereof, wherein, L 1 Yes-C 1-6 Alkylene-; Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6Alkylene] n -[X 1 ] p –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -[X 1 ] p –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -[X 1 ] p -, wherein at least one alkylene, alkenylene or alkynylene group in Y is replaced by one or more selected from R 50 Substituents are substituted, and wherein the alkylene, alkenylene or alkynylene in Y is optionally replaced by one or more selected from R 51 Substituents are substituted; R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, –C 2-6 Alkenylene-X 2 -[C 2-6 alkenylene] m -POLY, or –C 2-6 Alkynylidene-X 2 -[C 2-6 Alkynylidene] m -POLY, where R 50 Each alkylene, alkenylene or alkynylene group is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; R 51 independently selected from halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10)2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; X 1 and X 2 are independently selected from -N(R 10 )–, –C(O)–, and –N(R 10 )C(O)–; R 10 At each occurrence, independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; POLY is a water-soluble polymer; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0 and 1; p is an integer selected from 0 and 1; Su is the hexose form of a monosaccharide; D is a drug group moiety; and RL is the reactive linker group residue.

[0090] The present invention provides a compound having a structure shown in formula (I): or a pharmaceutically acceptable salt thereof, wherein L 1 Yes-C 1-6 Alkylene-; Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -X 1 –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C2-6 Alkynylidene] n -X 1 -, wherein at least one alkylene, alkenylene or alkynylene group in Y is replaced by one or more selected from R 50 Substituents are substituted; R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, –C 2-6 Alkenylene-X 2 -[C 2-6 alkenylene] m -POLY, or –C 2-6 Alkynylidene-X 2 -[C 2-6 Alkynylidene] m -POLY, where R 50 Each alkylene, alkenylene or alkynylene group is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; X 1 and X 2 are independently selected from -C(O)-, and -N(R 10 )C(O)–; R 10 At each occurrence, independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; POLY is a water-soluble polymer; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0 and 1; Su is the hexose form of a monosaccharide; D is a drug group moiety; and RL is the active linker group residue.

[0091] In some embodiments, the present invention provides a compound represented by formula (IA): or a pharmaceutically acceptable salt thereof.

[0092] In some embodiments, the compound represented by formula (I) has the structure represented by formula (IB): or a pharmaceutically acceptable salt thereof.

[0093] In some embodiments, L 1 Yes-C 1-3 In some embodiments, L 1 is -CH2-. In some embodiments, L 1 is -CH2CH2-. In some embodiments, L 1 It is -CH2CH2CH2-.

[0094] In some embodiments, p is 1. In some embodiments, Y is -X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 In some embodiments, Y is -X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -X 1 -, wherein at least one alkenylene group in Y is replaced by one or more selected from R 50 In some embodiments, Y is -X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -X 1 -, wherein at least one alkynylene group in Y is replaced by one or more selected from R 50 In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0095] In some embodiments, p is 1. In certain embodiments, Y is -X 1 -C 1-4 Alkylene-[X 1 -C 1-4 Alkylene]n -X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0096] In some embodiments, p is 1. In certain embodiments, Y is -X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 In certain embodiments, Y is -X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 In certain embodiments, Y is -X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 substituted by a substituent.

[0097] In some embodiments, p is 0. In some embodiments, Y is -X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 In some embodiments, Y is -X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -, wherein at least one alkenylene group in Y is replaced by one or more selected from R 50wherein the alkenylene group in Y is optionally substituted by one or more selected from R 51 In some embodiments, Y is -X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -, wherein at least one alkynylene group in Y is replaced by one or more selected from R 50 wherein the alkynylene group in Y is optionally substituted by one or more selected from R 51 In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0098] In some embodiments, p is 0. In some embodiments, Y is -X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 In some embodiments, Y is -X 1 -C 1-4 Alkylene-[X 1 -C 1-4 Alkylene] n -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 In some embodiments, Y is -X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-, wherein at least one alkylene in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, R 51 is independently selected from halogen, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)-. In some embodiments, R 51is halogen. In some embodiments, R 51 is -CN. In some embodiments, R 51 is -NO2. In some embodiments, R 51 In some embodiments, R 51 is -NH2. In some embodiments, R 51 is -C(O)NH2. In some embodiments, R 51 It is -C(O)-.

[0099] In some embodiments, X 1 and X 2 are independently selected from -N(R 10 )–, –C(O)–, and –N(R 10 )C(O)—. In some embodiments, X 1 and X 2 are independently selected from -NH-, -C(O)-, and -N(R 10 )C(O)—. In some embodiments, X 1 and X 2 are independently selected from -C(O)-, and -N(R 10 )C(O)—. In some embodiments, X 1 and X 2 are independently selected from -C(O)-, and -NHC(O)-.

[0100] In certain embodiments, R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, where R 50 Each alkylene group is optionally substituted by one or more substituents selected from the following: halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 In some embodiments, R 50 Yes – C 1-4 Alkylene-X 2 -[C 1-4 Alkylene] m -POLY, where R 50Each alkylene group is optionally substituted by one or more substituents selected from the following: halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 In some embodiments, R 50 Each alkylene group is optionally substituted by one or more substituents selected from the following: halogen, -OH, -N(R 10 )2、-C(O)N(R 10 )2、-C(O)-、C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 In some embodiments, m is 0. In some embodiments, m is 1.

[0101] In certain embodiments, R 50 Yes – C 2-6 Alkenylene-X 2 -[C 2-6 alkenylene] m -POLY, where R 50 Each alkenylene group is optionally substituted by one or more substituents selected from the following: halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 In some embodiments, m is 0. In some embodiments, m is 1.

[0102] In certain embodiments, R 50 Yes – C 2-6 Alkynylidene-X 2 -[C 2-6 Alkynylidene] m -POLY, where R 50Each alkynylene group is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 In some embodiments, m is 0. In some embodiments, m is 1.

[0103] In certain embodiments, POLY is polyethylene glycol (PEG), methoxy polyethylene glycol (mPEG), polypropylene glycol (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinicalcohols), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamides), poly(hydroxyalkylmethacrylates), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholine), polysarcosine, or combinations thereof. In some embodiments, POLY is polyethylene glycol (PEG). In some embodiments, POLY is methoxy polyethylene glycol (mPEG). In some embodiments, POLY is polypropylene glycol (PPG). In some embodiments, POLY is copolymers of ethylene glycol and propylene glycol. In some embodiments, POLY is poly(oxyethylated polyols). In some embodiments, POLY is poly(olefinicalcohols). In some embodiments, POLY is poly(vinylpyrrolidone). In some embodiments, POLY is poly(hydroxyalkyl methacrylamide). In some embodiments, POLY is poly(hydroxyalkyl methacrylate). In some embodiments, POLY is poly(saccharide). In some embodiments, POLY is poly(α-hydroxy acid). In some embodiments, POLY is poly(vinyl alcohol). In some embodiments, POLY is polyphosphazene. In some embodiments, POLY is polyoxazolines (POZ). In some embodiments, POLY is poly(N-acryloylmorpholine). In some embodiments, POLY is polysarcosine. In some embodiments, POLY is a non-peptide, water-soluble polymer. In some embodiments, POLY comprises polyethylene glycol (PEG) or methoxypolyethylene glycol (mPEG). In some embodiments, POLY is in represents the point of attachment to the rest of the compound, and wherein n1 is an integer from 1 to 20. In certain embodiments, n1 is an integer between 5 and 15. In some embodiments, n1 is 1. In some embodiments, n1 is 2. In some embodiments, n1 is 3. In some embodiments, n1 is 4. In some embodiments, n1 is 5. In some embodiments, n1 is 6. In some embodiments, n1 is 7. In some embodiments, n1 is 8. In some embodiments, n1 is 9. In some embodiments, n1 is 10. In some embodiments, n1 is 11. In some embodiments, n1 is 12. In some embodiments, n1 is 13. In some embodiments, n1 is 14. In some embodiments, n1 is 15. In some embodiments, n1 is 16. In some embodiments, n1 is 17. In some embodiments, n1 is 18. In some embodiments, n1 is 19. In some embodiments, n1 is 20. In some embodiments, n1 is 21. In some embodiments, n1 is 22. In some embodiments, n1 is 23. In some embodiments, n1 is 24. In some embodiments, n1 is 25. In some embodiments, n1 is 26. In some embodiments, n1 is 27. In some embodiments, n1 is 28. In some embodiments, n1 is 29. In some embodiments, n1 is 30.

[0104] In certain embodiments, the RL comprises an alkyne, a cyclooctyne, a strained olefin, a tetrazine, an amine, a methylcyclopropene, a thiol, a p-acetylphenylalanine residue, an oxyamine, a maleimide, or an azide. In some embodiments, the RL comprises an alkyne. In some embodiments, the RL comprises a cyclooctyne. In some embodiments, the RL comprises a strained olefin. In some embodiments, the RL comprises a tetrazine. In some embodiments, the RL comprises an amine. In some embodiments, the RL comprises a methylcyclopropene. In some embodiments, the RL comprises a thiol. In some embodiments, the RL comprises a p-acetylphenylalanine residue. In some embodiments, the RL comprises an oxyamine. In some embodiments, the RL comprises a maleimide. In some embodiments, the RL comprises an azide. In certain embodiments, the RL is selected from the group consisting of: –N3, –NH2, methylcyclopropene, and –SH; where R T It is C 1-6 Alkyl; and In some embodiments, RL is selected from the group consisting of: In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is Where R T It is C 1-6 Alkyl, and In some embodiments, R T is methyl, ethyl, or propyl. In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is In some embodiments, RL is -N3. In some embodiments, RL is -NH2. In some embodiments, RL is methylcyclopropene. In some embodiments, RL is -SH.

[0105] In some embodiments, Su is a sugar moiety. In some embodiments, Su is a hexose form of a monosaccharide. Su can be a glucuronic acid or mannose residue. In certain embodiments, Su is in represents the point of attachment to the rest of the compound. In certain embodiments, Su is in represents the point of attachment to the rest of the compound.

[0106] In some embodiments, D is an immunomodulatory payload. In some embodiments, the immunomodulatory payload is an agonist of stimulator of interferon genes (STING), Toll-like receptor 7 (TLR7), Toll-like receptor 7 / 8 (TLR7 / 8), or Toll-like receptor 8 (TLR8). In some embodiments, the immunomodulatory payload is an agonist of stimulator of interferon genes (STING). In some embodiments, the immunomodulatory payload is an agonist of Toll-like receptor 7 (TLR7). In some embodiments, the immunomodulatory payload is an agonist of Toll-like receptor 7 / 8 (TLR7 / 8). In some embodiments, the immunomodulatory payload is an agonist of Toll-like receptor 8 (TLR8). In some embodiments, the STING agonist is selected from the group consisting of: small molecule agonists of the STING pathway, antibodies that activate STING activity, recombinant proteins that activate the STING pathway, TTI-10001, DMXAA (ASA404), CDNs, c-di-GMP, 2′3′-cGAMP, MK-1454, ADU-S100 (MIW815), SB11285, ADU-V19, IACS-8779, IACS-8803, IMSA101, non-CDNs, E7766, MK-2118, diABZI, MSA-2, JNJ-'6196, bacterial vectors, SYNB1891, and STACT (see, e.g., Luo et al. Molecules 2022, 27, 4638, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the STING agonist is a small molecule agonist of the STING pathway. In some embodiments, the STING agonist is an antibody that activates STING activity. In some embodiments, the STING agonist is a recombinant protein that activates the STING pathway. In some embodiments, the STING agonist is TTI-10001. In some embodiments, the STING agonist is DMXAA (ASA404). In some embodiments, the STING agonist is a CDN class. In some embodiments, the STING agonist is c-di-GMP. In some embodiments, the STING agonist is 2′3′-cGAMP. In some embodiments, the STING agonist is MK-1454. In some embodiments, the STING agonist is ADU-S100 (MIW815). In some embodiments, the STING agonist is SB11285. In some embodiments, the STING agonist is ADU-V19. In some embodiments, the STING agonist is IACS-8779.In some embodiments, the STING agonist is IACS-8803. In some embodiments, the STING agonist is IMSA101. In some embodiments, the STING agonist is a non-CDN. In some embodiments, the STING agonist is E7766. In some embodiments, the STING agonist is MK-2118. In some embodiments, the STING agonist is diABZI. In some embodiments, the STING agonist is MSA-2. In some embodiments, the STING agonist is JNJ-'6196. In some embodiments, the STING agonist is a bacterial vector. In some embodiments, the STING agonist is SYNB1891. In some embodiments, the STING agonist is STACT.

[0107] In some embodiments, the Toll-like receptor 7 (TLR7) agonist is selected from the group consisting of: GS-986, PRTX-007, PRX-034, S-34240, MBS-8, and APR-002 (see, e.g., Bhagchandani et al. Advanced Drug Delivery Reviews 2021, 175, 113803, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the Toll-like receptor 7 (TLR7) agonist is GS-986. In some embodiments, the Toll-like receptor 7 (TLR7) agonist is PRTX-007. In some embodiments, the Toll-like receptor 7 (TLR7) agonist is PRX-034. In some embodiments, the Toll-like receptor 7 (TLR7) agonist is S-34240. In some embodiments, the Toll-like receptor 7 (TLR7) agonist is MBS-8. In some embodiments, the Toll-like receptor 7 (TLR7) agonist is APR-002.

[0108] In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is selected from the group consisting of imiquimod (R837), resiquimod (R848), 852-A (PF-4878691), vesatolimod (GS-9620), AZD8848, motolimod (VTX-2337), selgantolimod (GS-9688), NKTR-262, RG-7854 (RO 7020531), DSP-0509, BDB-001, BDC-1001, LHC-165, SHR-2150, JNJ-4964 (TQ-73334), RO-7119929, DN-1508052, VTX-1463, BNT-411 (SC1), APR-003, ALT-702, TRANSCON, VX-001, SNAPvax, R848-HA, SM360320, and GSK2245035 (see, e.g., Bhagchandani et al. Advanced Drug Delivery Reviews 2021, 175, 113803; and Evans et al. ACS Omega 2019, 4, 13, 15665, the contents of each of which are incorporated herein by reference in their entirety). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is imiquimod (R837). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is resiquimod (R848). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is 852-A (PF-4878691). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is visamod (GS-9620). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is AZD8848. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is motomod (VTX-2337). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is selgantolimod (GS-9688). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is NKTR-262. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is RG-7854 (RO 7020531). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is DSP-0509.In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is BDB-001. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is BDC-1001. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is LHC-165. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is SHR-2150. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is JNJ-4964 (TQ-73334). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is RO-7119929. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is DN-1508052. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is VTX-1463. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is BNT-411 (SC1). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is APR-003. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is ALT-702. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is TRANSCON. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is VX-001. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is SNAPvax. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is R848-HA. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is SM360320. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is GSK2245035.

[0109] In some embodiments, the Toll-like receptor 8 (TLR8) agonist is selected from the group consisting of: SBT-6050, SBT-6290, and ZM-TLR8 agonist. In some embodiments, the Toll-like receptor 8 (TLR8) agonist is SBT-6050. In some embodiments, the Toll-like receptor 8 (TLR8) agonist is SBT-6290. In some embodiments, the Toll-like receptor 8 (TLR8) agonist is a ZM-TLR8 agonist.

[0110] In certain embodiments, D is a cytotoxic agent or a cytotoxic payload. In certain embodiments, D is an alkylating agent or an alkylating payload. In certain embodiments, D is a bifunctional alkylating agent. In some embodiments, D is a bifunctional alkylating agent selected from the group consisting of cyclophosphamide, nitrogen mustard, chlorambucil, and melphalan. In some embodiments, D is cyclophosphamide. In some embodiments, D is nitrogen mustard. In some embodiments, D is chlorambucil. In some embodiments, D is melphalan. In some embodiments, D is a monofunctional alkylating agent. In some embodiments, D is a monofunctional alkylating agent selected from the group consisting of dacarbazine, nitrosoureas, and temozolomide. In some embodiments, D is dacarbazine. In some embodiments, D is nitrosoureas. In some embodiments, D is temozolomide. In certain embodiments, D is a cytoskeletal disruptor (e.g., a taxane). In some embodiments, D is a cytoskeletal disruptor selected from the group consisting of paclitaxel, docetaxel, albumin-bound paclitaxel (abraxane), and taxotere. In some embodiments, D is paclitaxel. In some embodiments, D is docetaxel. In some embodiments, D is albumin-bound paclitaxel (abraxane). In some embodiments, D is taxotere. In certain embodiments, D is an epothilone. In some embodiments, D is an epothilone selected from the group consisting of epothilone A, epothilone B, epothilone C, epothilone D, and ixabepilone. In some embodiments, D is epothilone A. In some embodiments, D is epothilone B. In some embodiments, D is epothilone C. In some embodiments, D is epothilone D. In some embodiments, D is ixabepilone. In certain embodiments, D is a histone deacetylase inhibitor. In some embodiments, D is a histone deacetylase inhibitor selected from the group consisting of vorinostat and romidepsin. In some embodiments, D is vorinostat. In some embodiments, D is romidepsin. In certain embodiments, D is a kinase inhibitor. In some embodiments, D is a kinase inhibitor selected from the group consisting of bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib. In some embodiments, D is bortezomib. In some embodiments, D is erlotinib. In some embodiments, D is gefitinib. In some embodiments, D is imatinib. In some embodiments, D is vemurafenib. In some embodiments, D is vismodegib. In certain embodiments, D is a nucleotide analog and / or precursor analog. In some embodiments, D is a nucleotide analog and / or precursor analog selected from the group consisting of azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine (formerly known as mercaptoguanine). In some embodiments, D is azacitidine. In some embodiments, D is azathioprine.In some embodiments, D is capecitabine. In some embodiments, D is cytarabine. In some embodiments, D is doxifluridine. In some embodiments, D is fluorouracil. In some embodiments, D is gemcitabine. In some embodiments, D is hydroxyurea. In some embodiments, D is mercaptopurine. In some embodiments, D is methotrexate. In some embodiments, D is thioguanine (formerly known as mercaptoguanine). In certain embodiments, D is a peptide antibiotic. In some embodiments, D is a peptide antibiotic selected from the group consisting of: bleomycin and actinomycin. In some embodiments, D is bleomycin. In some embodiments, D is actinomycin. In certain embodiments, D is a platinum-based drug or a platinum-based payload. In some embodiments, D is a platinum-based drug or a platinum-based payload selected from the group consisting of: carboplatin, cisplatin, and oxaliplatin. In some embodiments, D is carboplatin. In some embodiments, D is cisplatin. In some embodiments, D is oxaliplatin. In certain embodiments, D is a retinoid. In certain embodiments, D is a retinoid selected from the group consisting of tretinoin, alitretinoin, and bexarotene. In certain embodiments, D is tretinoin. In certain embodiments, D is alitretinoin. In certain embodiments, D is bexarotene. In certain embodiments, D is a vinca alkaloid and its derivatives. In certain embodiments, D is a vinca alkaloid and its derivatives selected from the group consisting of vinblastine, vincristine, vindesine, and vinorelbine. In certain embodiments, D is vinblastine. In certain embodiments, D is vincristine. In certain embodiments, D is vindesine. In certain embodiments, D is vinorelbine. In certain embodiments, the cytotoxic agent or cytotoxic payload is a tubulin inhibitor, a DNA topoisomerase I inhibitor, or a DNA topoisomerase II inhibitor. In certain embodiments, the cytotoxic agent or cytotoxic payload is a tubulin inhibitor. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase I inhibitor. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase I inhibitor selected from the group consisting of irinotecan, SN-38, topotecan, exatecan. In some embodiments, the cytotoxic agent or cytotoxic payload is irinotecan. In some embodiments, the cytotoxic agent or cytotoxic payload is SN-38. In some embodiments, the cytotoxic agent or cytotoxic payload is topotecan. In some embodiments, the cytotoxic agent or cytotoxic payload is exatecan. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase II inhibitor.In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase II inhibitor selected from the group consisting of: etoposide, teniposide, and tafluposide. In some embodiments, the cytotoxic agent or cytotoxic payload is etoposide. In some embodiments, the cytotoxic agent or cytotoxic payload is teniposide. In some embodiments, the cytotoxic agent or cytotoxic payload is tafluposide. In certain embodiments, D is selected from the group consisting of: hemiasterlins, camptothecins, and anthracyclines. Anthracyclines may include PNU-159682 and EDA PNU-159682 derivatives. In certain embodiments, the anthracycline is selected from the group consisting of: daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin. In some embodiments, the anthracycline is daunorubicin. In some embodiments, the anthracycline is doxorubicin. In some embodiments, the anthracycline is epirubicin. In some embodiments, the anthracycline is idarubicin. In some embodiments, the anthracycline is mitoxantrone. In some embodiments, the anthracycline is valrubicin. In some embodiments, D is hemicycline. In some embodiments, D is camptothecin. In some embodiments, D is an anthracycline. In some embodiments, D is PNU-159682. In some embodiments, D is an EDA PNU compound. In some embodiments, D is an EDA PNU-159682 derivative. In certain embodiments, D is hemicycline, exitecan, PNU-159682, or an EDA PNU-159682 derivative. In some embodiments, D is hemicycline. In some embodiments, D is exitecan. In some embodiments, D is PNU-159682. In some embodiments, D is an EDA PNU-159682 compound or derivative.

[0111] Representative compounds of the present invention include compounds represented by formula (I), (IA) or (IB), as shown in Table 1. Table 1: Representative compounds

[0112] In certain embodiments, the compound represented by formula (I), (IA) or (IB) is selected from the group consisting of: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: or a salt thereof.

[0113] In certain embodiments, the compound represented by formula (I), (IA) or (IB) is selected from In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: or a salt thereof.

[0114] In certain embodiments, the compound represented by formula (I), (IA) or (IB) is selected from In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: or a salt thereof.

[0115] In certain embodiments, the compound represented by formula (I), (IA) or (IB) is selected from or In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: In some embodiments, the compound represented by formula (I), (IA) or (IB) is: or a salt thereof.

[0116] In some embodiments, the compound represented by formula (I), (IA) or (IB) is or a pharmaceutically acceptable salt thereof. In some embodiments, the compound represented by formula (I), (IA) or (IB) is: or a pharmaceutically acceptable salt thereof. In some embodiments, the compound represented by formula (I), (IA) or (IB) is: or a pharmaceutically acceptable salt thereof. In some embodiments, the compound represented by formula (I), (IA) or (IB) is: or a pharmaceutically acceptable salt thereof. In some embodiments, the compound represented by formula (I), (IA) or (IB) is: or a pharmaceutically acceptable salt of any one thereof.

[0117] In some embodiments, the compound represented by formula (I), (IA) or (IB) is selected from or In some embodiments, the compound represented by formula (I), (IA) or (IB) is: or a pharmaceutically acceptable salt thereof. In some embodiments, the compound represented by formula (I), (IA) or (IB) is: or A pharmaceutically acceptable salt of any one thereof.

[0118] In some embodiments, the compound represented by formula (I), (IA) or (IB) is selected from or In some embodiments, the compound represented by formula (I), (IA) or (IB) is: or In some embodiments, the compound represented by formula (I), (IA) or (IB) is: or A pharmaceutically acceptable salt of any one thereof.

