Polymer-drug conjugates

Abstract

The polymer scaffolds useful for conjugating to targeting moieties can form targeting moiety-polymer-drug conjugates. The targeting moiety-polymer-drug conjugates are prepared from the polymer scaffolds. The compositions include the conjugates. Methods of their preparation and methods of treating various disorders using the conjugates or compositions thereof.

Description

[Technical field]

[0001]

[0001] The present disclosure relates generally to compounds, conjugates comprising those compounds, pharmaceutical compositions thereof, and methods of treating diseases or disorders using the conjugates or pharmaceutical compositions. [Background technology]

[0002]

[0002] Traditionally, therapeutic agents, mainly consisting of small molecules, are delivered to the body via oral / GI absorption or systemic injection, followed by blood circulation to the site of action. However, many challenges remain to be addressed. For example, many therapeutic agents generally exhibit limited or otherwise reduced efficacy and therapeutic effect, either because they are subject to partial degradation or accumulation in non-target tissues, or both, before reaching the desired target in the body. Summary of the Invention

[0003]

[0003] Thus, there is a need to deliver therapeutic agents intact to specific target areas of the body through a system that can stabilize drugs, control the movement of the therapeutic agent in vivo, and achieve maximum cytotoxicity for the therapeutic agent.

[0004]

[0004] The present disclosure relates to a polymer scaffold delivery system that exhibits high drug loading and strong binding to target antigens, thereby efficiently delivering and releasing drugs to target sites. The present disclosure also relates to a polymer scaffold that is useful for conjugating with a targeting moiety to obtain the polymer scaffold delivery system.

[0005] In one aspect, the present disclosure provides a polymer scaffold useful for conjugating a targeting moiety, the polymer scaffold having formula (I): [ka] where: The polymer scaffold comprises linear polyglycerol; L is a functional group W capable of forming a covalent bond with a targeting moiety. p is a linking moiety comprising M a is a stretcher connecting L to the -NH- moiety, each G 1 is independent, L p is a functional group that connects the linear polyglycerol Each L P are independently therapeutic moieties D and G 1 This is the drug release mechanism between each D is independently a therapeutic moiety; each G 2 are independently functional groups that can be converted to 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.

[0006] In another aspect, the present disclosure provides a polymer scaffold of formula (II): [ka] where: The polymer scaffold comprises linear polyglycerol; each G 2 are independently functional groups that can be converted to a charged state, each G 3 independently comprises a functional group capable of reacting with a reactive group in the drug release mechanism to connect 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; q is an integer from 0 to 1000.

[0007] In another aspect, the present disclosure provides a polymer scaffold of formula (III): [ka] where: The polymer scaffold comprises linear polyglycerol; each G 2 are independently functional groups that can be converted to a charged state, each G 3 independently comprises a functional group capable of reacting with a reactive group in the drug release mechanism to connect 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; q is an integer from 0 to 1000.

[0008] In another aspect, the present disclosure provides a polymer scaffold of formula (IV): [ka] where: The polymer scaffold comprises linear polyglycerol; Each L a independently, the targeting part is M a is a bivalent moiety that connects to Each M a is independent, L a is a stretcher that connects the -NH- moiety to each G 1 is independent, L p is a functional group that connects the linear polyglycerol Each L P are independently therapeutic moieties D and G 1 This is the drug release mechanism between each D is independently a therapeutic moiety; each G 2 are independently functional groups that can be converted to 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; s is an integer from 1 to 8.

[0009]

[0009] In a further aspect, the present disclosure provides a pharmaceutical composition comprising a polymer scaffold or conjugate described herein and a pharma ceutically acceptable carrier.

[0010]

[0010] In another aspect, the present disclosure provides a method for treating a disease in a subject in need thereof, the method 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]

[0011] [Figure 1] FIG. 1 shows cell-based binding of trastuzumab and test anti-HER2 ADCs to HER2-positive SKBR-3 cells (A) and NCI-N87 cells (B). [Diagram 2]

[0012] FIG. 1 shows internalization of test anti-HER2 ADCs into HER2-positive SKBR-3 cells (A) and NCI-N87 cells (B) over 0 to 24 hours. [Diagram 3]

[0013] FIG. 1 shows the in vitro cytotoxicity of tested anti-HER2 ADCs against HER2-expressing SKBR-3 cells (A) and NCI-N87 cells (B), and HER2-negative MCF-7 cells (C). [Figure 4]

[0014] FIG. 1 shows cell-based binding of Trop2 antibodies Trop2-1, Trop2-2, and datopotamab, as well as exemplary anti-Trop2 ADCs, to Trop2-positive SKBR-3 cells. [Diagram 5]

[0015] FIG. 1 shows internalization of tested anti-Trop2 ADCs into Trop2-positive SKBR-3 cells. [Figure 6]

[0016] Figure 1 shows in vitro cytotoxicity of 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). [Figure 7]

[0017] Figure 1 shows that ADC-5 and ADC-6 are effective in inhibiting tumor growth in BALB / c nude mice with established 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 28 days after administration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012]

[0018] Reference will now be made in detail to certain embodiments of the present invention, examples of which are illustrated in the accompanying structures and formulas. The present invention will be described in connection with the enumerated embodiments, but it will be understood that they are not intended to limit the present invention to those embodiments. On the contrary, the present invention is intended to cover all changes, modifications, and equivalents, which may be included in the scope of the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. In the event that one or more of the incorporated literature and similar materials, including but not limited to defined terms, term usage, described techniques, etc., differ or conflict with this application, this application will control.

[0013]

[0019] It will be appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, while being, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

[0014]

[0020] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms, unless the context clearly dictates otherwise. In this specification and the claims that follow, reference will be made to a number of terms, which will be defined to have the following meanings, unless a contrary intention is apparent.

[0015] definition

[0021] The definitions of specific functional groups and chemical terms are described in more detail below.For the purpose of this disclosure, chemical elements are identified according to the Periodic Table of Elements, CAS edition, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are generally defined as described therein.In addition, the general principles of organic chemistry and specific functional moieties and reactivity are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th edition, 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 edition, Cambridge University Press, Cambridge, 1987, the entire contents of each of which are incorporated herein by reference.

[0016]

[0022] At various points in this disclosure, linking substituents are described. Where a structure expressly requires a linking group, it is understood that the Markush variable listed for that group is the linking group. For example, where a structure requires a linking group and the Markush group definition for that variable lists "alkyl," it is understood that "alkyl" represents a linking alkylene group.

[0017]

[0023] Any variable part (e.g. R i When R occurs more than one time 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, a group may have 0 to 2 R i When a moiety is indicated to be substituted, the group may optionally be substituted with up to two R i may be substituted with a moiety, R i In each occurrence, R i Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0018]

[0024] As used herein, a dash "-" used for convenience before or at the end of a chemical group indicates the attachment point of the substituent. For example, -OH is attached via a carbon atom, and chemical groups can be drawn with or without one or more dashes without losing their normal meaning. A wavy line drawn through a straight line in a structure indicates the attachment point of the group. No direction is indicated or implied by the order in which chemical groups are written or named, unless chemically or structurally required. As used herein, a solid line emanating from the center of a ring indicates that the attachment point of a substituent on the ring can be any ring atom. When a substituent is listed without indicating the intermediate atom through which such a substituent is attached to the remainder of a compound of a given formula, such a substituent can be attached via any atom in such formula. Also, combinations of substituents and / or variables are permitted, but only if such combinations result in stable compounds.

[0019]

[0025] Any variable part (e.g. R i When R occurs more than one time 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, a group may have 0 to 2 R i When a moiety is indicated to be substituted, the group may optionally be substituted with up to two R imay be substituted with a moiety, R i In each occurrence, R i Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0020]

[0026] When used in the context of a numerical value, the term "about" is meant to include a population or range of values. For example, "about X", where X is a numerical value, includes a range of values ​​that are ±20%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2% or ±0.1% of X. In one embodiment, the term "about" refers to a range of values ​​that are 5% greater or less than a particular value. In another embodiment, the term "about" refers to a range of values ​​that are 2% greater or less than a particular value. In another embodiment, the term "about" refers to a range of values ​​that are 1% greater or less than a particular value.

[0021]

[0027] The recitation of a range of values ​​is intended to serve as a shorthand way of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. Ranges used herein include the two endpoints of the range, unless otherwise specified. For example, both the expressions "n is an integer between 1 and 6" and "n is an integer from 1 to 6" mean "x is 1, 2, 3, 4, 5 or 6."

[0022]

[0028] As used herein, "C i-j " refers to a range of carbon numbers where i and j are integers and the range includes the endpoints (e.g., i and j) and each integer point between, where j is greater than i. For example, C 1~6 represents a range of 1 to 6 carbon atoms, including 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, and 6 carbon atoms. In some embodiments, "C 1~12" denotes 1 to 12, particularly 1 to 10, particularly 1 to 8, particularly 1 to 6, particularly 1 to 5, particularly 1 to 4, particularly 1 to 3 or particularly 1 to 2 carbon atoms. In an analogous manner, the term "m- to n-membered" ring refers to a ring containing m to n atoms, where m and n are integers and n is greater than m.

[0023]

[0029] As used herein, the term "aliphatic" includes both saturated and unsaturated, straight-chain (i.e., unbranched) or branched aliphatic hydrocarbons, optionally substituted with one or more functional groups. As will be appreciated by those of skill in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, and alkynyl moieties.

[0024]

[0030] As used herein, the term "alkyl," whether used as part of another term or independently, refers to a saturated straight or branched chain hydrocarbon radical, which may be optionally substituted independently with one or more substituents described below. i~j The term "alkyl" refers to a straight or branched chain alkyl having i to j carbon atoms. For example, an alkyl group may contain 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. 1~6 Examples of "alkyl" include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, s-butyl, t-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.

[0025]

[0031] As used herein, the term "alkenyl," whether used as part of another term or independently, refers to a straight or branched chain hydrocarbon radical having at least one carbon-carbon double bond, which may be optionally substituted with one or more substituents described herein, and may include radicals having "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations. In some embodiments, an alkenyl group contains 2-12 carbon atoms. In some embodiments, an alkenyl group contains 2-11 carbon atoms. In some embodiments, an alkenyl group contains 2-11 carbon atoms, 2-10 carbon atoms, 2-9 carbon atoms, 2-8 carbon atoms, 2-7 carbon atoms, 2-6 carbon atoms, 2-5 carbon atoms, 2-4 carbon atoms, 2-3 carbon atoms, and in some embodiments, an alkenyl group contains 2 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethylenyl (ie, vinyl), propenyl (allyl), butenyl, pentenyl, 1-methyl-2 buten-1-yl, 5-hexenyl, and the like.

[0026]

[0032] As used herein, the term "alkynyl", whether used as part of another term or independently, refers to a linear or branched hydrocarbon radical having at least one carbon-carbon triple bond, and may be optionally independently substituted with one or more substituents described herein. In some embodiments, an alkenyl group contains 2-12 carbon atoms. In some embodiments, an alkynyl group contains 2-11 carbon atoms. In some embodiments, an alkynyl group contains 2-11 carbon atoms, 2-10 carbon atoms, 2-9 carbon atoms, 2-8 carbon atoms, 2-7 carbon atoms, 2-6 carbon atoms, 2-5 carbon atoms, 2-4 carbon atoms, 2-3 carbon atoms, and in some embodiments, an alkynyl group contains 2 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and the like.

[0027]

[0033] As used herein, the term "amino" refers to -NR a R b R refers to the a and R b is independently selected from the group consisting of hydrogen, alkyl, haloalkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl, each of which is optionally substituted.

[0028]

[0034] As used herein, the term "aryl", whether used as part of another term or independently, refers to monocyclic and polycyclic ring systems having a total of 5-20 ring members, where at least one ring of the system is aromatic and each ring of the system contains 3-12 ring members. Examples of "aryl" include, but are not limited to, phenyl, biphenyl, naphthyl, anthracyl, and the like, which may have one or more substituents. The term "aryl", as it is used herein, also includes groups in which an aromatic ring is fused to one or more additional rings. In the case of polycyclic systems, only one of the rings need be aromatic (e.g., 2,3-dihydroindole), although all rings may be aromatic (e.g., quinoline). The second ring may also be fused or bridged. Examples of polycyclic aryl include, but are not limited to, benzofuranyl, indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

[0029]

[0035] As used herein, the term "cycloalkyl", whether used as part of another term or independently, refers to monovalent non-aromatic, saturated or partially unsaturated, monocyclic and polycyclic ring systems in which all ring atoms are carbon and which contain at least three ring-forming carbon atoms. In some embodiments, a cycloalkyl group can contain 3-12 ring-forming carbon atoms, 3-10 ring-forming carbon atoms, 3-9 ring-forming carbon atoms, 3-8 ring-forming carbon atoms, 3-7 ring-forming carbon atoms, 3-6 ring-forming carbon atoms, 3-5 ring-forming carbon atoms, 4-12 ring-forming carbon atoms, 4-10 ring-forming carbon atoms, 4-9 ring-forming carbon atoms, 4-8 ring-forming carbon atoms, 4-7 ring-forming carbon atoms, 4-6 ring-forming carbon atoms, 4-5 ring-forming carbon atoms. A cycloalkyl group can be saturated or partially unsaturated. In some embodiments, a cycloalkyl group can be a saturated cyclic alkyl group. In some embodiments, a cycloalkyl group can be a partially unsaturated cyclic alkyl group containing at least one double or triple bond in its ring system.

[0030]

[0036] In some embodiments, the cycloalkyl group can be a saturated or partially 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.

[0031]

[0037] In some embodiments, the cycloalkyl group may be a saturated or partially unsaturated polycyclic (e.g., bicyclic and tricyclic) carbocyclic ring system, which may be arranged as a fused, spiro or bridged ring system. As used herein, the term "fused ring" refers to a ring system having two rings that share two adjacent atoms, the term "spiro ring" refers to a ring system in which two rings are connected through a single common atom, and the term "bridged ring" refers to a ring system in which two rings share three or more atoms. Examples of fused carbocyclic compounds include, but are not limited to, naphthyl, benzopyrenyl, anthracenyl, acenaphthenyl, fluorenyl, and the like. Examples of spiro carbocyclic compounds include, but are not limited to, spiro[5.5]undecanyl, spiro-pentadienyl, spiro[3.6]-decanyl, and the like. Examples of bridged carbocyclic compounds include, but are not limited to, bicyclo[1,1,1]pentenyl, bicyclo[2,2,1]heptenyl, bicyclo[2.2.1]heptanyl, bicyclo[2.2.2]octanyl, bicyclo[3.3.1]nonanyl, bicyclo[3.3.3]undecanyl, and the like.

[0032]

[0038] As used herein, the term "halo" or "halogen" refers to an atom selected from fluorine (or fluoro), chlorine (or chloro), bromine (or bromo), and iodine (or iodo).

[0033]

[0039] As used herein, the term "heteroatom" refers to nitrogen, oxygen, sulfur, or phosphorus, and includes any oxidized form of nitrogen, sulfur, or phosphorus, and any quaternized form of a basic nitrogen.

[0034]

[0040] As used herein, the term "heteroaliphatic" refers to an aliphatic moiety in which one or more carbon atoms in the backbone are replaced with a heteroatom. Thus, a heteroaliphatic group refers to an aliphatic chain that contains, for example, one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms in place of a carbon atom. The heteroaliphatic moiety may be branched or straight-chain unbranched. As will be appreciated by those skilled in the art, "heteroaliphatic" is intended herein to include, but is not limited to, heteroalkyl, heteroalkenyl, and heteroalkynyl moieties. In certain embodiments, heteroaliphatic moieties include, but are not limited to, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, alkoxy, aryloxy, heteroalkoxy, heteroaryloxy, alkylthio, arylthio, heteroalkylthio, heteroarylthio, F, Cl, Br, I, -NO2, -CN, -CF3, -CH2CF3, -CHC 12 , -CH2OH, -CH2CH2OH, -CH2NH2, -CH2SO2CH3 or -GR G1 (Here, G is -O-, -S-, -NR G2 -, -C(=O)-, -S(=O)-, -SO2-, -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 SO2NR G3 -, or -SO2NR G2 - (where RG1 , R G2 and R G3 is independently substituted by independent replacement of one or more hydrogen atoms thereof with one or more moieties including, but not limited to, hydrogen, halogen, or an optionally substituted aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety ("substituted heteroaliphatic"). Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples described herein.

[0035]

[0041] As used herein, the term "heteroalkyl" refers to an alkyl in which at least one carbon atom is replaced with a heteroatom selected from N, O, or S. Heteroalkyl can be a carbon or heteroatom radical (i.e., the heteroatom can occur at the center or end of the radical), and can be optionally substituted independently with one or more substituents described herein. The term "heteroalkyl" encompasses alkoxyl and heteroalkoxy radicals.

[0036]

[0042] As used herein, the term "heteroalkenyl" refers to an alkenyl having at least one carbon atom substituted with a heteroatom selected from N, O, or S. The heteroalkenyl can be a carbon or heteroatom radical (i.e., the heteroatom can occur at the center or at the end of the radical), and can be optionally substituted independently with one or more substituents described herein.

[0037]

[0043] As used herein, the term "heteroalkynyl" refers to an alkynyl in which at least one carbon atom is replaced with a heteroatom selected from N, O, or S. The heteroalkynyl can be a carbon or heteroatom radical (i.e., the heteroatom can occur at the center or at the end of the radical) and can be optionally substituted independently with one or more substituents described herein.

[0038]

[0044] 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 groups can be monocyclic. Examples of monocyclic heteroaryls 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 groups also include polycyclic groups in which a heteroaromatic ring is fused to one or more aryl, heteroaryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Examples of polycyclic heteroaryls include, but are not limited to, indolyl, isoindolyl, benzothienyl, benzofuranyl, benzo[1,3]dioxolyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0039]

[0045] As used herein, the term "heterocycloalkyl" refers to a saturated or partially unsaturated cycloalkyl group, in which one or more ring atoms are heteroatoms independently selected from oxygen, sulfur, nitrogen, phosphorus, etc., and the remaining ring atoms are carbon, in which one or more ring atoms may be optionally independently substituted with one or more substituents. In some embodiments, the heterocycloalkyl is a saturated heterocycloalkyl. In some embodiments, the heterocycloalkyl is a partially unsaturated heterocycloalkyl having one or more double bonds in its ring system. In some embodiments, the heterocycloalkyl may contain any oxidized form of carbon, nitrogen, or sulfur, and any quaternized form of a basic nitrogen. The heterocycloalkyl radical may be carbon-linked or nitrogen-linked, where possible. In some embodiments, the heterocycle is carbon-linked. In some embodiments, the heterocycle is nitrogen-linked. For example, the pyrrole-derived group may be pyrrol-1-yl (nitrogen-linked) or pyrrol-3-yl (carbon-linked). Additionally, the imidazole derived groups can be imidazol-1-yl (nitrogen-linked) or imidazol-3-yl (carbon-linked).

[0040]

[0046] Heterocycloalkyl groups can be monocyclic. Examples of monocyclic heterocycloalkyls include, but are not limited to, oxetanyl, 1,1-dioxothietanylpyrrolidyl, tetrahydrofuryl, tetrahydropyranyl, tetrahydrothienyl, azetidinyl, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, piperidyl, piperazinyl, morpholinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyrimidonyl, pyridazonyl, pyrrolidinyl, triazinonyl, and the like.

[0041]

[0047] Heterocycloalkyl groups can be polycyclic, and include fused, spiro, and bridged ring systems. Fused heterocycloalkyl groups include radicals in which the heterocycloalkyl radical is a saturated, partially unsaturated, or fully unsaturated (i.e., aromatic) carbocyclic or heterocyclic ring. Examples of fused heterocycloalkyls include, but are not limited to, phenyl- or pyridinyl-fused rings, such as quinolinyl, isoquinolinyl, quinoxalinyl, quinolidinyl, quinazolinyl, azaindolizinyl, pteridinyl, chromenyl, isochromenyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothienyl, benzothiazolyl, carbazolyl, phenazinyl, phenothiazinyl, phenanthridinyl, imidazo[1,2-a]pyridinyl, furo[3,4-d]pyrimidinyl, pyrrolo[3,4-d]pyrimidinyl, dihydrofuro[3,4-b]pyridinyl groups, and the like. Examples of spiroheterocycloalkyls include, but are not limited to, spiropyranyl, spirooxazinyl, 5-aza-spiro[2.4]heptanyl, 6-aza-spiro[2.5]octanyl, 6-aza-spiro[3.4]octanyl, 2-oxa-6-aza-spiro[3.3]heptanyl, 2-oxa-6-aza-spiro[3.4]octanyl, 6-aza-spiro[3.5]nonanyl, 7-aza-spiro[3.5]nonanyl, 1-oxa-7-aza-spiro[3.5]nonanyl, 3,8-dioxa-1-azaspiro[4.5]dec-1-enyl, and the like. Examples of bridged heterocycloalkyls include, but are not limited to, 3-aza-bicyclo[3.1.0]hexanyl, 8-aza-bicyclo[3.2.1]octanyl, 1-aza-bicyclo[2.2.2]octanyl, 2-aza-bicyclo[2.2.1]heptanyl, 1,4-diazabicyclo[2.2.2]octanyl, and the like.

[0042]

[0048] As used herein, the term "hydroxyl" refers to --OH.

[0043]

[0049] As used herein, the term "leaving group" refers to a molecular fragment that leaves with an electron pair in anisotropic bond decomposition. Leaving groups can be anions or neutral molecules. Leaving groups include, but are not limited to, halides, e.g., Cl. - , Br - and I - , sulfonic acid esters, such as paratoluenesulfonate ("tosylate", TsO - ) and R C(O)O, where R is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety. - Includes:

[0044]

[0050] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not occur.

