Homocamptothecin antibody conjugate drug, preparation method therefor, and use thereof

Abstract

Provided are a homocamptothecin antibody conjugate drug, a preparation method therefor, and use thereof. Particularly disclosed is an antibody-drug conjugate represented by formula I or a pharmaceutically acceptable salt thereof. The antibody-drug conjugate has relatively good cytotoxic activity and in vivo anti-tumor activity, and can significantly inhibit tumor growth.

Description

A homocamptothecin antibody-drug conjugate, preparation method and application thereof

[0001] This application claims the benefit of Chinese Patent Application No. 2023116270832, filed November 30, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The present invention relates to the field of biomedicine, and in particular to a homocamptothecin antibody-drug conjugate, a preparation method and application thereof. Background Art

[0003] Camptotheca (CPT) is a pentacyclic alkaloid isolated from the bark and stem of the Chinese tree Camptotheca acuminata. It inhibits topoisomerase I, leading to cell death. Due to its cytotoxic mechanism and broad-spectrum antitumor activity, research has focused on developing clinical analogs of CPT.

[0004] An antibody-drug conjugate (ADC) is a complex in which a cytotoxic small molecule drug is coupled to a monoclonal antibody through a rationally constructed linker. It can selectively deliver effective cytotoxic drugs into tumors, thereby increasing the drug's targeting, improving efficacy, and reducing toxic side effects. Recently, camptothecin compounds have been applied as small molecules in some antibody-drug conjugates (ADCs). For example, SN38 and DXd are used as warhead molecules in the marketed ADC drugs Trodelvy and Enhertu, respectively. Patent applications WO2020063676 and CN 112125915 have been modified on the compound of DXD. However, compared to traditional camptothecin, homocamptothecin (hCPT) compounds with a β-hydroxylactone ring structure retain high anti-tumor activity (Bailly, C. Crit. Rev. Oncol. Hematol. 2003, 45, 91), and diflomotecan has stronger Topo I inhibitory activity than camptothecin (Kroep, JR; Gelderblom, H. Expert. Opin. Investig. Drugs 2009, 18, 69). However, due to the lack of suitable conjugation sites, its application in ADCs has not yet been seen. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the existing technical limitations of homocamptothecin derivatives, which cannot be conjugated, and to develop novel homocamptothecin antibody-drug conjugates. The antibody-drug conjugates provided by the present invention have excellent cytotoxic activity and can inhibit the proliferation of tumor cells. The antibody-drug conjugates provided by the present invention also have excellent in vivo anti-tumor activity and can significantly inhibit tumor growth.

[0006] The present invention provides an antibody-drug conjugate as shown in Formula I or a pharmaceutically acceptable salt thereof,

[0007] Where T is or a stereoisomer thereof;

[0008] R 1 and R 2 Independently C 1-6 Alkyl, C 1-6 Alkoxy or halogen;

[0009] or R 1 and R 2 Formed with adjacent carbon atoms Indicates the connection position of the parallel ring;

[0010] L is The a end is connected to T, and the b end is connected to Q;

[0011] Q is a single amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue;

[0012] X is The two ends are connected to Z, and the one end is connected to connect;

[0013] R a and R b are independently H, D, halogen, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, amino, cyano, nitro, 3-10 membered cycloalkyl or 3-10 membered heterocyclic group;

[0014] Or, R a and R b Together with the carbon atom to which it is attached, it forms a 3-10 membered cycloalkyl group or a 3-10 membered heterocyclic group;

[0015] R c and R d Independently H, C 1-6 Alkyl, halogen, halogenated C 1-6 Alkyl, deuterated C 1-6 alkyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-14 membered aryl or 5-10 membered heteroaryl;

[0016] Or, R c and R dTogether with the carbon atom to which it is attached, it forms a 3-10 membered cycloalkyl group or a 3-10 membered heterocyclic group;

[0017] m1, m2, m3, m4, m5, m6 and m7 are independently an integer from 0 to 20;

[0018] n1, n2 and n3 are independently integers from 0 to 10;

[0019] q is an integer from 0 to 6;

[0020] r is an integer from 4 to 20;

[0021] Z is The c-end is connected to X, and the d-end is connected to G L connect;

[0022] p is 1-8;

[0023] G L For antibodies;

[0024] The heteroatoms in the 3-10 membered heterocyclic group and the 5-10 membered heteroaryl group are independently one or more of N, O or S, and the number of heteroatoms is independently 1, 2, 3 or 4.

[0025] In a certain embodiment of the present invention, in the antibody drug conjugate of Formula I or a pharmaceutically acceptable salt thereof, certain groups have the following definitions, and the definitions of the groups not mentioned are as described in any embodiment of the present invention (hereinafter referred to as "a certain embodiment of the present invention"), R 1 and R 2 Independently C 1-6 When alkyl, the C 1-6 Alkyl is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, for example methyl.

[0026] In one embodiment of the present invention, R 1 and R 2 Independently C 1-6 When alkoxy, the C 1-6 Alkoxy is methoxy, -OCH2CH3, -OCH(CH3)2 or tert-butoxy, for example methoxy;

[0027] In one embodiment of the present invention, R 1 and R 2 When independently halogen, the halogen is fluorine, chlorine, bromine or iodine, for example fluorine or chlorine.

[0028] In one embodiment of the present invention, the antibody-drug conjugate of Formula I has a structure as shown in Formula Ia:

[0029] Among them, R 1 、R 2 , Q, X, Z, G L and p are defined as above.

[0030] In one embodiment of the present invention, R 1 and R 2 Independently C 1-6 Alkyl or halogen, such as methyl or fluorine.

[0031] In a certain embodiment of the present invention, the single amino acid residue is Among them, the e end is connected to -NH-, and the f end is connected to -C(=O)-.

[0032] In a certain embodiment of the present invention, the dipeptide residue is C＝O -Lys-Phe- NH 、 C＝O -Ala-Val- NH 、 C＝O -Lys-Val- NH 、 C＝O -Lys-Ala- NH 、 C＝O -Cit-Val- NH 、 C＝O -Cit-Phe- NH 、 C＝O -Cit-Leu- NH 、 C＝O -Cit-Ile- NH 、 C＝O -Arg-Phe- NH 、 C＝O -Cit-Trp- NH or C＝O -Val-Gly- NH , preferably C＝O -Cit-Val- NH or C＝O -Ala-Val- NH .

[0033] In a certain embodiment of the present invention, the tripeptide residue is C＝O -Ala-Val-Glu- NH 、 C＝O -Cit-Val-Glu- NH 、 C＝O -Ala-Val-αGlu- NH or C＝O -Cit-Val-αGlu-NH .

[0034] In a certain embodiment of the present invention, the tetrapeptide residue is C＝O -Gly-Phe-(Gly)2- NH or C＝O –(Gly)2-Phe-Gly- NH .

[0035] The dipeptide residue, tripeptide residue or tetrapeptide residue C＝O The left side of the dipeptide residue, tripeptide residue or tetrapeptide residue is connected to the amino group of the antibody drug conjugate as shown in Formula I through its own carbonyl group, and the left side of the dipeptide residue, tripeptide residue or tetrapeptide residue is connected to the amino group of the antibody drug conjugate as shown in Formula I through its own carbonyl group. NH It means that the right side of the dipeptide residue, tripeptide residue or tetrapeptide residue is connected to the carbonyl group in the antibody drug conjugate as shown in Formula I through its own amino group.

[0036] In one embodiment of the present invention, Q is a dipeptide residue.

[0037] In a certain embodiment of the present invention, X is The two ends are connected to Z, and the one end is connected to The definitions of connections, m2, n2, r and q are as described above.

[0038] In one embodiment of the present invention, m2 is an integer from 6 to 14, such as 8 or 12.