[0119] In some aspects, the present invention provides a compound having a structure shown in formula (III): or a pharmaceutically acceptable salt thereof, wherein L 1 Yes-C 1-6 Alkylene-; Z is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 –, –X 1 -C2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -X 1 –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -X 1 -, wherein the alkylene, alkenylene or alkynylene in Z is optionally substituted by one or more substituents selected from the following: halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; X 1 and X 2 are independently selected from -C(O)-, and -N(R 10 )C(O)–; R 10 At each occurrence, independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; POLY is a water-soluble polymer; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0 and 1; Su is the hexose form of a monosaccharide; CYTO is a cytotoxic agent or a cytotoxic payload; and RL is the active linker group residue.

[0120] In some embodiments, the present invention provides a compound represented by formula (IIIA): or a pharmaceutically acceptable salt thereof.

[0121] In some embodiments, the compound represented by formula (III) has the structure represented by formula (IIIB): or a pharmaceutically acceptable salt thereof.

[0122] In some embodiments, L 1 Yes-C 1-3 In some embodiments, L 1 is -CH2-. In some embodiments, L 1 is -CH2CH2-. In some embodiments, L 1 It is -CH2CH2CH2-.

[0123] In some embodiments, Z is -X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -X 1 –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -X 1 -, wherein at least one alkylene, alkenylene or alkynylene in Z is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 In some embodiments, Z is -X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 -, wherein the alkylene in Z is optionally substituted by one or more substituents selected from the following: halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10)2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 In some embodiments, Z is -X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 –.

[0124] In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0125] In some embodiments, X 1 and X 2 are independently selected from -C(O)-, and -N(R 10 )C(O)–.

[0126] In certain embodiments, the RL comprises an alkyne, a cyclooctyne, a strained olefin, a tetrazine, an amine, a thiol, a p-acetylphenylalanine residue, an oxyamine, a maleimide, or an azide. In some embodiments, the RL comprises an alkyne. In some embodiments, the RL comprises a cyclooctyne. In some embodiments, the RL comprises a strained olefin. In some embodiments, the RL comprises a tetrazine. In some embodiments, the RL comprises an amine. In some embodiments, the RL comprises a thiol. In some embodiments, the RL comprises a p-acetylphenylalanine residue. In some embodiments, the RL comprises an oxyamine. In some embodiments, the RL comprises a maleimide. In some embodiments, the RL comprises an azide. In certain embodiments, the RL is selected from the group consisting of: –N3, –NH2, methylcyclopropene, and –SH; where R T It is C 1-6 Alkyl; and represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is Where R T It is C 1-6 Alkyl, and In some embodiments, R T is methyl, ethyl, or propyl. In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is In some embodiments, RL is -N3. In some embodiments, RL is -NH2. In some embodiments, RL is methylcyclopropene. In some embodiments, RL is -SH.

[0127] In some embodiments, Su is a sugar moiety. In some embodiments, Su is a hexose form of a monosaccharide. Su can be a glucuronic acid or mannose residue. In certain embodiments, Su is in represents the point of attachment to the rest of the compound. In certain embodiments, Su is in represents the point of attachment to the rest of the compound.

[0128] In certain embodiments, CYTO is a cytotoxic agent or a cytotoxic payload. In certain embodiments, the cytotoxic agent or cytotoxic payload is a tubulin inhibitor, a DNA topoisomerase I inhibitor, or a DNA topoisomerase II inhibitor. In some embodiments, the cytotoxic agent or cytotoxic payload is a tubulin inhibitor. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase I inhibitor. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase I inhibitor selected from the group consisting of irinotecan, SN-38, topotecan, exatecan. In some embodiments, the cytotoxic agent or cytotoxic payload is irinotecan. In some embodiments, the cytotoxic agent or cytotoxic payload is SN-38. In some embodiments, the cytotoxic agent or cytotoxic payload is topotecan. In some embodiments, the cytotoxic agent or cytotoxic payload is exatecan. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase II inhibitor. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase II inhibitor selected from the group consisting of: etoposide, teniposide, and tafluposide. In some embodiments, the cytotoxic agent or cytotoxic payload is etoposide. In some embodiments, the cytotoxic agent or cytotoxic payload is teniposide. In some embodiments, the cytotoxic agent or cytotoxic payload is tafluposide. In certain embodiments, CYTO is selected from the group consisting of: hemiasterlins, camptothecins, and anthracyclines. Anthracyclines may include PNU-159682 and EDA PNU-159682 derivatives. In certain embodiments, the anthracycline is selected from the group consisting of: daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin. In some embodiments, the anthracycline is daunorubicin. In some embodiments, the anthracycline is doxorubicin. In some embodiments, the anthracycline is epirubicin. In some embodiments, the anthracycline is idarubicin. In some embodiments, the anthracycline is mitoxantrone. In some embodiments, the anthracycline is valrubicin. In some embodiments, CYTO is hemicycline. In some embodiments, CYTO is camptothecin. In some embodiments, CYTO is an anthracycline. In some embodiments, CYTO is PNU-159682. In some embodiments, CYTO is an EDA PNU compound. In some embodiments, CYTO is an EDA PNU-159682 derivative.In certain embodiments, CYTO is hemiasterlin, exitecan, PNU-159682, or an EDAPNU-159682 derivative. In some embodiments, CYTO is hemiasterlin. In some embodiments, CYTO is exitecan. In some embodiments, CYTO is PNU-159682. In some embodiments, CYTO is an EDA PNU-159682 compound or derivative. In some embodiments, CYTO is not an immunostimulatory compound.

[0129] In some embodiments, the compound is or a pharmaceutically acceptable salt thereof.

[0130] In some embodiments, the compound is or a pharmaceutically acceptable salt thereof. Optically active compounds

[0131] In certain embodiments, the compound provided by the invention may have several chiral centers, and may exist and be separated in optically active form and racemic form. In certain embodiments, some compounds may show polymorphism. It will be appreciated by those skilled in the art that the compound provided by the invention may exist in any racemate, optically active form, diastereomer, polymorph or stereoisomer form, and / or its mixture. It will also be appreciated by those skilled in the art that the compound with useful properties described in the invention described in the present invention all belongs within the scope of the present invention. It will also be appreciated by those skilled in the art how to prepare the optically active form of the compound described in the invention, for example, by splitting the racemic form by recrystallization technique, by synthesizing by optically active starting materials, by chiral synthesis or by using chiral stationary phase for chromatographic separation. In addition, most amino acids are chiral (i.e., designated as L or D, wherein the L enantiomer is a naturally occurring configuration), and may exist as a single enantiomer.

[0132] Examples of methods for obtaining optically active substances are all known in the art and include at least the following: i) Physical separation of crystals - a technique by which macroscopic crystals of individual enantiomers are separated manually. This technique can be used if crystals of the individual enantiomers exist (ie the material is an aggregate and the crystals are visually distinct). ii) simultaneous crystallization - a technique by which the individual enantiomers are crystallized separately from a solution of the racemate, provided that the latter is an aggregate in the solid state; iii) Enzymatic resolution - a technique that partially or completely separates racemates by taking advantage of the different reaction rates of enantiomers in the presence of enzymes; iv) Enzymatic asymmetric synthesis - a synthetic technique that uses an enantiomerically pure or enriched synthetic precursor of the desired enantiomer by at least one step of the synthesis; v) Chemical asymmetric synthesis - a synthetic technique in which the desired enantiomer is synthesized from achiral precursors under conditions that result in asymmetry (i.e., chirality) of the product, which can be achieved using chiral catalysts or chiral auxiliaries; vi) Diastereoisomer separation - a technique in which a racemic compound is reacted with an enantiomerically pure reagent (chiral auxiliary) to convert the individual enantiomers into diastereomers. The resulting diastereomers are then separated by chromatography or crystallization, by virtue of their now more pronounced diastereomeric differences, and the chiral auxiliary is subsequently removed to obtain the individual enantiomers; vii) primary and secondary asymmetric transformations - techniques whereby the diastereomers of a racemate are allowed to equilibrate in solution to produce a predominance of a diastereomer over the desired enantiomer in solution, or a diastereomer preferentially crystallizes over the desired enantiomer to disturb the equilibrium so that ultimately in principle all material is converted to a crystalline diastereomer of the desired enantiomer. The desired enantiomer is then derived from said diastereomer; viii) Kinetic resolution - the technique is the partial or complete resolution of a racemate (or further resolution of a partially resolved compound) due to the unequal reaction rates of the enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions; ix) Stereospecific synthesis from non-racemic precursors - synthetic techniques by which the desired enantiomer is obtained from chiral starting materials and wherein the stereochemical integrity is not or only minimally compromised during the synthetic process; x) Chiral liquid chromatography - a technique that allows the enantiomers of a racemate to be separated in a liquid mobile phase by virtue of their different interactions with the stationary phase. The stationary phase may be made of a chiral material, or the mobile phase may contain additional chiral materials to induce different interactions; xi) Chiral gas chromatography – a technique whereby the racemate is volatilized and the enantiomers are separated by their differential interaction in a gaseous mobile phase with a chromatographic column containing a stationary non-racemic adsorbent phase; xii) Chiral solvent extraction - a technique that allows the separation of enantiomers by kinetically or thermodynamically dissolving one enantiomer in a specific chiral solvent; xiii) Transport across chiral membranes - a technique where the racemate is brought into contact with a thin membrane barrier. The barrier typically separates two miscible fluids, one of which contains the racemate, and a driving force such as concentration or pressure differential allows preferential transport across the membrane barrier. The separation occurs due to the non-racemic chiral nature of the membrane, which allows only one enantiomer of the racemate to pass.

[0133] In some embodiments, the present invention provides a composition of the compound of the present invention, comprising a compound shown in formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), (IIIB), (IV), (IVA), or (IVB), wherein the compound is substantially free of a specified stereoisomer of the compound. In certain embodiments, in the methods and compounds of the present invention, the compound is substantially free of other stereoisomers. In some embodiments, the composition comprises a compound that accounts for at least 85%, 90%, 95%, 98%, or 99% to 100% by weight of the compound, with the remainder comprising other chemical substances or enantiomers. In some embodiments, the present invention provides a composition of a compound shown in formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), (IIIB), (IV), (IVA), or (IVB), wherein the compound is substantially free of a specified enantiomer of the compound. In certain embodiments, in the methods and compounds of the invention, the compounds are substantially free of other enantiomers. In some embodiments, the composition comprises at least 85%, 90%, 95%, 98%, or 99% to 100% by weight of the compound, with the remainder comprising other chemical substances or enantiomers. Isotopically enriched compounds

[0134] The present invention also provides isotopically enriched compounds, including but not limited to isotopically enriched compounds of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), (IIIB), (IV), (IVA), or (IVB).

[0135] It has been previously demonstrated with certain classes of drugs that isotopic enrichment (eg, deuteration) of drugs can improve pharmacokinetic ("PK"), pharmacodynamic ("PD"), and / or toxicity profiles. See, eg, Lijinsky et.al., Food Cosmet. Toxicol., 20:393 (1982); Lijinsky et.al., J. Nat. Cancer Inst., 69:1127 (1982); Mangold et.al., Mutation Res. 308:33 (1994); Gordon et.al., Drug Metab. Dispos., 15:589 (1987); Zello et.al., Metabolism, 43:487 (1994); Gately et.al., J. Nucl. Med., 27:388 (1986); Wade D, Chem. Biol. Interact. 117:191 (1999).

[0136] Isotopic enrichment of a drug can be used, for example, to (1) reduce or eliminate unwanted metabolites, (2) increase the half-life of the parent drug, (3) reduce the number of doses required to achieve a desired effect, (4) reduce the amount of dose necessary to achieve a desired effect, (5) increase the formation of active metabolites (if any are formed), and / or (6) reduce the production of deleterious metabolites in specific tissues. Isotopic enrichment of a drug can also be used to prepare drugs for more effective and / or safer combination therapies, whether the combination therapy is intentional or unintentional.

[0137] Replacing an atom with one of its isotopes usually causes a change in the reaction rate of a chemical reaction. This phenomenon is called the kinetic isotope effect ("KIE"). For example, if a C-H bond breaks during the rate-determining step (i.e., the step with the highest transition state energy) in a chemical reaction, then replacing this active hydrogen with a (heavier) isotope will result in a decrease in the reaction rate. The deuterium kinetic isotope effect ("DKIE") is the most common form of KIE. (See, e.g., Foster et al., Adv. Drug Res., vol. 14, pp. 1-36 (1985); Kushner et al., Can. J. Physiol. Pharmacol., vol. 77, pp. 79-88 (1999)).

[0138] The magnitude of the DKIE can be expressed as the ratio between the rates of a given reaction in which a C—H bond is broken and the same reaction in which deuterium replaces hydrogen and the C—D bond is broken. The DKIE can range from about 1 (no isotope effect) to very large values, such as 50 or more, meaning that the reaction can be 50 or more times slower when deuterium replaces hydrogen.

[0139] Substitution of hydrogen with tritium ("T") produces a stronger bond than deuterium and a numerically larger isotope effect. Similarly, isotope substitution of other elements, including but not limited to, 13 C or 14 C replaces carbon, 33 S. 34 S, or 36 S replaces sulfur, 15 N replaces nitrogen, and 17 O or 18 O can replace oxygen and produce similar kinetic isotope effects.

[0140] Animals express various enzymes to remove foreign substances, such as therapeutic agents, from their circulatory systems. Examples of such enzymes include cytochrome P450 enzymes ("CYPs"), esterases, proteases, reductases, dehydrogenases, and monoamine oxidases to react with and convert these foreign substances into more polar intermediates or metabolites for renal excretion. Some of the most common metabolic reactions of drug compounds involve oxidation of carbon-hydrogen (CH) bonds to carbon-oxygen (CO) or carbon-carbon (C=C) π bonds. The resulting metabolites may be stable or unstable under physiological conditions and may have significantly different PK / PD and acute and long-term toxicity characteristics relative to the parent compound. For many drugs, such oxidation reactions are rapid. Therefore, these drugs typically require multiple or high-dose daily dosing.

[0141] Therefore, isotopic enrichment at certain positions of the compounds provided herein will produce a detectable KIE compared to similar compounds with natural isotopic compositions, which will affect the pharmacological, PK, PD, and / or toxicological characteristics of the compounds provided herein. Conjugates represented by formula (II), (IIA), (IIB), (IV), (IVA), (IVB)

[0142] The present invention provides a conjugate of a macromolecule and one of the compounds shown in formula (I), (IA), (IB), (III), (IIIA) or (IIIB) of the present invention. The conjugate is directly or indirectly covalently linked via a linker. In certain embodiments, the conjugate comprises a macromolecule coupled to one or more compounds shown in formula (I), (IA), (IB), (III), (IIIA) or (IIIB) of the present invention. In certain embodiments, the conjugate comprises more than one macromolecule. In certain embodiments, the macromolecule is connected to 1, 2, 3, 4, 5, 6, 7, 8 or more compounds shown in formula (I), (IA), (IB), (III), (IIIA) or (IIIB).

[0143] The linker can be any linker capable of forming at least one bond with a macromolecule and forming at least one bond with a compound of formula (I), (IA), (IB), (III), (IIIA) or (IIIB). Useful linkers are described in the present invention section and in the examples, particularly the linkers described below.

[0144] The macromolecule can be any macromolecule that a person skilled in the art finds suitable. In certain embodiments, the macromolecule is a second compound. In certain embodiments, COMP is a residue of the second compound. In certain embodiments, the macromolecule is a protein, peptide, antibody or its antigen-binding fragment, nucleic acid, carbohydrate, or other macromolecule composed of polymerized monomers. In certain embodiments, the macromolecule is a peptide with two or more residues. In certain embodiments, the macromolecule is a peptide with 10 or more residues. In certain embodiments, the mass of the macromolecule is at least 1000Da. In certain embodiments, the macromolecule comprises at least 1000 atoms. Useful macromolecules are described in the following sections.

[0145] In some embodiments, the present invention provides a conjugate represented by formula (II): in, COMP is the residue of the second compound; L 1 Yes-C 1-6 Alkylene-; Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -[X 1 ] p –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -[X 1 ] p –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -[X 1 ] p -, wherein at least one alkylene, alkenylene or alkynylene group in Y is replaced by one or more selected from R 50 Substituents are substituted, and wherein the alkylene, alkenylene or alkynylene in Y is optionally replaced by one or more selected from R 51 Substituents are substituted; R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, –C 2-6 Alkenylene-X 2 -[C 2-6 alkenylene] m -POLY, or –C 2-6 Alkynylidene-X 2 -[C 2-6 Alkynylidene] m -POLY, where R 50 Each alkylene, alkenylene or alkynylene group is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; R 51 independently selected from halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; X 1 and X 2 are independently selected from -N(R 10 )–, –C(O)–, and –N(R 10 )C(O)–; R 10 At each occurrence, independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; POLY is a water-soluble polymer; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0 and 1; p is an integer selected from 0 and 1; Su is the hexose form of a monosaccharide; D is a drug group moiety; and RL is the active linker group residue.

[0146] In some embodiments, the present invention provides a conjugate represented by formula (II): in, COMP is the residue of the second compound; L 1 Yes-C 1-6 Alkylene-; Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -X 1 –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -X 1 -, wherein at least one alkylene, alkenylene or alkynylene group in Y is replaced by one or more selected from R 50 Substituents are substituted; R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, –C 2-6 Alkenylene-X 2 -[C 2-6 alkenylene] m -POLY, or –C 2-6 Alkynylidene-X 2 -[C 2-6 Alkynylidene] m -POLY, where R 50Each alkylene, alkenylene or alkynylene group is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; X 1 and X 2 are independently selected from -C(O)-, and -N(R 10 )C(O)–; R 10 At each occurrence, independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; POLY is a water-soluble polymer; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0 and 1; Su is the hexose form of a monosaccharide; D is a drug group moiety; and RL is the active linker group residue.

[0147] In some embodiments, COMP is a residue of a polypeptide. In some embodiments, COMP is a residue of an antibody. In some embodiments, COMP is a residue of an antibody chain.

[0148] In some embodiments, the present invention provides a conjugate represented by formula (IIA): or a pharmaceutically acceptable salt thereof.

[0149] In some embodiments, the conjugate of formula (II) has the structure of formula (IIB): or a pharmaceutically acceptable salt thereof.

[0150] In some embodiments, L 1 Yes-C 1-3 In some embodiments, L 1is -CH2-. In some embodiments, L 1 is -CH2CH2-. In some embodiments, L 1 It is -CH2CH2CH2-.

[0151] In some embodiments, p is 1. In some embodiments, Y is -X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 In some embodiments, Y is -X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -X 1 -, wherein at least one alkenylene group in Y is replaced by one or more selected from R 50 In some embodiments, Y is -X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -X 1 -, wherein at least one alkynylene group in Y is replaced by one or more selected from R 50 In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0152] In some embodiments, p is 1. In certain embodiments, Y is -X 1 -C 1-4 Alkylene-[X 1 -C 1-4 Alkylene] n -X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0153] In some embodiments, p is 1. In certain embodiments, Y is -X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X1 -C 1-4 Alkylene-X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 In certain embodiments, Y is -X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 In certain embodiments, Y is -X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 substituted by a substituent.

[0154] In some embodiments, p is 0. In some embodiments, Y is -X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 In some embodiments, Y is -X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -, wherein at least one alkenylene group in Y is replaced by one or more selected from R 50 wherein the alkenylene group in Y is optionally substituted by one or more selected from R 51 In some embodiments, Y is -X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -, wherein at least one alkynylene group in Y is replaced by one or more selected from R 50 wherein the alkynylene group in Y is optionally substituted by one or more selected from R 51In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0155] In some embodiments, p is 0. In some embodiments, Y is -X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 In some embodiments, Y is -X 1 -C 1-4 Alkylene-[X 1 -C 1-4 Alkylene] n -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 In some embodiments, Y is -X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-, wherein at least one alkylene in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, R 51 is independently selected from halogen, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)-. In some embodiments, R 51 is halogen. In some embodiments, R 51 is -CN. In some embodiments, R 51 is -NO2. In some embodiments, R 51 In some embodiments, R 51 is -NH2. In some embodiments, R 51 is -C(O)NH2. In some embodiments, R 51 It is -C(O)-.

[0156] In some embodiments, X 1and X 2 are independently selected from -N(R 10 )–, –C(O)–, and –N(R 10 )C(O)—. In some embodiments, X 1 and X 2 are independently selected from -NH-, -C(O)-, and -N(R 10 )C(O)—. In some embodiments, X 1 and X 2 are independently selected from -C(O)-, and -N(R 10 )C(O)—. In some embodiments, X 1 and X 2 are independently selected from -C(O)-, and -NHC(O)-.

[0157] In certain embodiments, R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, where R 50 Each alkylene group is optionally substituted by one or more substituents selected from the following: halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 In some embodiments, R 50 Yes – C 1-4 Alkylene-X 2 -[C 1-4 Alkylene] m -POLY, where R 50 Each alkylene group is optionally substituted by one or more substituents selected from the following: halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10In some embodiments, R 50 Each alkylene group is optionally substituted by one or more substituents selected from the following: halogen, -OH, -N(R 10 )2、-C(O)N(R 10 )2、-C(O)-、C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 In some embodiments, m is 0. In some embodiments, m is 1.

[0158] In certain embodiments, R 50 Yes – C 2-6 Alkenylene-X 2 -[C 2-6 alkenylene] m -POLY, where R 50 Each alkenylene group is optionally substituted by one or more substituents selected from the following: halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 In some embodiments, m is 0. In some embodiments, m is 1.

[0159] In certain embodiments, R 50 Yes – C 2-6 Alkynylidene-X 2 -[C 2-6 Alkynylidene] m -POLY, where R 50 Each alkynylene group is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10In some embodiments, m is 0. In some embodiments, m is 1.

[0160] In certain embodiments, POLY is polyethylene glycol (PEG), methoxy polyethylene glycol (mPEG), polypropylene glycol (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinicalcohols), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamides), poly(hydroxyalkylmethacrylates), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholine), polysarcosine, or combinations thereof. In some embodiments, POLY is polyethylene glycol (PEG). In some embodiments, POLY is methoxy polyethylene glycol (mPEG). In some embodiments, POLY is polypropylene glycol (PPG). In some embodiments, POLY is copolymers of ethylene glycol and propylene glycol. In some embodiments, POLY is poly(oxyethylated polyols). In some embodiments, POLY is poly(olefinicalcohols). In some embodiments, POLY is poly(vinylpyrrolidone). In some embodiments, POLY is poly(hydroxyalkyl methacrylamide). In some embodiments, POLY is poly(hydroxyalkyl methacrylate). In some embodiments, POLY is poly(saccharide). In some embodiments, POLY is poly(α-hydroxy acid). In some embodiments, POLY is poly(vinyl alcohol). In some embodiments, POLY is polyphosphazene. In some embodiments, POLY is polyoxazolines (POZ). In some embodiments, POLY is poly(N-acryloylmorpholine). In some embodiments, POLY is polysarcosine. In some embodiments, POLY is a non-peptide, water-soluble polymer. In some embodiments, POLY comprises polyethylene glycol (PEG) or methoxypolyethylene glycol (mPEG). In some embodiments, POLY is in represents the point of attachment to the rest of the compound, and wherein n1 is an integer from 1 to 20. In certain embodiments, n1 is an integer between 5 and 15. In some embodiments, n1 is 1. In some embodiments, n1 is 2. In some embodiments, n1 is 3. In some embodiments, n1 is 4. In some embodiments, n1 is 5. In some embodiments, n1 is 6. In some embodiments, n1 is 7. In some embodiments, n1 is 8. In some embodiments, n1 is 9. In some embodiments, n1 is 10. In some embodiments, n1 is 11. In some embodiments, n1 is 12. In some embodiments, n1 is 13. In some embodiments, n1 is 14. In some embodiments, n1 is 15. In some embodiments, n1 is 16. In some embodiments, n1 is 17. In some embodiments, n1 is 18. In some embodiments, n1 is 19. In some embodiments, n1 is 20. In some embodiments, n1 is 21. In some embodiments, n1 is 22. In some embodiments, n1 is 23. In some embodiments, n1 is 24. In some embodiments, n1 is 25. In some embodiments, n1 is 26. In some embodiments, n1 is 27. In some embodiments, n1 is 28. In some embodiments, n1 is 29. In some embodiments, n1 is 30.

