Homocamptothecin antibody coupling medicine as well as preparation method and application thereof

Abstract

The invention discloses a homocamptothecin antibody conjugate drug as well as a preparation method and application thereof, and particularly discloses an antibody drug conjugate shown as a formula I or pharmaceutically acceptable salt thereof. The antibody drug conjugate has good cytotoxic activity and in-vivo anti-tumor activity, and can significantly inhibit the growth of tumors. # imgabs0 #

Description

[0001] This application claims the priority of Chinese Patent Application No. 2023116270832 with an application date of November 30, 2023. This application incorporates the entire text of the above-mentioned Chinese patent application by reference. Technical Field

[0002] The present invention relates to the field of biomedicine, and particularly to a high camptothecin antibody-drug conjugate, its preparation method and application. Background Art

[0003] Camptothecin (CPT) is a pentacyclic alkaloid isolated from the bark and stems of Camptotheca acuminata, which is native to China. Camptothecin can inhibit topoisomerase I, leading to cell death. Due to its cytotoxic mechanism and broad-spectrum antitumor activity, efforts have been made to develop clinical analogs of camptothecin.

[0004] Antibody-drug conjugates (ADCs) are complexes that conjugate cytotoxic small molecule drugs to monoclonal antibodies through a rationally constructed linker, which can selectively deliver effective cytotoxic drugs into tumors, thereby increasing the targeting of drugs, improving efficacy, and reducing side effects. Recently, camptothecin-like 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 made modifications to the DXD compound. However, compared with traditional camptothecin, high camptothecin (hCPT) compounds with a β-hydroxy lactone ring structure retain high antitumor activity (Bailly, C. Crit. Rev. Oncol. Hematol. 2003, 45, 91), and the Topo I inhibitory activity of Diflomotecan is stronger than that of camptothecin (Kroep, J. R.; Gelderblom, H. Expert.Opin. Investig. Drugs 2009, 18, 69). However, due to the lack of suitable conjugation sites, their application in ADCs has not been reported yet. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defect that high camptothecin derivatives cannot be conjugated in the prior art and develop new high camptothecin antibody-drug conjugates. The antibody-drug conjugates provided by the present invention have good cytotoxic activity and can inhibit the proliferation of tumor cells. The antibody-drug conjugates provided by the present invention also have good in vivo antitumor activity and can significantly inhibit tumor growth.

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

[0007]

[0008] wherein T is or a stereoisomer thereof;

[0009] R 1 and R 2 are independently C 1-6 alkyl, C 1-6 alkoxy or halogen;

[0010] Or R 1 and R 2 together with the adjacent carbon atoms form or ," " represents the connection position of the fused ring;

[0011] L is where the a-end is connected to T and the b-end is connected to Q;

[0012] Q is a single amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;

[0013] X is 、 、 、 、 、 、 or where the 2-end is connected to Z and the 1-end is connected to ;

[0014] R a and R b are independently H, D, halogen, C 1-6 alkyl, halo-C 1-6 alkyl, deuterated C 1-6 alkyl, C 1-6 alkoxy, hydroxy, amino, cyano, nitro, 3- to 10-membered cycloalkyl or 3- to 10-membered heterocyclic group;

[0015] Or, R a and R b together with the carbon atom to which they are attached form a 3- to 10-membered cycloalkyl or 3- to 10-membered heterocyclic group;

[0016] R c and R d are independently H, C 1-6 alkyl, halogen, halo-C 1-6 alkyl, deuterated C 1-6alkyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclic group, 6- to 14-membered aryl or 5- to 10-membered heteroaryl;

[0017] Alternatively, R c and R d together with the carbon atom to which they are attached form a 3- to 10-membered cycloalkyl or 3- to 10-membered heterocyclic group;

[0018] m1, m2, m3, m4, m5, m6 and m7 are independently integers from 0 to 20;

[0019] n1, n2 and n3 are independently integers from 0 to 10;

[0020] q is an integer from 0 to 6;

[0021] r is an integer from 4 to 20;

[0022] Z is or , where the c-terminus is connected to X and the d-terminus is connected to G L connected;

[0023] p is 1 - 8;

[0024] G L is an antibody;

[0025] In the heteroatoms in the 3- to 10-membered heterocyclic group and 5- to 10-membered heteroaryl are independently one or more of N, O or S, and the number of heteroatoms is independently 1, 2, 3 or 4.