[0039] In one embodiment of the present invention, n2 is an integer of 1-6, such as 2 or 3.

[0040] In one embodiment of the present invention, q is 4.

[0041] In one embodiment of the present invention, r is 16.

[0042] In one embodiment of the present invention, G L It is a HER2 antibody, such as Trastuzumab.

[0043] In a certain embodiment of the present invention, p may be an integer or a decimal, preferably 7-8, such as 7.6, 7.7, 7.8 or 8.0.

[0044] In a certain embodiment of the present invention, the d end of Z and the G L The G LIt can be obtained by reducing the disulfide bonds of an antibody containing disulfide bonds (such as Trastuzumab) to sulfhydryl groups under the action of a reducing agent. It will be understood by those skilled in the art that Z is connected to the sulfhydryl groups contained in the antibody itself after the disulfide bonds are opened (for example, the disulfide bonds of the antibody itself can be opened by reducing the disulfide bonds of the antibody itself to generate -SH). For example, in the antibody drug conjugate HER2-ADC1, -S- is not an additional external sulfur atom, but rather the sulfhydryl groups contained in the HER2 antibody itself after the disulfide bonds are opened. -S- is formed after the connection.

[0045] In one embodiment of the present invention, the pharmaceutically acceptable salt may be trifluoroacetate, hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, acetate, oxalate, maleate, tartrate, citrate, succinate or malonate, such as trifluoroacetate.

[0046] In a certain embodiment of the present invention, T is or a stereoisomer thereof;

[0047] R 1 and R 2 Independently C 1-6 Alkyl or halogen;

[0048] L is The a end is connected to T, and the b end is connected to Q;

[0049] Q is Among them, the e end is connected to -NH-, and the f end is connected to -C(=O)-;

[0050] X is The two ends are connected to Z, and the one end is connected to connection; m2 is an integer from 6 to 14; n2 is an integer from 1 to 6; q is 4; r is 16;

[0051] Z is The c-end is connected to X, and the d-end is connected to G L connect;

[0052] p is 1-8;

[0053] G L It is a HER2 antibody.

[0054] In a certain embodiment of the present invention, T is

[0055] In a certain embodiment of the present invention, T is

[0056] In one embodiment of the present invention, Q is Among them, the e end is connected to -NH-, and the f end is connected to -C(=O)-.

[0057] In a certain embodiment of the present invention, X is Among them, the 2 end is connected to Z, and the 1 end is connected to connect.

[0058] In one embodiment of the present invention, the antibody-drug conjugate as shown in Formula I is any of the following structures:

[0059] Among them G L Trastuzumab.

[0060] The present invention also provides a pharmaceutical composition comprising the antibody-drug conjugate as shown in Formula I or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical excipient.

[0061] The present invention also provides a use of an antibody-drug conjugate as shown in Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating cancer. The cancer is preferably breast cancer, lung cancer, kidney cancer, liver cancer, ovarian cancer, urethral cancer, prostate cancer, glioblastoma multiforme, pancreatic cancer, colorectal cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, melanoma, bladder cancer, gastric cancer or esophageal cancer, more preferably breast cancer.

[0062] The present invention also provides a use of an antibody-drug conjugate as shown in Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating a HER2-related or mediated disease. The disease may be cancer, preferably breast cancer, lung cancer, kidney cancer, liver cancer, ovarian cancer, urethral cancer, prostate cancer, glioblastoma multiforme, pancreatic cancer, colorectal cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, melanoma, bladder cancer, gastric cancer, or esophageal cancer, more preferably breast cancer.

[0063] The present invention also provides a linker-drug conjugate or a pharmaceutically acceptable salt thereof as shown in Formula III,

[0064] Where Y is T, L, Q and X are as defined above.

[0065] In a certain embodiment of the present invention, the linker-drug conjugate shown in Formula III is a structure shown in Formula IIIa.

[0066] Among them, R 1 、R 2 , Q, X and Y are as defined above.

[0067] In one embodiment of the present invention, the linker-drug conjugate as shown in Formula III is any of the following structures:

[0068] Definition of terms

[0069] Stereochemical definitions and conventions used herein generally follow SP 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.

[0070] Depending on the choice of starting materials and process, the compounds of the present invention may exist as one of the possible isomers or as a mixture thereof, such as a racemate and a diastereomeric mixture (depending on the number of asymmetric carbon atoms). Optically active (R)- or (S)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.

[0071] Any resulting mixture of stereoisomers can be separated into the pure or substantially pure geometric isomers, enantiomers, and diastereomers on the basis of the differences in the constituent physicochemical properties, for example, by chromatography and / or fractional crystallization.

[0072] In this specification, groups and substituents thereof can be selected by those skilled in the art to provide stable structural moieties and compounds. When substituents are described by conventional chemical formulas written from left to right, the substituents also include chemically equivalent substituents obtained when the structural formula is written from right to left.

[0073] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0074] The term "alkyl" refers to a straight or branched chain alkyl group having a specified number of carbon atoms (e.g., C1 to C6). Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl.

[0075] The term "aryl" refers to a group having a specified number of carbon atoms (e.g., C6 to C 10 ) cyclic groups consisting only of carbon atoms, which are monocyclic or polycyclic, and at least one ring is aromatic (in accordance with Huckel's rule). Aryl groups are connected to other fragments in the molecule through aromatic or non-aromatic rings. Aryl groups include but are not limited to phenyl, naphthyl, etc.

[0076] The "-" at the end of a group means that the group is connected to other fragments in the molecule through this site. For example, CH3-C(=O)- refers to acetyl.

[0077] As used herein, the term "alkylene," as a group or as part of another group, refers to a saturated divalent hydrocarbon group derived by removing two hydrogen atoms from a saturated straight-chain or branched hydrocarbon group; that is, one hydrogen atom of an alkyl group is replaced, as defined above. Examples of alkylene groups include methylene (-CH2-), ethylene {including -CH2CH2- or -CH(CH3)-}, isopropylene {including -CH(CH3)CH2- or -C(CH3)2-}, and the like.

[0078] In this application, the term "alkoxy" as a group or part of another group refers to -O-alkyl, and alkyl is as defined above.

[0079] The term "cycloalkyl" refers to a saturated monocyclic ring group consisting only of carbon atoms with a specified number of carbon atoms (e.g., C3 to C6). Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0080] The term "heterocyclyl" refers to a cyclic group having a specified number of ring atoms (e.g., 5-10 members), a specified number of heteroatoms (e.g., 1, 2, 3, or 4), and a specified heteroatom type (one or more of N, O, and S), which is monocyclic, bridged, or spirocyclic, and each ring is saturated. Heterocycloalkyl groups include, but are not limited to, azetidinyl, tetrahydropyrrolyl, tetrahydrofuranyl, morpholinyl, piperidinyl, and the like.

[0081] The term "aryl" refers to a group having a specified number of carbon atoms (e.g., C6 to C 10) cyclic groups consisting only of carbon atoms, which are monocyclic or polycyclic, and at least one ring is aromatic (in accordance with Huckel's rule). Aryl groups are connected to other fragments in the molecule through aromatic or non-aromatic rings. Aryl groups include but are not limited to phenyl, naphthyl, etc.

[0082] The term "heteroaryl" refers to a cyclic group having a specified number of ring atoms (e.g., 5-10 members), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatom (one or more of N, O, and S), which is monocyclic or polycyclic, and at least one ring is aromatic (in accordance with Huckel's rule). The heteroaryl group is connected to other fragments in the molecule through an aromatic ring or a non-aromatic ring. Heteroaryl includes, but is not limited to, furyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, indolyl, and the like.