[0161] In certain embodiments, the RL comprises an alkyne, a cyclooctyne, a strained olefin, a tetrazine, an amine, a methylcyclopropene, a thiol, a p-acetylphenylalanine residue, an oxyamine, a maleimide, or an azide. In some embodiments, the RL comprises an alkyne. In some embodiments, the RL comprises a cyclooctyne. In some embodiments, the RL comprises a strained olefin. In some embodiments, the RL comprises a tetrazine. In some embodiments, the RL comprises an amine. In some embodiments, the RL comprises a methylcyclopropene. In some embodiments, the RL comprises a thiol. In some embodiments, the RL comprises a p-acetylphenylalanine residue. In some embodiments, the RL comprises an oxyamine. In some embodiments, the RL comprises a maleimide. In some embodiments, the RL comprises an azide. In certain embodiments, the RL is selected from the group consisting of: –N3, –NH2, and –SH; among which R T It is C 1-6 Alkyl; and In some embodiments, RL is selected from the group consisting of: Where R T It is C 1-6 Alkyl; and In some embodiments, RL is selected from the group consisting of: In some embodiments, the RL is selected from the group consisting of: In certain embodiments, the RL is selected from the group consisting of: In some embodiments, RL is In some embodiments, RL is selected from the group consisting of: represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is Where R T It is C 1-6 Alkyl, and In some embodiments, R T is methyl, ethyl, or propyl. In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is In some embodiments, RL is -N3. In some embodiments, RL is -NH2. In some embodiments, RL is -SH.

[0162] In some embodiments, Su is a sugar moiety. In some embodiments, Su is a hexose form of a monosaccharide. Su can be a glucuronic acid or mannose residue. In certain embodiments, Su is in represents the point of attachment to the rest of the compound. In certain embodiments, Su is in represents the point of attachment to the rest of the compound.

[0163] In some embodiments, D is an immunomodulatory payload. In some embodiments, the immunomodulatory payload is an agonist of stimulator of interferon genes (STING), Toll-like receptor 7 (TLR7), Toll-like receptor 7 / 8 (TLR7 / 8), or Toll-like receptor 8 (TLR8). In some embodiments, the immunomodulatory payload is an agonist of stimulator of interferon genes (STING). In some embodiments, the immunomodulatory payload is an agonist of Toll-like receptor 7 (TLR7). In some embodiments, the immunomodulatory payload is an agonist of Toll-like receptor 7 / 8 (TLR7 / 8). In some embodiments, the immunomodulatory payload is an agonist of Toll-like receptor 8 (TLR8). In some embodiments, the STING agonist is selected from the group consisting of: small molecule agonists of the STING pathway, antibodies that activate STING activity, recombinant proteins that activate the STING pathway, TTI-10001, DMXAA (ASA404), CDNs, c-di-GMP, 2′3′-cGAMP, MK-1454, ADU-S100 (MIW815), SB11285, ADU-V19, IACS-8779, IACS-8803, IMSA101, non-CDNs, E7766, MK-2118, diABZI, MSA-2, JNJ-'6196, bacterial vectors, SYNB1891, and STACT (see, e.g., Luo et al. Molecules 2022, 27, 4638, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the STING agonist is a small molecule agonist of the STING pathway. In some embodiments, the STING agonist is an antibody that activates STING activity. In some embodiments, the STING agonist is a recombinant protein that activates the STING pathway. In some embodiments, the STING agonist is TTI-10001. In some embodiments, the STING agonist is DMXAA (ASA404). In some embodiments, the STING agonist is a CDN class. In some embodiments, the STING agonist is c-di-GMP. In some embodiments, the STING agonist is 2′3′-cGAMP. In some embodiments, the STING agonist is MK-1454. In some embodiments, the STING agonist is ADU-S100 (MIW815). In some embodiments, the STING agonist is SB11285. In some embodiments, the STING agonist is ADU-V19. In some embodiments, the STING agonist is IACS-8779.In some embodiments, the STING agonist is IACS-8803. In some embodiments, the STING agonist is IMSA101. In some embodiments, the STING agonist is a non-CDN. In some embodiments, the STING agonist is E7766. In some embodiments, the STING agonist is MK-2118. In some embodiments, the STING agonist is diABZI. In some embodiments, the STING agonist is MSA-2. In some embodiments, the STING agonist is JNJ-'6196. In some embodiments, the STING agonist is a bacterial vector. In some embodiments, the STING agonist is SYNB1891. In some embodiments, the STING agonist is STACT.

[0164] In some embodiments, the Toll-like receptor 7 (TLR7) agonist is selected from the group consisting of: GS-986, PRTX-007, PRX-034, S-34240, MBS-8, and APR-002 (see, e.g., Bhagchandani et al. Advanced Drug Delivery Reviews 2021, 175, 113803, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the Toll-like receptor 7 (TLR7) agonist is GS-986. In some embodiments, the Toll-like receptor 7 (TLR7) agonist is PRTX-007. In some embodiments, the Toll-like receptor 7 (TLR7) agonist is PRX-034. In some embodiments, the Toll-like receptor 7 (TLR7) agonist is S-34240. In some embodiments, the Toll-like receptor 7 (TLR7) agonist is MBS-8. In some embodiments, the Toll-like receptor 7 (TLR7) agonist is APR-002.

[0165] In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is selected from the group consisting of imiquimod (R837), resiquimod (R848), 852-A (PF-4878691), vesatolimod (GS-9620), AZD8848, motolimod (VTX-2337), selgantolimod (GS-9688), NKTR-262, RG-7854 (RO 7020531), DSP-0509, BDB-001, BDC-1001, LHC-165, SHR-2150, JNJ-4964 (TQ-73334), RO-7119929, DN-1508052, VTX-1463, BNT-411 (SC1), APR-003, ALT-702, TRANSCON, VX-001, SNAPvax, R848-HA, SM360320, and GSK2245035 (see, e.g., Bhagchandani et al. Advanced Drug Delivery Reviews 2021, 175, 113803; and Evans et al. ACS Omega 2019, 4, 13, 15665, the contents of each of which are incorporated herein by reference in their entirety). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is imiquimod (R837). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is resiquimod (R848). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is 852-A (PF-4878691). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is visamod (GS-9620). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is AZD8848. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is motomod (VTX-2337). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is selgantolimod (GS-9688). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is NKTR-262. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is RG-7854 (RO 7020531). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is DSP-0509.In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is BDB-001. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is BDC-1001. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is LHC-165. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is SHR-2150. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is JNJ-4964 (TQ-73334). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is RO-7119929. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is DN-1508052. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is VTX-1463. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is BNT-411 (SC1). In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is APR-003. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is ALT-702. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is TRANSCON. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is VX-001. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is SNAPvax. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is R848-HA. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is SM360320. In some embodiments, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist is GSK2245035.

[0166] In some embodiments, the Toll-like receptor 8 (TLR8) agonist is selected from the group consisting of: SBT-6050, SBT-6290, and ZM-TLR8 agonist. In some embodiments, the Toll-like receptor 8 (TLR8) agonist is SBT-6050. In some embodiments, the Toll-like receptor 8 (TLR8) agonist is SBT-6290. In some embodiments, the Toll-like receptor 8 (TLR8) agonist is a ZM-TLR8 agonist.

[0167] In certain embodiments, D is a cytotoxic agent or a cytotoxic payload. In certain embodiments, D is an alkylating agent or an alkylating payload. In certain embodiments, D is a bifunctional alkylating agent. In some embodiments, D is a bifunctional alkylating agent selected from the group consisting of cyclophosphamide, nitrogen mustard, chlorambucil, and melphalan. In some embodiments, D is cyclophosphamide. In some embodiments, D is nitrogen mustard. In some embodiments, D is chlorambucil. In some embodiments, D is melphalan. In some embodiments, D is a monofunctional alkylating agent. In some embodiments, D is a monofunctional alkylating agent selected from the group consisting of dacarbazine, nitrosoureas, and temozolomide. In some embodiments, D is dacarbazine. In some embodiments, D is nitrosoureas. In some embodiments, D is temozolomide. In certain embodiments, D is a cytoskeletal disruptor (e.g., a taxane). In some embodiments, D is a cytoskeletal disruptor selected from the group consisting of paclitaxel, docetaxel, albumin-bound paclitaxel (abraxane), and taxotere. In some embodiments, D is paclitaxel. In some embodiments, D is docetaxel. In some embodiments, D is albumin-bound paclitaxel (abraxane). In some embodiments, D is taxotere. In certain embodiments, D is an epothilone. In some embodiments, D is an epothilone selected from the group consisting of epothilone A, epothilone B, epothilone C, epothilone D, and ixabepilone. In some embodiments, D is epothilone A. In some embodiments, D is epothilone B. In some embodiments, D is epothilone C. In some embodiments, D is epothilone D. In some embodiments, D is ixabepilone. In certain embodiments, D is a histone deacetylase inhibitor. In some embodiments, D is a histone deacetylase inhibitor selected from the group consisting of vorinostat and romidepsin. In some embodiments, D is vorinostat. In some embodiments, D is romidepsin. In certain embodiments, D is a kinase inhibitor. In some embodiments, D is a kinase inhibitor selected from the group consisting of bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib. In some embodiments, D is bortezomib. In some embodiments, D is erlotinib. In some embodiments, D is gefitinib. In some embodiments, D is imatinib. In some embodiments, D is vemurafenib. In some embodiments, D is vismodegib. In certain embodiments, D is a nucleotide analog and / or precursor analog. In some embodiments, D is a nucleotide analog and / or precursor analog selected from the group consisting of azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine (formerly known as mercaptoguanine). In some embodiments, D is azacitidine. In some embodiments, D is azathioprine.In some embodiments, D is capecitabine. In some embodiments, D is cytarabine. In some embodiments, D is doxifluridine. In some embodiments, D is fluorouracil. In some embodiments, D is gemcitabine. In some embodiments, D is hydroxyurea. In some embodiments, D is mercaptopurine. In some embodiments, D is methotrexate. In some embodiments, D is thioguanine (formerly known as mercaptoguanine). In certain embodiments, D is a peptide antibiotic. In some embodiments, D is a peptide antibiotic selected from the group consisting of: bleomycin and actinomycin. In some embodiments, D is bleomycin. In some embodiments, D is actinomycin. In certain embodiments, D is a platinum-based drug or a platinum-based payload. In some embodiments, D is a platinum-based drug or a platinum-based payload selected from the group consisting of: carboplatin, cisplatin, and oxaliplatin. In some embodiments, D is carboplatin. In some embodiments, D is cisplatin. In some embodiments, D is oxaliplatin. In certain embodiments, D is a retinoid. In certain embodiments, D is a retinoid selected from the group consisting of tretinoin, alitretinoin, and bexarotene. In certain embodiments, D is tretinoin. In certain embodiments, D is alitretinoin. In certain embodiments, D is bexarotene. In certain embodiments, D is a vinca alkaloid and its derivatives. In certain embodiments, D is a vinca alkaloid and its derivatives selected from the group consisting of vinblastine, vincristine, vindesine, and vinorelbine. In certain embodiments, D is vinblastine. In certain embodiments, D is vincristine. In certain embodiments, D is vindesine. In certain embodiments, D is vinorelbine. In certain embodiments, the cytotoxic agent or cytotoxic payload is a tubulin inhibitor, a DNA topoisomerase I inhibitor, or a DNA topoisomerase II inhibitor. In certain embodiments, the cytotoxic agent or cytotoxic payload is a tubulin inhibitor. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase I inhibitor. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase I inhibitor selected from the group consisting of irinotecan, SN-38, topotecan, exatecan. In some embodiments, the cytotoxic agent or cytotoxic payload is irinotecan. In some embodiments, the cytotoxic agent or cytotoxic payload is SN-38. In some embodiments, the cytotoxic agent or cytotoxic payload is topotecan. In some embodiments, the cytotoxic agent or cytotoxic payload is exatecan. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase II inhibitor.In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase II inhibitor selected from the group consisting of: etoposide, teniposide, and tafluposide. In some embodiments, the cytotoxic agent or cytotoxic payload is etoposide. In some embodiments, the cytotoxic agent or cytotoxic payload is teniposide. In some embodiments, the cytotoxic agent or cytotoxic payload is tafluposide. In certain embodiments, D is selected from the group consisting of: hemiasterlins, camptothecins, and anthracyclines. Anthracyclines may include PNU-159682 and EDA PNU-159682 derivatives. In certain embodiments, the anthracycline is selected from the group consisting of: daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin. In some embodiments, the anthracycline is daunorubicin. In some embodiments, the anthracycline is doxorubicin. In some embodiments, the anthracycline is epirubicin. In some embodiments, the anthracycline is idarubicin. In some embodiments, the anthracycline is mitoxantrone. In some embodiments, the anthracycline is valrubicin. In some embodiments, D is hemicycline. In some embodiments, D is camptothecin. In some embodiments, D is an anthracycline. In some embodiments, D is PNU-159682. In some embodiments, D is an EDA PNU compound. In some embodiments, D is an EDA PNU-159682 derivative. In certain embodiments, D is hemicycline, exitecan, PNU-159682, or an EDA PNU-159682 derivative. In some embodiments, D is hemicycline. In some embodiments, D is exitecan. In some embodiments, D is PNU-159682. In some embodiments, D is an EDA PNU-159682 compound or derivative.

[0168] Representative conjugates of the present invention include conjugates represented by formula (II), (IIA) and (IIB), as shown in Table 2. Table 2

[0169] In certain embodiments, the conjugate of formula (II), (IIA) or (IIB) is selected from the group consisting of: or a pharmaceutically acceptable salt of any one thereof, wherein is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is and a pharmaceutically acceptable salt of any one thereof, wherein is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is in is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is in is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is in is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is in is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is in is the residue of the second compound.

[0170] In certain embodiments, the conjugate of formula (II), (IIA) or (IIB) is selected from the group consisting of: in is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is in is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is in is the residue of the second compound.

[0171] In certain embodiments, the conjugate of formula (II), (IIA) or (IIB) is selected from the group consisting of: in is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is in is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is in is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is in is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is in is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is in is the residue of the second compound. In some embodiments, the conjugate of formula (II), (IIA) or (IIB) is in is the residue of the second compound.

[0172] In certain embodiments, the conjugate of formula (II), (IIA) or (IIB) is selected from the group consisting of: or a pharmaceutically acceptable salt thereof, wherein COMP is the residue of the second compound. In certain embodiments, the conjugate of formula (II), (IIA) or (IIB) is selected from the group consisting of: or a pharmaceutically acceptable salt of any one thereof, wherein COMP is the residue of the second compound.

[0173] In certain embodiments, the conjugate of formula (II), (IIA) or (IIB) is selected from the group consisting of: or a pharmaceutically acceptable salt thereof, wherein COMP is the residue of the second compound. In certain embodiments, the conjugate of formula (II), (IIA) or (IIB) is selected from the group consisting of: or A pharmaceutically acceptable salt of any one thereof, wherein COMP is the residue of the second compound.

[0174] In some aspects, the present invention provides a conjugate having a structure shown in formula (IV): or a pharmaceutically acceptable salt thereof, wherein COMP is the residue of the second compound; L 1 Yes-C 1-6 Alkylene-; Z is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -X 1 –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -X 1 -, wherein at least one alkylene, alkenylene or alkynylene in Z is optionally substituted by one or more substituents selected from the following: halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; X 1 and X 2 are independently selected from -C(O)-, and -N(R 10 )C(O)–; R 10 At each occurrence, independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; POLY is a water-soluble polymer; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0 and 1; Su is the hexose form of a monosaccharide; CYTO is a cytotoxic agent or a cytotoxic payload; and RL is the active linker group residue.

[0175] In some embodiments, COMP is a residue of a polypeptide. In some embodiments, COMP is a residue of an antibody. In some embodiments, COMP is a residue of an antibody chain.

[0176] In some embodiments, the present invention provides a compound represented by formula (IVA): or a pharmaceutically acceptable salt thereof.

[0177] In some embodiments, the compound represented by formula (IV) has the structure represented by formula (IB): or a pharmaceutically acceptable salt thereof.

[0178] In some embodiments, L 1 Yes-C 1-3 In some embodiments, L 1 is -CH2-. In some embodiments, L 1 is -CH2CH2-. In some embodiments, L 1 It is -CH2CH2CH2-.

[0179] In some embodiments, Z is -X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 alkenylene] n -X1 –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -X 1 -, wherein at least one alkylene, alkenylene or alkynylene in Z is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 In some embodiments, Z is -X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 -, wherein the alkylene in Z is optionally substituted by one or more substituents selected from the following: halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 In some embodiments, Z is -X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 –.

[0180] In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0181] In some embodiments, X 1 and X 2 are independently selected from -C(O)-, and -N(R 10 )C(O)–.

[0182] In certain embodiments, the RL comprises an alkyne, a cyclooctyne, a strained olefin, a tetrazine, an amine, a thiol, a p-acetylphenylalanine residue, an oxyamine, a maleimide, or an azide. In some embodiments, the RL comprises an alkyne. In some embodiments, the RL comprises a cyclooctyne. In some embodiments, the RL comprises a strained olefin. In some embodiments, the RL comprises a tetrazine. In some embodiments, the RL comprises an amine. In some embodiments, the RL comprises a thiol. In some embodiments, the RL comprises a p-acetylphenylalanine residue. In some embodiments, the RL comprises an oxyamine. In some embodiments, the RL comprises a maleimide. In some embodiments, the RL comprises an azide. In certain embodiments, the RL is selected from the group consisting of: –N3, –NH2, methylcyclopropene, and –SH; where R T It is C 1-6 Alkyl; and represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is Where R T It is C 1-6 Alkyl, and In some embodiments, R T is methyl, ethyl, or propyl. In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, R T In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is represents the point of attachment to the rest of the compound. In some embodiments, RL is In some embodiments, RL is -N3. In some embodiments, RL is -NH2. In some embodiments, RL is methylcyclopropene. In some embodiments, RL is -SH.

[0183] In some embodiments, Su is a sugar moiety. In some embodiments, Su is a hexose form of a monosaccharide. Su can be a glucuronic acid or mannose residue. In certain embodiments, Su is in represents the point of attachment to the rest of the compound. In certain embodiments, Su is in represents the point of attachment to the rest of the compound.

[0184] In certain embodiments, CYTO is a cytotoxic agent or a cytotoxic payload. In certain embodiments, the cytotoxic agent or cytotoxic payload is a tubulin inhibitor, a DNA topoisomerase I inhibitor, or a DNA topoisomerase II inhibitor. In some embodiments, the cytotoxic agent or cytotoxic payload is a tubulin inhibitor. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase I inhibitor. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase I inhibitor selected from the group consisting of irinotecan, SN-38, topotecan, exatecan. In some embodiments, the cytotoxic agent or cytotoxic payload is irinotecan. In some embodiments, the cytotoxic agent or cytotoxic payload is topotecan. In some embodiments, the cytotoxic agent or cytotoxic payload is exatecan. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase II inhibitor. In some embodiments, the cytotoxic agent or cytotoxic payload is a DNA topoisomerase II inhibitor selected from the group consisting of: etoposide, teniposide, and tafluposide. In some embodiments, the cytotoxic agent or cytotoxic payload is etoposide. In some embodiments, the cytotoxic agent or cytotoxic payload is teniposide. In some embodiments, the cytotoxic agent or cytotoxic payload is tafluposide. In certain embodiments, CYTO is selected from the group consisting of: hemiasterlins, camptothecins, and anthracyclines. Anthracyclines may include PNU-159682 and EDA PNU-159682 derivatives. In some embodiments, the anthracycline is selected from the group consisting of: daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin. In some embodiments, the anthracycline is daunorubicin. In some embodiments, the anthracycline is doxorubicin. In some embodiments, the anthracycline is epirubicin. In some embodiments, the anthracycline is idarubicin. In some embodiments, the anthracycline is mitoxantrone. In some embodiments, the anthracycline is valrubicin. In some embodiments, CYTO is hemicycline. In some embodiments, CYTO is camptothecin. In some embodiments, CYTO is an anthracycline. In some embodiments, CYTO is PNU-159682. In some embodiments, CYTO is an EDA PNU compound. In some embodiments, CYTO is an EDA PNU-159682 derivative. In certain embodiments, CYTO is hemicycline, exitecan, PNU-159682, or an EDA PNU-159682 derivative. In some embodiments, CYTO is hemicycline.In some embodiments, CYTO is exotecan. In some embodiments, CYTO is PNU-159682. In some embodiments, CYTO is an EDAPNU-159682 compound or derivative. In some embodiments, CYTO is not an immunostimulatory compound.

[0185] In some embodiments, the conjugate is selected from: and a pharmaceutically acceptable salt of any one thereof, wherein is the residue of the second compound.

[0186] In some embodiments, the conjugate is and A pharmaceutically acceptable salt of any one thereof, in is the residue of the second compound. Macromolecules (COMP)

[0187] Macromolecule (COMP) can be any macromolecule that one skilled in the art considers suitable. In certain embodiments, the macromolecule is a protein, peptide, antibody or antigen-binding fragment thereof, nucleic acid, carbohydrate, or other macromolecule composed of polymerized monomers. In certain embodiments, the macromolecule is a protein. In certain embodiments, the macromolecule is an antibody or antigen-binding fragment thereof. In some embodiments, COMP is a residue of a polypeptide. In some embodiments, COMP is a residue of an antibody. In some embodiments, COMP is a residue of an antibody chain.