[0045]

[0051] As used herein, the term "partially unsaturated" refers to a radical that contains 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 (i.e., fully unsaturated) moieties.

[0046]

[0052] As used herein, the term "protecting group" means that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that reactions in a multifunctional compound can be selectively carried out at another reactive site. In some embodiments, the protecting group reacts selectively in good yields, resulting in a protected substrate that is stable to the anticipated reaction. The protecting group must be selectively removed in good yields by a reagent that is readily available, preferably non-toxic and does not attack other functional groups. The protecting group forms an easily separable derivative (more preferably, without creating new asymmetric centers). The protecting group has minimal additional functionality to avoid further reactive sites. As described herein, oxygen, sulfur, nitrogen, and carbon protecting groups can be utilized. For example, in some embodiments, certain exemplary oxygen protecting groups can be utilized. These oxygen-based 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 benzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilyl ether), TIPS (triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzylsilyl ether and TBDPS (t-butyldiphenylsilyl ether), esther, phenylsilyl ether ... Examples of suitable protecting groups include 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, carbamates (methyl, ethyl, and substituted ethyl carbamates (e.g., Troc), amides, cyclic imide derivatives, N-alkyl and N-aryl 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 of the oxygen protecting groups described above, as well as aliphatic carboxylic acids (e.g., acrylic acid), maleimide, vinylsulfonyl, and optionally substituted maleic acid. Certain other exemplary protecting groups are detailed herein, but it is understood that the present invention is not intended to be limited to these protecting groups, and a variety of additional equivalent protecting groups can be easily identified using the above criteria and utilized in the present invention. Additionally, a variety of protecting groups are described in "Protective Groups in Organic Synthesis," 3rd Edition, Greene, TW and Wuts, PG, John Wiley & Sons, eds., New York: 1999, the entire contents of which are incorporated herein by reference.

[0047]

[0053] As used herein, the term "leaving group" refers to a molecular fragment that leaves with an electron pair in anisotropic bond decomposition. Leaving groups can be anions or neutral molecules. Leaving groups include, but are not limited to, halides, e.g., Cl. - , Br - and I - , sulfonic acid esters, such as paratoluenesulfonate ("tosylate", TsO - ) and R C(O)O, where R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety. - Includes:

[0048]

[0054] As used herein, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogen atoms of the targeted moiety are replaced with a suitable substituent. "Substituted" or "substituted with" is understood to include, with the obvious proviso that such substitution is in accordance with the allowed valence of the atom being substituted, and that the substitution results in a stable or chemically suitable compound, e.g., a compound that does not undergo unintentional transformation by rearrangement, cyclization, elimination, or otherwise. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when multiple positions in any given structure may be substituted with multiple substituents selected from a specified group, the substituents may be the same or different at each position. Substituents may include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azide, carbamoyl, carboxyl, carboxyl ester, cyano, guanidino, halo, haloalkyl, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, alkylsulfinyl, sulfonic acid, alkylsulfonyl, thiocyanate, thiol, thione, or combinations thereof. Those skilled in the art will understand that the substituents themselves may also be substituted, where appropriate. Unless specifically stated as "unsubstituted," references to chemical moieties herein are understood to include substituted variants. For example, references to "aryl" groups or moieties unconditionally include both substituted and unsubstituted variants.

[0049]

[0055] 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-anchored protein, or a protoglycan. Examples of targeting moieties include, but are not limited to, antibodies or fragments thereof, lipocalins, proteins, peptides, or peptidomimetics, and the like. In addition to targeting the polymer scaffold to a specific cell, tissue, or location, the targeting moiety may also have a specific therapeutic effect, such as anti-proliferative (cytostatic and / or cytotoxic) activity, on the target cell or pathway. The targeting moiety may include or be engineered to include at least one chemically reactive group, such as -COOR, -SH, amine, or 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 to or complexes with a cell surface molecule, such as a cell surface receptor or antigen, for a given target cell population. Following specific binding or complexation of the ligand to its receptor, the cell becomes permissive for uptake of the ligand or ligand-drug conjugate, which is then internalized into the cell. As used herein, a ligand that "specifically binds to or complexes with" or "targets" a cell surface molecule binds preferentially to the cell surface molecule via intermolecular forces.

[0050]

[0056] As used herein, the term "ligand" refers to a variety of chemical or biological molecules that can have specific binding affinity to a selected target, where 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, a ligand can specifically bind to a protein or marker expressed on the surface of a target cell. In some embodiments, the ligands of the present disclosure bind to cell surface proteins or markers in a specific manner. -6 ~10 -11 M(K dIn some embodiments, the ligands of the present disclosure bind to a cell surface protein or marker 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 10 -6 Less than 10 -7 Less than and 10 -8 Less than M (K d In some embodiments, the ligands of the present disclosure bind to cell surface proteins or markers with a specific affinity, where specific affinity refers to the affinity of the ligand to the target cell surface protein or marker, which is at least 2, 3, 4, 5, 6, 8, 10, 20, 50, 100 or more times higher than to a non-target cell surface protein or marker. In some embodiments, the expression of the cell surface protein or marker of the present disclosure in a target cell (e.g., a cancer cell) is significantly higher than that in a standard cell. As used herein, the term "significantly" refers to a statistically significant difference, or a notable difference that can be recognized by one of ordinary skill in the art.

[0051]

[0057] As used herein, the term "targeting moiety" refers to a molecule, complex, or aggregate that specifically or selectively binds 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 antibody-based molecules or compounds. However, other examples of targeting moieties are known in the art and can be used, such as aptamers, avimers, 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 interchangeably herein.

[0052]

[0058] As used herein, the term "drug" refers to a compound (e.g., an active ingredient) that is biologically active and produces a desired physiological effect following administration to a subject in need thereof.

[0053]

[0059] As used herein, the term "antibody" includes any immunoglobulin, monoclonal, polyclonal, multivalent, multispecific, or bispecific (bivalent) antibody, or functional portion thereof that binds to a specific antigen. A natural intact antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a variable region (VH) and a first, second, and 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 the light and heavy chains are responsible for antigen binding. The variable regions of both chains are generally subdivided into three hypervariable regions called complementarity determining regions (CDRs) (the CDRs of the light chain (L) include LCDR1, LCDR2 and LCDR3, and the CDRs of the heavy chain (H) include HCDR1, HCDR2 and HCDR3). The scope of the CDRs for the antibodies and antigen-binding fragments disclosed herein may be defined or specified according to the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., 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, A.M., 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. (1991)). The three CDRs are interposed between adjacent segments known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. Thus, each VH and VL consists of three CDRs and four FRs, in the following order (amino acid residues N-terminal to C-terminal): FR1, CDR1, FR2, CDR2, FR3, DR3, FR4.The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Based on the amino acid sequence of the constant region of their heavy chains, antibodies are assigned to five major classes: IgA, IgD, IgE, IgG and IgM, which are characterized by the presence of α, δ, ε, γ and μ heavy chains, respectively. Some subclasses of the major antibody classes are, for example, IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain).

[0054]

[0060] As used herein, the term "Fab" with respect to an antibody refers to a monovalent antigen-binding fragment of an antibody consisting of a variable region and one light chain (both variable and constant regions) bound by a disulfide bond to the first constant region of one heavy chain. Fab can be obtained by papain digestion of an antibody at a residue proximal to the N-terminus of the disulfide bond between the hinge regions of the heavy chains.

[0055]

[0061] As used herein, the term "Fab" refers to a Fab fragment that can be obtained by pepsin digestion of an antibody at a residue proximal to the C-terminus of the disulfide bond between the hinge regions of the heavy chains, and thus contains a portion of the hinge region that differs from Fab in a small number of residues in the hinge region, including one or more cysteines.

[0056]

[0062] As used herein, the term "F(ab')2" refers to a dimer of Fab' containing two light chains and portions of two heavy chains.

[0057]

[0063] As used herein, the term "Fc" in reference to an antibody refers to the portion of an antibody that consists of the second and third constant regions of a first heavy chain and is bound to the second and third constant regions of a second heavy chain via disulfide bonds. The Fc region of IgG and IgM contains three heavy chain constant regions (the second, third and fourth heavy chain constant regions in each chain). It can be obtained by papain digestion of an antibody. The Fc portion of an antibody is responsible for various effector functions, such as ADCC and CDC, but does not function in antigen binding.

[0058]

[0064] As used herein, the term "Fv" with respect to an antibody refers to the smallest fragment of an antibody that has a complete antigen-binding site. An Fv fragment consists of the variable region of one light chain bound to the variable region of one heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of one light chain and the variable region of one heavy chain is a disulfide bond.

[0059]

[0065] As used herein, a "single chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected together directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)). An "scFv dimer" refers to a single chain containing two heavy chain variable regions and two light chain variable regions with a linker. An "scFv dimer" refers to a single chain containing two heavy chain variable regions and two light chain variable regions with a linker. H -V L dimerize with the V H is the other part's V L and V form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). H -V L The term "scFv dimer" may be a bivalent diabody or a bivalent ScFv (BsFv) comprising a V H1 and V L1 In cooperation with V H2 and V L2V cooperates with each other, such that each cooperated pair has a different antigen specificity. L1 -V H2 V attached to (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker).

[0060]

[0066] As used herein, the term "single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv linked to the Fc region of an antibody.

[0061]

[0067] As used herein, the term "camelized single domain antibody", "heavy chain antibody", "nanobody" or "HCAb" refers to a camelized single domain antibody having two V HIt refers to antibodies that contain heavy chains and do not contain 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. Patent No. 6,005,079). Heavy chain antibodies were originally obtained from the Camelidae family (camels, dromedaries and llamas). Although lacking light chains, camelized antibodies have a bona fide antigen-binding repertoire (Hamers-Casterman C. et al., Nature. June 3;363(6428):446-8 (1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation", Immunogenetics. April;54(1):39-47 (2002); Nguyen VK. et al., Immunology. May;109(1):93-101 (2003)). The variable domain of heavy-chain antibodies (VHH domains) represents the smallest known antigen-binding unit produced by the adaptive immune response (Koch-Nolte F. et al., FASEB J. November;21(13):3490-8 Epub 2007 June 15 (2007)). "Diabodies" refer to small antibody fragments with two antigen-binding sites, where the fragments are V L V connected to the domain H Domain Included (V H -V L or V L -V H ) (see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. July 15; 90(14):6444-8 (1993); EP404097; WO93 / 11161). The two domains on the same chain cannot pair because the linker is too short, so the domains are forced to pair with complementary domains on another chain, thereby generating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes).

[0062]

[0068] As used herein, the term "domain antibody" refers to an antibody fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In some embodiments, two or more V H The domains are covalently linked with peptide linkers to form bivalent or multivalent domain antibodies. H The domains may target the same or different antigens.

[0063]

[0069] As used herein, the term "(dsFv)2" refers to an antigen-binding fragment consisting of three peptide chains: two V connected by a peptide linker and separated by disulfide bridges. L Two V's attached to the part H portion.

[0064]

[0070] As used herein, the term "bispecific ds diabody" refers to a V L1 -V H2 (linked by a peptide linker) to V H1 and V L1 V is linked via a disulfide bridge between H1 -V L2 (also linked by a peptide linker).

[0065]

[0071] As used herein, the term "bispecific dsFv" or "dsFv-dsFv" refers to an antigen-binding fragment consisting of three peptide chains: the heavy chains are connected by peptide linkers (e.g., long flexible linkers) to each other via V L1 and V L2 V binds to and pairs with V via a disulfide bridge. H1 -V H2 Each of the disulfide-paired heavy and light chains has a different antigen specificity.

[0066]

[0072] In some embodiments, the antibody or antigen-binding fragment thereof is chimeric or humanized.

[0067]

[0073] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of the heavy and / or light chain derived from one species and the remaining heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody can contain a constant region derived from a human and a variable region derived from a non-human species, such as a mouse.

[0068]

[0074] As used herein, the term "humanized" refers to an antibody or antigen-binding fragment that comprises a non-human animal (e.g., rodent, rabbit, dog, goat, horse, or chicken) derived CDR, a human derived FR region, and, where applicable, a human derived constant region. In some embodiments, the constant region from a human antibody is fused to a non-human variable region. Humanized antibodies or antibody-binding fragments are useful as human therapeutics. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, hamster, or a non-human primate, such as a monkey (e.g., cynomolgus or rhesus) or an ape (e.g., chimpanzee, gorilla, monkey, or affen). In some embodiments, the humanized antibody or antigen-binding fragment is composed of substantially all human sequences, except for the CDR sequences, which are non-human. In some embodiments, the humanized antibody or antigen-binding fragment is modified to improve antibody performance, e.g., binding or binding affinity. For example, one or more amino acids in one or more non-human CDRs are modified to improve the antibody performance, e.g., binding or binding affinity. Acid residues are modified to reduce potential immunogenicity in humans, where the modified amino acid residues are not critical for immunospecific binding, or the modifications are conservative changes, so that the binding of the humanized antibody to the antigen is not significantly affected. In some embodiments, the FR region of human origin may contain the same amino acid sequence as the human antibody from which it is derived, or may contain some amino acid changes, such as not 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.

[0069]

[0075] As used herein, the term "natural amino acid" refers to any one 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). As used herein, the term "unnatural amino acid" as used herein refers to any amino acid that is not a natural amino acid. This includes, for example, amino acids that contain α-, β-, ω-, D-, L-aminoacyl residues. More generally, unnatural amino acids can be represented by the general formula: [ka] where the side chain R is different from a naturally occurring amino acid side chain. 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, allo-isoleucine, norleucine, α-methylleucine, cyclohexylglycine, β-cyclohexylalanine, β-cyclopentylalanine, α-methylproline, phenylglycine, α-methylphenylalanine, and homophenylalanine.

[0070]

[0076] As used herein, the term "polypeptide", "protein" or "peptide" may be a single amino acid or a polymer of amino acids. Polypeptides, proteins or peptides as described in this disclosure may include naturally occurring and non-naturally occurring amino acids, or analogs and mimetics thereof. Polypeptides, proteins or peptides may be obtained by any method well known in the art, including, but not limited to, separation and purification from natural materials, recombinant expression, chemical synthesis, etc.

[0071]

[0077] As used herein, the term "biocompatible" is intended to describe compounds that exert minimal disruptive or host reaction effects while in contact with body fluids or living cells or tissues. Thus, biocompatible groups, as used herein, refer to aliphatic, cycloalkyl, heteroaliphatic, heterocycloalkyl, aryl or heteroaryl moieties, which fall within the definition of the term biocompatible as defined above and herein. As used herein, the term "biocompatible" is also used to mean compounds that exhibit minimal interaction with recognition proteins, such as naturally occurring antibodies, cellular proteins, cells and other biological system components, unless such interaction is specifically desired. Thus, substances and functional groups, such as drugs and prodrugs, that are specifically intended to cause the above-mentioned minimal interactions are considered to be biocompatible. In some embodiments, compounds are "biocompatible" if they are added to normal cells in vitro at concentrations similar to their intended systemic concentrations in vivo, and cause 1% or less cell death during a time period equal to the compound half-life in vivo (e.g., the time required for 50% of the administered compound to be removed / cleared in vivo), and if their administration in vivo induces minimal and medically acceptable inflammation, body response to foreign substances, immunotoxicity, chemical toxicity and / or other such adverse effects.As used herein, the term "normal cells" refers to cells that are not intended to be destroyed or otherwise significantly affected by the test compound.

[0072]

[0078] As used herein, a "biodegradable" polymer is a polymer that is susceptible to biological processes in vivo. As used herein, a "biodegradable" compound or moiety, when taken up by a cell, can be degraded by lysosomes or other chemical mechanisms or hydrolysis into components that the cell can reuse or dispose of without significant toxic effects on the cell. The term "biocleavale" as used herein has the same meaning as "biodegradable". The degraded fragments preferably induce little or no organ or cell overload, or pathological processes caused by such overload, or other adverse effects in vivo. Examples of biodegradation processes include enzymatic and non-enzymatic hydrolysis, oxidation and reduction. Suitable conditions for non-enzymatic hydrolysis of the biodegradable protein-polymer-drug conjugates described herein (or their components, such as biodegradable polymer carriers and linkers between the carriers and antibodies or drug molecules) include, for example, exposure of the biodegradable conjugate to water at the temperature and pH of the lysosomal intracellular compartment. Biodegradation of some protein-polymer-drug conjugates (or components thereof, such as biodegradable polymer carriers and linkers between the carriers and antibodies or drug molecules) can also be enhanced extracellularly, for example in low pH regions of the animal's body, such as in areas of inflammation, in close proximity to activated macrophages or other cells that release degradation-promoting factors. In certain embodiments, the effective size of the polymer carrier at a pH of about 7.5 does not detectably change over 1-7 days and remains within 50% of the original polymer size for at least several weeks. Meanwhile, at a pH of about 5, the polymer carrier preferably degrades detectably over 1-5 days and is completely converted to low molecular weight fragments within a time frame of 2 weeks to several months. The integrity of the polymer in such tests can be measured, for example, by size exclusion HPLC. Although more rapid degradation may be preferred in some cases, it may generally be more desirable for the polymer to degrade intracellularly at a rate that does not exceed the rate of metabolism or excretion of the polymer fragments by the cell.In certain embodiments, the polymers and polymer biodegradation byproducts are biocompatible.

[0073]

[0079] As used herein, the term "bioavailability" refers to the systemic availability (i.e., blood / plasma levels) of a given amount of drug or compound administered to a subject. Bioavailability is an absolute term that indicates measurement of both the time (rate) and the total amount (extent) of drug or compound that reaches the general circulation from an administered dosage form.

[0074]

[0080] As used herein, the term "drug release mechanism" refers to a linking moiety that is biocleavable / biodegradable under intracellular conditions, such that cleavage of the linking moiety releases the drug into the intracellular environment. In some embodiments, the linking moiety is hydrolytically unstable, i.e., susceptible to hydrolysis at a particular pH, in water or aqueous solutions, including body fluids such as blood. In some embodiments, the linking moiety is enzymatically unstable, i.e., degradable by one or more enzymes. In some embodiments, the linking moiety is photolabile, making it useful on body surfaces and in many body cavities accessible to light. In some embodiments, the linking moiety is biocleavable under reducing conditions, where the activity of the drug is not affected.

[0075]

[0081] As used herein, the term "therapeutic agent" or "drug" refers to a compound (e.g., an active ingredient) that is biologically active and produces a desired physiological effect following administration to a subject in need thereof. In some embodiments, the therapeutic agent is a small molecule drug.

[0076]

[0082] As used herein, the term "small molecule" refers to a molecule having a relatively low molecular weight, whether naturally occurring or artificially created (e.g., via chemical synthesis). Preferred small molecules are biologically active in producing a localized or systemic effect in an animal, e.g., a mammal, e.g., a human. In certain embodiments, the small molecule is a drug, and the small molecule is referred to as a "drug molecule" or "drug" or "therapeutic agent." In certain embodiments, the drug molecule has a MW of about 5 kDa or less. In other embodiments, the drug molecule has a MW of about 1.5 kDa or less.

[0077]

[0083] Classes of drug molecules that may be used in the present disclosure include, but are not limited to, anti-cancer agents, radionuclides, vitamins, anti-AIDS agents, antibiotics, immunosuppressants, antivirals, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, lubricants, tranquilizers, anticonvulsants, muscle relaxants and anti-Parkinson agents, antispasmodics and muscle contractants including channel blockers, miotics and anticholinergics, anti-glaucoma compounds, anti-parasitic and / or anti-protozoal compounds, cell-extracellular matrix interaction modulators including cell proliferation inhibitors and anti-adhesion molecules, vasodilators, DNA, RNA or protein synthesis inhibitors, antihypertensives, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotic agents, antiemetics, contrast agents.

[0078]

[0084] In certain embodiments, polymers may be used as therapeutic agents in the present disclosure. Examples of suitable polymers include, but are not limited to, amino acid-based molecules such as peptides, polypeptides, enzymes, antibodies, immunoglobulins, or functional fragments thereof, among others.

[0079]

[0085] In some embodiments, the therapeutic agent used in the present disclosure is a therapeutic agent that has antiproliferative (cytostatic and / or cytotoxic) activity against a target cell or pathway. 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)2OR', -P(O)2OR', -CN, -NC, or -ONO, where R is an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety, and R' is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl moiety.

[0080]

[0086] As used herein, the term "cytotoxic" means toxic to a cell or a selected cell population (e.g., cancer cells). Toxic effects can cause cell death and / or lysis. In certain cases, toxic effects can be sublethal destructive effects on cells, such as slowing or stopping cell growth. To achieve cytotoxic effects, drugs or prodrugs can be selected from the group consisting of DNA damaging agents, microtubule disrupting agents, or cytotoxic proteins or polypeptides, among others.

[0081]

[0087] As used herein, the terms "specific binding" or "specifically binds" refer to a non-random binding reaction between two molecules, such as, for example, between an antibody and an antigen. In some embodiments, the antibodies or antigen-binding fragments provided herein have 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) for H7.4. D ) specifically binds to a target antigen. D is the ratio of the dissociation rate to the association rate (k off / k on ) and can be determined, for example, using surface plasmon resonance techniques using devices such as a Biacore.