[0026] In a certain embodiment of the present invention, in the antibody-drug conjugate of formula I or a pharmaceutically acceptable salt thereof, some groups have the following definitions, and the definitions of the groups not mentioned are as described in any embodiment of the present invention (the content of this paragraph will be hereinafter referred to as "in a certain embodiment of the present invention"), R 1 and R 2 are independently C 1-6 alkyl, the C 1-6 alkyl is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, such as methyl.

[0027] In a certain embodiment of the present invention, R 1 and R 2 are independently C 1-6 alkoxy, the C 1-6 alkoxy is methoxy, -OCH 2 CH 3 , -OCH(CH 3 ) 2 or tert-butoxy, such as methoxy;

[0028] In one embodiment of the present invention, R 1 and R 2 When independently being halogen, the halogen is fluorine, chlorine, bromine or iodine, such as fluorine or chlorine.

[0029] In one embodiment of the present invention, the antibody-drug conjugate shown in Formula I has the structure shown in Formula Ia:

[0030]

[0031] Wherein, R 1 , R 2 , Q, X, Z, G L and p are as defined above.

[0032] In one embodiment of the present invention, R 1 and R 2 are independently C 1-6 alkyl or halogen, such as methyl or fluorine.

[0033] In one embodiment of the present invention, the single amino acid residue is , , , , , , , or , wherein the e end is connected to -NH-, and the f end is connected to -C(=O)-.

[0034] In one 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 .

[0035] In one 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 .

[0036] In one embodiment of the present invention, the tetrapeptide residue is C=O -Gly-Phe-(Gly) 2 - NH or C=O -(Gly) 2 -Phe-Gly- NH .

[0037] In the dipeptide residue, tripeptide residue or tetrapeptide residue, C=O indicates that the left side of the dipeptide residue, tripeptide residue or tetrapeptide residue is connected to the amino group in the antibody-drug conjugate shown in Formula I through its own carbonyl group, and NH in the dipeptide residue, tripeptide residue or tetrapeptide residue indicates that the right side of the dipeptide residue, tripeptide residue or tetrapeptide residue is connected to the carbonyl group in the antibody-drug conjugate shown in Formula I through its own amino group.

[0038] In one embodiment of the present invention, Q is a dipeptide residue.

[0039] In one embodiment of the present invention, X is or , where the 2-end is connected to Z, and the 1-end is connected to is connected, and the definitions of m2, n2, r and q are as described above.

[0040] In one embodiment of the present invention, m2 is an integer from 6 to 14, such as 8 or 12.

[0041] In one embodiment of the present invention, n2 is an integer from 1 to 6, such as 2 or 3.

[0042] In one embodiment of the present invention, q is 4.

[0043] In one embodiment of the present invention, r is 16.

[0044] In one embodiment of the present invention, G L is a HER2 antibody, such as Trastuzumab.

[0045] In one embodiment of the present invention, p can be an integer or a decimal, preferably 7 - 8, such as 7.6, 7.7, 7.8 or 8.0.

[0046] In one embodiment of the present invention, the d - end of Z and the L G are connected by a thioether bond. The G L can be obtained by reducing the disulfide bond of an antibody containing a disulfide bond (such as Trastuzumab) to a mercapto group under the action of a reducing agent. Those skilled in the art can understand that Z is connected to the mercapto group contained in the antibody itself after opening the disulfide bond (for example, reducing the disulfide bond of the antibody itself by a reducing agent can open the disulfide bond to generate - SH). For example, in the antibody - drug conjugate HER2 - ADC1, the - S - is not an externally connected sulfur atom, but the mercapto group contained in the HER2 antibody itself after opening the disulfide bond is connected to to form - S -.

[0047] In one embodiment of the present invention, the pharmaceutically acceptable salt can be trifluoroacetate, hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, acetate, oxalate, maleate, tartrate, citrate, succinate or malonate, such as trifluoroacetate.

