Polymer-drug conjugates

Abstract

Disclosed herein is a polymeric scaffold for binding to a targeting moiety to form a targeting moiety-polymer-drug conjugate. Also disclosed herein are targeting moiety-polymer-drug conjugates prepared from the scaffold. Also disclosed herein are compositions comprising the conjugates, methods of making the same, and methods of treating various conditions with the conjugates or compositions thereof.

Description

Field of the Invention

[0001] The present disclosure generally relates to compounds, conjugates comprising these compounds, pharmaceutical compositions thereof, and methods of treating diseases or conditions with the conjugates or pharmaceutical compositions thereof. Background Art

[0002] Traditionally, therapeutic agents consisting primarily of small molecules are delivered into the body via oral / GI absorption or systemic injection and then delivered to the site of action via the blood circulation. However, many challenges still need to be addressed. For example, many therapeutic agents exhibit limited or reduced efficacy and therapeutic effects because they typically undergo partial degradation or accumulate in tissues other than the target, or both, before they reach the desired target in the body.

[0003] Therefore, there is a need for intact delivery of therapeutic agents to specific target areas of the body via a system that can stabilize the drug and control the in vivo transfer of the therapeutic agent to achieve maximum cytotoxicity of the therapeutic agent. Summary of the invention

[0004] The present disclosure relates to a polymer stent delivery system that exhibits high drug loading and strong binding to target antigens, thereby effectively delivering and releasing the drug to a target site. The present disclosure also relates to a polymer stent for combining with a targeting moiety to obtain a polymer stent delivery system.

[0005] In one aspect, the present disclosure provides a polymer scaffold of formula (I) for conjugation with a targeting moiety:

[0006]

[0007] in:

[0008] The polymer scaffold comprises linear polyglycerol;

[0009] L is a functional group W that can form a covalent bond with the targeting moiety. p The connecting part;

[0010] M a is an extending group connecting L to the -NH- portion;

[0011] Each G 1 Independently p a functional group attached to the linear polyglycerol;

[0012] Each L P Independently, the therapeutic agent moieties D and G 1 Drug release mechanism between;

[0013] Each D is independently a therapeutic agent moiety;

[0014] Each G 2 are independently functional groups capable of being transformed into a charged state;

[0015] n is an integer from 0 to 1000;

[0016] m is an integer from 0 to 1000;

[0017] p is an integer from 0 to 1000; and

[0018] q is an integer from 0 to 1000.

[0019] In another aspect, the present disclosure provides a polymer scaffold of formula (II):

[0020]

[0021] in,

[0022] The polymer scaffold comprises linear polyglycerol;

[0023] Each G 2 are independently functional groups capable of being transformed into a charged state;

[0024] Each G 3 independently comprising a functional group capable of reacting with a reactive group in a drug release mechanism to thereby link the drug release mechanism to the linear polyglycerol;

[0025] n is an integer from 0 to 1000;

[0026] m is an integer from 0 to 1000;

[0027] p is an integer from 0 to 1000; and

[0028] q is an integer from 0 to 1000.

[0029] In another aspect, the present disclosure provides a polymer scaffold of formula (III):

[0030]

[0031] in,

[0032] The polymer scaffold comprises linear polyglycerol;

[0033] Each G 2 are independently functional groups capable of being transformed into a charged state;

[0034] Each G 3 independently comprising a functional group capable of reacting with a reactive group in a drug release mechanism to thereby link the drug release mechanism to the linear polyglycerol;

[0035] n is an integer from 0 to 1000;

[0036] m is an integer from 0 to 1000;

[0037] p is an integer from 0 to 1000; and

[0038] q is an integer from 0 to 1000.

[0039] In another aspect, the present disclosure provides a polymer scaffold of formula (IV):

[0040]

[0041] in,

[0042] The polymer scaffold comprises linear polyglycerol;

[0043] Each L a Independently for attaching the targeting moiety to M a The divalent part of

[0044] Each M a Independently a an extending group attached to the -NH- moiety;

[0045] Each G 1 Independently p a functional group attached to the linear polyglycerol;

[0046] Each L P Independently, the therapeutic agent moieties D and G 1 Drug release mechanism between;

[0047] Each D is independently a therapeutic agent moiety;

[0048] Each G 2 are independently functional groups capable of being transformed into a charged state;

[0049] n is an integer from 0 to 1000;

[0050] m is an integer from 0 to 1000;

[0051] p is an integer from 0 to 1000;

[0052] q is an integer from 0 to 1000; and

[0053] s is an integer from 1 to 8.

[0054] In another aspect, the present disclosure provides a pharmaceutical composition comprising the polymer scaffold or conjugate described herein and a pharmaceutically acceptable carrier.

[0055] In another aspect, the present disclosure provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polymer scaffold or conjugate described herein or a pharmaceutical composition provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Shown are cell-based binding of Trastuzumab and test anti-HER2 ADCs to HER2-positive SKBR-3 cells (A) and NCI-N87 cells (B).

[0057] Figure 2 Internalization of the tested anti-HER2 ADCs into HER2-positive SKBR-3 cells (A) and NCI-N87 cells (B) from 0 to 24 hours is shown.

[0058] Figure 3 Shown are in vitro cytotoxicity of the tested anti-HER2 ADCs against HER2-expressing SKBR-3 cells (A) and NCI-N87 cells (B) and HER2-negative MCF-7 cells (C).

[0059] Figure 4 Shown are cell-based binding of the Trop2 antibody Trop2-1, antibody Trop2-2, and Datopotamab, as well as exemplary anti-Trop2 ADCs to Trop2-positive SKBR-3 cells.

[0060] Figure 5 Internalization of the tested anti-Trop2 ADC into Trop2-positive SKBR-3 cells is shown.

[0061] Figure 6 In vitro cytotoxicity of the tested anti-Trop2 ADCs against Trop2-expressing SKBR-3 cells (A), NCI-N87 cells (B), MCF-7 cells (C), and MDA-MB-468 cells (D) are shown.

[0062] Figure 7 ADC-5 and ADC-6 are shown to effectively inhibit tumor growth in BALB / c nude mice with existing NCI-N87 tumor cell xenografts. (A) Change in tumor volume over 28 days of treatment. (B) Change in body weight over 28 days of treatment. (C) Tumor weight measured on day 28 after administration. DETAILED DESCRIPTION

[0063] Now with reference to certain embodiments of the present invention in detail, examples of the embodiments are described in the accompanying structures and chemical formulas. Although the present invention will be described in conjunction with the enumerated embodiments, it should be understood that it is not intended to limit the present invention to those embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications and equivalent forms, which may be included in the scope of the present invention as defined in the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to the methods or materials described herein can be used to practice the present invention. The present invention is by no means limited to the methods and materials described. In the case where one or more of the incorporated documents and similar materials (including but not limited to defined terms, term usage, the technology, etc.) are different from or conflict with the present application, the present application shall prevail.

[0064] It should be understood that certain features of the present disclosure described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination.

[0065] It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an" and "the" include their plural forms unless the context clearly dictates otherwise. Certain terms that will be mentioned in this specification and the appended claims shall be defined as having the following meanings, unless there is an obvious contrary intention.

[0066] definition

[0067] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are generally defined as described herein. In addition, general principles of organic chemistry as well as specific functional moieties and reactivities are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March March, Advanced Organic Chemistry, 5th ed., John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd ed., Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0068] Throughout this disclosure, linking substituents are described. Where a structure clearly requires a linking group, the Markush variable listed for that group is understood to be a linking group. For example, if a structure requires a linking group and the Markush group definition for that variable lists "alkyl," it is understood that the "alkyl" means a linking alkylene group.

[0069] When any variable (e.g., R i ) occurs more than once in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is represented by 0 to 2 R i Partially substituted, the group may optionally be substituted with up to two R i Partially substituted, and R i Each occurrence is independently selected from Ri Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0070] As used herein, for convenience, a dash "-" is used in front of or at the end of a chemical group to indicate the point of attachment of a substituent. For example, -OH is attached through a carbon atom; a chemical group can be depicted with or without one or more dashes without losing its general meaning. A wavy line drawn through a line in a structure indicates the point of attachment of a group. Unless chemically or structurally required, directionality does not indicate or imply the order in which the chemical groups are written or named. As used herein, a solid line coming out of the center of a ring indicates that the point of attachment of a substituent on the ring can be at any ring atom. When a listed substituent does not indicate that the substituent is bonded to an atom of the rest of a compound of a given formula, then the substituent may be bonded via any atom in the formula. Combinations of substituents and / or variables are allowed, but only when the combination produces a stable compound.

[0071] When any variable (e.g., R i ) occurs more than once in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is represented by 0 to 2 R i Partially substituted, the group may optionally be substituted with up to two R i Partially substituted, and R i Each occurrence is independently selected from R i Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0072] The term "about" when used in conjunction with a numerical value is intended to include a set or range of values. For example, "about X" includes a range of values ​​of ±20%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X, where X is a numerical value. In one embodiment, the term "about" refers to a range of values ​​that is greater than or less than 5% of a specified value. In another embodiment, the term "about" refers to a range of values ​​that is greater than or less than 2% of a specified value. In another embodiment, the term "about" refers to a range of values ​​that is greater than or less than 1% of a specified value.

[0073] Unless otherwise indicated herein, the recitation of a range of values ​​is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if individually recited herein. Unless otherwise indicated, ranges used herein include both limits of the range. For example, the expressions "n is an integer between 1 and 6" and "n is an integer from 1 to 6" both mean "n is 1, 2, 3, 4, 5, or 6".

[0074] As used herein, the term “C i-j " indicates a range of carbon atoms, wherein i and j are integers, and the range of carbon atoms includes the endpoints (i.e., i and j) and every integer point therebetween, and wherein j is greater than i. For example, C 1-6 Indicates a range of one to six carbon atoms, including one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms, and six carbon atoms. In some embodiments, the term “C 1-12 ” indicates 1 to 12 carbon atoms, especially 1 to 10 carbon atoms, especially 1 to 8 carbon atoms, especially 1 to 6 carbon atoms, especially 1 to 5 carbon atoms, especially 1 to 4 carbon atoms, especially 1 to 3 carbon atoms or especially 1 to 2 carbon atoms. In a similar way, the term “m-membered to n-membered” ring (wherein m and n are integers and n is greater than m) refers to a ring containing m to n atoms.

[0075] As used herein, the term "aliphatic" includes saturated and unsaturated straight chain (i.e., non-branched) or branched aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by those of ordinary skill in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl moieties.

[0076] As used herein, the term "alkyl", whether used as part of another term or by itself, refers to a saturated straight or branched chain hydrocarbon group that may be optionally substituted independently with one or more substituents as described below. i-j "Alkyl" refers to a straight or branched chain alkyl group having i to j carbon atoms. For example, the alkyl group contains 1 to 10 carbon atoms, 1 to 9 carbon atoms, 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. "C 1-6 Examples of “alkyl” include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 2-ethyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, and the like.

[0077] As used herein, the term "alkenyl", whether used as part of another term or independently, refers to a straight or branched hydrocarbon group with at least one carbon-carbon double bond that can be optionally independently substituted by one or more substituents described herein, and includes groups with "cis" orientation and "trans" orientation or alternatively "E" orientation and "Z" orientation. In some embodiments, the alkenyl contains 2 to 12 carbon atoms. In some embodiments, the alkenyl contains 2 to 11 carbon atoms. In some embodiments, the alkenyl contains 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms, and in some embodiments, the alkenyl contains 2 carbon atoms. Examples of alkenyl include but are not limited to vinyl (ethylenyl / vinyl), propenyl (allyl), butenyl, pentenyl, 1-methyl-2-butyl-1-yl, 5-hexenyl, etc.

[0078] As used herein, the term "alkynyl", whether used as part of another term or independently, refers to a straight or branched hydrocarbon group with at least one carbon-carbon triple bond that can be optionally independently substituted by one or more substituents described herein. In some embodiments, alkenyl contains 2 to 12 carbon atoms. In some embodiments, alkynyl contains 2 to 11 carbon atoms. In some embodiments, alkynyl contains 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms, and in some embodiments, alkynyl contains 2 carbon atoms. Examples of alkynyl include but are not limited to ethynyl, 1-propynyl, 2-propynyl, etc.

[0079] As used herein, the term "amino" refers to the group -NR a R b , where R a and R b are independently selected from the group consisting of hydrogen, alkyl, haloalkyl, aryl, heteroaryl, cycloalkyl and heterocyclyl and each of which may be optionally substituted.

[0080] As used herein, the term "aryl", whether used as part of another term or independently, refers to monocyclic and polycyclic systems having a total of 5 to 20 ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 12 ring members. Examples of "aryl" include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, etc., which may carry one or more substituents. As used herein, groups in which an aromatic ring is fused to one or more additional rings are also included within the scope of the term "aryl". In the case of a polycyclic ring system, only one ring is required to be aromatic (e.g., 2,3-dihydroindole), but all rings may be aromatic (e.g., quinoline). The second ring may also be fused or bridged. Examples of polycyclic aromatic groups include, but are not limited to, benzofuranyl, indanyl, phthalimido, naphthylimide, phenanthracenyl, or tetrahydronaphthyl, etc.

[0081] As used herein, the term "cycloalkyl", whether used as part of another term or independently, refers to a monovalent non-aromatic saturated or partially unsaturated monocyclic and polycyclic system, wherein all ring atoms are carbon, and the system contains at least three ring-forming carbon atoms. In some embodiments, the cycloalkyl may contain 3 to 12 ring-forming carbon atoms, 3 to 10 ring-forming carbon atoms, 3 to 9 ring-forming carbon atoms, 3 to 8 ring-forming carbon atoms, 3 to 7 ring-forming carbon atoms, 3 to 6 ring-forming carbon atoms, 3 to 5 ring-forming carbon atoms, 4 to 12 ring-forming carbon atoms, 4 to 10 ring-forming carbon atoms, 4 to 9 ring-forming carbon atoms, 4 to 8 ring-forming carbon atoms, 4 to 7 ring-forming carbon atoms, 4 to 6 ring-forming carbon atoms, 4 to 5 ring-forming carbon atoms. Cycloalkyl can be saturated or partially unsaturated. In some embodiments, the cycloalkyl can be a saturated cyclic alkyl. In some embodiments, the cycloalkyl group may be a partially unsaturated cyclic alkyl group containing at least one double bond or triple bond in its ring system.

[0082] In some embodiments, the cycloalkyl group may be a saturated or unsaturated monocyclic carbocyclic ring system, examples of which include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl.

[0083] In some embodiments, cycloalkyl can be saturated or partially unsaturated polycyclic (for example, bicyclic and tricyclic) carbocyclic system, which can be arranged as condensed ring system, spiro ring system or bridged ring system. As used herein, the term "condensed ring" refers to a ring system with two rings sharing two adjacent atoms, the term "spiro ring" refers to a ring system with two rings connected by a single common atom, and the term "bridged ring" refers to a ring system with two rings sharing three or more atoms. The example of fused carbocyclic group includes but is not limited to naphthyl, benzopyrenyl, anthracenyl, acenaphthenyl, fluorenyl, etc. The example of spiral carbocyclic group includes but is not limited to spiral [5.5] undecyl, spiral-pentadienyl, spiral [3.6]-decyl, etc. Examples of bridged carbocyclic groups include, but are not limited to, bicyclo[1,1,1]pentenyl, bicyclo[2,2,1]heptenyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1]nonyl, bicyclo[3.3.3]undecyl, and the like.

[0084] As used herein, the term "halo" or "halogen" refers to an atom selected from fluorine (fluorine or fluoro), chlorine (chlorine or chloro), bromine (bromine or bromo), and iodine (iodine or iodo).

[0085] As used herein, the term "heteroatom" refers to nitrogen, sulfur, or phosphorus, and includes any oxidized form of the nitrogen, sulfur, or phosphorus and any quaternized form of a basic nitrogen.

[0086] As used herein, the term "heteroaliphatic" refers to an aliphatic moiety in which one or more carbon atoms in the main chain have been replaced by heteroatoms. Thus, a heteroaliphatic group refers to an aliphatic chain containing one or more oxygen atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, or silicon atoms (e.g., in place of carbon atoms). A heteroaliphatic moiety may be a branched chain or a straight unbranched chain. As will be understood by one of ordinary skill in the art, "heteroaliphatic" is intended herein to include, but is not limited to, heteroalkyl, heteroalkenyl, and heteroalkynyl moieties. In certain embodiments, a heteroaliphatic moiety is substituted ("substituted heteroaliphatic") by independently replacing one or more hydrogen atoms thereon with one or more moieties, including, but not limited to, aliphatic; heteroaliphatic; cycloalkyl; heterocycloalkyl; aryl; heteroaryl; alkaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; -NO 2 ;-CN;-CF 3 ; -CH 2 CF 3 ;-CHC 12 ; -CH 2 OH; -CH 2 CH 2OH; -CH 2 NH 2 ; -CH 2 SO 2 CH 3 ; or -GR G1 , where G is -O-, -S-, or -NR G2 -, -C(=O)-, -S(=O)-, -SO 2 -, -C(=O)O-, -C(=O)NR G2 -、-OC(=O)-、-NR G2 C(=O)-, -OC(=O)O-, -OC(=O)NR G2 -、-NR G2 C(=O)O-、-NR G2 C(=O)NR G2 -, -C(=S)-, -C(=S)S-, -SC(=S)-, -SC(=S)S-, -C(=NR G2 )-、-C(=NR G2 )O-、-C(=NR G2 )NR G3 -、-OC(=NR G2 )-、-NR G2 C(=NR G3 )-、-NR G2 SO2-、-NR G2 SO 2 NR G3 -or-SO 2 NR G2 -, where R G1 , R G2 and R G3 In each occurrence, independently includes, but is not limited to, hydrogen, halogen, or an optionally substituted aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkaryl, or alkylheteroaryl moiety. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples described herein.

[0087] As used herein, the term "heteroalkyl" refers to an alkyl group in which at least one of its carbon atoms is replaced by a heteroatom selected from N, O or S. A heteroalkyl group can be a carbon group or a heteroatom group (i.e., a heteroatom can appear in the middle or at a terminal end of the group), and can be optionally substituted independently with one or more substituents described herein. The term "heteroalkyl" encompasses alkoxy and heteroalkoxy groups.

[0088] As used herein, the term "heteroalkenyl" refers to an alkenyl group in which at least one of its carbon atoms is replaced with a heteroatom selected from N, O or S. The heteroalkenyl group may be carbonyl or heteroatomyl (i.e., the heteroatom may occur in the middle or at the terminal end of the group), and may be optionally substituted independently with one or more substituents described herein.

[0089] As used herein, the term "heteroalkynyl" refers to an alkynyl group in which at least one of its carbon atoms is replaced with a heteroatom selected from N, O or S. A heteroalkynyl group may be a carbon group or a heteroatom group (i.e., the heteroatom may occur in the middle or at a terminal end of the group), and may be optionally substituted independently with one or more substituents described herein.

[0090] As used herein, the term "heteroaryl", whether used as part of another term or independently, refers to an aryl group having one or more heteroatoms in addition to carbon atoms. Heteroaryl can be monocyclic. Examples of monocyclic heteroaryl include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, benzofuranyl, and pteridinyl. Heteroaryl also includes a polycyclic group to which a heteroaromatic ring is fused to one or more aryl, heteroaryl, cycloaliphatic or heterocyclic rings, wherein a linking group or point of attachment is located on the heteroaromatic ring. Examples of polycyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, benzothiophenyl, benzofuranyl, benzo[1,3]dioxolyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, dihydroquinolyl, dihydroisoquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, and the like.

[0091] As used herein, the term "heterocycloalkyl" refers to a saturated or partially unsaturated cycloalkyl group in which one or more ring atoms are independently selected from heteroatoms such as oxygen, sulfur, nitrogen, phosphorus, etc., and the remaining ring atoms are carbon, wherein one or more ring atoms may be optionally substituted independently by one or more substituents. In some embodiments, the heterocycloalkyl group is a saturated heterocycloalkyl group. In some embodiments, the heterocycloalkyl group is a partially unsaturated heterocycloalkyl group having one or more double bonds in its ring system. In some embodiments, the heterocycloalkyl group may contain any oxidized form of carbon, nitrogen or sulfur and any quaternized form of basic nitrogen. Where possible, the heterocycloalkyl group may be carbon-connected or nitrogen-connected. In some embodiments, the heterocycle is carbon-connected. In some embodiments, the heterocycle is nitrogen-connected. For example, a group derived from pyrrole may be pyrrole-1-yl (nitrogen-connected) or pyrrole-3-yl (carbon-connected). In addition, a group derived from imidazole may be imidazole-1-yl (nitrogen-connected) or imidazole-3-yl (carbon-connected).

[0092] The heterocycloalkyl group may be monocyclic. Examples of monocyclic heterocycloalkyl groups include, but are not limited to, oxetanyl, 1,1-dioxothietane, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, azetidinyl, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, piperidinyl, piperazinyl, morpholinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyrimidonyl, pyridazinonyl, pyrrolidinyl, triazinonyl, etc.

[0093] Heterocycloalkyl groups may be polycyclic, including fused ring systems, spiro ring systems, and bridged ring systems. Fused heterocycloalkyl groups include groups in which a heterocycloalkyl group is fused to a saturated, partially unsaturated, or fully unsaturated (ie, aromatic) carbocyclic or heterocyclic ring. Examples of fused heterocycloalkyl groups include, but are not limited to, phenyl fused rings or pyridyl fused rings, such as quinolyl, isoquinolyl, quinoxalinyl, quinolizinyl, quinazolinyl, azaindolizinyl, pteridinyl, chromenyl, isochromenyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothiophenyl, benzothiazolyl, carbazolyl, phenazinyl, phenothiazinyl, phenanthridinyl, imidazo[1,2-a]pyridinyl, furano[3,4-d]pyrimidinyl, pyrrolo[3,4-d]pyrimidinyl, dihydrofuro[3,4-b]pyridinyl, and the like. Examples of spiroheterocycloalkyl include, but are not limited to, spiropyranyl, spirooxazinyl, 5-aza-spiro[2.4]heptyl, 6-aza-spiro[2.5]octyl, 6-aza-spiro[3.4]octyl, 2-oxa-6-aza-spiro[3.3]heptyl, 2-oxa-6-aza-spiro[3.4]octyl, 6-aza-spiro[3.5]nonyl, 7-aza-spiro[3.5]nonyl, 1-oxa-7-aza-spiro[3.5]nonyl, 3,8-dioxa-1-azaspiro[4.5]dec-1-enyl, and the like. Examples of bridged heterocycloalkyl groups include, but are not limited to, 3-aza-bicyclo[3.1.0]hexyl, 8-aza-bicyclo[3.2.1]octyl, 1-aza-bicyclo[2.2.2]octyl, 2-aza-bicyclo[2.2.1]heptyl, 1,4-diazabicyclo[2.2.2]octyl, and the like.

[0094] As used herein, the term "hydroxy" refers to -OH.

[0095] As used herein, the term "leaving group" refers to a molecular fragment that takes away a pair of electrons during heterolytic bond cleavage. A leaving group can be an anionic molecule or a neutral molecule. Leaving groups include, but are not limited to, halides, such as Cl - Br - and I - ; sulfonates, such as p-toluenesulfonate ("toluenesulfonate", TsO - ) and RC(O)O-, where R is hydrogen, an aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl moiety.

[0096] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0097] As used herein, the term "partially unsaturated" refers to a group that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic (ie, fully unsaturated) moieties.

[0098] As used herein, the term "blocking group" means a specific functional part, such as O, S or N is temporarily blocked so that another reaction site in a multifunctional compound can be selectively reacted. In some embodiments, the blocking group reacts selectively with a good yield to obtain a protected substrate that is stable for the expected reaction. The blocking group must be selectively removed with a good yield by a readily available non-toxic reagent that preferably does not attack other functional groups. The blocking group forms an easily separable derivative (more preferably does not produce a new stereocenter). The blocking group has a minimum additional functionality to avoid further reaction sites. As described in detail herein, oxygen blocking groups, sulfur blocking groups, nitrogen blocking groups and carbon blocking groups can be utilized. For example, in some embodiments, certain exemplary oxygen blocking groups can be utilized. These oxygen protecting groups include, but are not limited to, methyl ethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM (methylthiomethyl ether), BOM (benzyloxymethyl ether), and PMBM (p-methoxybenzyloxymethyl ether)), substituted ethyl ethers, substituted anisole, silyl ethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilyl ether), TIPS (triisopropylsilyl ether), TBDMS (tert-butyldimethylsilyl ether), tritylsilyl ether, and TBDPS (tert-butyldiphenylsilyl ether), esters (e.g., formates, acetates, benzoates (Bz), trifluoroacetates, and dichloroacetates), carbonates, cyclic acetals, and ketals. In some other embodiments, nitrogen protecting groups are utilized. Nitrogen protecting groups and methods of protection and deprotection are known in the art. Nitrogen protecting groups include but are not limited to Limited to carbamates (including methyl carbamate, ethyl carbamate and substituted ethyl carbamate (e.g., Troc)), amides, cyclic imide derivatives, N- alkylamines and N- aromatic amines, imine derivatives and enamine derivatives. In yet other embodiments, certain exemplary sulfur protecting groups may be utilized. Sulfur protecting groups include, but are not limited to, those oxygen protecting groups described above, as well as aliphatic carboxylic acids (e.g., acrylic acid), maleimide, vinylsulfonyl and optionally substituted maleic acid. Some other exemplary protecting groups are described in detail herein, however, it should be understood that the present invention is not intended to be limited to these protecting groups; in fact, a variety of additional equivalent protecting groups can be easily identified using the above criteria and used in the present invention. In addition, a variety of protecting groups are described in "Protective Groups in Organic Synthesis", Third Edition, Greene, TW and Wuts, PG, John Wiley & Sons, New York: 1999, the entire contents of which are incorporated herein by reference.

[0099] As used herein, the term "leaving group" refers to a molecular fragment that takes away a pair of electrons when a heterogeneous bond is cleaved. A leaving group can be an anionic molecule or a neutral molecule. Leaving groups include, but are not limited to, halides such as Cl - Br - and I - ; sulfonates, such as p-toluenesulfonate ("toluenesulfonate", TsO - ) and RC(O)O-, where R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic or heterocycloalkyl moiety.

[0100] As used herein, the term "substituted", whether or not preceded by the term "optionally", means that one or more hydrogens of the designated moiety are replaced by suitable substituents. It should be understood that "substitution" or "substituted" includes the following implicit restrictions: the substitution is consistent with the allowed valence states of the substituted atom, and the substitution produces a stable or chemically feasible compound, such as a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, etc. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted by more than one substituent selected from a specified group, the substituent may be the same or different at each position. Substitution can include but is not limited to alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amide, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, guanidino, halo, haloalkyl, heteroalkyl, heteroaryl, heterocyclic radical, hydroxyl, hydrazino, imino, oxo, nitro, alkylsulfinyl, sulfonic acid, alkylsulfonyl, thiocyanate, mercaptan, thioketone or its combination. It will be appreciated by those skilled in the art that, if appropriate, the substituent itself can be substituted. Unless specifically stated as "unsubstituted", the reference to the chemical moiety herein should be understood to include substituted variants. For example, reference to "aryl" groups or parts implicitly includes both substituted variants and unsubstituted variants.