[0082]

[0088] As used herein, the term "tumor antigen" refers to an antigenic substance produced in tumor cells, i.e., tumor antigens induce an immune response in the host. Normal proteins in the body are not antigenic due to self-tolerance, a process in which autoreactive cytotoxic T lymphocytes (CTLs) and autoantibody-producing B lymphocytes are thinned out "centrally" in primary lymphoid tissues (BM) and "peripherally" in secondary lymphoid tissues (mostly the thymus for T cells and the spleen / lymph nodes for B cells). Thus, any protein that is not exposed to the immune system will induce an immune response. This may include normal proteins that are well isolated from the immune system, proteins that are normally produced in very small amounts, proteins that are normally produced only at certain stages of development, or proteins whose structure is altered due to mutation.

[0083]

[0089] As used herein, the term "effective amount" refers to the amount necessary to elicit a desired biological response. As will be understood by those skilled in the art, the effective amount of a drug or device can vary depending on, for example, the desired biological endpoint, the drug delivered, the composition of the encapsulating matrix, the target tissue, and other factors. For example, the effective amount of antigen-containing microparticles delivered to immunize an individual is the amount that produces a sufficient immune response to prevent infection with the organism that carries the administered antigen.

[0084]

[0090] As used herein, the "molecular weight" or "MW" of a polymer or polymeric carrier / scaffold or polymer conjugate refers to the weight average molecular weight, unless otherwise specified.

[0085]

[0091] The present disclosure is intended to include all isotopes of atoms present in the present compounds. Isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C 13 and C 14Includes:

[0086]

[0092] The present invention is intended to include all isomers of the compounds, which refers to and includes optical isomers and tautomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and optical isomers include separated optical isomers and mixtures of optical isomers, including racemic and non-racemic mixtures, where the isomers may be in separated form or in mixture with one or more other isomers.

[0087] Polymer Support

[0093] The conjugates of the present disclosure are useful for biomedical applications, such as drug delivery and tissue engineering, and the polymeric carriers used in the conjugates of the present disclosure are biocompatible and biodegradable. In some embodiments, the polymeric carriers are soluble polymers, nanoparticles, gels, liposomes, micelles, sutures, implants, and the like.

[0088]

[0094] In some embodiments, the polymeric carrier 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 100,000 Da, The weight average molecular weight Mw may be about 1,000 to about 90,000 Da, about 1,000 to about 80,000 Da, about 1,000 to about 70,000 Da, about 1,000 to about 60,000 Da, about 1,000 to about 50,000 Da, about 1,000 to about 40,000 Da, about 1,000 to about 30,000 Da, about 1,000 to about 20,000 Da, about 1,000 to about 10,000 Da, about 2,000 to about 10,000 Da, about 3,000 to about 10,000 Da, about 4,000 to about 10,000 Da, or about 5,000 to about 10,000 Da.

[0089]

[0095] In some embodiments, the polymeric carrier used in the present disclosure is polyglycerol. In some embodiments, the polymeric carrier used in the present disclosure is linear polyglycerol. In a specific embodiment, the linear polyglycerol has a molecular weight of about 400 to about 3,000,000 Da, for example, about 1,000 to about 2,000,000 Da, about 1,000 to about 1,000,000 Da, about 1,000 to about 900,000 Da, about 1,000 to about 800,000 Da, about 1,000 to about 700,000 Da, about 1,000 to about 600,000 Da, about 1,000 to about 500,000 Da, about 1,000 to about 400,000 Da, about 1,000 to about 300,000 Da, about 1,000 to about 200,000 Da, about 1,000 to about 100,000 Da, or the like. The weight average molecular weight Mw may be about 1,000 Da, about 1,000 to about 90,000 Da, about 1,000 to about 80,000 Da, about 1,000 to about 70,000 Da, about 1,000 to about 60,000 Da, about 1,000 to about 50,000 Da, about 1,000 to about 40,000 Da, about 1,000 to about 30,000 Da, about 1,000 to about 20,000 Da, about 1,000 to about 10,000 Da, about 2,000 to about 10,000 Da, about 3,000 to about 10,000 Da, about 4,000 to about 10,000 Da, or about 5,000 to about 10,000 Da.

[0090] Therapeutic Agents

[0096] In some embodiments, the therapeutic agent used in the conjugates of the present disclosure is a small molecule having a molecular weight of about 5 kDa or less, about 4 kDa or less, about 3 kDa or less, about 1.5 kDa or less, or about 1 kDa or less.

[0091]

[0097] In some embodiments, the therapeutic agent has an IC 50 has.

[0092]

[0098] IC > 1nM 50Some therapeutic agents having an IC of 0.01 to 0.05 are unsuitable for conjugation with targeting moieties using art-recognized conjugation techniques. Without wishing to be bound by theory, such therapeutic agents cannot be conjugated with sufficient copies (i.e., more than 8) of the drug using art-recognized techniques without reducing the pharmacokinetic and physiochemical properties of the conjugate, and have insufficient potency for use in moiety-drug conjugate targeting using conventional techniques. However, using the conjugation strategies described herein, sufficiently high loading of these less potent drugs can be achieved, thereby obtaining high therapeutic agent loading while maintaining the desired pharmacokinetic and physiochemical properties. Thus, in some embodiments, IC of greater than about 1 nM can be achieved. 50

[0043] Therapeutic agents having the following structure are useful in the targeting moiety-polymer-drug conjugates provided herein:

[0093]

[0099] Small molecule therapeutics (e.g., anti-proliferative (cytotoxic and cytostatic) agents that can be linked to polymeric carriers) for use in this disclosure include cytotoxic compounds (e.g., broad spectrum), angiogenesis inhibitors, cell cycle progression inhibitors, PI3K / m-TOR / AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperone inhibitors, HDAC inhibitors, PARP inhibitors, Wnt / Hedgehog signaling inhibitors, and RNA polymerase inhibitors.

[0094]

[0100] Broad spectrum 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.

[0095]

[0101] Exemplary DNA binding, intercalating or alkylating drugs include CC-1065 and its analogs, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin, nemorubicin and its derivatives, PNU-159682), bisnapththalimide compounds such as elinafide (LU79553) and its analogs, alkylating agents such as calicheamicin, dactinomycin, mitromycines, 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, bizeresin, carzelesin, seco-adozelesin, and related analogs and prodrug forms, examples of which are described in U.S. Patent 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. Patent No. 6,630,579. Calicheamicins include, for example, enediynes, such as esperamicin, and those described in U.S. Patent Nos. 5,714,586 and 5,739,116. Duocarmycins include those described in U.S. Patent Nos. 5,070,092, 5,101,038, 5,187,186, 6,548,530, 6,660,742, and 7,553,816(B2) and Li et al., Tet Letts., 50:2932-2935 (2009).

[0096]

[0102] Pyrrolobenzodiazepines (PBDs) and analogs thereof include those described in Denny, Exp. Opin. Ther. Patents., 10(4):459-474 (2000) and Antonow and Thurston, Chem Rev., 2815-2864 (2010).

[0097]

[0103] Exemplary microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel, tesetaxel, and carbazitaxel, maytansinoids, auristatins and their analogs, vinca alkaloid derivatives, epothilones, and cryptophycins.

[0098]

[0104] Exemplary maytansinoids or maytansinoid analogs include maytansinol and maytansinol analogs, such as maytansine or DM-1 and DM-4, ​​as described in U.S. Patent Nos. 5,208,020, 5,416,064, 6,333.410, 6,441,163, 6,716,821, RE39,151, and 7,276,497. In certain embodiments, the cytotoxic agent is a maytansinoid, another group of antitubulin agents (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52:127-131), a maytansinoid, or a maytansinoid analog. Examples of suitable maytansinoids 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.

[0099]

[0105] Exemplary auristatins include auristatin E (also known as a derivative of dolastatin-10), auristatin EB (AEB), auristatin EFP (AEFP), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), auristatin F, auristatin F phenylenediamine (AFP), auristatin F HPA, and dolastatin. Suitable auristatins also are described in U.S. Patent Application Publication Nos. 2003 / 0083263, 2011 / 0020343, and 2011 / 0070248, PCT Publication Nos. WO09 / 117531, WO2005 / 081711, WO04 / 010957, WO02 / 088172, and WO01 / 24763, and in U.S. Patent Nos. 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,124,431, 6,034,065, 5,780,588, 5,7 Nos. 67,237, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 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 entire disclosures of which are incorporated herein by reference.

[0100]

[0106] Exemplary vinca alkaloids include vincristine, vinblastine, vindesine and navelbine (vinorelbine). Suitable vinca alkaloids that can be used in the present disclosure are also disclosed in U.S. Patent Application Publication Nos. 2002 / 0103136 and 2010 / 0305149, and U.S. Patent No. 7,303,749(B1), the entire contents of which are incorporated herein by reference.

[0101]

[0107] Exemplary epothilone compounds include epothilones A, B, C, D, E and 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, as well as WO97 / 19086, WO98 / 08849, WO98 / 22461, WO98 / 25929, WO98 / 38192, WO99 / 01124, WO99 / 02514, WO99 / 03848, WO99 / 07692, WO99 / 27890 and WO99 / 28324, the entire disclosures of which are incorporated herein by reference.

[0102]

[0108] Exemplary cryptophycin compounds are described in US Pat. Nos. 6,680,311 and 6,747,021.

[0103]

[0109] Exemplary platinum compounds include cisplatin (PLATINOL®), carboplatin (PARAPLATIN®), oxaliplatin (ELOXATINE®), iproplatin, ormaplatin, and tetraplatin.

[0104]

[0110] Still other classes of compounds or compounds with these or other cytotoxic modes of action can be selected, including, for example, mitomycin C, mitomycin A, daunorubicin, doxorubicin, morpholino-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, cryptophycin, cemadotin, discodermolide, eleutherobin, and mitoxantrone.

[0105]

[0111] 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, ciratecan, lurtotecan, exatecan, diflomotecan, belotecan, lurtotecan and 539625. Other camptothecin compounds that can be used in the present invention include, for example, those described in J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554 (1980); J. Med. Chem., 30:1774 (1987).

[0106]

[0112] 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, which means any compound that contains fumagillin core structure, including fumagillamine, that inhibits MetAP-2's ability to remove NH2-terminal methionine from protein, 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 "fumagillol analogs" are disclosed in J. Org. Chem., 69, 357, 2004; J. Org. Chem., 70, 6870, 2005; European Patent Application No. 0 354 787; J. Med. Chem., 49, 5645, 2006; Bioorg. Med. Chem., 11, 5051, 2003; Bioorg. Med. Chem., 14, 91, 2004; Tet. Lett. 40, 4797, 1999; WO 99 / 61432, U.S. Patent Nos. 6,603,812, 5,789,405, 5,767,293, 6,566,541, and 6,207,704.

[0107]

[0113] 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 BI2536, BI6727 (vorasertib), GSK461364, ON-01910 (Estybon), and KSP inhibitors such as SB743921, SB715992 (ispinesib), MK-0731, AZD8477, AZ3146 and ARRY-520.

[0108]

[0114] 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.

[0109]

[0115] 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, Palomid529, Perifosine, PI-103, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, wortmannin, XL147 and XL765.

[0110]

[0116] Exemplary AKT inhibitors include, but are not limited to, AT7867.

[0111]

[0117] Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf and p38 MAPK inhibitors.

[0112]

[0118] 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.

[0113]

[0119] Exemplary B-raf inhibitors include CDC-0879, PLX-4032 and SB590885.

[0114]

[0120] Exemplary B p38 MAPK inhibitors include BIRB796, LY2228820 and SB202190.

[0115]

[0121] Receptor tyrosine kinases (RTKs) are cell surface receptors that are often associated with signal transduction pathways that stimulate the uncontrolled growth of cancer cells and angiogenesis.Many RTKs have been identified that are overexpressed or mutated, leading to constitutive activation of the receptor, 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.

[0116]

[0122] Exemplary ErbB2 receptor (EGFR family) inhibitors include, but are not limited to, AEE788 (NVP-AEE788), BIBW2992 (afatinib), lapatinib, erlotinib (Tarceva), and gefitinib (Iressa).

[0117]

[0123] Exemplary RTK inhibitors that target more than one signaling pathway (multiple 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 intracellular serine / threonine kinases of the VEGFR, PDGFR and Raf / Mek / Erk pathways.

[0118]

[0124] Exemplary protein chaperone inhibitors include HSP90 inhibitors. Exemplary HSP90 inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922 and KW-2478.

[0119]

[0125] 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 (NSC696085), SB939, Trichostatin A, and Vorinostat (SAHA).

[0120]

[0126] 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.

[0121]

[0127] Exemplary Wnt / Hedgehog signaling pathway inhibitors include vismodegib (RG3616 / GDC-0449), cyclopamine (11-deoxojervine) (a Hedgehog pathway inhibitor), and XAV-939 (a Wnt pathway inhibitor).

[0122]

[0128] Exemplary RNA polymerase inhibitors include amatoxins. Exemplary amatoxins include α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanulin, amanuric acid, amaninamide, amanine, and proamanitin.

[0123]

[0129] Exemplary protein synthesis inhibitors include trichothecene compounds.

[0124]

[0130] In some embodiments, the therapeutic agent of the disclosure is a topoisomerase inhibitor (such as, for example, a non-natural camptothecin compound), a vinca alkaloid, a kinase inhibitor (such as, for example, 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, a duocarmycin, an auristatin, a dolastatin, a calicheamicin, a topotecan, SN38, a camptothecin, an exatecan, a nemorubicin and its derivatives, PNU-159682, CC1065, an elinafide, a trichothecene, a pyrrolobenzodiazepine, a maytansinoid, a DNA binding drug or a platinum compound, and analogs thereof. In some embodiments, the therapeutic agent is a derivative of SN-38, camptothecin, topotecan, exatecan, calicheamicin, exatecan, nemorubicin, PNU-159682, anthracyclines, maytansinoids, taxanes, trichothecenes, CC1065, elinafide, vindesine, vinblastine, PI-103, AZD8330, dolastatins, auristatin E, auristatin F, duocarmycin compounds, ispinesib, pyrrolobenzodiazepines, ARRY-520, and stereoisomers, isosteres and analogs thereof.

[0125]

[0131] In some embodiments, the therapeutic agent used in the present disclosure is a combination of two or more drugs, such as a PI3 kinase inhibitor and a MEK inhibitor, a broad-spectrum cytotoxic compound and a platinum compound, a PARP inhibitor and a platinum compound, a broad-spectrum cytotoxic compound and a PARP inhibitor.

[0126]

[0132] In yet another embodiment, the therapeutic agent used in the present disclosure is auristatin F-hydroxypropylamide-L-alanine.

[0127]

[0133] Those skilled in the art will easily understand 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 understand that many of these compounds can be used in place of the therapeutic agents described herein.Therefore, the therapeutic agents of the present disclosure include analogs and derivatives of the compounds described herein.

[0128]

[0134] In some embodiments, the therapeutic agent has anti-proliferative activity against the target cell or pathway.

[0129]

[0135] In certain embodiments, the anti-proliferative activity is selected from cytostatic and / or cytotoxic activity.

[0130]

[0136] In certain embodiments, the therapeutic agent is selected from anti-cancer agents, cytotoxic drugs, radionuclides, vitamins, anti-AIDS agents, antibiotics, immunosuppressants, immunomodulatory compounds, therapeutic RNA, antiviral agents, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, tranquilizers, anticonvulsants, muscle relaxants and antiparkinsonian agents, antispasmodics and muscle contractants including channel blockers, miotics and anticholinergics, anti-glaucoma compounds, antiparasitic and / or antiprotozoal compounds, cell-extracellular matrix interaction modulators including cell proliferation inhibitors and anti-adhesion molecules, vasodilators, DNA, RNA or protein synthesis inhibitors, antihypertensives, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory agents, antiangiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotic agents, antiemetics, contrast agents.

[0131]

[0137] In certain embodiments, the therapeutic agent comprises an amino acid based molecule.

[0132]

[0138] In certain embodiments, the amino acid-based molecule includes a peptide, a polypeptide, an enzyme, an antibody, an immunoglobulin, or a functional fragment thereof.

[0133]

[0139] In certain embodiments, the therapeutic agent has a chemically reactive group.

[0134]

[0140] In certain embodiments, the chemically reactive group is -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 an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; R 15 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0135] Linker-Polymer-Drug Compounds

[0141] In one embodiment, a polymer scaffold of formula (I) useful for conjugating a targeting moiety: [ka] is provided, where The polymer scaffold comprises linear polyglycerol; L is a functional group W capable of forming a covalent bond with a targeting moiety. p A linking moiety comprising: M a is a stretcher connecting L to the -NH- moiety, each G 1 is independent, L p is a functional group that connects the linear polyglycerol Each L P are independently therapeutic moieties D and G 1 This is the drug release mechanism between each D is independently a therapeutic moiety; each G 2 are independently functional groups that can be converted to 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.

[0136]

[0142] In some embodiments, W p can react with a functional group on the targeting moiety via a click reaction.

[0137]

[0143] In certain embodiments, W p teeth, [ka] is selected from the group consisting of:

[0138]

[0144] In some embodiments, W p can react with an amino acid on the targeting moiety.

[0139]

[0145] In certain embodiments, W p can react with amino acids on the targeting moiety, the amino acids being natural amino acids, unnatural amino acids, or combinations thereof. In certain embodiments, the natural amino acids can include cysteine, lysine, tyrosine, aspartic acid, and glutamic acid.

[0140]

[0146] In some embodiments, W p can react with one or more cysteines on the targeting moiety.

[0141]

[0147] In certain embodiments, W p can react with one or more cysteines on the targeting moiety, and each W p teeth, [ka] wherein R 1 is a sulfur protecting group, and each R 2 are independently leaving groups.

[0142]

[0148] In certain embodiments, each R 2 are independently halo or R 2a C(O)O-, where R 2a is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0143]

[0149] In some embodiments, W p can react with one or more lysines on the targeting moiety.

[0144]

[0150] In certain embodiments, W p can react with one or more lysines on the targeting moiety, and each W p is independent, [ka] is selected from the group consisting of:

[0145]

[0151] In some embodiments, W p can react with one or more unnatural amino acids on the targeting moiety.

[0146]

[0152] In certain embodiments, W p can react with one or more unnatural amino acids on the targeting moiety, and each W p is independent, [ka] is selected from.

[0147]

[0153] In some embodiments, M a teeth, R 3 , [ka] wherein: * is the site of covalent attachment to L, ** is the site of covalent attachment to the -NH- moiety, R 3 is 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)-, 4-14 membered heterocycloalkyl, -C 1~10 Alkyl-(4-14 membered heterocycloalkyl)-, -(4-14 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-(4-14 membered heterocycloalkyl)-C(=O)-, -(4-14 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 1~10Alkyl-(C 3~8 Cycloalkyl)-NH-, -(C 3~8 Cycloalkyl)-C 1~10 Alkyl-NH-, -4 to 14 membered heterocycloalkyl-NH-, -C 1~10 Alkyl-(4-14 membered heterocycloalkyl)-NH-, -(4-14 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 Alkyl-(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 is alkyl, R 5 -C(O)-NR 5a or -NR 5a -C(O)-, R 5a is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is alkyl, R 6 is a bond or -NR 6a -(CR 6b R 6c )-C(O)-, R6a is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is 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, 5-12 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, n 4 is an integer from 1 to 4.

[0148]

[0154] In certain embodiments, M a teeth, [ka] is selected from the group consisting of:

[0149]

[0155] In some embodiments, G 1 teeth, [ka] wherein * is selected from the group consisting of L P Each R 7 is independently selected from a direct bond, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R 7ais selected from hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0150]

[0156] In some embodiments, G 1 teeth, [ka] is selected from the group consisting of

[0151]

[0157] In the formula, R 7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. p contains independently unstable structures.

[0152]

[0158] In certain embodiments, the labile structure is selected from a hydrolytically unstable structure or an enzymatically labile structure.

[0153]

[0159] In certain embodiments, the hydrolytically unstable structure is [ka] wherein * is selected from the group consisting of G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and R 8 is selected from hydrogen, alkyl or aryl; R 9 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0154]

[0160] In certain embodiments, the hydrolytically unstable structure is [ka] wherein * is selected from the group consisting of G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and R 8 is selected from hydrogen, alkyl or aryl; R9 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0155]

[0161] In some embodiments, G 1 teeth, [ka] where * is L P is a moiety covalently attached to R 7 is alkyl.

[0156]

[0162] In certain embodiments, -G 1 -L P -D is [ka] It is.

[0157]

[0163] In some embodiments, the enzymatically labile structure is sensitive to an enzyme selected from cathepsin B, a phosphatase, a sulfatase, or a glucuronidase.

[0158]

[0164] In certain embodiments, the enzymatically labile structure is sensitive to cathepsin B and is -Z- or [ka] wherein Z is a substrate for cathepsin B comprising 2 to 4 amino acids.

[0159]

[0165] In some embodiments, G 1 teeth, [ka] where * is L P is a moiety covalently attached to R 7 is alkyl.

[0160]

[0166] In certain embodiments, -G1 -L P -D is [ka] It is.

[0161]

[0167] In certain embodiments, the enzymatically labile structure is sensitive to glucuronidase; [ka] where * is G 1 is the site covalently bonded to, and ** is the site covalently bonded to D.

[0162]

[0168] In certain embodiments, G 1 teeth, [ka] and R 7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl.

[0163]

[0169] In some embodiments, G 1 teeth, [ka] where * is L P is a moiety covalently attached to R 7 is alkyl.

[0164]

[0170] In certain embodiments, -L P -D is [ka] is selected from.

[0165]

[0171] In certain embodiments, -G 1 -L P -D is [ka] is selected from.