[0048] In one embodiment of the present invention, T is or its stereoisomer;

[0049] R 1 and R 2 are independently C 1-6 alkyl or halogen;

[0050] L is , where the a - end is connected to T and the b - end is connected to Q;

[0051] Q is or , where the e - end is connected to - NH - and the f - end is connected to - C(=O)-;

[0052] X is or , where the 2 - end is connected to Z and the 1 - end is connected to ; m2 is an integer from 6 to 14; n2 is an integer from 1 to 6; q is 4; r is 16;

[0053] Z is or , where the c-terminus is connected to X and the d-terminus is connected to G L connected;

[0054] p is from 1 to 8;

[0055] G L is a HER2 antibody.

[0056] In one embodiment of the present invention, T is , , , , or .

[0057] In one embodiment of the present invention, T is .

[0058] In one embodiment of the present invention, Q is or , where the e-terminus is connected to -NH- and the f-terminus is connected to -C(=O)-.

[0059] In one embodiment of the present invention, X is , , or , where the 2-terminus is connected to Z and the 1-terminus is connected to connected.

[0060] In one embodiment of the present invention, the antibody-drug conjugate represented by Formula I is any of the following structures:

[0061] ,

[0062] ,

[0063] ,

[0064] ,

[0065] ,

[0066] or

[0067] , where G L is Trastuzumab.

[0068] The present invention also provides a pharmaceutical composition comprising the antibody-drug conjugate represented by Formula I as described above or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical excipient.

[0069] The present invention also provides the use of an antibody-drug conjugate as shown in Formula I or a pharmaceutically acceptable salt thereof as described above in the preparation of a drug 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.

[0070] The present invention also provides the use of an antibody-drug conjugate as shown in Formula I or a pharmaceutically acceptable salt thereof as described above in the preparation of a drug for preventing or treating HER2-related and mediated diseases. The disease can 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.

[0071] The present invention also provides a linker-drug conjugate as shown in Formula III or a pharmaceutically acceptable salt thereof,

[0072]

[0073] wherein Y is or , and the definitions of T, L, Q and X are as described above.

[0074] In one embodiment of the present invention, the linker-drug conjugate as shown in Formula III is a structure as shown in IIIa,

[0075]

[0076] wherein R 1 , R 2 , Q, X and Y are as defined above.

[0077] In one embodiment of the present invention, the linker-drug conjugate as shown in Formula III is any of the following structures:

[0078]

[0079] Ⅲ-1

[0080]

[0081] Ⅲ-2

[0082]

[0083] Ⅲ-3

[0084]

[0085] Ⅲ-4

[0086]

[0087] Ⅲ-5

[0088]

[0089] Ⅲ-6

[0090]

[0091] Ⅲ-7

[0092] Terminology Definition

[0093] The stereochemical definitions and rules used in the present invention generally follow S. P. 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.

[0094] Depending on the choice of starting materials and methods, the compounds of the present invention may exist in the form of one or a mixture of possible isomers, such as racemates and mixtures of diastereoisomers (depending on the number of asymmetric carbon atoms). The optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.

[0095] Any mixture of stereoisomers obtained can be separated into pure or substantially pure geometric isomers, enantiomers, diastereoisomers, for example, by chromatography and / or fractional crystallization, depending on the differences in the physicochemical properties of the components.

[0096] In this specification, groups and their substituents can be selected by those skilled in the art to provide stable structural moieties and compounds. When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes the chemically equivalent substituent obtained when the structural formula is written from right to left.

[0097] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0098] The term "alkyl" refers to a straight-chain or branched-chain alkyl group having a specified number of carbon atoms (e.g., C 1 ~C 6 )). 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, n-octyl, and the like.

[0099] The term "aryl" refers to a cyclic group consisting only of carbon atoms having a specified number of carbon atoms (e.g., C 6 ~C 10 ), which is monocyclic or polycyclic, and at least one ring is aromatic (complies with Hückel's rule). The aryl group is connected to other fragments in the molecule through an aromatic ring or a non-aromatic ring. Aryl groups include, but are not limited to, phenyl, naphthyl, and the like.

[0100] The "-" at the end of a group means that the group is connected to other fragments in the molecule through this site. For example, CH 3 -C(=O)- refers to an acetyl group.