[0101] As used herein, the term "targeting moiety" refers to a molecule that recognizes and binds to a cell surface marker or receptor (such as a transmembrane protein, a surface-fixed protein, or a proto-glycan). Examples of targeting moieties include, but are not limited to, antibodies or fragments thereof, lipocalins, proteins, peptides, or peptide mimetics, etc. In addition to targeting the polymer scaffold to a specific cell, tissue, or location, the targeting moiety may also have certain therapeutic effects, such as anti-proliferative (cell growth inhibition and / or cytotoxic) activity against a target cell or target pathway. The targeting moiety contains or may be engineered to contain at least one chemically reactive group, such as -COOR, -SH, an amine group; or a chemically reactive amino acid moiety or side chain, such as tyrosine, histidine, cysteine, or lysine. In some embodiments, the targeting moiety may be a ligand that specifically binds or complexes to a cell surface molecule (such as a cell surface receptor or antigen) of a given target cell population. After the ligand specifically binds or complexes to its receptor, the cell is allowed to absorb the ligand or ligand-drug conjugate, which is then internalized into the cell. As used herein, a ligand that "specifically binds or complexes" or "targets" a cell surface molecule preferentially associates with the cell surface molecule via intermolecular forces.

[0102] As used herein, the term "ligand" refers to a variety of chemical or biological molecules that can have a specific binding affinity to a selected target, wherein the selected target can be, for example, a cell surface receptor, a cell surface antigen, a cell, a tissue, an organ, etc. In some embodiments, the ligand can specifically bind to a protein or marker expressed on the surface of a target cell. In some embodiments, the ligand of the present disclosure is expressed in 10 -6 Up to 10 -11 M(K d In some embodiments, the ligands of the present disclosure bind to cell surface proteins or markers with an affinity of at least 10 -7 , at least 10 -8 and at least 10 -9 M(K d In some embodiments, the ligands of the present disclosure bind to cell surface proteins or markers with an affinity of less than 10 -6 , less than 10 -7 and less than 10 -8 M(K dIn some embodiments, the ligand of the present invention binds to a cell surface protein or marker with a certain affinity, wherein the certain affinity refers to the affinity of the ligand to the target cell surface protein or marker being at least two times, three times, four times, five times, six times, eight times, ten times, twenty times, fifty times, one hundred times or more higher than the affinity to the non-target cell surface protein or marker. In some embodiments, the expression of the cell surface protein or marker of the present invention in target cells (e.g., cancer cells) is significantly higher than that in normal cells. As used herein, the term "significant" refers to a statistically significant difference, or a significant difference that can be recognized by those skilled in the art.

[0103] As used herein, the term "targeting moiety" refers to a molecule, complex or aggregate that is specifically or selectively bound to a target molecule, cell, particle, tissue or aggregate. Examples of targeting moieties include, but are not limited to, antibodies, antibody binding fragments, bispecific antibodies, immunoglobulins or other molecules or compounds based on antibodies. However, other examples of targeting moieties are known in the art and can be used, such as aptamers, high-affinity polymers, receptor binding ligands, nucleic acids, biotin-avidin binding pairs, peptides, small molecules, nanoparticles or proteins, etc. The terms "targeting moiety" and "binding moiety" are used synonymously herein.

[0104] As used herein, the term "drug" refers to a compound (eg, an active pharmaceutical ingredient) that has biological activity and provides a desired physiological effect to a subject in need thereof after administration.

[0105] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, multispecific antibody or bispecific (bivalent) antibody or its functional part that binds to a specific antigen. A natural complete antibody comprises two heavy (H) chains and two light (L) chains interconnected by a disulfide bond. Each heavy chain consists of a variable region (VH) and a first constant region, a second constant region and a third constant region (CH1, CH2 and CH3, respectively), while each light chain consists of a variable region (VL) and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ and μ, and mammalian light chains are classified as λ or κ. The variable regions of light and heavy chains are responsible for antigen binding. The variable regions in the two chains are usually subdivided into three hypervariable regions, referred to as complementary determining regions (CDRs) (light (L) chain CDRs include LCDR1, LCDR2 and LCDR3, and heavy (H) chain CDRs include HCDR1, HCDR2, HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol, 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol, Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol, 196, 901 (1987); Chothia, C. et al., Nature Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, MD. Health, Bethesda, Md. (1991)). The three CDRs are inserted between flanking extensions called framework regions (FRs), which are more highly conserved than CDRs and form a scaffold to support the hypervariable loops. Each VH and VL includes three CDRs and four FRs (amino acid residues from N-terminus to C-terminus) in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains are not related to antigen binding, but exhibit a variety of effector functions. Antibodies are assigned to five major categories based on the amino acid sequence of the constant region of their heavy chains: IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α heavy chains, δ heavy chains, ε heavy chains, γ heavy chains, and μ heavy chains, respectively. There are several subclasses of the major antibody classes such as IgGl (γl heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgAl (α1 heavy chain) or IgA2 (α2 heavy chain).

[0106] As used herein, "Fab" with respect to an antibody refers to a monovalent antigen-binding fragment of an antibody consisting of a single light chain (both variable and constant regions) bound by disulfide bonds to the variable region and first constant region of a single heavy chain. Fab can be obtained by papain digestion of an antibody at the residues N-terminal to the disulfide bonds between the heavy chains in the hinge region.

[0107] As used herein, "Fab'" refers to a Fab fragment that includes a portion of the hinge region, which can be obtained by pepsin digestion of an antibody at the C-terminal residue close to the disulfide bond between the heavy chains in the hinge region and therefore differs from Fab in a few residues in the hinge region including one or more cysteines.

[0108] As used herein, "F(ab') 2 "" refers to a dimer of Fab' comprising two light chains and part of two heavy chains.

[0109] As used herein, "Fc" for antibodies refers to the part of the antibody consisting of the second constant region and the third constant region of the first heavy chain combined with the second constant region and the third constant region of the second heavy chain via a disulfide bond. IgG and IgM Fc regions include three heavy chain constant regions (the second heavy chain constant region, the third heavy chain constant region and the fourth heavy chain constant region in each chain). It can be obtained by papain digestion of antibodies. The Fc portion of an antibody is responsible for various effector functions, such as ADCC and CDC, but does not work in antigen binding.

[0110] As used herein, "Fv" with respect to an antibody refers to the smallest fragment of an antibody that carries a complete antigen binding site. An Fv fragment consists of the variable region of a single light chain combined with the variable region of a single heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment in which the connection between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.

[0111] As used herein, "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region, which are directly connected to each other or connected to each other through a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)). "scFv dimer" refers to a single chain comprising two heavy chain variable regions and two light chain variable regions with a linker. "scFv dimer" is a bivalent diabody or bivalent ScFv (BsFv), which includes VH-VL (connected by a peptide linker), which dimerizes with another VH-VL part, so that the VH of one part coordinates with the VL of the other part and forms two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). "ScFv dimer" can also be a bispecific diabody, wherein the bispecific diabody comprises a V L1 -V H2 (connected via a peptide linker) associated V H1 -V L2 (also linked via a peptide linker), such that V H1 With V L1 Coordinate and V H2 With V L2 The coordinated pairs have different antigenic specificities.

[0112] As used herein, "single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of a scFv linked to the Fc region of an antibody.

[0113] As used herein, "camelized single domain antibody", "heavy chain antibody" or "HCAb" refers to an antibody containing two VH domains without light chains (Riechmann L. and Muyldermans S., J Immunol Methods Dec 10; 231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Pat. No. 6,005,079). Heavy chain antibodies were originally obtained from Camelidae (camels, dromedaries and llamas). Although devoid of light chains, camelized antibodies have a robust antigen-binding repertoire (Hamers-Casterman C. et al., Nature Jun 3;363(6428):446-8 (1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation", Immunogenetics. Apr;54(1):39-47 (2002); Nguyen VK. et al., Immunology. May;109(1):93-101 (2003)). The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit produced by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)). "Diabodies" include small antibody fragments with two antigen-binding sites, wherein the fragments contain both VHH and VHH in a single polypeptide chain. L Domain-linked V H Domain (V H -V L or V L -V H ) (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. Jul. 15;90(14):6444-8 (1993); EP404097; WO93 / 11161). Because the linker is too short, the two domains on the same chain cannot pair, and thus are forced to pair with the complementary domains of another chain, thereby creating two antigen binding sites. The antigen binding sites can target the same or different antigens (or epitopes).

[0114] As used herein, "domain antibody" refers to an antibody fragment containing only the heavy chain variable region or the light chain variable region. H The domains are covalently linked via peptide linkers to form bivalent or multivalent domain antibodies. H The domains can target the same or different antigens.

[0115] As used herein, "(dsFv) 2 ” refers to an antigen-binding fragment consisting of three peptide chains: two V H The moieties are connected by a peptide linker and by disulfide bridges to the two V L Partial combination.

[0116] As used herein, "bispecific ds diabody" refers to a H1 -V L2 (connected via a peptide linker) via V H1 With V L1 The disulfide bridge between L1 -V H2 The antigen-binding fragments are combined (also connected by a peptide linker).

[0117] As used herein, "bispecific dsFv" or "dsFv-dsFv'" refers to an antigen-binding fragment composed of three peptide chains: V H1 -V H2 The heavy chain is linked to the V and V chains via disulfide bridges. L1 and V L2 Partial pairing. Each disulfide bond-paired heavy and light chain has a different antigenic specificity.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof is chimeric or humanized.

[0119] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of a heavy chain and / or a light chain derived from one species and the remainder of the heavy chain and / or the light chain derived from a different species. In an illustrative example, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human species (e.g., from a mouse).

[0120] As used herein, the term "humanization" for antibodies or antigen-binding fragments refers to antibodies or antigen-binding fragments including CDRs derived from non-human animals (e.g., rodents, rabbits, dogs, goats, horses, or chickens), FR regions derived from humans, and constant regions derived from humans when applicable. In some embodiments, the constant region from a human antibody is fused to a non-human variable region. Humanized antibodies or antigen-binding fragments are suitable for use as human therapeutic agents. In some embodiments, non-human animals are mammals, such as mice, rats, rabbits, goats, sheep, guinea pigs, hamsters, or non-human primates (e.g., monkeys (e.g., macaques or rhesus monkeys) or apes (e.g., chimpanzees, gorillas, apes, or monkeys)). In some embodiments, in addition to non-human CDR sequences, humanized antibodies or antigen-binding fragments are composed of substantially all human sequences. In some embodiments, humanized antibodies or antigen-binding fragments are modified to improve antibody performance, such as binding or binding affinity. For example, one or more amino acid residues in one or more non-human CDRs are changed to reduce the potential immunogenicity of the human, wherein the changed amino acid residues are not important for immunospecific binding or the changes are conservative changes, so that the binding of the humanized antibody to the antigen is not significantly affected. In some embodiments, the FR region derived from humans may include the same amino acid sequence as the human antibody from which it is derived, or it may include some amino acid changes, such as no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid changes. In some embodiments, such amino acid changes may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains.

[0121] As used herein, the term "natural amino acid" refers to any of the common naturally occurring L-amino acids found in naturally occurring proteins: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), lysine (Lys), arginine (Arg), histidine (His), proline (Pro), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine (Gln), cysteine ​​(Cys), and methionine (Met).

[0122] As used herein, the term "non-natural amino acid" as used herein refers to any amino acid that is not a natural amino acid. This includes, for example, amino acids containing α-aminoacyl residues, β-aminoacyl residues, ω-aminoacyl residues, D-aminoacyl residues, L-aminoacyl residues. More generally, non-natural amino acids contain the general formula The residues shown in the figure, wherein the side chain R is not an amino acid side chain that occurs in nature. Exemplary non-natural amino acids include, but are not limited to, sarcosine (N-methylglycine), citrulline (cit), homocitrulline, β-ureidoalanine, thiocitrulline, hydroxyproline, allothreonine, pipecolic acid (homoproline), α-aminoisobutyric acid, tert-butylglycine, tert-butylalanine, alloisoleucine, norleucine, α-methylleucine, cyclohexylglycine, β-cyclohexylalanine, β-cyclopentylalanine, α-methylproline, phenylglycine, α-methylphenylalanine, and homophenylalanine.

[0123] As used herein, the terms "polypeptide", "protein" or "peptide" may be a single amino acid or a polymer of amino acids. Polypeptides, proteins or peptides as described in the present disclosure may contain naturally occurring amino acids and non-naturally occurring amino acids, or analogs and mimetics thereof. Polypeptides, proteins or peptides can be obtained by any method well known in the art, such as, but not limited to, by separation and purification from natural materials, recombinant expression, chemical synthesis, etc.

[0124] As used herein, the term "biocompatible" as used herein is intended to describe compounds that exert minimal destructive or host-reactive effects when in contact with body fluids or living cells or tissues. Thus, as used herein, a biocompatible group refers to an aliphatic, cycloalkyl, heteroaliphatic, heterocycloalkyl, aryl or heteroaryl moiety that falls within the definition of the term biocompatible as defined above and herein. As used herein, the term "biocompatibility" is also used to refer to compounds exhibiting minimal interactions with recognition proteins (e.g., naturally occurring antibodies, cellular proteins, cells, and other components of biological systems), unless such interactions are particularly desirable. Thus, substances and functional groups (e.g., drugs and prodrugs) that are specifically intended to cause the above-mentioned minimal interactions are considered biocompatible. In some embodiments, a compound is "biocompatible" if its addition to normal cells in vitro at a concentration similar to the expected systemic in vivo concentration results in less than or equal to 1% cell death over a period of time equal to the in vivo half-life of the compound (e.g., the period of time required to eliminate / clear 50% of the compound administered in vivo), and its in vivo administration induces minimal and medically acceptable inflammation, foreign body response, immunotoxicity, chemotoxicity, and / or other such side effects. As used herein, the term "normal cells" refers to cells that are not intended to be destroyed or otherwise significantly affected by the compound to be tested.

[0125] As used herein, "biodegradable" polymers are polymers that are easy to be bioprocessed in vivo. As used herein, "biodegradable" compounds or parts are, when absorbed by cells, decomposed by lysosomes or other chemical mechanisms or hydrolyzed into components that can be reused or processed by cells, without causing significant toxic effects on cells. As used herein, the term "biodegradable" has the same meaning as "biodegradable". Degraded fragments preferably induce minimal or no organ or cell overload, or pathological processes caused by such overload or other side effects in vivo. Examples of biodegradable processes include enzymatic and non-enzymatic hydrolysis, oxidation and reduction. Suitable conditions for non-enzymatic hydrolysis of biodegradable protein-polymer-drug conjugates described herein (or their components, for example, biodegradable polymer carriers and the joints between the carriers and antibodies or drug molecules), for example, include exposing the biodegradable conjugate to water at the temperature and pH of the lysosomal intracellular compartment. The biodegradation of some protein-polymer-drug conjugates (or their components, such as biodegradable polymer carriers and the linkers between the carriers and antibodies or drug molecules) can also be enhanced outside the cell, such as in low pH areas (such as inflamed areas) of the animal body, near activated macrophages or other cells that release degradation-promoting factors. In some embodiments, at pH about 7.5, the effective size of the polymer carrier will not undergo detectable changes within 1 to 7 days, and will remain within 50% of the original polymer size for at least a few weeks. On the other hand, when pH is about 5, the polymer carrier is preferably detectably degraded within 1 to 5 days, and is completely converted into low molecular weight fragments within a period of two weeks to several months. The polymer integrity in this type of test can be measured by, for example, size exclusion HPLC. Although faster degradation may be preferred in some cases, in general, it may be more desirable that the rate of polymer degradation in the cell does not exceed the rate of cell metabolism or secretion of polymer fragments. In some embodiments, polymers and polymer biodegradation byproducts are biocompatible.

[0126] As used herein, the term "bioavailability" refers to the systemic availability (i.e., blood / plasma levels) of a given amount of a drug or compound administered to a subject. Bioavailability is an absolute term that indicates a measure of the time (rate) and total amount (extent) of a drug or compound that reaches systemic circulation from an administered dosage form.

[0127] As used herein, the term "drug release mechanism" refers to a linking portion that is biodegradable / biodegradable under intracellular conditions, such that the cleavage of the linking portion releases the drug in the intracellular environment. In some embodiments, the linking portion is hydrolytically unstable in water or in an aqueous solution (including, for example, body fluids, such as blood), i.e., sensitive to hydrolysis at certain pH. In some embodiments, the linking portion is enzyme-unstable, i.e., degradable by one or more enzymes. In some embodiments, the linking portion is photolabile and is used on the surface of the body and in many body cavities that can be exposed to light. In some embodiments, the linking portion is biodegradable under reducing conditions where the activity of the drug is not affected.

[0128] As used herein, the term "therapeutic agent" or "drug" refers to a compound (eg, an active pharmaceutical ingredient) that is biologically active and provides a desired physiological effect after administration to a subject in need thereof. In some embodiments, the therapeutic agent is a small molecule drug.

[0129] As used herein, the term "small molecule" refers to a naturally occurring or artificially produced (e.g., via chemical synthesis) molecule with a relatively low molecular weight. Preferred small molecules are biologically active because they produce local or systemic effects in animals, such as mammals, such as humans. In certain embodiments, small molecules are drugs and small molecules are referred to as "drug molecules" or "drugs" or "therapeutic agents." In certain embodiments, the MW of a drug molecule is less than or equal to about 5 kDa. In other embodiments, the MW of a drug molecule is less than or equal to about 1.5 kDa.

[0130] Classes of drug molecules useful in the present disclosure include, but are not limited to, anticancer substances, radionuclides, vitamins, anti-AIDS substances, antibiotics, immunosuppressants, antiviral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, lubricants, sedatives, anticonvulsants, muscle relaxants and anti-Parkinson substances, antispasmodics and muscle contractants (including channel blockers, miotics and anticholinergics), antiglaucoma compounds, antiparasitic compounds and / or antiprotozoal compounds, modulators of cell-extracellular matrix interactions (including cell growth inhibitors and anti-adhesion molecules), vasodilators, inhibitors of DNA, RNA or protein synthesis, antihypertensives, analgesics, antipyretics, steroidal anti-inflammatory agents and nonsteroidal anti-inflammatory agents, anti-angiogenic factors, antisecretory factors, anticoagulants and / or antithrombotics, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotic substances, antiemetics, imaging agents.

[0131] In certain embodiments, macromolecules can be used as therapeutic agents in the present disclosure. Examples of suitable macromolecules include, but are not limited to, amino acid-based molecules such as peptides, polypeptides, enzymes, antibodies, immunoglobulins, or functional fragments thereof, and the like.

[0132] In some embodiments, the therapeutic agents used in the present disclosure are therapeutic agents that have antiproliferative (cytostatic and / or cytotoxic) activity against target cells or target pathways. The drug may have a chemically reactive group such as -COOH, primary amine, secondary amine, -NHR, -OH, -SH, -C(O)H, -C(O)R, -C(O)NHR', -C(S)OH, -S(O) 2 OR', -P(O) 2 OR', -CN, -NC or -ONO, wherein R is an aliphatic, heteroaliphatic, carbocyclic or heterocycloalkyl moiety and R' is hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl moiety.

[0133] As used herein, the term "cytotoxic" means toxic to a cell or a selected cell population (e.g., cancer cells). The toxic effect may result in cell death and / or lysis. In some cases, the toxic effect may be a sublethal destructive effect on the cell, e.g., slowing or curbing cell growth. To achieve cytotoxic effects, the drug or prodrug may be selected from the group consisting of a DNA disruptor, a microtubule disruptor, or a cytotoxic protein or polypeptide.

[0134] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as an antibody and an antigen. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to the antigen at pH 7.4 with a binding affinity (K) of about 0.01 nM to about 100 nM, about 0.1 nM to about 100 nM, 0.01 nM to about 10 nM, about 0.1 nM to about 10 nM, 0.01 nM to about 1 nM, about 0.1 nM to about 1 nM, or about 0.01 nM to about 0.1 nM. D ) specifically binds to the target antigen. As used herein, K D The ratio of the dissociation rate to the association rate (k off / k on ), can be determined using surface plasmon resonance methods, for example using an instrument such as Biacore.

[0135] As used herein, the term "tumor antigen" refers to an antigenic substance produced in a tumor cell, i.e., it triggers an immune response in the host. Normal proteins in the body are not antigenic because of self-tolerance, a process in which autoreactive cytotoxic T lymphocytes (CTLs) and autoantibody-producing B lymphocytes are "centrally" eliminated in primary lymphoid tissues (BM) and "peripherally" eliminated in secondary lymphoid tissues (for T cells, mainly in the thymus, and for B cells, mainly in the spleen / lymph nodes). Therefore, any protein that is not exposed to the immune system will trigger an immune response. This may include normal proteins that are fully isolated from the immune system, proteins that are usually produced in very small amounts, proteins that are usually only produced at certain developmental stages, or proteins whose structures are modified due to mutations.

[0136] As used herein, the term "effective amount" refers to the amount necessary to elicit a desired biological response. As will be appreciated by one of ordinary skill in the art, the effective amount of an agent or device may vary depending on factors such as the desired biological endpoint, the agent to be delivered, the composition of the encapsulation matrix, the target tissue, etc. For example, an effective amount of microparticles containing an antigen to be delivered to immunize an individual is an amount that produces an immune response sufficient to prevent infection of an organism with the administered antigen.

[0137] As used herein, unless otherwise specified, "molecular weight" or "MW" of a polymer or polymer carrier / scaffold or polymer conjugate refers to the weight average molecular weight.

[0138] The present disclosure is intended to include all isotopes of atoms present in the compounds of the present invention. Isotopes include those atoms having the same atomic number, but different mass numbers. As a general example and not by way of limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C 13 and C 14 .

[0139] The present invention is intended to include all isomers of the compounds, which refers to and includes optical isomers and tautomers, wherein optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include isolated optical isomers and mixtures of optical isomers, including racemic mixtures and non-racemic mixtures; wherein the isomers may be in isolated form or in a mixture with one or more other isomers.

[0140] Polymer carrier

[0141] The conjugates of the present disclosure are suitable for biomedical applications, such as drug delivery and tissue engineering, and the polymer carriers used in the conjugates of the present disclosure are biocompatible and biodegradable. In some embodiments, the polymer carrier is a soluble polymer, nanoparticles, gel, liposome, micelle, suture, implant, etc.

[0142] In some embodiments, the weight average molecular weight Mw of the polymer carrier can be from about 400 to about 3,000,000 Da, for example, from about 1,000 to about 2,000,000 Da, from about 1,000 to about 1,000,000 Da, from about 1,000 to about 900,000 Da, from about 1,000 to about 800,000 Da, from about 1,000 to about 700,000 Da, from about 1,000 to about 600,000 Da, from about 1,000 to about 500,000 Da, from about 1,000 to about 400,000 Da, from about 1,000 to about 300,000 Da, from about 1,000 to about 200,000 Da, from about 1,000 to about From about 1,000 to about 100,000 Da, from about 1,000 to about 90,000 Da, from about 1,000 to about 80,000 Da, from about 1,000 to about 70,000 Da, from about 1,000 to about 60,000 Da, from about 1,000 to about 50,000 Da, from about 1,000 to about 40,000 Da, from about 1,000 to about 30,000 Da, from about 1,000 to about 20,000 Da, from about 1,000 to about 10,000 Da, from about 2,000 to about 10,000 Da, from about 3,000 to about 10,000 Da, from about 4,000 to about 10,000 Da, from about 5,000 to about 10,000 Da.

[0143] In some embodiments, the polymer carrier used in the present disclosure is polyglycerol. In certain embodiments, the polymer carrier used in the present disclosure is linear polyglycerol. In certain embodiments, the weight average molecular weight Mw of the linear polyglycerol may be from about 400 to about 3,000,000 Da, for example, from about 1,000 to about 2,000,000 Da, from about 1,000 to about 1,000,000 Da, from about 1,000 to about 900,000 Da, from about 1,000 to about 800,000 Da, from about 1,000 to about 700,000 Da, from about 1,000 to about 600,000 Da, from about 1,000 to about 500,000 Da, from about 1,000 to about 400,000 Da, from about 1,000 to about 300,000 Da, from about 1,000 to about 200,000 Da, from about 1,000 to about From about 1,000 to about 100,000 Da, from about 1,000 to about 90,000 Da, from about 1,000 to about 80,000 Da, from about 1,000 to about 70,000 Da, from about 1,000 to about 60,000 Da, from about 1,000 to about 50,000 Da, from about 1,000 to about 40,000 Da, from about 1,000 to about 30,000 Da, from about 1,000 to about 20,000 Da, from about 1,000 to about 10,000 Da, from about 2,000 to about 10,000 Da, from about 3,000 to about 10,000 Da, from about 4,000 to about 10,000 Da, from about 5,000 to about 10,000 Da.

[0144] Therapeutic agents

[0145] In some embodiments, the therapeutic agent used in the conjugates of the present disclosure is a small molecule with a molecular weight of no more than about 5 kDa, no more than about 4 kDa, no more than about 3 kDa, no more than about 1.5 kDa, or no more than about 1 kDa.

[0146] In some embodiments, the IC of the therapeutic agent is 50 Less than about 1 nM.

[0147] IC 50 Some therapeutic agents greater than about 1 nM are not suitable for binding to a targeting moiety using art-recognized binding techniques. Without wishing to be bound by theory, such therapeutic agents have insufficient potency for use in targeting moiety-drug conjugates using conventional techniques because sufficient copies of the drug (i.e., greater than 8) cannot be bound using art-recognized techniques without causing a reduction in the pharmacokinetic and physiochemical properties of the conjugate. However, using the binding strategies described herein, sufficiently high loadings of these less potent drugs can be achieved, thereby producing high loadings of the therapeutic agent while maintaining the desired pharmacokinetic and physiochemical properties. Thus, in some embodiments, IC 50Greater than about 1 nM of the therapeutic agent is suitable for use with the targeting moiety-polymer-drug conjugates provided herein.

[0148] Small molecule therapeutic agents used in the present disclosure (e.g., anti-proliferative (cytotoxic and cytostatic) agents that can be attached to a polymer carrier) include cytotoxic compounds (e.g., a wide range), angiogenesis inhibitors, cell cycle progression inhibitors, PI3K / m-TOR / AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, molecular chaperone protein inhibitors, HDAC inhibitors, PARP inhibitors, Wnt / hedgehog signaling pathway inhibitors and RNA polymerase inhibitors.

[0149] A wide range of cytotoxins include, but are not limited to, DNA-binding, intercalating or alkylating drugs, microtubule stabilizing and destabilizing agents, platinum compounds, topoisomerase I inhibitors, and protein synthesis inhibitors.