[0166]

[0172] In certain embodiments, the enzymatically labile structure is sensitive to a phosphatase; [ka] wherein * is selected from G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and each R 10 and R 11 is independently hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0167]

[0173] In certain embodiments, G 1 teeth, [ka] It is.

[0168]

[0174] In certain embodiments, -G 1 -L P -D is [ka] [ka] is selected from the group consisting of:

[0169]

[0175] In certain embodiments, the enzymatically labile structure is susceptible to sulfatase; [ka] where * is G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and each R 12 and R 13 is independently hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0170]

[0176] In some embodiments, G 1 teeth, [ka] where R 7 is selected from hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0171]

[0177] In some embodiments, G 1 teeth, [ka] where * is L P is a moiety covalently attached to R 7 is alkyl.

[0172]

[0178] In certain embodiments, -G 1 -L P -D is [ka] It is.

[0173]

[0179] In some embodiments, n is an integer from 1-100, m is an integer from 1-100, and p is an integer from 1-50.

[0174]

[0180] In some embodiments, the therapeutic agent has anti-proliferative activity against the target cell or pathway.

[0175]

[0181] In certain embodiments, the anti-proliferative activity is selected from cytostatic and / or cytotoxic activity.

[0176]

[0182] In certain embodiments, the therapeutic agent is selected from anti-cancer agents, cytotoxic drugs, radionuclides, vitamins, anti-AIDS agents, antibiotics, immunosuppressants, immunomodulatory compounds, therapeutic RNA, antiviral agents, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, tranquilizers, anticonvulsants, muscle relaxants and antiparkinsonian agents, antispasmodics and muscle contractants including channel blockers, miotics and anticholinergics, anti-glaucoma compounds, antiparasitic and / or antiprotozoal compounds, cell-extracellular matrix interaction modulators including cell proliferation inhibitors and anti-adhesion molecules, vasodilators, DNA, RNA or protein synthesis inhibitors, antihypertensives, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory agents, antiangiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotic agents, antiemetics, contrast agents.

[0177]

[0183] In certain embodiments, the therapeutic agent comprises an amino acid based molecule.

[0178]

[0184] In certain embodiments, the amino acid-based molecule includes a peptide, a polypeptide, an enzyme, an antibody, an immunoglobulin, or a functional fragment thereof.

[0179]

[0185] In certain embodiments, the therapeutic agent has a chemically reactive group.

[0180]

[0186] In certain embodiments, the chemically reactive group is -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; R 15 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0181]

[0187] In certain embodiments, G 2 teeth, [ka] wherein each R 16 is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0182]

[0188] In certain embodiments, G 2 teeth, [ka] It is.

[0183] Linker-Polymer Compounds

[0189] The present disclosure also relates to linker-polymer compounds which can be further attached to a drug moiety to form the linker-polymer-drug compounds provided herein.

[0184]

[0190] Thus, in a further aspect, the present disclosure provides a polymer scaffold of formula (II): [ka] where: The polymer scaffold comprises linear polyglycerol; L is a functional group W capable of forming a covalent bond with a targeting moiety. p is a linking moiety comprising M a is a stretcher connecting L to the -NH- moiety, each G 2 are independently functional groups that can be converted to a charged state, each G 3 independently comprises a functional group capable of reacting with a reactive group in the drug release mechanism to connect 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; q is an integer from 0 to 1000.

[0185]

[0191] In some embodiments, n is 2, m is 2, and p is 2.

[0186]

[0192] In some embodiments, q is an integer from 3 to 5.

[0187]

[0193] In some embodiments, W p can react with a functional group on the targeting moiety via a click reaction.

[0188]

[0194] In certain embodiments, W p teeth, [ka] is selected from the group consisting of:

[0189]

[0195] In some embodiments, W p can react with an amino acid on the targeting moiety.

[0190]

[0196] In certain embodiments, W p can react with amino acids on the targeting moiety, the amino acids being natural amino acids, unnatural amino acids, or combinations thereof. In certain embodiments, the natural amino acids can include cysteine, lysine, tyrosine, aspartic acid, and glutamic acid.

[0191]

[0197] In some embodiments, W p can react with one or more cysteines on the targeting moiety.

[0192]

[0198] In certain embodiments, W p can react with one or more cysteines on the targeting moiety, and each W p teeth, [ka] wherein R 1 is a sulfur protecting group, and each R 2 are independently leaving groups.

[0193]

[0199] In certain embodiments, each R 2 are independently halo or R 2a C(O)O-, where R 2a is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0194]

[0200] In some embodiments, W p can react with one or more lysines on the targeting moiety.

[0195]

[0201] In certain embodiments, W p can react with one or more lysines on the targeting moiety, and each W p is independent, [ka] is selected from the group consisting of:

[0196]

[0202] In some embodiments, W p can react with one or more unnatural amino acids on the targeting moiety.

[0197]

[0203] In certain embodiments, W p can react with one or more unnatural amino acids on the targeting moiety, and each W p is independent, [ka] is selected from.

[0198]

[0204] In some embodiments, M a teeth, R 3 , [ka] wherein: * is the site of covalent attachment to L, ** is the site of covalent attachment to the -NH- moiety, R 3 is 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)-, 4-14 membered heterocycloalkyl, -C 1~10 Alkyl-(4-14 membered heterocycloalkyl)-, -(4-14 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-(4-14 membered heterocycloalkyl)-C(=O)-, -(4-14 membered heterocycloalkyl)-C 1~10 Alkyl-C(=O)-, -C 1~10 Alkyl-NH-, -C 1~10 Heteroalkyl-NH-, -C 3~8Cycloalkyl-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-(4-14 membered heterocycloalkyl)-NH-, -(4-14 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 Alkyl-(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 is alkyl, R 5 -C(O)-NR 5a or -NR 5a -C(O)-, R 5a is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C1~6 is alkyl, R 6 is a bond or -NR 6a -(CR 6b R 6c )-C(O)-, R 6a is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is 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, 5-12 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, n 4 is an integer from 1 to 4.

[0199]

[0205] In certain embodiments, M a teeth, [ka] is selected from the group consisting of:

[0200]

[0206] In some embodiments, G 1 and G 2 is independent, [ka] wherein each R17 is independently selected from a direct bond, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0201]

[0207] In some embodiments, the polymer scaffolds provided herein have the structure of formula (IIa) or (IIb): [ka] .

[0202]

[0208] In some embodiments, the polymer scaffold provided herein has a structure of formula (IIc) or (IId): [ka] .

[0203] Modified polymer part

[0209] The present disclosure also relates to modified polymer moieties that can be further attached to linker moieties and drug moieties to form linker-polymer-drug compounds as provided herein.

[0204]

[0210] Thus, in another aspect, the present disclosure provides a polymer scaffold of formula (III): [ka] where: The polymer scaffold comprises linear polyglycerol; each G 2 are independently functional groups that can be converted to a charged state, each G 3 independently comprises a functional group capable of reacting with a reactive group in the drug release mechanism to connect 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; q is an integer from 0 to 1000.

[0205]

[0211] In some embodiments, n is 2, m is 2, and p is 2.

[0206]

[0212] In some embodiments, q is an integer from 3 to 5.

[0207]

[0213] In some embodiments, G 1 and G 2 is independent, [ka] wherein each R 17 is independently selected from a direct bond, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0208]

[0214] In some embodiments, the polymer scaffold has the structure of formula (IIIa) or (IIIb): [ka] .

[0209]

[0215] In some embodiments, the linker has the structure of formula (IIIc) or (IIId): [ka] .

[0210]

[0216] 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.

[0211]

[0217] In some embodiments, the polymer scaffold has a PDI of less than 1.5.

[0212] Targeting section

[0218] The targeting moiety directs the linker-polymer-drug conjugate to a particular tissue, cell, or subcellular location. The targeting moiety can direct the modified polymer in culture or in a whole organism, or both. In each case, the targeting moiety can bind to a ligand present on the cell surface of the target cell with effective specificity, affinity, and avidity. In some embodiments, the targeting moiety targets the modified polymer to a tissue other than the liver. In other embodiments, the targeting moiety targets the modified polymer to a particular tissue, such as the liver, kidney, lung, or pancreas. The targeting moiety can target the modified polymer to a receptor expressed on a target cell, such as a cancer cell, a matrix tissue, or a protein associated with cancer, such as a tumor antigen. Alternatively, cells containing tumor vasculature can be targeted. The targeting moiety can direct the modified polymer to specific targeting to a particular type of cell, such as hepatocytes of the liver, as opposed to Kupffer cells. In other cases, the targeting moiety can direct the modified polymer to reticuloendothelial or lymphoid cells, or to professional phagocytes, such as macrophages or eosinophils.

[0213]

[0219] In yet other embodiments, the targeting moiety can target the modified polymer to a subcellular location, such as the nucleus, cytoplasm, or endosome, etc. In certain embodiments, the targeting moiety can enhance binding to a cellular receptor, or intracytoplasmic transport and entry into the nucleus, or release from an endosome or other intracellular vesicle.

[0214]

[0220] In some embodiments, targeting moieties include antibodies, proteins and peptides or peptidomimetics.

[0215]

[0221] In some embodiments, the targeting moiety comprises a natural amino acid that can react with a functional group of the linking moiety 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.

[0216]

[0222] In certain embodiments, the targeting moiety comprises a cysteine ​​and the targeting moiety is covalently conjugated to the linker-polymer-drug conjugate via a sulfhydryl group and a functional group of the linking moiety of the linker-polymer-drug conjugate.

[0217]

[0223] In certain embodiments, the targeting moiety comprises a lysine, and the targeting moiety is covalently conjugated to the linker-polymer-drug conjugate via an amino group and a functional group of the linking moiety of the linker-polymer-drug conjugate.

[0218]

[0224] In some embodiments, the targeting moiety may comprise a non-natural amino acid that can react with a functional group of the linking moiety of the linker-polymer-drug conjugate to form a covalent bond. In certain embodiments, the targeting moiety is covalently conjugated to the linker-polymer-drug conjugate through the amino group and the functional group of the linking moiety of the linker-polymer-drug conjugate.

[0219]

[0225] In some embodiments, the targeting moiety may include a functional group that can react with a functional group of the linking moiety of the linker-polymer-drug conjugate via a click reaction to form a covalent bond.

[0220]

[0226] In some embodiments, the targeting moiety is selected from the group consisting of, 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, clumping factor, 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, T RPV1, CFTR, gpNMB, CA9, Cripto, c-KIT, c-MET, ACE, APP, adrenergic receptor-beta 2, claudin 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 (α4, α v β3, α v β5, α v β6, α1β4, α4β1, α4β7, α5β1, α6β4, α IIbβ3 integrin), 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 aeruginosa, hydrophobia, RANKL, respiratory syncytial virus, Rh 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, and the like.

[0221]

[0227] In certain embodiments, antibodies or Fab, Fab2, scFv derived antibodies 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, ErbB3, FA P, 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-beta2, claudin 3, mucin, MUC1, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, and integrin (α v β3, α v β5, α vThese include β6, α1β4, α4β1, α5β1, α6β4 integrins), tenascin-C, TRAIL-R2 and vimentin.

[0222]

[0228] Exemplary antibodies include 3F8, abagovomab, abciximab (REOPRO), adalimumab (HUMIRA), adecatumumab, afelimomab, afutuzumab, alacizumab, ALD518, alemtuzumab (CAMPATH), altumomab, amatuximab, anatumomab, anrukinzumab, apolizumab, arcitumomab (CEA-SCAN), acelizumab, atlizumab (tocilizumab, Actemra, Roactemra), atolimumab, bapineuzumab, basiliximab (Simulect), bavituximab, bectumomab (LYMPHOS CAN), belimumab (BENLYSTA), benralizumab, bertilimumab, besilesomab (SCINITIMUN), bevacizumab (AVASTIN), biciromab (FIBRISCINT), bivatuzumab, blinatumomab, brentuximab, briakinumab, canakinumab (ILARIS), cantuzumab, capromab, catumaxomab (REMOVAB), CC49, cedelizumab, certolizumab, cetuximab (ERBITUX), sitatuzumab, cixutumumab, clenoximab, clivatuzumab, conatumumab, CR6261, dace Tuzumab, daclizumab (ZENAPAX), daratumumab, denosumab (PROLIA), detumomab, dorlimomab, dorlixizumab, ecromeximab, eculizumab (SOLIRIS), edovacomab, edrecolomab (PANOREX), efalizumab (RAPTIVA), efungumab (MYCOGRAB), elotuzumab, ersilimomab, enlimomab, epitumomab, epratuzumab, erlizumab, ertumaxomab (REXOMUN), etaracizumab (ABEGRIN), exibirumab, fanolesomab (NEUTRO SPEC), faralimomab, farletuzumab, felvizumab, fezakinumab, fizitumumab, fontolizumab (HuZAF), foravirumab, fresolimumab, galiximab, gantenerumab, gavilimomab, gemtuzumab, girentuximab, glembatumumab, golimumab (SIMPONI), gomiliximab, ibalizumab, ibritumomab, igovomab (INDIMACIS-125), imuciromab (MYOSCINT), infliximab (REMICADE), intetumumab, inolimomab, inotuzumab, ipilimumab,Iratumumab, Keliximab, Labetuzumab (CEA-CIDE), Lebrikizumab, Remaresomab, Lerdelimumab, Lexatumumab, Ribivirumab, Lintuzumab, Lucatumumab, Rumiriximab, Mapatumumab, Maslimomab, Matuzumab, Mepolizumab (BOSATRIA), Metelimumab, Milatuzumab, Minletumomab, Mitumomab, Morolimumab, Motavizumab (NUMAX), Muromonab-CD3 (ORTHOCLONE) OKT3), nacolomab, naptumomab, natalizumab (TYSABRI), nebacumab, necitumumab, nerelimomab, nimotuzumab (THERACIM), nofetumomab, ocrelizumab, ozlimomab, ofatumumab (ARZERRA), olaratumab, omalizumab (XOLAIR), ontecizumab, oportuzumab, oregovomab (OVAREX), otelixizumab, pasivaximab, palivizumab (SYNAGIS), panitumumab, Tumumab (VECTIBIX), panobacumab, pascolizumab, pemtumomab (THERAGYN), pertuzumab (OMNITARG), pexelizumab, pintumomab, priliximab, pritumumab, PRO140, rafivirumab, ramucirumab, ranibizumab (LUCENTIS), raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab (RITUXAN), lobatumumab, rontalizumab, rovelizumab (L EUKARREST), ruplizumab (ANTOVA), sacituzumab, satumomab pendetide, sevirumab, sibrotuzumab, sifalimumab, siltuximab, siplizumab, solanezumab, sonepcizumab, sontuzumab, stamulumab, suresomab (LEUKOSCAN), tacatuzumab (AFP-CIDE), tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab (AUREXIS) , terimomab, tenatumomab, teneliximab, teplizumab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, tocilizumab (atlizumab, ACTEMRA), toralizumab, tositumomab (BEXXAR), trastuzumab (HERCEPTIN), tremelimumab, tucotuzumab, tuvilumab, urtoxazumab, ustekinumab (STELERA), bapaliximab, vedolizumab, veltuzumab,These include beparimomab, visilizumab (NUVION), volociximab (HUMASPECT), votumumab, zalutumumab (HuMEX-EGFr), zanolimumab (HuMAX-CD4), dillalimumab, and zolimomab.

[0223]

[0229] In some embodiments, the antibodies are directed to cell surface markers for 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, prostate cancer cells, PDGFRα, TAG-72, tenascin-C, TRAIL-R2, VEGF-A, and VEGFR2. In this embodiment, the antibody is selected from the group consisting of abagovomab, adecatumumab, alacizumab, altumomab, anatumomab, arcitumomab, bavituximab, bevacizumab (AVASTIN), bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, capromab, cetuximab, sitatuzumab, clivatuzumab, conatumumab, dacetuzumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, fizitumumab, gemtuzumab, glembatumumab, ibritumomab, igovomab, intetumumab, inotuzumab, labetuzumab, These include izumab, lexatumumab, lintuzumab, lucatumumab, matuzumab, mitumomab, naptumomab estafenatox, necitumumab, oportuzumab, oregovomab, panitumumab, pemtumomab, pertuzumab, pritumumab, rituximab (RITUXAN), rilotumumab, lobatumumab, satumomab, sibrotuzumab, taplitumomab, tenatumomab, tenatumomab, ticilimumab (tremelimumab), tigatuzumab, trastuzumab (HERCEPTIN), tositumomab, tremelimumab, tucotuzumab sermoreukin, volociximab, and zalutumumab.

[0224]

[0230] In certain embodiments, the antibody directed against the cell surface marker for HER2 is pertuzumab or trastuzumab, for EGFR (HER1) the antibody is cetuximab or panitumumab, for CD20 the antibody is rituximab, for VEGF-A the antibody is bevacizumab, for CD-22 the antibody is epratuzumab or veltuzumab, and for CEA the antibody is labetuzumab.

[0225]

[0231] 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.

[0226]

[0232] In certain embodiments, the peptides or peptidomimetics are LHRH receptor targeting peptides and ErbB2 (HER2) receptor targeting peptides.

[0227]

[0233] Exemplary proteins include insulin, transferrin, fibrinogen-gamma fragment, thrombospondin, claudins, apolipoprotein E, affibody molecules such as ABY-025, ankyrin repeat proteins, ankyrin-like repeat proteins, and synthetic peptides.

[0228]

[0234] In some embodiments, the targeting moiety-linker-polymer-drug conjugate comprises a broad spectrum cytotoxin in combination with a cell surface marker for HER2, e.g., pertuzumab or trastuzumab, for EGFR, e.g., cetuximab and panitumumab, for CEA, e.g., labetuzumab, for CD20, e.g., rituximab, for VEGF-A, e.g., bevacizumab, or for CD-22, e.g., epratuzumab or veltuzumab.

[0229]

[0235] In other embodiments, the targeting moiety-linker-drug-polymer conjugate comprises a combination of two or more targeting moieties, for example a combination of bispecific antibodies directed against EGF receptor (EGFR) on tumor cells and CD3 and CD28 on T cells, a combination of an antibody or an antibody or camelid antibody heavy chain fragment derived from a Fab, Fab2, scFv with a peptide or a peptide mimetic, a combination of an antibody or an antibody or camelid antibody heavy chain fragment derived from a Fab, Fab2, scFv with a protein, a combination of two bispecific antibodies, for example a CD3xCD19 plus CD28xCD22 bispecific antibody, etc.

[0230]

[0236] In other embodiments, the targeting moiety-linker-drug-polymer conjugate comprises a targeting moiety that is an antibody to antigen, such as, for example, trastuzumab, cetuximab, rituximab, bevacizumab, epratuzumab, veltuzumab, 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.

[0231]

[0237] In certain embodiments, the targeting moiety-linker-drug-polymer conjugate comprises a targeting moiety that is an antibody to 5T4, for example, a humanized anti-5T4 scFvFc antibody.

[0232]

[0238] Examples of suitable 5T4 targeting ligands or immunoglobulins include those commercially available or described in patent or non-patent literature, such as those described in U.S. Patent No. 8,044,178, U.S. Patent No. 8,309,094, U.S. Patent No. 7,514,546, EP1036091 (commercially available as TroVax™, Oxford Biomedica), EP2368914A1, WO2013041687A1 (Amgen), US2010 / 0173382, and P. Sapra et al., Mol. Cancer Ther. 2013, 12:38-47. Anti-5T4 antibodies are disclosed in U.S. Provisional Patent Application No. 61 / 877,439, filed September 13, 2013, and U.S. Provisional Patent Application No. 61 / 835,858, filed June 17, 2013. The entire contents of each patent and scientific publication are incorporated herein by reference.

[0233]

[0239] 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 form engineered from an anti-5T4 antibody. A single-chain variable region (scFv-Fc) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, which are connected to a linker peptide and further connected to the Fc region, which includes the hinge region and the CH2 and CH3 regions of an antibody (any combination of such antibody portions with each other or other peptide sequences is sometimes referred to herein as an "immunofusion" molecule). Within such a scFvFc molecule, the scFv section can be linked C-terminally to the N-terminus of the Fc section by a linker peptide.

[0234]

[0240] In some embodiments, the Fv portion of the 5T4 antigen binding portion may be engineered by well-known molecular biology techniques to contain one or more amino acid substitutions in the VH region. The Fc portion of the 5T4 antigen binding portion preferably comprises polypeptide sequences engineered from the human hinge, CH2 and CH3 regions of an anti-5T4 antibody.

[0235]

[0241] In some embodiments, the targeting moiety-linker-drug-polymer conjugate comprises a targeting moiety that is an antibody against TROP2, a member of the TACSTD family expressed in human trophoblasts and a single-pass transmembrane type 1 cell membrane protein involved in immune resistance common to human trophoblasts and cancer cells.

[0236]

[0242] The TROP2 antibody useful in the targeting moiety-linker-drug-polymer conjugate provided herein can be obtained using methods commonly practiced in the art, including immunizing animals with TROP2 or random polypeptides selected from the amino acid sequence of TROP2, and collecting and purifying the antibodies produced in vivo. The biological species of TROP2 used as an antigen is not limited to humans, and animals can also be immunized with TROP2 from non-human animals, such as mice or rats. In this case, the cross-reactivity between the obtained heterologous TROP2 and the antibody binding to human TROP2 can be investigated to select antibodies applicable to human diseases. Furthermore, monoclonal antibodies can be obtained from hybridomas established 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. et al., eds., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)).

[0237]

[0243] In some embodiments, Trop2 antibodies useful in the targeting moiety-linker-drug-polymer conjugates provided herein are commercially available from a number of sources, including but 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); MM0588-49D6, (Novus Biologicals, Littleton, CO); ab79976 and ab89928 (ABCAM®, Cambridge, MA).