[0188] In some embodiments, the macromolecule is an antibody or an antigen-binding fragment thereof. In some embodiments, the macromolecule is a known antibody. Useful antibodies include, but are not limited to, rituximab ( IDEC / Genentech / Roche) (see, e.g., U.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibody approved for the treatment of non-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently being developed by Genmab, an anti-CD20 antibody described in U.S. Pat. No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769 (PCT Application No. PCT / US2003 / 040426), trastuzumab ( Genentech (see, e.g., U.S. Pat. No. 5,677,171), a humanized anti-Her2 / neu antibody approved for the treatment of breast cancer; Pertuzumab (rhuMab-2C4, ), currently under development by Genentech; anti-Her2 antibody (U.S. Patent No. 4,753,894); cetuximab ( Imclone) (U.S. Pat. No. 4,943,533; PCT Publication No. WO 96 / 40210), a chimeric anti-EGFR antibody in clinical trials for a variety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883), currently being developed by Abgenix-Immunex-Amgen; HuMax-EGFR (U.S. Pat. No. 7,247,301), currently being developed by Genmab; 425, EMD55900, EMD62000 and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864; Murthy, et al. (1987) Arch. Biochem. Biophys. 252(2):549-60; Rodeck, et al. (1987) J. Cell. Biochem. 35(4):315-20; Kettleborough, et al. (1991) Protein Eng.4(7):773-83); ICR62 (Institute of Cancer Research) (PCT Publication No. WO 95 / 20045; Modjtahedi, et al. (1993) J. Cell. Biophys. 22 (I-3): 129-46; Modjtahedi, et al. (1993) Br.J.Cancer 67(2):247-53; Modjtahedi, et al. (1996) Br. J. Cancer 73(2):228-35; Modjtahedi, et al. (2003) Int. J. Cancer 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada; and Centrode Immunologia Molecular, Cuba) (U.S. Patent No. 5,891,996; U.S. Patent No. 6,506,883; Mateo, et al. (1997) Immunotechnol. 3 (1): 71-81); mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth, et al. (2003) Proc. Natl. Acad. Sci. USA.100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT Publication No. WO 01 / 62931A2); and SC100 (Scancell) (PCT Publication No. WO 01 / 88138); alemtuzumab (. Millenium), a humanized mAb currently approved for the treatment of B-cell chronic lymphocytic leukemia; muromonab CD3 (Orthoclone ), an anti-CD3 antibody developed by OrthoBiotech / Johnson & Johnson; Ibritumomab It is an anti-CD20 antibody developed by IDEC / Schering AG; gemtuzumab ozogamicin It is an anti-CD33 (P67 protein) antibody developed by Celltech / Wyeth; Afacept Anti-LFA-3 Fc fusion protein developed by Biogen; Abciximab Developed by Centocor / Eli Lilly; basiliximab Developed by Novartis; palivizumab Developed by Medimmune; infliximab An anti-TNFα antibody developed by Centocor; adalimumab Anti-TNFα antibody developed by Abbott; Anti-TNFα antibody developed by Celltech; golimumab (CNTO-148), a fully human TNF antibody developed by Centocor; etanercept p75 TNF receptor Fc fusion protein being developed by Immunex / Amgen; Ienercept, a p55 TNF receptor Fc fusion protein previously developed by Roche; ABX-CBL, an anti-CD147 antibody being developed by Abgenix; ABX-IL8, an anti-IL8 antibody being developed by Abgenix; ABX-MA1, an anti-MUC18 antibody being developed by Abgenix; Pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 antibody being developed by Antisoma; Therex (R1550), an anti-MUC1 antibody being developed by Antisoma; AngioMab (AS1405), being developed by Antisoma; HuBC-1, being developed by Antisoma; Thioplatin (AS1407), being developed by Antisoma; (natalizumab), an anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibody being developed by Biogen; VLA-1 mAb, an anti-VLA-1 integrin antibody being developed by Biogen; LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody being developed by Biogen; CAT-152, an anti-TGF-beta antibody being developed by Cambridge Antibody Technology; ABT 874 (J695), an anti-IL-12p40 antibody being developed by Abbott; CAT-192, an anti-TGFβ1 antibody being developed by Cambridge Antibody Technology and Genzyme; CAT-213, an anti-Eotaxin1 antibody being developed by Cambridge Antibody Technology; Anti-Blys antibody being developed by Cambridge Antibody Technology and Human Genome Sciences Inc.; TRAIL-R1 mAb, anti-TRAIL-R1 antibody being developed by Cambridge Antibody Technology and Human GenomeSciences Inc.; Bevacizumab, rhuMAb-VEGF, an anti-VEGF antibody being developed by Genentech; an anti-HER receptor family antibody being developed by Genentech; anti-tissue factor (ATF), an anti-tissue factor antibody being developed by Genentech; (omalizumab), an anti-IgE antibody being developed by Genentech; (efalizumab), an anti-CD11a antibody being developed by Genentech and Xoma; the MLN-02 antibody (formerly known as LDP-02), being developed by Genentech and Millenium Pharmaceuticals; HuMax CD4, an anti-CD4 antibody being developed by Genmab; HuMax-IL15, an anti-IL15 antibody being developed by Genmab and Amgen; HuMax-Inflam, being developed by Genmab and Medarex; HuMax-Cancer, an anti-heparanase I antibody being developed by Genmab, Medarex and Oxford GlycoSciences; HuMax-Lymphoma, being developed by Genmab and Amgen; HuMax-TAC, being developed by Genmab; IDEC-131, an anti-CD40L antibody being developed by IDEC Pharmaceuticals; IDEC-151 (cleliximab), an anti-CD40L antibody being developed by IDEC Pharmaceuticals; and an anti-CD4 antibody being developed by IDEC Pharmaceuticals; IDEC-114, an anti-CD80 antibody being developed by IDEC Pharmaceuticals; IDEC-152, an anti-CD23 antibody being developed by IDEC Pharmaceuticals; an anti-macrophage migration factor (MIF) antibody being developed by IDEC Pharmaceuticals; BEC2, an anti-idiotypic antibody being developed by Imclone; IMC-1C11, an anti-KDR antibody being developed by Imclone; DC101, an anti-flk-1 antibody being developed by Imclone; an anti-VE-cadherin antibody being developed by Imclone; (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibody being developed by Immunomedics; (epratuzumab), an anti-CD22 antibody being developed by Immunomedics; AFP-Cide being developed by Immunomedics; MyelomaCide being developed by Immunomedics; LkoCide being developed by Immunomedics; ProstaCide being developed by Immunomedics; MDX-010, an anti-CTLA4 antibody being developed by Medarex; MDX-060, an anti-CD30 antibody being developed by Medarex; MDX-070 being developed by Medarex; MDX-018 being developed by Medarex; and Immuno-Designed Molecules being developed by Medarex and Medarex. (IDM-1) and anti-Her2 antibody; -CD4, which is an anti-CD4 antibody being developed by Medarex and Genmab; HuMax-IL15, which is an anti-IL15 antibody being developed by Medarex and Genmab; CNTO 148, which is an anti-TNFα antibody being developed by Medarex and Centocor / J&J; CNTO 1275, which is an anti-cytokine antibody being developed by Centocor / J&J; MOR101 and MOR102, which are anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies being developed by MorphoSys; MOR201, which is an anti-fibroblast growth factor receptor 3 (FGFR-3) antibody being developed by MorphoSys; (Visilizumab), an anti-CD3 antibody being developed by Protein Design Labs; which is an anti-gamma interferon antibody being developed by Protein Design Labs; anti-alpha5beta1 interferon antibody being developed by Protein Design Labs; anti-IL-12 being developed by Protein Design Labs; ING-1, which is an anti-Ep-CAM antibody being developed by Xoma; (omalizumab), a humanized anti-IgE antibody being developed by Genentech and Novartis; and MLN01, an anti-β2 integrin antibody being developed by Xoma.

[0189] In another embodiment, the therapeutic agent includes KRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for Cancer Research); CNTO 95 (αV integrin, Centocor); MEDI-522 (αVβ3 integrin, Medimmune); volociximab (αVβ1 integrin, Biogen / PDL); human mAb 216 (B cell glycosylation antigen epitope, NCL); BiTE MT103 (bispecific CD19×CD3, MedImmune); 4G7×H22 (bispecific B cell×FcγR1, Medarex / Merck KGa); rM28 (bispecific CD28×MAPG, European Patent No. EP1444268); MDX447 (EMD 82633) (bispecific CD64 × EGFR, Medarex); catumaxomab (removab) (bispecific EpCAM × anti-CD3, Trion / Fres); ertuinomab (bispecific HER2 / CD3, Fresenius Biotech); oregovomab (OvaRex) (CA-125, ViRexx); (WX G250) (Carbonic Anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105 (CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Brystol Myers Squibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507) (CD2, Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituxan (CD20, Genentech); veltuzumab (hA20) (CD20, Immunomedics); Epratuzumab (CD22, Amgen); Ruximab (IDEC 152)(CD23, Biogen); muromonab-CD3(CD3, Ortho); HuM291(CD3 fc receptor, PDL Biopharma); HeFi-1(CD30, NCl); MDX-060(CD30, Medarex); MDX-1401(CD30, Medarex); SGN-30(CD30, Seattle Genentics); SGN-33(lintuzumab)(CD33, Seattle Genentics); zanolimumab(HuMax-CD4)(CD4, Genmab); HCD122(CD40, Novartis); SGN-40(CD40, Seattle =Genentics); MabCampath (alemtuzumab) (CD52, Genzyme); MDX-1411 (CD70, Medarex); hLL1 (EPB-1) (CD74.38, Immunomedics); Galiximab (IDEC-144) (CD80, Biogen); MT293 (TRC093 / D93) (cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma); ipilimumab (MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab (Ticilimumab, CP-675,2) (CTLA4, Pfizer); HGS-ETR1 (mapamumab) (DR4TRAIL-R1 agonist, Human Genome Science / Glaxo Smith Kline); AMG-655 (DR5, Amgen);Apomab (DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo); HGS-ETR2 (lexalimumab) (DR5TRAIL-R2 agonist, HGS); Cetuximab (Erbitux) (EGFR, Imclone); IMC-11F8, (EGFR, Imclone); Nimotuzumab (EGFR, YM Bio); Vectabix (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANTImmunotherapeutics); adecatumumab (MT201) (Epcam, Merck); edrecolomab (Panorex, 17-1A) (Epcam, Glaxo / Centocor); MORAb-003 (folate receptor a, Morphotech); KW-2871 (ganglioside GD3, Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex); trastuzumab (Herceptin) (HER2, Celldex); pertuzumab (rhuMAb 2C4)(HER2(DI), Genentech); apolizumab(HLA-DRβchain, PDL Pharma); AMG-479(IGF-1R, Amgen); anti-IGF-1R R1507(IGF1-R, Roche); CP751871(IGF1-R, Pfizer); IMC-A12(IGF1-R, Imclone); BIIB022(IGF-1R, Biogen); Mik-β-1(IL-2Rb(CD122), Hoffman LaRoche); CNTO328 (IL6, Centocor); anti-KIR (1-7F9) (Killer cell Ig-like receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute for Cancer Research); hCBE-11 (LTβR, Biogen); HuHMFG1 (MUC1, Antisoma / NCL); RAV12 (N-linked carbohydrate epitope, Raven); CAL (parathyroid hormone-related protein (PTH-RP), University of California); CT-011 (PD1, CureTech); MDX-1106 (ono-4538) (PD1, Medarex / Ono); Mab CT-011 (PD1, Curetech);IMC-3G3 (PDGFRa, Imclone); bavituximab (phosphatidylserine, Peregrine); huJ591 (PSMA, Cornell Research Foundation); muJ591 (PSMA, Cornell Research Foundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme); infliximab (Remicade) (TNFa, Centocor); A27.15 (transferrin receptor, Salk Institute, INSERN WO 2005 / 111082); E2.3 (transferrin receptor, Salk Institute); bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab, PCT Publication No. WO / 2000 / 034337, University ofTexas); IMC-18F1 (VEGFR1, Imclone); IMC-1121 (VEGFR2, Imclone). ;

[0190] Examples of useful bispecific antibodies include, but are not limited to, those having one antibody directed against a tumor cell antigen and another directed against a cytotoxic triggering molecule, such as anti-FcγRI / anti-CD15, anti-p185 HER2 / FcγRIII(CD16), anti-CD3 / anti-malignant B cells(1D10), anti-CD3 / anti-p185 HER2, anti-CD3 / anti-p97, anti-CD3 / anti-renal cell carcinoma, anti-CD3 / anti-OVCAR-3, anti-CD3 / L-D1 (anti-colon cancer), anti-CD3 / anti-melanocyte stimulating hormone analog, anti-EGF receptor / anti-CD3, anti-CD3 / anti-CAMA1, anti-CD3 / anti-CD19, anti-CD3 / MoV18, anti-neural cell adhesion molecule (NCAM) / anti-CD3, anti-folate binding protein (FBP) / anti-CD3, anti-pan-cancer associated antigen (AMOC-31) / anti-CD3; Bispecific antibodies with one antibody that specifically binds to a tumor antigen and another antibody that binds to a toxin, such as anti-saporin / anti-Id-1, anti-CD22 / anti-saporin, anti-CD7 / anti-saporin, anti-CD38 / anti-saporin, anti-CEA / anti-ricin A chain, anti-interferon alpha (IFN-α) / anti-hybridoma idiotype, anti-CEA / anti-vinca alkaloid; bispecific antibodies for converting enzyme-activated prodrugs, such as anti-CD30 / anti-alkaline phosphatase (which converts mitomycin phosphate to phosphate) prodrug catalyzed conversion into mitomycin alcohol); bispecific antibodies that can be used as fibrinolytic agents, such as anti-fibrinogen / anti-tissue plasminogen activator (tPA), anti-fibrinogen / anti-urokinase-type plasminogen activator (uPA); immune complex bispecific antibodies for targeting cell surface receptors, such as anti-low-density lipoprotein (LDL) / anti-Fc receptor (e.g., FcγRI, FcγRII or FcγRIII); bispecific antibodies for treating infectious diseases, such as anti-CD3 / anti-herpes simplex virus (HSV), anti-T cell receptor:CD3 complex / anti-influenza, anti-FcγR / anti-HIV; bispecific antibodies for in vitro or in vivo tumor detection, such as anti-CEA / anti-EOTUBE, anti-CEA / anti-DPTA, and anti-p185 HER2 / anti-half antibody; bispecific antibodies as vaccine adjuvants (see Fanger, MW et al., Crit Rev Immunol. 1992; 12 (34): 101-24, which is incorporated herein by reference); and bispecific antibodies as diagnostic tools, such as anti-rabbit IgG / anti-ferritin, anti-horseradish peroxidase (HRP) / anti-hormone, anti-somatostatin / anti-substance P, anti-HRP / anti-FITC, anti-CEA / anti-β-galactosidase (see Nolan, O. and O'Kennedy, R., Biochim Biophys Acta. 1990 Aug. 1; 1040 (1): 1-11, which is incorporated herein by reference). Examples of trispecific antibodies include anti-CD3 / anti-CD4 / anti-CD37, anti-CD3 / anti-CD5 / anti-CD37, and anti-CD3 / anti-CD8 / anti-CD37. Coupling

[0191] In certain embodiments, the conjugate may be formed by a macromolecule comprising one or more active (reactive) groups. In certain embodiments, the conjugate may be formed by a macromolecule comprising all naturally encoded amino acids. Those skilled in the art will recognize that several naturally encoded amino acids include active groups that can be coupled to compounds or linkers shown in formula (I), (IA), (IB), (III), (IIIA), or (IIIB). These active groups include cysteine ​​side chains, lysine side chains, and amino terminal groups. In these embodiments, the conjugate may include compounds or linkers shown in formula (I), (IA), (IB), (III), (IIIA), or (IIIB) that are connected to antibody active group residues. In these embodiments, compound precursors or linker precursors shown in formula (I), (IA), (IB), (III), (IIIA), or (IIIB) include active groups that can form bonds with antibody or antigen-binding fragment active groups thereof. Typical reactive groups include maleimide groups, activated carbonates (including but not limited to, p-nitrophenyl esters), activated esters (including but not limited to, N-hydroxysuccinimide, p-nitrophenyl esters, and aldehydes). Particularly useful reactive groups include maleimide and succinimide, such as N-hydroxysuccinimide, for forming bonds with cysteine ​​and lysine side chains. Further reactive groups will be described in the following sections and examples. Active Group

[0192] The reactive group can promote the coupling of the compound of formula (I), (IA), (IB), (III), (IIIA), or (IIIB) described in the present invention with a second compound (e.g., a macromolecule described in the present invention (i.e., COMP)). In certain embodiments, the reactive group is designated as RG in the present invention. The reactive group can react via any suitable reaction mechanism known to those skilled in the art. In certain embodiments, the reactive group reacts via a [3+2] alkyne-azide cycloaddition reaction, a reverse electron demand Diels-Alder ligation reaction, a thiol-electrophile reaction, or a carbonyl-oxamine reaction, as described in detail herein. In certain embodiments, the reactive group comprises an alkyne, a strained olefin, a tetrazine, a thiol, a para-acetylphenylalanine residue, an oxamine, a maleimide, or an azide. In certain embodiments, the reactive group is –N3, or –SH; where R 201 In certain embodiments, R 201 is methyl, ethyl or propyl. 201 In some embodiments, R 201 In some embodiments, R201 Other active groups are described in, for example, U.S. Patent Application Publication No. US2014 / 0356385, U.S. Patent Application Publication No. US2013 / 0189287, U.S. Patent Application Publication No. US2013 / 0251783, U.S. Patent No. US8,703,936, U.S. Patent No. US9,145,361, U.S. Patent No. US9,222,940 and U.S. Patent No. US8,431,558.

[0193] After coupling, a divalent residue of the reactive group (ie, RG') is formed and is linked to the residue of the second compound (eg, COMP). The structure of the divalent residue is determined by the type of coupling reaction used to form the conjugate.

[0194] [3+2] Alkyne-Azide Cycloaddition

[0195] Advantageously, compounds comprising a coupled alkyne group (e.g., compounds of formula (I), (IA), or (IB)) or compounds comprising an azide group of the present invention are conducive to selective and efficient reaction with a second compound comprising a complementary azide group or alkyne group. It is believed that the azide group and the alkyne group react in a 1,3-dipolar cycloaddition reaction to form a 1,2,3-triazolyl radical moiety, which connects the compound of formula (I), (IA), or (IB) or compound comprising an azide group of the present invention to the second compound. This reaction between the azide group and the alkyne group to form a triazole is generally referred to by those skilled in the art as a Huisgen cycloaddition reaction or a [3+2] alkyne-azide cycloaddition reaction.

[0196] The unique reactivity of azide and alkyne functional groups makes them useful for selective modification of peptides and other biomolecules. Organic azides (particularly aliphatic azides) and alkynes are generally stable to common reactive chemical conditions. In particular, both azide and alkyne functional groups are inert to the side chains of the 20 common amino acids found in naturally occurring peptides. It is believed that when azide and alkyne groups are brought into close proximity, their "spring-loaded" nature is revealed, and the azide and alkyne groups react selectively and efficiently via a [3+2] alkyne-azide cycloaddition reaction to generate the corresponding triazoles. See, e.g., Chin J., et al., Science 301:964-7 (2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin, JW, et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

[0197] Since the [3+2] alkyne-azide cycloaddition reaction involves a selective cycloaddition reaction [see, e.g., Padwa, A., in COMPREHENSIVE ORGANIC SYNTHESIS, Vol. 4, (ed. Trost, BM, 1991), pp. 1069-1109; Huisgen, R. in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), pp. 1-176] rather than a nucleophilic substitution reaction, the introduction of non-naturally encoded amino acids with azide and alkyne-containing side chains allows the resulting polypeptide to be selectively modified at the position of the non-naturally encoded amino acid. Cycloaddition reactions involving azide or alkyne-containing compounds can be carried out at room temperature under aqueous conditions by adding Cu(II) (including but not limited to a catalytic amount of CuSO4) and in the presence of a reducing agent to reduce Cu(II) in situ to Cu(I) in a catalytic amount. See, e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe, CW, et al., J. Org. Chem. 67: 3057-3064 (2002); Rostovtsev, et al., Angew. Chem. Int. Ed. 41: 2596-2599 (2002). Exemplary reducing agents include, but are not limited to, ascorbic acid, metallic copper, quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe 2+ 、Co 2+ , and the applied potential.

[0198] In certain embodiments, when the conjugate is formed by a [3+2] alkyne-azide cycloaddition reaction, the divalent residue of the reactive group (e.g., RG′) comprises a triazole ring or a fused cyclic group containing a triazole ring. In certain embodiments, when the conjugate is formed by a strain-promoted [3+2] alkyne-azide cycloaddition (SPAAC) reaction, the divalent residue of the reactive group (e.g., RG′) is and / or

[0199] Reverse electron demand ligation

[0200] Advantageously, compounds comprising a terminal tetrazine or strained olefin group can promote selective and efficient reaction with a second compound comprising a strained olefin or tetrazine group. It is believed that the tetrazine and strained olefin react in a reverse electron demand Diels-Alder reaction followed by a reverse Diels-Alder reaction, which connects the compound comprising a terminal tetrazine or strained olefin group to the second compound. It is believed that the reaction is specific and has little cross-reactivity with functional groups in biomolecules. The reaction can be carried out under mild conditions, such as at room temperature without a catalyst. This reaction between a tetrazine and a strained olefin is generally referred to as a tetrazine ligation reaction by those skilled in the art.

[0201] In certain embodiments, when the conjugate is formed by a tetrazine reverse electron demand Diels-Alder ligation reaction, the divalent residue of the reactive group (e.g., RG′) comprises a fused bicyclic ring having at least two adjacent nitrogen atoms in the ring. In certain embodiments, when the conjugate is formed by a tetrazine reverse electron demand Diels-Alder ligation reaction, the divalent residue of the reactive group (e.g., RG′) is

[0202] Thiol reaction

[0203] Advantageously, compounds containing a terminal thiol group or a suitable electrophilic group or disulfide forming group are favorable for selective and efficient reaction with a second compound containing a complementary electrophilic group or disulfide forming group or thiol group. It is believed that these reactions are selective with little cross-reactivity with functional groups within biomolecules. In some embodiments, the thiol reaction does not include the reaction of the maleimide group.

[0204] In certain embodiments, when the conjugate is formed by a thiol-maleimide reaction, the divalent residue of the reactive group comprises In certain embodiments, when the conjugate is linked via a thiol-maleimide When the reaction is formed, the divalent residue of the reactive group (e.g., RG′) is

[0205] Carbonyl-oxyamine reaction

[0206] Advantageously, compounds containing a terminal carbonyl or oxamine group are favorable for selective and efficient reaction with a second compound containing an oxamine or carbonyl group. It is believed that the carbonyl group reacts with the oxamine to form an oxime bond. It is believed that the reaction is specific with little cross-reactivity with functional groups within biomolecules.