[0150] Exemplary DNA-binding, intercalating or alkylating drugs include CC-1065 and its analogs, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin, nemorubicin and its derivatives, PNU-159682), bis-naphthoic acid imide compounds (such as elinafide (LU79553) and its analogs), alkylating agents (such as calicheamicin, dactinomycine, mitomycin, pyrrolobenzodiazepines), and the like. Exemplary CC-1065 analogs include duocarmycin SA, duocarmycin A, duocarmycin C1, duocarmycin C2, duocarmycin B1, duocarmycin B2, duocarmycin D, DU-86, KW-2189, adozelesin, bizelesin, carzelesin, scis-adozelesin and related analogs and prodrug forms, examples of which are described in U.S. Pat. Nos. 5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,586,618; 6,756,397 and 7,049,316. ​​Doxorubicin and its analogs include those described in U.S. Pat. No. 6,630,579. Calicinomycins include, for example, enediynes, such as esperamicin and those described in U.S. Pat. Nos. 5,714,586 and 5,739,116. Duocarmycins include those described in U.S. Pat. Nos. 5,070,092; 5,101,038; 5,187,186; 6,548,530; 6,660,742 and 7,553,816B2 and Li et al., Tet Letts., 50:2932-2935 (2009).

[0151] Pyrrolobenzodiazepines (PBDs) and their analogs include those described in Denny, Exp. Opin. Ther. Patents., 10(4):459-474 (2000) and Antonow and Thurston, Chem Rev., 2815-2864 (2010).

[0152] Exemplary microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel; tesetaxel and carbazitaxel, maytansinoids, auristatins and analogs thereof, vinca alkaloid derivatives, epothilones, and cryptophycins.

[0153] Exemplary maytansine or maytansine analogs include maytansinol and maytansinol analogs, and maytansine or DM-1 and DM-4 are U.S. Patent No. 5,208,020; No. 5,416,064; No. 6,333,410; No. 6,441,163; No. 6,716,821; Those described in No. RE39,151 and No. 7,276,497. In certain embodiments, cytotoxic agent is maytansine, another group of anti-tubulin agents (ImmunoGen company; See also Chari et al., 1992, " Cancer Research (Cancer Res.) " 52:127-131), maytansine or maytansine analogs. Suitable examples of maytansine include maytansinol and maytansinol analogs. Suitable maytansinoids are disclosed in U.S. Pat. Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

[0154] Exemplary auristatins include auristatin E (also known as a derivative of dolastatin-10), auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F, auristatin F phenylenediamine (AFP), auristatin F HPA, and dolastatin. Suitable auristatins are also described in U.S. Publication Nos. 2003 / 0083263, 2011 / 0020343, and 2011 / 0070248, PCT Application Publication Nos. WO 09 / 117531, WO 2005 / 081711, WO 04 / 010957, WO Nos. 02 / 088172 and WO01 / 24763 and U.S. Patents Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,124,431; 6,034,065; 5,780,588; 5,767,237; 5,665,860; 5,663,149; 5,635,483; 5,599,9 02; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444 and 4,486,414, the disclosures of which are incorporated herein by reference in their entirety.

[0155] Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine (vinorelbine). Suitable vinca alkaloids useful in the present disclosure are also disclosed in U.S. Publication Nos. 2002 / 0103136 and 2010 / 0305149 and U.S. Pat. No. 7,303,749B1, the disclosures of which are incorporated herein by reference in their entirety.

[0156] Exemplary epothilone compounds include epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, and epothilone F, and derivatives thereof. Suitable epothilone compounds and derivatives thereof are described, for example, in U.S. Pat. Nos. 6,956,036; 6,989,450; 6,121,029; 6,117,659; 6,096,757; 6,043,372; 5,969,145 and 5,886,026; and WO 97 / 19086; WO 98 / 08849; WO 98 / 22461; WO 98 / 25929; WO 98 / 38192; WO 99 / 01124; WO 99 / 02514; WO 99 / 03848; WO 99 / 07692; WO 99 / 27890 and WO No. 99 / 28324, the disclosure of which is incorporated herein by reference in its entirety.

[0157] Exemplary cryptophycin compounds are described in U.S. Pat. Nos. 6,680,311 and 6,747,021.

[0158] Exemplary platinum compounds include cisplatin Carboplatin Oxaliplatin Isopropylplatin, ormaplatin, and tetraplatin.

[0159] Other classes of compounds or compounds having these or other cytotoxic modes of action may be selected, including, for example, mitomycin C, mitomycin A, daunomycin, doxorubicin, azolidine-doxorubicin, cyanomorpholino-doxorubicin, aminopterin, bleomycin, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol, pyrrolobenzodiazepine (PBD) polyamides and dimers thereof. Other suitable cytotoxic agents include, for example, puromycin, topotecan, rhizoxin, echinomycin, combretastatin, netropsin, estramustine, cryptophysins, cemadotin, discodermolide, eleutherobin and mitoxantrone.

[0160] Exemplary topoisomerase I inhibitors include camptothecin, camptothecin derivatives, camptothecin analogs, and unnatural camptothecins, such as CPT-11 (irinotecan), SN-38, GI-147211C, topotecan, 9-aminocamptothecin, 7-hydroxymethylcamptothecin, 7-aminomethylcamptothecin, 10-hydroxycamptothecin, (20S)-camptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, diflomotecan, belotecan, lurtotecan, and 539625. Other camptothecin compounds useful in the present invention include those described in, for example, J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554 (1980); J. Med. Chem., 30:1774 (1987).

[0161] Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, MetAP2 inhibitors. Exemplary VGFR and PDGFR inhibitors include sorafenib (Nexavar), sunitinib (Sutent), and vatalanib. Exemplary MetAP2 inhibitors include fumagillol analogs, meaning any compound that includes the fumagillin core structure, including NH4+ that inhibits MetAP-2 from removing the protein. 2-Methionine-terminal fumagillamine as described in Rodeschini et al., J. Org. Chem., 69, 357-373, 2004 and Liu et al., Science, 282, 1324-1327, 1998. Non-limiting examples of "fumagillin analogs" are disclosed in Journal of Organic Chemistry, 69, 357, 2004; Journal of Organic Chemistry, 70, 6870, 2005; European Patent Application 0 354 787; Journal of Medicinal Chemistry, 49, 5645, 2006; Bioorg. Med. Chem., 11, 5051, 2003; Bioorg. Med. Chem., 14, 91, 2004; Tetrahedron Letters, 40, 4797, 1999; WO 99 / 61432; U.S. Patents Nos. 6,603,812; 5,789,405; 5,767,293; 6,566,541 and 6,207,704.

[0162] Exemplary cell cycle progression inhibitors include CDK inhibitors, such as BMS-387032 and PD0332991; Rho kinase inhibitors, such as GSK429286; checkpoint kinase inhibitors, such as AZD7762; Aurora kinase inhibitors, such as AZD1152, MLN8054, and MLN8237; PLK inhibitors, such as BI 2536, BI6727 (Volasertib), GSK461364, ON-01910 (Estybon); and KSP inhibitors, such as SB 743921, SB 715992 (ispinesib), MK-0731, AZD8477, AZ3146, and ARRY-520.

[0163] Exemplary PI3K / m-TOR / AKT signaling pathway inhibitors include phosphoinositide 3-kinase (PI3K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors, and PDK-1 inhibitors.

[0164] Exemplary PI3 kinase inhibitors are disclosed in U.S. Pat. No. 6,608,053, and include BEZ235, BGT226, BKM120, CAL101, CAL263, demethoxyviridin, GDC-0941, GSK615, IC87114, LY294002, Palomid 529, perifosine, PI-103, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, Wortmannin, XL147, and XL765.

[0165] Exemplary AKT inhibitors include, but are not limited to, AT7867.

[0166] Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf, and p38 MAPK inhibitors.

[0167] Exemplary MEK inhibitors are disclosed in U.S. Pat. No. 7,517,994 and include GDC-0973, GSK1120212, MSC1936369B, AS703026, RO5126766 and RO4987655, PD0325901, AZD6244, AZD8330, and GDC-0973.

[0168] Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.

[0169] Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820, and SB 202190.

[0170] Receptor tyrosine kinases (RTKs) are cell surface receptors that are often associated with signaling pathways that stimulate uncontrolled cancer cell proliferation and angiogenesis. Many RTKs that are overexpressed or have mutations that cause constitutive activation of receptors have been identified, including but not limited to VEGFR, EGFR, FGFR, PDGFR, EphR, and RET receptor family receptors. Exemplary specific RTK targets include ErbB2, FLT-3, c-Kit, and c-Met.

[0171] Exemplary inhibitors of the ErbB2 receptor (EGFR family) include, but are not limited to, AEE788 (NVP-AEE 788), BIBW2992 (Afatinib), Lapatinib, Erlotinib (Tarceva), and Gefitinib (Iressa).

[0172] Exemplary RTKRTK inhibitors that target more than one signaling pathway (multi-targeted kinase inhibitors) include AP24534 (Ponatinib), which targets FGFR, FLT-3, VEGFR-PDGFR, and Bcr-Abl receptors; ABT-869 (Linifanib), which targets FLT-3 and VEGFR-PDGFR receptors; AZD2171, which targets VEGFR-PDGFR, Flt-1, and VEGF receptors; CHR-258 (Dovitinib), which targets VEGFR-PDGFR, FGFR, Flt-3, and c-Kit receptors; Sunitinib (Sutent), which targets VEGFR, PDGFR, KIT, FLT-3, and CSF-IR; Sorafenib (Nexavar) and vatalanib, which target VEGFR, PDGFR, and intracellular serine / threonine kinases in the Raf / Mek / Erk pathway.

[0173] Exemplary chaperone protein inhibitors include HSP90 inhibitors. Exemplary HSP90 inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922 and KW-2478.

[0174] Exemplary HDAC inhibitors include Belinostat (PXD101), CUDC-101, Droxinostat, ITF2357 (Givinostat, Gavinostat), JNJ-26481585, LAQ824 (NVP-LAQ824, Dacinostat), LBH-589 (Panobinostat), MC1568, MGCD0103 (Mocetinostat), MS-275 (Entinostat), PCI-24781, Pyroxamide (NSC 696085), SB939, Trichostatin A, and Vorinostat (SAHA).

[0175] Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD-2281), ABT-888 (Veliparib), AG014699, CEP 9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3-aminobenzamide, A-966492, and AZD2461.

[0176] Exemplary Wnt / Hedgehog signaling pathway inhibitors include vismodegib (RG3616 / GDC-0449), cyclopamine (11-deoxykinamine) (hedgehog pathway inhibitor), and XAV-939 (Wnt pathway inhibitor).

[0177] Exemplary RNA polymerase inhibitors include amatoxins. Exemplary amatoxins include α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanullin, amanullic acid, amaninamide, amanin, and proamanullin.

[0178] Exemplary protein synthesis inhibitors include trichothecenes.

[0179] In some embodiments, the therapeutic agent of the present disclosure is a topoisomerase inhibitor (e.g., a non-natural camptothecin compound), a vinca alkaloid, a kinase inhibitor (e.g., a PI3 kinase inhibitor (GDC-0941 and PI-103)), a MEK inhibitor, a KSP inhibitor, an RNA polymerase inhibitor, a protein synthesis inhibitor, a PARP inhibitor, docetaxel, paclitaxel, doxorubicin, duocarmycin, auristatin, dolastatin, calicheamicin, topotecan, SN38, camptothecin, exatecan, nemorubicin and its derivatives, PNU-159682, CC1065, elinefand, a trichothecene, a pyrrolobenzodiazepine, a maytansine, a DNA-binding drug, or a platinum compound and its analogs. In some embodiments, the therapeutic agent is a derivative of SN-38, camptothecin, topotecan, exatecan, calicheamicin, exatecan, nemorubicin, PNU-159682, anthracycline, maytansine, taxane, trichothecene, CC1065, elinefand, vindesine, vinblastine, PI-103, AZD 8330, dolastatin, auristatin E, auristatin F, duocarmycin compounds, ispins, pyrrolobenzodiazepines, ARRY-520, and stereoisomers, isosteres, and analogs thereof.

[0180] In some embodiments, the therapeutic agent used in the present disclosure is a combination of two or more drugs, such as a combination of a PI3 kinase inhibitor and a MEK inhibitor; a combination of a broad-range cytotoxic compound and a platinum compound; a combination of a PARP inhibitor and a platinum compound; a combination of a broad-range cytotoxic compound and a PARP inhibitor.

[0181] In yet another embodiment, the therapeutic agent used in the present disclosure is auristatin F-hydroxypropylamide-L-alanine.

[0182] Those skilled in the art will readily appreciate that each of the therapeutic agents described herein can be modified in such a way that the resulting compound still retains the specificity and / or activity of the original compound. Those skilled in the art will also appreciate that many of these compounds can be used in place of the therapeutic agents described herein. Thus, the therapeutic agents of the present disclosure include analogs and derivatives of the compounds described herein.

[0183] In some embodiments, the therapeutic agent has anti-proliferative activity against a target cell or target pathway.

[0184] In certain embodiments, the anti-proliferative activity is selected from cytostatic activity and / or cytotoxic activity.

[0185] In certain embodiments, the therapeutic agent is selected from anticancer substances, cytotoxic drugs, radionuclides, vitamins, anti-AIDS substances, antibiotics, immunosuppressants, immunomodulatory compounds, therapeutic RNA, antiviral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, sedatives, anticonvulsants, muscle relaxants and anti-Parkinson substances, antispasmodics and muscle contractants (including channel blockers, miotics and anticholinergics), anti-glaucoma compounds, antiparasitic compounds and / or antiprotozoal compounds, regulators of cell-extracellular matrix interactions (including cell growth inhibitors and anti-adhesion molecules), vasodilators, inhibitors of DNA, RNA or protein synthesis, antihypertensives, analgesics, antipyretics, steroidal anti-inflammatory agents and non-steroidal anti-inflammatory agents, anti-angiogenic factors, antisecretory factors, anticoagulants and / or antithrombotics, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotic substances, antiemetics, imaging agents.

[0186] In certain embodiments, the therapeutic agent comprises an amino acid based molecule.

[0187] In certain embodiments, the amino acid-based molecule comprises a peptide, a polypeptide, an enzyme, an antibody, an immunoglobulin, or a functional fragment thereof.

[0188] In certain embodiments, the therapeutic agent has a chemically reactive group.

[0189] In certain embodiments, the chemically reactive group comprises -COOH, primary amine, secondary amine, -NHR, -OH, -SH, -C(O)H, C(O)R 14 、-C(O)NHR 15 、C(S)OH、-S(O) 2 OR 15 、-P(O) 2 OR 15 , -CN, -NC or -ONO, where R 14 is selected from aliphatic, heteroaliphatic, carbocyclic or heterocycloalkyl moieties, and R 15 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0190] Linker-polymer-drug compound

[0191] In one aspect, a polymer scaffold of formula (I) is provided for conjugation with a targeting moiety:

[0192]

[0193] in:

[0194] The polymer scaffold comprises linear polyglycerol;

[0195] L is a functional group W that can form a covalent bond with the targeting moiety. p The connecting part;

[0196] M a is an extending group connecting L to the -NH- portion;

[0197] Each G 1 Independently p a functional group attached to the linear polyglycerol;

[0198] Each L P Independently, the therapeutic agent moieties D and G 1 Drug release mechanism between;

[0199] Each D is independently a therapeutic agent moiety;

[0200] Each G 2 are independently functional groups capable of being transformed into a charged state;

[0201] n is an integer from 0 to 1000;

[0202] m is an integer from 0 to 1000;

[0203] p is an integer from 0 to 1000; and

[0204] q is an integer from 0 to 1000.

[0205] In some embodiments, W p Capable of reacting with functional groups on the targeting moiety via a click reaction.

[0206] In certain embodiments, W p Selected from the group consisting of:

[0207]

[0208] In some embodiments, W p Capable of reacting with amino acids on the targeting moiety.

[0209] In certain embodiments, W p Capable of reacting with an amino acid on a targeting moiety, and the amino acid is a natural amino acid, an unnatural amino acid, or a combination thereof. In certain embodiments, the natural amino acid may include cysteine, lysine, tyrosine, aspartic acid, and glutamic acid.

[0210] In some embodiments, W p Capable of reacting with one or more cysteines on the targeting moiety.

[0211] In certain embodiments, Wp Capable of reacting with one or more cysteines on the targeting moiety, and each W p Selected from the group consisting of:

[0212]

[0213]

[0214] Where R 1 is a sulfur protecting group, and each R 2 is independently a leaving group.

[0215] In certain embodiments, each R 2 are independently selected from halo or R 2a C(O)O-, where R 2a is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0216] In some embodiments, W p Capable of reacting with one or more lysines on the targeting moiety.

[0217] In certain embodiments, W p Capable of reacting with one or more lysines on the targeting moiety, and wherein each W p Independently selected from the group consisting of:

[0218]

[0219] In some embodiments, W p Capable of reacting with one or more unnatural amino acids on the targeting moiety.

[0220] In certain embodiments, W p capable of reacting with one or more unnatural amino acids on the targeting moiety, and each W p Independently selected from:

[0221]

[0222] In some embodiments, M a Selected from the group consisting of:

[0223] R 3 ,

[0224] in

[0225] * is the site of covalent attachment to L, ** is the site of covalent attachment to -NH- moiety;

[0226] R 3 C 1-10 Alkyl, C 1-10 Heteroalkyl, C 3-8 Cycloalkyl, -O-(C 1-8 alkyl)-, aryl, -C 1-10 Alkyl-aryl-, -aryl-C 1-10 Alkyl-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-, -(C 3-8 Cycloalkyl-C 1-10 alkyl)-, 4- to 14-membered heterocycloalkyl, -C 1-10 Alkyl-(4- to 14-membered heterocycloalkyl)-, -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-, -C 1-10 Alkyl-C(=O)-, -C 1-10 Heteroalkyl-C(=O)-, -C 3-8 Cycloalkyl-C(=O)-, -O-(C 1-8 alkyl)-C(=O)-, -aryl-C(=O)-, -C 1-10 Alkyl-aryl-C(=O)-, -aryl-C 1-10 Alkyl-C(=O)-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-C(=O)-, -(C 3-8 Cycloalkyl)-C 1-10 alkyl-C(═O)-, -4- to 14-membered heterocycloalkyl-C═(O)-, -C 1-10 alkyl-(4- to 14-membered heterocycloalkyl)-C(═O)-, -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-C(=O)-, -C 1-10 Alkyl-NH-, -C 1-10 Heteroalkyl-NH-, -C 3-8 Cycloalkyl-NH-, -O-(C 1-8 Alkyl)-NH-, -aryl-NH-, -C 1-10 Alkyl-aryl-NH-, -aryl-C 1-10 Alkyl-NH-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-NH-, -(C 3-8 Cycloalkyl)-C 1-10 Alkyl-NH-, -4- to 14-membered heterocycloalkyl-NH-, -C 1-10 Alkyl-(4- to 14-membered heterocycloalkyl)-NH-, -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-NH-, -C 1-10Alkyl-S-, -C 1-10 Heteroalkyl-S-, -C 3-8 Cycloalkyl-S-, -OC 1-8 Alkyl-S-, -aryl-S-, -C 1-10 Alkyl-aryl-S-, -aryl-C 1-10 Alkyl-S-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-S-, -(C 3-8 Cycloalkyl)-C 1-10 Alkyl-S-, -4- to 14-membered heterocycloalkyl-S-, -C 1-10 -(4- to 14-membered heterocycloalkyl)-S- or -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-S-;

[0227] Each R 4 are independently hydrogen, C 1-6 Alkyl, C 6-10 Aryl, C 3-8 Cycloalkyl, -COOH or -COO-C 1-6 alkyl;

[0228] R 5 -C(O)-NR 5a or -NR 5a -C(O)-;

[0229] R 5a For hydrogen, C 1-6 Alkyl, C 6-10 Aryl, C 3-8 Cycloalkyl, -COOH or -COO-C 1-6 alkyl;

[0230] R 6 is a bond or -NR 6a -(CR 6b R 6c )-C(O)-;

[0231] R 6a For hydrogen, C 1-6 Alkyl, C 6-10 Aryl, C 3-8 Cycloalkyl, -COOH or -COO-C 1-6 alkyl;

[0232] Each R 6b and R 6c are independently hydrogen, C 1-6 Alkyl, C 6-10 Aryl, hydroxylated C 6-10 Aryl, polyhydroxylated C 6-10 Aryl, 5- to 12-membered heterocycloalkyl, C3-8 Cycloalkyl, hydroxylated C 3-8 Cycloalkyl, polyhydroxylated C 3-8 Cycloalkyl, or the side chain of a natural or unnatural amino acid;

[0233] Each n 1 are independently an integer from 0 to 6;

[0234] n 2 is an integer from 0 to 8;

[0235] Each n 3 are independently an integer from 1 to 6; and

[0236] n 4 is an integer from 1 to 4.

[0237] In certain embodiments, M a Selected from the group consisting of:

[0238]

[0239] In some embodiments, G 1 Selected from the group consisting of:

[0240]

[0241] Where * is covalently linked to L P The site of each R 7 are independently selected from a direct bond, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, and R 7a is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl moieties.

[0242] In some embodiments, G 1 Selected from the group consisting of:

[0243]

[0244]

[0245] Where R 7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. p Independently contains unstable structures.

[0246] In certain embodiments, the labile structure is selected from a hydrolytically labile structure or an enzymatically labile structure.

[0247] In certain embodiments, the hydrolytically unstable structure is selected from the group consisting of:

[0248]

[0249] Where * is covalently linked to G 1 The site of covalent connection to D, R 8 is selected from hydrogen, alkyl or aryl, R 9 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0250] In certain embodiments, the hydrolytically unstable structure is selected from the group consisting of:

[0251]

[0252]

[0253] Where * is covalently linked to G 1 The site of covalent connection to D, R 8 is selected from hydrogen, alkyl or aryl, R 9 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0254] In some embodiments, G 1 for Where * is covalently linked to L P The site, R 7 It is an alkyl group.

[0255] In certain embodiments, -G 1 -L P -D is

[0256]

[0257] In some embodiments, the enzyme labile structure is labile to an enzyme selected from cathepsin B, a phosphatase, a sulfatase, or a glucuronidase.

[0258] In certain embodiments, the enzyme-labile structure is unstable to cathepsin B and is selected from -Z- or wherein Z is a substrate for cathepsin B comprising 2 to 4 amino acids. 1 for Where * is covalently linked to L P The site, R 7 In certain embodiments, -G 1 -L P -D is

[0259]

[0260] In certain embodiments, the enzyme-labile structure is labile to glucuronidase and is

[0261]

[0262] Where * is covalently linked to G 1 The site of covalent bonding to D.

[0263] In certain embodiments, G 1 for Where R 7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl.

[0264] In some embodiments, G 1 for Where * is covalently linked to L P The site, R 7 It is an alkyl group.

[0265] In certain embodiments, -L P -D is selected from:

[0266]

[0267] In certain embodiments, -G 1 -L P -D is selected from:

[0268]

[0269] In certain embodiments, the enzyme-labile structure is unstable to phosphatases and is selected from

[0270]

[0271] Where * is covalently linked to G 1 The site of covalent connection to D, R 10 and R 11 Each of is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl moiety.

[0272] In certain embodiments, G 1 for In certain embodiments, -G 1 -L P -D Select from the group consisting of:

[0273]

[0274]

[0275] In certain embodiments, the enzyme-labile structure is unstable to sulfatase and is

[0276] Where * is covalently linked to G 1 The site of covalent connection to D, R 12 and R 13 Each of is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl moiety.

[0277] In some embodiments, G 1 for Where R 7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl moieties.

[0278] In some embodiments, G 1 for Where * is covalently linked to L P The site, R 7 It is an alkyl group.

[0279] In certain embodiments, -G 1 -L P -D means:

[0280]

[0281] In some embodiments, n is an integer from 1 to 100; m is an integer from 1 to 100; and p is an integer from 1 to 50.

[0282] In some embodiments, the therapeutic agent has anti-proliferative activity against a target cell or target pathway.

[0283] In certain embodiments, the anti-proliferative activity is selected from cytostatic activity and / or cytotoxic activity.

[0284] In certain embodiments, the therapeutic agent is selected from anticancer substances, cytotoxic drugs, radionuclides, vitamins, anti-AIDS substances, antibiotics, immunosuppressants, immunomodulatory compounds, therapeutic RNA, antiviral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, sedatives, anticonvulsants, muscle relaxants and anti-Parkinson substances, antispasmodics and muscle contractants (including channel blockers, miotics and anticholinergics), anti-glaucoma compounds, antiparasitic compounds and / or antiprotozoal compounds, regulators of cell-extracellular matrix interactions (including cell growth inhibitors and anti-adhesion molecules), vasodilators, inhibitors of DNA, RNA or protein synthesis, antihypertensives, analgesics, antipyretics, steroidal anti-inflammatory agents and non-steroidal anti-inflammatory agents, anti-angiogenic factors, antisecretory factors, anticoagulants and / or antithrombotics, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotic substances, antiemetics, imaging agents.

[0285] In certain embodiments, the therapeutic agent comprises an amino acid based molecule.

[0286] In certain embodiments, the amino acid-based molecule comprises a peptide, a polypeptide, an enzyme, an antibody, an immunoglobulin, or a functional fragment thereof.

[0287] In certain embodiments, the therapeutic agent has a chemically reactive group.

[0288] In certain embodiments, the chemically reactive group comprises -COOH, primary amine, secondary amine, -NHR, -OH, -SH, -C(O)H, C(O)R 14 、-C(O)NHR 15 、C(S)OH、-S(O) 2 OR 15 、-P(O) 2 OR 15 , -CN, -NC or -ONO, where R 14 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, and R 15 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0289] In certain embodiments, G 2 Selected from the group consisting of:

[0290]

[0291] Each R 16 are independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0292] In certain embodiments, G2 for

[0293] Linker-polymer compounds

[0294] The present disclosure also relates to linker-polymer compounds that can be further conjugated to a drug moiety to form the linker-polymer-drug compounds provided herein.

[0295] Therefore, in another aspect, the present disclosure provides a polymer scaffold of formula (II):

[0296]

[0297] in,

[0298] The polymer scaffold comprises linear polyglycerol;

[0299] L is a functional group W that can form a covalent bond with the targeting moiety. p The connecting part;

[0300] M a is an extending group connecting L to the -NH- portion;

[0301] Each G 2 are independently functional groups capable of being transformed into a charged state;

[0302] Each G 3 independently comprising a functional group capable of reacting with a reactive group in a drug release mechanism to thereby link the drug release mechanism to the linear polyglycerol;

[0303] n is an integer from 0 to 1000;

[0304] m is an integer from 0 to 1000;

[0305] p is an integer from 0 to 1000; and

[0306] q is an integer from 0 to 1000.

[0307] In some embodiments, n is 2; m is 2, and p is 2.

[0308] In some embodiments, q is an integer from 3 to 5.

[0309] In some embodiments, W p Capable of reacting with functional groups on the targeting moiety via a click reaction.

[0310] In certain embodiments, W p Selected from the group consisting of:

[0311]

[0312]

[0313] In some embodiments, W p Capable of reacting with amino acids on the targeting moiety.

[0314] In certain embodiments, W p Capable of reacting with an amino acid on a targeting moiety, and the amino acid is a natural amino acid, an unnatural amino acid, or a combination thereof. In certain embodiments, the natural amino acid may include cysteine, lysine, tyrosine, aspartic acid, and glutamic acid.

[0315] In some embodiments, W p Capable of reacting with one or more cysteines on the targeting moiety.

