Conjugates and uses thereof

Abstract

Compounds of formula (I), or tautomers, stereoisomers or pharmaceutically acceptable salts thereof. The compound shown in the formula (I) or the tautomer, the stereoisomer or the pharmaceutically acceptable salt of the compound can be used as a novel linker compound and can be used for preparing ADC (Analog to Digital Converter) medicines with high DAR (Diaphragm Antigen) values. The invention also relates to an ADC drug prepared from the linker. (I)

Description

[0001] This application is a divisional application of Chinese invention patent application (application date: January 28, 2022; application number: 202280002773.6 (international application number: PCT / CN2022 / 074523); invention title: Conjugate and its use). Technical Field

[0002] This invention relates to the field of biomedicine, specifically to conjugates and their uses, and more specifically to compounds, conjugates, pharmaceutical compositions and their pharmaceutical uses. Background Technology

[0003] Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals that link cytotoxic molecules (cytotoxins) to antibodies using permanent or unstable chemical linkers. The antibody binds to specific antigens on the tumor cell membrane, inducing endocytosis, allowing the antibody and its attached cytotoxic molecule to enter the cell. Subsequently, through lysosomal degradation, the small drug molecule is released into the cell, inducing apoptosis.

[0004] The key technical aspects of ADC drugs include the selection of payload (toxin), linker, antibody, target, and conjugation technology.

[0005] The requirements for ADCs to bind to toxins include: 1. Sufficient water solubility and serum stability, as ADCs may circulate in vivo for several days; 2. The presence of functional groups capable of coupling with the linker; 3. Insensitivity to lysosomal enzymatic degradation; 4. Reduction of polymerization effects (lipophilic substances readily undergo polymerization) and alteration of the interaction between ADCs and pGp (permeability glycoprotein, drug efflux pump, readily binding to lipophilic substances), the latter being a major cause of multipotent drug resistance (MDR) in tumor cells. Furthermore, for cleavable linker ADCs, the bystander effect requires the toxin to kill the target cell and then enter and exit the cell membrane to kill surrounding cells, necessitating a certain lipid-water partition coefficient (LogP) and a positive / neutral charge. Currently, the most commonly used cytotoxic drugs in clinical practice can be divided into two main categories based on their mechanisms of action:

[0006] DNA damaging agents: Calcheamicin (CLM, an enediyne antibiotic) acts on DNA by binding to the minor groove of the DNA double helix, leading to DNA lysis and cell death.

[0007] Microtubule inhibitors: These inhibitors bind to microtubules, preventing their polymerization and arresting the cell cycle, thereby inducing tumor cell apoptosis. Major microtubule inhibitors include: auristatins (e.g., MMAE, MMAF, MMAD); maytansinoids (e.g., DM1, DM2, DM3, DM4); and leptospirin B and its derivatives (e.g., eribulin). Currently, the vast majority of ADCs in clinical trials utilize microtubule inhibitors, and some have been approved for marketing (Adcetris uses the auristatin MMAE, and Kadcyla uses the maytansin derivative DM1). Auristatins are dominant, accounting for over 50% of ADCs in development. However, these microtubule inhibitors have shown significant ocular toxicity and peripheral neuropathy, leading to treatment interruption or dose reduction in many cases. These results clearly indicate the need for further research into other cytotoxic agents and MTAs as ADC loadings, which may offer advantages in terms of toxicity and improved therapeutic index.

[0008] Linkers act as bridges connecting antibodies and cytotoxic drugs. Ideally, a linker must be stable in vitro or in the bloodstream to prevent systemic toxicity caused by premature release of the cytotoxic drug, while simultaneously enabling rapid release of the effective cytotoxic drug to kill cancer cells upon entry. An ideal linker plays a crucial role in the success of a drug treatment; its properties determine the drug's pharmacokinetic properties and therapeutic efficacy. Ideally, a linker should not release cytotoxins before reaching the target site, but only after reaching the cell. Linker release mechanisms are classified as cleavable and non-cleavable. In the non-cleavable mode, the linker remains connected to the cytotoxin even after the ADC has been digested by lysosomes. There are three types of cleavable linkers: First, acid-sensitive linkers, which trigger the hydrolysis of acid-dependent groups in the linker, such as hydrazone groups, at low pH; second, glutathione-sensitive linkers, where the intracellular glutathione concentration is higher than that in plasma, and linkers containing disulfide bonds are reduced and cleaved by glutathione after reaching the cell; and finally, lysosomal protease-sensitive linkers, where some proteases in lysosomes can recognize and cleave specific peptides in the linker to release the drug.

[0009] Traditional antibody-drug conjugates (ADCs) based on cysteine ​​sites are limited to using one drug per cysteine. However, some applications require a high drug-to-antibody ratio (DAR), such as when using low-efficiency loadings. Higher drug loadings can be achieved using classic cysteine ​​conjugation methods, but these can lead to heterogeneity, suboptimal efficacy, and pharmacokinetic issues.

[0010] Due to technological limitations, traditional high-DAR (drug-adsorption-reduction) ADCs often suffer from pharmacokinetic instability, increased drug metabolism rate, reduced half-life, and increased systemic toxicity. Therefore, ADC drug technology still requires further development and improvement. Summary of the Invention

[0011] The present invention aims to at least partially solve one of the technical problems in the related art, such as providing a high DAR value ADC drug that solves the problems of pharmacokinetic instability, increased drug metabolism rate, reduced half-life and increased systemic toxicity.

[0012] In a first aspect, the present invention relates to compounds of formula (I), or tautomers, stereoisomers, or pharmaceutically acceptable salts thereof.

[0013] (I)

[0014] in,

[0015] a, b, c, d, e, and f are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably 0, 1, 2, 3, 4, or 5; preferably 0, 1, 2, or 3; preferably 1 or 2;

[0016] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably 1, 2, 3, 4 or 5; preferably 1, 2 or 3; preferably 1;

[0017] m is 2 or 3;

[0018] X is C, N, or Si;

[0019] A represents -NH2, -NH-PG1. , or ;

[0020] R is -OH, -O-PG2 , , or ;

[0021] PG1, PG2, and PG3 are protecting groups;

[0022] n2 is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; preferably 7, 8, 9, 10 or 11;

[0023] Y represents a key or ;

[0024] Where n1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably 1, 2, 3, 4 or 5; preferably 1, 2 or 3;

[0025] b1, c1, and d1 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably 0, 1, 2, 3, 4, or 5; preferably 0, 1, 2, or 3; preferably 1 or 2;

[0026] When X is C or Si, m is 3;

[0027] When X is N, m is 2.

[0028] The compound of formula (I) according to embodiments of the present invention, or its tautomers, stereoisomers, or pharmaceutically acceptable salts, as a novel linker compound, can be used to prepare high DAR value ADC drugs in a site-specific manner with each cysteine ​​residue as a conjugate site. This reduces the in vivo exposure of small molecule drugs, improves the safety of small molecule drugs, and facilitates drug quality control. Simultaneously, the prepared high DAR value ADC drugs exhibit stronger cytotoxicity to target cells. Using the compound of formula (I) as a linker according to embodiments of the present invention, various types of water-soluble groups are introduced, effectively avoiding the phenomenon that ADCs easily polymerize and precipitate in plasma due to poor water solubility. This allows the toxin to kill target cells and then enter and exit the cell membrane to kill surrounding cells, thus demonstrating significant advantages in terms of efficacy and toxicity. The ADC drugs prepared using the compound of formula (I) as a linker according to embodiments of the present invention solve the problems of pharmacokinetic instability, increased drug metabolism rate, reduced half-life, and increased systemic toxicity caused by high DAR values.

[0029] According to embodiments of the present invention, the above-described compound may further include at least one of the following additional technical features:

[0030] According to embodiments of the present invention, it is the structure shown in formula (II) or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof.

[0031] (II).

[0032] According to an embodiment of the present invention, it is the structure shown in formula (III) or its tautomer, stereoisomer, or pharmaceutically acceptable salt.

[0033] (III).

[0034] According to embodiments of the present invention, R is -OH, -O-PG2, , , , , , , , , , , , , , , , .

[0035] According to an embodiment of the present invention, n2 is 7 or 11.

[0036] According to embodiments of the present invention, PG1 is a protecting group of an amino group; optionally, the protecting group of the amino group is selected from acetyl, trifluoroacetyl, tert-butoxycarbonyl (BOC, Boc), benzyloxycarbonyl (CBZ, Cbz) and 9-fluorenemethoxycarbonyl (Fmoc); PG2 is a protecting group of a hydroxyl group; optionally, the protecting group of the hydroxyl group is selected from acetyl and silyl; PG3 is a protecting group of a carboxyl group; optionally, the protecting group of the carboxyl group is selected from -CH2CH2SO2Ph, cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrobenzenesulfonyl)ethyl, 2-(diphenylphosphine)ethyl and nitroethyl.

[0037] In a second aspect, the present invention provides a conjugate. According to embodiments of the invention, the conjugate comprises the aforementioned compound and a pharmaceutical moiety, wherein the compound is covalently bonded to the pharmaceutical moiety via an R group. According to specific embodiments of the invention, the pharmaceutical moiety is a small molecule drug, also known as a payload.

[0038] According to an embodiment of the present invention, the conjugate in the second aspect further includes a targeting portion, wherein one or more of the conjugates are covalently bonded to the targeting portion via an A group. Furthermore, the conjugate in the second aspect can be directed to a target site under the guidance of the targeting movement of the targeting portion.

[0039] In a third aspect, the present invention provides a conjugate. According to embodiments of the invention, the conjugate comprises a targeting portion and one or more of the aforementioned compounds, the compounds being covalently bonded to the targeting portion via an A group. Furthermore, the conjugate of the third aspect can be directed to a target site under the mediation of the targeting movement of the targeting portion.

[0040] According to embodiments of the present invention, the drug is selected from at least one of eribulin, methylaurestatin E, and SN-38, and has the following structure:

[0041] , and .

[0042] According to embodiments of the present invention, the conjugate of the second aspect is selected from the following specific compounds or their tautomers, stereoisomers or pharmaceutically acceptable salts, represented by the structure shown in formula (IV).

[0043] (IV),

[0044] Where Rx is

[0045] And n2, *, and Payload are defined in the following table:

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] The following compounds are particularly preferred:

[0078] Where Rx is:

[0079] , , , , , , , , or .

[0080] According to embodiments of the present invention, the conjugate is selected from the following specific compounds, or their tautomers, stereoisomers, or pharmaceutically acceptable salts, represented by the structure shown in formula (V):

[0081] (V),

[0082] Where Rx is

[0083] And n2, *, and Payload are defined in the following table:

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] The following compounds are particularly preferred:

[0115] Where Rx is

[0116] , or .

[0117] According to an embodiment of the present invention, the targeting portion is based on a protein recognition molecule (PBRM).

[0118] According to an embodiment of the present invention, the recognition molecule is an internalized antibody or its internalized antigen-binding fragment targeting tumor cells. Furthermore, in a second aspect, the conjugate covalently binds to an internalized antibody or its internalized antigen-binding fragment targeting tumor cells via an A group, forming an antibody-drug conjugate (ADC).

[0119] According to embodiments of the present invention, the antibody or antigen-binding fragment is bound to anti-human epidermal growth factor receptor (HER2) antibody, EGFR, GPNMB, CD56, TACSTD2 (TROP2), CEACAM5, folate receptor-a, mesothelin, ENPP3, guanylate cyclase C, SLC44A4, NaPi2b, CD70, mucin 1, STEAP1, connexin 4, 5T4, SLTRK6, SC-16, LIV-1, P-cadherin, PSMA, fibronectin extracellular domain B, endothelin receptor ETB, tendinin c, collagen IV, VEGFR2, periostealin, CD30, CD79b, CD19, CD22, CD138, CD37, CD33, CD74, etc.

[0120] In a fourth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, the pharmaceutical composition comprises the aforementioned conjugate and a pharmaceutically acceptable carrier, the conjugate comprising a targeting portion, a compound described in the first aspect, and a pharmaceutical portion, the targeting portion being covalently linked to one or more of the compounds, the compounds being covalently linked to the pharmaceutical portion via R groups.

[0121] In a fifth aspect of the invention, the invention provides for the use of the aforementioned conjugates or pharmaceutical compositions in the preparation of a medicament for treating a patient with cancer expressing a target antigen or at risk of having cancer expressing a target antigen, the conjugate comprising a targeting portion, a compound described in the first aspect, and a pharmaceutical portion, the targeting portion being covalently linked to one or more of the compounds, the compounds being covalently linked to the pharmaceutical portion via an R group.

[0122] According to an embodiment of the present invention, the target antigen is human epidermal growth factor receptor 2.

[0123] According to an embodiment of the present invention, the cancer expresses high levels of human epidermal growth factor receptor 2.

[0124] According to an embodiment of the present invention, the cancer is breast cancer, gastric cancer, bladder cancer, or urothelial carcinoma.

[0125] In a sixth aspect, the invention provides a method for treating patients with cancer expressing a target antigen or at risk of developing cancer expressing a target antigen. According to an embodiment of the invention, the method includes administering a therapeutically effective amount of the aforementioned conjugate to the patient, the conjugate comprising a targeting portion, the compound described in the first aspect, and an ADC of the pharmaceutical portion.

[0126] In a seventh aspect of the invention, the invention provides a method for reducing or inhibiting the growth of tumors expressing target antigens, comprising administering a therapeutically effective amount of the aforementioned conjugate, said conjugate comprising a targeting portion, the compound described in the first aspect, and an ADC of pharmaceutical portion.

[0127] In an eighth aspect of the invention, the invention proposes the use of the aforementioned conjugate for treating cancers expressing a target antigen, said conjugate comprising a targeting portion, the compound described in the first aspect, and an ADC of pharmaceutical portion.

[0128] In a ninth aspect of the invention, the invention proposes the use of the aforementioned conjugate in a method of manufacturing a medicament for treating cancers expressing a target antigen, wherein the conjugate comprises a targeting portion, the compound described in the first aspect, and an ADC of the pharmaceutical portion.

[0129] In a tenth aspect of the invention, the invention provides a method for producing the aforementioned conjugate, comprising reacting an antibody or antigen-binding fragment with the aforementioned linker compound bound to a small molecule drug under conditions permissible coupling.

[0130] In an eleventh aspect of the invention, the invention provides a method for determining whether a patient will respond to treatment with the aforementioned conjugate, comprising providing a biological sample from the patient and contacting the biological sample with the aforementioned conjugate, the conjugate being an ADC comprising a targeting portion, a compound as described in the first aspect, and a pharmaceutical portion.

[0131] According to an embodiment of the present invention, the biological sample is a tumor biopsy derived from a patient with cancer expressing human epidermal growth factor receptor 2 or at risk of having cancer expressing human epidermal growth factor receptor 2, wherein the cancer is breast cancer, gastric cancer, bladder cancer, or urothelial carcinoma.

[0132] Specific implementation methods

[0133] The following embodiments are provided to further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0134] The terminology used in this article

[0135] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0136] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0137] Unless otherwise stated or there is a clear conflict in the context, the articles “a,” “an,” and “described” as used herein are intended to include “at least one” or “one or more.” Therefore, these articles as used herein refer to articles for one or more (i.e., at least one) objects. For example, “a component” refers to one or more components, meaning that more than one component may be considered for use or adoption in the implementation of the described embodiments.

[0138] As used in this invention, the term "test subject" refers to an animal. Typically, the animal is a mammal. Test subjects also include, for example, primates (e.g., humans, males or females), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In some embodiments, the test subject is a primate. In other embodiments, the test subject is a human.

[0139] As used in this invention, the term "patient" refers to a person (including adults and children) or other animal. In some embodiments, "patient" refers to a person.

[0140] The term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention, but does not exclude other aspects.

[0141] "Stereoisomers" are compounds that have the same chemical structure but whose atoms or groups are arranged differently in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and hindered isomers, etc.