[0101] In this application, as a group or part of other groups, the term "alkylene" represents a saturated divalent hydrocarbon group obtained by removing two hydrogen atoms from a saturated straight-chain or branched-chain hydrocarbon group; that is, one hydrogen in the alkyl group is substituted, and the alkyl group is defined as above. Examples of alkylene groups include methylene (-CH 2 -), ethylene {including -CH 2 CH 2 - or -CH(CH 3 )-}, isopropylidene {including -CH(CH 3 )CH 2 - or -C(CH 3 ) 2 -}, and so on.

[0102] In this application, as a group or part of other groups, the term "alkoxy" refers to -O-alkyl, where the alkyl group is defined as above.

[0103] The term "cycloalkyl" refers to a cyclic alkyl group having a specified number of carbon atoms (e.g., C 3 ~C 6)(which is) a saturated monocyclic group consisting only of carbon atoms. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0104] The term "heterocyclic group" refers to a cyclic group having a specified number of ring atoms (e.g., 5 - 10 membered), a specified number of heteroatoms (e.g., 1, 2, 3, or 4), and a specified type of heteroatoms (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, pyrrolidinyl, tetrahydrofuryl, morpholinyl, piperidinyl, etc.

[0105] The term "aryl" refers to a cyclic group having a specified number of carbon atoms (e.g., C 6 ~C 10 ) consisting only of carbon atoms, which is monocyclic or polycyclic, and at least one ring has aromaticity (complies with Hückel's rule). An aryl group is connected to other fragments in the molecule through an aromatic ring or a non - aromatic ring. Aryl groups include, but are not limited to, phenyl, naphthyl, etc.

[0106] The term "heteroaryl" refers to a cyclic group having a specified number of ring atoms (e.g., 5 - 10 membered), a specified number of heteroatoms (e.g., 1, 2, or 3), and a specified type of heteroatoms (one or more of N, O, and S), which is monocyclic or polycyclic, and at least one ring has aromaticity (complies with Hückel's rule). A heteroaryl group is connected to other fragments in the molecule through an aromatic ring or a non - aromatic ring. Heteroaryl groups include, but are not limited to, furyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, pyrimidinyl, indolyl, etc.

[0107] 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 in patients) acid or base. When a 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, etc. When a 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, hydrochloride salts, sulfate salts, mesylate salts, etc.

[0108] The term "pharmaceutical excipient" refers to excipients and additives used in the production of drugs and the formulation of prescriptions, which are all substances contained in a pharmaceutical preparation except for the active ingredient.

[0109] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0110] The reagents and raw materials used in the present invention are all commercially available.

[0111] The positive and progressive effects of the present invention are as follows: 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

[0112] Figure 1 It is for the anti-tumor efficacy detection of HER2-ADCs on the animal model of subcutaneous transplantation of NCI-N87 cells in NOG mice.

[0113] Figure 2 It is for the body weight change of HER2-ADCs on the animal model of subcutaneous transplantation of NCI-N87 cells in NOG mice. Detailed Description of the Invention

[0114] The present invention will be further illustrated below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions noted in the following examples are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0115] Example 1: Preparation of Compounds II-1 to II-6

[0116]

[0117]

[0118] Step 1: Synthesis of Compound 2

[0119] 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. In the ice-water bath, Compound 1 (12.5 g, 99.9 mmol) was added to the reaction system, and the reaction was maintained in the ice-water bath for 10 min. Then, chloroacetonitrile (13.5 mL, 213.3 mmol) and anhydrous aluminum chloride (17.5 g, 131.2 mmol) were added in sequence, and the reaction was maintained in the ice-water bath for 10 min. Then, the system was transferred to room temperature and reacted for 10 min, and then the system was heated to reflux and reacted for 40 h. After the reaction was completed, the system was cooled to room temperature, 200 mL of ice water was slowly added, and then 200 mL of 5% hydrochloric acid aqueous solution was added and stirred for 30 min. It was extracted with dichloromethane (300 mL × 3), and the organic phase was washed successively with water and saturated sodium chloride water, and anhydrous MgSO 4Dry. After rotary evaporation of the solvent, the crude product was obtained. It 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 (400 MHz, DMSO-d 6 ) δ 7.68 (d, J = 8.7 Hz, 1H), 7.31 (s, 2H), 6.53 (d, J = 12.5 Hz, 1H), 4.98 (s, 2H), 2.10 (s, 3H). MS (ESI) m / z = 202.5 (M + H + ).