[0316] In certain embodiments, W p Capable of reacting with one or more cysteines on the targeting moiety, and each W p Selected from the group consisting of:

[0317]

[0318] Where R 1 is a sulfur protecting group, and each R 2 is independently a leaving group.

[0319] In certain embodiments, each R 2 are independently selected from halo or R 2a C(O)O-, where R 2a is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0320] In some embodiments, W p Capable of reacting with one or more lysines on the targeting moiety.

[0321] In certain embodiments, W p Capable of reacting with one or more lysines on the targeting moiety, and wherein each W p Independently selected from the group consisting of:

[0322]

[0323] In some embodiments, W p Capable of reacting with one or more unnatural amino acids on the targeting moiety.

[0324] In certain embodiments, W p capable of reacting with one or more unnatural amino acids on the targeting moiety, and each Wp Independently selected from:

[0325]

[0326] In some embodiments, M a Selected from the group consisting of:

[0327] R 3 ,

[0328]

[0329]

[0330] in

[0331] * is the site of covalent attachment to L, ** is the site of covalent attachment to -NH- moiety;

[0332] R 3 C 1-10 Alkyl, C 1-10 Heteroalkyl, C 3-8 Cycloalkyl, -O-(C 1-8 alkyl)-, aryl, -C 1-10 Alkyl-aryl-, -aryl-C 1-10 Alkyl-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-, -(C 3-8 Cycloalkyl-C 1-10 alkyl)-, 4- to 14-membered heterocycloalkyl, -C 1-10 Alkyl-(4- to 14-membered heterocycloalkyl)-, -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-, -C 1-10 Alkyl-C(=O)-, -C 1-10 Heteroalkyl-C(=O)-, -C 3-8 Cycloalkyl-C(=O)-, -O-(C 1-8 alkyl)-C(=O)-, -aryl-C(=O)-, -C 1-10 Alkyl-aryl-C(=O)-, -aryl-C 1-10 Alkyl-C(=O)-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-C(=O)-, -(C 3-8 Cycloalkyl)-C 1-10 alkyl-C(═O)-, -4- to 14-membered heterocycloalkyl-C═(O)-, -C 1-10 alkyl-(4- to 14-membered heterocycloalkyl)-C(═O)-, -(4- to 14-membered heterocycloalkyl)-C 1-10Alkyl-C(=O)-, -C 1-10 Alkyl-NH-, -C 1-10 Heteroalkyl-NH-, -C 3-8 Cycloalkyl-NH-, -O-(C 1-8 Alkyl)-NH-, -aryl-NH-, -C 1-10 Alkyl-aryl-NH-, -aryl-C 1-10 Alkyl-NH-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-NH-, -(C 3-8 Cycloalkyl)-C 1-10 Alkyl-NH-, -4- to 14-membered heterocycloalkyl-NH-, -C 1-10 Alkyl-(4- to 14-membered heterocycloalkyl)-NH-, -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-NH-, -C 1-10 Alkyl-S-, -C 1-10 Heteroalkyl-S-, -C 3-8 Cycloalkyl-S-, -OC 1-8 Alkyl-S-, -aryl-S-, -C 1-10 Alkyl-aryl-S-, -aryl-C 1-10 Alkyl-S-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-S-, -(C 3-8 Cycloalkyl)-C 1-10 Alkyl-S-, -4- to 14-membered heterocycloalkyl-S-, -C 1-10 -(4- to 14-membered heterocycloalkyl)-S- or -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-S-;

[0333] Each R 4 are independently hydrogen, C 1-6 Alkyl, C 6-10 Aryl, C 3-8 Cycloalkyl, -COOH or -COO-C 1-6 alkyl;

[0334] R 5 -C(O)-NR 5a or -NR 5a -C(O)-;

[0335] R 5a For hydrogen, C 1-6 Alkyl, C 6-10 Aryl, C 3-8 Cycloalkyl, -COOH or -COO-C 1-6 alkyl;

[0336] R 6 is a bond or -NR 6a -(CR 6b R 6c )-C(O)-;

[0337] R 6a For hydrogen, C 1-6 Alkyl, C 6-10 Aryl, C 3-8 Cycloalkyl, -COOH or -COO-C 1-6 alkyl;

[0338] Each R 6b and R 6c are independently hydrogen, C 1-6 Alkyl, C 6-10 Aryl, hydroxylated C 6-10 Aryl, polyhydroxylated C 6-10 Aryl, 5- to 12-membered heterocycloalkyl, C 3-8 Cycloalkyl, hydroxylated C 3-8 Cycloalkyl, polyhydroxylated C 3-8 Cycloalkyl, or the side chain of a natural or unnatural amino acid;

[0339] Each n 1 are independently an integer from 0 to 6;

[0340] n 2 is an integer from 0 to 8;

[0341] Each n 3 are independently an integer from 1 to 6; and

[0342] n 4 is an integer from 1 to 4.

[0343] In certain embodiments, M a Selected from the group consisting of:

[0344]

[0345]

[0346] In some embodiments, G 1 and G 2 Independently selected from the group consisting of:

[0347]

[0348] Among them, each R 17 Independently selected from direct bonds, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. In some embodiments, the polymer scaffold provided herein has a structure of formula (IIa) or formula (IIb):

[0349] In some embodiments, the polymer scaffolds provided herein have the structure of Formula (IIc) or (IId):

[0350]

[0351] Modified polymer part

[0352] The present disclosure also relates to modified polymer moieties that can be further conjugated to linker moieties and drug moieties to form linker-polymer-drug compounds provided herein.

[0353] Therefore, in another aspect, the present disclosure provides a polymer scaffold of formula (III):

[0354]

[0355] in,

[0356] The polymer scaffold comprises linear polyglycerol;

[0357] Each G 2 are independently functional groups capable of being transformed into a charged state;

[0358] Each G 3 independently comprising a functional group capable of reacting with a reactive group in a drug release mechanism to thereby link the drug release mechanism to the linear polyglycerol;

[0359] n is an integer from 0 to 1000;

[0360] m is an integer from 0 to 1000;

[0361] p is an integer from 0 to 1000; and

[0362] q is an integer from 0 to 1000.

[0363] In some embodiments, n is 2; m is 2, and p is 2.

[0364] In some embodiments, q is an integer from 3 to 5.

[0365] In some embodiments, G 1 and G 2 Independently selected from the group consisting of:

[0366]

[0367] Each R 17is independently selected from a direct bond, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group. In some embodiments, the polymer scaffold has a structure of formula (IIIa) or formula (IIIb):

[0368]

[0369] In some embodiments, the linker has a structure of formula (IIIc) or (IIId)

[0370]

[0371] In some embodiments, the polymer scaffold has a weight average molecular weight Mw of 1 to 100,000. In certain embodiments, the polymer scaffold has a weight average molecular weight Mw of 10,000 to 15,000. In certain embodiments, the polymer scaffold has a weight average molecular weight Mw of 5,000 to 10,000.

[0372] In some embodiments, the polymer scaffold has a PDI of less than 1.5.

[0373] Targeting moiety

[0374] The targeting moiety directs the linker-polymer-drug conjugate to a location in a specific tissue, cell or cell. The targeting moiety can direct the modified polymer in the culture or in the whole organism or in both. In each case, the targeting moiety can effectively bind to the ligand presented on the cell surface of one or more target cells with specificity, affinity and avidity. In some embodiments, the targeting moiety targets the modified polymer to tissues other than the liver. In other embodiments, the targeting moiety targets the modified polymer to specific tissues, such as the liver, kidney, lung or pancreas. The targeting moiety can target the modified polymer to target cells (such as cancer cells), such as receptors expressed on cells (such as cancer cells), matrix tissues or proteins associated with cancer, such as tumor antigens. Alternatively, cells containing tumor vasculature can be targeted. The targeting moiety can direct the modified polymer to specific types of cells, such as hepatocytes in the liver instead of Kupffer cells (Kupffer cell) that specifically target the liver. In other cases, the targeting moiety can direct the modified polymer to cells of the reticuloendothelial or lymphatic system, or to professional phagocytes, such as macrophages or eosinophils.

[0375] In other embodiments, the targeting moiety can be targeted to a position in the cell, such as the nucleus, cytoplasm or endosome through a modified polymer. In certain embodiments, the targeting moiety can enhance the binding of the cell to a receptor, or the cytoplasm is delivered to the nucleus, and the nucleus enters the endosome or other intracellular vesicle or is released from the endosome or other intracellular vesicle.

[0376] In some embodiments, targeting moieties include antibodies, proteins, and peptides or peptide mimetics.

[0377] In some embodiments, the targeting moiety comprises a natural amino acid capable of reacting with a functional group in the linking portion of the linker-polymer-drug conjugate to form a covalent bond. In certain embodiments, the natural amino acids include cysteine, lysine, tyrosine, aspartic acid and glutamic acid.

[0378] In certain embodiments, the targeting moiety comprises a cysteine ​​and the targeting moiety is bound to the linker-polymer-drug conjugate by a covalent bond through a thiol group to a functional group of the linking moiety in the linker-polymer-drug conjugate.

[0379] In certain embodiments, the targeting moiety comprises a lysine and the targeting moiety is bound to the linker-polymer-drug conjugate by a covalent bond through the amino group to a functional group of the linking moiety in the linker-polymer-drug conjugate.

[0380] In some embodiments, the targeting moiety may comprise a natural amino acid capable of reacting with a functional group in the linking portion of the linker-polymer-drug conjugate to form a covalent bond. In certain embodiments, the targeting moiety is bound to the linker-polymer-drug conjugate by a covalent bond via an amino group to a functional group in the linking portion of the linker-polymer-drug conjugate.

[0381] In some embodiments, the targeting moiety may comprise a functional group that is capable of reacting with a functional group in the linking moiety of the linker-polymer-drug conjugate to form a covalent bond via a click reaction.

[0382] In some embodiments, the targeting moiety can be one or more antibodies derived from Fab, Fab2, ScFv or camelid antibody heavy chain fragments that are specific for cell surface markers, including but not limited to 5T4, AOC3, ALK, AXL, C242, CA-125, CCL11, CCR 5, CD2, CD3, CD4, CD5, CD15, CA15-3, CD18, CD19, CA19-9, CD20, CD22, CD23, CD25, CD28, CD30, CD31, CD33, CD37, CD38, CD40, CD41, CD44, CD44 v6, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD74, CD79-B, CD80, CD125, CD138, CD141, CD147, CD152, CD 154, CD326, CEA, aggregation factors, CTLA-4, CXCR2, EGFR (HER1), ErbB2, ErbB3, EpCAM, EPHA2, EPHB2, EPHB4, FGFR (i.e., FGFR1, FGFR2, FGFR3, FGFR4), FLT3, folate receptor, FAP, GD2, GD3, GPNMB, HGF, HMI.24, ICAM, ICOS-L, IGF-1 receptor, VEGFR1, EphA2, TRP V1, CFTR, gpNMB, CA9, Cripto, c-KIT, c-MET, ACE, APP, adrenergic receptor-β2, tight junction protein (Claudine) 3, mesothelin, MUC1, NaPi2b, NOTCH1, NOTCH2, NOTCH3, NOTCH4, RON, ROR1, PD-L1, PD-L2, B7-H3, B7-B4, IL-2 receptor, IL-4 receptor, IL-13 receptor, Trop-2, integrin (including α 4 , α v β 3 , α v β 5 , α v β 6 , α 1 β 4 , α 4 β 1 , α 4 β 7 , α 5 β 1 , α 6 β 4 , α IIb β 3Integrin), IFN-α, IFN-γ, IgE, IgE, IGF-1 receptor, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, ITGB2 (CD18), LFA-1 (CD11a), L-selectin (CD62L), mucin, MUC1, myostatin, NCA-90, NGF, PDGFRα, phosphatidylserine, prostate cancer cells, Pseudomonas aeruginosa, rabies, RANKL, respiratory syncytial virus, rhesus factor, SLAMF7, sphingosine-1-phosphate, TAG-72, T cell receptor, tenascin C, TGF-1, TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR2, vimentin, etc.

[0383] In certain embodiments, one or more antibodies derived from Fab, Fab2, scFv or camelid antibody heavy chain fragments specific for cell surface markers include CA-125, C242, CD3, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD138, CD141, CD326, CEA, CTLA-4, EGFR (HER1), ErbB2, E rbB3, FAP, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, TAG-72, tenascin C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate cancer cells, adrenergic receptor-β2, tight junction protein 3, mucin, MUC1, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, and integrin (including α v β 3 , α v β 5 , α v β 6 , α 1 β 4 , α 4 β 1 , α 5 β 1 , α 6 β 4 integrin), tenascin C, TRAIL-R2, and vimentin.

[0384] Exemplary antibodies include 3F8, abagovomab, abciximab (REOPRO), adalimumab (HUMIRA), adecatumumab, afelimomab, afutuzumab, alacizumab, ALD518, alemtuzumab (CAMPATH), altumomab, amatuximab, anatumomab, and anrulimumab. anrukinzumab), apolizumab, arcitumomab (CEA-SCAN), aselizumab, atlizumab (tocilizumab, Actemra, RoActemra), torolimumab, bapineuzumab, basiliximab (Simulect), bavituximab, bectumomab (LYMPHOS CAN), belimumab (BENLYSTA), benralizumab, bertilimumab, besilesomab (SCINITIMUN), bevacizumab (AVASTIN), biciromab (FIBRISCINT), bivatuzumab, blinatumomab, brentuximab, briakinumab, canakinumab ab)(ILARIS), cantuzumab, capromab, catumaxomab(REMOVAB), CC49, cedelizumab, certolizumab, cetuximab(ERBITUX), citatuzumab, cixutumumab, clenoliximab, clivatuzumab, conatumumab, CR6261,dacetuzumab, daclizumab (ZENAPAX), daratumumab, denosumab (PROLIA), detumomab, dorlimomab, dorlixizumab, ecoromeximab, eculizumab (SOLIRIS), edobacomab, edrecolomab (PANOREX), efalizumab (PENOREX), ab)(RAPTIVA), efungumab(MYCOGRAB), elotuzumab, elsilimomab, enlimomab, epitumomab, epratuzumab, erlizumab, ertumaxomab(REXOMUN), etaracizumab(ABEGRIN), exbivirumab, fanolesomab(NEUTROSPE C), faralimomab, farletuzumab, felvizumab, fezakinumab, figitumumab, fontolizumab (HuZAF), foravirumab, fresolimumab, galiximab, gantenerumab, gavilimomab, gemtuzumab, girentuximab ximab), glembatumumab, golimumab (SIMPONI), gomiliximab, ibalizumab, ibritumomab, igovomab (INDIMACIS-125), imciromab (MYOSCINT), infliximab (REMICADE), intetumumab, inolimomab, inotuzumab,ipilimumab, iratumumab, keliximab, labetuzumab (CEA-CIDE), lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, lintuzumab, lucatumumab, lumiliximab mab), mapatumumab, maslimomab, matuzumab, mepolizumab (BOSATRIA), metelimumab, milatuzumab, minretumomab, mitumomab, morolimumab, motavizumab (NUMAX), morolimumab-CD3 (ORTHOCLONE OKT3), nacolomab, naptumomab, natalizumab (TYSABRI), nebacumab, necitumumab, nerelimomab, nimotuzumab (THERACIM), nofetumomab, ocrelizumab, odulimomab, ofatumumab (ARZERRA), olaratumab, omali zumab (XOLAIR), ontecizumab, oportuzumab, oregovomab (OVAREX), otelixizumab, pagibaximab, palivizumab (SYNAGIS), panitumumab (VECTIBIX), panobacumab, pascolizumab, pemtumomab (THERAGYN), pertuzumab (OMNITARG),pexelizumab, pintumomab, priliximab, pritumumab, PRO 140, rafivirumab, ramucirumab, ranibizumab (LUCENTIS), raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab (RITUXAN), robatumumab, rontalizumab, rovelizumab (LEUKARREST), ruplizumab (ANTOVA), sacituzumab, satumomab pendetide, sevirumab, sibrotuzumab, sifalimumab, siltuximab, siplizumab, solanezumab, sonepcizumab, sontuzumab, stamulumab, sulesomab (LEUKOSCAN), tacatuzumab (AFP-CIDE), tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox), tefibazumab (AUREXIS), telimomab, tenatumomab, teneliximab, teplizumab, TGN1412, ticilimumab / tremelimumab, tigatuzumab, TNX-650, tocilizumab (atlizumab), ACTEMRA), toralizumab, tositumomab (BEXXAR), trastuzumab (HERCEPTIN),tremelimumab, tucotuzumab, tuvirumab, urtoxazumab, ustekinumab (STELERA), vapaliximab, vedolizumab, veltuzumab, vepalimomab, visilizumab (NUVION), volociximab (HUMASPECT), votumumab, zalutumumab (HuMEX-EGFr), zanolimumab (HuMAX-CD4), ziralimumab, and zolimomab.

[0385] In some embodiments, the antibodies are against 5T4, CA-125, CEA, CD3, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD51, CTLA-4, EpCAM, HER2, EGFR (HER1), FAP, folate receptor, HGF, integrin αvβ3, integrin α5β1, IGF-1 receptor, GD3, GPNMB, mucin, MUC1, phosphatidylserine, cell surface markers of prostate cancer cells, PDGFRα, TAG-72, tenascin C, TRAIL-R2, VEGF-A, and VEGFR2. In this embodiment, the antibody is abavotumab, adelimumab, alacizumab, atumomab, maanatumomab, acitumomab, bavituximab, bevacizumab (AVASTIN), bivacumab, blinatumomab, brentuximab, cantuzumab, captoximab, caproumab, cetuximab, cetuzumab, clituzumab, canatumumab, darizumab, edrecolomab, epratuzumab, ertuinumab, edazinumab, fatutumomab, phentolumab, gemtuzumab, gebatumumab, ibritumomab tiuxetan, igovotumab, intumumab, inotuzumab, labetuzumab, lexalimumab, linduzumab, linduzumab, linduzumab, darizumab, edrecolomab, epratuzumab, ertuinumab, edazinumab, fatutumomab, fentolumab, gemtuzumab, gebatumumab, ibritumomab tiuxetan, igovotumab, intumumab, inotuzumab, labetuzumab, lexalimumab, linduz ... tocilizumab, rucamumab, matuzumab, mitumomab, nacipitumomab, necituzumab, oplitumumab, ogavuzumab, panitumumab, pantumomab, pertuzumab, plintumumab, rituximab (RITUXAN), rituximab, rotuximab, satumumab, sirolimusumab, talitumomab, tetuzumab, tetuzumab, tremelimumab (ticilimumab / tremelimumab), tegalizumab, trastuzumab (HERCEPTIN), tositumomab, tremelimumab, tutuuzumab simoleukin, volociximab, and zalutumumab.

[0386] In certain embodiments, the antibody to a cell surface marker for HER2 is pertuzumab or trastuzumab, and for EGFR (HER1), the antibody is cetuximab or panitumumab; and for CD20, the antibody is rituximab; and for VEGF-A, the antibody is bevacizumab; and for CD-22, the antibody is epratuzumab or velutuzumab; and for CEA, the antibody is labetuzumab.

[0387] Exemplary peptides or peptide mimetics include integrin targeting peptides (RGD peptides), LHRH receptor targeting peptides, ErbB2 (HER2) receptor targeting peptides, prostate-specific membrane-bound antigen (PSMA) targeting peptides, lipoprotein receptor LRP1 targeting, ApoE protein-derived peptides, ApoA protein peptides, somatostatin receptor targeting peptides, chlorotoxin-derived peptides, and bombesin.

[0388] In a specific embodiment, the peptide or peptide mimetic is an LHRH receptor targeting peptide and an ErbB2 (HER2) receptor targeting peptide.

[0389] Exemplary proteins include insulin, transferrin, fibrinogen-gamma fragment, thrombin, tight junction proteins, apolipoprotein E, affibody molecules (eg, ABY-025), ankyrin repeat proteins, ankyrin-like repeat proteins, and synthetic peptides.

[0390] In some embodiments, the targeting moiety-linker-polymer-drug conjugate comprises a broad range of cytotoxins in combination with the following cell surface markers: for HER2, such as pertuzumab or trastuzumab; for EGFR, such as cetuximab and panitumumab; for CEA, such as labetuzumab; for CD20, rituximab; for VEGF-A, such as bevacizumab; or for CD-22, such as epratuzumab or velutuzumab.

[0391] In other embodiments, the targeting moiety-linker-drug-polymer conjugate comprises a combination of two or more targeting moieties, such as a combination of a bispecific antibody directed against the EGF receptor (EGFR) on tumor cells and a bispecific antibody directed against CD3 and CD28 on T cells; a combination of one or more antibodies derived from Fab, Fab2, scFv or camel antibody heavy chain fragments and a peptide or peptide mimetic; a combination of one or more antibodies derived from Fab, Fab2, scFv or camel antibody heavy chain fragments and a protein; a combination of two bispecific antibodies, such as a CD3×CD19 bispecific antibody plus a CD28×CD22 bispecific antibody.

[0392] In other embodiments, the targeting moiety-linker-drug-polymer conjugate comprises a targeting moiety that is an antibody to an antigen, such as trastuzumab, cetuximab, rituximab, bevacizumab, epratuzumab, velutuzumab, labetuzumab, B7-H4, B7-H3, CA125, CD33, CXCR2, EGFR, FGFR1, FGFR2, FGFR3, FGFR4, Her2, NaPi2b, c-Met, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PD-L1, c-Kit, MUC1, and 5T4.

[0393] In certain embodiments, the targeting moiety-linker-drug-polymer conjugate comprises a targeting moiety that is an antibody against 5T4, such as a humanized anti-5T4 scFvFc antibody.

[0394] Examples of suitable 5T4 targeting ligands or immunoglobulins include those commercially available or described in the following patent or non-patent literature: for example, U.S. Pat. No. 8,044,178, U.S. Pat. No. 8,309,094, U.S. Pat. No. 7,514,546, EP1036091 (commercially available as TroVax TM , Oxford Biomedica, UK), EP2368914A1, WO 2013041687 A1 (Amgen), US2010 / 0173382 and P. Sapra et al., Mol. Cancer Ther. 2013, 12:38-47. Anti-5T4 antibodies are disclosed in U.S. Provisional Application No. 61 / 877,439 filed on September 13, 2013 and U.S. Provisional Application No. 61 / 835,858 filed on June 17, 2013. The contents of each of the patent documents and scientific publications are incorporated herein by reference in their entirety.

[0395] As used herein, the term "5T4 antigen binding portion" refers to a polypeptide sequence that can selectively bind to the 5T4 antigen. In an exemplary conjugate, the 5T4 antigen binding portion generally comprises a single-chain scFv-Fc format engineered from an anti-5T4 antibody. The single-chain variable fragment (scFv-Fc) is a fusion protein in which the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin are connected to a linker peptide, and further connected to an Fc region comprising a hinge region and a CH2 region and a CH3 region of an antibody (any such combination of antibody portions with each other or with other peptide sequences is sometimes referred to herein as an "immunofusion" molecule). In such scFvFc molecules, the scFv portion can be connected to the N-terminus of the Fc portion via a linker peptide at the C-terminus.

[0396] In some embodiments, the Fv portion of the 5T4 antigen binding portion can be engineered by recognized molecular biology techniques to include one or more amino acid substitutions in the VH region. The Fc portion of the 5T4 antigen binding portion preferably comprises a polypeptide sequence engineered from the human hinge region, CH2 region, and CH3 region of an anti-5T4 antibody.

[0397] In some embodiments, the targeting moiety-linker-drug-polymer conjugate comprises a targeting moiety that is an antibody against TROP2. TROP2 is a member of the TACSTD family expressed in human trophoblasts and is a single-pass transmembrane type 1 cell membrane protein involved in immune resistance, which is common to human trophoblasts and cancer cells.

[0398] The TROP2 antibodies used for the targeting moiety-linker-drug-polymer conjugates provided herein can be obtained using methods commonly performed in the art, which involve immunizing animals with TROP2 or any polypeptide selected from the amino acid sequence of TROP2, and collecting and purifying antibodies produced in vivo. The biological species of TROP2 used as an antigen are not limited to humans, and animals can be immunized with TROP2 derived from animals other than humans (such as mice or rats). In this case, by checking the cross-reactivity between the antibody bound to the obtained heterologous TROP2 and human TROP2, antibodies suitable for human diseases can be selected. In addition, monoclonal antibodies can be obtained from fusion tumors formed by fusing antibody-producing cells that produce antibodies against TROP2 with myeloma cells according to known methods (e.g., Kohler and Milstein, Nature, (1975) 256, pp. 495-497; Rennet, R. ed., Monoclonal Antibodies, pp. 365-367, New York Prenham Press, NY (1980)).

[0399] In some embodiments, the Trop2 antibodies used in the targeting moiety-linker-drug-polymer conjugates provided herein can be purchased from a number of sources and include, but are not limited to, LS-C126418, LS-C178765, LS-C126416, LS-C126417 (LifeSpan Biosciences, Inc., Seattle, WA); 10428-MM01, 10428-MM02, 10428-R001, 10428-R030 (Sino Biological Inc., Beijing, China); MR54 (eBioscience, San Diego, CA); sc-376181, sc-376746 (Santa Cruz Biotechnology, Santa Cruz, CA); Cruz, CA); MM0588-49D6 (Novus Biologicals, Littleton, CO); ab79976 and ab89928 ( Cambridge, MA).

[0400] Other Trop2 antibodies for use in the targeting moiety-linker-drug-polymer conjugates provided herein have been disclosed in the patent literature. For example, U.S. Publication No. 2013 / 0089872 discloses Trop2 antibodies K5-70 (Accession No. FERM BP-11251), K5-107 (Accession No. FERM BP-11252), K5-116-2-1 (Accession No. FERM BP-11253), T6-16 (Accession No. FERM BP-11346) and T5-86 (Accession No. FERM BP-11254), deposited in the International Patent Organism Depositary, Tsukuba, Japan. U.S. Patent No. 5,840,854 discloses Trop2 monoclonal antibody BR110 (ATCC No. HB11698). U.S. Patent No. 7,420,040 discloses a Trop2 antibody produced by the hybridoma cell line AR47A6.4.2, which is deposited at IDAC (International Depository Authority of Canada, Winnipeg, Canada) under accession number 141205-05. U.S. Patent No. 7,420,041 discloses a Trop2 antibody produced by the hybridoma cell line AR52A301.5, which is deposited at IDAC under accession number 141205-03. U.S. Publication No. 2013 / 0122020 discloses Trop2 antibodies 3E9, 6G11, 7E6, 15E2, 18B1. Hybridomas encoding representative antibodies are deposited at the American Type Culture Collection (ATCC) under accession numbers PTA-12871 and PTA-12872. U.S. Patent No. 8,715,662 discloses Trop2 antibodies produced by fusion tumors, deposited with AID-ICLC (Genoa, Italy) under the deposit numbers PD 08019, PD 08020 and PD 08021. U.S. Patent Application Publication No. 20120237518 discloses Trop2 antibodies 77220, KM4097 and KM4590. U.S. Patent No. 8,309,094 (Wyeth) discloses antibodies A1 and A3 identified by the sequence listing. U.S. Patent No. 10,227,417 discloses a number of Trop2 antibodies identified by the sequence listing. The examples section of each patent or patent application cited in this paragraph above is incorporated herein by reference. Regarding non-patent publications, Lipinski et al. (1981, Proc. Natl. Acad. Sci. USA, 78:5147-50) disclosed Trop-2 antibodies 162-25.3 and 162-46.2.The Pr1E11 Trop2 antibody was reported to recognize a unique epitope on Trop2 (Ikeda et al., Biochem Biophys Res Comm 458:877-82).