[0142] "Chirality" refers to molecules that have the property that they cannot be superimposed on their mirror image; while "chirality" refers to molecules that can be superimposed on their mirror image.

[0143] "Enantiomers" refer to two non-overlapping but mirror-image isomers of a compound.

[0144] A diastereomer is a stereoisomer that has two or more chiral neutral molecules that are not mirror images of each other. Diastereomers possess different physical properties, such as melting point, boiling point, spectral properties, and reactivity. Mixtures of diastereomers can be separated using high-resolution analytical techniques such as electrophoresis and chromatography, for example, HPLC.

[0145] The stereochemical definitions and rules used in this invention generally follow those of S. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds,” John Wiley & Sons, Inc., New York, 1994.

[0146] Many organic compounds exist in an optically active form, meaning they possess the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to one or more of its chiral centers. The prefixes d and l or (+) and (-) are symbols used to specify the plane-polarized light rotation caused by the compound, where (-) or l indicates that the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. A specific stereoisomer is an enantiomer, and a mixture of such isomers is called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process.

[0147] Any asymmetric atom (e.g., carbon, etc.) in the compounds disclosed in this invention can exist in a racemic or enantiomerically enriched form, such as in (R)-, (S)-, or (R, S)- configurations. In some embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (R)- or (S)- configuration.

[0148] Depending on the choice of starting materials and methods, the compounds of this invention can exist as one or a mixture of possible isomers, such as racemic mixtures and mixtures of non-corresponding isomers (depending on the number of asymmetric carbon atoms). Optically active (R)- or (S)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituents may be E or Z configurations; if the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituents may be cis or trans configurations.

[0149] Any mixture of stereoisomers obtained can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0150] Racemates of any resulting end product or intermediate can be separated into optical enantiomers using known methods, such as by separating the obtained diastereomeric salts. Racemate products can also be separated by chiral chromatography, such as high-performance liquid chromatography (HPLC) using chiral adsorbents. In particular, enantiomers can be prepared by asymmetric synthesis, for example, see Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2 nd Ed. Robert E. Gawley, Jeffrey Aubé, Elsevier, Oxford, UK, 2012); Eliel, EL Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); 1972); Chiral Separation Techniques: A Practical Approach (Subramanian, G.Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007).

[0151] The terms "tautomer" or "tautomer form" refer to structural isomers with different energies that can interconvert through a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium can be achieved in the tautomer. For example, proton tautomers (also called prototropic tautomers) involve interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers involve interconversions via the rearrangement of some bonding electrons. A specific example of a keto-enol tautomer is the interconversion between pentane-2,4-dione and 4-hydroxypent-3-en-2-one. Another example of tautomerism is phenol-keto tautomerism. A specific example of a phenol-keto tautomer is the interconversion between pyridine-4-ol and pyridine-4(1H)-keto. Unless otherwise stated, all tautomer forms of the compounds of this invention are within the scope of this invention.

[0152] As described in this invention, the compounds of this invention may optionally be substituted with one or more substituents, such as the general formula compounds above, or as the specific examples in the embodiments, and the class of compounds included in this invention.

[0153] The term "protecting group" or "PG" refers to a substituent that, when reacting with other functional groups, is typically used to block or protect specific functionalities. For example, "amino protecting group" refers to a substituent attached to an amino group to block or protect the functionality of the amino group in a compound. Suitable amino protecting groups include acetyl, trifluoroacetyl, tert-butoxycarbonyl (BOC, Boc), benzyloxycarbonyl (CBZ, Cbz), and 9-fluorenemethoxycarbonyl (Fmoc). Similarly, "hydroxyl protecting group" refers to a substituent of a hydroxyl group used to block or protect its functionality; suitable protecting groups include acetyl and silyl. "Carboxyl protecting group" refers to a substituent of a carboxyl group used to block or protect its functionality. Common carboxyl protecting groups include -CH2CH2SO2Ph, cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrobenzenesulfonyl)ethyl, 2-(diphenylphosphine)ethyl, nitroethyl, etc. For a general description of protecting groups, please refer to: T W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991; and PJ Kocienski, Protecting Groups, Thieme, Stuttgart, 2005.

[0154] As used in this invention, "pharmaceutically acceptable salts" refers to the organic and inorganic salts of the compounds of this invention. Pharmaceutically acceptable salts are well-known in the field, as described in the literature: SM Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. Pharmaceutically acceptable salts formed from non-toxic acids include, but are not limited to, inorganic acid salts formed by reactions with amino groups, such as hydrochlorides, hydrobromic acids, phosphates, sulfates, and perchlorates, and organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, or these salts obtained by other methods described in the literature, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, transbutenedioic acid, glucono-p-gluconate, glyceryl phosphate, gluconate, hemisulfate, heptanate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pyruvate, pectinate, persulfate, 3-phenylpropionate, picrate, pentanoate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts obtained by means of appropriate bases include alkali metals, alkaline earth metals, ammonium, and nitrogen. + Salts of (C1-C4 alkyl)4. This invention also contemplates quaternary ammonium salts formed from any compound containing an N group. Water-soluble or oil-soluble or dispersed products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts, and amine cations resistant to the formation of equilibrium ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C 1-8 Sulfonates and aromatic sulfonates.

[0155] The term "conjugate" refers to a substance formed by the covalent bonding of two or more compounds, such as the conjugate formed by the covalent bonding of the compound shown in formula (I) of this application with a small molecule drug, or the conjugate formed by the covalent bonding of the compound shown in formula (I) with an antibody or antigen binding fragment, or the conjugate formed by the covalent bonding of the compound shown in formula (I) with an antibody or antigen binding fragment and a small molecule drug. In this case, the conjugate is an antibody-drug conjugate (ADC).

[0156] The terms “antibody-drug conjugate,” “antibody-drug conjugate,” “conjugate,” “immunoconjugate,” and “ADC” are used interchangeably and refer to compounds or derivatives thereof attached to an antibody (e.g., an anti-HER2 antibody).

[0157] The term "antibody," used in its broadest sense, refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or a combination thereof, through an antigen recognition site within the variable region of the immunoglobulin molecule. The antibody heavy chain consists of a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The light chain consists of a light chain variable domain (VL) and a light chain constant domain (CL). For the purposes of this application, the mature heavy and light chain variable domains each contain three complementarity-determining regions (CDR1, CDR2, and CDR3) within four framework regions (FR1, FR2, FR3, and FR4) arranged from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. "Antibodies" can be naturally occurring or artificial, such as monoclonal antibodies produced using conventional hybridoma techniques. The term "antibody" includes full-length monoclonal antibodies and full-length polyclonal antibodies, as well as antibody fragments such as Fab, Fab′, F(ab′)2, Fv, and single-chain antibodies. Antibodies can be any of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (e.g., isotypes IgG1, IgG2, IgG3, and IgG4). The term further encompasses human antibodies, chimeric antibodies, humanized antibodies, and any modified immunoglobulin molecule containing an antigen recognition site, provided that it exhibits the desired biological activity.

[0158] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., HER2). The antigen-binding fragment preferably also retains the ability to be internalized into cells expressing the antigen. In some embodiments, the antigen-binding fragment also retains immune effector activity. Fragments of full-length antibodies have been shown to perform the antigen-binding function of full-length antibodies. Examples of binding fragments encompassed within the term "antigen-binding fragment" or "antigen-binding moiety" of antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds in hinge regions; (iii) Fd fragments, consisting of VH and CH1 domains; (iv) Fv fragments, consisting of VL and VH domains in a single arm of the antibody; (v) dAb fragments, which contain a single variable domain, such as the VH domain (see, for example, Ward et al. (1989) Nature 341:544-6; and Winter et al., WO 90 / 05144); and (vi) separate complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be conjugated using recombination methods via synthetic linkers capable of producing a single protein chain (called a single-chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule. See, for example, Bird et al. (1988) Science 242:423-6; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-83. This type of single-chain antibody is also intended to be encompassed within the term "antigen-binding fragment" or "antigen-binding moiety" of antibody and is recognized in the art as an exemplary type of binding fragment that can be internalized into the cell upon binding. See, for example, Zhu et al. (2010) 9:2131-41; He et al. (2010) J. Nucl. Med. 51:427-32; and Fitting et al. (2015) MAbs 7:390-402. In some implementations, the scFv molecule may be incorporated into the fusion protein. Other forms of single-chain antibodies are also covered, such as bifunctional antibodies. Bifunctional antibodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to pair between the two domains on the same chain, thus forcing the domains to pair with complementary domains on another chain and creating two antigen-binding sites (see, for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-8; and Poljak et al. (1994) Structure 2: 1121-3).Antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and are screened for utility (e.g., binding affinity, internalization) in the same manner as intact antibodies. Antigen-binding fragments can be prepared, for example, by cleaving intact proteins using proteases or chemical cleavage.

[0159] As used herein with respect to antibody or antigen-binding fragments, "internalization" refers to the ability of an antibody or antigen-binding fragment, upon binding to a cell, to pass through the cell's lipid bilayer membrane into its internal compartments (i.e., "internalization"), preferably into the degradation compartments within the cell. For example, an internalized anti-HER2 antibody is an antibody that, after binding to HER2 on the cell membrane, is able to enter the cell.

[0160] The terms “human epidermal growth factor receptor 2,” “her2,” or “her2 / neu” refer to any naturally occurring form of human her2. The term encompasses full-length her2 (e.g., NCBI reference sequence: NP_004439.2; SEQ ID NO: 21) and any form of human her2 produced by cellular processing. The term also encompasses naturally occurring her2 variants, including (but not limited to) splice variants, allelic variants, and isoforms. Her2 can be isolated from humans or can be produced recombinantly or synthetically.

[0161] The term "anti-HER2 antibody" or "antibody that specifically binds to HER2" refers to any form of antibody or fragment thereof that specifically binds to HER2, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that the fragment specifically binds to HER2. U.S. Patent No. 5,821,337 (incorporated herein by reference) provides exemplary HER2 binding sequences, including exemplary anti-HER2 antibody sequences. Anti-HER2 antibodies used in the ADCs disclosed herein are preferably internalizing antibodies or fragments of internalizing antibodies. Trastuzumab is an exemplary internalizing anti-human HER2 antibody.

[0162] The terms "kon" or "ka" refer to the association rate constant of an antibody and an antigen to form an antibody / antigen complex. This rate can be measured using standard assays such as Biacore or ELISA.

[0163] The terms "koff" or "kd" refer to the dissociation rate constant of an antibody from its antibody / antigen complex. This rate can be measured using standard assays such as Biacore or ELISA.

[0164] Term "K" D"KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. KD is calculated from ka / kd. The rate can be determined using standard assays such as Biacore or ELISA. The antibody or antigen-binding fragment in the conjugate of this application can be determined at the equilibrium dissociation constant (K... D ≤1 mM, ≤100 nM, or ≤10 nM, or any amount in between, binds to the target antigen. In some embodiment schemes, K D The range is from 1 pM to 500 pM. In some implementations, K D Between 500 pM and 1 μM.

[0165] The term "antibody:drug ratio," "drug-antibody ratio," or "DAR" refers to the number of drug portions attached to each antibody, i.e., the drug load.

[0166] The terms “therapeutic agent,” “drug,” or “drug fraction” refer to agents that can modulate biological processes and / or have biological activity.

[0167] The term "cytotoxic agent" refers to a substance that causes cell death primarily by interfering with the expression activity and / or function of cells. Examples of cytotoxic agents include (but are not limited to) antimitotic agents such as eribulin and oliquistatin (e.g., monomethyloliquistatin E (MMAE)).

[0168] The term "cancer" refers to a physiological condition in mammals characterized by uncontrolled cell growth in a population of cells.

[0169] "Pharmaceutical composition" means a formulation which is in a form permissible for administration and subsequently provides the desired biological activity and / or achieves the therapeutic effect of the active ingredient and does not contain any other components whose toxicity is unacceptable to the subject to which the formulation is administered. Pharmaceutical compositions may be sterile.

[0170] "Drug excipients" include substances such as adjuvants, carriers, pH adjusters and buffers, tension adjusters, wetting agents, and preservatives.

[0171] "Pharmaceutical acceptable" means that, within the bounds of reliable medical judgment, it is suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and that the benefits / risks are proportionate to a reasonable ratio.

[0172] As disclosed herein, an "effective amount" of an ADC is sufficient to perform the specifically stated purpose, such as producing a therapeutic effect after administration, for example, reducing tumor growth rate or tumor volume, reducing cancer symptoms, or some other sign of therapeutic efficacy. An effective amount can be determined using conventional methods for the stated purpose. The term "therapeutic effective amount" refers to the amount of ADC that is effective in treating a subject's disease or condition. In the case of cancer, a therapeutically effective amount of ADC can reduce the number of cancer cells, decrease tumor size, inhibit (e.g., slow or terminate) tumor metastasis, inhibit (e.g., slow or terminate) tumor growth, and / or alleviate one or more symptoms. A "preventive effective amount" refers to the amount required to effectively achieve preventive results at the necessary dose and for the required time period. Typically, because the preventive dose is administered to the subject before or in the early stages of disease, the preventive effective amount will be less than the therapeutic effective amount.

[0173] As disclosed herein, an "effective amount" of an ADC is sufficient to perform the specifically stated purpose, such as producing a therapeutic effect after administration, for example, reducing tumor growth rate or tumor volume, reducing cancer symptoms, or some other sign of therapeutic efficacy. An effective amount can be determined using conventional methods for the stated purpose. The term "therapeutic effective amount" refers to the amount of ADC that is effective in treating a subject's disease or condition. In the case of cancer, a therapeutically effective amount of ADC can reduce the number of cancer cells, decrease tumor size, inhibit (e.g., slow or terminate) tumor metastasis, inhibit (e.g., slow or terminate) tumor growth, and / or alleviate one or more symptoms. A "preventive effective amount" refers to the amount required to effectively achieve preventive results at the necessary dose and for the required time period. Typically, because the preventive dose is administered to the subject before or in the early stages of disease, the preventive effective amount will be less than the therapeutic effective amount.

[0174] Certain embodiments of the invention will now be described in more detail, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to cover all alternatives, modifications, and equivalents, all of which are included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials can be used to practice the invention. The invention is by no means limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0175] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention, for brevity, have been described in a single embodiment, but may also be provided individually or in any suitable sub-combination.

[0176] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0177] Unless otherwise stated, the following definitions shall apply as used herein. For the purposes of this invention, chemical elements are consistent with the periodic table (CAS edition) and the Handbook of Chemistry and Physics, 75th edition, 1994. Furthermore, general principles of organic chemistry can be found in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference.

[0178] Preparation Examples

[0179] Example 1 Synthesis of MS-1

[0180]

[0181] Step 1: Synthesis of Cbz-Ms-1

[0182]

[0183] Add SM2 (8 g, 23.5 mmol) and 150 mL DMF to a 250 mL three-necked flask, cool to 0 °C in an ice-salt bath under argon protection. Add SM1 (9 g, 23.5 mmol) and HBTU (13.3 g, 35.2 mmol), then add DIPEA (4.5 g, 35.2 mmol) dropwise. After completion, react at room temperature (20 °C) for 1 h. Monitor the reaction by TLC (DCM:MeOH = 10:1), and observe the disappearance of SM2.

[0184] The reaction mixture was added to 500 mL of water and extracted three times with 200 mL of EA. The organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was passed through a silica gel column and eluted with 2%-3% MeOH / DCM to give 11 g (15.9 mmol, 68.0%) of a colorless oily product.

[0185] 1H NMR (300 MHz, DMSO-d6) δ 7.95-7.91 (m, 1H), 7.53 (d, J=8.7Hz, 1H), 737-7.34 (m, 5H), 5.10-4.98 (m, 2H), 4.38-4.30 (m, 1H), 3.51 – 3.44 (m, 26H), 3.43 – 3.38 (m, 4H), 3.24 – 3.18 (m, 5H), 2.67 – 2.60 (m, 1H), 2.48 – 2.43(m, 1H), 1.40 (s, 9H).