[0207] In certain embodiments, when the conjugate is formed by an oxime coupling reaction, the divalent residue of the reactive group comprises a divalent residue of an unnatural amino acid. In certain embodiments, when the conjugate is formed by an oxime coupling reaction, the divalent residue of the reactive group (e.g., RG′) is In certain embodiments, when the conjugate is formed by an oxime coupling reaction, the divalent residue of the reactive group comprises an oxime bond. In certain embodiments, when the conjugate is formed by an oxime coupling reaction, the divalent residue of the reactive group (e.g., RG′) is

[0208] Other reactions

[0209] Other suitable coupling reactions are described in the literature. See, for example, Lang, K. and Chin, J. 2014, Bioorthogonal Reactions for Labeling Proteins, ACS Chem Biol 9, 16-20; Paterson, DM et al. 2014, Finding the Right (Bioorthogonal) Chemistry, ACS Chem Biol 9, 592-605; King, M. and Wagner, A. 2014, Developments in the Field of Bioorthogonal Bond Forming Reactions–Past and Present Trends, Bioconjugate Chem., 2014, 25(5), pp825-839; and Ramil, CP and Lin, Q., 2013, Bioorthogonal chemistry: strategies and recent developments, Chem Commun 49, 11007-11022.

[0210] Release reaction

[0211] The release reaction is a reaction for releasing the biologically active part of the compound or conjugate of the present invention from the compound or conjugate in vivo and / or in vitro. In certain embodiments, the released biologically active part is a compound (e.g., a cytotoxic agent or a cytotoxic payload) described elsewhere in the present invention or a pharmaceutically acceptable salt, solvate, stereoisomer or tautomer thereof. An example of a release reaction is an intramolecular reaction between an eliminator group and a release trigger group of the compound or conjugate of the present invention to release the biologically active part of the compound or conjugate of the present invention. The elimination group itself can be decomposed into two reactive components, as shown in these reactions, where X is a drug with a heteroatom of N or O for connection. An exemplary release reaction is shown in the following scheme: Water soluble polymer

[0212] In certain embodiments, the conjugate comprises one or more water-soluble polymers. A variety of macromolecular polymers and other molecules can be connected to the polypeptide of the present invention to regulate the biological properties of the polypeptide, and / or provide new biological properties for the polypeptide. These macromolecular polymers can be connected to the polypeptide via naturally encoded amino acids, via non-naturally encoded amino acids, or any functional substituents of natural or modified amino acids, or any substituents or functional groups added to natural or modified amino acids. The molecular weight of the polymer may include a wide range, including but not limited to about 100Da to about 100,000Da, or larger.

[0213] The selected polymer may be water soluble so that the protein to which it is attached will not precipitate in an aqueous environment such as a physiological environment. The polymer may be branched or unbranched. In certain embodiments, the polymer will be pharmaceutically acceptable for therapeutic use of the final product formulation.

[0214] In certain embodiments, the ratio of polyethylene glycol molecules to polypeptide molecules will vary, and their concentrations in the reaction mixture will also vary. Generally, the optimal ratio (in terms of reaction efficiency, there is a minimum excess of unreacted protein or polymer) can be determined by the molecular weight of the selected polyethylene glycol and the number of available reactive groups. In terms of molecular weight, generally the higher the molecular weight of the polymer, the fewer the number of polymer molecules that can be attached to the protein. Similarly, when optimizing these parameters, the branching of the polymer should be considered. Generally, the higher the molecular weight (or the more branches), the higher the polymer: protein ratio.

[0215] The water-soluble polymer can be in any structural form, including but not limited to straight chain, forked or branched. Typically, the water-soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), but other water-soluble polymers may also be used. For example, PEG is used to describe certain embodiments.

[0216] PEG is a well-known water-soluble polymer that can be purchased commercially or prepared by ring-opening polymerization of ethylene oxide according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). The term "PEG" is used broadly to include any polyethylene glycol molecule, regardless of the size or modification of the PEG terminus, and can be represented as being attached to a polypeptide by the formula: X'O-(CH2CH2O) n –CH2CH2–Y′, wherein n is an integer selected from 2 to 10,000, and X′ is H or a terminal modification, including but not limited to C 1-4 alkyl, and Y' is the point of attachment to the polypeptide.

[0217] In some cases, PEG is terminated at one end with a hydroxyl or methoxy group, i.e., X' is H or CH3 (also known as "methoxy PEG"). Alternatively, the PEG can be terminated with a PEG active (reactive) group to form a bifunctional polymer. Typical PEG reactive groups may include those reactive groups that are commonly used to react with the following functional groups: functional groups present in the 20 common amino acids (including but not limited to maleimide groups, activated carbonates (including but not limited to p-nitrophenyl esters), activated esters (including but not limited to N-hydroxysuccinimide, p-nitrophenyl esters and aldehydes); and functional groups that are inert to the 20 common amino acids but specifically react with complementary functional groups present in non-naturally encoded amino acids (including but not limited to azide groups and / or alkyne groups). It should be noted that the other end of the PEG, represented by Y' in the above formula, will be directly or indirectly linked to the polypeptide via a naturally occurring or non-naturally encoded amino acid. For example, Y' can be an amide, carbamate or urea bond of an amine group of a polypeptide (including but not limited to the epsilon amine or N-terminus of lysine). Alternatively, Y' can be a thiol group (including but not limited to cysteine) Alternatively, Y' can be a bond of a residue that is not usually available via 20 common amino acids. For example, an azide group on PEG can react with an alkyne group on a polypeptide to form a Huisgen [3+2] cycloaddition product. Alternatively, an alkyne group on PEG can react with an azide group present in a non-naturally encoded amino acid (e.g., a modified amino acid described in the present invention) to form a similar product. In some embodiments, as applicable, a strong nucleophile (including but not limited to, hydrazine, hydrazide, hydroxylamine, semicarbazide) can react with an aldehyde or ketone group present in a non-naturally encoded amino acid to form a hydrazone, oxime or semicarbazide, which, in some cases, can be further reduced by treatment with an appropriate reducing agent. Alternatively, the strong nucleophile can be incorporated into a polypeptide via a non-naturally encoded amino acid and used to preferentially react with a ketone or aldehyde group present in a water-soluble polymer.

[0218] Depending on the actual needs, any molecular weight PEG can be used, including but not limited to, from about 100 Daltons (Da) to 100,000 Da or greater (including but not limited to, in certain embodiments, 0.1-50 kDa or 10-40 kDa) as required. Branched PEGs include, but are not limited to, PEG molecules where each chain has a molecular weight (MW) ranging from 1-100 kDa (including but not limited to, 1-50 kDa or 5-20 kDa). A wide range of PEG molecules are described in the product catalog of Shearwater Polymers and the product catalog of Nektar Therapeutics, both of which are incorporated herein by reference.

[0219] Typically, at least one end of the PEG molecule can be used to react with the rest of the compound shown in formula (I), (IA), or (IB). For example, PEG derivatives having alkyne and azide moieties for reacting with amino acid side chains can be used to attach PEG to non-naturally encoded amino acids as described herein. If the non-naturally encoded amino acid comprises an azide, PEG will typically either contain an alkyne moiety to achieve formation of a [3+2] cycloaddition product, or an activated PEG species (i.e., ester, carbonate) containing a phosphine group to achieve formation of an amide bond. Alternatively, if the non-naturally encoded amino acid comprises an alkyne, PEG will typically contain an azide moiety to achieve formation of a [3+2] Huisgen cycloaddition product. If the non-naturally encoded amino acid comprises a carbonyl group, the PEG will typically contain a nucleophile (including but not limited to, hydrazide, hydrazine, hydroxylamine, or semicarbazide functional group) to achieve formation of the corresponding hydrazone, oxime, and semicarbazide bonds, respectively. In other alternatives, the reverse orientation of the active (reactive) groups described herein can be used (ie, the azide portion of the non-naturally encoded amino acid can be reacted with the alkyne-containing PEG derivative).

[0220] In some embodiments, the polypeptide variant with a PEG derivative comprises a chemical functionality that reacts with a chemical functionality present on the side chain of the non-naturally encoded amino acid.

[0221] In certain embodiments, the water-soluble polymer is a polymer containing an azide or acetylene, comprising a water-soluble polymer backbone, having an average molecular weight from about 800Da to about 100,000Da. The polymer backbone of the water-soluble polymer can be polyethylene glycol. However, it should be understood that a variety of water-soluble polymers including but not limited to polyethylene glycol and other related polymers (including poly (dextran)) and polypropylene glycol) are also suitable for use, and the use of the term "PEG" or "polyethylene glycol" is intended to cover and include all such molecules. The term "PEG" further includes but is not limited to any form of polyethylene glycol, including bifunctional PEG, multi-arm PEG, derivatized PEG, forked PEG, branched PEG, pendant PEG (i.e., PEG or related polymers having one or more functional groups pendant to the polymer backbone), or PEG having a degradable bond therein.

[0222] The polymer backbone can be linear or branched. Branched polymer backbones are generally known in the art. Typically, branched polymers have a central branching core group portion and multiple linear polymer chains attached to the central branching core. PEG is typically used in a branched form that can be prepared by adding ethylene oxide to various polyols, such as glycerol, glycerol oligomers, pentaerythritol, and sorbitol. The central branching group portion can also be derived from several amino acids, such as lysine. The branched polyethylene glycol can be represented in the general form as R-(-PEG-OH) m , wherein R is derived from a core group portion, such as glycerol, glycerol oligomers, or pentaerythritol, and m represents the number of arms. Multi-arm PEG molecules, such as those described in the following documents, can also be used as the polymer backbone: U.S. Patent Nos. 5,932,462, 5,643,575, 5,229,490, and 4,289,872; U.S. Patent Application No. US2003 / 0143596; and WO96 / 21469 and WO 93 / 21259, each of which is incorporated into the present invention in its entirety by reference.

[0223] Branched PEG can also be PEG(-Y″CHZ2) n The forked PEG form represented by PEG is a linking group, and Z is an activated terminal group connected to CH with a chain of atoms of defined length.

[0224] Another branching form, pendant PEG, has PEG active (reactive) groups, such as carboxyl groups, along the PEG backbone rather than at the termini of the PEG chains.

[0225] In addition to these forms of PEG, the polymers can also be prepared using weak or degradable bonds in the backbone. For example, PEG can be prepared using ester bonds in the polymer backbone that are subject to hydrolysis. As shown in the present invention, the hydrolysis allows the polymer to be cut into lower molecular weight fragments: -PEG-CO2-PEG-+H2O→PEG-CO2H+HO-PEG-. It will be understood by those skilled in the art that the term "polyethylene glycol" or PEG represents or includes all forms known in the art, including but not limited to those disclosed in the present invention.

[0226] Many other polymers are also suitable for use. In some embodiments, a water-soluble polymer backbone having from 2 to about 300 terminals is particularly suitable. Examples of suitable polymers include, but are not limited to, other poly(alkylene glycols) (e.g., polypropylene glycol ("PPG")), copolymers thereof (including but not limited to copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like. Although the molecular weight of each chain of the polymer backbone can vary, it is generally in the range of about 800 Da to about 100,000 Da, typically about 6,000 Da to about 80,000 Da.

[0227] Those of ordinary skill in the art will recognize that the foregoing list of substantially water-soluble backbones is by no means exhaustive, but is merely exemplary, and all polymeric materials having the properties described herein are considered suitable for use.

[0228] In some embodiments, the polymer derivative is "multifunctional", meaning that the polymer backbone has at least two termini, and potentially up to about 300 termini, functionalized or activated with functional groups. Multifunctional polymer derivatives include, but are not limited to, linear polymers having two termini, wherein each terminus is attached to a functional group that may be the same or different. Composition and use Pharmaceutical composition and administration method

[0229] The conjugates provided by the present invention can be formulated into pharmaceutical compositions using methods available in the art and those disclosed in the present invention. Any conjugate provided by the present invention can be provided in a suitable pharmaceutical composition and administered by a suitable route of administration.

[0230] The method provided by the present invention includes administering a pharmaceutical composition comprising at least one conjugate provided by the present invention and one or more compatible and pharmaceutically acceptable carriers. In this case, the term "pharmaceutically acceptable" refers to those approved by the regulatory agency of the federal or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias for animals, and in certain embodiments for humans. The term "carrier" includes a diluent, adjuvant (such as Freund's adjuvant (complete and incomplete)), auxiliary material or vehicle used together with the therapeutic agent. Such drug carriers can be sterile liquids, such as water and oil, including oils from petroleum, animals, plants or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water can be used as a carrier. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Examples of suitable pharmaceutical carriers are described in Martin, EW, Remington's Pharmaceutical Sciences.

[0231] In clinical practice, the pharmaceutical composition or conjugate provided by the present invention can be administered by any route known in the art. Exemplary routes of administration include, but are not limited to, oral, inhaled, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes. In some embodiments, the pharmaceutical composition or conjugate provided by the present invention is administered orally. In some embodiments, the pharmaceutical composition or conjugate provided by the present invention is administered parenterally.

[0232] Compositions for parenteral administration can be emulsions or sterile solutions. Parenteral compositions can include, for example, propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters (e.g., ethyl oleate). These compositions can also include wetting agents, isotonic agents, emulsifiers, dispersants and stabilizers. Sterilization can be carried out in several ways, for example, using a bacterial filter, by irradiation or by heating. Parenteral compositions can also be prepared in the form of sterile solid compositions, which can be dissolved in sterile water or any other injectable sterile medium when used.

[0233] In some embodiments, the compositions provided herein are pharmaceutical compositions or single unit dosage forms. The pharmaceutical compositions and single unit dosage forms provided herein contain a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic conjugates.

[0234] The pharmaceutical composition may include one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, wherein a person of ordinary skill in the art is able to select a suitable pharmaceutical excipient. Non-limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art, including but not limited to the manner in which the dosage form is applied to the subject and the specific conjugate in the dosage form. If desired, the composition or single unit dosage form may also include a small amount of a wetting agent or emulsifier, or a pH buffer. Therefore, the pharmaceutical excipients provided below are exemplary only and are not intended to be limiting. Other pharmaceutical excipients include, for example, those described in Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), which are incorporated herein by reference in their entirety.

[0235] In some embodiments, the pharmaceutical composition comprises a defoamer. Any suitable defoamer can be used. In some aspects, the defoamer is selected from alcohol, ether, oil, wax, polysiloxane, surfactant, and a combination thereof. In some aspects, the defoamer is selected from mineral oil, vegetable oil, vinyl bis stearamide, paraffin, ester wax, fatty alcohol wax, long-chain fatty alcohol, fatty acid soap, fatty acid ester, silicon glycol, fluorosilicone, polyethylene glycol-polypropylene glycol copolymer, polydimethylsiloxane-silicon dioxide, ether, octanol, caprylic alcohol, sorbitan trioleate, ethanol, 2-ethyl-hexanol, dimethicone, oleyl alcohol, dimethicone, and a combination thereof.

[0236] In some embodiments, the pharmaceutical composition comprises a co-solvent. Illustrative examples of co-solvents include ethanol, poly(ethylene) glycol, butylene glycol, dimethylacetamide, glycerol, and propylene glycol.

[0237] In some embodiments, the pharmaceutical composition comprises a buffer. Illustrative examples of buffers include acetate, borate, carbonate, lactate, malate, phosphate, citrate, hydroxide, diethanolamine, monoethanolamine, glycine, methionine, guar gum, and monosodium glutamate.

[0238] In some embodiments, the pharmaceutical composition comprises a carrier or filler. Illustrative examples of carriers or fillers include lactose, maltodextrin, mannitol, sorbitol, chitosan, stearic acid, xanthan gum, and guar gum.

[0239] In some embodiments, the pharmaceutical composition comprises a surfactant. Illustrative examples of surfactants include d-alpha tocopherol, benzalkonium chloride, benzethonium chloride, cetrimonium bromide, cetylpyridinium chloride, docusate sodium, behenyl glyceryl, monooleyl glyceryl, lauric acid, macrogol 15hydroxystearate, myristyl alcohol, phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxyglycerol esters, sodium lauryl sulfate, sorbitan esters, and vitamin E polyethylene glycol succinate.

[0240] In some embodiments, the pharmaceutical composition comprises an anti-caking agent. Illustrative examples of anti-caking agents include calcium phosphate (trivalent), hydroxymethylcellulose, hydroxypropylcellulose, and magnesium oxide.

[0241] Other excipients that can be used together with the pharmaceutical composition include, for example, albumin, antioxidants, antibacterial agents, antifungal agents, bioabsorbable polymers, chelating agents, controlled release agents, diluents, dispersants, dissolution enhancers, emulsifiers, gelling agents, ointment bases, penetration enhancers, preservatives, solubilizers, solvents, stabilizers, and sugars. Specific examples of each of these agents are described in, for example, Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), The Pharmaceutical Press, which is incorporated herein by reference in its entirety.

[0242] In some embodiments, the pharmaceutical composition comprises a solvent. In some aspects, the solvent is a saline solution, such as a sterile isotonic saline solution or a glucose solution. In some aspects, the solvent is water for injection.

[0243] In some embodiments, the pharmaceutical composition is in the form of particles, such as microparticles or nanoparticles. Microparticles and nanoparticles can be formed from any suitable material, such as a polymer or lipid. In some aspects, the microparticles or nanoparticles are micelles, liposomes, or polymer vesicles.

[0244] The present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the conjugates, since, in some embodiments, water can cause degradation of certain antibodies or antigen-binding fragments thereof.

[0245] The anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture content ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms comprising lactose and at least one active ingredient comprising a primary or secondary amine can be anhydrous if significant contact with moisture and / or humidity is expected during manufacture, packaging and / or storage.

[0246] Anhydrous pharmaceutical compositions should be prepared and stored in a manner that maintains their anhydrous nature. Thus, anhydrous compositions may be packaged using materials known to prevent exposure to water so that they can be included in appropriately formulated kits. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0247] The lactose-free compositions provided by the present invention may include excipients well known in the art, and include, for example, those listed in U.S. Pharmacopoeia (USP) SP (XXI) / NF (XVI). Typically, the lactose-free compositions include pharmaceutically compatible and pharmaceutically acceptable amounts of active ingredients, binders / fillers, and lubricants. Exemplary lactose-free dosage forms include active ingredients, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

[0248] The present invention also provides pharmaceutical compositions and dosage forms, which contain one or more excipients that can reduce the decomposition rate of the conjugate. Such excipients are referred to as "stabilizers" in the present invention, including but not limited to antioxidants such as ascorbic acid, pH buffers, or salt buffers. Parenteral Dosage Forms

[0249] In certain embodiments, the present invention provides parenteral dosage forms. Parenteral dosage forms can be administered to subjects by various routes, including but not limited to subcutaneous, intravenous (including bolus injection), intramuscular and intra-arterial administration. Because its administration usually bypasses the natural defense of the subject against pollutants, parenteral dosage forms are usually sterile or can be sterilized before being applied to the subject. Examples of parenteral dosage forms include but are not limited to ready-to-use injection solutions, dry products that can be immediately dissolved or suspended in pharmaceutically acceptable injection vehicles, ready-to-use injection suspensions and emulsions.

[0250] Suitable vehicles that can be used to provide parenteral dosage forms are known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0251] Excipients that increase the solubility of one or more of the antibodies disclosed herein may also be included in the parenteral dosage forms. Dosage and unit dosage form

[0252] In human therapy, the physician will determine the dosing that he considers most appropriate according to the prophylactic or therapeutic treatment and according to the age, weight, condition and other particular factors of the subject to be treated.

[0253] In certain embodiments, the compositions provided herein are pharmaceutical compositions or single unit dosage forms. The pharmaceutical compositions and single unit dosage forms provided herein contain a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic antibodies or antigen-binding fragments thereof.

[0254] The amount of the conjugate or composition that is effective in preventing or treating a disorder or one or more symptoms of the disorder will vary with the nature and severity of the disease or disorder, and with the route of administration of the conjugate. The frequency and dosage will also vary according to factors specific to each subject, which depend on the specific therapy (e.g., therapeutic or prophylactic agent or therapeutic or prophylactic payload) administered, the severity of the disorder, disease or condition, the route of administration, and the age, body, weight, response and previous medical history of the subject. The effective dose can be extrapolated from a dose-response curve derived from an in vitro or animal model test system.

[0255] Different therapeutically effective amounts may be applicable to different diseases and conditions, which are obvious to those of ordinary skill in the art. Similarly, amounts sufficient to prevent, treat, treat or improve such conditions, but insufficient to cause or sufficient to reduce adverse reactions associated with the antibodies or antigen-binding fragments thereof provided by the present invention are also encompassed within the dosage and dosage frequency time course described herein. In addition, when multiple doses of the compositions provided by the present invention are administered to a subject, not all doses need to be the same. For example, the dose administered to a subject may be increased to improve the preventive or therapeutic effect of the compositions, or it may be reduced to reduce one or more adverse reactions experienced by a particular subject.

[0256] In certain embodiments, treatment or prevention can be initiated with one or more loading doses of a conjugate or composition provided herein and then maintained with one or more maintenance doses.

[0257] In certain embodiments, the dosage of the conjugate or composition provided by the invention can be administered so that the conjugate reaches a steady-state concentration in the subject's blood or serum. The steady-state concentration can be determined by the assay techniques available to the technician, or can be determined based on the subject's physical characteristics such as height, weight and age. Therapeutic applications

[0258] For therapeutic applications, the conjugates of the invention are administered to mammals (in certain embodiments, humans) in pharmaceutically acceptable dosage forms (such as those known in the art and as discussed herein). For example, the conjugates of the invention may be administered to humans by intravenous bolus or by continuous infusion for a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, or intratumoral routes. The conjugates may also be appropriately administered by peri-tumor, intralesional, or perilesional routes to exert local as well as systemic therapeutic effects. The intraperitoneal route may be particularly useful, for example, in the treatment of ovarian tumors.

[0259] The conjugates provided by the present invention can be used to treat any disease or condition described herein (e.g., inflammatory and / or proliferative diseases or conditions). In some embodiments, the disease or condition is a disease or condition that can be diagnosed by overexpression of an antigen. In some embodiments, the disease or condition is a disease or condition that can benefit from macromolecular therapy. In some embodiments, the disease or condition is cancer. Diagnostic Applications

[0260] In some embodiments, the conjugates provided herein are used for diagnostic applications. These assays can be used, for example, to diagnose and / or prognose diseases such as cancer.

[0261] In some diagnostic and prognostic applications or embodiments, the conjugate may be labeled with a detectable moiety. Suitable detectable moieties include, but are not limited to, radioisotopes, fluorescent labels, and enzyme-substrate labels. In another embodiment, the conjugate need not be labeled, and the presence of the conjugate may be detected using a labeled antibody or antigen-binding fragment thereof that specifically binds to the conjugate. Reagent test kit

[0262] In some embodiments, the conjugates provided herein are provided in the form of a kit (i.e., a packaged combination of reagents in predetermined amounts with instructions for use to perform a procedure). In some embodiments, the procedure is a diagnostic assay. In other embodiments, the procedure is a therapeutic procedure.

[0263] In some embodiments, the kit further comprises a solvent for reconstituting the conjugate. In some embodiments, the conjugate is provided in the form of a pharmaceutical composition.

[0264] In some embodiments, the kit may include a conjugate or composition provided by the present invention, an optional second agent or composition, and instructions for providing information about the usage of the treatment of a disease to a medical service staff. Instructions may be provided in printed form or in the form of an electronic medium (e.g., a floppy disk, a CD, or a DVD), or in the form of a website address from which such instructions may be obtained. The unit dose of a conjugate or composition provided by the present invention or a second agent or composition may include such a dose, i.e., when applied to a subject, the treatment or prevention effective plasma level of the compound or composition may be maintained in the subject for at least 1 day. In some embodiments, the compound or composition may be included as a sterile aqueous pharmaceutical composition or a dry powder (e.g., lyophilized) composition.