[0083] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for use by patients) acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, ammonium salts, and the like. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochlorides, sulfates, methanesulfonates, and the like.

[0084] The term "pharmaceutical excipients" refers to excipients and additives used in the production of medicines and the preparation of prescriptions. It is all substances contained in pharmaceutical preparations in addition to the active ingredients.

[0085] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0086] The reagents and raw materials used in the present invention are commercially available.

[0087] The positive progress of the present invention is that the present invention provides a class of antibody-drug conjugates with novel structures, which can inhibit the proliferation of tumor cells and have good in vivo anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 shows the anti-tumor efficacy test of HER2-ADCs in the NOG mouse subcutaneously transplanted NCI-N87 cell animal model.

[0089] Figure 2 shows the changes in body weight of the NOG mouse subcutaneously transplanted NCI-N87 cell animal model with HER2-ADCs. DETAILED DESCRIPTION

[0090] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0091] Example 1: Preparation of Compounds II-1 to II-6

[0092] Step 1: Synthesis of compound 2

[0093] Under nitrogen protection, 80 mL of a dichloromethane solution of boron trichloride (1 mol / L) was dissolved in 400 mL of anhydrous 1,2-dichloromethane and the system was cooled to 0°C in an ice-water bath. Compound 1 (12.5 g, 99.9 mmol) was added to the reaction system in an ice-water bath and the reaction was maintained in an ice-water bath for 10 minutes. Chloroacetonitrile (13.5 mL, 213.3 mmol) and anhydrous aluminum chloride (17.5 g, 131.2 mmol) were then added in sequence and the reaction was maintained in an ice-water bath for 10 minutes. The system was then moved to room temperature and reacted for 10 minutes. The system was then heated to reflux and reacted for 40 hours. After the reaction was completed, the system was cooled to room temperature, 200 mL of ice water was slowly added, followed by 200 mL of 5% aqueous hydrochloric acid solution, stirred for 30 minutes, and extracted with dichloromethane (300 mL × 3). The organic phase was washed with water and saturated sodium chloride water in sequence and dried over anhydrous MgSO4. The solvent was dried and the crude product was separated and purified by column chromatography (petroleum ether:ethyl acetate=20:1) to obtain 7.0 g of compound 2 with a yield of 35%. 1 H NMR (400MHz, DMSO-d6) δ7.68(d,J=8.7Hz,1H),7.31(s,2H),6.53(d,J=12.5Hz,1H),4.98(s,2H),2.10(s,3H).MS(ESI)m / z=202.5(M+H + ).

[0094] Step 2: Synthesis of compound 3

[0095] Compound 2 (2.7g, 13.4mmol) was dissolved in 20mL of anhydrous dichloromethane, and DCC (5.5g, 26.8mmol) and DMAP (160mg, 1.3mmol) were added sequentially. Under an ice-water bath, ethyl malonate (2.4mL, 20.1mmol) was slowly added dropwise to the system. After completion of the addition, the reaction was continued at room temperature for 12 hours. After completion of the reaction, the filtrate was filtered and the filtrate was spin-dried to obtain 4.0g of crude product, which was dissolved in 20mL of ethanol. Sodium ethoxide (1.4g, 20.6mmol) was added to the system in batches and the reaction was continued at room temperature for 2 hours. After completion of the reaction, the solid precipitated in the system was filtered out, and the solid was pulped with dichloromethane and ether to obtain 2.5g of compound 3 in a yield of 63%. 1 H NMR(400MHz,Chloroform-d)δ7.90(d,J=7.3Hz,1H),7.62(d,J=9.9Hz,1H),4.86(s,2 H),4.53(q,J=6.2Hz,2H),2.51(s,3H),1.46(t,J=6.7Hz,3H).MS(ESI)m / z=298.6(M+H + ).

[0096] Step 3: Synthesis of compound 4

[0097] Compound 3 (2.0 g, 6.7 mmol) was dispersed in 30 mL of anhydrous acetonitrile, and phosphorus oxybromide (2.8 g, 10.1 mol) was added to the system. After the addition was complete, the temperature was raised and refluxed for 12 h. After the reaction was complete, the system was poured into 100 mL of ice water and extracted with ethyl acetate (100 mL × 3). The organic phase was washed with water and saturated sodium chloride solution in sequence, and dried over anhydrous MgSO4. The solvent was spin-dried to obtain a crude product, which was separated and purified by column chromatography (petroleum ether:ethyl acetate = 90:1) to obtain 2.0 g of compound 4, with a yield of 75%. 1 H NMR(400MHz,Chloroform-d)δ7.89(d,J=7.6Hz,1H),7.68–7.61(m,1H),4.74(d,J=6.2Hz ,2H),4.54(q,J=6.7Hz,2H),2.51(s,3H),1.48(t,J=7.0Hz,3H).MS(ESI)m / z=406.4(M+H + ).

[0098] Step 4: Synthesis of compound 5

[0099] Compound 4 (2.0 g, 4.9 mmol) was dissolved in 40 mL of anhydrous dichloromethane under an ice-water bath. A 1 M solution of diisobutylaluminum hydride in n-hexane (25 mL, 24.5 mmol) was added dropwise to the reaction system. After the addition was complete, the reaction mixture was kept in an ice-water bath for 2 hours. After completion of the reaction, the reaction solution was slowly added to 40 mL of saturated sodium tartaric acid aqueous solution. After stirring for 4 hours, the mixture was extracted with dichloromethane (20 mL × 3). The organic phase was washed with saturated sodium chloride aqueous solution and dried over anhydrous MgSO4. The solvent was spin-dried to obtain a crude product, which was separated and purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain 1.4 g of compound 5 with a yield of 80%. 1 H NMR(400MHz,Chloroform-d)δ7.85(d,J=7.8Hz,1H),7.61(d,J=10.1Hz,1H),5.06(s,2H),4.98(s,2H),2.50(s,3H),2.40(s,1H).MS(ESI)m / z=364.1(M+H + ).

[0100] Step 5: Synthesis of compound 6

[0101] Compound 5 (1.4 g, 3.9 mmol) was dissolved in 15 mL of dimethyl sulfoxide, and sodium azide (300 mg, 4.7 mmol) was added. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, 150 mL of water was added. Solid precipitated and was directly filtered to obtain 1.2 g of compound 6. The solid was not further isolated and was directly used for the next step. MS (ESI) m / z = 326.2 (M+H + ).

[0102] Step 6: Synthesis of compound 7

[0103] Compound 6 (1.2 g, 3.7 mmol) was dissolved in a mixture of tetrahydrofuran and water (3:1, 40 mL). A 1.0 M solution of trimethylphosphine in tetrahydrofuran (6.5 mL, 5.6 mmol) was added dropwise to the mixture under ice-water bath conditions. After completion of the addition, the mixture was allowed to react in the ice-water bath for 3 hours. After completion of the reaction, the reaction mixture was dried to obtain the crude product, which was then isolated and purified by column chromatography (dichloromethane:methanol = 30:1) to afford 1.0 g of compound 7, in a 90% yield. 1 H NMR (400MHz, DMSO-d6) δ8.23(d,J=8.2Hz,1H),7.65(d,J=10.6Hz,1H),4.83(s,2H),4.25(s,2H),2.45(s,3H).MS(ESI)m / z=300.2(M+H + ).