[0186] Step 2: Synthesis of Ms-1

[0187]

[0188] Palladium on carbon (0.2 g) was added to a methanol solution of Cbz-Ms-1 (1.0 g, 1.5 mmol, 1.0 equivalent). The reaction mixture was purged five times with hydrogen and stirred at 25 °C for 4 hours. The reaction solution was filtered, concentrated under reduced pressure to obtain 650 mg of a colorless oily substance, Ms-1 (81%). This product can be used directly in the next step without further purification.

[0189] MS m / z [M+H]+(ESI): 555.45.

[0190] Example 2 Synthesis of CS-2

[0191]

[0192] Step 1: Synthesis of INA

[0193]

[0194] 300 mL of DCM was added to a three-necked flask, followed by SM3 (30 g, 0.0885 mol), SM4 (13.2 g, 0.115 mol), and EDCI (22.1 g, 0.115 mol). The mixture was stirred at room temperature for 6 hours. The reaction was monitored by TLC; the DCM / MeOH ratio was 10:1, indicating complete conversion of the starting material. The reaction solution was diluted to 600 mL with dichloromethane, then washed twice with 200 mL of 0.25 M dilute hydrochloric acid, once with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to obtain 35 g (0.08 mol, 90.39%) of a white, foamy solid. No purification was required; this solid was used directly in the next step.

[0195] Step 2: Synthesis of INB

[0196]

[0197] INA (35 g, 0.08 mmol) was dissolved in 400 mL of DMF, and L-citrulline (30.9 g, 0.177 mol) and sodium bicarbonate (18.5 g, 0.221 mol) were added. Then, 200 mL of water was added, and the reaction was carried out at 25 °C for 16 hours. TLC monitoring showed that DCM / MeOH = 10 / 1, indicating no reactants remained, and the reaction was complete. DMF was removed by rotary evaporation, and 500 mL of water and 50 g of citric acid were added. The mixture was stirred for 1 hour, filtered, and the solid was evaporated to dryness. The mixture was then stirred with 500 mL of dichloromethane for 1 hour, filtered, and the solid was dried using an oil pump to obtain 34.5 g (0.07 mmol, 87.5%) of the target product as a white solid.

[0198] 1 H NMR (300 MHz, DMSO-d6) δ 12.40 (s, 1H), 8.15 (d, J = 7.3 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.80 – 7.69 (m, 2H), 7.46 – 7.29 (m, 6H), 6.00 –5.88 (m, 1H), 5.37 (s, 2H), 4.30 – 4.14 (m, 4H), 3.95 – 3.89 (m, 1H), 2.98 –2.89 (m, 2H), 2.05 – 1.87 (m, 1H), 1.83 – 1.50 (m, 2H), 1.45 – 1.31 (m, 2H),0.87 (dd, J = 9.3, 6.7 Hz, 6H).

[0199] Step 3: Synthesis of INC

[0200]

[0201] In a 3L single-necked flask, INB (26.9 g, 54.23 mmol) was added, followed by a 2:1 mixture of DCM / MeOH (2 L), and stirred until dissolved. SM5 (8 g, 65.08 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (24.1 g, 97.6 mmol) were added. The mixture was protected from light and reacted at 20°C for 16 hours, followed by stirring in a 30°C oil bath for 5 hours. TLC monitoring showed a DCM / MeOH ratio of 5:1, indicating complete conversion of the starting material. Most of the reaction mixture was removed by rotary evaporation, transferred to a 1L single-necked flask, and evaporated to dryness. The mixture was then slurried with 500 mL of methyl tert-butyl ether for 1 hour, filtered, and then slurried with 300 mL of tetrahydrofuran for 16 hours. Filtering yielded 20 g (33.2 mmol, 61.3%) of a white solid.

[0202] 1 H NMR (300 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.10 (d, J = 7.1 Hz, 1H), 7.89 (d, J = 7.3 Hz, 2H), 7.78 – 7.65 (m, 2H), 7.54 (d, J = 8.1 Hz, 2H), 7.42(t, J = 7.6 Hz, 3H), 7.32 (t, J = 7.3 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 6.04– 5.88 (m, 1H), 5.47 – 5.30 (m, 2H), 5.15 – 5.00 (m, 1H), 4.50 – 4.17 (m,6H), 3.93 (t, J = 7.5 Hz, 1H), 3.10 – 2.88 (m, 2H), 2.05 – 1.95 (m, 1H), 1.76 – 1.57 (m, 2H), 1.50 – 1.34 (m, 2H), 0.96 – 0.81 (m, 6H).

[0203] Step 4: Synthesis of CS-2

[0204]

[0205] Add INC (20g, 33mmol) and 1L DMF to a single-necked flask, stir until completely dissolved, then add SM6 (63g, 209mmol). Cool to 0-5℃ in an ice bath, add DIPEA (27g, 209mmol) dropwise, and then react in an ice bath for 3 hours.

[0206] TLC monitoring showed complete conversion of dichloromethane to methanol (10:1) and INC. The reaction was stopped, the mixture was pumped dry at 55°C, crushed, and slurried with 100 mL of methyl tert-butyl ether for 1 hour, then filtered. It was then slurried with 300 mL of a 1:1 DCM / PE mixture for 1 hour, and filtered again. The mixture was then slurried again with 300 mL of a 1:1 DCM / PE mixture, and filtered to obtain 15.5 g (20.2 mmol, 61.3%) of a yellowish-brown solid.

[0207] 1H NMR (300 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.37 – 8.26 (m, 2H), 8.14(d, J = 7.4 Hz, 1H), 7.89 (d, J = 7.3 Hz, 2H), 7.74 (t, J = 6.6 Hz, 2H), 7.65(d, J = 8.4 Hz, 2H), 7.60 – 7.52 (m, 2H), 7.47 – 7.37 (m, 5H), 7.32 (t, J =7.1 Hz, 2H), 6.02 – 5.90 (m, 1H), 5.41 (s, 2H), 5.25 (s, 2H), 4.49 – 4.37 (m,1H), 4.34 – 4.18 (m, 3H), 3.97 – 3.88 (m, 1H), 3.09 – 2.91 (m, 2H), 2.07 –1.92 (m, 1H), 1.76 – 1.54 (m, 2H), 1.52 – 1.30 (m, 2H), 0.93 – 0.79 (m, 6H).

[0208] Example 3 Synthesis of FS-1

[0209]

[0210] Step 1: Synthesis of IND

[0211]

[0212] In a single-necked flask, SM7 (9 g, 36.24 mmol), 1,4-dioxane (45 mL), and Na2CO3 aqueous solution (10%, 90 mL) were added. Under ice bath conditions, a 1,4-dioxane (45 mL) solution of Fmoc-Cl (10.3 g, 39.87 mmol) was added dropwise, and the reaction was continued under ice bath conditions. The reaction was monitored by TLC (DCM:MeOH = 10:1), and the starting material was completely converted. 300 mL of EA was added to the reaction mixture, and the solution was washed twice with 100 mL of water and twice with 100 mL of saturated brine. The solution was then concentrated. The crude product was passed through a silica gel column and eluted with EA / PE at 30%-50% to give 16.5 g (35.09 mmol, 96.8%) of a colorless oily product.

[0213] 1H NMR (300 MHz, DMSO-d6) δ 8.00 – 7.76 (m, 3H), 7.72 – 7.65 (m, 2H), 7.44 – 7.30 (m, 5H), 4.47 – 4.10 (m, 3H), 3.52 – 3.42 (m, 4H), 3.42 – 3.32 (m, 4H), 3.23 – 3.02 (m, 4H), 1.37 (s, 9H).

[0214] Step 2: Synthesis of INE

[0215]

[0216] Add IND (16.5 g, 35.09 mmol) to a 250 mL single-necked flask, and add HCl-dioxane solution (4 N, 50 mL) while stirring. React at room temperature for 1 h. TLC monitoring showed that PE:EA = 1:1, indicating complete conversion of the starting material. Remove the solvent by rotary evaporation to give 14 g (34.4 mmol, 98.05%) of a colorless oily product.

[0217] 1 H NMR (300 MHz, DMSO-d6) δ 8.05 (s, 3H), 7.90 (d, J = 7.4 Hz, 2H), 7.73 – 7.61 (m, 2H), 7.38 (dt, J = 13.8, 7.3 Hz, 5H), 4.44 – 4.14 (m, 3H), 3.56 – 3.51 (m, 4H), 3.49 – 3.26 (m, 4H), 3.15 (dd, J = 11.7, 5.8 Hz, 2H), 2.94 (dd, J = 10.1, 5.0 Hz, 2H).

[0218] Step 3: Synthesis of FS-1

[0219]

[0220] In a 500 mL single-necked flask, add DCM (250 mL), INE (14 g, 34.4 mmol), and succinic anhydride (11.4 g, 114.2 mmol), and cool in an ice bath. Add triethylamine (3.86 g, 38.1 mmol) dropwise, and allow to react at room temperature for 3 hours. Monitor the reaction by TLC; DCM / MeOH = 10 / 1, indicating complete conversion of the starting material. Dilute the reaction solution with 800 mL of dichloromethane, wash twice with 200 mL of water, wash once with 200 mL of saturated brine, dry to anhydrous sodium sulfate, filter, and concentrate the filtrate. Pass the crude product through a silica gel column using DCM / MeOH = 20:1 as the eluent to obtain 15.5 g (32.9 mmol, 95.9%) of a pale yellow oil. 1 H NMR (300 MHz, DMSO-d6) δ12.08 (s, 1H), 7.98 – 7.80 (m, 3H), 7.73 – 7.60 (m, 2H), 7.37 (dt, J = 13.9,7.3 Hz, 5H), 4.46 – 4.14 (m, 3H), 3.55 – 3.43 (m, 4H), 3.42 – 3.34 (m, 4H), 3.25 – 3.06 (m, 4H), 2.44 – 2.39 (m, 2H), 2.37 – 2.26 (m, 2H).

[0221] Example 4 Synthesis of Bn-LS-1

[0222]

[0223] Step 1: Synthesis of INF

[0224]

[0225] DMF (450 mL) was added to a 1 L single-necked flask, followed by SM9 (50 g, 0.413 mol), benzyl bromide (155 g, 0.908 mol), and K2CO3 powder (143 g, 1.038 mol). The mixture was refluxed in an oil bath at 155 °C for 20 hours. The reaction was monitored by LCMS. The solvent was removed by rotary evaporation, followed by the addition of 1 L of dichloromethane. The mixture was washed twice with water (300 mL), twice with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness. 40 mL of ethyl acetate was added to the crude product, and the mixture was heated under reflux until completely dissolved. The mixture was then cooled and allowed to crystallize overnight. After filtration and vacuum drying, 55 g (0.182 mmol, 44.07%) of off-white crystals were obtained.

[0226] 1H NMR (300 MHz, DMSO-d6) δ 7.31 – 7.00 (m, 10H), 4.29 (t, J = 5.1 Hz, 3H), 3.97 (s, 4H), 3.52 (d, J = 5.1 Hz, 6H).

[0227] Step 2: Synthesis of Bn-LS-1

[0228]

[0229] In a 500 mL three-necked flask, add 80 mL of water, 80 g of sodium hydroxide (1.99 mol), then add 160 mL of DCM, 3.2 g of tetrabutylammonium bromide (10 mmol), and 20 g of INF (66.36 mmol), and stir mechanically. Cool to below 10 °C in an ice bath, then add dropwise tert-butyl bromoacetate (52 g, 265.44 mmol). After the addition is complete, allow to rise naturally to 20 °C and stir overnight (16 h). Monitor the reaction by TLC (PE:EA = 1:1), and no starting material remains. Add 1 L of DCM to the reaction system, wash twice with water, then twice with saturated sodium bicarbonate solution, and concentrate. Elute the crude product by silica gel column chromatography with PE:EA = 20:1 to obtain 17 g of product Bn-LS-1 (26.40 mmol, 39.78%).

[0230] 1 H NMR (300 MHz, DMSO-d6) δ 7.29 – 7.19 (m, 4H), 7.17 – 7.00 (m, 6H), 3.98 (s, 4H), 3.88 (s, 6H), 3.63 (s, 6H), 1.41 (s, 27H).

[0231] Example 5: Synthesis of HG-PL1

[0232]

[0233] Step 1: Synthesis of PH-HG-001-7

[0234]

[0235] Add MMAE (740.2 mg, 1.05 mmol, 1.5 equivalence), HOBt (18.6 mg, 0.14 mmol, 0.2 equivalence), and pyridine (16.3 mg, 0.21 mmol, 0.3 equivalence) in portions to a DMF (10.0 mL) solution of CS-2 (527.0 mg, 0.7 mmol, 1.0 equivalence). The reaction solution was heated to 25 °C.o The mixture was stirred overnight under nitrogen protection. 100 mL of water was added to the reaction mixture, and the mixture was extracted with dichloromethane (3 × 100 mL). The combined organic phases were washed with saturated brine (3 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative HPLC (X Bridge Prep OBD C18 column; mobile phase: water (10 mmol ammonium bicarbonate) and acetonitrile (from 42.0% acetonitrile to 72.0% acetonitrile, 10 min); detector: UV 254 nm) to give 550 mg (59%) of white solid PH-HG-001-7.

[0236] MS m / z [M+H] + (ESI): 1345.45.

[0237] Step 2: Synthesis of PH-HG-001-8

[0238]

[0239] Diethylamine (4 mL) was added to 8.0 mL of DMF containing PH-HG-001-7 (600.0 mg, 0.4 mmol, 1.0 equivalent) under nitrogen protection. The reaction mixture was stirred at 25 °C for 3 hours and then concentrated by rotary evaporation. The crude product was prepared by Flash-Prep-HPLC (C8 column, mobile phase: water (10 mmol ammonium bicarbonate) and acetonitrile (10% to 80%), detector UV 210 nm) to obtain 340 mg (68%) of white solid PH-HG-001-8.

[0240] MS m / z [M+H] + (ESI): 1123.75. 1H NMR (300 MHz, DMSO-d6) δ: 0.78-0.92(m, 24H), 0.99-1.06 (m, 7H), 1.21-1.27 (m, 1H), 1.31-1.37 (m, 3H), 1.49-1.86(m, 7H), 1.90-2.21 (m, 4H), 2.37-2.45 (m, 1H), 2.81-3.01 (m, 6H), 3.10-3.22(m, 9H), 3.51-3.81 (m, 3H), 3.90-4.08 (m, 2H), 4.14-4.28 (m, 2H), 4.34-4.80(m, 4H), 4.84-5.17 (m, 2H), 5.39-5.58 (m, 2H), 6.01-6.35 (m, 1H), 7.10-7.37(m, 7H), 7.55-7.92 (m, 3H), 7.99-8.41 (m, 1H), 10.12-10.52 (m, 1H).

[0241] Step 3: Synthesis of PH-HG-001-1

[0242]

[0243] Palladium hydroxide on carbon (0.2 g) was added to a methanol (20 mL, chromatographic grade) solution of Bn-LS-1 (1.0 g, 1.6 mmol, 1.0 equivalent). The reaction mixture was purged with hydrogen five times and stirred at 25 °C for 4 hours. The reaction solution was filtered, concentrated under reduced pressure to obtain 650 mg of a colorless oily substance, PH-HG-001-1 (90%). This product can be used directly in the next step without further purification.

[0244] MS m / z [M+H] + (ESI): 464.30.