[0265] In some embodiments, the invention provides suitable packaging. As used in the present invention, "packaging" includes solid substrates or materials that are generally used in systems, and can accommodate compounds provided by the invention and / or be suitable for being applied to a second medicament for a subject within fixed limits. Such materials include glass and plastic (e.g., polyethylene, polypropylene and polycarbonate) bottles, vials, paper, plastic and plastic foil laminated envelopes, etc. If electron beam sterilization technology is used, packaging should have a sufficiently low density to allow contents to be sterilized. Preparation and Synthesis Procedure

[0266] The following provides a general scheme for synthesizing compounds of formula (I), (IA) or (IB). All other groups or variables are as defined in the Abstract or any embodiment of the invention.

[0267] The following provides a general scheme for synthesizing compounds of formula (III), (IIIA), or (IIIB). All other groups or variables are as defined in the Abstract or any embodiment of the invention. Coupling

[0268] The conjugates can be prepared by standard techniques. In certain embodiments, the macromolecule is contacted with a compound of formula (I), (IA), (IB), (III), (IIIA) or (IIIB) under conditions suitable for the formation of a bond between the macromolecule and the compound of formula (I), (IA), (IB), (III), (IIIA) or (IIIB) to form a conjugate. In certain embodiments, the macromolecule is contacted with a linker precursor under conditions suitable for the formation of a bond between the macromolecule and the linker. The resulting macromolecule-linker is contacted with a compound or a drug group portion under conditions suitable for the formation of a bond between the macromolecule-linker and the compound or drug group portion to form a conjugate. In certain embodiments, the compound or drug group portion is contacted with a linker precursor under conditions suitable for the formation of a bond between the compound or drug group portion and the linker. The resulting compound-linker or drug group portion-linker is contacted with a macromolecule under conditions suitable for the formation of a bond between the compound-linker or drug group portion-linker and the macromolecule to form a conjugate. For example, in certain embodiments, the second compound comprises a tetrazine; and RL comprises a strained olefin. In some embodiments, RL is In certain embodiments, the second compound comprises an azide; and RL comprises an alkyne. In some embodiments, RL is In certain embodiments, the second compound comprises an alkyne; and RL comprises an azide. In certain embodiments, the second compound comprises a strained olefin; and RL comprises a tetrazine. In certain embodiments, the second compound comprises a thiol; and RL comprises a maleimide. In some embodiments, RL is In certain embodiments, the second compound comprises maleimide; and RL comprises a thiol. In some embodiments, the second compound comprises In certain embodiments, the second compound comprises a carbonyl group; and RL comprises an oxamine. In certain embodiments, RL is In some embodiments, the second compound comprises In certain embodiments, the second compound comprises an oxamine; and RL comprises a carbonyl group. In certain embodiments, RL is or In some embodiments, RL is In some embodiments, RL is In certain embodiments, the second compound comprises In certain embodiments, the second compound is a polypeptide. In certain embodiments, the second compound is an antibody. In certain embodiments, the second compound is an antibody chain. Suitable linkers for preparing conjugates are disclosed herein, and exemplary conditions for conjugation are described in the Examples below. Example

[0269] The compounds provided by the present invention can be prepared, isolated or obtained by any method obvious to those skilled in the art. The compounds provided by the present invention can be prepared according to the exemplary preparation schemes provided below. The reaction conditions, steps and reactants not provided in the exemplary preparation schemes are obvious to those skilled in the art and are known. The symbols and conventions used in the present invention for the process methods, schemes and embodiments, whether or not specific abbreviations are specifically defined, are consistent with those used in contemporary scientific literature, such as the Journal of the American Chemical Society or the Journal of Biological Chemistry. Specifically, but not limited to, the following abbreviations can be used in the embodiments and throughout the specification: g (gram); mg (milligram); mL (milliliter); μL (microliter); mM (millimolar concentration); μM (micromolar concentration); Hz (hertz); MHz (megahertz); mmol (millimole); h, hr or hrs (hours); min (minutes); MS (mass spectrometry); ESI (electrospray ionization); LCMS (liquid chromatography-mass spectrometry); TLC (thin layer chromatography); HPLC (high performance liquid chromatography); rt (room temperature); atm (atmospheric pressure); calc d (calculated); equiv (equivalent); CDCl3 (deuterated chloroform); DBCO (dibenzocyclooctynamine); DCE (dichloroethane); DCM (dichloromethane); DIPEA (diisopropylethylamine); DMSO (dimethyl sulfoxide); DMSO-d6 (deuterated dimethyl sulfoxide); EtOAc (ethyl acetate); EtOH (ethanol); MeCN (acetonitrile); MeOH (methanol); RB (round-bottom flask); TFA (trifluoroacetic acid); THF (tetrahydrofuran); DMF (dimethylformamide); and BOC (tert-butyloxycarbonyl).

[0270] For all of the following examples, standard post-treatment and purification methods known to those skilled in the art may be used. Unless otherwise stated, all temperatures are expressed in ° C (Celsius). Unless otherwise stated, all reactions are carried out at room temperature. The synthetic methodology shown in the present invention is intended to illustrate applicable chemistry by using specific examples, rather than to indicate the scope of the present invention disclosed.

[0271] Unless otherwise stated, all anhydrous solvents were commercially available and stored in Sure-Seal bottles under nitrogen. All other reagents and solvents were purchased of the highest grade and used without further purification. NMR spectra were recorded on an Avance II HD (500 MHz) spectrometer equipped with a 5 mm Prodiogy H / F-BBO cryogenic probe and a BCU-I temperature controller. Chemical shifts (δ) are reported in parts per million (ppm) with reference to tetramethylsilane of δ 0.00, and coupling constants (J) are reported in Hz. Low-resolution mass spectral data were acquired by LC-MS on an Agilent G6125B spectrometer connected to an Agilent 1260 high-performance liquid chromatograph. The product was purified by RP-HPLC method, system: Shimadzu LC equipped with CTC IFC, Phenomenex Gemini NX 5μ, C18, 150 x 50 mm reverse phase column, using mobile phase B (CH3CN) / A (water containing 0.1% TFA) linear gradient, flow rate of 50 mL / min. Analytical HPLC was performed on a Waters 2695 instrument. For analytical HPLC, the stationary phases used were Phenomenex Gemini NX 5μ, C18, 150x 4.6mm RP column. The product was eluted on an acidic linear gradient (designated Gradient A) of mobile phase B (CH3CN with 0.05% TFA; 5% to 95% in 20 minutes) / mobile phase A (0.05% TFA / water) at a flow rate of 1.0 mL / min. Preparative HPLC purifications were performed on a Shimadzu LC equipped with a CTC IFC. All other preparative normal phase purifications were performed by standard flash silica gel chromatography using an ISCO fast liquid system.

[0272] Example 1: Synthesis of DBCOβ-glucuronide-PEG12-exitecan (107)

[0273] Scheme 1: Synthesis of β-Glu-exitecan benzylamine (19)

[0274] Scheme 2: Synthesis of compound β-Glu-PEG12-Ixitecan (107)

[0275] Scheme 3: Synthesis of β-Glu PNP linker (8)

[0276] Scheme 4: Synthesis of m-PEG12-DBCO-PFP linker (16)

[0277] Synthesis of β-Glu PNP Linker Fragment (8)

[0278] Synthesis of compound (4)

[0279] Concentrated H2SO4 (32 mL) was slowly added to a suspension of 4-hydroxybenzaldehyde (1) (5.3 g, 43.4 mmol) and 2-chloro-N-(hydroxymethyl)acetamide (2) (5 g, 40.6 mmol) in AcOH (20 mL). The mixture was stirred at room temperature (22°C) for 16 hours. The resulting viscous liquid was poured into ice water (300 mL) and then extracted with EtOAc (100 mL x 5). The organic layer was dried over Na2SO4 and concentrated under reduced pressure to give crude compound (3), which was dissolved in 1,4-dioxane (40 mL). Concentrated hydrochloric acid (40 mL) was added to the resulting solution. The mixture was heated to reflux for one hour and then concentrated in vacuo to give a residue. The residue was dissolved in dioxane:H2O (1:1, 50 mL). Et3N (9 mL) was added to the resulting mixture, followed by Boc2O (10 g, 46 mmol). The reaction mixture was stirred at room temperature for 16 hours and then partitioned between EtOAc (300 mL) and water (100 mL). The organic layer was dried over Na2SO4 and concentrated to dryness under reduced pressure. The residue was purified by silica gel chromatography (220 g column, 20-30% EtOAc:hexane in 30 minutes, flow rate: 40 mL / min) to afford compound (4) as an off-white solid (5.3 g). MS calculated value C 13 H 17 NO4, 251.3; Measured value 252.2 [M+H] + ; 1 H NMR(500MHz,DMSO-d6)δ10.75(s,1H),9.77(s,1H),7.69–7.62(m,2H),7.31 (t, J=6.2Hz, 1H), 6.96 (d, J=8.1Hz, 1H), 4.11 (d, J=6.1Hz, 2H), 1.41 (s, 9H).

[0280] Synthesis of (7)

[0281] To a solution of compound (4) (2.2 g, 8.8 mmol) and (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (5) (3.2 g, 8.1 mmol) in anhydrous acetonitrile (50 mL) was added Ag2O (3.7 g, 16 mmol). The suspension was stirred for 16 hours under an argon atmosphere. The solid was filtered off and washed with acetonitrile (10 mL). i-PrOH (10 mL) and NaBH4 (300 mg, 8.1 mmol) were added to the combined acetonitrile solution, and the mixture was stirred at room temperature for reaction. After 30 minutes, the reaction was quenched with water (100 mL) and then extracted with EtOAc (3 x 100 mL). The organic layer was dried over Na2SO4 and concentrated to dryness under reduced pressure. The residue was purified by silica gel chromatography (220 g column, 40-70% EtOAc:hexanes in 30 minutes, flow rate: 40 mL / min) to afford compound (7) as a white foam (3.0 g, 5.2 mmol). MS calculated value C 26 H 35 NO 13 ,569.2; Found 570.4[M+H] + ; 1 H NMR (500MHz, DMSO-d6) δ7.15(q,J=6.0Hz,3H),6.98(d,J=8.7Hz,1H),5.61–5.41(m,2H),5.25–5.03(m,3H),4.73(d,J=9.9Hz ,1H),4.42(d,J=5.3Hz,2H),4.03(tt,J=16.4,8.3Hz,2H),3.65(s,3H),2.14–1.92(m,10H),1.41(s,9H),1.33–1.14(m,2H).

[0282] Synthesis of (8)

[0283] DIEA (1.1 mL) and bis-PNP carbonate (2 g, 6.57 mmol) were added to a solution of compound (7) (2.6 g, 4.56 mmol) in anhydrous DMF (15 mL). The mixture was stirred at room temperature for 16 hours and then directly purified by reverse phase HPLC (Phenomenex Gemini NX 5μ, C18, 150x 50mm, mobile phase: A: 0.1% TFA / water, B: acetonitrile, gradient: 20-90% B, lasted 20 minutes, flow rate: 50mL / min), after lyophilization, compound (8) (1.8g) was obtained as a white solid. MS calculated value C 33 H38 N2O 17 ,734.2; Found 735.5[M+H] + ; 1 H NMR(500MHz,DMSO-d6)δ8.35–8.29(m,2H),7.60–7.53(m,2H),7.36(dd,J=8.4,2.2Hz,1H),7.29(d, J=2.3Hz,1H),7.22(t,J=6.2Hz,1H),7.09(d,J=8.4Hz,1H),5.65(d,J=7.9Hz,1H),5.52(t,J=9.6Hz ,1H),5.26(s,2H),5.19(dd,J=9.8,7.9Hz,1H),5.10(t,J=9.7Hz,1H),4.76(d,J=9.9Hz,1H),4.03( qd,J=16.7,6.2Hz,3H),3.66(s,3H),2.06(s,3H),2.02(d,J=2.6Hz,6H),1.39(s,8H),1.30(s,1H).

[0284] Synthesis of m-PEG12-DBCO-PFP(16)

[0285] Synthesis of (11)

[0286] To a solution of Fmoc-Dap(Boc)-COOH(9)(853mg, 2mmol) in anhydrous DMF(10mL) was added N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU)(608mg, 2mmol) and then DIPEA(0.7mL). The mixture was stirred at room temperature for 10 minutes. A solution of β-alanine(0.2g) in acetonitrile:water(1:1,3mL) was added and then DIPEA(0.4mL) was added. The reaction mixture was stirred at room temperature for 30 minutes and then acidified with 0.5N hydrochloric acid(50mL). The mixture was extracted with EtOAc(200mL), the organic layer was dried over Na2SO4 and concentrated to dryness under reduced pressure to give crude compound(10) as a white powder. The crude compound(10) was treated with TFA:DCM(1:4, v / v, 20mL) at room temperature for one hour. The mixture was evaporated to dryness under reduced pressure to give crude compound (11), which was used directly in the next step. MS calculated value C 21 H 23 N3O5, 397.16; Found 398.2 [M+H] + .

[0287] Synthesis of (14)

[0288] m-PEG12-acid (12) (1.18 g, 2 mmol) was dissolved in DMF (10 mL), to which TSTU (610 mg, 2 mmol) was added, followed by DIPEA (700 μL). After 5 minutes, a solution of compound (11) in DMF (10 mL) and DIPEA (0.7 mL) were added. The reaction mixture was stirred at room temperature for 30 minutes and then directly purified by reverse phase HPLC to give compound (14) as a viscous foam (1.27 g). MS calculated value C 47 H 73 N3O 18 ,967.5; Found 968.8[M+H] + ; 1 H NMR(500MHz,DMSO-d6)δ8.35–8.29(m,2H),7.60–7.53(m,2H),7.36(dd,J=8.4,2.2Hz,1H),7.29(d, J=2.3Hz,1H),7.22(t,J=6.2Hz,1H),7.09(d,J=8.4Hz,1H),5.65(d,J=7.9Hz,1H),5.52(t,J=9.6Hz ,1H),5.26(s,2H),5.19(dd,J=9.8,7.9Hz,1H),5.10(t,J=9.7Hz,1H),4.76(d,J=9.9Hz,1H),4.03( qd,J=16.7,6.2Hz,3H),3.66(s,3H),2.06(s,3H),2.02(d,J=2.6Hz,6H),1.39(s,8H),1.30(s,1H).

[0289] Synthesis of (15)

[0290] i-Pr2NH(DIPA) (10 mL) was added to a solution of compound (14) (1.24 g) in DMF (10 mL), and the reaction mixture was stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure to about 9 mL. DBCO-C6-NHS (0.55 g) was added to the solution, followed by DIPEA (0.23 mL). The mixture was stirred at room temperature for 2 hours and then directly purified by reverse phase HPLC to give compound (15) as a colorless syrup (1.17 g). MS calculated value C 53 H 80 N4O 18 ,1060.6; Found 1061.9[M+H] + .

[0291] Synthesis of m-PEG12-DBCO-PFP linker (16)

[0292] To a solution of compound (15) (1.1 g) in DMF (8 mL) was added pentafluorophenol tetramethyluronium hexafluorophosphate (PfTU) (0.5 g) followed by DIEA (0.4 mL) and the reaction mixture was stirred at room temperature for 10 minutes. The mixture was directly purified by RP-HPLC to give compound (16) as a colorless syrup (1.0 g). MS calculated value C 59 H 79 F5N4O 18 ,1226.5; Found 1227.8[M+H] + Compound (16) was immediately used in the next reaction.

[0293] Synthesis of (107)

[0294] Synthesis of (19)

[0295] DIPEA (350 μL) was added to a DMF (10 mL) solution of compound (8) (735 mg, 1 mmol) and isotecan mesylate (531 mg, 1 mmol). The reaction mixture was stirred at room temperature (22°C) for 5 hours and then diluted with EtOAc (200 mL). The mixture was washed with 0.5N hydrochloric acid (100 mL), water (100 mL) and brine (50 mL). The organic layer was dried over Na2SO4 and concentrated to dryness under reduced pressure to obtain crude compound (18), which was suspended in acetonitrile: water (2:1, 50 mL). 1N NaOH aqueous solution (7 mL) was added to the resulting mixture and the reaction was stirred at room temperature. After 3 hours, 1N hydrochloric acid (7 mL) was added and the mixture was evaporated to dryness under reduced pressure. The resulting residue was treated with TFA:DCM (1:4, v / v, 20 mL) at room temperature for one hour. The mixture was diluted with toluene (30 mL) and then evaporated to dryness under reduced pressure. The residue was purified by reverse phase HPLC to give compound (19) as a yellow solid (745 mg). MS calculated value C 39 H 39 FN4O 13 ,790.3; Found 791.6[M+H] + ; 1H NMR (500MHz, DMSO-d6) δ8.09(d,J=8.0Hz,4H),7.73(dd,J=10.7,3.2Hz,1H),7.54(d,J=2.1Hz,1H),7.49(dd,J=8.5,2.2Hz,1H ),7.32(s,1H),7.22(d,J=8.5Hz,1H),6.55(s,1H),5.75(s,1H),5.48(d,J=16.3Hz,1H),5.46–5.37(m,3H),5.31–5.13(m,4H), 5.08–5.02(m,2H),4.19–4.07(m,2H),3.95(d,J=9.5Hz,1H),3.44(dd,J=8.8,2.6Hz,1H),3.28–3.19(m,2H),3.14–3.04(m,1H ), 2.34 (s, 3H), 2.26 (dd, J = 12.5, 6.4Hz, 1H), 2.12 (qd, J = 9.0, 4.6Hz, 1H), 1.87 (dh, J = 21.5, 7.2Hz, 2H), 0.89 (t, J = 7.2Hz, 3H).

[0296] Synthesis of (107)

[0297] Compound β-Glu-exitecan benzylamine (19) (540 mg, 0.68 mmol) was dissolved in DMF (4 mL), compound m-PEG12-DBCO-PFP (16) (750 mg, 0.6 mmol) was added, and then DIPEA (0.32 mL) was added. The reaction mixture was stirred at room temperature for 30 minutes and then directly purified by reverse phase HPLC (Phenomenex Gemini NX 5μ, C18, 150 x 50 mm, mobile phase: A: 0.1% TFA / water, B: acetonitrile, gradient: 20-90% B over 20 minutes, flow rate: 50 mL / min) to afford compound (107) as a light yellow solid (704 mg). HRMS m / z (ESI + ): Calculated value C 92 H 117 F8O 30 ,1832.78; Measured value 1833.79[M+H] + ; 1H NMR (500MHz, DMSO-d6) δ12.84(s,1H),8.20(t,J=6.2Hz,1H),8.02(d,J=8.8Hz,1H),7.75(td,J=6.1,3.4Hz,2H),7.62–7.52(m,1H),7.5 0–7.39(m,2H),7.32–7.25(m,2H),6.52(s,1H),5.50–5.39(m,2H),5.27(d,J=13.6Hz,3H),5.01–4.94(m,1H),4.29(t,J=6.3Hz,2H),3.5 7(d,J=14.0Hz,1H),3.56–3.46(m,35H),3.48–3.40(m,5H),3.35(td,J=12.8,6.5Hz,5H),3.24(s,2H),3.22(s,3H),2.36(d,J=1.8Hz,3 H), 2.27 (dq, J=13.4, 6.3Hz, 3H), 2.16 (ddd, J=21.5, 11.0, 6.0Hz, 2H), 1.87 (dp, J=21.0, 7.2Hz, 4H), 1.17 (s, 3H), 0.88 (t, J=7.3Hz, 3H).

[0298] Synthesis of β-Glucuronide-cleavable Hemipteratin (114)

[0299] Solution 5

[0300] (5) Synthesis

[0301] A solution of compound (1) DBCO-C6-NHS ester (150 mg, 0.35 mmol), compound (2) β-alanine (32 mg, 0.70 mmol) and DIEA (112 mg, 0.87 mmol) in 50% CH3CN:H2O (5 mL) was stirred at room temperature for 20 minutes. The resulting solution was extracted with dichloromethane (50 mL), and the organic layer was concentrated to obtain crude compound (3), which was redissolved in anhydrous DCM (5 mL). N-hydroxysuccinimide (129 mg, 0.70 mmol) and EDC·HCl (200 mg, 1.05 mmol) were added to the resulting solution in sequence, and the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo. The residue was dissolved in DMF (3 mL) and purified by reverse phase HPLC to obtain compound (5) as an amorphous solid (136 mg).

[0302] Solution 6

[0303] Scheme 7: Synthesis of (7)

[0304] Synthesis of (114)

[0305] DIPEA (75 μL) was added to a solution of compound (6) (100 mg, 136 μmol) and compound (7) (TFA salt, 100 mg, 134 μmol) in DMF (2 mL). The reaction mixture was stirred at room temperature (22°C) for 3 days, and the crude product was purified by reverse phase HPLC and lyophilized to obtain compound (8) as a white solid (143 mg, 86%). Compound (8) (143 mg, 117 μmol) was treated with TFA:DCM (1:4, v / v, 3 mL) at room temperature for 30 minutes. The reactant was concentrated to dryness under reduced pressure to obtain a residue. The residue was dissolved in acetonitrile:water (6:4, v / v, 3 mL) and an aqueous NaOH solution (1 M, 0.8 mL) was added. The reaction mixture was stirred at room temperature for 2 days and acidified with hydrochloric acid (1 M, 0.5 mL). The mixture was purified by reverse phase preparative HPLC and lyophilized to obtain compound (9) (87 mg, two-step yield 69%) as a white solid. Compound (9) (87 mg) was dissolved in DMF (2 mL), compound (5) (41 mg, 82 μmol) was added, and then DIPEA (70 μL) was added. The reaction mixture was stirred at room temperature for 20 minutes and then directly purified by reverse phase preparative HPLC (Phenomenex Gemini NX 5μ, C18, 150 x 50 mm, mobile phase: A: 0.1% TFA / water, B: acetonitrile, gradient: 5-60% B over 30 minutes, flow rate: 50 mL / min) to give compound (144) as a white solid (87 mg, 84%). MS calculated value C 66 H 83 N7O 16 ,1229.59; found m / z 1230.7[M+H] + . 1H NMR(500MHz,DMSO-d6)δ9.65(s,1H),8.70(s,1H),7.99(s,1H),7.66(t,J=5.6Hz,1H) ,7.61(dd,J=7.6,1.5Hz,1H),7.57–7.40(m,5H),7.40–7.24(m,6H),7.20(t,J=7.9Hz ,1H),7.09(dd,J=13.4,8.1Hz,2H),6.64(dd,J=9.6,1.7Hz,1H),5.48(s,1H),5.17(s ,1H),5.07–4.99(m,3H),4.91(t,J=10.1Hz,1H),4.76(d,J=8.8Hz,2H),4.37(dd,J=1 4.7, 6.5Hz, 1H), 4.23 (dd, J=14.8, 5.6Hz, 1H), 3.59 (d, J=13.8Hz, 2H), 3.18 (qd, J=6. 8,2.6Hz,3H),2.98(s,3H),2.27(t,J=7.2Hz,2H),2.13(ddd,J=13.9,8.0,5.6Hz,1H) ,2.06–1.93(m,4H),1.83–1.75(m,5H),1.71(dtd,J=14.5,5.9,2.7Hz,1H),1.26(s,4 H), 1.16 (d, J = 20.8Hz, 6H), 0.93 (s, 9H), 0.79 (d, J = 6.6Hz, 3H), 0.73 (d, J = 6.5Hz, 3H).