[0401] In some embodiments, the targeting moiety-linker-drug-polymer conjugate comprises a targeting moiety as an antibody against HER2. HER2 is one of the oncogene products of the typical growth factor receptor oncogene identified as human epidermal growth factor receptor 2-related oncogene, and is a transmembrane receptor protein with a molecular weight of 185kDa and a tyrosine kinase domain. HER2 is a member of the EGFR family consisting of HER1 (EGFR, ErbB-1), HER2 (neu, ErbB-2), HER3 (ErbB-3) and HER4 (ErbB-4) and is known to be autophosphorylated at intracellular tyrosine residues by homodimer formation or heterodimer formation with another EGFR receptor HER1, HER3 or HER4, and is activated in the above manner, thereby playing an important role in cell growth, differentiation and survival of normal cells and tumor cells.

[0402] The HER2 antibody for the targeting moiety-linker-drug-polymer conjugate provided herein is not particularly limited. HER2 antibody can be obtained according to the method commonly performed in the field, for example, which involves immunizing animals with antigen polypeptides and collecting and purifying antibodies produced in vivo. The source of the antigen is not limited to humans, and animals can be immunized with antigens derived from non-human animals (such as mice, rats, etc.). Alternatively, the antibody production cells for producing antibodies for antigens are fused with myeloma cells according to methods known in the field (for example, Kohler and Milstein, "Nature" (1975) 256, pp. 495-497; and Kennet, R, "Monoclonal Antibodies", pp. 365-367, New York Prenham Press (1980)) to form a fusion tumor, and then monoclonal antibodies can be obtained from the fusion tumor.

[0403] Examples of HER2 antibodies as used herein may include, but are not limited to, Pertuzumab (International Patent Publication No. WO 01 / 00245), Trastuzumab (U.S. Patent No. 5,821,337), and antibodies identified by sequence listing in U.S. Publication No. 2019 / 0077880. However, the HER2 antibodies applicable herein are not limited thereto, as long as they are specifically bound to HER2 and more preferably have an active HER2 antibody internalized in HER2-expressing cells by binding to HER2.

[0404] Conjugate

[0405] In one aspect, a polymer scaffold of formula (IV) is provided:

[0406]

[0407] in

[0408] The polymer scaffold comprises linear polyglycerol;

[0409] Each L a Independently for attaching the targeting moiety to M a The divalent part of

[0410] Each M a Independently a an extending group attached to the -NH- moiety;

[0411] Each G 1 Independently p a functional group attached to the linear polyglycerol;

[0412] Each L P Independently, the therapeutic agent moieties D and G 1 Drug release mechanism between;

[0413] Each D is independently a therapeutic agent moiety;

[0414] Each G 2 are independently functional groups capable of being transformed into a charged state;

[0415] n is an integer from 0 to 1000;

[0416] m is an integer from 0 to 1000;

[0417] p is an integer from 0 to 1000;

[0418] q is an integer from 0 to 1000; and

[0419] s is an integer from 1 to 8.

[0420] In some embodiments, W p Capable of reacting with functional groups on the targeting moiety via a click reaction.

[0421] In certain embodiments, W p Selected from the group consisting of:

[0422]

[0423] In some embodiments, W p Capable of reacting with amino acids on the targeting moiety.

[0424] In certain embodiments, W p Capable of reacting with an amino acid on a targeting moiety, and the amino acid is a natural amino acid, an unnatural amino acid, or a combination thereof. In certain embodiments, the natural amino acid may include cysteine, lysine, tyrosine, aspartic acid, and glutamic acid.

[0425] In some embodiments, W p Capable of reacting with one or more cysteines on the targeting moiety.

[0426] In certain embodiments, W p Capable of reacting with one or more cysteines on the targeting moiety, and each W p Selected from the group consisting of:

[0427]

[0428]

[0429] Where R 1 is a sulfur protecting group, and each R 2 is independently a leaving group.

[0430] In certain embodiments, each R 2 are independently selected from halo or R 2a C(O)O-, where R 2a is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0431] In some embodiments, W p Capable of reacting with one or more lysines on the targeting moiety.

[0432] In certain embodiments, W p Capable of reacting with one or more lysines on the targeting moiety, and wherein each W p Independently selected from the group consisting of:

[0433]

[0434] In some embodiments, W p Capable of reacting with one or more unnatural amino acids on the targeting moiety.

[0435] In certain embodiments, W p capable of reacting with one or more unnatural amino acids on the targeting moiety, and each W p Independently selected from:

[0436]

[0437] In some embodiments, M a Selected from the group consisting of:

[0438] R 3 ,

[0439] in

[0440] * is the site of covalent attachment to L, ** is the site of covalent attachment to -NH- moiety;

[0441] R 3 C 1-10 Alkyl, C 1-10 Heteroalkyl, C 3-8 Cycloalkyl, -O-(C 1-8 alkyl)-, aryl, -C 1-10 Alkyl-aryl-, -aryl-C 1-10 Alkyl-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-, -(C 3-8 Cycloalkyl-C 1-10 alkyl)-, 4- to 14-membered heterocycloalkyl, -C 1-10 Alkyl-(4- to 14-membered heterocycloalkyl)-, -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-, -C 1-10 Alkyl-C(=O)-, -C 1-10 Heteroalkyl-C(=O)-, -C 3-8 Cycloalkyl-C(=O)-, -O-(C 1-8 alkyl)-C(=O)-, -aryl-C(=O)-, -C 1-10 Alkyl-aryl-C(=O)-, -aryl-C 1-10 Alkyl-C(=O)-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-C(=O)-, -(C 3-8 Cycloalkyl)-C 1-10 alkyl-C(═O)-, -4- to 14-membered heterocycloalkyl-C═(O)-, -C 1-10 alkyl-(4- to 14-membered heterocycloalkyl)-C(═O)-, -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-C(=O)-, -C 1-10 Alkyl-NH-, -C 1-10 Heteroalkyl-NH-, -C 3-8 Cycloalkyl-NH-, -O-(C 1-8 Alkyl)-NH-, -aryl-NH-, -C 1-10Alkyl-aryl-NH-, -aryl-C 1-10 Alkyl-NH-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-NH-, -(C 3-8 Cycloalkyl)-C 1-10 Alkyl-NH-, -4- to 14-membered heterocycloalkyl-NH-, -C 1-10 Alkyl-(4- to 14-membered heterocycloalkyl)-NH-, -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-NH-, -C 1-10 Alkyl-S-, -C 1-10 Heteroalkyl-S-, -C 3-8 Cycloalkyl-S-, -OC 1-8 Alkyl-S-, -aryl-S-, -C 1-10 Alkyl-aryl-S-, -aryl-C 1-10 Alkyl-S-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-S-, -(C 3-8 Cycloalkyl)-C 1-10 Alkyl-S-, -4- to 14-membered heterocycloalkyl-S-, -C 1-10 -(4- to 14-membered heterocycloalkyl)-S- or -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-S-;

[0442] Each R 4 are independently hydrogen, C 1-6 Alkyl, C 6-10 Aryl, C 3-8 Cycloalkyl, -COOH or -COO-C 1-6 alkyl;

[0443] R 5 -C(O)-NR 5a or -NR 5a -C(O)-;

[0444] R 5a For hydrogen, C 1-6 Alkyl, C 6-10 Aryl, C 3-8 Cycloalkyl, -COOH or -COO-C 1-6 alkyl;

[0445] R 6 is a bond or -NR 6a -(CR 6b R 6c )-C(O)-;

[0446] R 6a For hydrogen, C1-6 Alkyl, C 6-10 Aryl, C 3-8 Cycloalkyl, -COOH or -COO-C 1-6 alkyl;

[0447] Each R 6b and R 6c are independently hydrogen, C 1-6 Alkyl, C 6-10 Aryl, hydroxylated C 6-10 Aryl, polyhydroxylated C 6-10 Aryl, 5- to 12-membered heterocycloalkyl, C 3-8 Cycloalkyl, hydroxylated C 3-8 Cycloalkyl, polyhydroxylated C 3-8 Cycloalkyl, or the side chain of a natural or unnatural amino acid;

[0448] Each n 1 are independently an integer from 0 to 6;

[0449] n 2 is an integer from 0 to 8;

[0450] Each n 3 are independently an integer from 1 to 6; and

[0451] n 4 is an integer from 1 to 4.

[0452] In certain embodiments, M a Selected from the group consisting of:

[0453]

[0454] In some embodiments, G 1 Selected from the group consisting of:

[0455]

[0456] Where * is covalently linked to L P The site of each R 7 are independently selected from a direct bond, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, and R 7a is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl moieties.

[0457] In some embodiments, G 1 Selected from the group consisting of:

[0458]

[0459]

[0460] Where R 7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0461] In some embodiments, each L p Independently contains unstable structures.

[0462] In certain embodiments, the labile structure is selected from a hydrolytically labile structure or an enzymatically labile structure.

[0463] In certain embodiments, the hydrolytically unstable structure is selected from the group consisting of:

[0464]

[0465] Where * is covalently linked to G 1 The site of covalent connection to D, R 8 is selected from hydrogen, alkyl or aryl, R 9 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0466] In certain embodiments, the hydrolytically unstable structure is selected from the group consisting of:

[0467]

[0468]

[0469] Where * is covalently linked to G 1 The site of covalent connection to D, R 8 is selected from hydrogen, alkyl or aryl, R 9 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0470] In some embodiments, G 1 for Where * is covalently linked to L P The site, R 7 It is an alkyl group.

[0471] In some embodiments, -G 1 -L P -D is

[0472]

[0473] In some embodiments, the enzyme labile structure is labile to an enzyme selected from cathepsin B, a phosphatase, a sulfatase, or a glucuronidase.

[0474] In certain embodiments, the enzyme-labile structure is unstable to cathepsin B and is selected from -Z- or wherein Z is a substrate for cathepsin B comprising 2 to 4 amino acids. 1 for Where * is covalently linked to L P The site, R 7 In certain embodiments, -G 1 -L P -D is

[0475]

[0476] In certain embodiments, the enzyme-labile structure is labile to glucuronidase and is

[0477]

[0478] Where * is covalently linked to G 1 The site of covalent bonding to D.

[0479] In some embodiments, G 1 for Where * is covalently linked to L P The site, R 7 It is an alkyl group.

[0480] In certain embodiments, G 1 for Where R 7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl.

[0481] In certain embodiments, -L P -D is selected from:

[0482]

[0483] In certain embodiments, -G 1 -L P -D is selected from:

[0484]

[0485] In certain embodiments, the enzyme-labile structure is unstable to phosphatases and is selected from

[0486]

[0487] Where * is covalently linked to G 1 The site of covalent connection to D, R 10 and R 11 Each of is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl moiety.

[0488] In certain embodiments, G 1 for In certain embodiments, -G 1 -L P -D Select from the group consisting of:

[0489]

[0490]

[0491] In certain embodiments, the enzyme-labile structure is unstable to sulfatase and is

[0492] Where * is covalently linked to G 1 The site of covalent connection to D, R 12 and R 13 Each of is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl moiety.

[0493] In some embodiments, G 1 for Where R 7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl moieties.

[0494] In some embodiments, G 1 for Where * is covalently linked to L P The site, R 7 It is an alkyl group.

[0495] In certain embodiments, -G 1 -L P -D means:

[0496]

[0497] In some embodiments, n is an integer from 1 to 100; m is an integer from 1 to 100; and p is an integer from 1 to 50.

[0498] In some embodiments, the therapeutic agent has anti-proliferative activity against a target cell or target pathway.

[0499] In certain embodiments, the anti-proliferative activity is selected from cytostatic activity and / or cytotoxic activity.

[0500] In certain embodiments, the therapeutic agent is selected from anticancer substances, cytotoxic drugs, radionuclides, vitamins, anti-AIDS substances, antibiotics, immunosuppressants, immunomodulatory compounds, therapeutic RNA, antiviral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, sedatives, anticonvulsants, muscle relaxants and anti-Parkinson substances, antispasmodics and muscle contractants (including channel blockers, miotics and anticholinergics), anti-glaucoma compounds, antiparasitic compounds and / or antiprotozoal compounds, regulators of cell-extracellular matrix interactions (including cell growth inhibitors and anti-adhesion molecules), vasodilators, inhibitors of DNA, RNA or protein synthesis, antihypertensives, analgesics, antipyretics, steroidal anti-inflammatory agents and non-steroidal anti-inflammatory agents, anti-angiogenic factors, antisecretory factors, anticoagulants and / or antithrombotics, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotic substances, antiemetics, imaging agents.

[0501] In certain embodiments, the therapeutic agent comprises an amino acid based molecule.

[0502] In certain embodiments, the amino acid-based molecule comprises a peptide, a polypeptide, an enzyme, an antibody, an immunoglobulin, or a functional fragment thereof.

[0503] In certain embodiments, the therapeutic agent has a chemically reactive group.

[0504] In certain embodiments, the chemically reactive group comprises -COOH, primary amine, secondary amine, -NHR, -OH, -SH, -C(O)H, C(O)R 14 、-C(O)NHR 15 、C(S)OH、-S(O) 2 OR 15 、-P(O) 2 OR 15 , -CN, -NC or -ONO, where R 14 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, and R 15 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0505] In certain embodiments, G 2 Selected from the group consisting of:

[0506]

[0507] Each R 16 are independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

[0508] In some embodiments, the targeting moiety is an antibody and / or fragment thereof.

[0509] In certain embodiments, the targeting moiety is antibody IgG1, antibody IgG2, antibody IgG3, and antibody IgG4.

[0510] In certain embodiments, the targeting moiety is selected from the group consisting of: Fab, Fab', F(ab') 2 、Fd、Fv fragment、disulfide bond stabilized Fv fragment (dsFv)、(dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies or bivalent domain antibodies.

[0511] Synthesis method

[0512] Any available technique can be used to prepare the conjugates provided herein or compositions comprising the same, and intermediates and components (eg, carriers and modifiers) used to prepare the same. For example, semi-synthetic and total synthetic methods can be used.

[0513] The synthetic process disclosed herein can tolerate a variety of functional groups, and thus can use a variety of substituted starting materials. The process generally provides the desired final compound at or near the end of the overall process, but in some instances it may be necessary to further convert the compound into a pharmaceutically acceptable salt, ester or prodrug thereof.

[0514] In some embodiments, the linker-polymer compounds provided herein can be conjugated to both a targeting moiety and a therapeutic agent (D). The linker-polymer compounds provided herein comprise a linking moiety for linking a targeting moiety and a linking moiety for linking a drug (D).

[0515] In some embodiments, the conjugates provided herein are formed in several steps, including (1) modifying a polymer carrier so that the polymer carrier contains functional groups that can react with functional groups of a targeting moiety or a derivative thereof and functional groups that can react with functional groups of a drug or a derivative thereof; (2) reacting the modified polymer with the drug or a derivative thereof so that the drug is attached to the modified polymer; and (3) reacting the modified polymer-drug conjugate with the targeting moiety or a derivative thereof to form the conjugate provided herein.

[0516] In another embodiment, the conjugate is formed in several steps: (1) modifying a polymer carrier so that the polymer carrier contains a functional group that can react with the functional group of the targeting portion or its derivative and a functional group that can react with the functional group of the first drug or its derivative; (2) reacting the modified polymer with the first drug or its derivative so that the first drug is attached to the modified polymer; (3) modifying the resulting polymer-drug conjugate so that it contains a different functional group that can react with the functional group of the second drug or its derivative; (4) reacting the modified polymer-drug conjugate with the second drug or its derivative so that the second drug is attached to the modified polymer-drug conjugate; (5) reacting the modified polymer-drug conjugate of step (4) with the targeting portion or its derivative to form the conjugate provided herein.

[0517] The synthetic process of the present invention can tolerate various functional groups, and thus can use various substituted starting materials. The process generally provides the desired final compound at the end of the overall process or near the end of the overall process, but in some cases it may be necessary to further convert the compound into its pharmaceutically acceptable salt, ester or prodrug.

[0518] The drug compounds for the conjugates provided herein can be prepared in various ways by using standard synthesis methods and procedures known to those skilled in the art or clear to those skilled in the art according to the teachings herein, using commercially available starting materials, compounds known in the literature, or from easily prepared intermediates. Standard synthesis methods and steps for preparing organic molecules and functional group conversion and control can be obtained from relevant scientific literature or from standard textbooks in the field. Although not limited to any one or several sources, the following are useful and recognized as reference textbooks for organic synthesis by those skilled in the art: classic textbooks such as Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition, John Wiley & Sons: New York, 2001; and Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999.

[0519] The conjugates of the present disclosure can be conveniently prepared by various methods familiar to those skilled in the art. The conjugates of the present disclosure with various formulas described herein can be prepared from commercially available starting materials or starting materials that can be prepared using literature steps. The steps show the preparation of representative conjugates of the present disclosure.

[0520] After the binding substances designed, selected and / or optimized by the methods described above are produced, they can be characterized using various analyses known to those skilled in the art for determining whether the binding substances have biological activity. For example, binding substances can be characterized by conventional analyses, including but not limited to analyses as described below for determining whether they have predicted activity, binding activity and / or binding specificity.

[0521] In addition, high throughput screening can be used to accelerate the analysis using such assays. Therefore, the activity of the conjugate molecules described herein can be quickly screened using techniques known in the art. The general method for performing high throughput screening is described in, for example, Devlin 1998) "High Throughput Screening", Marcel Dekker; and U.S. Patent No. 5,763,263. High throughput analysis can use one or more different analytical techniques, including but not limited to those described below.

[0522] Pharmaceutical composition

[0523] For administration purposes, in some embodiments, the conjugates provided herein are administered as raw chemical substances or formulated into pharmaceutical compositions.

[0524] Thus, in one aspect, the present disclosure provides a pharmaceutical composition comprising one or more conjugates as disclosed herein and acceptable carriers, such as stabilizers, buffers, and the like. The conjugate can be administered and introduced to a subject in a standard manner, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. Administration can be topical (including ocular and transmucosal, including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer); intratracheal, intranasal, epidermal and transdermal, oral and parenteral administration, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion or intracranial (e.g., intrathecal) or intraventricular administration. The conjugate can be formulated and used as a sterile solution and / or suspension for injectable administration; a lyophilized powder for reconstitution prior to injection / infusion; a topical composition; a tablet, capsule or elixir for oral administration; or a suppository for rectal administration, and other compositions known in the art.

[0525] A pharmacological composition or formulation refers to a composition or formulation in a form for administration to a cell or subject (including, for example, humans), such as systemic administration. Suitable forms depend in part on the route of use or entry, such as oral, inhaled, transdermal, or by injection / infusion. Such forms should not prevent compositions and formulations from reaching target cells (i.e., cells to which the drug needs to be delivered). For example, a pharmacological composition injected into the bloodstream should be soluble. Other factors are known in the art and include, for example, the following considerations: toxicity and form that prevent the composition or formulation from exerting its effect.

[0526] As used herein, the term "systemic administration" means the in vivo systemic absorption or accumulation of the modified polymer in the bloodstream, followed by distribution throughout the body. Routes of administration that cause systemic absorption include, but are not limited to, intravenous, subcutaneous, intraperitoneal, inhaled, oral, intrapulmonary, and intramuscular. Each of these routes of administration exposes the modified polymer to accessible diseased tissue. The rate at which the active agent enters the circulation has been shown to be a function of molecular weight or size. Using the conjugates provided herein, drug delivery in certain cells, such as cancer cells, can be achieved via the specific targeting of the targeting moiety.

[0527] As used herein, the term "pharmaceutically acceptable formulation" means that the composition or formulation allows for effective distribution of the conjugate in the physical location most suitable for its desired activity. In some embodiments, effective delivery is performed before clearance by the reticuloendothelial system or off-target binding (which can result in reduced efficacy or toxicity). Non-limiting examples of agents for formulation with the conjugate include: P-glycoprotein inhibitors (such as Pluronic P85), which can enhance the entry of active agents into the CNS; biodegradable polymers, such as poly (DL-lactide-co-glycolide) microspheres for sustained release delivery after implantation in the brain; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver active agents across the blood-brain barrier and can alter neuronal uptake mechanisms.

[0528] Also included herein are pharmaceutical compositions prepared for storage or administration, which include a pharmaceutically effective amount of the desired conjugate in a pharmaceutically acceptable carrier or diluent. Acceptable carriers, diluents and / or excipients for therapeutic use are well known in the pharmaceutical field. For example, buffers, preservatives, bulking agents, dispersants, stabilizers, dyes can be provided. In addition, antioxidants and suspending agents can be used. Examples of suitable carriers, diluents and / or excipients include, but are not limited to: (1) Dulbecco's phosphate buffered saline, pH about 6.5, which will contain about 1 mg / ml to 25 mg / ml human serum albumin, (2) 0.9% saline (0.9% w / v NaCl), and (3) 5% (w / v) glucose.

[0529] As used herein, the term "pharmaceutically effective amount" refers to the amount of a pharmaceutical agent that is used to treat, improve or prevent an identified disease or condition or that shows a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend on the subject's weight, size, and health; the nature and extent of the condition; and the treatment or therapeutic combination selected for administration. The pharmaceutically effective amount for a given situation can be determined by routine experimentation within the skill and judgment of the clinician.

[0530] For any conjugate, the pharmaceutically effective amount can be estimated initially in, for example, cell culture assays of neoplastic cells or in animal models (usually rats, mice, rabbits, dogs or pigs). Animal models can also be used to determine appropriate concentration ranges and routes of administration. This information can then be used to determine useful doses and routes for human administration. Therapeutic / prophylactic efficacy and toxicity can be assessed by standard pharmaceutical procedures in cell cultures or experimental animals, such as EDTA. 50 (the dose that is therapeutically effective in 50% of the population) and LD 50 The dose ratio between toxic effect and therapeutic effect is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit a large therapeutic index are preferred. The dosage can vary within this range, depending on the dosage form employed, the sensitivity of the patient, and the route of administration.

[0531] For example, Cell Potency Glo can be used to assess the ability of a linker-polymer-drug compound or targeting moiety-linker-polymer-drug conjugate to inhibit tumor growth in several cell lines. Dose response curves can be generated using SoftMax Pro software, and the IC can be determined based on a four-parameter curve fit. 50The cell lines employed may include those that are the target of the targeting moiety, and control cell lines that are not the target of the targeting moiety may also be included in the test conjugate.

[0532] In some embodiments, the conjugate is formulated for parenteral administration by injection, including the use of conventional catheter insertion techniques or infusion. The formulation for injection can exist in unit dosage form, for example in an ampoule or in a multi-dose container, optionally with the addition of a preservative. The conjugate can also be administered parenterally by a sterile medium. Depending on the vehicle and concentration used, the conjugate can be suspended or dissolved in the vehicle. Advantageously, adjuvants, such as local anesthetics, preservatives and buffers can be dissolved in the vehicle. As used herein, the term "parenteral" includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular or intrathecal injection or infusion techniques, etc. In addition, a pharmaceutical formulation comprising a conjugate and a pharmaceutically acceptable carrier is provided. One or more conjugates can be associated with one or more nontoxic pharmaceutically acceptable carriers and / or diluents and / or adjuvants, and other active ingredients as necessary.

[0533] Sterile injectable preparations can also be in the form of sterile injectable solutions or suspensions in non-toxic parenteral acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are routinely used as solvents or suspension media. For this purpose, mild fixed oils can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids, such as oleic acid can be used to prepare injections.

[0534] The conjugate and composition described herein can be in a suitable form, preferably parenterally, more preferably intravenously. For parenteral administration, the conjugate or composition can be an aqueous or non-aqueous sterile solution, suspension or emulsion. Propylene glycol, vegetable oil and injectable organic esters (such as ethyl oleate) can be used as solvents or vehicles. The composition can also contain an adjuvant, an emulsifier or a dispersant.

[0535] The order of magnitude dosage level between about 0.001mg and about 140mg per kilogram of body weight per day is applicable to the treatment of the above indicated condition (between about 0.05mg and about 7g per subject per day). In some embodiments, the dosage administered to the patient is between about 0.001mg per kilogram of subject weight and about 100mg per kilogram of subject weight. In some embodiments, the dosage administered to the patient is between about 0.01mg per kilogram of subject weight and about 15mg per kilogram of subject weight. In some embodiments, the dosage administered to the patient is between about 0.1mg per kilogram of subject weight and about 15mg per kilogram of subject weight. In some embodiments, the dosage administered to the patient is between about 0.1mg per kilogram of subject weight and about 20mg per kilogram of subject weight. In some embodiments, the dosage administered to the patient is between about 0.1mg per kilogram of subject weight and about 5mg per kilogram of subject weight or about 0.1mg per kilogram of subject weight and about 10mg per kilogram of subject weight. In some embodiments, the dosage is between about 1 mg per kilogram of subject weight and about 15 mg per kilogram of subject weight. In some embodiments, the dosage is between about 1 mg per kilogram of subject weight and about 10 mg per kilogram of subject weight. The amount of the conjugate that can be combined with the formulation material to produce a single dosage form will vary according to the subject being treated and the specific mode of administration. The unit dosage form generally may contain between about 0.001 mg and about 100 mg; between about 0.01 mg and about 75 mg; or between about 0.01 mg and about 50 mg; or between about 0.01 mg and about 25 mg.

[0536] For intravenous administration, dosage levels may include the ranges described above, or about 0.01 to about 200 mg of the conjugate per kilogram of animal body weight. In some embodiments, the composition may include about 1 to about 100 mg of the conjugate per kilogram of animal body weight. In some embodiments, the amount administered will be within the range of about 0.1 to about 25 mg of the compound per kilogram of body weight.

[0537] In some embodiments, the conjugate may be administered as follows: The conjugate may be administered intravenously daily for about 5 days, as a bolus injection daily for about 5 days, or as a continuous infusion for about 5 days.

[0538] Alternatively, the conjugate may be administered once a week for six weeks or longer. As another alternative, the conjugate may be administered once every two or three weeks. The bolus dose is administered in about 50 to about 400 ml of normal saline to which about 5 to about 10 ml of human serum albumin may be added. The continuous infusion is administered in about 250 to about 500 ml of normal saline to which about 25 to about 50 ml of human serum albumin may be added per 24 hour period.

[0539] In some embodiments, about one week to about four weeks after treatment, the patient may receive a second course of treatment. Specific clinical protocols regarding routes of administration, excipients, diluents, dosages, and timing may be determined by a person skilled in the art based on clinical circumstances.