[0245] Step 4: Synthesis of PH-HG-001-2

[0246]

[0247] FS-1 (650.3 mg, 1.4 mmol, 1.1 equivalent), N,N-diisopropylethylamine (494.4 mg, 3.8 mmol, 3.0 equivalent), and 1H-benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP) (995.4 mg, 1.9 mmol, 1.5 equivalent) were added to a nitrogen-protected solution of PH-HG-001-1 (600.0 mg, 1.3 mmol, 1.0 equivalent) at 0 °C. The reaction mixture was stirred at 25 °C for 2 hours, quenched with 100 mL of water, and extracted with ethyl acetate (3 × 100 mL). The combined organic phases were washed with saturated brine (3 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was passed through a silica gel column and eluted with dichloromethane:methanol = 30:1 to give 900 mg (77%) of a colorless oily substance PH-HG-001-2.

[0248] MS m / z [M+H] + (ESI): 916.50.

[0249] Step 5: Synthesis of PH-HG-001-3

[0250]

[0251] Formic acid (5.0 mL) was added to a nitrogen-protected solution of PH-HG-001-2 (1.1 g, 1.2 mmol, 1.0 equivalent) in dichloromethane (5.0 mL). The reaction mixture was stirred overnight at 25 °C and concentrated by rotary evaporation. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase: water and acetonitrile (10% to 80% acetonitrile, 20 min); detector: UV 210 nm) to obtain 720 mg (80%) of colorless oily PH-HG-001-3.

[0252] MS m / z [M+H] + (ESI): 748.15. 1H NMR (400 MHz, methanol-d4) δ: 2.42-2.58 (m,4H), 3.36-3.40 (m, 4H), 3.47-3.55 (m, 4H), 3.57-3.64 (m, 4H), 3.89 (s, 6H), 4.09 (s, 6H), 4.20-4.24 (m, 1H), 4.37-4.39 (m, 2H), 7.30-7.32 (m, 2H), 7.38-7.42 (m, 2H), 7.66 (d, J = 7.2 Hz, 2H), 7.81 (d, J = 7.2 Hz, 2H).

[0253] Step 6: Synthesis of PH-HG-001-14

[0254]

[0255] At 0 °C, Ms-1 (648.7 mg, 1.2 mmol, 3.3 equivalence) was added sequentially to a nitrogen-protected solution of PH-HG-001-3 (265.0 mg, 0.35 mmol, 1.0 equivalent) in DMF (10.0 mL), followed by N,N-diisopropylethylamine (206.1 mg, 1.6 mmol, 4.5 equivalent) and benzotriazole-N,N,N',N'-tetramethylureaium hexafluorophosphate (HBTU) (604.8 mg, 1.6 mmol, 4.5 equivalent). The reaction mixture was stirred at 25 °C for 2 hours, diluted with 100 mL of water, and extracted with dichloromethane (3 × 200 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was purified by Flash-Prep-HPLC (conditions: C18 silica column; mobile phase: water and acetonitrile (10% to 100% acetonitrile, 20 min); detector: UV 210 nm) to obtain 550 mg (66%) of colorless oil PH-HG-001-14.

[0256] MS m / z [M / 2+H] + (ESI): 1179.45.

[0257] Step 7: Synthesis of PH-HG-001-6

[0258]

[0259] Formic acid (2.5 mL) was added to a nitrogen-protected solution of PH-HG-001-14 (550.0 mg, 0.2 mmol, 1.0 equivalent) in dichloromethane (2.5 mL). The reaction mixture was stirred overnight at 25 °C and then concentrated by rotary evaporation. The crude product was purified by Flash-Prep-HPLC (conditions: C18 silica column; mobile phase: water and acetonitrile (10% to 80% acetonitrile, 20 min); detector: UV 210 nm) to obtain 360 mg (70%) of colorless oil PH-HG-001-6.

[0260] MS m / z [M / 2+H] + (ESI): 1095.40. 1 H NMR (300 MHz, methanol-d4) δ: 2.48-2.55(m, 4H), 2.85-2.87 (m, 6H), 3.35-3.40 (m, 19H), 3.53-3.60 (m, 17H), 3.62-3.64(m, 81H), 3.89 (s, 6H), 4.09 (m, 6H), 4.38 (m, 3H), 4.77-4.81 (m, 3H), 7.33-7.42 (m, 4H), 7.68 (d, J = 7.2 Hz, 2H), 7.83 (d, J = 7.5 Hz, 2H).

[0261] Step 8: Synthesis of PH-HG-001-9

[0262]

[0263] At 0 °C, PH-HG-001-8 (254.0 mg, 0.23 mmol, 3.3 equivalents), N-methylimidazole (50.6 mg, 0.63 mmol, 9.0 equivalents), and N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH) (69.2 mg, 0.25 mmol, 3.6 equivalents) were added sequentially to a nitrogen-protected solution of PH-HG-001-6 (150.0 mg, 0.07 mmol, 1.0 equivalents) in 3.0 mL of acetonitrile. The reaction solution was stirred at 25 °C for 4 hours and then concentrated by rotary evaporation. The crude product was purified by preparative HPLC (conditions: XSelect CSH Prep C18 OBD™ column; size 19 mm × 250 mm, 5 µm; mobile phase, water (0.05% formic acid) and acetonitrile (54.0% to 68.0% acetonitrile, 10 min); detector, UV 254 nm) to give 160 mg (42%) of white solid product PH-HG-001-9.

[0264] MS m / z [M / 4+H] + (ESI): 1377.00.

[0265] Step 9: Synthesis of PH-HG-001-10

[0266]

[0267] Piperidine (0.5 mL) was added to a nitrogen-protected solution of PH-HG-001-9 in DMF (1.5 mL). The reaction mixture was stirred at 25 °C for 3 hours and then concentrated by rotary evaporation. The crude product was purified by preparative HPLC (conditions: XSelect CSH Prep C18 OBD™ column; size 19 mm × 250 mm, 5 µm; mobile phase, water (0.05% formic acid) and acetonitrile (41.0% to 50.0% acetonitrile, 10 min); detector, UV 254 nm) to obtain 90 mg (43%) of white solid product PH-HG-001-10.

[0268] MS m / z [M / 4+H] + (ESI): 1321.45.

[0269] Step 10: Synthesis of HG-PL1

[0270]

[0271] To a 4.0 mL solution of PH-HG-001-10 (215.0 mg, 0.04 mmol, 1.0 equivalent) in DMF, hydroxysuccinimide 3-maleimide propionate (21.7 mg, 0.08 mmol, 2.0 equivalent) and N,N-diisopropylethylamine (15.8 mg, 0.12 mmol, 3.0 equivalent) were added. The reaction mixture was stirred at 25 °C for 4 hours and then concentrated by rotary evaporation. The crude product was purified by preparative HPLC (conditions: X Select CSH Prep C18 OBD™ column; dimensions 19 mm × 250 mm, 5 µm; mobile phase, water (0.05% formic acid) and acetonitrile (41.0% to 71.0% acetonitrile, 10 min); detector, UV 254 nm) to obtain 126.4 mg (57%) of white solid product HG-PL1.

[0272] MS m / z [M / 3+H] + (ESI): 1811.90. 1H NMR (400 MHz, DMSO-d6) δ: 0.75-0.77(m, 30H), 0.83-0.84 (m, 49H), 0.88-0.97 (m, 15H), 0.98-0.99 (m, 6H), 1.01-1.03 (m, 7H), 1.04-1.05 (m, 9H), 1.10-1.20 (m, 2H), 1.30-1.48 (m, 14H), 1.50-1.73 (m, 7H), 1.99-2.01 (m, 5H), 2.32-2.34 (m, 8H), 2.30-2.39 (m, 5H), 2.40-2.50 (m, 3H), 2.83-2.85 (m, 11H), 2.87-2.87 (m, 11H), 2.88-2.97 (m, 9H), 3.12-3.15 (m, 7H), 3.17-3.19 (m, 8H), 3.23-3.24 (m, 24H), 3.33-3.35 (m, 2H),3.40-3.41 (m, 9H), 3.42-3.43 (m, 4H), 3.45-3.49 (m, 64H), 3.57-3.59 (m, 5H),3.60-3.61 (m, 7H), 3.70-3.76 (m, 13H), 3.92-3.96 (m, 7H), 4.00-4.27 (m, 10H),4.40-4.49 (m, 6H), 5.00-5.10 (m, 5H), 5.40-5.43 (m, 9H), 5.99 (s, 3H), 6.99(s, 2H), 7.26-7.28 (m, 4H), 7.29-7.32 (m, 22H), 7.60-7.62 (m, 9H), 8.02-8.10(m, 18H), 8.11-8.20 (m, 4H), 9.80 (s, 3H).

[0273] Example 6: Synthesis of HG-PL2

[0274]

[0275] Step 1: Synthesis of PH-HG-002-1

[0276]

[0277] To a nitrogen-protected solution of PH-HG-001-3 (3.8 g, 5.1 mmol, 1.0 equivalent) in 60 mL of DMF, N,N-diisopropylethylamine (3.0 g, 23.2 mmol, 4.5 equivalent), SM11 (5.7 g, 17.0 mmol, 3.3 equivalent), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) (12.1 g, 23.3 mmol, 4.5 equivalent) were added sequentially. The reaction mixture was stirred at 25 °C for 5 hours, diluted with 300 mL of water, and extracted with ethyl acetate (3 × 300 mL). The combined organic phases were washed with saturated brine (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica column; mobile phase: water and acetonitrile (10% to 100% acetonitrile, 20 min); detector: UV 210 nm) to obtain 6.1 g (70%) of colorless oily PH-HG-002-1.

[0278] MS m / z [M+H] + (ESI): 1702.93. 1 H NMR (400 MHz, DMSO-d6) δ: 1.30-1.42(s, 27H), 2.25-2.40 (m, 4H), 3.03-3.08 (m, 6H), 3.12-3.20 (m, 4H), 3.25-3.29(m, 6H), 3.35-3.44 (m, 18H), 3.49-3.50 (m, 38H), 3.71 (s, 6H), 3.88 (s, 6H), 4.21-4.30 (m, 3H), 6.71-6.73 (m, 3H), 7.33 (t, J = 7.6 Hz, 3H), 7.42 (t, J =7.6 Hz, 2H), 7.68-7.75 (m, 6H), 7.88-7.90 (m, 3H).

[0279] Step 2: Synthesis of PH-HG-002-2

[0280]

[0281] Trifluoroacetic acid (15 mL) was added to a nitrogen-protected solution of PH-HG-002-1 (3.1 g, 1.8 mmol, 1.0 equivalent) in dichloromethane (15 mL). The reaction mixture was stirred at 25 °C for 3 hours and then concentrated by rotary evaporation. The crude product was purified by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase: water and acetonitrile (10% to 80% acetonitrile, 20 min); detector: UV 210 nm) to obtain 1.6 g (64%) of colorless oil PH-HG-002-2.

[0282] MS m / z [M / 2+H]+ (ESI): 1402.77.

[0283] Step 3: Synthesis of PH-HG-002-3

[0284]

[0285] At 0 °C, succinic anhydride (1.9 g, 19.3 mmol, 9.0 equivalence), triethylamine (2.6 g, 25.7 mmol, 12.0 equivalence), and 4-dimethylaminopyridine (0.8 g, 6.4 mmol, 3.0 equivalence) were added to a nitrogen-protected solution of PH-HG-002-2 in 45 mL of dichloromethane. The reaction mixture was stirred at 25 °C for 12 hours and then concentrated by rotary evaporation. The crude product was purified by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase: water and acetonitrile (10% to 80% acetonitrile, 30 min); detector: UV 210 nm) to obtain 1.6 g (44%) of colorless oily PH-HG-002-3.

[0286] MS m / z [M / 2+H] + (ESI): 1402.77. 1 H NMR (300 MHz, DMSO-d6) δ: 2.34-2.39(m, 10H), 2.41-2.44 (m, 6H), 3.13-3.28 (m, 16H), 3.37-3.51 (m, 56H), 3.72 (s,6H), 3.89 (s, 6H), 4.19-4.31 (m, 3H), 7.32 (t, J = 7.5 Hz, 3H), 7.43 (t, J =7.5 Hz, 2H), 7.68-7.77 (m, 6H), 7.88-7.91 (m, 6H), 12.00-12.06 (m, 2H).

[0287] Step 4: Synthesis of PH-HG-002-4

[0288]

[0289] At 0 °C, Ms-1 (1.7 g, 3.1 mmol, 3.3 equivalents), N,N-diisopropylethylamine (546.5 mg, 4.2 mmol, 4.5 equivalents), and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) (1.6 g, 4.2 mmol, 4.5 equivalents) were added to a nitrogen-protected solution of PH-HG-002-3 (1.6 g, 0.9 mmol, 1.0 equivalents) in 30 mL of DMF. The reaction mixture was stirred at 25 °C for 5 hours, diluted with 300 mL of water, and extracted with ethyl acetate (3 × 300 mL). The combined organic phases were washed with saturated brine (3 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica column; mobile phase: water (containing 0.1% formic acid) and acetonitrile (10% to 100% acetonitrile, 20 min); detector: UV 210 nm) to obtain 1.7 g (55%) of yellow oily substance PH-HG-002-4.

[0290] MS m / z [M / 2+H] + (ESI): 1657.15.

[0291] Step 5: Synthesis of PH-HG-002-5

[0292]

[0293] Formic acid (5.0 mL) was added to a nitrogen-protected solution of PH-HG-002-4 (1.0 g, 0.3 mmol, 1.0 equivalent) in dichloromethane (5 mL). The reaction mixture was stirred at 25 °C for 48 hours and then concentrated by rotary evaporation. The crude product was purified by Flash-Prep-HPLC (conditions: C18 silica column; mobile phase: water (containing 0.1% formic acid) and acetonitrile (10% to 80% acetonitrile, 30 min); detector: UV 210 nm) to obtain 700 mg (74%) of colorless oily PH-HG-002-5.

[0294] MS m / z [M / 2+H] + (ESI): 1573.20. 1H NMR (300 MHz, Methanol-d4) δ: 2.48-2.58 (m, 16H), 2.65-2.84 (m, 6H), 3.20-3.30 (m, 3H), 3.33-3.47 (m, 30H), 3.53-3.65 (m, 144H), 3.88 (s, 6H), 4.03 (s, 6H), 4.20-4.40 (m, 3H), 4.73-4.85(m, 3H), 7.34-7.42 (m, 4H), 7.67 (m, 2H), 7.83 (d, J = 7.5 Hz, 2H).

[0295] Step 6: Synthesis of PH-HG-002-6

[0296]

[0297] Diethylamine (0.1 mL) was added to a nitrogen-protected DMF solution of PH-HG-002-5 (300 mg, 0.1 mmol, 1.0 equivalent) in 4 mL of DMF at 0 °C. After stirring at 0 °C for 2 hours, the reaction solution was purified by preparative HPLC (conditions: X Select CSH PrepC18 OBD column; size 19 mm × 250 mm, 5 µm; mobile phase, water (0.05% formic acid) and acetonitrile (8.0% to 23.0% acetonitrile, 11 min); detector, UV 200 nm) to obtain 140 mg (50%) of colorless oily product PH-HG-002-6.

[0298] MS m / z [M / 2+H] + (ESI): 1461.95.

[0299] Step 7: Synthesis of PH-HG-002-7

[0300]

[0301] To a nitrogen-protected solution of PH-HG-002-6 in DMF (5 mL), hydroxysuccinimide 3-maleimide propionate (19.1 mg, 0.07 mmol, 1.5 equivalence) and N,N-diisopropylethylamine (10.5 mg, 0.14 mmol, 3.0 equivalence) were added. After stirring at 0 °C for 4 hours, the reaction mixture was purified by preparative HPLC (conditions: XBridgePrep C18 OBD column; 30 mm × 150 mm, 5 µm; mobile phase: water (0.05% formic acid) and acetonitrile (8.0% to 38.0% acetonitrile, 7 min); detector: UV 200 nm) to obtain 75 mg (51%) of a colorless oily product, PH-HG-002-7.