[0306] Synthesis of (108)

[0307] Solution 8

[0308] Compound (9) (60 mg, 55 μmol) was dissolved in anhydrous DMF (2 mL), compound (16) (74 mg, 60 μmol) was added, and then DIPEA (60 μL) was added. The reaction mixture was stirred at room temperature for 20 minutes and then directly purified by reverse phase preparative HPLC (Phenomenex Gemini NX 5μ, C18, 150 x 50 mm, mobile phase: A: 0.1% TFA / water, B: acetonitrile, gradient: 5-60% B over 30 minutes, flow rate: 50 mL / min) to give compound (108) as a white solid (93 mg, 84%). HRMS m / z (ESI + ): Calculated value C 95 H139 N9O 30 ,1885.96; Found 1886.97[M+H] + ; 1 H NMR(500MHz,DMSO-d6)δ12.61(s,2H),9.77(s,1H),8.76(d,J=8.2Hz,2H),8.20(t,J=6.1Hz,1H), 7.85(t,J=5.8Hz,1H),7.76(t,J=5.9Hz,2H),7.67(dd,J=8.0,2.7Hz,1H),7.64–7.52(m,3H),7.53 –7.41(m,3H),7.41–7.25(m,6H),7.25–7.15(m,2H),7.07(d,J=8.5Hz,1H),6.67(dd,J=9.6,1.7Hz ,1H),5.57(s,2H),5.17–4.99(m,3H),5.00–4.87(m,2H),4.77(d,J=8.1Hz,1H),4.30(ddt,J=31.2 ,20.7,7.9Hz,3H),4.22–4.10(m,1H),3.91(d,J=9.7Hz,1H),3.69–3.37(m,70H),3.37–3.28(m,5 H),3.23(s,9H),3.02(s,3H),2.28(q,J=5.9Hz,7H),2.17(ddd,J=16.6,9.0,3.8Hz,1H),2.01(ddd ,J=10.5,7.9,5.0Hz,1H),1.95–1.85(m,2H),1.79(d,J=1.4Hz,3H),1.74(ddd,J=14.2,7.0,2.8Hz ,1H),1.33(s,4H),1.26–1.10(m,6H),0.98(s,10H),0.80(d,J=6.5Hz,3H),0.77(d,J=6.5Hz,3H).

[0309] Synthesis of (109)

[0310] Solution 9

[0311] To a solution of compound (1) (50 mg) in DCM (2 mL) was added pentafluorophenol (20 mg), followed by DIC (8 mL), and the reaction mixture was stirred at room temperature for 5 minutes under an argon atmosphere. A solution of compound (19) (45 mg) in DMF (2 mL) was added to the resulting solution, followed by DIPEA (0.04 mL). The reaction mixture was stirred at room temperature for 1 hour. LCMS showed that the reaction was complete. DCM was removed under reduced pressure, and the residue was directly purified by reverse phase HPLC to give compound (109) as a light yellow solid (44 mg). LCMS m / z (ESI + ): Calculated value C 89 H 112 F7O 29 ,1761.75; Found 1762.8[M+H] + ; 1 H NMR(500MHz,DMSO-d6)δ12.80(s,1H),8.29–8.14(m,1H),8.00(d,J=8.8Hz,1H),7.78(tt,J=14.3,6.8Hz,3H),7.58(ddd,J=7.4,4.5,1.7Hz,1H), 7.53(dd,J=7.0,1.8Hz,1H),7.44(ttd,J=7.6,5.0,2.7Hz,3H),7.38–7.2 2(m,5H),7.22–7.14(m,1H),7.07(d,J=8.2Hz,1H),6.53(d,J=5.0Hz,1H) ,5.57–5.32(m,4H),5.27(s,4H),5.13–4.86(m,4H),4.44–4.16(m,3H),3 .90(d,J=9.7Hz,1H),3.60–3.52(m,2H),3.53–3.37(m,42H),3.38(s,51H ),3.24(s,5H),2.38(d,J=1.9Hz,3H),2.29–2.02(m,5H),2.00–1.77(m,4 H),1.77–1.62(m,1H),1.23(dd,J=57.4,11.9Hz,4H),0.94–0.75(m,3H).

[0312] Synthesis of (110)

[0313] Solution 10

[0314] To a solution of compound (1) (88 mg) in DMF (2 mL) was added PfTU (35 mg) and then DIPEA (0.03 mL), and the reaction mixture was stirred at room temperature for 10 minutes. The mixture was directly purified by reverse phase HPLC to give compound (2) as a colorless syrup (85 mg).

[0315] Compound (9) (45 mg) was dissolved in DMF (2 mL), compound (2) (64 mg, 0.05 mmol) was added, and then DIPEA (0.04 mL) was added. The reaction mixture was stirred at room temperature for 30 minutes and then directly purified by reverse phase HPLC to obtain compound (110) as a light yellow solid (57 mg). LCMS m / z (ESI + ): Calculated value C 95 H 123 F8O 30 ,1874.83; Measured value 1875.9[M+H] + ; 1 H NMR (500MHz, DMSO-d6) δ12.82(s,2H),8.18(t,J=6.0Hz,1H),8.03(d,J=8.8Hz,1H),7.84( t,J=5.8Hz,1H),7.76(dt,J=11.3,4.1Hz,2H),7.70(d,J=8.0Hz,1H),7.60(dd,J=7.5,1.5H z,1H),7.55(dt,J=6.8,1.9Hz,1H),7.51–7.39(m,3H),7.39–7.18(m,6H),7.09(d,J=8.5H z,1H),6.52(s,1H),5.55(s,1H),5.50–5.32(m,3H),5.27(s,4H),5.13–4.86(m,4H),4.40– 4.19(m,2H),4.06(tdd,J=8.3,5.1,3.0Hz,1H),3.91(d,J=9.7Hz,1H),3.17–3.01(m,1H), 2.96(dd,J=10.4,4.5Hz,2H),2.37(d,J=1.9Hz,3H),2.27(q,J=6.6Hz,4H),2.16(ddd,J=21 .5,10.9,5.8Hz,3H),1.88(tp,J=14.0,6.2Hz,4H),1.79–1.64(m,1H),1.51(s,1H),1.38(d ,J=9.3Hz,1H),1.29(d,J=8.9Hz,3H),1.16(dq,J=15.0,7.3Hz,4H),0.88(t,J=7.4Hz,3H).

[0316] Synthesis of (111)

[0317] Solution 11

[0318] (111) was synthesized in a similar manner using the same method as above. LCMS m / z (ESI + ): Calculated value C 95 H 123 F8O 30 ,1804.75; measured value 1804.90[M+H] + ; 1 H NMR(500MHz,DMSO-d6)δ12.82(s,1H),8.39–8.10(m,1H),8.11–7.93(m,1H),7.93–7.54(m,6H),7.54–7.36(m,3H),7.40–7.16(m,6H),7.10(dd,J =8.6,4.3Hz,1H),6.53(d,J=5.2Hz,2H),5.61–5.30(m,4H),5.26(d,J=9. 2Hz,4H),5.15–4.69(m,4H),4.42–4.22(m,2H),4.22–4.07(m,1H),3.91( d,J=9.5Hz,1H),3.66–3.53(m,3H),3.54–3.45(m,36H),3.45–3.39(m,6 H),3.39–3.28(m,63H),3.24(s,5H),3.16–2.96(m,2H),2.60(dt,J=15.8 ,7.2Hz,1H),2.41–2.27(m,5H),2.23(t,J=6.7Hz,3H),2.21–2.11(m,2H) ,2.03(dq,J=14.3,7.4Hz,1H),1.94–1.71(m,3H),0.88(t,J=7.3Hz,3H).

[0319] Synthesis of Maleimidocaproyl-PEGylated β-Glucuronide Cleavable Linker Payload (111a) Scheme 11a

[0320] Preparation of (15a)

[0321] i-Pr2NH (2 mL) was added to a solution of compound (14a) (0.2 g) in DMF (2 mL), and the reaction mixture was stirred at room temperature for 2 hours. The mixture was then concentrated under reduced pressure to about 2 mL, and MC-Pfp (76 mg, CAS 692739-25-6) was added to the solution, followed by DIEA (0.035 mL). The mixture was stirred at room temperature for 2 hours, and the crude material was directly purified by reverse phase preparative HPLC to give compound (15a) as a colorless syrup (0.14 g). LCMS m / z (ESI + ): Calculated value C 42 H 74 N4O 19 ,938.49; Found 939.63[M+H] + .

[0322] Preparation of (16a)

[0323] To a solution of compound (15a) (0.14 g) in DMF (2 mL) was added PfTU (TCI-US, 0.065 g) and then DIEA (0.06 mL), and the reaction mixture was stirred at room temperature for 10 minutes. LCMS showed the formation of the desired product, and the crude mixture was directly purified by reverse phase preparative HPLC to give compound (16a) as a colorless syrup (0.12 g). LCMS m / z (ESI + ): Calculated value C 48 H 73 F5N4O 19 ,1104.48; Found 1105.6[M+H] + .

[0324] Preparation of (111a)

[0325] Compound (9a) (92 mg) was dissolved in DMF (2 mL), compound (16a) (110 mg, 0.1 mmol) was added, and then DIPEA (0.07 mL) was added. The reaction mixture was stirred at room temperature for 30 minutes, after which LCMS showed the formation of the desired product. The crude material was directly purified by reverse phase preparative HPLC to give compound 111a as a light yellow solid (120 mg). 11H NMR (500 MHz, DMSO-d6) δ 12.82 (s, 2H), 8.21 (t, J = 6.1 Hz, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.89 (t, J = 5.7 Hz, 1H), 7.83–7.69 (m, 3H), 7.31 (d, J = 7.0 Hz, 2H), 7.25 (d, J = 2.2 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 6.99 (s, 2H), 6.51 (s, 1H), 5.45 (d, J = 4.1 Hz, 3H), 5.28 (s, 4H), 5.07 (d, J = 6.1 Hz, 3H), 4.97 (d, J = 6.9 Hz, 1H), 4.45–4.12 (m, 4H), 3.92 (d, J = 9.7 Hz, 2H), 3.56 (s, 2H), 3.54–3.40 (m, 45H), 3.40–3.27 (m, 13H), 3.24 (s, 8H), 3.16–3.04 (m, 2H), 2.38 (d, J = 1.9 Hz, 3H), 2.31 (q, J = 5.5 Hz, 4H), 2.07 (d, J = 7.4 Hz, 2H), 1.88 (dd, J = 13.1, 7.1 Hz, 2H), 1.51–1.41 (m, 4H), 1.18 (s, 2H), 0.89 (t, J = 7.3 Hz, 3H); 13 13C NMR (126 MHz, DMSO-d6) δ 172.92, 172.54, 171.53, 171.08, 170.98, 170.52, 170.23, 163.11, 161.13, 157.23, 156.53, 155.10, 153.01, 150.49, 148.51, 148.40, 145.69, 141.39, 136.94, 134.90, 130.91, 128.91, 128.76, 125.89, 124.20, 124.04, 122.05, 119.60, 115.55, 110.20, 101.57, 97.18, 76.10, 75.94, 73.57, 72.84, 71.84, 71.76, 70.26, 70.24, 70.12, 70.06, 69.95, 67.19, 66.16, 65.76, 58.52, 53.26, 50.28, 47.77, 36.49, 35.93, 35.59, 35.52, 31.16, 30.80, 28.24, 26.30, 24.99, 24.21, 11.46, 11.42, 8.22; LCMS m / z (ESI + ): Calculated for C 81 H 111 FN8O31 ,1710.73; Found 1711.9[M+H] + .

[0326] Synthesis of mDPR PEGylated β-Glucuronide Cleavable Linker Payload (111b)

[0327] (111b) was synthesized in a similar manner to (111a) above (e.g., using Instead of (15a)), purification by reverse phase preparative HPLC gave Boc protected (111b), which was treated with TFA / CH3CN and purified by preparative HPLC to give (111b). 1 H NMR(500MHz,DMSO-d6)δ8.05–7.99(m,2H),7.91(s,2H),7.32(d,J=6.8Hz,2H),7.18–7.08(m,3H),5 .51–5.40(m,3H),5.28(s,3H),5.12–5.03(m,2H),4.29–4.19(m,1H),3.60–3.54(m,3H),3.54–3.47 (m,47H),3.47–3.40(m,10H),3.40–3.27(m,6H),3.25(s,8H),2.39(d,J=1.9Hz,3H),2.36–2.26(m, 4H), 2.19 (dt, J=22.6, 6.3Hz, 2H), 1.88 (dtt, J=21.2, 14.2, 7.3Hz, 2H), 0.89 (t, J=7.3Hz, 3H).LCMS m / z(ESI + ): Calculated value C 78 H 106 FN9O 31 ,1683.70; Found 1684.71[M+H] + .

[0328] Synthesis of β-Glucuronide-cleavable EDA PNU-159682(112)

[0329] Solution 12

[0330] Mono-Fmoc-ethylenediamine (1) (HCl salt, 64 mg, 0.2 mmol) was added to anhydrous DMF (2 mL) solution of compound (8) (147 mg, 0.2 mmol), followed by DIPEA (0.07 mL). The mixture was stirred at room temperature for 1 hour, then distributed between EtOAc (50 mL) and water (50 mL). The organic layer was dried over Na2SO4 and concentrated to dryness to give crude compound (2). Crude compound (2) was treated with TFA:DCM (1:4, v / v, 4 mL) at room temperature for 30 minutes. The mixture was evaporated under reduced pressure to dryness to give crude compound 3, which was directly used in the next step.

[0331] The crude compound (3) was dissolved in DMF (2 mL), DBCO-PEG12-Pfp ester (240 mg, 0.2 mmol) was added, followed by DIPEA (70 μL). The reaction mixture was stirred at room temperature for 2 hours and then directly purified by reverse phase HPLC to give compound (4) as a viscous syrup (240 mg).

[0332] To a solution of compound 4 (240 mg) in ACN:water (3:2, v / v, 6 mL) was added NaOH (aqueous solution, 1 N, 1.2 mL), and the reaction mixture was stirred at room temperature for 6 hours. The mixture was then acidified with 1 N hydrochloric acid (0.6 mL) and directly purified by reverse phase HPLC to give compound (5) as a colorless syrup (138 mg).

[0333] To a solution of compound (6) (36.4 mg, 50 μmol) in anhydrous DMF (1 mL) was added TSTU (15 mg, 50 μmol) and DIPEA (18 μL, 0.1 mmol). The mixture was stirred at room temperature for 10 minutes, and then a solution of compound (5) (78.6 mg, 50 μmol) in anhydrous DMF (1 mL) was added. The reaction mixture was stirred for another 10 minutes and then directly purified by reverse phase HPLC. The freeze-dried fractions gave compound (112) as a red solid (42 mg). LCMS m / z (ESI + ): Calculated value C 101 H 134 N8O 38 :2066.88; Measured value 2067.9[M+H] + .

[0334] In certain embodiments, the present invention also contemplates a linker-payload compound and / or conjugate thereof containing or carrying an immunomodulatory payload. The linker-immunomodulatory payload and / or conjugate thereof may be synthesized according to standard techniques known in the art, and / or may be synthesized by methods described herein for preparing a linker-payload and / or conjugate thereof (i.e., synthesized by methods described herein for preparing a linker-payload and / or conjugate thereof containing or carrying a cytotoxic agent or cytotoxic payload, or a linker-cytotoxic agent or a linker-cytotoxic payload), which will be appreciated by those of ordinary skill in the art.

[0335] Coupling method: (114), (107) and (108) linker-payload were dissolved in DMSO at a final concentration of 5 mM. Coupling was performed in 1x PBS at an antibody concentration of 1 mg / mL and a drug linker:pAMF ratio of 3 with 15% DMSO. The reaction mixture was incubated at 30°C overnight. Figure 1 As shown, the coupling efficiency was determined by MALDI. Uncoupled drug linker was removed by desalting. The purity of the conjugate was determined by Sepax SEC-300. The conjugate was prepared in 1x PBS.

[0336] Results: Figure 1 As shown, (114), (107) and (108) were conjugated to aFolR mAb, where eight pAMF sites were incorporated at the Y180F404 site of the heavy chain and the K42E161 site of the light chain. Following the conjugation conditions described in the methods section, the conjugation efficiencies of the three different linkers-payloads were all over 94%. The results obtained by analytical SEC showed that all conjugates were of high purity with a monomer content of >99%. # Bioreg Id mAbs Compound No. DAR monomer% 1 202 1848-H01 HC-Y180F404 / LC-K42E161 114 7.64 100.0 2 205 1848-H01 HC-Y180F404 / LC-K42E161 107 7.84 99.7 3 203 1848-H01 HC-Y180F404 / LC-K42E161 108 7.52 100.0

[0337] In vitro cell-killing activity of β-glucuronide linker payload antibody-drug conjugates (ADCs)

[0338] To evaluate the cell killing activity of ADCs with different β-Glu linker payloads, anti-FolRα antibodies were conjugated with (114), (107) and (108) at DAR=8 and tested in an in vitro cell killing assay. Anti-FolRα antibodies conjugated with hemiasterlin having a cathepsin-cleavable ValCit linker were used as controls. FolRα-negative A549 and MC38 cells were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA), and FolRα-positive Igrov1 cells were purchased from NCI (Frederick National Cancer Institute, Maryland). MC38-hFolRα cells were generated at Sutro by stable transfection of a vector expressing human FolRα. All cell lines were maintained in DMEM:F12 (1:1), high glucose (Corning, Corning, NY), supplemented with 10% heat-inactivated fetal bovine serum (Thermo Scientific, Grand Island, NY), 2 mM glutamax (Thermo Scientific, Grand Island, NY), and 1x penicillin / streptomycin (Corning, Corning, NY). The cytotoxic effects of the ADCs were determined using a cell proliferation assay. A total of 625 Igrov1 and A549 cells and 312 MC38-hFolRα cells were seeded in 384-well flat-bottom white polystyrene plates in a volume of 25 μL one day before the actual assay. The ADCs were prepared in cell culture medium at 2x the starting concentration and filtered through a MultiScreen HTS 96-well filter plate (Millipore, Billerica, Massachusetts). The filter-sterilized samples were serially diluted (1:3) under sterile conditions and added to the cells in triplicate. The plates were incubated in a CO2 incubator at 37°C for 120 hours (for Igrov1 and A549 cells) and 72 hours (for MC38-hFolRα cells). To determine cell viability, 30 μL Cell Reagent (Promega Corp, Madison, WI) was added to each well, and the plates were processed according to the manufacturer's instructions. Relative luminescence was measured on a plate reader (Perkin-Elmer; Waltham, MA). Untreated cells were used as controls and relative luminescence readings were converted to percent viability (%). Data were analyzed by nonlinear regression using GraphPad Prism using log(inhibitor) vs. response, variable slope, 4-parameter fit equation.

[0339] As free payloads, exatecan and hemiasterlin showed good cell killing effects against all three cell lines tested. Free exatecan (207) was approximately 3 times more potent than free hemiasterlin (201), as shown in Tables 1A and Figure 2D-2F As expected, the anti-FolRα ADCs with β-Glu or β-Glu-PEG12 linkers showed potent cell killing against both FolRα-positive Igrov1 and MC38-hFolRα cells, while no cell killing was observed against hFolRα-negative A549 cells. The results indicate that there is no nonspecific release of free payload to kill target-negative cells, which means that both β-Glu and β-Glu-PEG12 linkers are stable in cell culture medium for 5 days.

[0340] As shown in Table 1A and Figure 2A-2C As shown, ADC (202) coupled to hemicastelin with β-Glu or ADC (203) coupled to hemicastelin with β-Glu-PEG12 linker showed good and similar killing activity, slightly weaker than hemicastelin ADC (204) with ValCit linker. For Igrov1 cells, the potency of exotecan ADC (205) with β-Glu-PEG12 linker was much weaker than that of hemicastelin ADC (203) with the same linker, and was inactive against both MC38-hFolRα cells. Table 1A

[0341] β-Glucuronidase reactivity

[0342] like Figure 3A-3DAs shown, the sensitivity of the β-glucuronide linker-payload to enzymatic cleavage was evaluated by treating (107) and (108) linker-payload with β-glucuronidase. This experiment confirmed the expected release of exotecan (207) and 3-aminohamiltonine (201). The cleavage products were monitored by LCMS. 945 μL of 50 mM sodium acetate, 5 μL of 2 mM (107) / (108) linker-payload stock solution (final concentration of 10 μM) and 50 μL of 6 KU / mL Escherichia coli (E. coli) β-glucuronidase in 0.2% NaCl solution (final concentration of 300 U / mL) were mixed and incubated at 37°C. Aliquots (50 μL) were taken at t = 0 h, 0.5 h, 1 h, 2 h, 4 h and 24 h, treated with 3 volumes of quenching solution (methanol: acetonitrile 5:95 v / v), and analyzed by LCMS. After 30 minutes, most of (107) and (108) disappeared from the reaction mixture, while (207) and (201) appeared. The remaining (107) and (108) were cleaved after 24 hours.

[0343] β-Glucuronidase cleavage of (107)

[0344] Synthesis of DBCO-Valcit-pAB-Hamitelin (208):

[0345] The 208DBCO-Valcit-pAB-Hamitelin linker payload was synthesized as described in WO 2020 / 252015 Al.

[0346] β-Glucuronidase cleavage of (108) Equivalent

[0347] The content of the present invention described above may include multiple different embodiments with independent practicality. Although each of these embodiments is disclosed, the specific embodiments disclosed and shown in the present invention should not be regarded as limiting, because various variations are possible. The subject matter of the embodiments of the present invention includes all novel and non-obvious combinations and sub-combinations of various elements / elements, features, functions and / or properties disclosed in the present invention. The following claims specifically point out certain combinations and sub-combinations that are considered novel and non-obvious. Inventions embodied in other combinations and sub-combinations of features, functions, elements and / or properties may be claimed in this application, an application claiming priority to this application, or a related application. This claim, whether it is about different embodiments or about the same embodiment and whether the scope is broader, narrower, equal or different than the original claim, is also considered to be included in the subject matter of the invention of the present invention.

[0348] One or more features of any embodiment described herein or in the accompanying drawings may be combined with one or more features of any other embodiment described herein or in the accompanying drawings without departing from the scope of the invention.