[0120] Step 2: Synthesis of compound 3

[0121] Compound 2 (2.7 g, 13.4 mmol) was dissolved in 20 mL of anhydrous dichloromethane. DCC (5.5 g, 26.8 mmol) and DMAP (160 mg, 1.3 mmol) were added successively. Under an ice-water bath, monoethyl malonate (2.4 mL, 20.1 mmol) was slowly added dropwise to the system. After the addition was complete, the reaction was carried out at room temperature for 12 hours. After the reaction was completed, filtration was carried out. The filtrate was rotary evaporated to obtain 4.0 g of the crude product. The crude product was dissolved in 20 mL of ethanol. Sodium ethoxide (1.4 g, 20.6 mmol) was added to the system in batches, and the reaction was continued at room temperature for 2 hours. After the reaction was completed, the solid precipitated in the system was filtered out. The solid was slurried with dichloromethane and ether to obtain 2.5 g of compound 3 with a yield of 63%. 1 H NMR (400 MHz, Chloroform-d) δ 7.90 (d, J = 7.3 Hz, 1H), 7.62 (d, J = 9.9 Hz, 1H), 4.86 (s, 2H), 4.53 (q, J = 6.2 Hz, 2H), 2.51 (s, 3H), 1.46 (t, J = 6.7 Hz, 3H). MS (ESI) m / z = 298.6 (M + H + ).

[0122] Step 3: Synthesis of compound 4

[0123] Compound 3 (2.0 g, 6.7 mmol) was dispersed in 30 mL of anhydrous acetonitrile. Phosphorus oxychloride (2.8 g, 10.1 mol) was added to the system. After the addition was complete, the temperature was raised to reflux for 12 h. After the reaction was completed, the system was poured into 100 mL of ice water and extracted with ethyl acetate (100 mL × 3). The organic phase was washed successively with water and saturated sodium chloride aqueous solution, and anhydrous MgSO 4Dry. After rotary evaporation of the solvent, the crude product was obtained. It 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 (400 MHz, Chloroform-d) δ 7.89 (d, J = 7.6 Hz, 1H), 7.68 -7.61 (m, 1H), 4.74 (d, J = 6.2 Hz, 2H), 4.54 (q, J = 6.7 Hz, 2H), 2.51 (s,3H), 1.48 (t, J = 7.0 Hz, 3H). MS(ESI)m / z = 406.4 (M + H + )。

[0124] Step 4: Synthesis of compound 5

[0125] Under an ice-water bath, compound 4 (2.0 g, 4.9 mmol) was dissolved in 40 mL of anhydrous dichloromethane. A 1M solution of diisobutylaluminum hydride in n-hexane (25 mL, 24.5 mmol) was added dropwise to the reaction system. After the addition, the reaction was maintained under the ice-water bath for 2 hours. After the reaction was completed, the reaction solution was slowly added to 40 mL of saturated sodium tartrate aqueous solution. After stirring for 4 hours, it was extracted with dichloromethane (20 mL×3). The organic phase was washed with saturated sodium chloride aqueous solution and dried over anhydrous MgSO 4 Dry. After rotary evaporation of the solvent, the crude product was obtained. It 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 (400 MHz, Chloroform-d) δ 7.85 (d, J = 7.8 Hz, 1H), 7.61 (d, J =10.1 Hz, 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 + )。

[0126] Step 5: Synthesis of compound 6

[0127] Compound 5 (1.4 g, 3.9 mmol) was dissolved in 15 mL of dimethyl sulfoxide. Sodium azide (300 mg, 4.7 mmol) was added, and the reaction was stirred at room temperature for 12 hours. After the reaction was completed, 150 mL of water was added, and a solid precipitated. It was directly filtered to obtain 1.2 g of compound 6, which was not further separated and directly used for the next reaction. MS(ESI)m / z = 326.2 (M + H+ )。