[0302] MS m / z [M / 2+H] + (ESI): 1537.55.

[0303] Step 8: Synthesis of HG-PL2

[0304]

[0305] At 0 °C, PH-HG-001-8 (144.8 mg, 0.13 mmol, 3.3 equivalent), N,N-diisopropylethylamine (22.7 mg, 0.18 mmol, 4.5 equivalent), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) (91.4 mg, 0.18 mmol, 4.5 equivalent) were added to a nitrogen-protected DMF (4 mL) solution of PH-HG-002-7 (120 mg, 0.039 mmol, 1.0 equivalent). After stirring at 0°C for 2 hours, the reaction solution was purified by preparative HPLC (conditions: XBridgePrep C18 OBD column; size 30 mm × 150 mm, 5 µm; mobile phase, water (0.01% formic acid) and acetonitrile (35.0% to 65.0% acetonitrile, 7 min); detector, UV 254 nm) to obtain 23.4 mg (9%) of white solid product HG-PL2.

[0306] MS m / z [M / 4+H] + (ESI): 1598.50. 1H NMR (400 MHz, DMSO-d6) δ: 0.75-1.05(m, 101H), 1.24-1.39 (m, 7H), 1.42-1.54 (m, 11H), 1.64-1.82 (m, 16H), 1.90-2.15 (m, 13H), 2.21-2.47 (m, 31H), 2.67-2.78 (m, 5H), 2.85-3.09 (m, 23H), 3.12-3.30 (m, 76H), 3.38-3.60 (m, 96H), 3.62-3.76 (m, 11H), 3.88-4.05 (m,14H), 4.14-4.37 (m, 11H), 4.39-4.55 (m, 11H), 4.62-4.77 (m, 4H), 4.93-5.12(m, 7H), 5.37-5.45 (m, 10H), 5.97-6.02 (m, 3H), 7.00 (s, 2H), 7.18-7.32 (m,23H), 7.59-7.63 (m, 8H), 7.74-7.77 (m, 4H), 7.88-8.08 (m, 16H), 8.19-8.34 (m,8H), 9.72 (s, 3H).

[0307] Example 7 Synthesis of HG-PL3

[0308]

[0309] Step 1: Synthesis of PH-HG-003-1

[0310]

[0311] Diethylamine (0.25 mL) was added to a nitrogen-protected solution of PH-HG-001-6 (1.0 g, 0.1 mmol, 1.0 equivalent) in DMF (10 mL) at 0 °C. After stirring at 0 °C for 2 hours, the reaction solution was purified by preparative HPLC (conditions: XSelect CSH PrepC18 OBD column; size 19 mm × 250 mm, 5 µm; mobile phase, water (0.1% formic acid) and acetonitrile (8.0% to 23.0% acetonitrile, 11 min); detector, UV 200 nm) to obtain 400 mg (45%) of colorless oily product PH-HG-003-1.

[0312] MS m / z [M+H] + (ESI):1966.55.

[0313] Step 2: Synthesis of PH-HG-003-2

[0314]

[0315] To a nitrogen-protected solution of PH-HG-003-1 (100 mg, 0.05 mmol, 1.0 equivalent) in DMF (2.0 mL), hydroxysuccinimide 3-maleimide propionate (20.3 mg, 0.08 mmol, 1.5 equivalent) and N,N-diisopropylethylamine (19.7 mg, 0.15 mmol, 3.0 equivalent) were added. After stirring at 0 °C for 4 hours, the reaction mixture was purified by preparative HPLC (conditions: XBridge Prep C18 OBD column; 30 mm × 150 mm, 5 µm; mobile phase: water (0.1% formic acid) and acetonitrile (17.0% to 27.0% acetonitrile, 10 min); detector: UV 200 nm) to obtain 50 mg (46%) of colorless oily product PH-HG-003-2.

[0316] MS m / z [M / 2+H] + (ESI): 1059.95.

[0317] Step 3: Synthesis of PH-HG-003-3

[0318]

[0319] To a nitrogen-protected solution of CS-2 (88 mg, 0.115 mmol, 1.0 equivalent) in DMF (2.0 mL), eribulin (100.5 mg, 0.138 mmol, 1.2 equivalent), 1-hydroxybenzotriazole (HOBT) (3.1 mg, 0.023 mmol, 0.2 equivalent), and pyridine (2.7 mg, 0.035 mmol, 0.3 equivalent) were added. The reaction mixture was stirred overnight at 25 °C, diluted with 20 mL of water, and extracted with dichloromethane (3 × 20 mL). The combined organic phases were washed with saturated brine (3 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica column; mobile phase: water and acetonitrile (10% to 100% acetonitrile, 30 min); detector: UV 254 nm) to obtain 60 mg (39%) of white solid PH-HG-003-3.

[0320] MS m / z [M+H] + (ESI): 1357.68.

[0321] Step 4: Synthesis of PH-HG-003-4

[0322]

[0323] Diethylamine (0.05 mL) was added to a nitrogen-protected solution of PH-HG-003-3 (100 mg, 0.07 mmol, 1.0 equivalent) in DMF (2.0 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 2 hours and then concentrated. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase: water and acetonitrile (10% to 100% acetonitrile, 30 min); detector: UV 210 nm) to obtain 70 mg (84%) of white solid PH-HG-003-4.

[0324] MS m / z [M+H] + (ESI): 1135.61; 1 H NMR (300 MHz, DMSO-d6) δ: 0.81 (d, J= 6.6 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H), 1.02-1.05 (m, 3H), 1.1.12-1.20 (m,2H), 1.27-1.40 (m, 4H), 1.47-1.58 (m, 4H), 1.62-1.75 (m, 7H), 1.88-2.05 (m,7H), 2.11-2.13 (m, 1H), 2.18-2.28 (m, 4H), 2.32-2.35 (m, 1H), 2.58-2.61 (m,1H), 2.65-2.75 (m, 2H), 2.81-2.87 (m, 2H), 2.93-3.05 (m, 4H), 3.07-3.11 (m,1H), 3.21-3.24 (m, 4H), 3.45-3.57 (m, 4H), 3.68-3.83 (m, 3H), 4.01-4.13 (m,4H), 4.18-4.31 (m, 2H), 4.48-4.65 (m, 4H), 4.75-4.85 (m, 2H), 4.91-5.09 (m,4H), 5.42 (s, 2H), 5.98-6.02 (m, 1H), 7.09-7.11 (m, 1H), 7.28 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.4 Hz, 2H), 8.21-8.25 (m, 1H), 10.14 (s, 1H).

[0325] Step 5: Synthesis of HG-PL3

[0326]

[0327] At 0 °C, PH-HG-003-4 (70.8 mg, 0.063 mmol, 3.3 equivalents), N,N-diisopropylethylamine (11.0 mg, 0.085 mmol, 4.5 equivalents), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) (44.2 mg, 0.085 mmol, 4.5 equivalents) were added to a nitrogen-protected DMF (1 mL) solution of PH-HG-003-2 (40 mg, 0.02 mmol, 1.0 equivalents). After stirring at 0°C for 2 hours, the reaction solution was purified by preparative HPLC (conditions: XBridge Shield RP18OBD column; size 30 mm × 150 mm, 5 µm; mobile phase, water and acetonitrile (36.0% to 66.0% acetonitrile, 10 min); detector, UV 254 nm) to obtain 14.2 mg (14%) of white solid product HG-PL3.

[0328] MS m / z [M / 3+H] + (ESI): 1823.65.

[0329] Example 8: Synthesis of HG-PL4

[0330]

[0331] Step 1: Synthesis of HG-PL4

[0332]

[0333] At 0 °C, PH-HG-003-4 (79.2 mg, 0.069 mmol, 3.3 equivalent), N,N-diisopropylethylamine (12.3 mg, 0.095 mmol, 4.5 equivalent), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) (49.5 mg, 0.095 mmol, 4.5 equivalent) were added to a nitrogen-protected solution of PH-HG-002-7 (65 mg, 0.021 mmol, 1.0 equivalent) in DMF (2.0 mL). After stirring at 0°C for 2 hours, the reaction solution was purified by preparative HPLC (conditions: XBridgeShield RP18 OBD column; size 19 mm × 150 mm, 5 µm; mobile phase, water and acetonitrile (35.0% to 65.0% acetonitrile, 10 min); detector, UV 254 nm) to obtain 20.9 mg (15%) white solid product HG-PL4.

[0334] MS m / z [M / 4+H] + (ESI): 1606.80. 1 H NMR (400 MHz, DMSO-d6) δ: 0.75-0.80(m, 34H), 0.90-0.97 (m, 22H), 1.12-1.43 (m, 51H), 1.59-1.70 (m, 38H), 1.84-1.97 (m, 35H), 2.19-2.25 (m, 41H), 2.67-2.77 (m, 21H), 2.90-2.95 (m, 24H), 3.10-3.18 (m, 53H), 3.64-3.68 (m, 23H), 3.76-3.82 (m, 16H), 3.98-4.11 (m,30H), 4.19-4.26 (m, 10H), 4.49-4.59 (m, 17H), 4.66-4.78 (m, 6H), 4.85-4.98(m, 14H), 5.31-5.38 (m, 5H), 6.97-7.23 (m, 2H), 7.58-7.95 (m, 32H), 8.10-8.23(m, 10H), 9.61 (m, 6H).

[0335] Example 9: Synthesis of HG-PL5

[0336]

[0337] Step 1: Synthesis of PH-HG-005-1

[0338]

[0339] At 0 °C, 4-(dimethylamino)pyridine (77.8 mg, 0.64 mmol, 0.1 equivalence), triethylamine (1.9 g, 19.1 mmol, 3.0 equivalence), and di-tert-butyl dicarbonate (1.67 g, 7.7 mmol, 1.2 equivalence) were added to a nitrogen-protected solution of SN-38 (2.5 g, 6.4 mmol, 1.0 equivalence) in 35 mL of tetrahydrofuran. The reaction mixture was stirred at 25 °C for 3 hours and then concentrated. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase: water and acetonitrile (10% to 100% acetonitrile, 30 min); detector: UV 254 nm) to obtain 2.1 g (64%) of yellow solid PH-HG-005-1.

[0340] MS m / z [M+H] +(ESI): 493.19. 1 H NMR (300 MHz, DMSO-d6) δ: 0.91 (t, J =6.9 Hz, 3H), 1.31 (t, J = 7.5 Hz, 3H), 1.55 (s, 9H), 1.84-1.93 (m, 2H), 3.18-3.25 (m, 2H), 5.34 (s, 2H), 5.45 (s, 2H), 6.54 (s, 1H), 7.34 (s, 1H), 7.74(d, J = 9.3 Hz, 1H), 8.10 (s, 1H), 8.21 (d, J = 9.3 Hz, 1H).

[0341] Step 2: Synthesis of PH-HG-005-2

[0342]

[0343] At 0°C, 4-(dimethylamino)pyridine (620.1 mg, 5.1 mmol, 5.0 equivalent) and triphosgene (301.3 mg, 1.0 mmol, 1.0 equivalent) were added to a nitrogen-protected solution of PH-HG-005-1 in 10.0 mL of dichloromethane. The reaction mixture was stirred at 25°C for 4 hours and then concentrated. This product can be used directly in the next step without further purification.

[0344] Step 3: Synthesis of PH-HG-005-3

[0345]

[0346] At 0 °C, INC (650.5 mg, 1.1 mmol, 1.1 equivalent) and 4A molecular sieve (500 mg) were added to a nitrogen-protected solution of PH-HG-005-2 (crude product from step 2) in dichloromethane (10.0 mL). The reaction solution was stirred at 25 °C for 12 hours and then concentrated. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase: water and acetonitrile (10% to 100% acetonitrile, 30 min); detector: UV 254 nm) to obtain 240 mg (21% two-step yield) of yellow solid PH-HG-005-3.

[0347] MS m / z [M+H]+ (ESI): 1120.46; 1H NMR (300 MHz, DMSO-d6) δ: 0.86-0.91(m, 9H), 1.28-1.30 (m, 3H), 1.39-1.43 (m, 2H), 1.55 (s, 9H), 1.62-1.75 (m,2H), 1.98-2.07 (m, 1H), 2.11-2.23 (m, 2H), 2.90-3.10 (m, 2H), 3.18-3.23 (m,2H), 3.91-3.97 (m, 1H), 4.21-4.32 (m, 3H), 4.41-4.47 (m, 1H), 5.06-5.16 (m,2H), 5.37-5.42 (m, 4H), 5.54 (s, 2H), 5.98-6.02 (m, 1H), 7.06 (s, 1H), 7.33-7.37 (m, 4H), 7.40-7.42 (m, 3H), 7.54-7.63 (m, 2H), 7.75-7.78 (m, 3H), 7.87-7.91 (m, 2H), 8.12-8.15 (m, 2H), 8.24-8.26 (m, 1H), 10.11 (s, 1H).

[0348] Step 4: Synthesis of PH-HG-005-4

[0349]

[0350] Diethylamine (0.1 mL) was added to a nitrogen-protected solution of PH-HG-005-3 (400.0 mg, 0.36 mmol, 1.0 equivalent) in DMF (4.0 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 2 hours and then concentrated. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase: water (10 mmol ammonium bicarbonate) and acetonitrile (10% to 80% acetonitrile, 20 min); detector: UV 210 nm) to obtain 170 mg (53%) of yellow solid PH-HG-005-4.

[0351] MS m / z [M+H] + (ESI): 898.39.

[0352] Step 5: Synthesis of PH-HG-005-5

[0353]

[0354] Diethylamine (0.1 mL) was added to a nitrogen-protected solution of PH-HG-005-4 (100.0 mg, 0.11 mmol, 1.0 equivalent) in hexafluoroisopropanol (2.0 mL). The reaction mixture was stirred at 45 °C for 2 hours and then concentrated. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase: water and acetonitrile (10% to 80% acetonitrile, 30 min); detector: UV 210 nm) to obtain 45 mg (51%) of yellow solid PH-HG-005-5.

[0355] MS m / z [M+H] + (ESI): 798.34.

[0356] Step 6: Synthesis of HG-PL5

[0357]

[0358] At 0 °C, PH-HG-005-5 (24.9 mg, 0.03 mmol, 3.3 equivalence), N,N-diisopropylethylamine (5.5 mg, 0.04 mmol, 4.5 equivalence), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) (22.1 mg, 0.04 mmol, 4.5 equivalence) were added to a nitrogen-protected DMF (1.0 mL) solution of PH-HG-003-2 (20 mg, 0.01 mmol, 1.0 equivalence). After stirring at 0°C for 2 hours, the reaction solution was purified by preparative HPLC (conditions: XBridge Prep OBD C18 column; size 19 mm × 250 mm, 5 µm; mobile phase, water (containing 0.01% formic acid) and acetonitrile (20.0% to 55.0% acetonitrile, 13 min); detector, UV 254 nm) to obtain 2.9 mg (7%) of yellow solid product HG-PL5.

[0359] MS m / z [M / 3+H] + (ESI): 1486.35.

[0360] Example 10 Synthesis of HG-PL6

[0361]

[0362] Step 1: Synthesis of HG-PL6

[0363]

[0364] At 0 °C, PH-HG-005-5 (21.4 mg, 0.026 mmol, 3.3 equivalents), N,N-diisopropylethylamine (4.7 mg, 0.036 mmol, 4.5 equivalents), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) (19.1 mg, 0.036 mmol, 4.5 equivalents) were added to a nitrogen-protected DMF (1.0 mL) solution of PH-HG-002-7 (25 mg, 0.0081 mmol, 1.0 equivalents). After stirring at 0°C for 2 hours, the reaction solution was purified by preparative HPLC (conditions: XSelect CSH Prep C18 column; size 19 mm × 250 mm, 5 µm; mobile phase, water (containing 0.01% formic acid) and acetonitrile (30.0% to 48.0% acetonitrile, 12 min); detector, UV 254 nm) to obtain 2.6 mg (5%) of yellow solid product HG-PL6.