[0540] In other embodiments, the therapeutically effective amount can be provided on a regular schedule, that is, every day, every week, every month or every year, or with an irregular schedule with different days, weeks, months, etc. of application. Alternatively, the therapeutically effective amount to be applied can vary. In some embodiments, the therapeutically effective amount of the first dose is higher than the therapeutically effective amount of one or more subsequent doses. In some embodiments, the therapeutically effective amount of the first dose is lower than the therapeutically effective amount of one or more subsequent doses. Equivalent doses can be applied in various time periods, including but not limited to about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every two weeks, about every three weeks, about every month and about every two months. The number of doses and frequency corresponding to the complete process of treatment will be determined according to the judgment of the relevant regulatory agencies and health care practitioners. The therapeutically effective amount described herein refers to the total amount applied in a given time period; that is, if more than one different conjugate described herein is applied, the therapeutically effective amount corresponds to the total amount applied. It should be understood that the specific dosage level for a particular subject will depend on a variety of factors, including the activity of the specific conjugate, age, weight, health status, sex, diet, time of administration, route of administration and excretion rate, combination with other active agents and the severity of the specific disease undergoing therapy.

[0541] For administration to non-human animals, the composition may also be added to the animal feed or drinking water. Such animal feed and drinking water are preferably formulated so that the animal ingests a therapeutically appropriate amount of the conjugate with its diet. It is also appropriate to present the conjugate in the form of a premix to be added to the feed or drinking water.

[0542] The conjugate can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat indications can increase the beneficial effects while reducing the presence of side effects. In some embodiments, the conjugate is used in combination with a chemotherapeutic agent (such as the chemotherapeutic agent disclosed in U.S. Patent No. 7,303,749). In other embodiments, the chemotherapeutic agent includes but is not limited to letrozole, oxaliplatin, docetaxel, 5-FU, lapatinib, capecitabine, leucovorin, erlotinib, patuximab, bevacizumab and gemcitabine. The present disclosure also provides a pharmaceutical kit, which includes one or more containers filled with one or more conjugates and / or compositions (including one or more chemotherapeutic agents) of the present disclosure. Such kits can also include, for example, other compounds and / or compositions, devices for administering compounds and / or compositions, and written instructions in the form of regulations for the manufacture, use or sale of governmental agencies regulating drugs or biological products. The compositions described herein can be packaged in a single dose or used for continuous or periodic discontinuous administration. For continuous administration, the package or kit may include each dosage unit containing the conjugate (e.g., a solution or other unit described above or utilized in drug delivery), and optional instructions for daily, weekly or monthly administration of the dose for a predetermined length of time or in a prescribed form. When it is desired that the concentration of the composition, the concentration of the components in the composition, or the relative ratio of the conjugate or agent within the composition vary over time, the package or kit may contain a series of dosage units that provide the desired variation.

[0543] A variety of packages or kits are known in the art to distribute medicaments for periodic oral use. In some embodiments, the package has an indicator for each time period. In some embodiments, the package is a labeled blister package, a scaled dispenser package, or a bottle. The packaging device of the kit itself can be adjusted according to the application, such as a syringe, pipette, eye dropper or other such device, whereby the formulation can be applied to the infected area of ​​the body, injected into the subject, or even applied to and mixed with other components of the kit.

[0544] Methods of treating diseases

[0545] The targeting moiety-linker-polymer-drug conjugates provided herein can be used in methods of treating animals (eg, mammals, such as humans, and including males, females, infants, children, and adults).

[0546] In some embodiments, the conjugates provided herein can be used in methods for treating animals, comprising administering the conjugates disclosed herein to the animals. The conjugates of the present invention can be used as drug carriers and drug carrier components in systems for controlling drug release, preparations for low-invasive surgical procedures, etc. The drug formulations can be injectable, implantable, etc.

[0547] In yet another aspect, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of at least one conjugate provided herein, wherein the conjugate releases one or more therapeutic agents upon biodegradation.

[0548] In some embodiments, the conjugates provided herein can be administered in vitro, in vivo, and / or ex vivo to treat a subject and / or modulate the growth of a selected cell population (including, for example, cancer). In some embodiments, specific types of cancer that can be treated with the conjugates provided herein include, but are not limited to: (1) solid tumors, including, but not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphoendotheliosarcoma, synovialoma, mesothelioma, Ewing's tumor (Ewing's tumor), leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, laryngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial cancer, renal cell carcinoma, hepatobiliary cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, lung cancer, epithelial cancer, glioma, glioblastoma, multiforme astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma (2) blood-borne cancers, including but not limited to acute lymphoblastic leukemia "ALL", acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia "AML", acute promyelocytic leukemia "APL", acute monoblastic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute non-lymphocytic leukemia, acute undifferentiated leukemia, chronic myeloid leukemia "CML", chronic lymphocytic leukemia "CLL", hairy cell leukemia, multiple myeloma, acute leukemias and chronic leukemias, such as lymphoblastic myeloid leukemia and lymphocytic myeloid leukemia; and (3) lymphomas, such as Hodgkin's disease (Hodgkin's disease), non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and polycythemia vera.

[0549] In some embodiments, the conjugates provided herein can be administered in vitro, in vivo, and / or ex vivo to treat a subject and / or modulate the growth of a selected cell population in a patient suffering from anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esophageal cancer, eye cancer, laryngeal cancer, oral cancer, mesothelioma, skin cancer, myeloma, oral cancer, rectal cancer, laryngeal cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, or gastric cancer.

[0550] In a certain embodiment, the cancer is selected from the group consisting of breast cancer, gastric cancer, non-small cell lung cancer (NSCLC), and ovarian cancer.

[0551] In some embodiments, the conjugates provided herein can be administered in vitro, in vivo and / or ex vivo to treat, prevent, reduce the risk of, and / or delay the onset of certain lesions (e.g., cancer). For example, the conjugates provided herein are used to treat, prevent, delay the progression of, or otherwise alleviate the symptoms of a cancer selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esophageal cancer, eye cancer, laryngeal cancer, oral cancer, mesothelioma, skin cancer, myeloma, oral cancer, rectal cancer, laryngeal cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), colon cancer, pancreatic cancer, kidney cancer, and gastric cancer.

[0552] In some embodiments, the conjugates provided herein can be administered in vitro, in vivo and / or ex vivo to treat autoimmune diseases, such as systemic lupus, rheumatoid arthritis, psoriasis and multiple sclerosis; transplant rejection, such as kidney transplant rejection, liver transplant rejection, lung transplant rejection, heart transplant rejection and bone marrow transplant rejection; graft-versus-host disease; viral infections, such as CMV infection, HIV infection and AIDS; and parasitic infections, such as giardiasis, amoebiasis, schistosomiasis, etc.

[0553] In some embodiments, the conjugates provided herein can also be used in the manufacture of a medicament for treating or lessening the severity of a disorder, such as characterized by abnormal cell growth (eg, cancer).

[0554] In some embodiments, the therapeutic agent is delivered locally to specific target cells, tissues, or organs.

[0555] In certain embodiments, the conjugates provided herein may further comprise or be associated with a diagnostic marker. In certain embodiments, the diagnostic marker is selected from the group consisting of: radiopharmaceuticals or radioisotopes for gamma scintigraphy and PET, contrast agents for magnetic resonance imaging (MRI), contrast agents for computed tomography, contrast agents for X-ray imaging methods, agents for ultrasound diagnostic methods, agents for neutron activation, moieties that can reflect, scatter or affect X-rays, ultrasound, radio waves and microwaves, and fluorophores. In certain exemplary embodiments, the conjugate is further monitored in vivo.

[0556] Examples of diagnostic labels include, but are not limited to, diagnostic radiopharmaceuticals or radioisotopes for gamma scintigraphy and PET, contrast agents for magnetic resonance imaging (MRI) (e.g., paramagnetic atoms and superparamagnetic nanocrystals), contrast agents for computed tomography, contrast agents for X-ray imaging methods, agents for ultrasound diagnostic methods, agents for neutron activation, and moieties that can reflect, scatter, or affect X-rays, ultrasound, radio waves and microwaves, fluorophores in various optical procedures, etc. Diagnostic radiopharmaceuticals include gamma-emitting radionuclides, such as indium-111, technetium-99m, and iodine-131, etc. Contrast agents for MRI (magnetic resonance imaging) include magnetic compounds, such as paramagnetic ions, iron, manganese, gadolinium, lanthanides, organic paramagnetic moieties, and superparamagnetic, ferromagnetic, and antiferromagnetic compounds, such as iron oxide colloids, ferrite colloids, etc. Contrast agents for computed tomography and other X-ray-based imaging methods include compounds that absorb X-rays, such as iodine, barium, etc. Contrast agents for ultrasound-based methods include compounds that absorb, reflect and scatter ultrasound waves, such as latex, crystals, bubbles, etc. Other examples include substances for neutron activation, such as boron and gadolinium. In addition, labels that can reflect, refract, scatter or otherwise affect X-rays, ultrasound, radio waves, microwaves and other rays used in diagnostic procedures can be used. Fluorescent labels can be used for optical imaging. In certain embodiments, the modifier comprises a paramagnetic ion or group.

[0557] In another aspect, the present disclosure provides a method of treating a disease or condition in a subject, comprising preparing an aqueous formulation of at least one Conjugate provided herein and parenterally injecting the formulation into the subject.

[0558] In another aspect, the present disclosure provides a method of treating a disease or condition in a subject, comprising preparing an implant comprising at least one conjugate provided herein, and implanting the implant into the subject. In certain embodiments, the implant is a biodegradable gel matrix.

[0559] In another aspect, the present disclosure provides a method of treating an animal in need thereof, comprising administering a conjugate according to the method described above.

[0560] In another aspect, the present disclosure provides a method of eliciting an immune response in an animal, comprising administering a conjugate as described above.

[0561] In another aspect, the present disclosure provides a method for diagnosing a disease in an animal, comprising the steps of: administering a conjugate as described above, wherein the conjugate comprises a detectable molecule; and detecting the detectable molecule.

[0562] In some embodiments, the step of detecting the detectable molecule is performed non-invasively. In some embodiments, the step of detecting the detectable molecule is performed using a suitable imaging device.

[0563] In some embodiments, a method of treating an animal comprises administering to the animal a conjugate provided herein in a packaged form for use in a surgical wound from which a tumor or growth has been removed. The conjugate package will replace the tumor site during recovery and degrade and dissipate as the wound heals.

[0564] In certain embodiments, the conjugates provided herein are associated with a diagnostic marker for in vivo monitoring.

[0565] The conjugates provided herein can be used for therapeutic, prophylactic and analytical (diagnostic) treatment of animals. The conjugates are generally intended for parenteral administration, but may be administered by other routes in some cases.

[0566] In some embodiments, soluble or colloidal conjugates are administered intravenously. In some embodiments, soluble or colloidal conjugates are administered via local (e.g., subcutaneous, intramuscular) injection. In some embodiments, solid conjugates (e.g., particles, implants, drug delivery systems) are administered via implantation or injection.

[0567] In some embodiments, a conjugate comprising a detectable label is administered to study the pattern and kinetics of label distribution in an animal.

[0568] In certain embodiments, any one or more of the conjugates provided herein can be used to practice any of the methods described above.

[0569] Throughout the specification, when a composition is described as having, containing or including a particular component, it is contemplated that the composition also consists essentially of or consists of said component. Similarly, when a method or process is described as having, containing or including a particular process step, the process also consists essentially of or consists of said process step. Further, it is to be understood that the order of steps or the order in which certain actions are performed is immaterial as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0570] All publications and patent documents cited herein are incorporated by reference as if each such publication or document was specifically and individually indicated to be incorporated by reference. The citation of publications and patent documents is not intended to be an admission of any relevant prior art nor does it constitute any admission of their contents or date.

[0571] Example

[0572] For the purpose of illustration, the following examples are included herein. The examples provided herein describe the synthesis of compounds and conjugates disclosed herein and intermediates for preparing compounds and conjugates. However, it should be understood that these examples do not limit the present disclosure and are intended only to show the method of practicing the present disclosure. Those skilled in the art will recognize that the described chemical reactions can be easily adapted to prepare a variety of other compounds or conjugates of the present disclosure, and the alternative methods for preparing compounds and conjugates of the present disclosure are considered to be within the scope of the present disclosure. In addition, those skilled in the art will also understand that the individual steps described herein or in a separate batch of compounds can be combined. Alternatively, other reactions disclosed herein or known in the art will be considered to be applicable to preparing other compounds of the present disclosure. Therefore, the following description is not intended to limit the scope of the present disclosure, but is specified by the appended claims.

[0573] abbreviation

[0574] The following abbreviations are used in the reaction schemes and synthetic examples that follow. This list is not meant to be an all-inclusive list of abbreviations used in this application as additional standard abbreviations (which are readily understood by organic synthesis technicians in the field) may also be used in the synthetic schemes and examples.

[0575] DMSO Dimethyl sulfoxide DMF N,N-Dimethylformamide HATU O-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate THF Tetrahydrofuran DIPEA N,N-Diisopropylethylamine IPA Isopropyl alcohol DCM Dichloromethane EDCI N-(3-Dimethylaminopropyl)-n"-ethylcarbodiimide hydrochloride DMAP 4-Dimethylaminopyridine TFA Trifluoroacetic acid EEDQ 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline HOB N-Hydroxybenzotriazole TBAF Tetra-n-butylammonium fluoride EGE Ethoxyethyl glycidyl ether t-BGA Tert-Butyl Glycidyloxyacetate EA Ethyl acetate PS Polystyrene NHS N-Hydroxysuccinimide PEG Polyethylene glycol PB Phosphate buffered saline TCEP Tris(2-carboxyethyl)phosphine EDTA EDTA

[0576] Example 1. Synthesis of copolymer linker compound 1

[0577]

[0578] In N 2 DownwardN 3 NBu 4 To a solution of 1.98 g, 6.98 mmol) in diethylene glycol dimethyl ether (200 mL) was added EGE (61 g, 418.6 mmol) and t-BGA (39.3 g, 209.3 mmol). The mixture was cooled to -40 °C, and then Al(iBu) was slowly added. 3 (30 ml, 29.7 mmol, 1 M in hexanes). The reaction was warmed to room temperature and heated under N 2The reaction was quenched with EtOH, diluted with water and extracted with EA. The combined organic phases were washed with brine and washed with Na 2 SO 4 Dried, filtered and concentrated under reduced pressure to obtain compound 1-1 (103 g) as a colorless oil, whose Mw was determined to be 27,000 by GPC using PS as a standard.

[0579] HCl (200 mL, 1 M in 1,4-dioxane) was slowly added to a solution of compound 1-1 (50 g, 1.85 mmol) in DCM (500 mL), and the reaction mixture was stirred at room temperature for 16 hours. After completion, the solvent was removed under reduced pressure, and the residue was dissolved with 10% NaOH solution. After the pH was adjusted to 7 with 4N HCl, the total volume of the solution was adjusted to 500 mL with water to obtain compound 1-2 in the form of an aqueous solution.

[0580] Add Na to the solution of 1-2 2 S·9H 2 O (30 g, 125 mmol) was added and the reaction mixture was heated at 100° C. for 18 hours. After completion, the reaction was cooled to room temperature and the pH was adjusted to 10 with 4N HCl. The mixture was dialyzed with 3KD MWCO to give compound 1-3 (300 mL) as an aqueous solution.

[0581] To the solution of 1-3 was added NaOH (5 g, 125 mmol) and (Boc) 2 O (30 g, 125 mmol) was added and the reaction mixture was stirred under outdoor temperature for 18 hours. After completion, the mixture was dialyzed with 3KD MWCO to obtain compound 1 (800 mL, 25 mg / mL) in the form of aqueous solution.

[0582] 1H NMR (400MHz, D 2 O)δ3.95(s,100H),3.87-3.57(br,800H),1.43(s,9H).

[0583] Example 2. Synthesis of Compound 2

[0584]

[0585] To a solution of 2-1 in THF (200 mL) were added pyridine (12 mL, 148 mmol) and Boc 2 O (1.44 g, 6.59 mmol). The reaction mixture was stirred at 15 °C for 18 h. The reaction mixture was washed with 0.5 N HCl (3 × 100 mL) and saturated NaHCO 3(100 mL). The organic phase was washed with MgSO 4 Drying, filtering and concentrating in vacuo gave the desired product 2-2 (2.45 g) as a white solid.

[0586] To a solution of 2-2 (2.5 g, 5.38 mmol) in DCM (50 mL) was added (tert-butoxycarbonyl)-L-alanine (2.5 g, 13.2 mmol), EDCI (1.99 g, 10.4 mmol) and DMAP (197.3 mg, 1.61 mmol). The reaction mixture was stirred at 15 °C for 16 h. The reaction mixture was washed with 0.5% NaHCO 3 (2 × 400 mL), water (400 mL) and 0.1 N HCl (2 × 400 mL). The organic phase was washed with anhydrous MgSO 4 Dry, filter and evaporate under vacuum.The residue was purified by silica gel column chromatography to give the desired product 2-3 (3.0 g, 87.7% yield) as a white solid.

[0587] To a solution of 2-3 (1.5 g, 2.36 mmol) in DCM (30 mL) was added TFA (10 mL). After stirring at 15 °C for 16 h, the solvent was removed under vacuum and the residue was purified by preparative HPLC (TFA) to give the desired product 2-4 (627 mg) as a yellow solid.

[0588] NHS (0.12 mg, 1.05 μmol), EDC (20 mg, 105.4 μmol) and DMF solution of 2-4 (23.4 mg, 42.2 μmol) were slowly added to the aqueous solution of compound 1 at 0°C and the reaction mixture was stirred at room temperature for 16 hours. After completion, the mixture was dialyzed with 5KD MWCO to give compound 2-5 (25 mL, 5 mg / mL) in the form of an aqueous solution.

[0589] 4N HCl (1.6 mL) was slowly added to an aqueous solution of compound 2-5 (25 mL, 5 mg / mL) at 0°C, and the reaction mixture was stirred at room temperature for 16 hours. After completion, saturated NaHCO 3(aq) The pH of the mixture was adjusted to 7 to give compound 2-6 which was used directly in the next step.

[0590] Slowly add NHS-PEG to the aqueous solution of compound 2-6 at 0°C 4 -Mal (32.6 mg, 5.25 μmol) in DMF (10 mL), the reaction mixture was stirred at room temperature for 16 hours. After completion, the mixture was dialyzed using 5KD MWCO to obtain compound 2 (15 mL, 5 mg / mL) in the form of an aqueous solution.

[0591] Example 3. Synthesis of Compound 3

[0592]

[0593] In N 2 To a solution of 3-1 (10 g, 26.6 mmol) in DCM (100 mL) was added TiBr 4 (10.7 g, 29.2 mmol). The reaction mixture was stirred at room temperature for 15 hours. The mixture was filtered and the filtrate was concentrated. The residue was purified by silica gel column chromatography with petroleum ether / EtOAc=10:1 to give compound 3-2 (8.0 g, 20.1 mmol, 75.7% yield) as a white solid.

[0594] In N 2 To a solution of 3-2 (8.0 g, 20.1 mmol) in MeCN (160 mL) were added 3-3 (3.39 g, 20.1 mmol) and Ag. 2 O (5.12 g, 22.1 mmol). After stirring at room temperature in the dark for 15 hours, the mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with petroleum ether / DCM=10:1 to give compound 3-4 (4.8 g, 9.94 mmol, 49.4% yield) as a white solid.

[0595] At 0°C in N 2 3-4 (4.8 g, 9.94 mmol) and silica gel (2.12 g) were dissolved in CHCl 3 To a mixture of 2-nitropropene (50 mL) and iPrOH (10 mL) was added NaBH 4 (564 mg, 14.91 mmol). After stirring at 25°C for 1.5 hours, the mixture was filtered and the solvent was evaporated to give the product 3-5 (5.0 g, 10.3 mmol, crude) as a colorless oil.

[0596] In N 2 To a solution of 3-5 (5.0 g, 10.3 mmol) in DMF (50 mL) was added imidazole (5.2 g, 9.26 mmol) and DMAP (282 mg, 2.31 mmol), followed by a solution of TBSCl (2.33 g, 15.45 mmol) in DMF (30 mL). After stirring at room temperature for 16 h, the reaction mixture was diluted with EtOAc (100 mL) and washed with NH 4 The combined organic layers were washed with NaCl (3×50 mL) and brine (3×50 mL). 2 SO 4Dry, filter and remove the solvent in vacuo.The crude product was purified by silica gel column chromatography (PE:EA=4:1) to give 3-6 (6.2 g, 10.3 mmol, 100% yield) as a white solid.

[0597] To a solution of 3-6 (4.2 g, 7 mmol) in EtOH (50 mL) was added 10% Pd / C (420 mg) at room temperature. The mixture was stirred at 25 °C under H 2 The mixture was stirred at 4% 4% (1 atm) for 16 h. After completion, the mixture was filtered through celite and washed with EtOH (50 mL). The filtrate was concentrated under vacuum to give the desired product 3-7 (3.8 g, 6.67 mmol, 95% yield) as a white solid.

[0598] To a solution of 3-7 (3.8 g, 6.67 mmol) and 3-8 (1.39 g, 7.3 mmol) in DCM (50 ml) was added EEDQ (6.6 g, 26.68 mmol). At room temperature under N 2 After stirring for 16 hours at 4 °C, the reaction mixture was diluted with water (100 mL) and extracted with DCM (3×100 mL). The organic phase was washed with brine (100 mL) and purified by Na 2 SO 4 Drying and concentration under vacuum gave the crude product, which was purified by flash chromatography to give the product 3-9 (2.4 g, 3.24 mmol, 48.6% yield) as a white solid.

[0599] TEA·3HF (2 mL) was added to a solution of 3-9 (2.4 g, 3.24 mmol) in THF (20 mL). The reaction mixture was stirred at room temperature for 1.5 hours. After completion, the solvent was removed and the residue was purified by silica gel column chromatography to obtain the desired product 3-10 (1.75 g, 2.79 mmol, 86% yield) as a white solid.

[0600] In N 2 To a solution of 3-10 (1.1 g, 1.76 mmol) and TEA (356 mg, 3.52 mmol) in DCM (50 mL) was added 3-11 (532 mg, 2.64 mmol). After stirring at 25 °C for 16 hours, the solvent was removed and the residue was purified by silica gel column chromatography to give the desired product 3-12 (800 mg, 1.01 mmol, 57% yield) as a white solid.

[0601] In N 2To a solution of 3-12 (200 mg, 0.25 mmol) in DMF (5 mL) was added (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamide (181 mg, 0.25 mmol), HOBT (6.7 mg, 0.025 mmol) and TEA (50.6 mg, 0.5 mmol). After stirring at 25 °C for 16 h, the mixture was diluted with water and extracted with EtOAc (20 mL×3). The combined organic layers were washed with brine (20 mL x 2) and purified by Na 2 SO 4 Drying, filtering and concentration in vacuo gave the crude product, which was purified by preparative HPLC to give the desired product 3-13 (202 mg, 0.147 mmol, 59% yield).

[0602] To a solution of 3-13 (400 mg, 0.29 mmol) in THF (10 mL) and water (2 mL) was added LiOH (27.7 mg, 1.16 mmol) at 0°C. After stirring at 25°C for 4 hours, the reaction was diluted with water (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (20 mL) and purified by Na 2 SO 4 Drying, filtering and concentrating in vacuo afforded 3-14 (300 mg, 0.24 mmol, 84% yield) as a colorless oil.

[0603] To a solution of 3-14 (300 mg, 0.24 mmol) in DCM (3 mL) was added TFA (1 mL) at room temperature. After stirring at room temperature for 16 hours, the reaction was concentrated in vacuo to give a crude product, which was purified by preparative HPLC (TFA) to give the desired product 3-15 (120 mg, 0.106 mmol, 44% yield).

[0604] To an aqueous solution of compound 1 (150 mg, 6 mL, 25 mg / mL) was slowly added NHS (0.12 mg, 1.05 μmol), EDCI (20 mg, 105.4 μmol) and 3-15 (23.4 mg, 42.2 μmol) in DMF at 0°C, and the reaction solution was stirred at room temperature for 16 hours. After completion, the mixture was dialyzed with 5KD MWCO to give compound 3-16 (25 mL, 5 mg / mL) in the form of an aqueous solution.

[0605] 4N HCl (1.6 mL) was slowly added to an aqueous solution of compound 3-16 (25 mL, 5 mg / mL) at 0°C, and the reaction mixture was stirred at room temperature for 16 hours. After completion, saturated NaHCO 3(aq) The pH of the mixture was adjusted to 7 to give solution compound 3-17 which was used directly in the next step.

[0606] Slowly add NHS-PEG to the aqueous solution of compound 3-17 at 0°C 4 -Mal (32.6 mg, 5.25 μmol) in DMF (10 mL), the reaction mixture was stirred at room temperature for 16 hours. After completion, the mixture was dialyzed using 5KD MWCO to obtain compound 3 (19 mL, 5 mg / mL) in the form of an aqueous solution.

[0607] Example 4: Synthesis of Compound 4

[0608]

[0609] To a solution of 4-1 (15 g, 77 mmol) and TBAF (92.7 mL, 1 M in THF, 92.7 mL) in DMF (105 mL) was added BnBr (11.97 g, 0.07 mol). The reaction mixture was stirred at room temperature for 16 hours. After completion, the solvent was evaporated under vacuum and the residue was purified by silica gel column chromatography (DCM: MeOH = 10: 1) to give 4-2 (20.1 g, 70 mmol, 96% yield) as a yellow oil.

[0610] To a solution of 4-2 (20 g, 70 mmol) in pyridine (60 mL) was added Ac 2 O (30 mL). The reaction mixture was stirred at room temperature for 16 hours. After completion, the solvent was removed in vacuo, and the residue was dissolved with DCM (400 mL), followed by washing with water (400 mL) and brine (2×400 ml). The organic phase was washed with anhydrous Na 2 SO 4Dry, filter and evaporate under vacuum.The residue was purified by silica gel column chromatography to give the desired product 4-3 (10 g, 31.6% yield) as a white solid.

[0611] To a solution of 4-3 (5.0 g, 11 mmol) in DCM (10 mL) was added HBr (11 mL, 48% in HBr, 44 mmol) dropwise. The reaction mixture was stirred at room temperature for 1.5 hours. The solvent was co-evaporated with toluene (3×500 mL) under vacuum. The residue was dissolved in DCM (500 mL), then washed with water (500 mL) and brine (2×500 mL). The organic phase was purified by anhydrous Na 2 SO 4 Dry, filter and evaporate under vacuum.The residue was purified by silica gel column chromatography to give the desired product 4-4 (1.8 g, 3.8 mmol, 34.5% yield) as a white solid.

[0612] To a solution of 4-4 (5.0 g, 10.57 mol) and 4-5 (1.76 g, 10.57 mmol) in MeCN (50 mL) was added Ag. 2 O (2.45 g, 10.57 mmol). The reaction mixture was stirred at room temperature in the dark for 2 hours. The solvent was removed under vacuum and the residue was purified by silica gel column chromatography to give 4-6 (4.2 g, 9.3 mmol, 88.1% yield) as a white solid.

[0613] 4-6 (5.1 g, 9.12 mmol) and silica gel (2.12 g) were dissolved in CHCl 3 To a mixture of 2-nitropropene (50 mL) and iPrOH (10 mL) was added NaBH 4 (517 mg, 13.68 mmol). After stirring at 25 °C for 1.5 hours, the reaction mixture was filtered and the solvent was removed under vacuum. The residue was purified by silica gel column chromatography (PE:EA=1:1) to give 4-7 (4.8 g, 8.5 mmol, 93.8% yield) as a white solid.