[0128] Step 6: Synthesis of Compound 7

[0129] Dissolve Compound 6 (1.2 g, 3.7 mmol) in a mixed solvent of tetrahydrofuran and water (3:1, 40 mL). Add a 1.0 M tetrahydrofuran solution of trimethylphosphine (6.5 mL, 5.6 mmol) to the system under an ice-water bath. After the addition, maintain the reaction at the ice-water bath temperature for 3 hours. After the reaction is completed, rotary evaporate the reaction system to obtain the crude product, and purify it by column chromatography (dichloromethane:methanol = 30:1) to obtain 1.0 g of Compound 7 with a yield of 90%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.23 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 10.6 Hz, 1H), 4.83 (s, 2H), 4.25 (s, 2H), 2.45(s, 3H). MS (ESI) m / z = 300.2 (M + H + )。

[0130] Step 7: Synthesis of Compound 8

[0131] Under an ice-water bath, dissolve Compound 7 (1.0 g, 3.3 mmol) in 20 mL of anhydrous N,N-dimethylformamide, add Boc anhydride (1.0 g, 4.6 mmol), and gradually raise the temperature of the system to room temperature and react for 4 hours. After the reaction is completed, add 200 mL of water and extract with ethyl acetate (50 mL×3). The organic phase is washed successively with water and saturated sodium chloride solution, and dried over anhydrous MgSO 4 Dry. After rotary evaporation of the solvent, the crude product is obtained, and purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain 1.0 g of Compound 8 with a yield of 80%. 1 H NMR (400 MHz, Chloroform-d) δ 7.94 (d, J = 7.8 Hz, 1H), 7.59 (d, J =10.2 Hz, 1H), 5.25 (s, 1H), 5.09 (d, J = 4.0 Hz, 2H), 4.87 (d, J = 6.1 Hz,2H), 3.39 (s, 1H), 2.47 (s, 3H), 1.41 (s, 9H). MS (ESI) m / z = 400.3 (M + H + )。

[0132] Step 8: Synthesis of Compound 10

[0133] Compound 9 was prepared according to the reference (Bioorganic & Medicinal Chemistry, 2015, 23(9), 1950 - 1962).

[0134] 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 transferred to room temperature and reacted for 6 hours. After the reaction was completed, the solvent was evaporated to dryness to obtain the 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%. 1 H NMR (400 MHz, Chloroform - d) δ 8.15 (d, J = 8.2 Hz, 1H),7.58 (d, J = 10.2 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 6.50 (d, J = 7.7 Hz,1H), 5.88 (s, 1H), 5.61 - 5.33 (m, 3H), 5.19 (d, J = 15.2 Hz, 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, 7.4 Hz, 1H), 1.73 (dt, J = 14.9, 7.5 Hz, 1H), 1.45 (s, 9H),0.85 (t, J = 7.5 Hz, 3H). MS (ESI) m / z = 635.3 (M + H + )

[0135] Step 9: Synthesis of compound 11

[0136] Under nitrogen protection, compound 10 (250 mg, 0.39 mmol), palladium acetate (90 mg, 0.39 mmol), potassium acetate (155 mg, 1.58 mmol), tris(ortho-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 temperature was raised to reflux for 12 hours. After the reaction was completed, the solvent was evaporated to dryness to obtain the crude product, which was separated and purified by column chromatography (dichloromethane:methanol = 80:1) to obtain 150 mg of compound 11 with a yield of 69%. MS (ESI) m / z = 554.4 (M + H + ).

[0137] Step 10: Synthesis of compound II-1

[0138] 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 was completed, the solvent was evaporated to dryness to obtain the crude product, which was slurried with ether to obtain 110 mg of compound II-1 with a yield of 90%. 1 HNMR (400 MHz, DMSO-d 6 ) δ 8.54 - 8.35 (m, 4H), 7.97 (d, J = 10.5 Hz, 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), 2.54 (s, 3H), 2.29 (dd, J = 14.3, 7.5 Hz, 1H), 1.81 (dd, J = 14.3,7.4 Hz, 1H), 0.74 (t, J = 7.1 Hz, 3H). MS (ESI) m / z = 454.2 (M + H + ).

[0139] Using the same method as above, compound II-2 can be obtained by using 3-chloro-4-methylaniline instead of 3-fluoro-4-methylaniline.

[0140]

[0141] 1 H NMR (400 MHz, DMSO-d 6) δ 8.52 - 8.36 (m, 4H), 7.97 (d, J = 10.5 Hz,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), 2.54 (s, 3H), 2.29 - 1.81 (m, 2H), 0.74 (t, J = 7.1 Hz,3H). MS (ESI) m / z = 470.2 (M + H + ).