[0365] MS m / z [M / 4+H] + (ESI): 1354.40.

[0366] Example 11 Synthesis of HG-PL7

[0367]

[0368] Step 1: Synthesis of PH-HG-007-1

[0369]

[0370] Add (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethylsuccinimide carbonate (BCN-Osu) (57.8 mg, 0.2 mmol, 1.5 equivalence) and N,N-diisopropylethylamine (51.3 mg, 0.4 mmol, 3.0 equivalence) to a nitrogen-protected solution of PH-HG-003-1 (260 mg, 0.13 mmol, 1.0 equivalence) in DMF (4.0 mL). After stirring at 25°C for 4 hours, the reaction solution was purified by preparative HPLC (conditions: XBridge Prep OBD C18 column; size 30 mm × 250 mm, 5 µm; mobile phase, water (containing 0.1% formic acid) and acetonitrile (19.0% to 49.0% acetonitrile, 7 min); detector, UV 200 nm) to obtain 100 mg (35%) of colorless oily substance PH-HG-007-1.

[0371] MS m / z [M / 2+H] + (ESI): 1072.35.

[0372] Step 2: Synthesis of HG-PL7

[0373]

[0374] At 0 °C, PH-HG-001-8 (86.5 mg, 0.08 mmol, 3.3 equivalent), N,N-diisopropylethylamine (13.6 mg, 0.103 mmol, 4.5 equivalent) and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) (54.6 mg, 0.103 mmol, 4.5 equivalent) were added to a nitrogen-protected DMF (2.0 mL) solution of PH-HG-007-1 (50 mg, 0.023 mmol, 1.0 equivalent). After stirring at 0°C for 2 hours, the reaction solution was purified by preparative HPLC (conditions: XBridgeShield Prep OBD column; size 30 mm × 150 mm, 5 µm; mobile phase, water (containing 0.01% formic acid) and acetonitrile (29.0% to 59.0% acetonitrile, 7 min); detector, UV 254 nm) to obtain 10.4 mg (8%) white solid HG-PL7.

[0375] MS m / z [M / 3+H] + (ESI): 1820.90. 1 H NMR (300 MHz, DMSO-d6) δ: 0.75-0.90(m, 84H), 0.95-1.05 (m, 23H), 1.15-1.24 (m, 1H), 1.25-1.52 (m, 17H), 1.60-1.88 (m, 14H), 1.90-2.24 (m, 18H), 2.38-2.42 (m, 6H), 2.80-2.90 (m, 7H), 3.07-3.13 (m, 13H), 3.17-3.28 (m, 36H), 3.48-3.52 (m, 89H), 3.72-3.81 (m,13H), 3.91-4.06 (m, 20H), 4.20-4.56 (m, 21H), 4.62-4.78 (m, 7H), 4.94-5.17(m, 7H), 5.35-5.43 (m, 10H), 5.95-6.03 (m, 3H), 7.14-7.35 (m, 22H), 7.60-7.75(m, 8H), 7.89-8.35 (m, 21H), 9.81 (s, 3H).

[0376] Example 12 Synthesis of HG-PL8

[0377]

[0378] Step 1: Synthesis of HG-PL8

[0379]

[0380] At 0 °C, PH-HG-003-4 (174.8 mg, 0.155 mmol, 3.3 equivalents), N,N-diisopropylethylamine (27.1 mg, 0.211 mmol, 4.5 equivalents), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) (109.3 mg, 0.211 mmol, 4.5 equivalents) were added to a nitrogen-protected DMF (4.0 mL) solution of PH-HG-007-1 (100 mg, 0.047 mmol, 1.0 equivalents). After stirring at 0°C for 2 hours, the reaction solution was purified by preparative HPLC (conditions: XBridgeShield RP18 OBD column; size 19 mm × 150 mm, 10 µm; mobile phase, water and acetonitrile (53.0% to 65.0% acetonitrile, 11 min); detector, UV 254 nm) to obtain 21.2 mg (8%) white solid HG-PL8.

[0381] MS m / z [M / 3+H]+(ESI): 1832.75. 1H NMR (300 MHz, DMSO-d6) δ: 0.86-0.89(m, 22H), 0.97-1.06 (m, 13H), 1.22-1.40 (m, 27H), 1.51-1.60 (m, 14H), 1.67-1.75 (m, 22H), 1.94-2.02 (m, 21H), 2.13-2.36 (m, 28H), 2.62-2.87 (m, 25H), 2.99-3.11 (m, 14H), 3.24-3.26 (m, 27H), 3.51-3.55 (m, 76H), 3.70-3.89 (m,20H), 3.94-4.40 (m, 35H), 4.57-4.77 (m, 15H), 4.84-5.07 (m, 15H), 5.44 (s,6H), 5.96-6.04 (m, 3H), 7.09-7.11 (m, 3H), 7.27-7.29 (m, 8H), 7.61-7.64 (m,7H), 7.80-8.20 (m, 15H), 9.78 (s, 3H).

[0382] Example 13 Synthesis of HG-PL9

[0383]

[0384] Step 1: Synthesis of PH-HG-009-1

[0385]

[0386] At 0 °C, aminododecyl monomethyl ether (1.4 g, 2.5 mmol, 1.0 equivalent), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (0.7 g, 3.8 mmol, 1.5 equivalent), and N-methylmorpholine (0.56 g, 5.5 mmol, 2.2 equivalent) were added sequentially to a nitrogen-protected solution of SM2 (0.81 g, 2.8 mmol, 1.1 equivalent) in dichloromethane (15.0 mL). The reaction mixture was stirred at 25 °C for 24 h, diluted with 100 mL of water, and extracted with dichloromethane (3 × 30 mL). The combined organic phases were washed with saturated brine (3 × 30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica column; mobile phase: water and acetonitrile (10% to 100% acetonitrile, 20 min); detector: UV 210 nm) to obtain 1.2 g (56%) of colorless oily PH-HG-009-1.

[0387] MS m / z [M+H] + (ESI): 865.45; 1 H NMR (400 MHz, Methanol-d4) δ: 1.41 (s,9H), 2.52-2.58 (m, 1H), 2.74-2.79 (m, 1H), 3.34-3.37 (m, 9H), 3.51-3.54 (m,4H), 3.60-3.62 (m, 38H), 4.48-4.51 (m, 1H), 5.05-5.15 (m, 2H), 7.29-7.38 (m,5H).

[0388] Step 2: Synthesis of PH-HG-009-2

[0389]

[0390] Palladium / carbon (0.2 g) was added to a methanol (15.0 mL) solution of PH-HG-009-1 (1.2 g, 1.3 mmol, 1.0 equivalent). The reaction mixture was purged five times with hydrogen and stirred at 25 °C for 4 hours. The reaction solution was filtered, concentrated under reduced pressure, and yielded 780.0 mg of a colorless oily substance, PH-HG-009-2 (77%). This product can be used directly in the next step without further purification.

[0391] MS m / z [M+H] + (ESI): 731.35

[0392] Step 3: Synthesis of PH-HG-009-3

[0393]

[0394] At 0 °C, PH-HG-001-3 (240 mg, 0.3 mmol, 1.0 equivalence), N,N-diisopropylethylamine (187 mg, 1.4 mmol, 4.5 equivalence), and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (548.0 mg, 1.4 mmol, 4.5 equivalence) were added sequentially to a nitrogen-protected solution of PH-HG-009-2 (774.0 mg, 1.1 mmol, 3.3 equivalence) in 8.0 mL of DMF. The reaction mixture was stirred at 25 °C for 5 h, diluted with 100 mL of water, and extracted with dichloromethane (3 × 30 mL). The combined organic phases were washed with saturated brine (3 × 30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica column; mobile phase: water and acetonitrile (10% to 100% acetonitrile, 20 min); detector: UV 210 nm) to obtain 450 mg (49%) of colorless oily PH-HG-009-3.

[0395] MS m / z [M / 3+H] + (ESI): 963.05.

[0396] Step 4: Synthesis of PH-HG-009-4

[0397]

[0398] Formic acid (2.5 mL) was added to a nitrogen-protected solution of PH-HG-009-3 (450.0 mg, 0.2 mmol, 1.0 equivalent) in dichloromethane (2.5 mL). The reaction mixture was stirred at 25 °C for 12 h and then concentrated. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase: water and acetonitrile (10% to 80% acetonitrile, 20 min); detector: UV 210 nm) to obtain 200 mg (47%) of colorless oily PH-HG-009-4.

[0399] MS m / z [M / 2+H] + (ESI): 1359.85.

[0400] Step 5: Synthesis of PH-HG-009-5

[0401]

[0402] Diethylamine (0.1 mL) was added to a nitrogen-protected solution of PH-HG-009-4 (200 mg, 0.07 mmol, 1.0 equivalent) in DMF (4.0 mL) at 0 °C. After stirring at 0 °C for 2 hours, the reaction solution was purified by preparative HPLC (conditions: XSelect CSH Prep C18 OBD column; size 19 mm × 250 mm, 5 µm; mobile phase, water (containing 0.1% formic acid) and acetonitrile (8.0% to 23.0% acetonitrile, 11 min); detector, UV 200 nm) to obtain 96 mg (52%) of colorless oily PH-HG-009-5.

[0403] MS m / z [M / 3+H] + (ESI): 832.80

[0404] Step 6: Synthesis of PH-HG-009-6

[0405]

[0406] To a nitrogen-protected solution of PH-HG-009-5 in DMF (2.0 mL), hydroxysuccinimide 3-maleimide propionate (15.4 mg, 0.06 mmol, 1.5 equivalence) and N,N-diisopropylethylamine (14.9 mg, 0.11 mmol, 3.0 equivalence) were added. After stirring at 25 °C for 4 hours, the reaction mixture was purified by preparative HPLC (conditions: XBridge Prep C18 OBD column; 30 mm × 150 mm, 5 µm; mobile phase: water (0.01% formic acid) and acetonitrile (8.0% to 38.0% acetonitrile, 7 min); detector: UV 200 nm) to obtain 51 mg (50%) of colorless oily product PH-HG-009-6.

[0407] MS m / z [M / 2+H] + (ESI): 1323.85

[0408] Step 7: Synthesis of HG-PL9

[0409]

[0410] Add PH-HG-001-8 (98 mg, 0.086 mmol, 3.3 equivalence) and N,N-diisopropylethylamine (15.4 mg, 0.12 mmol, 4.5 equivalence) and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) (61.9 mg, 0.12 mmol, 4.5 equivalence) to a nitrogen-protected DMF (4.0 mL) solution of PH-HG-009-6 (70 mg, 0.026 mmol, 1.0 equivalence). After stirring at 0°C for 2 hours, the reaction solution was purified by preparative HPLC (conditions: XBridge Prep OBD C18 column; size 30 mm × 150 mm, 5 µm; mobile phase, water (containing 0.01% formic acid) and acetonitrile (35.0% to 65.0% acetonitrile, 7 min); detector, UV 254 nm) to obtain 23.6 mg (15%) white solid HG-PL9.

[0411] MS m / z [M / 4+H] + (ESI): 1491.45; 1H NMR (300 MHz, DMSO-d6) δ: 0.74-1.05(m, 108H), 1.10-1.80 (m, 35H), 1.90-2.45 (m, 27H), 2.60-2.75 (m, 5H), 2.82-3.05 (m, 24H), 3.12-3.19 (m, 27H), 3.21-3.48 (m, 108H), 3.51-3.80 (m, 47H), 3.88-4.07 (m, 15H), 4.19-4.52 (m, 17H), 4.59-5.14 (m, 13H), 5.92-6.08 (m,2H), 6.90-7.07 (m, 3H), 7.12-7.20 (m, 3H), 7.22-7.35 (m, 20H), 7.57-7.67 (m,7H), 7.75-8.37 (m, 20H), 9.78 (s, 3H).

[0412] Example 14 Synthesis of HG-PL10

[0413]

[0414] Step 1: Synthesis of PH-HG-010-1

[0415]

[0416] At 0 °C, aminooctaglycol monomethyl ether (1.4 g, 3.65 mmol, 1.0 equivalent), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.1 g, 5.84 mmol, 1.5 equivalent), and N-methylmorpholine (553.5 mg, 5.48 mmol, 1.5 equivalent) were added sequentially to a nitrogen-protected solution of (R)-2-(((benzyloxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutyric acid (EDCI) (1.3 g, 4.0 mmol, 1.1 equivalent) and N-methylmorpholine (553.5 mg, 5.48 mmol, 1.5 equivalent). The reaction mixture was stirred at 25 °C for 24 h, diluted with 100 mL of water, and extracted with dichloromethane (3 × 30 mL). The combined organic phases were washed with saturated brine (3 × 30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica column; mobile phase: water and acetonitrile (10% to 100% acetonitrile, 20 min); detector: UV 210 nm) to obtain 1.2 g (48%) of colorless oily PH-HG-010-1.

[0417] MS m / z [M+H] +(ESI): 689.25.

[0418] Step 2: Synthesis of PH-HG-010-2

[0419]

[0420] Palladium / carbon (0.25 g) was added to a methanol (15.0 mL) solution of PH-HG-010-1 (1.2 g, 1.7 mmol, 1.0 equivalent). The reaction mixture was purged five times with hydrogen and stirred at 25 °C for 4 hours. The reaction solution was filtered, concentrated under reduced pressure to obtain 800.0 mg of a colorless oily substance PH-HG-010-2 (83%). This product can be used directly in the next step without further purification.

[0421] MS m / z [M+H] + (ESI): 555.45

[0422] Step 3: Synthesis of PH-HG-010-3

[0423]

[0424] At 0 °C, PH-HG-001-3 (300 mg, 0.3 mmol, 1.0 equivalence), N,N-diisopropylethylamine (230.4 mg, 1.8 mmol, 4.5 equivalence), and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (686.7 mg, 1.8 mmol, 4.5 equivalence) were added sequentially to a nitrogen-protected solution of PH-HG-010-2 (735.4.0 mg, 1.3 mmol, 3.3 equivalence) in 10.0 mL of DMF. The reaction mixture was stirred at 25 °C for 5 h, diluted with 100 mL of water, and extracted with dichloromethane (3 × 30 mL). The combined organic phases were washed with saturated brine (3 × 30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica column; mobile phase: water and acetonitrile (10% to 100% acetonitrile, 20 min); detector: UV 210 nm) to obtain 470 mg (50%) of colorless oily PH-HG-010-3.

[0425] MS m / z [M / 2+H] + (ESI): 1179.45

[0426] Step 4: Synthesis of PH-HG-010-4

[0427]

[0428] Formic acid (2.5 mL) was added to a nitrogen-protected solution of PH-HG-010-3 (470.0 mg, 0.2 mmol, 1.0 equivalent) in dichloromethane (2.5 mL). The reaction mixture was stirred at 25 °C for 12 h and then concentrated. The crude product was prepared by Flash-Prep-HPLC (conditions: C18 silica gel column; mobile phase: water and acetonitrile (10% to 80% acetonitrile, 20 min); detector: UV 210 nm) to obtain 210 mg (48%) of colorless oily PH-HG-010-4.

[0429] MS m / z [M / 2+H] + (ESI): 1095.40

[0430] Step 5: Synthesis of PH-HG-010-5

[0431]

[0432] Diethylamine (0.1 mL) was added to a nitrogen-protected solution of PH-HG-010-4 (210 mg, 0.1 mmol, 1.0 equivalent) in DMF (4.0 mL) at 0 °C. After stirring at 0 °C for 2 hours, the reaction solution was purified by preparative HPLC (conditions: XSelect CSHPrep C18 OBD column; size 19 mm × 250 mm, 5 µm; mobile phase, water (containing 0.1% formic acid) and acetonitrile (8.0% to 23.0% acetonitrile, 11 min); detector, UV 200 nm) to obtain 100 mg (53%) of colorless oily PH-HG-010-5.