[0238]

[0244] Other Trop2 antibodies useful in the targeting moiety-linker-drug-polymer conjugates provided herein are disclosed in patent literature.For example, US Patent Application 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), which are deposited at the Patent Biological Depository Center in Tsukuba, Japan.US ​​Patent No. 5,840,854 discloses Trop2 monoclonal antibody BR110 (ATCC No. HB11698). U.S. Patent No. 7,420,040 discloses Trop2 antibodies produced by hybridoma cell line AR47A6.4.2, deposited with IDAC (International Depository Authority of Canada, Winnipeg, Canada) under accession number 141205-05. U.S. Patent No. 7,420,041 discloses Trop2 antibodies produced by hybridoma cell line AR52A301.5, deposited with IDAC under accession number 141205-03. U.S. Patent Application Publication No. 2013 / 0122020 disclosed Trop2 antibodies 3E9, 6G11, 7E6, 15E2, 18B1. Hybridomas encoding representative antibodies were deposited with 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 hybridomas and deposited at AID-ICLC (Genoa, Italy) under accession numbers PD08019, PD08020 and PD08021. 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 a sequence listing.U.S. Patent No. 10,227,417 discloses a number of Trop2 antibodies identified by a sequence listing. The Examples section of each patent or patent application cited above in this paragraph is incorporated herein by reference. In non-patent literature, 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).

[0239]

[0245] In some embodiments, the targeting moiety-linker-drug-polymer conjugate comprises a targeting moiety that is an antibody against HER2. HER2 is one of the oncogene products of typical growth factor receptor oncogenes, identified as human epidermal growth factor receptor 2-associated oncogenes, and has a molecular weight of 185 kDa and is a transmembrane receptor protein with 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 autophosphorylate at intracellular tyrosine residues by homodimerization or heterodimerization with another EGFR receptor, HER1, HER3 or HER4, and is activated in such a manner, thereby playing an important role in cell proliferation, differentiation and survival in normal cells and tumor cells.

[0240]

[0246] The HER2 antibody useful in the targeting moiety-linker-drug-polymer conjugate provided herein is not particularly limited. The HER2 antibody can be obtained, for example, according to the method commonly practiced in the art, which includes the steps of immunizing an animal with an antigen polypeptide, and recovering and purifying the antibody produced in vivo. The source of the antigen is not limited to human, and the animal can be immunized with an antigen from a non-human animal, for example, a mouse, a rat, etc. Alternatively, the antibody-producing cell that produces an antibody against the antigen can be fused with a myeloma cell according to the method known in the art (e.g., Kohler and Milstein, Nature (1975) 256, 495-497; and Kennet, R., ed., Monoclonal Antibodies, 365-367, Plenum Press, NY (1980)) to establish a hybridoma, from which a monoclonal antibody can be subsequently obtained.

[0241]

[0247] Examples of HER2 antibodies used herein may include, but are not limited to, pertuzumab (International Patent Application Publication No. WO01 / 00245), trastuzumab (U.S. Patent No. 5,821,337), and antibodies identified by the sequence listing in U.S. Patent Application Publication No. 2019 / 0077880. However, the HER2 antibodies useful herein are not limited thereto, as long as they are HER2 antibodies that specifically bind to HER2, and more preferably have the activity of internalizing by binding to HER2 in HER2-expressing cells.

[0242] Conjugates

[0248] In one embodiment, a polymer scaffold of formula (IV): [ka] is provided, where The polymer scaffold comprises linear polyglycerol; Each L a independently, the targeting part is M a is a bivalent moiety that connects to Each Ma is independent, L a is a stretcher that connects the -NH- moiety to each G 1 is independent, L p is a functional group that connects the linear polyglycerol Each L P are independently therapeutic moieties D and G 1 This is the drug release mechanism between each D is independently a therapeutic moiety; each G 2 are independently functional groups that can be converted to 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; s is an integer from 1 to 8.

[0243]

[0249] In some embodiments, W p can react with a functional group on the targeting moiety via a click reaction.

[0244]

[0250] In certain embodiments, W p teeth, [ka] is selected from the group consisting of:

[0245]

[0251] In some embodiments, W p can react with an amino acid on the targeting moiety.

[0246]

[0252] In certain embodiments, W p can react with amino acids on the targeting moiety, the amino acids being natural amino acids, unnatural amino acids, or combinations thereof. In certain embodiments, the natural amino acids can include cysteine, lysine, tyrosine, aspartic acid, and glutamic acid.

[0247]

[0253] In some embodiments, W p can react with one or more cysteines on the targeting moiety.

[0248]

[0254] In certain embodiments, W p can react with one or more cysteines on the targeting moiety, and each W p teeth, [ka] wherein R 1 is a sulfur protecting group, and each R 2 are independently leaving groups.

[0249]

[0255] In certain embodiments, each R 2 are independently halo or R 2a C(O)O-, where R 2a is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0250]

[0256] In some embodiments, W p can react with one or more lysines on the targeting moiety.

[0251]

[0257] In certain embodiments, W p can react with one or more lysines on the targeting moiety, and each W p is independent, [ka] is selected from the group consisting of:

[0252]

[0258] In some embodiments, W p can react with one or more unnatural amino acids on the targeting moiety.

[0253]

[0259] In certain embodiments, W pcan react with one or more unnatural amino acids on the targeting moiety, and each W p is independent, [ka] is selected from.

[0254]

[0260] In some embodiments, M a teeth, R 3 , [ka] is selected from the group consisting of During the ceremony, * is the site of covalent attachment to L, ** is the site of covalent attachment to the -NH- moiety, R 3 is 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)-, 4-14 membered heterocycloalkyl, -C 1~10 Alkyl-(4-14 membered heterocycloalkyl)-, -(4-14 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 3~8Cycloalkyl)-C 1~10 Alkyl-C(=O)-, -4 to 14 membered heterocycloalkyl-C=(O)-, -C 1~10 Alkyl-(4-14 membered heterocycloalkyl)-C(=O)-, -(4-14 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-(4-14 membered heterocycloalkyl)-NH-, -(4-14 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 Alkyl-(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 3~8Cycloalkyl, -COOH or -COO-C 1~6 is alkyl, R 5 -C(O)-NR 5a or -NR 5a -C(O)-, R 5a is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is alkyl, R 6 is a bond or -NR 6a -(CR 6b R 6c )-C(O)-, R 6a is hydrogen, C 1~6 Alkyl, C 6~10 Aryl, C 3~8 Cycloalkyl, -COOH or -COO-C 1~6 is 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, 5-12 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, n 4 is an integer from 1 to 4.

[0255]

[0261] In certain embodiments, M a teeth, [ka] Selected from the group consisting of

[0256]

[0262] In some embodiments, G 1 teeth, [ka] wherein * is selected from the group consisting of L P Each R 7 is independently selected from a direct bond, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R 7a is selected from hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0257]

[0263] In some embodiments, G 1 teeth, [ka] wherein R 7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0258]

[0264] In some embodiments, each L p contains independently unstable structures.

[0259]

[0265] In certain embodiments, the labile structure is selected from a hydrolytically unstable structure or an enzymatically labile structure.

[0260]

[0266] In certain embodiments, the hydrolytically unstable structure is [ka] wherein * is selected from the group consisting of G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and R 8is selected from hydrogen, alkyl or aryl; R 9 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0261]

[0267] In certain embodiments, the hydrolytically unstable structure is [ka] wherein * is selected from the group consisting of G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and R 8 is selected from hydrogen, alkyl or aryl; R 9 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0262]

[0268] In some embodiments, G 1 teeth, [ka] where * is L P is a moiety covalently attached to R 7 is alkyl.

[0263]

[0269] In certain embodiments, -G 1 -L P -D is [ka] It is.

[0264]

[0270] In some embodiments, the enzymatically labile structure is sensitive to an enzyme selected from cathepsin B, a phosphatase, a sulfatase, or a glucuronidase.

[0265]

[0271] In certain embodiments, the enzymatically labile structure is sensitive to cathepsin B and is -Z- or [ka] wherein Z is a substrate for cathepsin B comprising 2 to 4 amino acids.

[0266]

[0272] In some embodiments, G 1 teeth, [ka] where * is L P is a moiety covalently attached to R 7 is alkyl.

[0267]

[0273] In certain embodiments, -G 1 -L P -D is [ka] It is.

[0268]

[0274] In certain embodiments, the enzymatically labile structure is sensitive to glucuronidase; [ka] where * is G 1 is the site covalently bonded to, and ** is the site covalently bonded to D.

[0269]

[0275] In some embodiments, G 1 teeth, [ka] where * is L P is a moiety covalently attached to R 7 is alkyl.

[0270]

[0276] In certain embodiments, G 1 teeth, [ka] and R 7is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl.

[0271]

[0277] In certain embodiments, -L P -D is [ka] is selected from.

[0272]

[0278] In certain embodiments, -G 1 -L P -D is [ka] is selected from.

[0273]

[0279] In certain embodiments, the enzymatically labile structure is sensitive to a phosphatase; [ka] wherein * is selected from G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and each R 10 and R 11 is independently hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0274]

[0280] In certain embodiments, G 1 teeth, [ka] It is.

[0275]

[0281] In certain embodiments, -G 1 -L P -D is [ka] [ka] is selected from the group consisting of:

[0276]

[0282] In certain embodiments, the enzymatically labile structure is susceptible to sulfatase; [ka] where * is G 1 is the site covalently bonded to D, ** is the site covalently bonded to D, and each R 12 and R 13 is independently hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0277]

[0283] In some embodiments, G 1 teeth, [ka] where R 7 is selected from hydrogen, an aliphatic, heteroaliphatic, cycloalkyl, or heterocycloalkyl moiety.

[0278]

[0284] In some embodiments, G 1 teeth, [ka] where * is L P is a moiety covalently attached to R 7 is alkyl.

[0279]

[0285] In certain embodiments, -G 1 -L P -D is [ka] It is.

[0280]

[0286] In some embodiments, n is an integer from 1-100, m is an integer from 1-100, and p is an integer from 1-50.

[0281]

[0287] In some embodiments, the therapeutic agent has anti-proliferative activity against the target cell or pathway.

[0282]

[0288] In certain embodiments, the anti-proliferative activity is selected from cytostatic and / or cytotoxic activity.

[0283]

[0289] In certain embodiments, the therapeutic agent is selected from anti-cancer agents, cytotoxic drugs, radionuclides, vitamins, anti-AIDS agents, antibiotics, immunosuppressants, immunomodulatory compounds, therapeutic RNA, antiviral agents, enzyme inhibitors, neurotoxins, opioids, hypnotics, antihistamines, tranquilizers, anticonvulsants, muscle relaxants and antiparkinsonian agents, antispasmodics and muscle contractants including channel blockers, miotics and anticholinergics, anti-glaucoma compounds, antiparasitic and / or antiprotozoal compounds, cell-extracellular matrix interaction modulators including cell proliferation inhibitors and anti-adhesion molecules, vasodilators, DNA, RNA or protein synthesis inhibitors, antihypertensives, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory agents, antiangiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotic agents, antiemetics, contrast agents.

[0284]

[0290] In certain embodiments, the therapeutic agent comprises an amino acid based molecule.

[0285]

[0291] In certain embodiments, the amino acid-based molecule includes a peptide, a polypeptide, an enzyme, an antibody, an immunoglobulin, or a functional fragment thereof.

[0286]

[0292] In certain embodiments, the therapeutic agent has a chemically reactive group.

[0287]

[0293] In certain embodiments, the chemically reactive group is -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; R 15 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0288]

[0294] In certain embodiments, G 2 teeth, [ka] wherein each R 16 is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0289]

[0295] In some embodiments, the targeting moiety is an antibody and / or a fragment thereof.

[0290]

[0296] In certain embodiments, the targeting moieties are antibodies IgG1, IgG2, IgG3 and IgG4.

[0291]

[0297] In certain embodiments, the targeting moiety is selected from the group consisting of a Fab, a Fab', a F(ab')2, a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single chain antibody molecule (scFv), a scFv dimer, a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody or a bivalent domain antibody.

[0292] Synthesis method

[0298] Any available technique can be used to make the conjugates provided herein or compositions comprising them, as well as intermediates and components (e.g., carriers and modifiers) useful for making them. For example, semi-synthetic and total synthetic methods can be used.

[0293]

[0299] The synthetic processes of the present disclosure can tolerate a wide variety of functional groups, and therefore can employ a wide variety of substituted starting materials. Although the processes generally provide the desired final compound at or near the end of the overall process, in certain cases it may be desirable to further convert the compound to a pharma- ceutically acceptable salt, ester, or prodrug thereof.

[0294]

[0300] 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 include a linking moiety suitable for connecting a targeting moiety and a linking moiety suitable for connecting a drug (D).

[0295]

[0301] In some embodiments, the conjugates provided herein are formed in several steps, including: (1) modifying the polymeric carrier such that it contains a functional group capable of reacting with a functional group of a targeting moiety or a derivative thereof and a functional group capable of reacting with a functional group of a drug or a derivative thereof; (2) reacting the modified polymer with a drug or a derivative thereof to link the drug to the modified polymer; and (3) reacting the modified polymer-drug conjugate with a targeting moiety or a derivative thereof to form a conjugate provided herein.

[0296]

[0302] In another embodiment, the conjugate is formed in several steps: (1) modifying the polymeric carrier such that it contains a functional group capable of reacting with a functional group of a targeting moiety or a derivative thereof and a functional group capable of reacting with a functional group of a first drug or a derivative thereof; (2) reacting the modified polymer with a first drug or a derivative thereof to link the first drug to the modified polymer; (3) modifying the polymer-drug conjugate such that the resulting polymer-drug conjugate contains a different functional group capable of reacting with a functional group of a second drug or a derivative thereof; (4) reacting the modified polymer-drug conjugate with a second drug or a derivative thereof to link the second drug to the modified polymer-drug conjugate; and (5) reacting the modified polymer-drug conjugate of step (4) with a targeting moiety or a derivative thereof to form a conjugate provided herein.

[0297]

[0303] The synthetic processes of the present invention can tolerate a wide variety of functional groups, and therefore can employ a wide variety of substituted starting materials. Although the processes generally provide the desired final compound at or near the end of the overall process, in certain cases it may be desirable to further convert the compound to a pharma- ceutically acceptable salt, ester, or prodrug thereof.

[0298]

[0304] The drug compounds used for the conjugates provided herein can be prepared in a variety of ways by using commercially available starting materials, compounds known in the literature, or from easily prepared intermediates, utilizing standard synthetic methods and procedures known to those skilled in the art or that will become apparent to those skilled in the art in light of the teachings herein.Standard synthetic methods and procedures for organic molecule preparation and functional group transformation and manipulation can be obtained from relevant scientific literature or from standard textbooks in the field.Without being limited to any one or several sources, 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 are useful and recognized reference textbooks of organic synthesis known to those skilled in the art, which are incorporated herein by reference.

[0299]

[0305] The conjugates of the present disclosure can be conveniently prepared by a variety of methods familiar to those skilled in the art. The conjugates of the present disclosure having each formula described herein can be prepared from commercially available starting materials or starting materials that can be prepared using literature procedures. The procedures show the preparation of representative conjugates of the present disclosure.

[0306] Once produced, the conjugates designed, selected and / or optimized by the methods described above can be characterized using a variety of assays known to those of skill in the art to determine whether the conjugate has biological activity. For example, the conjugates can be characterized by conventional assays, including but not limited to the assays described below, to determine whether the conjugate has the predicted activity, binding activity and / or binding specificity.

[0300]

[0307] Furthermore, high-throughput screening can be used to speed up the analysis using such assay.As a result, it is possible to rapidly screen the conjugate molecules described herein for activity using techniques known in the art.The general methodology for carrying out high-throughput screening is described, for example, in Devlin (1998) High Throughput Screening, Marcel Dekker, and U.S. Patent No. 5,763,263.High-throughput assay can use one or more different assay techniques, including but not limited to those described below.

[0301] Pharmaceutical Compositions

[0308] For purposes of administration, in some embodiments, the conjugates provided herein are administered as raw chemicals or formulated as pharmaceutical compositions.

[0302]

[0309] Thus, in one aspect, the disclosure provides a pharmaceutical composition comprising one or more conjugates disclosed herein and acceptable carriers, such as stabilizers, buffers, etc. The conjugates can be administered and introduced into a subject by standard methods, with or without stabilizers, buffers, etc., to form a pharmaceutical composition. Administration can be topical (including to the eye and mucous membranes, including vaginal and rectal delivery), pulmonary, such as by inhalation or insufflation of powders or aerosols, including nebulizers, intratracheal, intranasal, epithelial and transdermal, oral or parenteral, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, or intracranial, such as intrathecal or intraventricular administration. The conjugates can be formulated and used as sterile solutions and / or suspensions for injectable administration; lyophilized powders for reconstitution prior to injection / infusion; topical compositions; tablets, capsules or elixirs for oral administration; or suppositories for rectal administration, as well as other compositions known in the art.

[0303]

[0310] A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., to a cell or systemically to a subject, including, e.g., a human. The suitable form depends, in part, on the use or route of entry, e.g., oral, inhalation, transdermal, or injection / infusion. Such a form should not prevent the composition or formulation from reaching the target cell (i.e., the cell to which the drug is desired 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, toxicity, and consideration of forms that prevent the composition or formulation from exerting its effect.

[0304]

[0311] As used herein, the term "systemic administration" refers to the distribution throughout the body following systemic absorption or accumulation of modified polymer in the bloodstream in vivo. Administration routes that lead to systemic absorption include, but are not limited to, intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the modified polymer to accessible diseased tissue. It has been shown that the rate of entry of active agents into the bloodstream is a function of molecular weight or size. The use of the conjugates provided herein allows for localization of drug delivery in specific cells, such as cancer cells, via the specificity of the targeting moiety.

[0305]

[0312] As used herein, the term "pharmaceutical acceptable formulation" refers to a composition or formulation that allows effective distribution of the conjugate in the physical location that is most suitable for the desired activity of the conjugate. In some embodiments, effective delivery occurs before the generation of off-target binding that may lead to clearance by the reticuloendothelial system or reduced efficacy or toxicity. Non-limiting examples of agents suitable for formulation with the conjugate include P-glycoprotein inhibitors (e.g., Pluronic P85), which can enhance the entry of active agents into the CNS, biodegradable polymers, such as poly(DL-lactide-co-glycolide) microparticles for sustained release delivery after intracerebral implantation, and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver active agents across the blood-brain barrier and alter neuronal uptake mechanisms.

[0306]

[0313] Also included herein are pharmaceutical compositions prepared for storage or administration, comprising a pharma- ceutically effective amount of the desired conjugate in a pharma- ceutically acceptable carrier or diluent. Acceptable carriers, diluents, and / or excipients for therapy are known in pharmacology. For example, buffers, preservatives, bulking agents, dispersants, stabilizers, dyes may be provided. In addition, antioxidants and suspending agents may be used. Suitable carriers, diluents, and / or excipients include, but are not limited to, (1) Dulbecco's phosphate buffered saline, pH about 6.5, containing about 1 mg / ml to 25 mg / ml of human serum albumin, (2) 0.9% saline (0.9% w / v NaCl), and (3) 5% (w / v) dextrose.

[0307]

[0314] As used herein, the term "pharmaceutical effective amount" refers to an amount of pharmaceutical agent for treating, improving or preventing a specified disease or condition, or exhibiting a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The exact effective amount for a subject depends on the subject's weight, size and health; the nature and extent of the condition; and the therapeutic agent or combination of therapeutic agents selected for administration. The pharmaceutical effective amount for a given situation can be determined by routine experimental methods that are within the skill and judgment of the clinician.

[0308]

[0315] For any conjugate, the pharmacologic effective amount can be estimated first either in cell culture assays, e.g., tumor 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. Such information can then be used to determine doses and routes useful for administration in humans. Therapeutic / prophylactic efficacy and toxicity can be determined in cell cultures or experimental animals using standard pharmaceutical procedures, e.g., ED 50 (the dose that is therapeutically effective in 50% of the population) and LD 50 The dose ratio between toxic and therapeutic effects is the therapeutic index (the dose that is lethal to 50% of the population), and it can be expressed as a ratio, LD50 / ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage can vary within this range, depending on the dosage form utilized, sensitivity of the patient, and the route of administration.

[0309]

[0316] For example, linker-polymer-drug compounds or targeting moiety-linker-polymer-drug conjugates can be evaluated for their ability to inhibit tumor growth in several cell lines using Cell titer Glo. Dose-response curves can be generated using SoftMax Pro software, and IC 50Values ​​can be determined from a four parameter curve fit. Cell lines utilized can include a cell line that is the target of the targeting moiety and a control cell line that is not the target of the targeting moiety contained in the conjugate being tested.

[0310]

[0317] In some embodiments, the conjugate is formulated for parenteral administration by injection, including using conventional catheter techniques or infusion. The formulation for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with the addition of preservatives. The conjugate can be administered parenterally in a sterile medium. The conjugate can be suspended or dissolved in the vehicle, depending on the vehicle and concentration used. Advantageously, adjuvants, such as local anesthetics, preservatives and buffering agents, 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, and the like. In addition, pharmaceutical formulations comprising the conjugate and a pharmaceutically acceptable carrier are provided. One or more conjugates can be presented with one or more non-toxic pharmaceutically acceptable carriers and / or diluents and / or adjuvants, and other active ingredients, if desired.