[0614] In N 2 Imidazole (5.2 g, 9.26 mmol) and DMAP (282 mg, 2.31 mmol) were added to a solution of 4-7 (5.2 g, 1.85 mmol) in DMF (30 mL) under a 4% CO atmosphere. After stirring for 5 minutes, a solution of TBSCl (2.08 g, 13.9 mmol) in DMF (30 mL) was added, and the reaction solution was stirred at room temperature for 16 hours. The mixture was washed with CH 2 Cl 2 The organic phase was diluted with NH 4The combined organic layers were washed with Cl(aq) (3×150 mL) and brine (3×150 mL). 2 SO 4 Dry, filter and remove the solvent in vacuo.The residue was purified by silica gel column chromatography (PE:EA=4:1) to give compound 4-8 (5 g, 79.9% yield) as a white solid.

[0615] 4-8 (3.87 g, 5.7 mmol) was dissolved in EtOH (20 mL) and H 2 To the solution in O (5 mL) were added Fe (1.59 g, 28.5 mmol) and NH 4 Cl (aq) (1.54 g, 28.5 mmol). After stirring at 65°C for 2 hours, the reaction mixture was washed with NH 4 The product was quenched with Cl(aq) (150 mL) and extracted with EA (2×150 mL). The combined organic layers were washed with brine (2×150 mL), dried over anhydrous Na2SO4, filtered and evaporated under vacuum. The residue was purified by flash silica gel column chromatography to afford 4-9 (3.0 g, 81.5% yield) as a yellow solid.

[0616] To a stirred solution of 4-9 (3.0 g, 4.65 mmol) and 4-10 (4.38 g, 3.75 mmol) in DCM (50 mL) was added EEDQ (5.7 g, 23.2 mmol). The mixture was stirred under N 2 The mixture was stirred at room temperature for 16 hours under an atmosphere of 4% paraformaldehyde. The reaction was quenched with water (100 mL) and extracted with DCM (3×100 mL). The combined organic layers were washed with brine (3×100 mL) and purified by Na 2 SO 4 Dry, filter and evaporate under vacuum.The residue was purified by silica gel column chromatography to give 4-11 (3.2 g, 3.92 mmol, 84.3% yield) as a white solid.

[0617] TEA3HF (5 mL) was added to a solution of 4-11 in THF (20 mL). The reaction mixture was stirred at room temperature for 1.5 hours. After completion, the solvent was removed and the residue was purified by silica gel column chromatography to obtain 4-12 (2 g, 66.4% yield) as a white solid.

[0618] To a solution of 4-12 (1.0 g, 1.42 mmol) and TEA (0.39 mL, 2.84 mol) in DCM (10 mL) was added 4-13 (2.16 g, 7.12 mmol). After stirring at 25 °C for 16 hours, the reaction was concentrated and the residue was purified by silica gel column chromatography to give 4-14 (930 mg, 1.07 mmol, 75.5% yield) as a yellow oil.

[0619] To a solution of 4-14 (954 mg, 1.1 mmol) in DMF (10 mL) was added 4-15 (950 mg, 1.1 mmol), HOBT (148 mg, 1.1 mmol) and TEA (222 mg, 2.2 mmol). After stirring at 25 °C for 16 hours, the mixture was quenched with water and extracted with EtOAc (200 mL×3). The combined organic layers were washed with brine (100 mL×2) and purified by Na 2 SO 4 Dry, filter and concentrate.The residue was purified by preparative HPLC to afford 4-16 (300 mg, 2 mmol, 18.0% yield).

[0620] To a solution of 4-16 (300 mg, 0.2 mmol) in MeOH (3 mL) was added 10% Pd / C (60 mg). The reaction mixture was stirred at 25 °C under 1 atm of H 2 The mixture was filtered and the solvent was evaporated to give 4-17 (250 mg, 0.17 mmol, 88.0% yield).

[0621] To a solution of 4-17 (250 mg, 0.17 mmol) in MeOH (3 mL) was added Na 2 CO 3 (60 mg, 0.6 mmol). The reaction mixture was stirred at 25°C for 16 hours. After filtration, the solvent was removed to give 4-18 (250 mg, 0.17 mmol, 99% yield).

[0622] To a solution of 4-18 (250 mg, 0.17 mmol) in DCM (5 mL) was added TFA (2 mL). After stirring at 25 °C for 1 hour, the solvent was removed and the residue was purified by preparative HPLC to give the desired product 4-19 (100 mg, 0.08 mmol, 44.4% yield).

[0623] To an aqueous solution of compound 1 (150 mg, 6 mL, 25 mg / mL) was slowly added NHS (0.12 mg, 1.05 μmol), EDCI (20 mg, 105.4 μmol) and 4-19 (23.4 mg, 42.2 μmol) in DMF at 0°C, and the reaction mixture was stirred at room temperature for 16 hours. After completion, the mixture was dialyzed with 5KD MWCO to give compound 4-20 (25 mL, 5 mg / mL) in the form of an aqueous solution.

[0624] 4N HCl (1.6 mL) was slowly added to an aqueous solution of compound 4-20 (25 mL, 5 mg / mL) at 0°C, and the reaction mixture was stirred at room temperature for 16 hours. After completion, saturated NaHCO 3 (aq) The pH of the mixture was adjusted to 7 to give an aqueous solution of compound 4-21 which was used directly in the next step.

[0625] Slowly add NHS-PEG to the aqueous solution of compound 4-21 at 0°C. 4 -Mal (32.6 mg, 5.25 μmol) in DMF (10 mL) was added and the reaction mixture was stirred at room temperature for 16 hours. After completion, the mixture was dialyzed using 5KD MWCO to obtain compound 4 (24 mL, 5 mg / mL) in the form of an aqueous solution.

[0626] Example 5: Synthesis of Compound 5

[0627]

[0628] To a solution of 5-1 (500 mg, 0.36 mmol) in DMF (5 mL) was added piperidine (60.7 mg, 0.71 mmol) at 20°C. The reaction was then stirred at room temperature for 18 hours. Upon completion, the mixture was purified by preparative HPLC (TFA) to give the desired product 5-2 (200 mg, 0.169 mmol, 47.5% yield) as a white solid.

[0629] To an aqueous solution of compound 1 (150 mg, 6 mL, 25 mg / mL) was slowly added NHS (0.12 mg, 1.05 μmol), EDCI (20 mg, 105.4 μmol) and 5-2 (23.4 mg, 42.2 μmol) in DMF at 0°C, and the reaction mixture was stirred at room temperature for 16 hours. After completion, the mixture was dialyzed with 5KD MWCO to give compound 5-3 (25 mL, 5 mg / mL) in the form of an aqueous solution.

[0630] 4N HCl (1.6 mL) was slowly added to an aqueous solution of compound 5-3 (25 mL, 5 mg / mL) at 0°C, and the reaction mixture was stirred at room temperature for 16 hours. After completion, saturated NaHCO 3 (aq) The pH of the mixture was adjusted to 7 to give an aqueous solution of compound 5-4 which was used directly in the next step.

[0631] Slowly add NHS-PEG to the aqueous solution of compound 5-4 at 0°C. 4 -Mal (32.6 mg, 5.25 μmol) in DMF (10 mL), the reaction mixture was stirred at room temperature for 16 hours. After completion, the mixture was dialyzed using 5KD MWCO to obtain compound 5 (16 mL, 5 mg / mL) in the form of an aqueous solution.

[0632] Example 6: Synthesis of Compound 6

[0633]

[0634] In N 2 6-2 (5.55 g, 27.6 mmol) and TEA (10 mL) were added to a solution of 6-1 (2.5 g, 6.87 mmol) in THF (400 mL) under reduced pressure. The reaction was stirred at room temperature for 16 hours. The reaction was diluted with water (100 mL) and extracted with EtOAc (50 mL×3). The combined organic phase was washed with brine (100 mL) and purified by Na 2 SO 4 Drying and concentration under vacuum gave a residue which was purified by silica gel column chromatography to give the desired product 6-3 (2.5 g, 4.7 mmol, 69% yield) as an off-white solid.

[0635] In N 2 6-4 (1.5 g, 7.9 mmol) and TEA (815 mg, 8.05 mmol) were added to a solution of 6-3 (3.3 g, 6.24 mmol) in THF (300 mL) under reduced pressure. The reaction was stirred at room temperature for 16 hours. The reaction was quenched with water (100 mL) and extracted with EtOAc (100 mL×3). The combined organic phase was washed with brine (100 mL) and purified by Na 2 SO 4 Drying and concentration under vacuum gave a residue, which was purified by silica gel column chromatography to give the desired product 6-5 (2.1 g, 3.6 mmol, 58% yield) as a white solid.

[0636] At room temperature in N 2To a solution of 6-5 (1.0 g, 1.73 mmol) in DCM (50 mL) was added tetrazole (242 mg, 3.46 mmol). After stirring for 0.5 hours, 6-6 (1.07 g, 3.11 mmol) was added dropwise at 0 ° C. The resulting solution was stirred at room temperature for 16 hours. After completion, mCPBA (559 mg, 2.59 mmol) was added at 0 ° C and stirred for another 16 hours. The reactant was then diluted with water (100 mL) and the organic layer was washed with sodium metabisulfite aqueous solution, sodium bicarbonate solution and filtered through Na 2 SO 4 The mixture was filtered and the solvent was evaporated, the residue was purified by preparative HPLC (water / ACN with 0.1% TFA) to give the desired product 6-7 (1.0 g, 69% yield) as a yellow solid.

[0637] At 0°C in N 2 To a solution of 6-7 (1.0 g, 1.19 mmol) in DCM (20 mL) was added TFA (20 mL). The reaction was stirred at room temperature for 16 hours. The reaction was concentrated under vacuum to give a crude product, which was purified by preparative HPLC (TFA) to give the desired product 6 as a yellow solid.

[0638] 1 H NMR (400MHz, CDCl3) δ8.21(s,1H),7.93(dd,J=25.8,9.1Hz,2H),7.59(s,1H),7.43(s,1H),5.60(d,J=16.5Hz,1H),5.40(d,J=16.6Hz,1H),5 .18(s,2H),3.94(s,1H),3.40(s,1H),3.29-3.17(m,2H),3.08(d,J=15.4Hz,3H),2.78(s,3H),2.14(d,J=7.3Hz,2H),1.03(t,J=7.2Hz,3H).

[0639] Example 7. Synthesis of Compound 7

[0640]

[0641] 2,2'-azanediylbis(ethane-1-ol) (39.04 g, 125.41 mmol), DMAP (1.39 g, 11.4 mmol) and DCC (3.56 g, 136.81 mmol) were added to a solution of 7-1 (25.0 g, 114.01 mmol) in DCM (250 mL). After stirring at room temperature for 16 hours, the reaction mixture was diluted with water (400 mL) and extracted with EA (700 mL), and the organic phase was washed with Na 2 SO 4 Dried, filtered and concentrated. The residue was purified by silica gel column chromatography to give the desired product 7-2 (25 g, 114 mmol, 89%) as a yellow oil.

[0642] To a solution of 7-2 (25.0 g, 114.01 mmol) in DCM (250 mL) was added (((9H-fluoren-9-yl)methoxy)carbonyl)-L-alanine (39.04 g, 125.41 mmol), DMAP (1.39 g, 11.4 mmol) and DCC (3.56 g, 136.81 mmol). After stirring at room temperature for 16 h, the reaction mixture was diluted with water (400 mL) and washed with EA (700 mL). The organic phase was purified by Na 2 SO 4 Dried, filtered and concentrated. The residue was purified by silica gel column chromatography to give the desired product 7-3 (25.5 g, 49.7 mmol, 43%) as a colorless oil.

[0643] To a solution of 7-3 (23 g, 44.87 mmol) in THF (200 mL) was added TBDPSCl (13.57 g, 49.36 mmol) and imidazole (6.11 g, 89.74 mmol). After stirring at room temperature for 12 hours, the reaction mixture was concentrated to give the crude product 7-4 (39 g, 51.9 mmol, 100%) as a colorless oil, which was used in the next step without further purification.

[0644] To a solution of 7-4 (39 g, 51.93 mmol) in DCM (150 mL) was added TFA (150 mL) and stirred at room temperature for 12 hours. The solvent was removed under vacuum and the residue was purified by flash column chromatography with DCM / MeOH (20 / 1, v / v) to give the desired product 7-5 (21 g, 30.2 mmol, 58%) as a white solid.

[0645] To a solution of 7-5 (5 g, 7.2 mmol) in DCM (50 mL) was added 1-hydroxypyrrolidine-2,5-dione (1.24 g, 10.79 mmol) and DCC (5.94 g, 28.8 mmol) at room temperature and stirred for 16 hours. The solvent was removed under vacuum and the residue was purified by flash column chromatography with PE / EtOAc (1 / 1, v / v) to give the desired product 7-6 (5 g, 6.3 mmol, 88%) as a colorless oil.

[0646] To a solution of 7-6 in DMF (20 mL) was added 7-7 (1.2 g, 1.52 mmol) and DIEA (653 mg). The reaction mixture was stirred at room temperature for 16 hours. The mixture was diluted with water and extracted with EA. The organic phase was purified by Na 2 SO 4 Dry, filter and evaporate under reduced pressure.The residue was purified by flash column chromatography on silica gel with DCM / MeOH (20 / 1, v / v) to give the desired product 7-8 (750 mg, 0.67 mmol, 67%) as a green solid.

[0647] To a solution of 7-8 (750 mg, 0.67 mmol) in THF (8 mL) was added dropwise TBAF (2 mL, 1 M in THF, 2 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. The mixture was concentrated under vacuum and the residue was purified by flash silica gel column chromatography with DCM / MeOH (20 / 1, v / v) to give the desired product 7-9 (500 mg, 0.57 mmol, 85% yield) in a black solid state.

[0648] To a solution of 7-9 (500 mg, 0.57 mmol) in pyridine (5 mL) was added SO 3 Pyridine (455 mg, 2.86 mmol) was added and the mixture was stirred for 16 hours. The solvent was then removed under vacuum and the product was purified by silica gel column chromatography using MeOH / H 2 The residue was purified by HPLC-MS / MS (1000 x 400 μL) O (1 / 1, v / v) to afford 7-10 (500 mg, 0.52 mmol, 92%) as a yellow solid.

[0649] To a solution of 7-10 (500 mg, 0.52 mmol) in DMF (3 mL) was added piperidine (89.26 mg, 1.05 mmol) at room temperature and the mixture was stirred for 16 hours. The solvent was removed under vacuum and the residue was purified by C18 column chromatography (MeOH / H2O (1 / 1, v / v)) to obtain the desired product 7 (229 mg, 0.31 mmol, 60%) as a yellow solid.

[0650] 1H NMR (400MHz, DMSO) δ8.92 (s, 1H), 8.31 (s, 3H), 7.78 (d, J = 10.9Hz, 1H), 7.30 (s, 1H), 5.67-5.12 (m, 5H), 4.44 (s, 2H), 3.96 (d, J=62.4Hz, 6H), 3.59-2.98 (m, 6H), 2.44-2.04 (m, 5H), 1.85 (tt, J= 14.0, 7.1Hz, 2H), 1.46-1.17 (m, 3H), 0.86 (t, J= 7.3Hz, 3H).

[0651] Example 8: Synthesis of Compound 8

[0652]

[0653] To a solution of 8-1 (500 mg, 0.36 mmol) in DMF (5 mL) was added piperidine (60.7 mg, 0.71 mmol) at 20°C. The reaction was then stirred at room temperature for 18 hours. Upon completion, the mixture was purified by preparative HPLC (TFA) to give the desired product 8-2 (200 mg, 0.169 mmol, 47.5% yield) as a white solid.

[0654] To an aqueous solution of compound 1 (150 mg, 6 mL, 25 mg / mL) was slowly added NHS (0.12 mg, 1.05 μmol), EDCI (20 mg, 105.4 μmol) and 8-2 (23.4 mg, 42.2 μmol) in DMF at 0°C, and the reaction mixture was stirred at room temperature for 16 hours. After completion, the mixture was dialyzed with 5KD MWCO to give compound 8-3 (25 mL, 5 mg / mL) in the form of an aqueous solution.

[0655] 4N HCl (1.6 mL) was slowly added to an aqueous solution of compound 8-3 (25 mL, 5 mg / mL) at 0°C, and the reaction mixture was stirred at room temperature for 16 hours. After completion, saturated NaHCO 3 (aq) The pH of the mixture was adjusted to 7 to give an aqueous solution of compound 8-4 which was used directly in the next step.

[0656] Slowly add NHS-PEG to the aqueous solution of compound 8-4 at 0°C. 4 -Mal (32.6 mg, 5.25 μmol) in DMF (10 mL), the reaction mixture was stirred at room temperature for 16 hours. After completion, the mixture was dialyzed using 5KD MWCO to obtain compound 8 (16 mL, 5 mg / mL) in the form of an aqueous solution.

[0657] Example 9. Synthesis of ADC-1

[0658] TCEP (8 μL, 17.4 mM, 2 equivalents) was added to a trastuzumab (5 mg, 2.5 mg / ml) buffer solution (PB, histidine, TEAA, sodium acetate buffer). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 2 were added and reacted for another 1.5 hours. 50 equivalents of L-cysteine ​​were then added to quench the reaction. The mixture was purified via a cation exchange column to obtain ADC-1 (3 mg, 2 mg / ml). DAR was determined by UV spectrometer.

[0659] Example 10. Synthesis of ADC-2

[0660] TCEP (8 μL, 17.4 mM, 2 equivalents) was added to a trastuzumab (5 mg, 2.5 mg / ml) buffer solution (PB, histidine, TEAA, sodium acetate buffer). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 3 were added and reacted for another 1.5 hours. 50 equivalents of L-cysteine ​​were then added to quench the reaction. The mixture was purified via a cation exchange column to give ADC-2 (3 mg, 2 mg / ml). DAR was determined by UV spectrometer.

[0661] Example 11. Synthesis of ADC-3

[0662] TCEP (8 μL, 17.4 mM, 2 equivalents) was added to a trastuzumab (5 mg, 2.5 mg / ml) buffer solution (PB, histidine, TEAA, sodium acetate buffer). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 4 were added and reacted for another 1.5 hours. 50 equivalents of L-cysteine ​​were then added to quench the reaction. The mixture was purified via a cation exchange column to obtain ADC-3 (3 mg, 2 mg / ml). DAR was determined by UV spectrometer.

[0663] Example 12. Synthesis of ADC-4

[0664] TCEP (8 μL, 17.4 mM, 2 equivalents) was added to a Trop2 antibody Trop2-1 (5 mg, 2.5 mg / ml) buffer solution (PB, histidine, TEAA, sodium acetate buffer). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 2 were added and reacted for another 1.5 hours. Then 50 equivalents of L-cysteine ​​were added to quench the reaction. The mixture was purified via a cation exchange column to obtain ADC-4 (3 mg, 2 mg / ml). DAR was determined by UV spectrometer.

[0665] Example 13. Synthesis of ADC-5

[0666] TCEP (8 μL, 17.4 mM, 2 equivalents) was added to a Trop2 antibody Trop2-1 (5 mg, 2.5 mg / ml) buffer solution (PB, histidine, TEAA, sodium acetate buffer). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 3 were added and reacted for another 1.5 hours. Then 50 equivalents of L-cysteine ​​were added to quench the reaction. The mixture was purified via a cation exchange column to obtain ADC-5 (3 mg, 2 mg / ml). DAR was measured by UV spectrometer.

[0667] Example 14. Synthesis of ADC-6

[0668] TCEP (8 μL, 17.4 mM, 2 equivalents) was added to a Trop2 antibody Trop2-1 (5 mg, 2.5 mg / ml) buffer solution (PB, histidine, TEAA, sodium acetate buffer). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 4 were added and reacted for another 1.5 hours. Then 50 equivalents of L-cysteine ​​were added to quench the reaction. The mixture was purified via a cation exchange column to obtain ADC-6 (3 mg, 2 mg / ml). DAR was determined by UV spectrometer.

[0669] Example 15. Synthesis of ADC-7

[0670] TCEP (8 μL, 17.4 mM, 2 equivalents) was added to a Trop2 antibody Trop2-2 (5 mg, 2.5 mg / ml) buffer solution (PB, histidine, TEAA, sodium acetate buffer). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 2 were added and reacted for another 1.5 hours. Then 50 equivalents of L-cysteine ​​were added to quench the reaction. The mixture was purified via a cation exchange column to obtain ADC-7 (3 mg, 2 mg / ml). DAR was determined by UV spectrometer.

[0671] Example 16. Synthesis of ADC-8

[0672] TCEP (8 μL, 17.4 mM, 2 equivalents) was added to a Trop2 antibody Trop2-2 (5 mg, 2.5 mg / ml) buffer solution (PB, histidine, TEAA, sodium acetate buffer). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 3 were added and reacted for another 1.5 hours. Then 50 equivalents of L-cysteine ​​were added to quench the reaction. The mixture was purified via a cation exchange column to obtain ADC-8 (3 mg, 2 mg / ml). DAR was determined by UV spectrometer.

[0673] Example 17. Synthesis of ADC-9

[0674] TCEP (8 μL, 17.4 mM, 2 equivalents) was added to a Trop2 antibody Trop2-2 (5 mg, 2.5 mg / ml) buffer solution (PB, histidine, TEAA, sodium acetate buffer). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 4 were added and reacted for another 1.5 hours. Then 50 equivalents of L-cysteine ​​were added to quench the reaction. The mixture was purified via a cation exchange column to obtain ADC-9 (3 mg, 2 mg / ml). DAR was measured by UV spectrometer.

[0675] Example 18. Cell-based binding of anti-HER2 antibodies and anti-HER2 ADCs to HER2-expressing tumor cell lines

[0676] HER2 positive cell lines SKBR-3 cells (breast cancer, ATCC No. HTB30) and NCI-N87 cells (gastric cancer, ATCC No. CRL-5822) were used for in vitro cell binding evaluation of exemplary anti-HER2 ADCs ADC-2 (two batches, i.e., ADC-2-1 and ADC-2-2) and ADC-3. Commercially available anti-HER2 ADC drugs, DS-8201a from Daiichi Sankyo and T-DM1 from Genentech (Roche) were used as positive control ADCs, and native antibody trastuzumab (Genentech (Roche)) was used for free antibody control.

[0677] Briefly, SKBR-3 cells and NCI-N87 cells were maintained in McCoy's 5a medium (Cat. No. 30-2007, ATCC) and RPMI-1640 medium (Cat. No. abs9468, Absin), respectively, supplemented with 10% fetal bovine serum (FBS) (Cat. No. FSP500, Excell Bio), 2 mM L-glutamine (Cat. No. 25030081, ThermoFisher) and 1% penicillin-streptomycin solution (BL505A, Biosharp Life Sciences). Cells were cultured at 37°C in 5% CO 2 100 μL of 2×10 cells / well was added to each well of a 96-well cell culture plate. 6Cells at 100 cells / ml were incubated on ice for 30 to 60 minutes with 100 μL of 1× secondary antibody PE anti-human IgG Fc antibody (Catalog No. 366904, Biolegend) and trastuzumab and 5-fold serial dilutions from 100 nM to 0.00128 nM. Cells were washed twice with 200 μL / well FACS buffer (PBS with 1% FBS). Cells were incubated on ice for 30 to 60 minutes with 100 μL of 1× secondary antibody PE anti-human IgG Fc antibody (Catalog No. 366904, Biolegend). Cells were washed twice with 200 μL FACS buffer and resuspended with 200 μL FACS buffer to be further detected by CytoFlex cell measurement (Beckman Coulter). Data were analyzed with CytoFlex's built-in analysis software.

[0678] exist Figure 1 The cell binding results are shown in . The binding ability of the tested anti-HER2 ADC to SKBR-3 cells and NCI-N87 cells was efficient and compatible with trastuzumab.

[0679] Example 19. Analysis of Antibody-Mediated Internalization in HER2-Expressing Tumor Cells Using FACS

[0680] HER2 positive cell lines SKBR-3 cells and NCI-N87 cells as used in Example 18 and test anti-HER2 ADC (exemplary anti-HER2 ADCs ADC-2-1, ADC-2-2 and ADC-3, and positive control ADCs DS-8201a and T-DM1) were also used in the following internalization analysis. The culture conditions of the two cell lines were the same as in Example 18.

[0681] The cells were harvested with 0.25% trypsin / EDTA (T1320, Solarbio) and the cell concentration was adjusted to 4×10 with 1×PBS containing 1% FBS buffer. 6 100 μL of cells / well were then seeded in a 96-well cell culture plate and incubated with trastuzumab and the test anti-HER2 ADC at a final concentration of 100 nM for seven time points (0, 0.5 hours, 2 hours, 4 hours, 8 hours, 16 hours, and 24 hours). The mixture was incubated at 4°C in a 5% CO 2 At specific time points, the cells were immediately washed twice and suspended in 100 μL of cold wash buffer containing 2% paraformaldehyde for 30 minutes. The cells were washed twice and then incubated with 1×PE anti-human IgG Fc antibody (Catalog No. 366904, Biogen Biotech) at 4°C and 5% CO 2The internalization rate at several time points was calculated with the median fluorescence intensity (MFI) value at a specific time point relative to the loss rate of the value at 0 hours. The internalization rate was plotted with Graphpad Prism software relative to the curve of incubation time (hours).

[0682] The results for SKBR-3 cells and NCI-N87 cells are shown in Tables 1 and 2 and Figure 2 All tested anti-HER2 ADCs can effectively mediate internalization into HER2-positive cells (SKBR-3 cells and NCI-N87 cells), with internalization rates >60% (SKBR-3 cells) and >30% (NCI-N87 cells) within 24 hours. The internalization results show that binding does not change target-mediated internalization.

[0683] Table 1. MFI values ​​for internalization analysis of SKBR-3 cells

[0684] MFI ADC-2-1 ADC-2-2 ADC-3 DS-8201a T-DM1 Trastuzumab 0h 81296.3 70886.1 58383.9 111493.1 89030.9 114620.8 0.5h 51815.9 42850.6 36842.6 84105.2 54683.9 80551.7 2h 48758.2 41203.7 32890.5 91504.5 51777.7 88422.6 4h 37648.4 30356.6 24160.4 59683.6 39019.6 66067.7 8h 31700.9 25784.3 19417.6 55324.7 31583.1 53596.8 16h 27502.4 22283.5 18533.7 48587.4 29393.7 61201.3 24h 20177 17657.4 16436.4 37770 20671.3 43793.5

[0685] Table 2. MFI values ​​for internalization analysis of NCI-N87 cells

[0686] MFI ADC-2-1 ADC-2-2 ADC-3 DS-8201a T-DM1 Trastuzumab 0h 382098.4 322860.4 302930 470434.6 455385 494263.3 0.5h 389864.7 332284.1 306071.1 485012.7 452742.7 505817.1 2h 386741.6 319953.5 279387.2 453325.2 475100.1 523528.4 4h 311931.9 260945 238134.4 399661.6 369328.5 412925.1 8h 301151.8 234073.9 222454.3 361668.5 345220.2 398055.9 16h 282719.9 228232.9 201278 357611.1 362105.3 408260.5 24h 255656.2 209783 199868.8 312219.9 317532.7 347311

[0687] Example 20. In vitro cytotoxicity analysis of anti-HER2 ADC on HER2 expressing cells

[0688] The cytotoxicity of the exemplary anti-HER2 ADCs was studied using SKBR-3 cells (breast cancer, ATCC No. HTB30) and NCI-N87 cells (gastric cancer, ATCC No. CRL-5822) and compared with positive control ADCs DS-8201a and T-DM1.