[0433] MS m / z [M+H] + (ESI): 1967.17

[0434] Step 6: Synthesis of PH-HG-010-6

[0435]

[0436] To a nitrogen-protected solution of PH-HG-010-5 in DMF (5.0 mL), hydroxysuccinimide 3-maleimide propionate (20.2 mg, 0.07 mmol, 1.5 equivalence) and N,N-diisopropylethylamine (19.6 mg, 0.14 mmol, 3.0 equivalence) were added. After stirring at 25 °C for 4 hours, the reaction mixture was purified by preparative HPLC (conditions: XBridge Prep C18 OBD column; dimensions 30 mm × 150 mm, 5 µm; mobile phase, water (0.01% formic acid) and acetonitrile (8.0% to 38.0% acetonitrile, 7 min); detector, UV 200 nm) to obtain 55 mg (51%) of colorless oily product PH-HG-010-6.

[0437] MS m / z [M / 2+H] + (ESI): 1060.00

[0438] Step 7: Synthesis of HG-PL10

[0439]

[0440] At 0 °C, PH-HG-001-8 (85.4 mg, 0.076 mmol, 3.3 equivalents), N,N-diisopropylethylamine (12.9 mg, 0.10 mmol, 4.5 equivalents), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) (52.0 mg, 0.10 mmol, 4.5 equivalents) were added to a nitrogen-protected DMF (4.0 mL) solution of PH-HG-010-6 (48 mg, 0.023 mmol, 1.0 equivalents). After stirring at 0°C for 2 hours, the reaction solution was purified by preparative HPLC (conditions: XBridgePrep OBD C18 column; size 30 mm × 150 mm, 5 µm; mobile phase, water (containing 0.01% formic acid) and acetonitrile (35.0% to 65.0% acetonitrile, 7 min); detector, UV 254 nm) to obtain 18.2 mg (15%) white solid HG-PL10.

[0441] MS m / z [M / 4+H] + (ESI): 1359.35; 1H NMR (300 MHz, DMSO-d6) δ: 0.73-0.89(m,70H), 0.96-1.05 (m, 21H), 1.05-2.31 (m, 34H), 2.31-2.32 (m, 26H), 2.24-2.51 (m, 9H), 2.84-2.88 (m, 9H), 3.13-3.31 (m, 46H), 3.38-3.45 (m, 14H), 3.64-3.49 (m, 95H), 3.76-3.86 (m, 8H), 3.96-4.07 (m, 12H), 4.30-4.21 (m, 6H),4.36-4.52 (m, 9H), 4.71-4.78 (m, 6H), 4.91-5.15 (m, 6H), 6.01-6.35 (m, 1H), 7.48-5.60 (m, 6H), 6.98 (s, 3H), 7.26 (s, 2H), 7.12-7.35 (m, 21H), 7.50-7.80(m, 9H), 7.80-8.19 (m, 15H), 8.25-8.53 (m, 5H), 9.95 (s, 3H).

[0442] Biological Examples

[0443] The following abbreviations are used throughout this invention:

[0444]

[0445] Example 15 Synthesis of trastuzumab conjugate (HG-ADC-1) using HG-PL1 as a scaffold prepared in the example

[0446] 1. Preparation of HG-ADC-1-D2

[0447] The antibody Herceptin was transferred to PB buffer (40 mM PB, 2 mM EDTA, pH 7.0), and then 1.0 equivalent of tris(2-carboxyethyl)phosphine (TCEP) was added for reduction. The reaction solution was incubated at 22°C. o The reaction was carried out at C for 3 hours. The pH of the reaction solution was adjusted to 5 using 400 mM acetic acid solution, and then directly used for the next coupling reaction. The reduced antibody was slowly added to water and an aqueous solution of the linker-payload HG-PL1 (10 mg / mL, 3 equivalents) under ice bath conditions. After thorough mixing, the coupling reaction solution was incubated at 4°C. oThe reaction was carried out at C for 2 hours. After the coupling reaction, the ADC solution was filtered through a 0.22 μm PVDF pinhole filter, and then analyzed on an AKTA instrument using the Hydrohobic Interaction Chromatography column (Thermo SCIENTIFIC ProPac). TM ADC was purified using an HIC-10 desalting column (5 μm, 300 A, 7.8 * 75 mm). Before purification, the desalting column was equilibrated with 30 mL of buffer A (20 mM histidine, 2.0 M NaCl, pH 5.5). The NaCl concentration in the ADC solution was then adjusted to 2.0 M using buffer (20 mM histidine, 5.0 M NaCl, pH 5.5) to ensure ADC binding to the desalting column packing material. After loading the sample, the desalting column was washed with buffer A until baseline equilibration was achieved at an absorbance of 280 nm. Subsequently, over 20 minutes (buffer flow rate 1 mL / min), the buffer system was continuously replaced with 100% buffer B (20 mM histidine, pH 5.5) from 100% buffer A (20 mM histidine, 2.0 M NaCl, pH 5.5) for elution. The purified sample (HG-ADC-1-D2) was then concentrated to 20 mM histidine buffer using an Amicon ultrafiltration tube (50 kDa). The ADC product was obtained by filtration through a 0.2 μm PVDF pinhole filter and sent for testing.

[0448] 2. Preparation of HG-ADC-1-D4

[0449] In PB buffer (40 mM PB, 2 mM EDTA, pH 7.0), the antibody Herceptin was reduced with 2.2 equivalents of TCEP. o The reaction proceeds at C for 3 hours. Without removing excess TCEP, the reaction solution is used directly for the next coupling reaction. The pH of the reaction solution is adjusted to pH = 5 using 400 mM acetic acid solution. Then, under ice bath conditions, water and HG-PL1 solution (10 mg / mL, dissolved in water, 6 equivalents) are slowly added to the reaction solution. After thorough mixing, 4 o The reaction was carried out at C for 2 hours. After the reaction, the reaction solution was concentrated into 20 mM histidine buffer using an Amicon ultrafiltration tube (50 kDa), and then filtered through a 0.2 μm PVDF pinhole filter to obtain the ADC product. Samples were then sent for testing.

[0450] 3. Preparation of DS8201

[0451] Herceptin antibody was reduced with 7.5 equivalents of TCEP in PB buffer (40 mM PB, 2 mM EDTA, pH 7.0) on a shaker at 32°C.o The reaction mixture was shaken for 3 hours (shaking speed 60 rpm). Without removing excess TCEP, the reaction solution was used directly for the next coupling reaction. DMA (dimethylacetamide) and Deruxtecan solution (10 mg / mL, dissolved in DMA, 20 equivalents) were slowly added to the reaction solution. After thorough mixing, the mixture was heated to 22... o The reaction was carried out at C for 2 hours. After the reaction, the reaction solution was replaced with 20 mM histidine buffer using a desalting column (40 K), and then filtered through a 0.2 μm PVDF pinhole filter to obtain the ADC product. Samples were then sent for testing.

[0452] Deruxtecan

[0453] 4. Summary of Antibody-Drug Conjugate Data

[0454] RP-DAR Measurement

[0455] Add 37.5 μL of 8.0 mol / L guanidine hydrochloride solution, 2.5 μL of 1.0 mol / L Tris-HCl and 1.0 μL of 1 mol / L dithiothreitol to 10.0 μL ADC solution, mix well and send the sample for high performance liquid chromatography detection.

[0456] The chromatographic conditions are shown in the table below:

[0457]

[0458] Test results:

[0459] The DAR of antibody-drug conjugate HG-ADC-1-D2 was 6.54, the DAR of HG-ADC-1-D4 was 12.60, and the DAR of DS8201 was 8.00.

[0460] Example 16 Synthesis of trastuzumab conjugate (HG-ADC-2) using HG-PL2 as a scaffold prepared in the example

[0461] 1. Preparation of HG-ADC-2-D1

[0462] Herceptin antibody was reduced with 1 equivalent of tris(2-carboxyethyl)phosphine (TCEP) in PB buffer (40 mM PB, 2 mM EDTA, pH 7.0) on a shaker at 22°C. oThe reaction mixture was subjected to shaking for 3 hours (shaking speed 60 rpm). Without removing excess TCEP, the reaction solution was used directly for the next coupling reaction. The reduced antibody was slowly added to PB buffer, dimethylacetamide (DMA), and a dimethylacetamide (DMA) solution (6.39 mg / mL, 1 equivalent) of the linker-payload HG-PL2 in an ice bath. After thorough mixing, the coupling reaction solution was incubated at 4°C. o The reaction was carried out at C for 1.5 hours. L-cysteine ​​(1.21 mg / ml aqueous solution, 4 equivalents) was added to the reaction solution, and after thorough mixing, the mixture was incubated at 4°C. o The reaction was carried out at C for 0.5 hours. After the reaction, the ADC solution was filtered through a 0.22 μm PVDF pinhole filter and then purified using a hydrophobic interaction column (GE HiTrapButyl HP column, 1 mL) on an AKTA protein purifier. Before purification, the column was equilibrated with 10 mL of buffer A (20 mM Histidine, 1.5 M NaCl, pH 5.5). The NaCl concentration in the ADC solution was adjusted to 2.0 M with buffer (20 mM Histidine, 5.0 M NaCl, pH 5.5) to allow the ADC to bind to the column packing material. After loading the sample, the column was washed with buffer A until baseline equilibration was achieved at an absorbance of 280 nm. Subsequently, over a 2-minute period (buffer flow rate of 1 mL / min), the buffer system was continuously replaced with 100% buffer A (20 mM Histidine, 1.5 M NaCl, pH 5.5) and eluted with 100% buffer B (20 mM Histidine, pH 5.5). The purified HG-ADC-2-D1 was then concentrated to 20 mM Histidine buffer using an Amicon ultrafiltration tube (10 kDa). Finally, the ADC product was obtained by filtration through a 0.2 μm PVDF pinhole filter and sampled for analysis.

[0463] 2. Preparation of HG-ADC-2-D2

[0464] Herceptin antibody was reduced with 1 equivalent of tris(2-carboxyethyl)phosphine (TCEP) in PB buffer (40 mM PB, 2 mM EDTA, pH 7.0) on a shaker at 22°C. oThe reaction mixture was subjected to shaking for 3 hours (shaking speed 60 rpm). Without removing excess TCEP, the reaction solution was used directly for the next coupling reaction. The reduced antibody was slowly added to PB buffer, dimethylacetamide (DMA), and a dimethylacetamide (DMA) solution (6.43 mg / mL, 3 equivalents) of the linker-payload HG-PL2 under ice conditions. After thorough mixing, the coupling reaction solution was incubated at 4°C. o The reaction was carried out at C for 2 hours. After the reaction, the ADC solution was filtered through a 0.22 μm PVDF pinhole filter and then purified using a hydrophobic interaction column (Polar MC30 HICEther column, 1 mL) on an AKTA protein purifier. Before purification, the column was equilibrated with 10 mL of buffer A (20 mM Histidine, 4 M NaCl, pH 5.5). The NaCl concentration in the ADC solution was adjusted to 4 M with buffer (20 mM Histidine, 5.0 M NaCl, pH 5.5) to allow the ADC to bind to the column packing material. After loading the sample, the column was washed with buffer A until baseline equilibration was achieved at an absorbance of 280 nm. Subsequently, the buffer system was continuously replaced with 100% buffer A (20 mM Histidine, 4 M NaCl, pH 5.5) with 100% buffer B (20 mM Histidine, pH 5.5) over 2 minutes (buffer flow rate 1 mL / min) for elution. The purified HG-ADC-2-D2 was then concentrated to 20 mM Histidine buffer using an Amicon ultrafiltration tube (10 kDa). Finally, the ADC product was obtained by filtration through a 0.2 μm PVDF pinhole filter and samples were sent for testing.

[0465] Example 17 Synthesis of trastuzumab conjugate (HG-ADC-3) using HG-PL3 as a scaffold prepared in the example

[0466] 1. Preparation of HG-ADC-3-D1

[0467] Herceptin antibody was reduced with 1 equivalent of tris(2-carboxyethyl)phosphine (TCEP) in PB buffer (40 mM PB, 2 mM EDTA, pH 7.0) on a shaker at 22°C. oThe reaction mixture was subjected to shaking for 3 hours (shaking speed 60 rpm). Without removing excess TCEP, the reaction solution was used directly for the next coupling reaction. The reduced antibody was then slowly added to PB buffer, dimethylacetamide (DMA), and linker-payload HG-PL3 solution (water:DMA = 3:1 (v / v), 5.47 mg / mL, 1 equivalent) on ice. After thorough mixing, the coupling reaction solution was incubated at 4°C. o The reaction was carried out at C for 1.5 hours. L-cysteine ​​(1.21 mg / ml aqueous solution, 4 equivalents) was added to the reaction solution, and after thorough mixing, the mixture was incubated at 4°C. o The reaction was carried out at C for 0.5 hours. After the reaction, the ADC solution was filtered through a 0.22 μm PVDF pinhole filter and then purified using a hydrophobic interaction column (GE HiTrap Octyl FF column, 1 mL) on an AKTA protein purifier. Before purification, the column was equilibrated with 10 mL of buffer A (20 mM Histidine, 1.5 M NaCl, pH 5.5). The NaCl concentration in the ADC solution was adjusted to 2 M using buffer (20 mM Histidine, 5.0 M NaCl, pH 5.5) to allow the ADC to bind to the column packing material. After loading the sample, the column was washed with buffer A until baseline equilibration was achieved at an absorbance of 280 nm. Subsequently, over a 2-minute period (buffer flow rate of 1 mL / min), the buffer system was continuously replaced with 100% buffer A (20 mM Histidine, 1.5 M NaCl, pH 5.5) and eluted with 100% buffer B (20 mM Histidine, pH 5.5). The purified HG-ADC-3-D1 was then concentrated to 20 mM Histidine buffer using an Amicon ultrafiltration tube (10 kDa). Finally, the ADC product was obtained by filtration through a 0.2 μm PVDF pinhole filter and sent for testing.

[0468] 2. Preparation of HG-ADC-3-D2

[0469] Herceptin antibody was reduced with 1 equivalent of tris(2-carboxyethyl)phosphine (TCEP) in PB buffer (40 mM PB, 2 mM EDTA, pH 7.0) on a shaker at 22°C. oThe reaction mixture was subjected to shaking for 3 hours (shaking speed 60 rpm). Without removing excess TCEP, the reaction solution was used directly for the next coupling reaction. The reduced antibody was then slowly added to PB buffer, dimethylacetamide (DMA), and linker-payload HG-PL3 solution (water:DMA = 3:1 (v / v), 5.47 mg / mL, 3 equivalents) on ice. After thorough mixing, the coupling reaction solution was incubated at 4°C. o The reaction was carried out at C for 2 hours. After the reaction, the ADC solution was filtered through a 0.22 μm PVDF pinhole filter and then purified using a hydrophobic interaction column (GEHiTrap Octyl FF column, 1 mL) on an AKTA protein purifier. Before purification, the column was equilibrated with 10 mL of buffer A (20 mM Histidine, 1.2 M NaCl, pH 5.5). The NaCl concentration in the ADC solution was adjusted to 1.5 M with buffer (20 mM Histidine, 5.0 M NaCl, pH 5.5) to allow the ADC to bind to the column packing material. After loading the sample, the column was washed with buffer A until baseline equilibration was achieved at an absorbance of 280 nm. Subsequently, over a 2-minute period (buffer flow rate of 1 mL / min), the buffer system was continuously replaced with 100% buffer A (20 mM Histidine, 1.2 M NaCl, pH 5.5) and eluted with 100% buffer B (20 mM Histidine, pH 5.5). The purified HG-ADC-3-D2 was then concentrated to 20 mM Histidine buffer using an Amicon ultrafiltration tube (10 kDa). Finally, the ADC product was obtained by filtration through a 0.2 μm PVDF pinhole filter and sent for testing.