[0311]

[0318] Sterile injectable preparations can be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are commonly used as solvents or suspending media. For this purpose, sterile, fixed oils can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used to prepare injectable solutions.

[0312]

[0319] The conjugate and composition described herein can be administered 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 adjuvants, emulsifiers or dispersants.

[0313]

[0320] Dosage levels on the order of about 0.001 mg to about 140 mg per kilogram of body weight per day are useful in treating the above-indicated conditions (about 0.05 mg to about 7 g per subject per day). In some embodiments, the dose administered to the patient is about 0.001 mg / kg to about 100 mg / kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 0.01 mg / kg to about 15 mg / kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 0.1 mg / kg to about 15 mg / kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 0.1 mg / kg to about 20 mg / kg of the subject's body weight. In some embodiments, the dose administered is about 0.1 mg / kg to about 5 mg / kg or about 0.1 mg / kg to about 10 mg / kg of the subject's body weight. In some embodiments, the dose administered is about 1 mg / kg to about 15 mg / kg of the subject's body weight. In some embodiments, the dose administered is about 1 mg / kg to about 10 mg / kg of the subject's body weight. The amount of conjugate that can be combined with a carrier material to produce a single dosage form varies depending on the host treated and the particular mode of administration. A unit dosage form can generally contain about 0.001 mg to about 100 mg, about 0.01 mg to about 75 mg, or about 0.01 mg to about 50 mg, or about 0.01 mg to about 25 mg of conjugate.

[0314]

[0321] For intravenous administration, dosage levels may range as described above, or may comprise from about 0.01 to about 200 mg of conjugate per kg of the animal's body weight. In some embodiments, the composition may comprise from about 1 to about 100 mg of conjugate per kg of the animal's body weight. In some embodiments, the amount administered ranges from about 0.1 to about 25 mg of compound per kg of body weight.

[0315]

[0322] In some embodiments, the conjugate can be administered as follows: The conjugate can be given either daily for about 5 days, iv, as a bolus each day for about 5 days, or as a continuous infusion for about 5 days.

[0316]

[0323] Alternatively, the conjugate can be administered once a week for six weeks or more. As another alternative, the conjugate can be administered once every two or three weeks. A bolus dose can be given in about 50 to about 400 ml of saline to which about 5 to about 10 ml of human serum albumin can be added. A continuous infusion can be given in about 250 to about 500 ml of saline to which about 25 to about 50 ml of human serum albumin can be added every 24 hours.

[0317]

[0324] In some embodiments, the patient can receive a second course of treatment about 1 to about 4 weeks after treatment. Specific clinical protocols regarding route, excipients, diluents, doses and times of administration can be determined by one skilled in the art as required by the clinical situation.

[0318]

[0325] In other embodiments, the therapeutically effective amount can be provided on another regular schedule, i.e., on a daily, weekly, monthly, or yearly basis, or on varying dosing days, weeks, months, etc. of an irregular schedule. Alternatively, the therapeutically effective amount administered can vary. In some embodiments, the therapeutically effective amount of the first administration is greater than the therapeutically effective amount of one or more subsequent administrations. In some embodiments, the therapeutically effective amount of the first administration is less than the therapeutically effective amount of one or more subsequent administrations. Equivalent doses can be administered over a variety of 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 2 weeks, about every 3 weeks, about every month, and about every 2 months. The number and frequency of administrations corresponding to a complete course of treatment are determined according to the recommendations of relevant regulatory authorities and the judgment of medical personnel. The therapeutically effective amount described herein refers to the total amount administered during a given time, i.e., if more than one different conjugate described herein is administered, the therapeutically effective amount corresponds to the total amount administered. It will be understood that the specific dose level for a particular subject will depend on a variety of factors, including the activity of the specific conjugate, age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, combination with other active agents, and the severity of the particular disease under treatment.

[0319]

[0326] For administration to non-human animals, the conjugate can also be added to animal feed or drinking water. It may be convenient to formulate animal feed and drinking water so that the animal takes in a therapeutically effective amount of the conjugate with its diet. It may also be convenient to present the conjugate as a mixture for addition to feed or drinking water.

[0320]

[0327] The conjugates 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 a condition can increase the beneficial effects while reducing the presence of side effects. In some embodiments, the conjugates are used in combination with chemotherapeutic agents, such as those disclosed in U.S. Pat. No. 7,303,749. In other embodiments, the chemotherapeutic agents include, but are not limited to, letrozole, oxaliplatin, docetaxel, 5-FU, lapatinib, capecitabine, leucovorin, erlotinib, pertuzumab, bevacizumab, and gemcitabine. The present disclosure also provides pharmaceutical kits that include one or more containers filled with a composition of the present disclosure that includes one or more conjugates and / or one or more chemotherapeutic agents. Such kits can also include, for example, other compounds and / or compositions, devices for administering the compounds and / or compositions, and instructions in a format prescribed by a government agency that oversees the manufacture, use, or sale of pharmaceutical or biological products. The compositions described herein can be packaged as a single dose or for continuous or periodic discontinuous administration.For continuous administration, the package or kit can include the conjugate in each dosage unit (e.g., solution or other above-described or drug delivery units), and optionally instructions for administering the dose daily, weekly, or monthly, for a given period or as prescribed.If it is desired to change the composition, the concentration of the components of the composition, or the relative ratio of the conjugate or drug in the composition over time, the package or kit can contain dosage units in a sequence that provides the desired variety.

[0321]

[0328] In the art, there are many packages or kits known for dispensing pharmaceuticals for periodic oral use. In some embodiments, the package has an indication for each period. In some embodiments, the package is a labeled blister package, a dial dispenser package, or a bottle. The packaging means of the kit can be adapted for administration, for example, as a syringe, a pipette, an eye dropper, or other such device, from which the formulation is applied to the affected area of ​​the body, injected into the subject, or further applied and mixed with other components of the kit. Methods for Treating Disease

[0322]

[0329] The targeting moiety-linker-polymer-drug conjugates provided herein can be used in methods of treating animals (eg, mammals, such as humans, including men, women, infants, children and adults).

[0323]

[0330] In some embodiments, the conjugates provided herein can be used in a method of treating an animal, comprising administering to the animal a conjugate of the present disclosure. The conjugates of the present invention can be used as drug carriers and drug carrier components, in controlled drug release systems, as preparations for minimally invasive surgical procedures, and the like. The pharmaceutical formulations can be injectable, implantable, and the like.

[0324]

[0331] In yet another aspect, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of at least one conjugate provided herein, wherein the conjugate biodegrades to release one or more therapeutic agents.

[0325]

[0332] In some embodiments, the conjugates provided herein can be administered in vitro, in vivo and / or ex vivo to treat a subject and / or to modulate the proliferation of selected cell populations, including, for example, cancer.In some embodiments, specific cancer types that can be treated with the conjugates provided herein include, but are not limited to, (1) fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovium, mesothelioma, 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, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, renal cell carcinoma, hepatoma bile duct carcinoma, carcinoma, choriomas, seminomas, embryonal carcinomas, nephroblastomas, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, lung cancer, epithelial carcinomas, gliomas, glioblastomas, multiforme astrocytomas, medulloblastomas, craniopharyngiomas, ependymoma, pinealomas, hemangioblastomas, acoustic neuromas, oligodendroglioma, meningiomas, skin cancers, melanomas, neuroblastomas, and retinoblastomas; (2) solid tumors, including, but not limited to, acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroleukemic leukemia (AL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocytic leukemia, acute anaplastic leukemia, chronic myelogenous leukemia "CML", chronic lymphocytic leukemia "CLL", hairy cell leukemia, multiple myeloma, acute and chronic leukemias, e.g., lymphoblastic myelogenous and lymphocytic myelocytic leukemia, and (3) lymphomas, e.g., Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, and polycythemia vera.

[0326]

[0333] In some embodiments, the conjugates provided herein can be administered to treat a subject in vitro, in vivo and / or ex vivo and / or to modulate the proliferation of select cell populations in patients with anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma, esophageal cancer, eye cancer, laryngeal cancer, mouth cancer, mesothelioma, skin cancer, myeloma, oral cancer, rectal cancer, pharyngeal cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, pancreatic cancer, renal cancer, or gastric cancer.

[0327]

[0334] In certain embodiments, the cancer is selected from the group consisting of breast cancer, gastric cancer, non-small cell lung cancer (NSCLS) and ovarian cancer.

[0328]

[0335] 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 developing, and / or delay the onset of a particular condition, such as cancer. For example, the conjugates provided herein are useful in treating, preventing, delaying, or otherwise ameliorating 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, pharyngeal cancer, mouth cancer, mesothelioma, skin cancer, myeloma, oral cancer, rectal cancer, pharyngeal 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.

[0329]

[0336] 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, amebiasis, schistosomiasis, and the like.

[0330]

[0337] In some embodiments, the conjugates provided herein can also be used to manufacture a medicament useful, for example, for treating or lessening the severity of a disorder characterized by abnormal proliferation of cells (e.g., cancer).

[0331]

[0338] In some embodiments, the therapeutic agent is delivered locally to a specific target cell, tissue or organ.

[0332]

[0339] In certain embodiments, the conjugate provided herein can further comprise or be associated with diagnostic label.In certain embodiments, diagnostic label 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, agents for ultrasound diagnostics, neutron activators, moieties that can reflect, scatter or act on X-rays, ultrasound, radio waves and microwaves, and fluorophores.In certain exemplary embodiments, the conjugate is further monitored in vivo.

[0333]

[0340] 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, agents for ultrasound diagnostics, neutron activators, and moieties that can reflect, scatter, or affect X-rays, ultrasound, radio waves, and microwaves, fluorophores in various optical procedures, and others. Diagnostic radiopharmaceuticals include gamma-ray emitting radionuclides, such as indium-111, technetium-99m, and iodine-131, and others. Contrast agents for MRI (magnetic resonance imaging) include magnetic compounds, such as paramagnetic ions, iron, manganese, gadolinium, lanthanides, organic paramagnetic moieties, and superparamagnetic, ferroparamagnetic, and antiferromagnetic compounds, such as iron oxide colloids, ferrite colloids, and others. Contrast agents for computed tomography and other X-ray-based imaging include X-ray absorbing compounds such as iodine, barium, etc. Contrast agents for ultrasound-based methods include compounds that can absorb, reflect, and scatter ultrasound, such as emulsions, crystals, bubbles, etc. Still other examples include substances useful for neutron activation, such as boron and gadolinium. In addition, labels can be utilized that can reflect, refract, scatter, or otherwise affect radiation useful in X-ray, ultrasound, radio waves, microwaves, and other diagnostic procedures. Fluorescent labels can be used for photographic imaging. In certain embodiments, the modifier includes a paramagnetic ion or group.

[0334]

[0341] In another aspect, the disclosure provides a method of treating a disease or disorder in a subject, the method comprising preparing an aqueous formulation of at least one conjugate provided herein and parenterally injecting the formulation in a subject.

[0335]

[0342] In another aspect, the present disclosure provides a method of treating a disease or disorder 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.

[0336]

[0343] In another aspect, the disclosure provides a method of treating an animal in need thereof comprising administering a conjugate according to the methods described above.

[0337]

[0344] In another aspect, the disclosure provides a method of raising an immune response in an animal comprising administering a conjugate as in the methods described above.

[0338]

[0345] In another aspect, the disclosure provides a method of diagnosing a disease in an animal comprising administering a conjugate as in the methods described above, wherein the conjugate comprises a detectable molecule, and detecting the detectable molecule.

[0339]

[0346] In some embodiments, the step of detecting the detectable molecule is performed non-invasively, hi some embodiments, the step of detecting the detectable molecule is performed using a suitable imaging device.

[0340]

[0347] In some embodiments, the method of treating an animal includes administering a conjugate provided herein as a packing for a surgical wound from which a tumor or growth is removed. The packing of the conjugate is replaced at the tumor site during recovery and degrades and dissipates as the wound heals.

[0341]

[0348] In certain embodiments, the conjugates provided herein are associated with a diagnostic label for in vivo monitoring.

[0342]

[0349] The conjugates provided herein can be used for therapeutic, preventative and analytical (diagnostic) treatment of animals. The conjugates are generally intended for parenteral administration, although in some cases they can be administered by other routes.

[0343]

[0350] 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.

[0344]

[0351] In some embodiments, conjugates containing a detectable label are administered to examine patterns and kinetics of label distribution within an animal.

[0345]

[0352] In certain embodiments, any one or more of the conjugates provided herein can be used in practicing any of the methods described above.

[0346]

[0353] Throughout the description, when a composition is described as having, including, or containing a specific compound, it is also contemplated that the composition consists essentially of or consists of the recited components. Similarly, when a method or process is described as having, including, or containing specific process steps, the process also consists essentially of or consists of the recited process steps. Furthermore, it should be understood that the order of steps or the order in which certain actions are performed is not important so long as the invention remains operable. Furthermore, two or more steps or actions can be performed simultaneously.

[0347]

[0354] All publications and patent documents cited in this specification are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. The citation of publications and patent documents is not intended as an admission that any are pertinent prior art, and does not constitute any admission as to the contents or date of the same. EXAMPLES

[0348]

[0355] For illustrative purposes, the following examples are included. The examples provided herein describe the synthesis of the compounds and conjugates disclosed herein, as well as intermediates used to prepare the compounds and conjugates. However, it is understood that these examples do not limit the disclosure, but are only meant to suggest a method of practicing the disclosure. Those skilled in the art will recognize that the described chemical reactions can be easily adopted to prepare many other compounds or conjugates of the disclosure, and alternative methods of preparing the compounds or conjugates of the disclosure are considered to be within the scope of the disclosure. Those skilled in the art will also understand that the individual steps or separate batches of compounds described herein can be combined. Alternatively, it will be understood that other reactions disclosed herein or known in the art have applicability to prepare other compounds of the disclosure. Therefore, the following description is not intended to limit the scope of the disclosure, which is defined by the claims appended hereto.

[0349]

[0356] Abbreviation

[0357] The following abbreviations are used in the reaction schematics and synthetic examples which follow: This list is not meant to be an all-inclusive list of abbreviations used herein, and additional standard abbreviations readily understood by those skilled in the art of organic synthesis may also be used in the synthetic schematics and examples.

[0350] [Table 1]

[0351] Example 1 Synthesis of copolymer linker compound 1 [ka]

[0352]

[0358] A solution of N3NBu4 (1.98 g, 6.98 mmol), EGE (61 g, 418.6 mmol) and t-BGA (39.3 g, 209.3 mmol) in diglyme (200 mL) was added under N2. The mixture was cooled to -40 °C, then Al(iBu)3 (30 ml, 29.7 mmol, 1 M in hexanes) was added slowly. The reaction was allowed to warm to room temperature and stirred under N2 for 16 h. The reaction was quenched with EtOH, diluted with water and extracted with EA. The combined organic layers were washed with brine, dried with Na2SO4, filtered and concentrated under reduced pressure to give compound 1-1 (103 g) as a colorless oil with Mw 27,000 as determined by GPC using PS as standard.

[0353]

[0359] To a solution of compound 1-1 (50 g, 1.85 mmol) in DCM (500 mL), HCl (200 mL, 1 M in 1,4-dioxane) was added slowly and the reaction mixture was stirred at room temperature for 16 h. After completion, the solvent was removed under reduced pressure and the residue was dissolved in 10% NaOH solution. After adjusting the pH to 7 with 4N HCl, the total volume of the solution was adjusted to 500 mL with water to give compound 1-2 as an aqueous solution.

[0354]

[0360] To solution 1-2, Na2S·9H2O (30 g, 125 mmol) was added and the reaction mixture was heated at 100 °C for 18 h. Upon completion, the reaction was cooled to room temperature and the pH was adjusted using 4N HCl. The mixture was dialyzed using a 3 KD MWCO to give compound 1-3 as an aqueous solution (300 mL).

[0355]

[0361] To solution 1-3 was added NaOH (5 g, 125 mmol) and (Boc)2O (30 g, 125 mmol) and the reaction mixture was stirred at room temperature for 18 h. Upon completion, the mixture was dialyzed using a 3 KD MWCO to give compound 1 as an aqueous solution (800 mL, 25 mg / mL).

[0356]

[0362] 1H NMR (400 MHz, D2O) δ 3.95 (s, 100H), 3.87-3.57 (br, 800H), 1.43 (s, 9H).

[0357] Example 2 Synthesis of compound 2 [ka]

[0358]

[0363] To a solution of 2-1 in THF (200 mL) was added pyridine (12 mL, 148 mmol) and Boc2O (1.44, 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 x 100 mL) and saturated NaHCO3 (100 mL). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo to give the desired product 2-2 as a white solid (2.45 g).

[0359]

[0364] 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% NaHCO3 (2 x 400 mL), water (400 mL) and 0.1 N HCl (2 x 400 mL). The organic layer was dried over anhydrous MgSO4, filtered and evaporated under vacuum. The residue was purified by silica gel column chromatography to give the desired product 2-3 as a white solid (3.0 g, 87.7% yield).

[0360]

[0365] 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 in vacuo and the residue was purified by prep HPLC (TFA) to give the desired product 2-4 as a yellow solid (627 mg).

[0361]

[0366] To the aqueous solution of compound 1, a solution of NHS (0.12 mg, 1.05 μmol), EDC (20 mg, 105.4 μmol) and 2-4 (23.4 mg, 42.2 μmol) in DMF was slowly added at 0° C., and the reaction mixture was stirred at room temperature for 16 h. After completion, the mixture was dialyzed using a 5 KD MWCO to give compound 2-5 as an aqueous solution (25 mL, 5 mg / mL).

[0362]

[0367] To a solution of compound 2-5 in water (25 mL, 5 mg / mL) was slowly added 4N HCl (1.6 mL) at 0° C. and the reaction mixture was stirred at room temperature for 16 h. After completion, saturated NaHCO 3(aq) The pH of the mixture was adjusted to 7 using to give compound 2-6, which was used directly in the next step.

[0363]

[0368] To the aqueous solution of compound 2-6, NHS-PEG4-Mal (32.6 mg, 5.25 μmol) in DMF (10 mL) was slowly added at 0° C., and the reaction mixture was stirred at room temperature for 16 h. After completion, the mixture was dialyzed using a 5 KD MWCO to give compound 2 as an aqueous solution (15 mL, 5 mg / mL).

[0364] Example 3 Synthesis of compound 3 [ka]

[0365]

[0369] To a solution of 3-1 (10 g, 26.6 mmol) in DCM (100 mL) was added TiBr4 (10.7 g, 29.2 mmol) under N2. The reaction mixture was stirred at room temperature for 15 h. The mixture was filtered, the filtrate was concentrated, and the residue was purified using silica gel column chromatography with petroleum ether / EtOAc = 10:1 to give compound 3-2 as a white solid (8.0 g, 20.1 mmol, 75.7% yield).

[0370] To a solution of 3-2 (8.0 g, 20.1 mmol) in MeCN (160 mL) was added 3-3 (3.39 g, 20.1 mmol) and Ag2O (5.12 g, 22.1 mmol) under N2. After stirring at room temperature in the dark for 15 h, the mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified using silica gel column chromatography with petroleum ether / DCM=10:1 to give compound 3-4 as a white solid (4.8 g, 9.94 mmol, 49.4% yield).

[0366]

[0371] To a mixture of 3-4 (4.8 g, 9.94 mmol) and silica gel (2.12 g) in CHCl3 (50 mL) and iPrOH (10 mL) was added NaBH4 (564 mg, 14.91 mmol) under N2 at 0 °C. After stirring at 25 °C for 1.5 h, the mixture was filtered and the solvent was evaporated to give the product 3-5 as a colorless oil (5.0 g, 10.3 mmol, crude).

[0367]

[0372] 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) under N2, 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 NH4Cl (3 x 50 mL) and brine (3 x 50 mL). The combined organic layers were dried over Na2SO4, filtered and the solvent removed in vacuo. The crude product was purified by silica gel column chromatography (PE:EA = 4:1) to give 3-6 as a white solid (6.2 g, 10.3 mmol, 100% yield).

[0368]

[0373] 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 under H2 (1 atm) at 25 °C 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 as a white solid (3.8 g, 6.67 mmol, 95% yield).

[0369]

[0374] 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). After stirring at room temperature under N2 atmosphere for 16 h, the reaction mixture was diluted with water (100 mL) and extracted with DCM (3 x 100 mL). The organic layer was washed with brine (100 mL), dried over Na2SO4, and concentrated in vacuo to give the crude product, which was purified by flash chromatography to give the product 3-9 as a white solid (2.4 g, 3.24 mmol, 48.6% yield).

[0370]

[0375] To a solution of 3-9 (2.4 g, 3.24 mmol) in THF (20 mL) was added TEA·3HF (2 mL). The reaction mixture was stirred at room temperature for 1.5 h. After completion, the solvent was removed and the residue was purified by silica gel column chromatography to give the desired product 3-10 as a white solid (1.75 g, 2.79 mmol, 86% yield).

[0371]

[0376] 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) under N2. After stirring at 25 °C for 16 h, the solvent was removed and the residue was purified by silica gel column chromatography to give the desired product 3-12 as a white solid (800 mg, 1.01 mmol, 57% yield).

[0372]

[0377] To 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-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide (181 mg, 0.25 mmol), HOBT (6.7 mg, 0.025 mmol) and TEA (50.6 mg, 0.5 mmol) under N2. 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×2), dried over Na2SO4, filtered, and concentrated in vacuo to give the crude product, which was purified by pre-HPLC to give the desired product 3-13 (202 mg, 0.147 mmol, 59% yield).