[0689] Briefly, 5,000 cells / well of SKBR-3 cells and NCI-N87 cells were seeded in 90 μL of basal medium (McCoy's 5a medium for SKBR-3 cells and RPMI-1640 medium for NCI-N87 cells) containing 10% FBS, 1% penicillin-streptomycin solution (BL505A, Biosharp Life Sciences, Inc.) and 2 mM L-glutamine (Catalog No. 25030081, Thermo Fisher Scientific) in a white clear 96-well assay plate (Catalog No. 6005182, PerkinElmer) (excluding the edge wells containing only medium) and incubated at 37°C in the presence of 5% CO 2After overnight incubation, each tested anti-HER2 ADC or trastuzumab was added to individual wells at 10 μL of 10× concentrated test substance ranging from 100 nM to 0.015 nM. After another 72 hours of incubation, the plates were removed from the incubator and allowed to equilibrate to room temperature. Approximately 30 minutes later, 50 μL of 2.0 Luminescent Cell Viability Reagent (Catalog No. G7573, Promega). The plate was shaken at 450 rpm for 3 minutes, followed by incubation for 10 minutes without shaking, and luminescence was measured on an Envision plate reader (Equipment No. 2104, PerkinElmer) with an integration time of 250 milliseconds per well. The curve of luminescence versus ADC concentration (μM) was fitted using GraphPad Prism software.

[0690] The results of in vitro cytotoxicity assays were Figure 3 In the Figure, exemplary anti-HER2 ADCs (ADC-2-1, ADC-2-2, and ADC-3) showed activity against HER-positive NCI-N87 cells ( Figure 3 A) and SKBR-3 cells ( Figure 3 B) showed better cytotoxic activity than the positive control ADC DS-8201a and T-DM1, while in HER2-negative MCF-7 cells ( Figure 3 In C), cytotoxic activity was observed only at very high concentrations of ADC (particularly ADC-3). The control native antibody trastuzumab had only limited cytotoxic activity even at high concentrations.

[0691] Example 21. Cell-based binding of anti-Trop2 antibodies and anti-Trop2 ADCs to Trop2-expressing tumor cells

[0692] Trop2 positive SKBR-3 cells (breast cancer, ATCC No. HTB30) were used for in vitro cell binding assessment of exemplary anti-Trop2 ADCs ADC-4, ADC-5, ADC-6, ADC-7, ADC-8 and ADC-9. Human native IgG1 antibody dabigastatin from DS-1062 of Daiichi Sankyo Co., Ltd. was used for free antibody control. The protocol for binding analysis was the same as in Example 18.

[0693] Briefly, SKBR-3 cells were maintained in McCoy's 5a medium (Cat. No. 30-2007, ATCC) supplemented with 10% FBS (Cat. No. FSP500, Ecosine Biotech), 2 mM L-glutamine (Cat. No. 25030081, Thermo Fisher Scientific) and 1% penicillin-streptomycin solution (BL505A, Biosharp Life Sciences, Inc.). The cells were incubated at 37°C in a 5% CO 2 100 μL of 2×10 cells / well was added to each well of a 96-well cell culture plate. 6 Cells at 100 cells / ml were incubated on ice for one hour with Trop2 antibodies Trop2-1, Trop2-2 and Dabotuzumab and 5-fold serial dilutions of exemplary anti-Trop2 ADC starting from 100 nM to 0.00128 nM. After washing twice with 200 microliters / well of FACS buffer, the cells were incubated on ice for 30 to 60 minutes with 100 μL of 1× secondary antibody PE anti-human IgG Fc antibody (Catalog No. 366904, Baijin Biotech). The cells were washed twice with 200 μL FACS buffer and resuspended with 200 μL FACS buffer for further detection by CytoFlex cytometry (Beckman Coulter). The data were analyzed with the built-in analysis software of CytoFlex.

[0694] exist Figure 4 The cell binding results are shown in . The exemplary anti-Trop2 ADC has comparable binding capacity to its native Trop2 antibodies Trop2-1 and Trop2-2 and can effectively bind to SKBR-3 cells, which indicates that the binding has minimal effect on the binding activity of the target.

[0695] Example 22. Analysis of Antibody-Mediated Internalization in Trop2-Expressing Tumor Cells Using FACS

[0696] Trop2 positive cell line SKBR-3 cells (breast cancer, ATCC No. HTB30) were used for the following internalization analysis. The culture conditions were the same as in Example 21.

[0697] The cells were harvested with 0.25% trypsin / EDTA (T1320, Solebro) and the cell concentration was adjusted to 4×10 cells / mL with 1×PBS containing 1% FBS buffer. 6Cells / mL. Then 100 μL of cells per well were seeded in a 96-well cell culture plate and incubated with Trop2 antibodies Trop2-1, Trop2-2, daptomazolidinone and gosartumomab and exemplary anti-Trop2 ADCs (ADC-4, ADC-5, ADC-6, ADC-7, ADC-8 and ADC-9) at a final concentration of 100 nM for seven time points (0, 0.5 hours, 2 hours, 4 hours, 8 hours, 16 hours and 24 hours). The mixture was incubated at 4°C in a 5% CO 2 At specific time points, the cells were immediately washed twice and suspended in 100 μL of cold wash buffer containing 2% paraformaldehyde for 30 minutes. The cells were washed twice and then incubated with 1×PE anti-human IgG Fc antibody (Catalog No. 366904, Biogen Biotech) at 4°C and 5% CO 2 The internalization rate at several time points was calculated with the MFI value at a specific time point relative to the missing rate of the value at 0 hours. The internalization rate was plotted with Graphpad Prism software relative to the curve of incubation time (hours).

[0698] The results are shown in Table 3 and Figure 5 The exemplary anti-Trop2 ADC can effectively mediate internalization into SKBR-3 cells, with an internalization rate of >60% within 8 hours. The internalization results indicate that binding may not alter target-mediated internalization.

[0699]

[0700] Example 23: In vitro cytotoxicity analysis of anti-Trop2 ADC against Trop2-expressing cancer cells

[0701] The cytotoxicity of exemplary free linker-payloads (Compound 2, Compound 3, and Compound 4) as positive controls, free Trop2 antibodies (Trop2-1, Trop2-2, daptomazumab, and gosatuzumab), exemplary anti-Trop2 ADCs (ADC-4, ADC-5, ADC-6, ADC-7, ADC-8, and ADC-9), and Trodelvy (an FDA-approved anti-Trop2 ADC) was evaluated against SKBR-3 cells (breast cancer, ATCC No. HTB30), NCI-N87 cells (gastric cancer, ATCC No. CRL-5822), MCF-7 cells (breast cancer, ATCC No. HTB-22), and MDA-MB-468 cells (breast cancer, ATCC No. HTB-132).

[0702] Briefly, 5,000 cells / well were seeded in 90 μL of basal medium (McCoy's 5a medium for SKBR-3 cells, RPMI-1640 medium for NCI-N87 cells, EMEM medium for MCF-7 cells, and Leibovitz's L-15 medium for MDA-MB-468 cells) containing 10% FBS, 1% penicillin-streptomycin solution (BL505A, Biosharp Life Sciences, Inc.), and 2 mM L-glutamine (Catalog No. 25030081, Thermo Fisher Scientific) in a white clear 96-well assay plate (Catalog No. 6005182, PerkinElmer) (excluding the edge wells containing only medium) and incubated at 37°C in the presence of 5% CO 2 After overnight incubation, the exemplary anti-Trop2 ADC, Todawi, free Trop2 antibody, and free linker-payload were added to individual wells at 10 μL of 10× concentrated test substance ranging from 100 nM to 0.015 nM. After another 72 hours of incubation, the plate was removed from the incubator and allowed to equilibrate to room temperature. After approximately 30 minutes, 50 μL of 2.0 Luminescent Cell Viability Reagent (Catalog No. G7573, Promega). The plate was shaken at 450 rpm for 3 minutes, followed by incubation for 10 minutes without shaking, and luminescence was measured on an Envision plate reader (Equipment No. 2104, PerkinElmer) with an integration time of 250 milliseconds per well. GraphPad Prism software was used to fit the curve of luminescence versus ADC concentration (μM).

[0703] The results of in vitro cytotoxicity assays were Figure 6and shown in Table 4. All exemplary anti-Trop2 ADCs showed cytotoxic activity against the test cell lines. ADC-6, ADC-9 and ADC-5 showed better cytotoxic activity against the test cell lines than Tondawei, and ADC-4 showed target cell toxicity against the test cell lines comparable to Tondawei. Free linker-payload compound 2-4 had only limited cytotoxic activity even at high concentrations. In addition, natural antibodies gosartuzumab, Trop2-1 and Trop2-2 did not show target cell killing effects on these four Trop-2 expressing cells.

[0704]

[0705] Example 24: In vivo therapeutic efficacy of anti-Trop2 ADC in the NCI-N87 cell xenograft mouse model

[0706] On day 0, NCI-N87 cells (1×10 7 , 1:1 matrix gel) were injected subcutaneously into the right upper flank of 6- to 8-week-old BALB / c female nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). On day 6, when the average tumor volume was 178 mm 3 At the same time, mice were randomly divided into 8 groups (n=6). Treatment was started on the same day (defined as PG-D0). Mice in the Todawi group received 5.0 mg / kg (intravenous, BIW) for 3 weeks; mice in the ADC-5 treatment group were injected with 1.0, 2.5 and 5.0 mg / kg (intravenous, BIW) ADC-5 for 3 weeks; mice in the ADC-6 treatment group were injected (iv) 1.0 and 2.5 mg / kg ADC-6 for three doses per week (QW), or 5.0 mg / kg (single dose on the PG-D0 day). Tumor volume and body weight were measured and recorded twice a week. At the termination of the 28th day (defined as PG-D28), the tumor weight was dissected and measured. Tumor growth inhibition rate (%) was calculated as (TV CR -TV TR ) / TV CR (%), among which TV CR and TV TR The relative tumor volumes of the vehicle control group and the experimental group are shown respectively. Once the tumor grows larger than 2,500 mm 3 , mice were euthanized and considered dead from disease. Statistical analysis by one-way ANOVA was performed using GraphPad Prism 6.0 software.

[0707] Figure 7 The results are shown in Table 5. All three ADC-5 treatment groups (1.0, 2.5 and 5.0 mg / kg, BIW*3) showed better anti-tumor responses than mice in the treatment group of TuoDaWei (5.0 mg / kg, BIW*3). In addition, the mice in the ADC-5 treatment group showed complete tumor response (CR) at medium (2.5 mg / kg, BIW) and high (5.0 mg / kg, BIW) doses, wherein the tumor growth inhibition (TGI) at the 28th day was 100.37% and 106.43% respectively. For the ADC-6 treatment group, the results of medium doses (2.5 mg / kg, QW*3) and high doses (5.0 mg / kg, single dose) under low frequency and / or dosage showed that ADC-6 also showed great tumor inhibition efficacy, but low doses (1.0 mg / kg, QW*3) of ADC-6 showed slightly weaker anti-tumor efficacy due to frequency and dosage lower than TuoDaWei. At the same time, the results of body weight changes showed that ADC-5 and ADC-6 had no obvious potential toxicity risk in this in vivo efficacy study, which further provided strong evidence for the extended therapeutic window of ADC-5 and ADC-6 compared with Tondawi ( Figure 7 B).

[0708] Table 5. Tumor growth inhibition rate (TGI, %) of exemplary anti-Trop2 ADC in gastric cancer NCI-N87 cell xenograft mouse model

[0709]

[0710]

[0711] The foregoing description is considered to be only an illustration of the principles of the present disclosure. Further, since many modifications and variations will be apparent to those skilled in the art, it is not desirable to limit the present invention to the exact configuration and process shown as described above. Therefore, all suitable modifications and equivalents may be considered to fall within the scope of the present invention as defined by the appended claims.

[0712] The words “comprise”, “comprising”, “include”, “including” and “includes” when used in this specification and the following claims are intended to specify the presence of stated features, integers, components or steps, but do not exclude the presence or addition of one or more other features, integers, components, steps or groups thereof.

Claims

1. A polymer scaffold of formula (I) for binding to a targeting moiety: in: The polymer scaffold comprises linear polyglycerol; L is a functional group W that can form a covalent bond with the targeting moiety. p The connecting part; M a is an extending group connecting L to the -NH- moiety; Each G 1 Independently p a functional group attached to the linear polyglycerol; Each L P Independently, the therapeutic agent moieties D and G 1 Drug release mechanism between; Each D is independently a therapeutic agent moiety; Each G 2 are independently functional groups capable of being transformed into a charged state; n is an integer from 0 to 1000; m is an integer from 0 to 1000; p is an integer from 0 to 1000; and q is an integer from 0 to 1000.

2. The polymer stent according to claim 1, wherein W p Capable of reacting with a functional group on the targeting moiety via a click reaction.

3. The polymer stent according to claim 2, wherein W p Selected from the group consisting of:

4. The polymer stent according to claim 1, wherein W p Capable of reacting with an amino acid on the targeting moiety.

5. The polymer scaffold of claim 4, wherein the amino acid is a natural amino acid, an unnatural amino acid, or a combination thereof.

6. The polymer scaffold of claim 5, wherein the natural amino acids include cysteine, lysine, tyrosine, aspartic acid, and glutamic acid.

7. The polymer stent of claim 4, wherein W p Capable of reacting with one or more cysteines on the targeting moiety.

8. The polymer stent according to claim 7, wherein W p Selected from the group consisting of: Where R 1 is a sulfur protecting group, each R 2 is independently a leaving group.

9. The polymer stent according to claim 8, wherein each R 2 are independently selected from halogen or R 2a C(O)O-, where R 2a is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

10. The polymer stent of claim 4, wherein W p Capable of reacting with one or more lysines on the targeting moiety, wherein each W p Independently selected from the group consisting of:

11. The polymer stent of claim 4, wherein W p Capable of reacting with one or more unnatural amino acids on the targeting moiety.

12. The polymer stent of claim 11, wherein W p Selected from:

13. The polymer stent of claim 1, wherein M a Selected from the group consisting of: R 3 , Wherein * is the site of covalent attachment to L, ** is the site of covalent attachment to -NH- moiety, R 3 C 1-10 Alkyl, C 1-10 Heteroalkyl, C 3-8 Cycloalkyl, -O-(C 1-8 alkyl)-, aryl, -C 1-10 Alkyl-aryl-, -aryl-C 1-10 Alkyl-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-, -(C 3-8 Cycloalkyl-C 1-10 alkyl)-, 4- to 14-membered heterocycloalkyl, -C 1-10 Alkyl-(4- to 14-membered heterocycloalkyl)-, -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-, -C 1-10 Alkyl-C(=O)-, -C 1-10 Heteroalkyl-C(=O)-, -C 3-8 Cycloalkyl-C(=O)-, -O-(C 1-8 alkyl)-C(=O)-, -aryl-C(=O)-, -C 1-10 Alkyl-aryl-C(=O)-, -aryl-C 1-10 Alkyl-C(=O)-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-C(=O)-, -(C 3-8 Cycloalkyl)-C 1-10 alkyl-C(═O)-, -4- to 14-membered heterocycloalkyl-C═(O)-, -C 1-10 alkyl-(4- to 14-membered heterocycloalkyl)-C(═O)-, -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-C(=O)-, -C 1-10 Alkyl-NH-, -C 1-10 Heteroalkyl-NH-, -C 3-8 Cycloalkyl-NH-, -O-(C 1-8 Alkyl)-NH-, -aryl-NH-, -C 1-10 Alkyl-aryl-NH-, -aryl-C 1-10 Alkyl-NH-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-NH-, -(C 3-8 Cycloalkyl)-C 1-10 Alkyl-NH-, -4- to 14-membered heterocycloalkyl-NH-, -C 1-10 Alkyl-(4- to 14-membered heterocycloalkyl)-NH-, -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-NH-, -C 1-10 Alkyl-S-, -C 1-10 Heteroalkyl-S-, -C 3-8 Cycloalkyl-S-, -OC 1-8 Alkyl-S-, -aryl-S-, -C 1-10 Alkyl-aryl-S-, -aryl-C 1-10 Alkyl-S-, -C 1-10 Alkyl-(C 3-8 Cycloalkyl)-S-, -(C 3-8 Cycloalkyl)-C 1-10 Alkyl-S-, -4- to 14-membered heterocycloalkyl-S-, -C 1-10 -(4- to 14-membered heterocycloalkyl)-S- or -(4- to 14-membered heterocycloalkyl)-C 1-10 Alkyl-S-; Each R 4 are independently hydrogen, C 1-6 Alkyl, C 6-10 Aryl, C 3-8 Cycloalkyl, -COOH or -COO-C 1-6 alkyl; R 5 -C(O)-NR 5a or -NR 5a -C(O)-; R 5a For hydrogen, C 1-6 Alkyl, C 6-10 Aryl, C 3-8 Cycloalkyl, -COOH or -COO-C 1-6 alkyl; R 6 is a bond or -NR 6a -(CR 6b R 6c )-C(O)-; R 6a For hydrogen, C 1-6 Alkyl, C 6-10 Aryl, C 3-8 Cycloalkyl, -COOH or -COO-C 1-6 alkyl; Each R 6b and R 6c are independently hydrogen, C 1-6 Alkyl, C 6-10 Aryl, hydroxylated C 6-10 Aryl, polyhydroxylated C 6-10 Aryl, 5- to 12-membered heterocycloalkyl, C 3-8 Cycloalkyl, hydroxylated C 3-8 Cycloalkyl, polyhydroxylated C 3-8 Cycloalkyl, or the side chain of a natural or unnatural amino acid; Each n 1 are independently an integer from 0 to 6; n 2 is an integer from 0 to 8; Each n 3 are independently an integer from 1 to 6; and n 4 is an integer from 1 to 4.

14. The polymer stent of claim 13, wherein M a Selected from the group consisting of:

15. The polymer stent of claim 1, wherein G 1 Selected from the group consisting of: Where * is covalently linked to L P The site of each R 7 are independently selected from a direct bond, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, and R 7a is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl moieties.

16. The polymer stent of claim 1, wherein G 1 Selected from the group consisting of: Where R 7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl moieties.

17. The polymer stent of claim 1, wherein each L p Independently contains unstable structures.

18. The polymer scaffold of claim 17, wherein the labile structure is selected from a hydrolytically labile structure or an enzymatically labile structure.

19. The polymer stent of claim 18, wherein the hydrolytically unstable structure is selected from the group consisting of: Where * is covalently linked to G 1 The site of covalent bonding to D, R 8 is selected from hydrogen, alkyl or aryl, R 9 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

20. The polymer stent of claim 18, wherein the hydrolytically unstable structure is selected from the group consisting of: Where * is covalently linked to G 1 The site of covalent bonding to D, R 8 is selected from hydrogen, alkyl or aryl, R 9 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

21. The polymer stent of claim 20, wherein G 1 for Where * is covalently linked to L P The site, R 7 It is an alkyl group.

22. The polymer stent of claim 21, wherein -G 1 -L P -D is 23. The polymer scaffold of claim 17, wherein the enzyme labile structure is labile to an enzyme selected from cathepsin B, phosphatase, sulfatase, or glucuronidase.

24. The polymer scaffold of claim 19, wherein the enzyme labile structure is labile to cathepsin B and is selected from -Z- or wherein Z is a substrate for cathepsin B comprising 2 to 4 amino acids.

25. The polymer stent of claim 24, wherein G 1 for Where * is covalently linked to L P The site, R 7 It is an alkyl group.

26. The polymer stent of claim 24, wherein -G 1 -L P -D is 27. The polymer scaffold of claim 23, wherein the enzyme-labile structure is unstable to glucuronidase and is Where * is covalently linked to G 1 The site of covalent bonding to D.

28. The polymer stent of claim 27, wherein G 1 for Where R 7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

29. The polymer stent of claim 27, wherein G 1 for Where * is covalently linked to L P The site, R 7 It is an alkyl group.

30. The polymer stent of claim 27, wherein -L P -D is selected from:

31. The polymer stent of claim 27, wherein -G 1 -L P -D is selected from:

32. The polymer scaffold of claim 23, wherein the enzyme-labile structure is unstable to phosphatases and is selected from Where * is covalently linked to G 1 The site of covalent bonding to D, R 10 and R 11 Each of is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

33. The polymer stent of claim 32, wherein G 1 for 34. The polymer stent of claim 33, wherein -G 1 -L P -D Select from the group consisting of:

35. The polymer scaffold of claim 23, wherein the enzyme-labile structure is unstable to sulfatase and is Where * is covalently linked to G 1 The site of covalent bonding to D, R 12 and R 13 Each of is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

36. The polymer stent of claim 35, wherein G 1 for Where R 7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

37. The polymer stent of claim 35, wherein G 1 for Where * is covalently linked to L P The site, R 7 It is an alkyl group.

38. The polymer stent of claim 37, wherein -G 1 -L P -D means:

39. The polymer stent of claim 1, wherein n is an integer from 1 to 100; m is an integer from 1 to 100; and p is an integer from 1 to 50.

40. The polymeric scaffold of claim 1, wherein the therapeutic agent has anti-proliferative activity on a target cell or target pathway.

41. The polymer scaffold of claim 40, wherein the anti-proliferative activity is selected from cytostatic activity and / or cytotoxic activity.

42. The polymeric stent of claim 1, wherein the therapeutic agent is selected from anticancer substances, cytotoxic drugs, radionuclides, vitamins, anti-AIDS substances, antibiotics, immunosuppressants, immunomodulatory compounds, therapeutic RNA, antiviral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, sedatives, anticonvulsants, muscle relaxants and anti-Parkinson substances, antispasmodics and muscle contractants (including channel blockers, miotics and anticholinergics), antiglaucoma compounds, antiparasitic compounds and / or antiprotozoal compounds, regulators of cell-extracellular matrix interactions (including cell growth inhibitors and anti-adhesion molecules), vasodilators, inhibitors of DNA, RNA or protein synthesis, antihypertensives, analgesics, antipyretics, steroidal anti-inflammatory agents and nonsteroidal anti-inflammatory agents, antiangiogenic factors, antisecretory factors, anticoagulants and / or antithrombins, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotic substances, antiemetics, imaging agents.

43. The polymeric stent of claim 1, wherein the therapeutic agent comprises an amino acid based molecule.

44. The polymer scaffold of claim 43, wherein the amino acid-based molecule comprises a peptide, a polypeptide, an enzyme, an antibody, an immunoglobulin, or a functional fragment thereof.

45. The polymeric stent of claim 1, wherein the therapeutic agent has a chemically reactive group.

46. ​​The polymer stent of claim 45, wherein the chemically reactive groups include -COOH, primary amines, secondary amines, -NHR, -OH, -SH, -C(O)H, C(O)R 14 、-C(O)NHR 15 、C(S)OH、-S(O)2OR 15 、-P(O)2OR 15 , -CN, -NC or -ONO, where R 14 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, and R 15 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

47. The polymer stent of claim 1, wherein G 2 Selected from the group consisting of: Each R 16 are independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

48. The polymer stent of claim 1, wherein G 2 for 49. A polymer scaffold of formula (II): in, The polymer scaffold comprises linear polyglycerol; L is a functional group W that can form a covalent bond with the targeting moiety. p The connecting part; M a is an extending group connecting L to the -NH- moiety; Each G 2 are independently functional groups capable of being transformed into a charged state; Each G 3 independently comprising a functional group capable of reacting with a reactive group in a drug release mechanism to thereby link the drug release mechanism to the linear polyglycerol; n is an integer from 0 to 1000; m is an integer from 0 to 1000; p is an integer from 0 to 1000; and q is an integer from 0 to 1000.

50. The polymer stent of claim 49, wherein n is 2; m is 2; and p is 2.

51. The polymeric stent of claim 49, wherein q is an integer from 3 to 5.

52. The polymer stent of claim 49, wherein G 2 and G 3 Independently selected from the group consisting of: Each R 17 are independently selected from a direct bond, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group.

53. The polymer scaffold of claim 49, having a structure of Formula (IIa) or Formula (IIb):

54. The polymer scaffold of claim 52, having a structure of Formula (IIc) or Formula (IId):

55. A polymer scaffold of formula (III): in, The polymer scaffold comprises linear polyglycerol; Each G 2 are independently functional groups capable of being transformed into a charged state; Each G 3 independently comprising a functional group capable of reacting with a reactive group in a drug release mechanism to thereby link the drug release mechanism to the linear polyglycerol; n is an integer from 0 to 1000; m is an integer from 0 to 1000; p is an integer from 0 to 1000; and q is an integer from 0 to 1000.

56. The polymer stent of claim 55, wherein n is 2; m is 2; and p is 2.

57. The polymeric stent of claim 55, wherein q is an integer from 3 to 5.

58. The polymer stent of claim 55, wherein G 2 and G 3 Independently selected from the group consisting of: Each R 17 are independently selected from a direct bond, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group.

59. The polymer scaffold of claim 55, having a structure of Formula (IIIa) or Formula (IIIb):

60. The polymer stent of claim 58, having a structure of Formula (IIIc) or (IIId):

61. The polymer stent of claim 55, having a weight average molecular weight Mw of 1 to 100,000.

62. The polymer stent of claim 55, having a weight average molecular weight Mw of 10,000 to 15,000.

63. The polymer stent of claim 55, having a weight average molecular weight Mw of 5,000 to 10,000.

64. The polymeric stent of claim 55, having a PDI of less than 1.

5.

65. A polymer scaffold of formula (IV): in, The polymer scaffold comprises linear polyglycerol; Each L a Independently for attaching the targeting moiety to M a The divalent part of Each M a Independently a an extending group attached to the -NH- moiety; Each G 1 Independently p a functional group attached to the linear polyglycerol; Each L P Independently, the therapeutic agent fragments D and G 1 Drug release mechanism between; Each D is independently a fragment of a therapeutic agent; Each G 2 are independently functional groups capable of being transformed into a charged state; n is an integer from 0 to 1000; m is an integer from 0 to 1000; p is an integer from 0 to 1000; q is an integer from 0 to 1000; and s is an integer from 1 to 8.

66. The polymeric scaffold of claim 65, wherein the targeting moiety is an antibody and / or fragment thereof.

67. The polymer scaffold of claim 65, wherein the targeting moiety is antibody IgGl, antibody IgG2, antibody IgG3, and antibody IgG4.

68. The polymer scaffold of claim 65, wherein the targeting moiety is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies.

69. A pharmaceutical composition comprising one or more of the polymeric scaffolds according to any one of claims 1 to 48 and 65 to 68 and an acceptable carrier.

70. A method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polymer scaffold according to any one of claims 1 to 48 and 65 to 68 or a pharmaceutical composition according to claim 69.

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