[0104] Step 7: Synthesis of compound 8

[0105] Compound 7 (1.0 g, 3.3 mmol) was dissolved in 20 mL of anhydrous N,N-dimethylformamide under an ice-water bath. Boc anhydride (1.0 g, 4.6 mmol) was added, and the mixture was gradually warmed to room temperature for 4 hours. After completion of the reaction, 200 mL of water was added and the mixture was extracted with ethyl acetate (50 mL x 3). The organic phase was washed sequentially with water and saturated sodium chloride solution, and dried over anhydrous MgSO₄. The solvent was evaporated to obtain the crude product, which was separated and purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain 1.0 g of compound 8 in an 80% yield. 1 H NMR(400MHz,Chloroform-d)δ7.94(d,J=7.8Hz,1H),7.59(d,J=10.2Hz,1H),5.25(s,1H),5.09(d,J =4.0Hz,2H),4.87(d,J=6.1Hz,2H),3.39(s,1H),2.47(s,3H),1.41(s,9H).MS(ESI)m / z=400.3(M+H + ).

[0106] Step 8: Synthesis of compound 10

[0107] Compound 9 was prepared according to the literature (Bioorganic & Medicinal Chemistry, 2015, 23(9), 1950-1962).

[0108] Under nitrogen protection, compound 8 (380 mg, 0.95 mmol), compound 9 (240 mg, 0.95 mmol) and triphenylphosphine (300 mg, 1.14 mmol) were dissolved in 20 mL of anhydrous dichloromethane. Diethyl azodicarboxylate (180 μL, 1.14 mmol) was added dropwise to the system under an ice-water bath. After the addition was complete, the system was moved to room temperature and reacted for 6 hours. After completion of the reaction, the solvent was dried to obtain a crude product, which was separated and purified by column chromatography (dichloromethane:methanol=80:1) to obtain 400 mg of compound 10, with a yield of 67%. 1H NMR(400MHz,Chloroform-d)δ8.15(d,J=8.2Hz,1H),7.58(d,J=10.2Hz,1H),7.43(d,J=7.5H z,1H),6.50(d,J=7.7Hz,1H),5.88(s,1H),5.61–5.33(m,3H),5.19(d,J=15.2Hz,1H),4.98– 4.78(m,2H),4.45(s,1H),3.55(s,3H),3.16(s,1H),2.46(s,3H),2.07(dd,J=14.4,7.4Hz,1 H),1.73(dt,J=14.9,7.5Hz,1H),1.45(s,9H),0.85(t,J=7.5Hz,3H).MS(ESI)m / z=635.3(M+H + ).

[0109] Step 9: Synthesis of compound 11

[0110] Under nitrogen, compound 10 (250 mg, 0.39 mmol), palladium acetate (90 mg, 0.39 mmol), potassium acetate (155 mg, 1.58 mmol), tri(o-methylphenyl)phosphine (120 mg, 0.39 mmol), and tetrabutylammonium chloride (110 mg, 0.39 mmol) were dissolved in 25 mL of anhydrous acetonitrile and the mixture was heated to reflux for 12 hours. After completion of the reaction, the solvent was dried to obtain a crude product, which was separated and purified by column chromatography (dichloromethane:methanol=80:1) to obtain 150 mg of compound 11 in a yield of 69%. MS (ESI) m / z=554.4 (M+H + ). Step 10: Synthesis of Compound II-1

[0111] 150 mg of compound 11 was dissolved in 15 mL of dichloromethane containing 5% trifluoroacetic acid and stirred at room temperature for 6 hours. After the reaction, the solvent was dried to obtain a crude product, which was then slurried with ether to obtain 110 mg of compound II-1, with a yield of 90%. 1 H NMR (400MHz, DMSO-d6) δ8.54–8.35(m,4H),7.97(d,J=10.5Hz,1H),7.37(s,1H),5.96(s,1H),5.63–5.44(m,4H),4.98(s,1H),4.71(s,2 H),3.47(s,3H),2.54(s,3H),2.29(dd,J=14.3,7.5Hz,1H),1.81(dd,J=14.3,7.4Hz,1H),0.74(t,J=7.1Hz,3H).MS(ESI)m / z=454.2(M+H +).

[0112] Using the same method as above, using 3-chloro-4-methylaniline instead of 3-fluoro-4-methylaniline, compound II-2 can be obtained.

[0113] 1 H NMR(400MHz, DMSO-d6)δ8.52–8.36(m,4H),7.97(d,J=10.5Hz,1H),7.37(s,1H),5.96(s,1H),5.63–5.4 4(m,4H),4.98(s,1H),4.71(s,2H),3.47(s,3H),2.54(s,3H),2.29–1.81(m,2H),0.74(t,J=7.1Hz,3H). MS(ESI)m / z=470.2(M+H + ).

[0114] Using the same method as above, compound II-3 can be obtained by replacing 3-fluoro-4-methylaniline with 3,4-difluoroaniline.

[0115] 1 H NMR (400MHz, DMSO-d6) δ8.54–8.35(m,4H),7.97(d,J=10.5Hz,1H),7.37(s,1H),5.96(s,1H),5.63–5.44(m,4H),4.98 (s,1H),4.71(s,2H),3.47(s,3H),2.29(dd,J=14.3,7.5Hz,1H),1.81(dd,J=14.3,7.4Hz,1H),0.74(t,J=7.1Hz,3H). MS(ESI)m / z=458.2(M+H + ).

[0116] Using the same method as above, compound II-4 can be obtained by replacing 3-fluoro-4-methoxyaniline with 3-fluoro-4-methylaniline.

[0117] 1 H NMR (400MHz, DMSO-d6) δ8.54–8.35(m,4H),7.97(d,J=10.5Hz,1H),7.37(s,1H),5.96(s,1H),5.63–5.44(m,4H),4. 98(s,1H),4.71(s,2H),3.92(s,3H),3.47(s,3H),2.21–1.83(m,2H),0.72(t,J=7.1Hz,3H).MS(ESI)m / z=470.2(M+H+ ).

[0118] Using the same method as above, compound II-5 can be obtained by replacing 3-fluoro-4-methylaniline with 3,4-methylenedioxyaniline.

[0119] 1 H NMR(400MHz,DMSO-d6)δ8.32(s,3H),7.82(s,1H),7.56(s,1H),7.26(s,1H),6.31(s,2H),5.91(s,1H),5.60–5.38(m,4H), 4.96 (s, 1H), 4.60 (s, 2H), 3.44 (s, 3H), 2.26 (dd, J = 14.1, 7.4Hz, 1H), 1.78 (dq, J = 14.9, 7.5Hz, 1H), 0.70 (t, J = 7.3Hz, 3H). MS(ESI)m / z=466.2(M+H + ).

[0120] Using the same method as above, compound II-6 can be obtained by replacing 3-fluoro-4-methylaniline with 6-amino-1,4-benzodioxetane.

[0121] 1 H NMR(400MHz,DMSO-d6)δ8.32(s,3H),7.82(s,1H),7.56(s,1H),7.26(s,1H),6.31(s,2H),5.91(s,1H) ,5.60–5.38(m,4H),4.96(s,1H),4.62(s,4H),3.44(s,3H),2.26–1.78(m,2H),0.70(t,J=7.3Hz,3H). MS(ESI)m / z=480.2(M+H + ).