[0373]

[0378] 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 h, the reaction was diluted with water (50 mL) and extracted with EtOAc (50 mL×3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated in vacuo to give 3-14 (300 mg, 0.24 mmol, 84% yield) as a colorless oil.

[0374]

[0379] 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 h, the reaction was concentrated in vacuo to give the crude product, which was purified by prep HPLC (TFA) to give the desired product 3-15 (120 mg, 0.106 mmol, 44% yield).

[0375]

[0380] To an aqueous solution of compound 1 (150 mg, 6 mL, 25 mg / mL), a solution of NHS in DMF (0.12 mg, 1.05 μmol), EDCI (20 mg, 105.4 μmol) and 3-15 (23.4 mg, 42.2 μmol) were slowly added at 0° C., and the reaction solution was stirred at room temperature for 16 hours. Upon completion, the mixture was dialyzed with a 5 KD MWCO to give compound 3-16 as an aqueous solution (25 mL, 5 mg / mL).

[0376]

[0381] To a solution of compound 3-16 in water (25 mL, 5 mg / mL) was slowly added 4N HCl (1.6 mL) at 0° C. and the reaction mixture was stirred at room temperature for 16 h. After completion, the pH of the mixture was adjusted to 7 using saturated NaHCO3(aq) to give compound 3-17, which was used directly in the next step.

[0377]

[0382] To the aqueous solution of compound 3-17, NHS-PEG4-Mal (32.6 mg, 5.25 μmol) in DMF (10 mL) was slowly added at 0° C. and the reaction mixture was stirred at room temperature for 16 h. After completion, the mixture was dialyzed using a 5 KD MWCO to give compound 3 as an aqueous solution (19 mL, 5 mg / mL).

[0378] Example 4 Synthesis of compound 4 [ka]

[0379]

[0383] 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 h. 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 as a yellow oil (20.1 g, 70 mmol, 96% yield).

[0380]

[0384] To a solution of 4-2 (20 g, 70 mmol) in pyridine (60 mL) was added Ac2O (30 mL). The reaction mixture was stirred at room temperature for 16 h. After completion, the solvent was removed in vacuo and the residue was dissolved with DCM (400 mL) and then washed with water (400 mL) and brine (2 x 400 mL). The organic layer was dried over anhydrous Na2SO4, filtered and evaporated under vacuum. The residue was purified by silica gel column chromatography to give the desired product 4-3 as a white solid (10 g, 31.6% yield).

[0381]

[0385] 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 h. The solvent was co-evaporated under vacuum with toluene (3×500 mL). The residue was dissolved with DCM (500 mL) and then washed with water (500 mL) and brine (2×500 mL). The organic layer was dried over anhydrous Na2SO4, filtered and evaporated under vacuum. The residue was purified by silica gel column chromatography to give the desired product 4-4 as a white solid (1.8 g, 3.8 mmol, 34.5% yield).

[0382]

[0386] 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 Ag2O (2.45 g, 10.57 mmol). The reaction mixture was stirred at room temperature for 2 h in the dark. The solvent was removed in 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.

[0383]

[0387] To a mixture of 4-6 (5.1 g, 9.12 mmol) and silica gel (2.12 g) in CHCl3 (50 mL) and iPrOH (10 mL) was added NaBH4 (517 mg, 13.68 mmol). After stirring at 25 °C for 1.5 h, 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 as a white solid (4.8 g, 8.5 mmol, 93.8% yield).

[0384]

[0388] 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 N2 atmosphere. After stirring for 5 min, 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 h. The mixture was diluted with CHCl2 and the organic layer was washed with NH4Cl(aq) (3×150 mL) and brine (3×150 mL). The combined organic layers were dried over Na2SO4, filtered and the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography (PE:EA=4:1) to give compound 4-8 as a white solid (5 g, 79.9% yield).

[0385]

[0389] To a solution of 4-8 (3.87 g, 5.7 mmol) in EtOH (20 mL) and HO (5 mL) was added Fe (1.59 g, 28.5 mmol) and NHCl (aq)(1.54 g, 28.5 mmol) was added. After stirring at 65 °C for 2 h, the reaction mixture was quenched with NH4Cl(aq) (150 mL) and extracted with EA (2 x 150 mL). The combined organic layers were washed with brine (2 x 150 mL), dried over anhydrous Na2SO4, filtered and evaporated in vacuo. The residue was purified by flash silica gel column chromatography to give 4-9 as a yellow solid (3.0 g, 81.5% yield).

[0386]

[0390] 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 at room temperature under N2 atmosphere for 16 h. The reaction was quenched with water (100 mL) and extracted with DCM (3 x 100 mL). The combined organic layers were washed with brine (3 x 100 mL), dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by silica gel column chromatography to give 4-11 as a white solid (3.2 g, 3.92 mmol, 84.3% yield).

[0387]

[0391] To a solution of 4-11 in THF (20 mL) was added TEA3HF (5 mL). The reaction mixture was stirred at room temperature for 1.5 h. After completion, the solvent was removed and the residue was purified by silica gel column chromatography to give 4-12 as a white solid (2 g, 66.4% yield).

[0388]

[0392] 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 h, the reaction was concentrated and purified by silica gel column chromatography to give 4-14 as a yellow oil (930 mg, 1.07 mmol, 75.5% yield).

[0389]

[0393] 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 h, the mixture was quenched with water and extracted with EtOAc (200 mL x 3). The combined organic layers were washed with brine (100 mL x 2), dried over Na2SO4, filtered and concentrated. The residue was purified by pre-HPLC to give 4-16 (300 mg, 2 mmol, 18.0% yield).

[0390]

[0394] 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 for 1 h under 1 atm H2. The mixture was filtered and the solvent was evaporated to give 4-17 (250 mg, 0.17 mmol, 88.0% yield).

[0391]

[0395] To a solution of 4-17 (250 mg, 0.17 mmol) in MeOH (3 mL) was added Na2CO3 (60 mg, 0.6 mmol). The reaction mixture was stirred at 25 °C for 16 h. After filtration, the solvent was removed to give 4-18 (250 mg, 0.17 mmol, 99% yield).

[0392]

[0396] 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 h, the solvent was removed and the residue was purified by pre-HPLC to give the desired product 4-19 (100 mg, 0.08 mmol, 44.4% yield).

[0393]

[0397] To an aqueous solution of compound 1 (150 mg, 6 mL, 25 mg / mL), NHS (0.12 mg, 1.05 μmol), EDC (20 mg, 105.4 μmol) and 4-19 (23.4 mg, 42.2 μmol) in DMF were slowly added at 0° C., and the reaction mixture was stirred at room temperature for 16 h. After completion, the mixture was dialyzed using a 5 KD MWCO to give compound 4-20 as an aqueous solution (25 mL, 5 mg / mL).

[0394]

[0398] To a solution of compound 4-20 in water (25 mL, 5 mg / mL) was slowly added 4N HCl (1.6 mL) at 0° C. and the reaction mixture was stirred at room temperature for 16 h. After completion, the pH of the mixture was adjusted to 7 using saturated NaHCO3(aq) to give an aqueous solution of compound 4-21, which was used directly in the next step.

[0395]

[0399] To the aqueous solution of compound 4-21, NHS-PEG4-Mal (32.6 mg, 5.25 μmol) in DMF (10 mL) was slowly added at 0° C. and the reaction mixture was stirred at room temperature for 16 h. After completion, the mixture was dialyzed using a 5 KD MWCO to give compound 4 as an aqueous solution (24 mL, 5 mg / mL).

[0396] Example 5 Synthesis of compound 5 [ka]

[0397]

[0400] 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 h. Upon completion, the mixture was purified by prep HPLC (TFA) to give the desired product 5-2 as a white solid (200 mg, 0.169 mmol, 47.5% yield).

[0398]

[0401] To an aqueous solution of compound 1 (150 mg, 6 mL, 25 mg / mL), a DMF solution of NHS (0.12 mg, 1.05 μmol), EDC (20 mg, 105.4 μmol) and 5-2 (23.4 mg, 42.2 μmol) was slowly added at 0° C., and the reaction mixture was stirred at room temperature for 16 h. Upon completion, the mixture was dialyzed using a 5 KD MWCO to give compound 5-3 as an aqueous solution (25 mL, 5 mg / mL).

[0399]

[0402] To a solution of compound 5-3 in water (25 mL, 5 mg / mL) was slowly added 4N HCl (1.6 mL) at 0° C. and the reaction mixture was stirred at room temperature for 16 h. After completion, the pH of the mixture was adjusted to 7 using saturated NaHCO3(aq) to give an aqueous solution of compound 5-4, which was used directly in the next step.

[0400]

[0403] To the aqueous solution of compound 5-4, NHS-PEG4-Mal (32.6 mg, 5.25 μmol) in DMF (10 mL) was slowly added at 0° C., and the reaction mixture was stirred at room temperature for 16 h. After completion, the mixture was dialyzed using a 5 KD MWCO to give compound 5 as an aqueous solution (16 mL, 5 mg / mL).

[0401] Example 6 Synthesis of compound 6 [ka]

[0402]

[0404] To a solution of 6-1 (2.5 g, 6.87 mmol) in THF (400 mL) was added 6-2 (5.55 g, 27.6 mmol) and TEA (10 mL) under N2. The reaction was stirred at room temperature for 16 h. The reaction was diluted with water (100 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, and concentrated in vacuo to give a residue which was purified by silica gel column chromatography to give the desired product 6-3 as an off-white solid (2.5 g, 4.7 mmol, 69% yield).

[0403]

[0405] To a solution of 6-3 (3.3 g, 6.24 mmol) in THF (300 mL) was added 6-4 (1.5 g, 7.9 mmol) and TEA (815 mg, 8.05 mmol) under N2. The reaction was stirred at room temperature for 16 h. The reaction was quenched with water (100 mL) and extracted with EtOAc (100 mL x 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, and concentrated in vacuo to give a residue which was purified by silica gel column chromatography to give the desired product 6-5 as a white solid (2.1 g, 3.6 mmol, 58% yield).

[0404]

[0406] To a solution of 6-5 (1.0 g, 1.73 mmol) in DCM (50 mL) was added tetrazole (242 mg, 3.46 mmol) at room temperature under N2. After stirring for 0.5 h, 6-6 (1.07 g, 3.11 mmol) was added dropwise at 0 °C. The resulting solution was stirred at room temperature for 16 h. After completion, mCPBA (559 mg, 2.59 mmol) was added at 0 °C and stirred for another 16 h. The reaction was then diluted with water (100 mL) and the organic layer was washed with sodium metabisulfite, aqueous sodium bicarbonate, and dried over Na2SO4. The mixture was filtered, the solvent was evaporated, and the residue was purified by prep HPLC (water / ACN with 0.1% TFA) to give the desired product 6-7 as a yellow solid (1.0 g, 69% yield).

[0405]

[0407] To a solution of 6-7 (1.0 g, 1.19 mmol) in DCM (20 mL) was added TFA (20 mL) at 0 °C under N2. The reaction was stirred for 16 h at room temperature. The reaction was concentrated in vacuo to give the crude product, which was purified by prep HPLC (TFA) to give the desired product 6 as a yellow solid.

[0406]

[0408] 1H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H), 7.93 (dd, J = 25.8, 9.1 Hz, 2H), 7.59 (s, 1H), 7.43 (s, 1H), 5.60 (d, J = 16.5 Hz, 1H), 5.40 (d, J = 16.6 Hz, 1H), 5.18 (s, 2H), 3.94 (s, 1H), 3.40 (s, 1H), 3.29 - 3.17 (m, 2H), 3.08 (d, J = 15.4 Hz, 3H), 2.78 (s, 3H), 2.14 (d, J = 7.3 Hz, 2H), 1.03 (t, J = 7.2 Hz, 3H).

[0407] Example 7 Synthesis of compound 7 [ka]

[0408]

[0409] To a solution of 7-1 (25.0 g, 114.01 mmol) in DCM (250 mL) was added 2,2'-azanediylbis(ethan-1-ol) (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), extracted with EA (700 mL) and the organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography to give the desired product 7-2 as a yellow oil (25 g, 114 mmol, 89%).

[0409]

[0410] 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 extracted with EA (700 mL). The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography to give the desired product 7-3 as a colorless oil (25.5 g, 49.7 mmol, 43%).

[0410]

[0411] 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 h, the reaction mixture was concentrated to give the crude product 7-4 as a colorless oil (39 g, 51.9 mmol, 100%), which was used in the next step without further purification.

[0411]

[0412] 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 h. The solvent was removed in vacuo and the residue was purified by flash column chromatography with DCM / MeOH (20 / 1, v / v) to give the desired product 7-5 as a white solid (21 g, 30.2 mmol, 58%).

[0412]

[0413] To a solution of 7-5 (5 g, 7.2 mmol) in DCM (50 mL), 1-hydroxypyrrolidine-2,5-dione (1.24 g, 10.79 mmol) and DCC (5.94 g, 28.8 mmol) were added at room temperature and stirred for 16 h. The solvent was removed in vacuum and the residue was purified by flash column chromatography with PE / EtOAc (1 / 1, v / v) to give the desired product 7-6 as a colorless oil (5 g, 6.3 mmol, 88%).

[0413]

[0414] 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 h. The mixture was diluted with water and extracted with EA. The organic layer was dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was purified by flash silica gel column chromatography with DCM / MeOH (20 / 1, v / v) to give the desired product 7-8 as a green solid (750 mg, 0.67 mmol, 67%).

[0414]

[0415] To a solution of 7-8 (750 mg, 0.67 mmol) in THF (8 mL) was added TBAF (2 mL, 1 M in THF, 2 mmol) dropwise at room temperature. The reaction mixture was stirred at room temperature for 16 h. The mixture was concentrated in vacuo 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 as a black solid (500 mg, 0.57 mmol, 85% yield).

[0415]

[0416] To a solution of 7-9 (500 mg, 0.57 mmol) in pyridine (5 mL) was added SO3pyridine (455 mg, 2.86 mmol) at room temperature and the mixture was stirred for 16 h. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography with MeOH / H2O (1 / 1, v / v) to give 7-10 (500 mg, 0.52 mmol, 92%) as a yellow solid.

[0416]

[0417] 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 h. The solvent was removed under vacuum and the residue was purified by C18 column chromatography (MeOH / HO (1 / 1, v / v)) to give the desired product 7 as a yellow solid (229 mg, 0.31 mmol, 60%).

[0417]

[0418] 1H NMR (400 MHz, DMSO) δ 8.92 (s, 1H), 8.31 (s, 3H), 7.78 (d, J = 10.9 Hz, 1H), 7.30 (s, 1H), 5.67 - 5.12 (m, 5H), 4.44 (s, 2H), 3.96 (d, J = 62.4 Hz, 6H), 3.59 - 2.98 (m, 6H), 2.44 - 2.04 (m, 5H), 1.85 (tt, J = 14.0, 7.1 Hz, 2H), 1.46 - 1.17 (m, 3H), 0.86 (t, J = 7.3 Hz, 3H).

[0418] Example 8 Synthesis of compound 8 [ka]

[0419] 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 h. After completion, the mixture was purified by prep HPLC (TFA) to give the desired product 8-2 as a white solid (200 mg, 0.169 mmol, 47.5% yield).

[0420] To an aqueous solution of compound 1 (150 mg, 6 mL, 25 mg / mL), a DMF solution of NHS (0.12 mg, 1.05 μmol), EDC (20 mg, 105.4 μmol) and 8-2 (23.4 mg, 42.2 μmol) was slowly added at 0° C., and the reaction mixture was stirred at room temperature for 16 h. After completion, the mixture was dialyzed using a 5 KD MWCO to give compound 8-3 as an aqueous solution (25 mL, 5 mg / mL).

[0421] To a solution of compound 8-3 in water (25 mL, 5 mg / mL) was slowly added 4N HCl (1.6 mL) at 0° C. and the reaction mixture was stirred at room temperature for 16 h. After completion, the pH of the mixture was adjusted to 7 using saturated NaHCO3(aq) to give an aqueous solution of compound 8-4, which was used directly in the next step.

[0422] To the aqueous solution of compound 8-4, NHS-PEG4-Mal (32.6 mg, 5.25 μmol) in DMF (10 mL) was slowly added at 0° C. and the reaction mixture was stirred at room temperature for 16 h. After completion, the mixture was dialyzed using a 5 KD MWCO to give compound 8 as an aqueous solution (16 mL, 5 mg / mL).

[0423] Example 9 Synthesis of ADC-1

[0423] TCEP (8 μL, 17.4 mM, 2 eq) was added to 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. The reaction was then quenched by adding 50 equivalents of L-cysteine. The mixture was purified through a cation exchange column to obtain ADC-1 (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy.

[0424] Example 10 Synthesis of ADC-2

[0424] TCEP (8 μL, 17.4 mM, 2 eq) was added to 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. The reaction was then quenched by adding 50 equivalents of L-cysteine. The mixture was purified through a cation exchange column to obtain ADC-2 (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy.

[0425] Example 11 Synthesis of ADC-3

[0425] TCEP (8 μL, 17.4 mM, 2 eq) was added to 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. The reaction was then quenched by adding 50 equivalents of L-cysteine. The mixture was purified through a cation exchange column to obtain ADC-3 (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy.

[0426] Example 12 Synthesis of ADC-4

[0426] TCEP (8 μL, 17.4 mM, 2 eq) was added to a buffer solution (PB, histidine, TEAA, sodium acetate buffer) of Trop2 antibody Trop2-1 (5 mg, 2.5 mg / ml). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 2 were added and reacted for another 1.5 hours. The reaction was then quenched by adding 50 equivalents of L-cysteine. The mixture was purified through a cation exchange column to obtain ADC-4 (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy.

[0427] Example 13 Synthesis of ADC-5

[0427] TCEP (8 μL, 17.4 mM, 2 eq) was added to a buffer solution (PB, histidine, TEAA, sodium acetate buffer) of Trop2 antibody Trop2-1 (5 mg, 2.5 mg / ml). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 3 were added and reacted for another 1.5 hours. The reaction was then quenched by adding 50 equivalents of L-cysteine. The mixture was purified through a cation exchange column to obtain ADC-5 (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy.

[0428] Example 14 Synthesis of ADC-6

[0428] TCEP (8 μL, 17.4 mM, 2 eq) was added to a buffer solution (PB, histidine, TEAA, sodium acetate buffer) of Trop2 antibody Trop2-1 (5 mg, 2.5 mg / ml). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 4 were added and reacted for another 1.5 hours. The reaction was then quenched by adding 50 equivalents of L-cysteine. The mixture was purified through a cation exchange column to obtain ADC-6 (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy.

[0429] Example 15 Synthesis of ADC-7

[0429] TCEP (8 μL, 17.4 mM, 2 eq) was added to a buffer solution (PB, histidine, TEAA, sodium acetate buffer) of Trop2 antibody Trop2-2 (5 mg, 2.5 mg / ml). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 2 were added and reacted for another 1.5 hours. The reaction was then quenched by adding 50 equivalents of L-cysteine. The mixture was purified through a cation exchange column to obtain ADC-7 (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy.

[0430] Example 16 Synthesis of ADC-8

[0430] TCEP (8 μL, 17.4 mM, 2 eq) was added to a buffer solution (PB, histidine, TEAA, sodium acetate buffer) of Trop2 antibody Trop2-2 (5 mg, 2.5 mg / ml). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 3 were added and reacted for another 1.5 hours. The reaction was then quenched by adding 50 equivalents of L-cysteine. The mixture was purified through a cation exchange column to obtain ADC-8 (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy.

[0431] Example 17 Synthesis of ADC-9

[0431] TCEP (8 μL, 17.4 mM, 2 eq) was added to a buffer solution (PB, histidine, TEAA, sodium acetate buffer) of Trop2 antibody Trop2-2 (5 mg, 2.5 mg / ml). After reacting at room temperature for 1.5 hours, 6 equivalents of compound 4 were added and reacted for another 1.5 hours. The reaction was then quenched by adding 50 equivalents of L-cysteine. The mixture was purified through a cation exchange column to obtain ADC-9 (3 mg, 2 mg / ml). The DAR was determined by UV spectroscopy.

[0432] Example 18 Cell-based binding of anti-HER2 antibodies and anti-HER2 ADCs to tumor cell line-expressed HER2 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, namely ADC-2-1 and ADC-2-2) and ADC-3. Commercially available anti-HER2 ADC drugs, DS-8201a from Daiichi Sankyo Co., Ltd. and T-DM1 from Genentech (Roche), were used as positive control ADCs, and the natural antibody trastuzumab (Genentech (Roche)) was used as a free antibody control.

[0433] Briefly, SKBR-3 and NCI-N87 cells were maintained in McCoy's 5a medium (catalog no. 30-2007, ATCC) and RPMI-1640 medium (catalog no. abs9468, Absin), respectively, supplemented with 10% fetal bovine serum (FBS) (catalog no. FSP500, Excell Bio), 2 mM L-glutamine (catalog no. 25030081, ThermoFisher), and 1% penicillin-streptomycin solution (BL505A, Biosharp Life Sciences). Cells were cultured at 37°C in a 5% CO2 atmosphere in air and harvested from flasks for assay when they reached 70%-80% confluence. 2 × 10 cells were cultured per well in a 96-well cell culture plate.6 100 μL of cells / mL were incubated with trastuzumab and the test anti-HER2 ADCs in a 5-fold serial dilution starting from 100 nM to 0.00128 nM for 1 h on ice. After washing twice with 200 μL of FACS buffer (PBS with 1% FBS) per well, cells were incubated with 100 μL of 1× secondary antibody PE anti-human IgG Fc antibody (Cat. No. 366904, Biolegend) for 30–60 min on ice. Cells were washed twice with 200 μL of FACS buffer and resuspended in 200 μL of FACS buffer for further detection by CytoFlex Cytometry (Beckman Coulter). Data were analyzed using the built-in analysis software CytoFlex.