[0142] Using the same method as above, compound II-3 can be obtained by using 3,4-difluoroaniline instead of 3-fluoro-4-methylaniline.

[0143]

[0144] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.54 - 8.35 (m, 4H), 7.97 (d, J = 10.5 Hz,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, 7.5 Hz, 1H), 1.81 (dd, J = 14.3, 7.4Hz, 1H), 0.74 (t, J = 7.1 Hz, 3H). MS (ESI) m / z = 458.2 (M + H + ).

[0145] Using the same method as above, compound II-4 can be obtained by using 3-fluoro-4-methoxyaniline instead of 3-fluoro-4-methylaniline.

[0146]

[0147] 1 H NMR (400 MHz, DMSO-d 6) δ 8.54 - 8.35 (m, 4H), 7.97 (d, J = 10.5 Hz,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 = 7.1 Hz,3H). MS(ESI)m / z = 470.2 (M + H + )。

[0148] Using the same method as above, compound II-5 can be obtained by replacing 3-fluoro-4-methylaniline with 3,4-methylenedioxyaniline.

[0149]

[0150] 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.32 (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, 7.4 Hz, 1H), 1.78 (dq, J =14.9, 7.5 Hz, 1H), 0.70 (t, J = 7.3 Hz, 3H). MS(ESI)m / z = 466.2 (M + H + )。

[0151] Using the same method as above, compound II-6 can be obtained by replacing 3-fluoro-4-methylaniline with 6-amino-1,4-benzodioxane.

[0152]

[0153] 1 H NMR (400 MHz, DMSO-d 6) δ 8.32 (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 = 7.3 Hz, 3H). MS (ESI) m / z = 480.2 (M + H + )。

[0154] Example 2: Preparation of Compounds Ⅲ-1 to Ⅲ-4

[0155] 1. Synthesis of Compound Ⅲ-1

[0156]

[0157] Step 1: Synthesis of Compound 12

[0158] Under argon protection, dissolve Compound Ⅱ-1 (55 mg, 0.1 mmol) in N,N-dimethylformamide (5 mL). At room temperature, sequentially add 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), and react at room temperature for 2 hours. After detecting the complete reaction of the raw materials by TLC, remove the solvent under reduced pressure with an oil pump and separate by column chromatography to obtain 97 mg of a pale yellow solid with a yield of 90%. 1 HNMR (400 MHz, DMSO-d 6) δ 10.05 (s, 1H), 8.30 (d, J = 8.3 Hz, 1H), 8.17 (t, J= 5.6 Hz, 1H), 8.10 (d, J = 7.3 Hz, 1H), 7.88 (d, J = 8.5 Hz, 3H), 7.73 (t, J= 8.1 Hz, 2H), 7.61 - 7.53 (m, 2H), 7.47 - 7.20 (m, 8H), 5.97 (t, J = 5.5 Hz,1H), 5.90 (s, 1H), 5.71 - 5.27 (m, 6H), 4.96 (d, J = 4.0 Hz, 3H), 4.77 (d, J= 5.7 Hz, 2H), 4.49 - 4.37 (m, 1H), 4.35 - 4.15 (m, 3H), 3.93 (t, J = 7.9 Hz,1H), 3.47 (s, 3H), 3.08 - 2.89 (m, 2H), 2.48 (s, 3H), 2.36 - 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 = 7.4 Hz, 3H). MS (ESI) m / z = 1081.4 (M +H + )。

[0159] Step 2: Synthesis of Compound 13

[0160] Under argon protection, dissolve Compound 12 (97 mg, 0.09 mmol) in N,N-dimethylformamide (3 mL), add diethylamine (0.5 mL) at room temperature, and react at room temperature for 2 hours. After detecting the complete reaction of the raw materials by LC-MS, remove the solvent under reduced pressure with an oil pump, add methyl tert-butyl ether (10 mL), and a solid precipitates. Filter and dry to obtain 65 mg of a pale yellow solid with a yield of 65%. 1 H NMR(400 MHz, DMSO-d 6) δ 10.00 (s, 1H), 8.30 (d, J = 8.2 Hz, 1H), 8.17 (t, J =5.7Hz, 1H), 8.09 (d, J = 7.5 Hz, 1H), 7.99 (t, J = 5.3 Hz, 1H), 7.93 - 7.79(m, 2H), 7.57 (d, J = 8.3 Hz, 2H), 7.34 (s, 1H), 7.27 (d, J = 8.4 Hz, 2H),5.98 (t, J = 5.5 Hz, 1H), 5.90 (s, 1H), 5.64 - 5.34 (m, 6H), 4.95 (d, J = 4.6Hz, 3H), 4.77 (d, J = 5.8 Hz, 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 = 7.4 Hz, 3H). MS (ESI) m / z = 859.4 (M + H + )。