[0349] All publications, patents and patent applications cited in this specification are incorporated herein by reference, just as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the above invention has been described in detail by way of illustration and example for the purpose of clear understanding, it is obvious to those skilled in the art based on the teachings of the present invention that certain changes / variations and modifications / modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

1. A compound having a structure represented by formula (I): or a pharmaceutically acceptable salt thereof, wherein L 1 Yes-C 1-6 Alkylene-; Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -[X 1 ] p –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 [Alkenylene] n -[X 1 ] p –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -[X 1 ] p -, wherein at least one alkylene, alkenylene or alkynylene group in Y is replaced by one or more selected from R 50 Substituents are substituted, and wherein the alkylene, alkenylene or alkynylene in Y is optionally replaced by one or more selected from R 51 Substituents are substituted; R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, –C 2-6 Alkenylene-X 2 -[C 2-6 [Alkenylene] m -POLY, or –C 2-6 Alkynylidene-X 2 -[C 2-6 Alkynylidene] m -POLY, where R 50 Each alkylene, alkenylene or alkynylene group is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; R 51 independently selected from halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; X 1 and X 2 are independently selected from -N(R 10 )–, –C(O)–, and –N(R 10 )C(O)–; R 10 At each occurrence, independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; POLY is a water-soluble polymer; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0 and 1; p is an integer selected from 0 and 1; Su is the hexose form of a monosaccharide; D is a drug group moiety; and RL is the reactive linker group residue.

2. The compound according to claim 1, wherein L 1 Yes-C 1-6 Alkylene-; Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 [Alkenylene] n -X 1 –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene] n -X 1 -, wherein at least one alkylene, alkenylene or alkynylene group in Y is replaced by one or more selected from R 50 Substituents are substituted; R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, –C 2-6 Alkenylene-X 2 -[C 2-6 [Alkenylene] m -POLY, or –C 2-6 Alkynylidene-X 2 -[C 2-6 Alkynylidene m -POLY, where R 50 Each alkylene, alkenylene or alkynylene group is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; X 1 and X 2 are independently selected from -C(O)-, and -N(R 10 )C(O)–; R 10 At each occurrence, independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; POLY is a water-soluble polymer; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0 and 1; Su is the hexose form of a monosaccharide; D is a drug group moiety; and RL is the active linker group residue.

3. The compound or salt according to claim 1 or 2, wherein The compound represented by formula (I) has the structure represented by formula (IA): or a pharmaceutically acceptable salt thereof.

4. A compound or salt according to any one of the preceding claims, wherein L 1 Yes-C 1-3 Alkylene-.

5. The compound or salt according to claim 1 or 3, wherein p is 1.

6. A compound or salt according to any one of the preceding claims, wherein Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 substituted by a substituent.

7. A compound or salt according to any one of the preceding claims, wherein Y is – X 1 -C 1-4 Alkylene-[X 1 -C 1-4 Alkylene] n -X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 substituted by a substituent.

8. The compound or salt according to claim 7, wherein Y is – X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 substituted by a substituent.

9. The compound or salt according to claim 7, wherein Y is – X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 substituted by a substituent.

10. The compound or salt according to any one of claims 1, 3 or 4, wherein p is 0.

11. The compound or salt according to any one of claims 1, 3 or 4, wherein Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 substituted by a substituent.

12. The compound or salt according to any one of claims 1, 3 or 4, wherein Y is – X 1 -C 1-4 Alkylene-[X 1 -C 1-4 Alkylene] n -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 substituted by a substituent.

13. The compound or salt according to claim 12, wherein Y is – X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-, wherein at least one alkylene in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 substituted by a substituent.

14. A compound or salt according to any one of the preceding claims, wherein R 51 Independently selected from halogen, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)-.

15. A compound or salt according to any one of the preceding claims, wherein R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, where R 50 Each alkylene group is optionally substituted by one or more substituents selected from the following: halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Halogenated alkyl.

16. A compound or salt according to any one of the preceding claims, wherein m is 1.

17. A compound or salt according to any one of the preceding claims, wherein POLY is polyethylene glycol (PEG), methoxypolyethylene glycol (mPEG), polypropylene glycol (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohol), poly(vinyl pyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohol), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholine), polysarcosine, or a combination thereof.

18. The compound or salt according to claim 17, wherein POLY comprises polyethylene glycol (PEG) or methoxypolyethylene glycol (mPEG).

19. A compound or salt according to any one of the preceding claims, wherein POLY is in represents the point of attachment to the rest of the compound, and wherein n1 is an integer from 1 to 20.

20. The compound or salt according to claim 19, wherein n1 is an integer between 5 and 15.

21. A compound or salt according to any one of the preceding claims, wherein RL comprises an alkyne, a cyclooctyne, a strained olefin, a tetrazine, a methylcyclopropene, a thiol, a p-acetylphenylalanine residue, an oxyamine, a maleimide, or an azide.

22. A compound or salt according to any one of the preceding claims, wherein The RL is selected from the group consisting of: –N3, –NH2 and –SH; among them, R T It is C 1-6 Alkyl; and represents the point of attachment to the rest of the compound.

23. The compound or salt according to claim 22, wherein R T It is methyl, ethyl, or propyl.

24. The compound or salt according to claim 23, wherein R T It's methyl.

25. The compound or salt according to claim 22, wherein The RL is selected from the group consisting of:

26. The compound or salt according to claim 22, wherein RL is 27. A compound or salt according to any one of the preceding claims, wherein Su is in represents the point of attachment to the rest of the compound.

28. A compound or salt according to any one of the preceding claims, wherein Su is in represents the point of attachment to the rest of the compound.

29. A compound or salt according to any one of the preceding claims, wherein D is the immunomodulatory payload.

30. The compound or salt according to claim 29, wherein The immunomodulatory payload is an agonist of stimulator of interferon genes (STING), Toll-like receptor 7 (TLR7), Toll-like receptor 7 / 8 (TLR7 / 8), or Toll-like receptor 8 (TLR8).

31. The compound or salt according to claim 30, wherein The STING agonist is selected from the group consisting of: small molecule agonists of the STING pathway, antibodies that activate STING activity, recombinant proteins that activate the STING pathway, TTI-10001, DMXAA (ASA404), CDNs, c-di-GMP, 2′3′-cGAMP, MK-1454, ADU-S100 (MIW815), SB11285, ADU-V19, IACS-8779, IACS-8803, IMSA101, non-CDNs, E7766, MK-2118, diABZI, MSA-2, JNJ-'6196, bacterial vectors, SYNB1891, and STACT.

32. The compound or salt according to claim 30, wherein The Toll-like receptor 7 (TLR7) agonist is selected from the group consisting of GS-986, PRTX-007, PRX-034, S-34240, MBS-8, and APR-002.

33. The compound or salt according to claim 30, wherein The Toll-like receptor 7 / 8 (TLR7 / 8) agonist is selected from the group consisting of: imiquimod (R837), resiquimod (R848), 852-A (PF-4878691), vesatolimod (GS-9620), AZD8848, motolimod (VTX-2337), selgantolimod (GS-9688), NKTR-262, RG-7854 (RO 7020531), DSP-0509, BDB-001, BDC-1001, LHC-165, SHR-2150, JNJ-4964 (TQ-73334), RO-7119929, DN-1508052 , VTX-1463, BNT-411(SC1), APR-003, ALT-702, TRANSCON, VX-001, SNAPvax, R848-HA, SM360320, and GSK2245035.

34. The compound or salt according to claim 30, wherein The Toll-like receptor 8 (TLR8) agonist is selected from the group consisting of SBT-6050, SBT-6290, and ZM-TLR8 agonist.

35. A compound or salt according to any one of the preceding claims, wherein D is the cytotoxic payload.

36. The compound or salt according to claim 35, wherein The cytotoxic payload is a tubulin inhibitor, a DNA topoisomerase I inhibitor, or a DNA topoisomerase II inhibitor.

37. A compound or salt according to any one of the preceding claims, wherein D is selected from the group consisting of: Hemiptera (hemiasterlins), camptothecins, anthracyclines, PNU-159682, and EDA PNU-159682 derivatives.

38. A compound or salt according to any one of the preceding claims, wherein D is hemiasterlin, exatecan, PNU-159682, or EDA PNU-159682 derivatives.

39. A compound or salt according to any one of the preceding claims, wherein The compound is selected from or a pharmaceutically acceptable salt of any one thereof.

40. A compound or salt according to any one of the preceding claims, wherein The compound is selected from or a pharmaceutically acceptable salt of any one thereof.

41. A compound or salt according to any one of the preceding claims, wherein The compound is selected from or a pharmaceutically acceptable salt of any one thereof.

42. A compound or salt according to any one of claims 1, 3, 4 or 10-38, wherein The compound is selected from or a pharmaceutically acceptable salt of any one thereof.

43. A compound or salt according to any one of claims 1, 3, 4 or 10-38, wherein The compound is selected from or a pharmaceutically acceptable salt of any one thereof.

44. A compound or salt according to any one of claims 1 to 9 or 15 to 38, wherein The compound is selected from or a pharmaceutically acceptable salt of any one thereof.

45. A compound or salt according to any one of claims 1 to 9 or 15 to 38, wherein The compound is selected from or a pharmaceutically acceptable salt of any one thereof.

46. ​​Compounds having the following chemical structure: or a pharmaceutically acceptable salt thereof.

47. Compounds having the following chemical structure: or a pharmaceutically acceptable salt thereof.

48. A conjugate comprising a compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, linked to a second compound.

49. The conjugate according to claim 48 has a structure shown in formula II: in, COMP is the residue of the second compound; L 1 Yes-C 1-6 Alkylene-; Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -[X 1 ] p –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 [Alkenylene] n -[X 1 ] p –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene n -[X 1 ] p -, wherein at least one alkylene, alkenylene or alkynylene group in Y is replaced by one or more selected from R 50 Substituents are substituted, and wherein the alkylene, alkenylene or alkynylene in Y is optionally replaced by one or more selected from R 51 Substituents are substituted; R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, –C 2-6 Alkenylene-X 2 -[C 2-6 [Alkenylene] m -POLY, or –C 2-6 Alkynylidene-X 2 -[C 2-6 Alkynylidene m -POLY, where R 50 Each alkylene, alkenylene or alkynylene group is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; R 51 independently selected from halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; X 1 and X 2 are independently selected from -N(R 10 )–, –C(O)–, and –N(R 10 )C(O)–; R 10 At each occurrence, independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; POLY is a water-soluble polymer; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0 and 1; p is an integer selected from 0 and 1; Su is the hexose form of a monosaccharide; D is a drug group moiety; and RL is the active linker group residue.

50. The conjugate according to claim 49, wherein COMP is the residue of the second compound; L 1 Yes-C 1-6 Alkylene-; Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 –, –X 1 -C 2-6 Alkenylene-[X 1 -C 2-6 [Alkenylene] n -X 1 –, or –X 1 -C 2-6 Alkynylidene-[X 1 -C 2-6 Alkynylidene n -X 1 -, wherein at least one alkylene, alkenylene or alkynylene group in Y is replaced by one or more selected from R 50 Substituents are substituted; R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, –C 2-6 Alkenylene-X 2 -[C 2-6 [Alkenylene] m -POLY, or –C 2-6 Alkynylidene-X 2 -[C 2-6 Alkynylidene] m -POLY, where R 50 Each alkylene, alkenylene or alkynylene group is optionally substituted by one or more substituents selected from the group consisting of halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; X 1 and X 2 are independently selected from -C(O)-, and -N(R 10 )C(O)–; R 10 At each occurrence, independently selected from H, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Haloalkyl; POLY is a water-soluble polymer; n is an integer selected from 0, 1, 2 and 3; m is an integer selected from 0 and 1; Su is the hexose form of a monosaccharide; D is a drug group moiety; and RL is the active linker group residue.

51. The conjugate according to claim 49 or 50, wherein COMP is a residue of a polypeptide.

52. The conjugate according to claim 49 or 50, wherein COMP is the residue of antibody.

53. The conjugate according to claim 49 or 50, wherein COMP is the residue of the antibody chain.

54. The conjugate according to claim 49 or 50, wherein The conjugate represented by formula (II) has the structure represented by formula (IIA):

55. The conjugate according to any one of claims 49 to 54, wherein L 1 Yes-C 1-3 Alkylene-.

56. The conjugate according to claim 49 or 54, wherein p is 1.

57. The conjugate according to any one of claims 50 to 56, wherein Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 substituted by a substituent.

58. The conjugate according to any one of claims 50 to 57, wherein Y is – X 1 -C 1-4 Alkylene-[X 1 -C 1-4 Alkylene] n -X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 substituted by a substituent.

59. The conjugate according to claim 58, wherein Y is – X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 substituted by a substituent.

60. The conjugate according to claim 58, wherein Y is – X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 substituted by a substituent.

61. The conjugate according to claim 49, 54 or 55, wherein p is 0.

62. The conjugate according to claim 49, 54 or 55, wherein Y is – X 1 -C 1-6 Alkylene-[X 1 -C 1-6 Alkylene] n -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 substituted by a substituent.

63. The conjugate according to claim 49, 54 or 55, wherein Y is – X 1 -C 1-4 Alkylene-[X 1 -C 1-4 Alkylene] n -, wherein at least one alkylene group in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 substituted by a substituent.

64. The conjugate according to claim 63, wherein Y is – X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-X 1 -C 1-4 Alkylene-, wherein at least one alkylene in Y is replaced by one or more selected from R 50 wherein the alkylene group in Y is optionally substituted by one or more selected from R 51 substituted by a substituent.

65. A conjugate according to any one of the preceding claims, wherein R 51 Independently selected from halogen, -CN, -NO2, -OH, -NH2, -C(O)NH2, and -C(O)-.

66. The conjugate according to any one of claims 49 to 60, wherein R 50 Yes – C 1-6 Alkylene-X 2 -[C 1-6 Alkylene] m -POLY, where R 50 Each alkylene group is optionally substituted by one or more substituents selected from the following: halogen, -CN, -NO2, -OH, -N(R 10 )2、-C(O)N(R 10 )2, -C(O)-, -C(S)-, -C(O)OCH2C6H5, -NHC(O)OCH2C6H5, C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, C 3-12 Carbocyclic rings, heterocyclic rings of 3 to 12 atoms, and C 1-10 Halogenated alkyl.

67. The conjugate according to any one of claims 49 to 66, wherein m is 1.

68. The conjugate according to any one of claims 49 to 67, wherein POLY is polyethylene glycol (PEG), methoxypolyethylene glycol (mPEG), polypropylene glycol (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(enols), poly(vinyl pyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohol), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholine), polysarcosine, or a combination thereof.

69. The conjugate according to claim 66, wherein POLY comprises polyethylene glycol (PEG) or methoxypolyethylene glycol (mPEG).

70. The conjugate according to any one of claims 49 to 69, wherein POLY is in represents the point of attachment to the rest of the compound, and wherein n1 is an integer from 1 to 20.

71. The conjugate according to claim 70, wherein n1 is an integer between 5 and 15.

72. The conjugate according to any one of claims 49 to 71, wherein The RL comprises an alkyne, a cyclooctyne, a strained olefin, a tetrazine, a thiol, a p-acetylphenylalanine residue, an oxyamine, an amine, a maleimide, or an azide.

73. The conjugate according to any one of claims 49 to 72, wherein The RL is selected from the group consisting of: Among them, R T It is C 1-6 Alkyl; and represents the point of attachment to the rest of the compound.

74. The conjugate according to any one of claims 49 to 73, wherein R T It is methyl, ethyl, or propyl.

75. The conjugate according to any one of claims 49 to 73, wherein R T It's methyl.

76. The conjugate according to claim 73, wherein The RL is selected from the group consisting of:

77. The conjugate according to claim 76, wherein The RL is selected from the group consisting of:

78. The conjugate according to any one of claims 49 to 76, wherein Su is in represents the point of attachment to the rest of the compound.

79. The conjugate according to any one of claims 49 to 78, wherein Su is in represents the point of attachment to the rest of the compound.

80. A conjugate according to any one of the preceding claims, wherein D is the immunomodulatory payload.

81. The conjugate according to claim 80, wherein The immunomodulatory payload is an agonist of stimulator of interferon genes (STING), Toll-like receptor 7 (TLR7), Toll-like receptor 7 / 8 (TLR7 / 8), or Toll-like receptor 8 (TLR8).

82. The conjugate according to claim 81, wherein The STING agonist is selected from the group consisting of: small molecule agonists of the STING pathway, antibodies that activate STING activity, recombinant proteins that activate the STING pathway, TTI-10001, DMXAA (ASA404), CDNs, c-di-GMP, 2′3′-cGAMP, MK-1454, ADU-S100 (MIW815), SB11285, ADU-V19, IACS-8779, IACS-8803, IMSA101, non-CDNs, E7766, MK-2118, diABZI, MSA-2, JNJ-'6196, bacterial vectors, SYNB1891, and STACT.

83. The conjugate according to claim 81, wherein The Toll-like receptor 7 (TLR7) agonist is selected from the group consisting of GS-986, PRTX-007, PRX-034, S-34240, MBS-8, and APR-002.

84. The conjugate according to claim 81, wherein The Toll-like receptor 7 / 8 (TLR7 / 8) agonist is selected from the group consisting of: imiquimod (R837), resiquimod (R848), 852-A (PF-4878691), vesatolimod (GS-9620), AZD8848, motolimod (VTX-2337), selgantolimod (GS-9688), NKTR-262, RG-7854 (RO 7020531), DSP-0509, BDB-001, BDC-1001, LHC-165, SHR-2150, JNJ-4964 (TQ-73334), RO-7119929, DN-1508052 , VTX-1463, BNT-411(SC1), APR-003, ALT-702, TRANSCON, VX-001, SNAPvax, R848-HA, SM360320, and GSK2245035.

85. The conjugate according to claim 81, wherein The Toll-like receptor 8 (TLR8) agonist is selected from the group consisting of: SBT-6050, SBT-6290 and ZM-TLR8 agonists.

86. The conjugate according to any one of claims 49 to 79, wherein D is the cytotoxic payload.

87. The conjugate according to claim 86, wherein The cytotoxic payload is a tubulin inhibitor, a DNA topoisomerase I inhibitor, or a DNA topoisomerase II inhibitor.

88. The conjugate according to any one of claims 49 to 87, wherein D is selected from the group consisting of: hemiasterlins, camptothecins, anthracyclines, PNU-159682, and EDA PNU-159682 derivatives.

89. The conjugate according to any one of claims 49 to 88, wherein D is hemiasterlin, exatecan, PNU-159682, or EDA PNU-159682 derivatives.

90. The conjugate according to any one of claims 49 to 89, wherein The compound is selected from or a pharmaceutically acceptable salt of any one thereof, wherein is the residue of the second compound.

91. The conjugate according to any one of claims 50 to 90, wherein The compound is selected from or a pharmaceutically acceptable salt of any one thereof, wherein is the residue of the second compound.

92. The conjugate according to any one of claims 50 to 91, wherein The compound is selected from or a pharmaceutically acceptable salt of any one thereof, in is the residue of the second compound.

93. The conjugate according to any one of claims 49, 51-55 or 61-89, wherein The conjugate is selected from or a pharmaceutically acceptable salt of any one thereof, wherein COMP is said residue of said second compound.

94. The conjugate according to any one of claims 49, 51-55 or 61-89, wherein The conjugate is selected from or a pharmaceutically acceptable salt of any one thereof, wherein COMP is said residue of said second compound.

95. The conjugate according to any one of claims 49-60 or 66-89, wherein The conjugate is selected from or a pharmaceutically acceptable salt of any one thereof, wherein COMP is said residue of said second compound.

96. The conjugate according to any one of claims 49-60 or 66-89, wherein The conjugate is selected from or a pharmaceutically acceptable salt of any one thereof, wherein COMP is said residue of said second compound.

97. A conjugate having the following chemical structure: A pharmaceutically acceptable salt of any one thereof, in is the residue of the second compound.

98. A conjugate having the following chemical structure: A pharmaceutically acceptable salt of any one thereof, in is the residue of the second compound.

99. A pharmaceutical composition comprising the compound of any one of claims 1-47, or the conjugate of any one of claims 48-98; and a pharmaceutically acceptable excipient, carrier, or diluent.

100. A compound according to any one of claims 1-47, a conjugate according to any one of claims 48-98, or a pharmaceutical composition according to claim 99 for use in therapy.

101. A compound according to any one of claims 1-47, a conjugate according to any one of claims 48-98, or a pharmaceutical composition according to claim 99 for use in treating cancer.

102. A compound according to any one of claims 1-47, a conjugate according to any one of claims 48-98, or a pharmaceutical composition according to claim 99 for use as a medicament.

103. A method of inhibiting tubulin polymerization in a subject in need thereof, comprising administering to the subject an effective amount of a compound of any one of claims 1-47, a conjugate of any one of claims 48-98, or a pharmaceutical composition of claim 99.

104. A method of reducing cell proliferation in a subject in need thereof, comprising administering to the subject an effective amount of a compound of any one of claims 1-47, a conjugate of any one of claims 48-98, or a pharmaceutical composition of claim 99.

105. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a compound of any one of claims 1-47, a conjugate of any one of claims 48-98, or a pharmaceutical composition of claim 99.

106. The method of claim 105, wherein: The cancer is small cell lung cancer, non-small cell lung cancer, ovarian cancer, platinum-resistant ovarian cancer, ovarian adenocarcinoma, endometrial cancer, breast cancer, breast cancer overexpressing HER2, triple-negative breast cancer, lymphoma, large cell lymphoma, diffuse mixed histiocytic and lymphocytic lymphoma, follicular B-cell lymphoma, colon cancer, colon carcinoma (colon carcinoma, colon adenocarcinoma, colorectal adenocarcinoma, melanoma, prostate cancer, or multiple myeloma.

107. A method for preparing a conjugate, comprising contacting a compound as described in any one of claims 1-47 with a second compound under conditions suitable for coupling the compound as described in any one of claims 1-47 with the second compound; wherein the second compound comprises an alkyne, a cyclooctyne, a strained olefin, a tetrazine, a methylcyclopropene, a thiol, a maleimide, a carbonyl, an amine, an oxyamine, or an azide.

108. The method of claim 107, wherein: The second compound comprises a tetrazine; and RL comprises a strained olefin.

109. The method according to claim 107 or 108, wherein: RL is 110. The method of claim 107, wherein: The second compound comprises an azide; and RL comprises an alkyne.

111. The method according to claim 107 or 110, wherein: RL is 112. The method of claim 107, wherein: The second compound comprises an alkyne; and RL comprises an azide.

113. The method of claim 107, wherein: The second compound comprises a strained olefin; and RL comprises a tetrazine.

114. The method of claim 107, wherein: The second compound comprises a thiol; and the RL comprises a maleimide.

115. The method of claim 107 or 114, wherein: RL is 116. The method of claim 107, wherein: The second compound comprises a maleimide; and RL comprises a thiol.

117. The method of claim 107 or 116, wherein: The second compound comprises 118. The method of claim 107, wherein: The second compound comprises a carbonyl group; and the RL comprises an oxyamine.

119. The method of claim 107 or 118, wherein: RL is 120. The method of claim 107, 118 or 119, wherein: The second compound comprises 121. The method of claim 107, wherein: The second compound comprises an oxyamine; and RL comprises a carbonyl group.

122. The method of claim 107 or 121, wherein: RL is 123. The method of claim 107, 121 or 122, wherein: The second compound comprises 124. The method according to any one of claims 107-123, wherein: The second compound is a polypeptide.

125. The method according to any one of claims 107-123, wherein: The second compound is an antibody.

126. The method according to any one of claims 107-123, wherein: The second compound is an antibody chain.

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