[0122] Example 2: Preparation of Compounds III-1 to III-4

[0123] 1. Synthesis of Compound III-1

[0124] Step 1: Synthesis of compound 12

[0125] Under argon, compound II-1 (55 mg, 0.1 mmol) was dissolved in N,N-dimethylformamide (5 mL). Fmoc-Val-Cit-PAB-PNP (CAS No. 863971-53-3, 92 mg, 0.12 mmol), 1-hydroxybenzotriazole (16 mg, 0.12 mmol), and N,N-diisopropylethylamine (26 mg, 0.2 mmol) were added sequentially at room temperature. The mixture was allowed to react for 2 hours. After TLC analysis, the reaction was complete. The solvent was removed under reduced pressure using an oil pump, and the product was separated by column chromatography to obtain 97 mg of a pale yellow solid (90% yield). 1 H NMR (400MHz, DMSO-d6) δ10.05(s,1H),8.30(d,J=8.3Hz,1H),8.17(t,J=5.6Hz,1H),8.10(d,J=7.3Hz,1H),7.88(d,J=8.5Hz,3H),7.7 3(t,J=8.1Hz,2H),7.61–7.53(m,2H),7.47–7.20(m,8H),5.97(t,J=5.5Hz,1H),5.90(s,1H),5.71–5.27(m,6H),4.96(d,J=4.0Hz,3H) ,4.77(d,J=5.7Hz,2H),4.49–4.37(m,1H),4.35–4.15(m,3H),3.93(t,J=7.9Hz,1H),3.47(s,3H),3.08–2.89(m,2H),2.48(s,3H),2.3 6–2.20(m,1H),2.07–1.91(m,1H),1.90–1.75(m,1H),1.75–1.53(m,2H),1.51–1.29(m,2H),0.90–0.82(m,6H),0.74(t,J=7.4Hz,3H). MS(ESI)m / z=1081.4(M+H + ).

[0126] Step 2: Synthesis of compound 13

[0127] Under argon, compound 12 (97 mg, 0.09 mmol) was dissolved in N,N-dimethylformamide (3 mL). Diethylamine (0.5 mL) was added at room temperature and allowed to react for 2 hours. LC-MS analysis confirmed the complete reaction of the starting material. The solvent was removed under reduced pressure using an oil pump, and methyl tert-butyl ether (10 mL) was added. A solid precipitated, which was filtered and dried to afford 65 mg of a pale yellow solid (65% yield). 1H NMR (400MHz, DMSO-d6) δ10.00(s,1H),8.30(d,J=8.2Hz,1H),8.17(t,J=5.7Hz,1H),8.09(d,J=7.5Hz,1H),7.99(t,J=5.3Hz,1H),7. 93–7.79(m,2H),7.57(d,J=8.3Hz,2H),7.34(s,1H),7.27(d,J=8.4Hz,2H),5.98(t,J=5.5Hz,1H),5.90(s,1H),5.64–5.34(m,6H),4. 95(d,J=4.6Hz,3H),4.77(d,J=5.8Hz,2H),4.45–4.29(m,1H),4.28–4.17(m,1H),3.48(s,3H),3.06–2.91(m,2H),2.49(s,3H),2.36 –2.20(m,1H),2.05–1.91(m,1H),1.90–1.75(m,1H),1.75–1.53(m,2H),1.51–1.29(m,2H),0.90–0.82(m,6H),0.74(t,J=7.4Hz,3H). MS(ESI)m / z=859.4(M+H + ).

[0128] Step 3: Synthesis of Compound III-1

[0129] Under argon, compound 13 (65 mg, 0.075 mmol) was dissolved in N,N-dimethylformamide (3 mL). Mal-amido-PEG8-NHS ester (CAS No. 756525-93-6, 58 mg, 0.09 mmol) was added at room temperature and allowed to react overnight. After the reaction was complete as determined by LC-MS, the solvent was removed under reduced pressure using a pump. Preparative liquid chromatography (HPLC) was used to obtain 70 mg of a pale yellow oil (70% yield). 1H NMR(400MHz, DMSO-d6)δ9.97(s,1H),8.30(d,J=8.1Hz,1H),8.17(t,J=5.8Hz,1H),8.09(d,J=7.5Hz,1H),7.99(t,J=5.3Hz,1H),7.93–7.79(m,2H), 7.57(d,J=8.3Hz,2H),7.34(s,1H),7.27(d,J=8.4Hz,2H),6.99(s,2H),5. 98(t,J=5.5Hz,1H),5.90(s,1H),5.64–5.34(m,6H),4.95(d,J=4.6Hz,3H) ,4.77(d,J=5.8Hz,2H),4.45–4.29(m,1H),4.28–4.17(m,1H),3.63–3.43( m,35H),3.38–3.34(m,2H),3.17–3.11(m,2H),3.05–2.90(m,2H),2.49–2. 43(m,5H),2.41–2.24(m,3H),2.04–1.90(m,1H),1.87–1.76(m,1H),1.76– 1.53(m,2H),1.49–1.24(m,2H),0.88–0.80(m,6H),0.74(t,J=7.4Hz,3H). MS (ESI) m / z = 1433.7 (M+H + ).

[0130] 2. Synthesis of Compound III-2

[0131] Step 1: Synthesis of compound 14

[0132] Under argon, compound II-1 (55 mg, 0.1 mmol) was dissolved in N,N-dimethylformamide (5 mL). Fmoc-Val-Ala-PAB-PNP (CAS No. 1394238-92-6, 82 mg, 0.12 mmol), 1-hydroxybenzotriazole (16 mg, 0.12 mmol), and N,N-diisopropylethylamine (26 mg, 0.2 mmol) were added sequentially at room temperature. The mixture was allowed to react for 2 hours. After TLC analysis, the reaction was complete, and the solvent was removed under reduced pressure using an oil pump. The product was then separated by column chromatography to afford 90 mg of a pale yellow solid in a 90% yield. 1H NMR (400MHz, DMSO-d6) δ10.05(s,1H),8.30(d,J=8.3Hz,1H),8.17(t,J=5.6Hz,1H),8.10(d,J=7.3Hz,1H),7 .88(d,J=8.5Hz,3H),7.73(t,J=8.1Hz,2H),7.61–7.53(m,2H),7.47–7.20(m,8H),5.97(t,J=5.5Hz,1H),5. 90(s,1H),5.71–5.27(m,6H),4.49–4.37(m,1H),4.35–4.15(m,3H),3.93(t,J=7.9Hz,1H),3.47(s,3H),2.4 8(s,3H),2.36–2.20(m,1H),1.75–1.53(m,2H),1.51–1.29(m,3H),0.90–0.82(m,6H),0.74(t,J=7.4Hz,3H). MS(ESI)m / z=995.4(M+H + ).

[0133] Step 2: Synthesis of compound 15

[0134] Under argon, compound 14 (90 mg, 0.09 mmol) was dissolved in N,N-dimethylformamide (3 mL). Diethylamine (0.5 mL) was added at room temperature and allowed to react for 2 hours. LC-MS analysis confirmed the complete reaction, followed by removal of the solvent under reduced pressure using an oil pump and the addition of methyl tert-butyl ether (10 mL). Solid precipitated, which was filtered and dried to afford 49 mg of a pale yellow solid (70% yield). 1 H NMR (400MHz, DMSO-d6) δ10.00(s,1H),8.30(d,J=8.2Hz,1H),8.17(t,J=5.7Hz,1H),8.09(d,J=7.5Hz,1H), 7.99(t,J=5.3Hz,1H),7.93–7.79(m,2H),7.57(d,J=8.3Hz,2H),7.34(s,1H),7.27(d,J=8.4Hz,2H),5.98( t,J=5.5Hz,1H),5.90(s,1H),5.64–5.34(m,6H),4.45–4.29(m,1H),4.28–4.17(m,1H),3.48(s,3H),2.49( s,3H),2.36–2.20(m,1H),1.75–1.53(m,3H),1.51–1.29(m,2H),0.90–0.82(m,6H),0.74(t,J=7.4Hz,3H). MS(ESI)m / z=773.4(M+H +).