[0434]

[0434] The cell binding results are shown in Figure 1. The binding capacity of the tested anti-HER2 ADCs to SKBR-3 and NCI-N87 cells is efficient and compatible with trastuzumab.

[0435] Example 19 Antibody-mediated internalization effect using FACS analysis in HER2-expressing tumor cells The HER2-positive cell lines SKBR-3 and NCI-N87 cells and the test anti-HER2 ADCs (exemplary HER2 ADCs ADC-2-1, ADC-2-2, and ADC-3, and positive control ADCs DS-8201a and T-DM1) used in Example 18 were also used in the following internalization assay. The culture conditions for the two cell lines were the same as those in Example 18.

[0436] Cells were harvested using 0.25% Tricine / EDTA (T1320, Solarbio) and the cell concentration was adjusted to 4×10 using 1× PBS containing 1% FBS buffer. 6The concentration of cells was adjusted to 100 nM cells / mL. Then, 100 μL of cells per well were plated in a 96-well cell culture plate and incubated with trastuzumab and the tested anti-HER2 ADCs at a final concentration of 100 nM for 7 time points (0, 0.5, 2, 4, 8, 16 and 24 hours). The mixture was incubated at 4°C in a 5% CO2 atmosphere in air. At the specified time points, the cells were immediately washed twice and suspended in 100 μL of cold washing buffer containing 2% paraformaldehyde for 30 minutes. The cells were washed twice and then incubated with 1×PE anti-human IgG Fc antibody (Cat. No. 366904, Biolegend) for an additional hour at 4°C and 5% CO2. After two steps of washing with washing buffer, the cells were resuspended in 100 μL of FACS buffer and the fluorescent signal of the cells was detected using CytoFlex Cytometry (Beckman Coulter). Percent internalization at several time points was calculated as the percentage of median fluorescence intensity (MFI) value lost at a particular time point at time 0. Internalization rate versus incubation time (hr) curves were plotted using Graphpad Prism Software.

[0437] The results for SKBR-3 and NCI-N87 cells are shown in Tables 1 and 2, and Figure 2. All tested anti-HER2 ADCs effectively mediated internalization into HER2-positive cells (SKBR-3 and NCI-N87 cells), with internalization rates of >60% (SKBR-3 cells) and >30% (NCI-N87 cells) within 24 hours. The internalization results indicated that conjugation could not alter target-mediated internalization.

[0438] [Table 2]

[0439] [Table 3]

[0440] Example 20 In vitro cytotoxicity assay of anti-HER2 ADCs against HER2-expressing cells

[0438] The cytotoxicity of exemplary anti-HER2 ADCs was investigated 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.

[0441]

[0439] Briefly, 5,000 cells / well of SKBR-3 and NCI-N87 cells were plated (except for the edge wells which contained medium only) in white clear 96-well assay plates (catalog no. 6005182, PerkinElmer) in 90 μL of basal culture 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) and 2 mM L-glutamine (catalog no. 25030081, ThermoFisher) and grown at 37°C in a humidified incubator with a 5% CO2 atmosphere. After overnight incubation, each test anti-HER2 ADC or trastuzumab was added to each well in a volume of 10 μL of 10× concentrations tested, ranging from 100 nM to 0.015 nM. After an additional 72-hour incubation, the plates were removed from the incubator and equilibrated to room temperature. After approximately 30 minutes, 50 μL of CellTiter-Glo® 2.0 Luminescent Cell Viability Reagent (Catalog No. G7573, Promega) was added to each well. Plates were shaken at 450 rpm for 3 minutes, followed by a 10-minute incubation without shaking, after which fluorescence was measured on an Envision plate reader (Instrument No. 2104, PerkinElmer) using an integration time of 250 ms per well. Fluorescence vs. ADC concentration (μM) curves were fitted using GraphPad Prism Software.

[0442]

[0440] The results of the in vitro cytotoxicity assay are shown in Figure 3. The exemplary anti-HER2 ADCs (ADC-2-1, ADC-2-2 and ADC-3) showed better cytotoxic activity than the positive control ADCs DS-8201a and T-DM1 against both HER-positive NCI-N87 cells (Figure 3A) and SKBR-3 cells (Figure 3B), whereas in HER2-negative MCF-7 cells (Figure 3C), cytotoxic activity was observed only at very high concentrations of the ADCs, especially ADC-3. The control native antibody trastuzumab had only limited cytotoxic activity even at high concentrations.

[0443] Example 21 Cell-based binding of anti-Trop2 antibodies and anti-Trop2 ADCs to tumor cell-expressed Trop2 Trop2 positive SKBR-3 cells (breast cancer, ATCC No. HTB30) were used for in vitro cell binding evaluation of exemplary anti-Trop2 ADCs ADC-4, ADC-5, ADC-6, ADC-7, ADC-8 and ADC-9. Natural human IgG1 antibody Datopotamab DS-1062 from Daiichi Sankyo Co., Ltd. was used for free antibody control. The binding assay protocol was the same as in Example 18.

[0444] Briefly, SKBR-3 cells were maintained in McCoy's 5a medium (catalog no. 30-2007, ATCC) supplemented with 10% FBS (catalog no. FSP500, Excell Bio), 2 mM L-glutamine (catalog no. 25030081, ThermoFisher), and 1% penicillin-streptomycin solution (BL505A, Biosharp Life Sciences). Cells were cultured at 37°C in an atmosphere of 5% CO2 in air and harvested from flasks for assay when they reached 70%-80% confluence. 2 × 10 cells were cultured in 100 μL of ... 6Cells at 1000 μL / mL were incubated with Trop2 antibodies Trop2-1, Trop2-2 and datopotamab, as well as exemplary anti-Trop2 ADCs, in 5-fold serial dilutions starting from 100 nM to 0.00128 nM for 1 hour on ice. After washing twice with 200 μL FACS buffer per well, cells were incubated with 100 μL 1× secondary antibody PE anti-human IgG Fc antibody (Cat. No. 366904, Biolegend) for 30-60 minutes on ice. Cells were washed twice with 200 μL FACS buffer and resuspended in 200 μL FACS buffer for further detection by CytoFlex Cytometry (Beckman Coulter). Data were analyzed using the built-in analysis software CytoFlex.

[0445]

[0443] Cell binding results are shown in Figure 4. The exemplary anti-Trop2 ADCs were able to effectively bind to SKBR-3 cells with binding capacities comparable to their native Trop2 antibodies Trop2-1 and Trop2-2, indicating that conjugation had no significant effect on binding to target binding activity.

[0446] Example 22 Antibody-mediated internalization effect using FACS analysis in Trop2-expressing tumor cells

[0444] Trop2 positive cell line SKBR-3 cells (breast cancer, ATCC No. HTB30) were used for the following internalization assay. The culture conditions were the same as those in Example 21.

[0447] Cells were harvested using 0.25% Tricine / EDTA (T1320, Solarbio) and the cell concentration was adjusted to 4×10 using 1× PBS containing 1% FBS buffer. 6The cells were adjusted to cells / mL. 100 μL of cells per well were then plated in a 96-well cell culture plate and incubated with Trop2 antibodies Trop2-1, Trop2-2, datopotamab and sacituzumab, as well as 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 7 time points (0, 0.5, 2, 4, 8, 16 and 24 hours). The mixtures were incubated at 4° C. in an atmosphere of 5% CO2 in air. At the specified 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 (Cat. No. 366904, Biolegend) for an additional hour at 4° C. and 5% CO2. After two-step washing with washing buffer, the cells were resuspended in 100 μL of FACS buffer, and the fluorescent signals of the cells were detected using CytoFlex Cytometry (Beckman Coulter). The internalization rate at several time points was calculated as the percentage loss of the MFI value at a particular time point at 0 h. The internalization rate versus incubation time (hours) curves were plotted using Graphpad Prism Software.

[0448] The results are shown in Table 3 and Figure 5. The exemplary anti-Trop2 ADCs were able to effectively mediate internalization into SKBR-3 cells, with the internalization rate being >60% within 8 hours. The internalization results indicated that conjugation could not alter target-mediated internalization.

[0449] [Table 4]

[0450] Example 23 In vitro cytotoxicity assay of anti-Trop2 ADCs against Trop2-expressing cancer cells The cytotoxicity of exemplary free linker-payloads (compounds 2, 3 and 4), free Trop2 antibodies (Trop2-1, Trop2-2, datopotamab and sacituzumab), exemplary anti-Trop2 ADCs (ADC-4, ADC-5, ADC-6, ADC-7, ADC-8 and ADC-9) and, as a positive control, Trodelvy (an FDA approved anti-Trop2 ADC) was evaluated in 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).

[0451] Briefly, 5,000 cells / well were plated in white clear 96-well assay plates (catalog no. 6005182, PerkinElmer) in 90 μL of basal culture medium (McCoy's 5a for SKBR-3 cells, RPMI-1640 for NCI-N87 cells, EMEM for MCF-7 cells, and Leibovitz's L-15 for MDA-MB-468 cells) containing 10% FBS, 1% penicillin-streptomycin solution (BL505A, Biosharp Life Sciences), and 2 mM L-glutamine (catalog no. 25030081, ThermoFisher) (except for the edge wells which contained medium only) and grown at 37° C. in a humidified incubator with a 5% CO2 atmosphere. After overnight incubation, the exemplary anti-Trop2 ADC, Trodelvi, free Trop2 antibody and free linker-payload were added to each well in a volume of 10 μL for 10× concentrations tested from 100 nM to 0.015 nM. After an additional 72 hours of incubation, the plates were removed from the incubator and equilibrated to room temperature. After approximately 30 minutes, 50 μL of CellTiter-Glo® 2.0 Luminescent Cell Viability Reagent (Catalog No. G7573, Promega) was added to each well. The plates were shaken at 450 rpm for 3 minutes, followed by a 10 minute incubation without shaking, after which fluorescence was measured on an Envision plate reader (Instrument No. 2104, PerkinElmer) using an integration time of 250 ms per well. Fluorescence vs. ADC concentration (μM) curves were fitted using GraphPad Prism Software.

[0452] The results of the in vitro cytotoxicity assay are shown in FIG. 6 and Table 4. All exemplary anti-Trop2 ADCs showed cytotoxic activity against the tested cell lines. ADC-6, ADC-9, and ADC-5 showed better cytotoxic activity than Trodelvy against the tested cell lines, and ADC-4 exhibited comparable target cell cytotoxicity to Trodelvy against the tested cell lines. The free linker-payload compound 2-4 had only limited cytotoxic activity even at high concentrations. In addition, the natural antibodies sacituzumab, Trop2-1, and Trop2-2 did not exhibit target cell killing effects against these four Trop-2 expressing cells.

[0453] [Table 5]

[0454] Example 24 In vivo therapeutic efficacy of anti-Trop2 ADCs in NCI-N87 cell xenograft mouse model On day 0, NCI-N87 cells (1 × 10 7 , 1:1 Matrigel) was subcutaneously injected 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, the mice were randomized into 8 groups (n = 6), and the average tumor volume at this time was 178 mm. 3Treatment was initiated on this day (defined as PG-D0). Mice in the Trodelvy group received 5.0 mg / kg (iv, BIW) for 3 weeks, mice in the ADC-5 treatment group were injected with 1.0, 2.5 and 5.0 mg / kg ADC-5 for 3 weeks (iv, BIW), and mice in the ADC-6 treatment group were injected with 1.0 and 2.5 mg / kg ADC-6 weekly (QW) in three doses or with 5.0 mg / kg (single dose on PG-D0) (iv). Tumor volumes and body weights were measured twice weekly and recorded. On the final day 28 (defined as PG-D28), tumor weights were dissected and measured. Tumor growth inhibition (%) was calculated using the TV CR -TV TR ) / TV CR (%), where TV CR and TV TR are the relative tumor volumes of the vehicle control and experimental groups, respectively. Mice were euthanized and tumors were allowed to grow to 2,500 mm 3 A plant was considered to have the disease if it grew to a size greater than 1. For statistical analysis using one-way ANOVA, GraphPad Prism 6.0 Software was used.

[0455] The results are shown in Figure 7 and Table 5. Mice in all three ADC-5 treatment groups (1.0, 2.5 and 5.0 mg / kg, BIW*3) demonstrated significantly better antitumor responses than mice in the Trodelvy (5.0 mg / kg, BIW*3) treatment group. In addition, mice in the ADC-5 treatment groups showed complete tumor response (CR) at the medium (2.5 mg / kg, BIW) and high (5.0 mg / kg, BIW) doses, with tumor growth inhibition (TGI) at day 28 of 100.37% and 106.43%, respectively. For the ADC-6 treatment group, the results at low frequency and / or low doses at medium dose (2.5 mg / kg, QW*3) and high dose (5.0 mg / kg, single dose) showed that ADC-6 also exhibited great tumor inhibition efficacy, but ADC-6 showed slightly weaker antitumor efficacy at low dose (1.0 mg / kg, QW*3) due to less frequent and less administered dose than Trodelvy. Meanwhile, the results of body weight change showed no obvious potential toxicity risk of ADC-5 and ADC-6 in this in vivo efficacy study, which further provided solid evidence for the therapeutic window expansion of ADC-5 and ADC-6 compared with Trodelvy (Figure 7B).

[0456] [Table 6]

[0457]

[0452] The preceding description is considered as illustrative only of the principles of the disclosure. Moreover, since numerous modifications and changes will be readily apparent to those skilled in the art, it is not desired to limit the invention to the exact construction and process shown as described above. Accordingly, all suitable modifications and equivalents may be considered to be within the scope of the invention as defined by the following claims.

[0458]

[0453] The words "comprise," "comprising," "include," "including," and "includes," when used in this specification and in the claims that follow, are intended to specify the presence of stated features, integers, components or steps, but they 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) useful for conjugating with a targeting portion: 【Chemistry 1】 And, The polymer scaffold contains linear polyglycerol, L is a linking portion containing a functional group W p that can form a covalent bond with the targeting portion. Ma is a stretcher that connects L to the -NH- section. Each G1 is an independent functional group that connects L P to a linear polyglycerol. Each L P is independently a drug release mechanism between therapeutic portion D and G 1. Each D is independently a therapeutic agent portion. Each G2 is a functional group that can be independently converted to a charged state. n is an integer between 0 and 1000. m is an integer between 0 and 1000. p is an integer between 0 and 1000. q is an integer between 0 and 1000. Polymer scaffolding.

2. W p can react with the functional group on the targeting portion by a click reaction, The aforementioned W p is, 【Chemistry 2】 A polymer scaffold according to claim 1, selected from the group consisting of the following.

3. W p can react with amino acids on the targeting portion, The amino acids are natural amino acids, unnatural amino acids, or combinations thereof. The polymer scaffold according to claim 1, wherein the natural amino acids include cysteine, lysine, tyrosine, aspartic acid, and glutamic acid.

4. (i) Wp can react with one or more cysteines on the targeting portion, and the Wp is 【Transformation 3】 Selected from the group consisting of, where R1 is a sulfur protecting group, Each R2 is independently a leaving group selected from halo or R2a C(O)O-, where R2a is hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. (ii) W p can react with one or more lysines on the targeting region, and each W p can react independently 【Chemistry 4】 Selected from the group consisting of, or (iii) W p can react with one or more non-natural amino acids on the targeting portion, and said W p, 【Transformation 5】 Selected from the group consisting of, The polymer scaffold according to claim 3.

5. M a is 【Transformation 6】 A polymer scaffold according to claim 1, selected from the group consisting of the following.

6. G1 is, 【Transformation 7】 The polymer scaffold according to claim 1, wherein R7 is selected from the group consisting of, where R7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl or heterocycloalkyl moiety.

7. Each L p independently comprises an unstable structure selected from hydrolytically unstable structures or enzymatically unstable structures, wherein (i) Structures that are hydrolyzably unstable, 【Transformation 8】 A group consisting of the following is selected, where * is a site covalently bonded to G 1, ** is a site covalently bonded to D, R 8 is selected from hydrogen, alkyl, or aryl, and R 9 is selected from aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. G1 is, 【Chemistry 9】 In the formula, * represents a site covalently bonded to L P, and R 7 is an alkyl group. (ii) Enzymatically unstable structures are sensitive to cathepsin B, and -Z- or 【Chemistry 10】 Selected from, where Z is a cathepsin B substrate containing 2 to 4 amino acids, G1 is, 【Chemistry 11】 In the formula, * represents a site covalently bonded to L P, and R 7 is an alkyl group. (iii) Enzymatically unstable structures are sensitive to glucuronidase, 【Chemistry 12】 In the formula, * represents the site covalently bonded to G1, and ** represents the site covalently bonded to D, where, G1 is, 【Chemistry 13】 In the formula, R7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, or G1 is, 【Chemistry 14】 In the formula, * represents a site covalently bonded to L P, and R 7 is an alkyl group. (iv) Enzymatically unstable structures are sensitive to phosphatases, 【Chemistry 15】 Selected from the following, where * is a site covalently bonded to G1, ** is a site covalently bonded to D, and each R10 and R11 is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. G1 is, 【Chemistry 16】 And, R7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. or (v) Enzymatically unstable structures are sensitive to sulfatase, 【Chemistry 17】 In the formula, * represents a site covalently bonded to G1, ** represents a site covalently bonded to D, and each R12 and R13 is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. G1 is, [Chemistry 18] In the formula, R7 is selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, or G1 is, 【Chemistry 19】 In the formula, * represents a site covalently bonded to L P, and R 7 is an alkyl group. The polymer scaffold according to claim 1.

8. -G 1 -L P -D is 【Chemistry 20-1】 【Chemistry 20-2】 【Chemistry 20-3】 【Chemistry 20-4】 【Chemistry 20-5】 A polymer scaffold according to claim 7, selected from the group consisting of the following.

9. n is an integer from 1 to 100, m is an integer between 1 and 100, p is an integer between 1 and 50. The polymer scaffold according to claim 1.

10. (i) 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, tranquilizers, anticonvulsants, muscle relaxants and antiparkinsonian substances, antispasmodics and muscle contractions including channel blockers, miotics and anticholinergics, antiglaucoma compounds, antiparasitic and / or antiprotozoan compounds, cell-extracellular matrix interaction modifiers including cell proliferation inhibitors and anti-adhesion molecules, vasodilators, DNA, RNA or protein synthesis inhibitors, antihypertensives, analgesics, antipyretics, steroidal and nonsteroidal anti-inflammatory drugs, anti-angiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, eye drops, prostaglandins, antidepressants, antipsychotics, antiemetics, contrast agents, (ii) The therapeutic agent comprises an amino acid system molecule selected from peptides, polypeptides, enzymes, antibodies, immunoglobulins, or functional fragments thereof, (iii) The therapeutic agent 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 has a chemically reactive group selected from hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. The polymer scaffold according to claim 1.

11. G 2 is, 【Chemistry 21】 The polymer scaffold according to claim 1, selected from the group consisting of, where each R 16 is independently hydrogen, aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

12. Polymer scaffold of formula (II): 【Chemistry 22】 And, The polymer scaffold contains linear polyglycerol, L is a linking portion containing a functional group W p that can form a covalent bond with the targeting portion. Ma is a stretcher that connects L to the -NH- section. Each G2 is a functional group that can be independently converted to a charged state. Each G3 independently contains a functional group that reacts with a reactive group in the drug release mechanism and can connect the drug release mechanism to a linear polyglycerol. n is an integer between 0 and 1000. m is an integer between 0 and 1000. p is an integer between 0 and 1000. q is an integer between 0 and 1000. Polymer scaffolding.

13. n is 2, m is 2, The polymer scaffold according to claim 12, wherein p is 2.

14. The polymer scaffold according to claim 12, wherein q is an integer from 3 to 5.

15. G2 and G3 independently 【Chemistry 23】 The polymer scaffold according to claim 12, wherein each R 17 is independently selected from the group consisting of, where R 17 is independently selected from directly bonded, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

16. Structure of formula (IIa), (IIb), (IIc), or (IId): 【Chemistry 24】 A polymer scaffold according to claim 12, having the following features.

17. Polymer scaffold of formula (IV): 【Chemistry 25】 And, The polymer scaffold contains linear polyglycerol, Each L a is an independent divalent part that connects the targeting part to M a, Each Ma is an independent stretcher that connects La to the -NH- section. Each G1 is an independent functional group that connects L P to a linear polyglycerol. Each L P is independently a drug release mechanism between the fragment of therapeutic agent D and G 1. Each D is independently a fragment of the therapeutic agent. Each G2 is a functional group that can be independently converted to a charged state. n is an integer between 0 and 1000. m is an integer between 0 and 1000. p is an integer between 0 and 1000. q is an integer between 0 and 1000. s is an integer between 1 and 8. Polymer scaffolding.

18. The polymer scaffold according to claim 17, wherein the targeting portion is an antibody and / or a fragment thereof.

19. The polymer scaffold according to claim 17, wherein the targeting portion is antibodies IgG1, IgG2, IgG3, and IgG4.

20. The polymer scaffold according to claim 17, wherein the targeting portion 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 diabody (dsdiabody), single-chain antibody molecule (scFv), scFv dimer, polyspecific antibody, camelized single-domain antibody, nanobody, domain antibody, or bivalent domain antibody.

21. A pharmaceutical composition comprising one or more polymer scaffolds and an acceptable carrier according to any one of claims 1 to 20.

Images