[0470] Example 18 Synthesis of trastuzumab conjugate (HG-ADC-4) using HG-PL4 as a scaffold prepared in the example

[0471] 1. Preparation of HG-ADC-4-D1

[0472] Herceptin antibody was reduced with 1.0 equivalent of tris(2-carboxyethyl)phosphine (TCEP) in PB buffer (40 mM PB, 2 mM EDTA, pH 7.0) on a shaker at 22°C. oThe reaction mixture was subjected to shaking for 3 hours (shaking speed 60 rpm). Without removing excess TCEP, the reaction solution was used directly for the next coupling reaction. The reduced antibody was slowly added to PB buffer, dimethylacetamide (DMA), and a dimethylacetamide (DMA) solution (6.43 mg / mL, 1 equivalent) of the linker-payload HG-PL4 in an ice bath. After thorough mixing, the coupling reaction solution was incubated at 4°C. o The reaction was carried out at C for 1.5 hours. L-cysteine ​​(1.21 mg / ml aqueous solution, 4 equivalents) was added to the reaction solution, and after thorough mixing, the mixture was incubated at 4°C. o The reaction was carried out at C for 0.5 hours. After the reaction, the ADC solution was filtered through a 0.22 μm PVDF pinhole filter and then purified using a hydrophobic interaction column (GE HiTrap Butyl HP column, 1 mL) on an AKTA protein purifier. Before purification, the column was equilibrated with 10 mL of buffer A (20 mM Histidine, 1.5 M NaCl, pH 5.5). The NaCl concentration in the ADC solution was adjusted to 2.0 M with buffer (20 mM Histidine, 5.0 M NaCl, pH 5.5) to allow the ADC to bind to the column packing material. After loading the sample, the column was washed with buffer A until baseline equilibration was achieved at an absorbance of 280 nm. Subsequently, the buffer system was continuously eluted from 100% buffer A (20 mM Histidine, 1.5 M NaCl, pH 5.5) to 100% buffer B (20 mM Histidine, pH 5.5) over 2 minutes (buffer flow rate 1 mL / min). The purified HG-ADC-4-D1 was then concentrated to 20 mM Histidine buffer using an Amicon ultrafiltration tube (10 kDa). Finally, the ADC product was obtained by filtration through a 0.2 μm PVDF pinhole filter and sampled for analysis.

[0473] 2. Preparation of HG-ADC-4-D2

[0474] Herceptin antibody was reduced with 1 equivalent of tris(2-carboxyethyl)phosphine (TCEP) in PB buffer (40 mM PB, 2 mM EDTA, pH 7.0) on a shaker at 22°C. oThe reaction mixture was incubated at 4°C with shaking for 3 hours (shaking speed 60 rpm). Without removing excess TCEP, the reaction solution was used directly for the next coupling reaction. The reduced antibody was then slowly added to PB buffer, dimethylacetamide (DMA), and a dimethylacetamide (DMA) solution (6.43 mg / mL, 3 equivalents) of the linker-payload HG-PL4 in an ice bath. After thorough mixing, the coupling reaction solution was incubated at 4°C with shaking for 3 hours (shaking speed 60 rpm). o The reaction was carried out at C for 2 hours. After the reaction, the ADC solution was filtered through a 0.22 μm PVDF pinhole filter and then purified using a hydrophobic interaction column (GE HiTrap OctylFF column, 1 mL) on an AKTA protein purifier. Before purification, the column was equilibrated with 10 mL of buffer A (20 mM Histidine, 1.2 M NaCl, pH 5.5). The NaCl concentration in the ADC solution was adjusted to 1.5 M with buffer (20 mM Histidine, 5.0 M NaCl, pH 5.5) to allow the ADC to bind to the column packing material. After loading the sample, the column was washed with buffer A until baseline equilibration was achieved at an absorbance of 280 nm. Subsequently, over a 2-minute period (buffer flow rate of 1 mL / min), the buffer system was continuously replaced with 100% buffer A (20 mM Histidine, 1.2 M NaCl, pH 5.5) and eluted with 100% buffer B (20 mM Histidine, pH 5.5). The purified HG-ADC-4-D2 was then concentrated to 20 mM Histidine buffer using an Amicon ultrafiltration tube (10 kDa). Finally, the ADC product was obtained by filtration through a 0.2 μm PVDF pinhole filter and sent for testing.

[0475] Summary of antibody-drug conjugate data:

[0476] Methods for determining the level of free linker-load (free drug):

[0477] The level of free linker-payload in ADC products was determined using reverse high performance liquid chromatography (RP-HPLC).

[0478] 1) First, dilute the linker-loador with DMA to 20 µg / mL, then dilute the solvent with 20 mM histidine buffer (pH 5.5) and DMA to 2 X mol / L and 0.8 X mol / L, respectively, so that the final solution contains 10% DMA.

[0479]

[0480] 2) The antibody was diluted to 2 mg / mL with 20 mM histidine buffer (pH 5.5) and DMA, resulting in a final solution containing 10% DMA.

[0481] 3) Add 12.5 µL of solution 2) to 12.5 µL of solution 1) to obtain a standard solution with an antibody concentration of 1 mg / mL and a linker-load concentration of 5% or 2% molar percentage.

[0482] 4) Dilute the ADC with 20 mM histidine buffer (pH 5.5) and DMA to 1 mg / mL, so that the final solution contains 10% DMA.

[0483] 5) The prepared ADC solution and the standard solution of 5% or 2% molar percentage linker-load were detected by LC-MS.

[0484]

[0485] The DAR of the antibody-drug conjugates HG-ADC-2-D1 was 2.85, HG-ADC-2-D2 was 7.17, HG-ADC-3-D1 was 4.50, HG-ADC-3-D2 was 7.80, HG-ADC-4-D1 was 3.81, and HG-ADC-4-D2 was 6.51.

[0486] Example 19 In vitro characterization example

[0487] Sample information:

[0488]

[0489] Instruments and equipment

[0490]

[0491] Reagents and consumables

[0492]

[0493] Experimental methods

[0494] FACS binding specificity assay

[0495] When the confluence of cultured NCI-N87 cells reached 70-90%, adherent cells were digested with trypsin, and digestion was terminated with RPMI 1640 medium (Gibco, 22400-089) containing 10% FBS (ExCell Bio, FND500). The cell suspension was centrifuged at 1500 rpm for 5 minutes at 4°C (Eppendorf, 5810R). The cells were then resuspended in PBS containing 1% BSA, and the cell suspension density was adjusted to 1×10⁻⁶. 6 Cells / mL. Add 100 μL (1×10⁶ cells / mL) to each well of a 96-well round-bottom microplate (Corning, 3799). 6 Cell suspension at a concentration of cells / mL was centrifuged and the supernatant was discarded. ADC or monoclonal samples were serially diluted 1:5 (maximum concentration 100 nM) in PBS containing 1% BSA. 100 μL of the diluted sample was added to each well and incubated at 4°C for 1 hour. Human IgG1 antibody was used as an isotype control. After incubation, each well was washed twice with 160 μL of PBS containing 1% BSA, and then 100 μL of goat anti-human-IgG Fc-Alexa 647 antibody diluted 1:500 in PBS containing 1% BSA was added as a secondary antibody. The cells were incubated at 4°C in the dark for 30 minutes. After incubation, the cells were washed twice and resuspended in 80 μL of PBS containing 1% BSA. Fluorescence intensity was detected using a flow cytometer (BD Biosciences, FACS CantoII), and the fluorescence value is expressed as mean fluorescence intensity (MFI).

[0496] Raw data obtained from FACS experiments were analyzed using FlowJo software. Using sample wells without antibodies or incubated only with secondary antibodies as the cell background fluorescence intensity, EC was calculated using a GraphPad Prism 6 nonlinear four-parameter nonlinear regression. 50 value.

[0497] Cytotoxicity assay

[0498] When the confluence of cultured NCI-N87 cells reaches 70-90%, the cells are digested with trypsin, the trypsin is removed by centrifugation, and the cells are resuspended in RPMI 1640 medium containing 10% FBS to a final volume of 1×10⁻⁶. 5Cells / mL: 50 μL of cell suspension was added to each well of a 96-well black-walled microplate (Greinier, 655090) and incubated overnight at 37°C in a 5% CO2 incubator to allow cell adhesion. The next day, 50 μL of the diluted ADC or linker-toxin sample was serially diluted 1:5 (maximum concentration 50 nM) in RPMI 1640 medium containing 10% FBS and added to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 6 days. After incubation, cell viability was assessed using CellTiter-Glo reagent (Promega, G7573), and specific cell viability values ​​were measured using a multi-mode microplate reader (PerkinElmer, EnVision).

[0499] The cytotoxicity of each sample well is determined by comparing the cell viability values ​​of the sample wells with those of the control wells containing only cells. The calculation formula is: Cytotoxicity = 100 * (Cell viability value) 仅有细胞孔 – Cell viability 样品孔 Cell viability value 仅有细胞孔 The IC was calculated using GraphPad Prism 6 nonlinear four-parameter nonlinear regression. 50 value.

[0500] Experimental results

[0501] FACS binding specificity assay

[0502] This experiment analyzed the specific binding of DS8201, HG-ADC-1-D2, HG-ADC-1-D4, HG-ADC-2-D1, HG-ADC-2-D2, HG-ADC-3-D1, HG-ADC-3-D2, HG-ADC-4-D1, HG-ADC-4-D2, mAb-Herceptin, and hIgG1_Isotype to HER2-positive NCI-N87 cells using FACS. The results showed that the binding curves and EC50 values ​​of the nine ADC samples (DS8201, HG-ADC-1-D2, HG-ADC-1-D4, HG-ADC-2-D1, HG-ADC-2-D2, HG-ADC-3-D1, HG-ADC-3-D2, HG-ADC-4-D1, and HG-ADC-4-D2) to NCI-N87 cells were consistent with those of IgG1. 50 The values ​​were comparable to those of the naked Herceptin antibody (i.e., mAb-Herceptin in the table), and the binding curves of these samples all reached the upper plateau. However, the hIgG1_Isotype control (an antibody without antigen-binding properties) did not bind to NCI-N87 cells. The corresponding sample information is consistent with EC... 50 See Tables 1 through 3.

[0503] Table 1: Summary of FACS detection of ADC1 and corresponding monoclonal antibody binding to NCI-N87 cells

[0504]

[0505] Note: Neg, parallel control wells incubated only with secondary antibody, background fluorescence value.

[0506] Table 2: Summary of FACS detection of ADC2 and corresponding monoclonal antibody binding to NCI-N87 cells

[0507]

[0508] Table 3: Summary of FACS detection of ADC3, ADC4 and corresponding monoclonal antibodies binding to NCI-N87 cells

[0509]

[0510] Cytotoxicity assay

[0511] This experiment used the cytotoxicity methods described above to detect the cytotoxicity of DS8201, HG-ADC-1-D2, HG-ADC-1-D4, HG-ADC-2-D1, HG-ADC-2-D2, HG-ADC-3-D1, HG-ADC-3-D2, HG-ADC-4-D1, HG-ADC-4-D2, the scaffold HG-PL1, and the DS8201 scaffold against NCI-N87 cells. The results showed that, compared to DS8201, HG-ADC-1-D2, HG-ADC-1-D4, HG-ADC-2-D1, HG-ADC-2-D2, HG-ADC-3-D1, HG-ADC-3-D2, HG-ADC-4-D1, and HG-ADC-4-D2 were more sensitive to cytotoxicity against NCI-N87 cells. The results are shown in Table 4.

[0512] Table 4: Summary of ADC cytotoxicity against NCI-N87 cells

[0513]

[0514] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature having the same, equivalent, or similar purpose. Therefore, unless otherwise expressly stated, each disclosed feature is merely an example of a series of equivalent or similar features.

[0515] Based on the above description, those skilled in the art can readily identify the essential features of the present invention, and various changes and modifications can be made to adapt it to various uses and conditions without departing from the spirit and scope of the invention. Therefore, other embodiments are also within the scope of the appended claims.

Claims

1. The structure shown in formula (II) or its tautomers, stereoisomers or pharmaceutically acceptable salts, (II) in, R is , , , , , , , , n² is 7, 8, 9, 10, or 11; PG1 is a protecting group for an amino group, selected from acetyl, trifluoroacetyl, tert-butyloxycarbonyl (BOC, Boc), benzyloxycarbonyl (CBZ, Cbz) and 9-fluorenemethoxycarbonyl (Fmoc); PG2 is a protecting group for the hydroxyl group, selected from acetyl and silyl groups; PG3 is a carboxyl protecting group selected from -CH2CH2SO2Ph, cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrobenzenesulfonyl)ethyl, 2-(diphenylphosphine)ethyl and nitroethyl.

2. The compound according to claim 1, wherein n2 is 7 or 11.

3. A conjugate having the structure represented by formula (IV). (IV), Where Rx is: ， ， 。 4. The conjugate of claim 3, further comprising a targeting portion, wherein one or more of the conjugates are... The group is covalently bound to the target portion, which is an antibody or antigen-binding fragment that binds to the human epidermal growth factor receptor (HER2).

5. A conjugate comprising a targeting moiety and one or more compounds according to claim 1 or 2, wherein said compound is transmitted through... The group is covalently bound to the target portion, which is an antibody or antigen-binding fragment that binds to the human epidermal growth factor receptor (HER2).

6. A pharmaceutical composition comprising the conjugate of claim 3 or 4 and a pharmaceutically acceptable carrier.

7. Use of the conjugate of claim 3 or 4 or the pharmaceutical composition of claim 6 in the preparation of a medicament for treating a patient with cancer expressing a target antigen or at risk of having cancer expressing a target antigen.

8. The use according to claim 7, wherein the target antigen is human epidermal growth factor receptor 2, preferably, wherein the cancer expresses a high level of human epidermal growth factor receptor 2, preferably, wherein the cancer is selected from breast cancer, gastric cancer, bladder cancer or urothelial carcinoma.

9. A method for synthesizing Bn-LS-1, comprising the following steps: In a 500 mL three-necked flask, add 80 mL of water, 80 g of sodium hydroxide (1.99 mol), then add 160 mL of DCM, 3.2 g of tetrabutylammonium bromide (10 mmol), and 20 g of INF (66.36 mmol), and stir mechanically. Cool the mixture to below 10 °C in an ice bath, and add 52 g of tert-butyl bromoacetate (265.44 mmol) dropwise. After the addition is complete, allow the mixture to rise naturally to 20 °C and stir overnight (16 h). Monitor the reaction by TLC (PE:EA = 1:1), and no reactants remain. Add 1 L of DCM to the reaction system, wash twice with water, wash twice with saturated sodium bicarbonate solution, and concentrate. Pass the crude product through a silica gel column and elute with PE:EA = 20:1 to obtain product Bn-LS-1.

10. A method for detecting the level of free linker-payload in an ADC product using reversed-performance liquid chromatography (RP-HPLC), comprising the following steps: 1) First, dilute the linker-loador with DMA to 20 µg / mL, then dilute the solvent with 20 mM histidine buffer (pH 5.5) and DMA to 2 X mol / L and 0.8 X mol / L, respectively, so that the final solution contains 10% DMA; 2) The antibody was diluted to 2 mg / mL with 20 mM histidine buffer (pH 5.5) and DMA, resulting in a final solution containing 10% DMA. 3) Add 12.5 µL of solution 2) to 12.5 µL of solution 1) to obtain a standard solution with an antibody concentration of 1 mg / mL and a linker-load concentration of 5% or 2% molar percentage. 4) Dilute the ADC with 20 mM histidine buffer (pH 5.5) and DMA to 1 mg / mL, so that the final solution contains 10% DMA; 5) The prepared ADC solution and the standard solution of 5% or 2% molar percentage linker-load were detected by LC-MS.

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