[0135] Step 3: Synthesis of compound III-2

[0136] Under argon, compound 15 (58 mg, 0.075 mmol) was dissolved in N,N-dimethylformamide (3 mL). Mal-amido-PEG8-NHS ester (58 mg, 0.09 mmol) was added at room temperature and allowed to react overnight. After the reaction was complete as determined by LC-MS, the solvent was removed under reduced pressure using an oil pump. Preparative liquid chromatography (HPLC) was used to obtain 70 mg of a pale yellow oil (70% yield). 1 H NMR (400MHz, DMSO-d6) δ9.97(s,1H),8.30(d,J=8.1Hz,1H),8.17(t,J=5.8Hz,1H),8.09(d,J=7.5Hz,1H),7.99(t,J=5.3Hz,1H),7.93 –7.79(m,2H),7.57(d,J=8.3Hz,2H),7.34(s,1H),7.27(d,J=8.4Hz,2H),6.99(s,2H),5.98(t,J=5.5Hz,1H),5.90(s,1H),5.64–5.34( m,6H),4.95(d,J=4.6Hz,3H),4.77(d,J=5.8Hz,2H),4.45–4.29(m,1H),4.28–4.17(m,1H),3.63–3.43(m,35H),3.38–3.34(m,2H),3.1 7–3.11(m,2H),2.49–2.43(m,5H),2.41–2.24(m,3H),1.76–1.53(m,2H),1.49–1.24(m,3H),0.88–0.80(m,6H),0.74(t,J=7.4Hz,3H). MS(ESI)m / z=1347.6(M+H + ).

[0137] 3. Synthesis of Compound III-3

[0138] Referring to the synthesis method of compound III-1, Mal-amido-PEG12-NHS ester (CAS No.: 326003-46-7) was used instead of Mal-amido-PEG8-NHS ester to obtain compound III-3. MS (ESI) m / z = 1609.8 (M+H + ).

[0139] 4. Synthesis of Compound III-4

[0140] Referring to the synthesis method of compound III-2, compound III-4 was obtained by replacing Mal-amido-PEG8-NHS ester with Mal-amido-PEG12-NHS ester. MS (ESI) m / z = 1523.7 (M+H + ).

[0141] Example 3: Preparation of III-5 and III-6

[0142] 1. Synthesis of Compound III-5

[0143] Step 1: Synthesis of compound 17

[0144] Under argon, Fmoc-Lys-OH (370 mg, 1.0 mmol) was dissolved in anhydrous dichloromethane (20 mL). m-PEG17-NHS-ester (910 mg, 1.0 mmol) and N,N-diisopropylethylamine (650 mg, 1.0 mmol) were added at room temperature. The mixture was allowed to react overnight. LC-MS analysis confirmed the complete reaction. The solvent was removed under reduced pressure using an oil pump and the product was separated by column chromatography to yield 1.0 g of a colorless liquid (86% yield). 1 H NMR(400MHz,Chloroform-d)δ7.73–7.68(m,3H),7.63–7.59(m,2H),7.38–7.31(m,2H),7.26–7.21(m,3H),4.36–4.31 (m,1H),4.16–4.10(m,2H),3.63–3.56(m,54H),3.38(s,3H),3.20–3.14(m,2H),2.47–2.42(m,5H),1.48–1.42(m,6H). MS(ESI)m / z=1159.6(M+H + ).

[0145] Step 2: Synthesis of compound 18

[0146] Under argon protection, compound 17 (580 mg, 0.5 mmol) was dissolved in N,N-dimethylformamide (10 mL), and diethylamine (1.0 mL) was added at room temperature. The mixture was allowed to react for 2 hours. After the reaction was complete, LC-MS analysis showed that the solvent was removed by oil pump under reduced pressure. Preparative liquid phase separation was performed to obtain 420 mg of a colorless oil with a yield of 90%. MS (ESI) m / z = 937.6 (M+H + ).

[0147] Step 3: Synthesis of compound 19

[0148] Under argon, compound 18 (240 mg, 0.25 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL). 6-(maleimido)hexanoic acid succinimidyl ester (105 mg, 0.5 mmol) and N,N-diisopropylethylamine (65 mg, 0.5 mmol) were added at room temperature and allowed to react overnight. After the reaction was complete as determined by LC-MS, the solvent was removed under reduced pressure using an oil pump. Preparative liquid chromatography (HPLC) was used to obtain 220 mg of a colorless oil (80% yield). 1 H NMR(400MHz,Chloroform-d)δ6.86–6.83(m,1H),6.71(s,2H),6.54–6.52(m,1H),4.56–4.51(m,1H),3.75–3.53(m,68H),3.4 0(s,3H),3.30–3.25(m,2H),2.50–2.47(m,2H),2.27–2.23(m,2H),1.90–1.76(m,2H),1.70–1.47(m,6H),1.39–1.26(m,4H). MS(ESI)m / z=1130.6(M+H + ).

[0149] Step 4: Synthesis of compound III-5

[0150] Under argon protection, compound 19 (110 mg, 0.1 mmol) was dissolved in N,N-dimethylformamide (5 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19 mg, 0.1 mmol) and N-hydroxysuccinimide (12 mg, 0.1 mmol) were added at room temperature. The reaction was continued at room temperature for 1 hour. Compound 13 (44 mg, 0.05 mmol) was added at room temperature and the reaction was continued at room temperature overnight. After the reaction of the raw materials was complete, LC-MS detected that the solvent was removed under reduced pressure by oil pump and preparative liquid phase separation was performed to obtain 50 mg of a light yellow solid with a yield of 50%. MS (ESI) m / z = 1971.1 (M+H + ).

[0151] 2. Synthesis of Compound III-6 Referring to the synthesis method of Compound III-5, Compound 15 was used instead of Compound 13 in Step 4 to obtain Compound III-6. MS (ESI) m / z = 1885.0 (M+H + ).

[0152] Example 4: Preparation of Compound III-7

[0153] Step 1: Synthesis of compound 21

[0154] Under argon protection, the compound 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (100 mg, 0.37 mmol) was dissolved in dichloromethane (10 mL). 1-Hydroxybenzotriazole (60 mg, 0.45 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (86 mg, 0.45 mmol) were added sequentially at room temperature, and the reaction was continued at room temperature for 1 hour. Amino-dodecapolyethylene glycol-tert-butyl propionate (250 mg, 0.375 mmol) was added at room temperature, and the reaction was continued at room temperature overnight. After TLC detection of the complete reaction of the raw material 20, the solvent was removed under reduced pressure and separated by chromatographic column to obtain 300 mg of a colorless liquid with a yield of 87%. 1 H NMR(400MHz,Chloroform-d)δ9.39(s,2H),3.73(t,J=7.9Hz,2H),3.67(t,J=7.3Hz,2H),3.52–3.40(m,44H),3.28(t,J=7.3 Hz,2H),2.95(s,3H),2.53(t,J=7.9Hz,2H),2.34(t,J=5.4Hz,2H),2.18(t,J=7.8Hz,2H),1.95–1.85-3(m,2H),1.42(s,9H). MS(ESI)m / z=924.4(M+H + ).

[0155] Step 2: Synthesis of compound 22

[0156] Under argon protection, compound 21 (300 mg, 0.32 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (1 mL) was added at room temperature, and the reaction was continued at room temperature for 4 hours. After TLC detection, the reaction of the raw material was complete, and the solvent was removed under reduced pressure to obtain 270 mg of a light yellow liquid with a yield of 96%. It was used directly in the next step without purification. MS (ESI) m / z = 868.4 (M+H + ).

[0157] Step 3: Synthesis of compound III-7

[0158] Under argon, compound 22 (100 mg, 0.12 mmol) was dissolved in N,N-dimethylformamide (5 mL). N-hydroxysuccinimide (14 mg, 0.12 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23 mg, 0.12 mmol) were added sequentially at room temperature, and the reaction was continued at room temperature for 1 hour. Compound 13 (50 mg, 0.06 mmol) was added at room temperature, and the reaction was continued at room temperature overnight. After the reaction of starting material 13 was complete as determined by LC-MS, the solvent was removed under reduced pressure by oil pump and the mixture was purified on a C18 reverse-phase column to obtain compound III-7 as a light yellow solid (55 mg, 55% yield).1 H NMR (400MHz, DMSO-d6) δ9.97 (s, 1H), 9.39 (s, 2H), 8.30 (d, J = 8.1Hz, 1H), 8. 17(t,J=5.8Hz,1H),8.09(d,J=7.5Hz,1H),7.99(t,J=5.3Hz,1H),7.93–7.79 (m,2H),7.57(d,J=8.3Hz,2H),7.34(s,1H),7.27(d,J=8.4Hz,2H),5.98(t, J=5.5Hz,1H),5.90(s,1H),5.64–5.34(m,6H),4.95(d,J=4.6Hz,2H),4.77(d ,J=5.8Hz,2H),4.45–4.29(m,1H),4.28–4.17(m,1H),3.63–3.43(m,49H),3 .38–3.34(m,2H),3.17–3.11(m,2H),3.05–2.90(m,5H),2.49(s,3H),2.41–2 .24(m,5H),2.18(t,J=7.8Hz,2H),2.04–1.90(m,3H),1.87–1.76(m,1H),1.7 6–1.53(m,2H),1.49–1.24(m,2H),0.88–0.80(m,6H),0.74(t,J=7.4Hz,3H). MS (ESI) m / z = 1709.8 (M+H + ).

[0159] Example 5: Synthesis of compounds HER2-ADC1 to HER2-ADC7

[0160] At 37°C, a solution of 0.05M PBS buffer solution (pH = 6.5; 10.0 mg / mL, 1.0 mL, 67.6 nmol) of the antibody Trastuzumab (HER2 antibody, CAS: 180288-69-1) purchased from Hangzhou Haoyang Biotechnology Co., Ltd.) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 37.2 μL, 372 nmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours before stopping the reaction. The reaction solution was cooled to 25°C in a water bath. Compound III-1 (1.45 mg, 1014 nmol) was dissolved in 50 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (mobile phase: 0.05 M PBS buffer, pH 6.5, containing 0.001 M EDTA) to obtain the conjugate HER2-ADC1 in PBS buffer (1.1 mg / mL, 7.5 mL), which was stored at 4°C. SEC-HPLC analysis revealed a purity of 96.68%, and the average drug loading calculated by RP-HPLC was p = 7.8.

[0161] Referring to the same synthesis method, III-1 was replaced with III-2, III-3, III-4, III-5, III-6, III-7, and MC-GGFG-DXD (CAS No. 1599440-13-7, purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.) and coupled with the antibody Trastuzumab to obtain the corresponding conjugates: HER2-ADC2 (DAR=7.7), HER2-ADC3 (DAR=7.7), HER2-ADC4 (DAR=7.8), HER2-ADC5 (DAR=8.0), HER2-ADC6 (DAR=8.0), HER2-ADC7 (DAR=7.8), and HER2-GGFG-DXD (DS-8201, DAR=7.9).

[0162] Biological evaluation

[0163] Experimental Example 1. Determination of the inhibitory activity of compounds on tumor cell proliferation

[0164] The effects of homocamptothecin derivatives II-1, II-2, II-3, II-4, II-5, II-6, and DXd (CAS No. 1599440-33-1, purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.) on tumor cell proliferation were evaluated using the SRB assay. Cells in the logarithmic growth phase were seeded into 96-well culture plates at the appropriate concentrations and cultured overnight with 100 μL of complete medium per well. Compounds were added at varying concentrations, with triplicate wells set up for each concentration. Positive control wells without compound treatment and negative control wells without cells were also included. Cells were incubated at 37°C and 5% CO₂ for 72 hours. After drug treatment, the inhibitory effect of the compounds on cell proliferation was assessed using sulforhodamine B (SRB) protein staining. The specific steps were as follows: the culture medium was decanted, the cells were fixed with 10% trichloroacetic acid, incubated at 4°C for 1 hour, washed five times with distilled water, and dried in an oven. Add 100 μL / well of a 4 mg / ml SRB solution prepared in 1% glacial acetic acid and stain at room temperature for 15 minutes. Remove the supernatant, wash five times with 1% glacial acetic acid, and dry in an oven. Finally, add 150 μL / well of Tris solution, shake to mix, and measure the OD at 560 nm using a SpectraMax 190 full-wavelength microplate reader. Calculate the inhibitory rate of the compound on cell proliferation using the following formula:

[0165] Inhibition rate (%) = [1-(OD of drug-administered well-OD of negative control well) / (OD of positive control well-OD of negative control well)] x 100%.

[0166] The cell lines used in this screening are shown in Table 1, and the results of the inhibition of tumor cell proliferation by compounds II-1, II-2, II-3, II-4, II-5, II-6 and DXD are shown in Table 2.

[0167] Table 1. Overview of cell lines used in this screening

[0168] Table 2. Effects of compounds on tumor cell proliferation

[0169] Experimental Example 2. Method for testing the tumor cell proliferation ability of HER2-ADC1-ADC7 and HER2-GGFG-DXD:

[0170] After digestion, resuspend NCI-N87 cells and count them. Centrifuge and remove the supernatant, then adjust the cell density. Count the cells using a cell counter. Aspirate 20 μL of the cell suspension and mix with 20 μL of VisStain AOPI staining solution. Count the cells using a 20 μL aliquot. Plate the cells in a 96-well cell culture plate, adding 180 μL per well to seed 1000 or 2000 cells per well. Gently tap to mix, and incubate the cell culture plate at 37°C for 16 hours. Dilute the compound in RPMI1640 medium / 10% FBS, adding 20 μL to each well to a maximum concentration of 100 nM. Plate 10 points with 3-fold dilutions. For a vehicle control using DMSO, add 20 μL of RPMI1640 medium / 10% FBS to the blank. Gently tap to mix, and incubate the cell culture plate at 37°C for 7 days. After 7 days of culture, remove the cell plate from the incubator, aspirate the culture medium, add 20 μL of culture medium, and then add 20 μL of CTG to each well. Oscillate on a shaker for 5 minutes, let it stand at room temperature for 10 minutes, and transfer 30 μL of the reaction solution to a Viewplate-96white, white, clear-bottomed microtiter plate for data analysis using an EnVision plate reader.

[0171] Calculation formula: The inhibition rate (IR) of the test antibody was calculated using the following formula: IR (%) = (1 – (RLU antibody – RLU blank control) / (RLU vehicle control – RLU blank control)) * 100%. The inhibition rate at different antibody concentrations was calculated in Excel, and inhibition curves were plotted and relevant parameters were calculated using GraphPad Prism software. The results are shown in Table 3.

[0172] Table 3. Effects of ADC on the proliferation of NCl-N87 tumor cells

[0173] Experimental Example 3. Anti-tumor efficacy test of HER2-ADCs in NOG mouse subcutaneously transplanted NCI-N87 cell animal model:

[0174] In this study, NCI-N87 cells were subcutaneously inoculated into NOG mice to determine the anti-tumor effect of HER2-ADCs.

[0175] NOG mice: Female NOG mice (6 weeks old) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. The mice were acclimated for 7 days after arrival before the study began.

[0176] Cells: Human gastric cancer NCI-N87 cells (ATCC source, catalog number CRL-5822) were routinely subcultured according to the manufacturer's instructions. Cells were resuspended in serum-free medium and the cell density was adjusted. On day 0, the cell suspension was subcutaneously inoculated into the right axilla of female NOG mice to establish the NCI-N87 tumor-bearing mouse model.

[0177] Dosage:

[0178] On the 10th day after tumor cell inoculation, the tumor volume of each mouse was detected, and mice with a tumor volume of 130 mm were selected. 3 ~250mm 3 Mice within the range were evenly divided into groups according to tumor volume (5 mice per group) and administered on the 11th day after inoculation, respectively, with negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP067-F1), positive control antibody Trastuzumab (HER2 antibody, CAS: 180288-69-1, purchased from Hangzhou Haoyang Biotechnology Co., Ltd.), and positive controls HER2-GGFG-DXd, HER2-ADC3 and HER2-ADC5. During the administration period, the changes in tumor volume and body weight of mice in each group were monitored at a frequency of 2 times / week for 3 consecutive weeks. Body weight and tumor volume were measured before each administration. Tumor volume inhibition rate (TGI%) was calculated on day 30 after inoculation using the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)] × 100; where Ti: mean tumor volume of the administration group, T0: mean tumor volume of the administration group on day D0, Vi: mean tumor volume of the isotype control group, and V0: mean tumor volume of the isotype control group on day D0. Tumor volume measurement: The long diameter (a) and wide diameter (b) of the tumor were measured using a vernier caliper. Tumor volume was calculated using the following formula: TV = 1 / 2 × a × b 2 Body weight was measured using an electronic balance.

[0179] The dosage and method of administration are shown in Table 4.

[0180] Table 4. Dosage study design

[0181] As shown in Table 5 and Figure 1, on the 30th day after inoculation, the tumor volume inhibition rate of the positive control antibody HER2-GGFG-DXd was 107.03%; HER2-ADC3 and HER2-ADC5 also significantly inhibited tumor growth at a dose of 4 mg / kg, with tumor volume inhibition rates of 115.16% and 114.52%, respectively.

[0182] Table 5. Inhibitory effect of ADC on tumor growth in vivo on day 23

[0183] Note: P value is compared with negative control antibody

[0184] The above experiments showed that HER2-ADC3 and HER2-ADC5 significantly inhibited the growth of tumors in the NCI-N87 cell animal model transplanted subcutaneously in NOG mice, and their efficacy was better than that of the control HER2-GGFG-DXD, and had no significant effect on the body weight of NOG mice (Figure 2).

Claims

1. An antibody-drug conjugate as shown in formula I or a pharmaceutically acceptable salt thereof, in, T is or its stereoisomers; R 1 and R 2 Independently for C 1-6 Alkyl, C 1-6 Alkoxy or halogen; or R 1 and R 2 With adjacent carbon atoms Indicates the connection position of the parallel ring; L is The a end is connected to T, and the b end is connected to Q; Q is a single amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; X is The 2 ends are connected to Z and the 1 end is connected to connect; R a and R b are independently H, D, halogen, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, amino, cyano, nitro, 3-10 membered cycloalkyl or 3-10 membered heterocyclic group; Or, R a and R b Together with the carbon atom to which it is attached, it forms a 3-10 membered cycloalkyl group or a 3-10 membered heterocyclic group; R c and R d Independently H, C 1-6 Alkyl, halogen, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, 6-14 membered aryl or 5-10 membered heteroaryl; Or, R c and R d Together with the carbon atom to which it is attached, it forms a 3-10 membered cycloalkyl group or a 3-10 membered heterocyclic group; m1, m2, m3, m4, m5, m6 and m7 are independently integers of 0-20; n1, n2 and n3 are independently integers from 0 to 10; q is an integer from 0 to 6; r is an integer from 4 to 20; Z is The c-terminal is connected to X, and the d-terminal is connected to G L connect; p is 1-8; G L For antibodies; The heteroatoms in the 3-10 membered heterocyclic group and the 5-10 membered heteroaryl group are independently one or more of N, O or S, and the number of the heteroatoms is independently 1, 2, 3 or 4.

2. The antibody-drug conjugate of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antibody drug conjugate as shown in Formula I has a structure as shown in Formula Ia: Among them, R 1 , R 2 , Q, X, Z, G L and p are as defined in claim 1.

3. The antibody-drug conjugate of formula I or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: The antibody-drug conjugate of Formula I or a pharmaceutically acceptable salt thereof satisfies one or more of the following conditions: (1)R 1 and R 2 Independently for C 1-6 Alkyl or halogen; (2) The single amino acid residue is Among them, the e end is connected to -NH-, and the f end is connected to -C(=O)-; (3) The dipeptide residue is C＝O -Lys-Phe- NH , C＝O -Ala-Val- NH , C＝O -Lys-Val- NH , C＝O -Lys-Ala- NH , C＝O -Cit-Val- NH , C＝O -Cit-Phe- NH , C＝O -Cit-Leu- NH , C＝O -Cit-Ile- NH , C＝O -Arg-Phe- NH , C＝O -Cit-Trp- NH or C＝O -Val-Gly- NH , preferably C＝O -Cit-Val- NH or C＝O -Ala-Val- NH ; (4) The tripeptide residue is C＝O -Ala-Val-Glu- NH , C＝O -Cit-Val-Glu- NH , C＝O -Ala-Val-αGlu- NH or C＝O -Cit-Val-αGlu- NH ; (5) The tetrapeptide residue is C＝O -Gly-Phe-(Gly)2- NH or C＝O –(Gly)2-Phe-Gly- NH ; (6)X is The 2 ends are connected to Z and the 1 end is connected to connection; preferably, m2 is an integer of 6-14, such as 8 or 12; n2 may be an integer of 1-6, such as 2 or 3; q may be 4; r may be 16; (7)G L HER2 antibodies, such as Trastuzumab; (8) p is 7-8, for example, 7.6, 7.7, 7.8 or 8.

0.

4. The antibody-drug conjugate of formula I or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: The antibody-drug conjugate of Formula I or a pharmaceutically acceptable salt thereof satisfies one or more of the following conditions: (1) T is (2) Q is Among them, the e end is connected to -NH-, and the f end is connected to -C(=O)-; (3)X is Among them, the 2nd end is connected to Z, and the 1st end is connected to connect.

5. The antibody-drug conjugate of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antibody-drug conjugate as shown in Formula I is any of the following structures: Among them G L For Trastuzumab.

6. A pharmaceutical composition, characterized in that It comprises an antibody-drug conjugate as shown in formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, and at least one pharmaceutical excipient.

7. Use of an antibody-drug conjugate as shown in formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, or a pharmaceutical composition according to claim 6 in the preparation of a medicament for preventing or treating cancer; the cancer is preferably breast cancer, lung cancer, kidney cancer, liver cancer, ovarian cancer, urethral cancer, prostate cancer, glioblastoma multiforme, pancreatic cancer, colorectal cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, melanoma, bladder cancer, gastric cancer or esophageal cancer.

8. Use of an antibody drug conjugate as shown in formula I or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 5, or a pharmaceutical composition as described in claim 6 in the preparation of a medicament for preventing or treating a HER2-related or mediated disease; the disease may be cancer, preferably breast cancer, lung cancer, kidney cancer, liver cancer, ovarian cancer, urethral cancer, prostate cancer, glioblastoma multiforme, pancreatic cancer, colorectal cancer, gastrointestinal stromal tumor, cervical cancer, squamous cell carcinoma, peritoneal cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, melanoma, bladder cancer, gastric cancer or esophageal cancer.

9. A linker-drug conjugate as shown in formula III or a pharmaceutically acceptable salt thereof, in, Y is T, L, Q and X are as defined in any one of claims 1-5.

10. The linker-drug conjugate of formula III or a pharmaceutically acceptable salt thereof according to claim 9, characterized in that: The linker-drug conjugate shown in formula III is a structure shown in formula IIIa, Among them, R 1 , R 2 , Q, X and Y are defined as in any one of claims 1-5; Preferably, the linker-drug conjugate as shown in Formula III is any of the following structures:

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