[0161] Step 3: Synthesis of Compound Ⅲ-1

[0162] Under argon protection, dissolve Compound 13 (65 mg, 0.075 mmol) in N,N-dimethylformamide (3 mL), add Mal-amido-PEG8-NHS ester (CAS No.: 756525-93-6, 58 mg, 0.09 mmol) at room temperature, and react overnight at room temperature. After detecting the complete reaction of the raw materials by LC-MS, remove the solvent under reduced pressure with an oil pump and separate by preparative liquid phase to obtain 70 mg of a pale yellow oil, with a yield of 70%. 1 H NMR (400 MHz, DMSO-d 6) δ 9.97 (s, 1H), 8.30 (d, J = 8.1 Hz, 1H),8.17 (t, J = 5.8 Hz, 1H), 8.09 (d, J = 7.5 Hz, 1H), 7.99 (t, J = 5.3 Hz, 1H),7.93 - 7.79 (m, 2H), 7.57 (d, J = 8.3 Hz, 2H), 7.34 (s, 1H), 7.27 (d, J = 8.4Hz, 2H), 6.99 (s, 2H), 5.98 (t, J = 5.5 Hz, 1H), 5.90 (s, 1H), 5.64 - 5.34(m, 6H), 4.95 (d, J = 4.6 Hz, 3H), 4.77 (d, J = 5.8 Hz, 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 = 7.4 Hz, 3H). MS (ESI) m / z = 1433.7 (M + H + ).

[0163] 2. Synthesis of Compound Ⅲ-2

[0164]

[0165] Step 1: Synthesis of Compound 14

[0166] Under argon protection, compound II-1 (55 mg, 0.1 mmol) was dissolved in N,N-dimethylformamide (5 mL). At room temperature, 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 successively. The reaction was carried out at room temperature for 2 hours. After TLC detection showed that the raw materials had completely reacted, the solvent was removed under reduced pressure with an oil pump and the product was separated by a chromatographic column to obtain 90 mg of a pale yellow solid with a yield of 90%. 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.05 (s, 1H), 8.30 (d, J = 8.3 Hz, 1H), 8.17 (t,J = 5.6 Hz, 1H), 8.10 (d, J = 7.3 Hz, 1H), 7.88 (d, J = 8.5 Hz, 3H), 7.73 (t,J = 8.1 Hz, 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 = 7.9 Hz, 1H), 3.47 (s, 3H), 2.48 (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 = 7.4 Hz, 3H). MS (ESI) m / z = 995.4 (M + H + )。

[0167] Step 2: Synthesis of Compound 15

[0168] Under argon protection, 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 the reaction was carried out at room temperature for 2 hours. After LC-MS detection showed that the raw materials had completely reacted, the solvent was removed under reduced pressure with an oil pump and methyl tert-butyl ether (10 mL) was added. A solid precipitated out. After filtration and drying, 49 mg of a pale yellow solid was obtained with a yield of 70%. 1 H NMR(400 MHz, DMSO-d6 ) δ 10.00 (s, 1H), 8.30 (d, J = 8.2 Hz, 1H), 8.17 (t, J =5.7Hz, 1H), 8.09 (d, J = 7.5 Hz, 1H), 7.99 (t, J = 5.3 Hz, 1H), 7.93 - 7.79(m, 2H), 7.57 (d, J = 8.3 Hz, 2H), 7.34 (s, 1H), 7.27 (d, J = 8.4 Hz, 2H),5.98 (t, J = 5.5 Hz, 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 + ).

[0169] Step 3: Synthesis of Compound Ⅲ-2

[0170] Under argon protection, dissolve Compound 15 (58 mg, 0.075 mmol) in N,N-dimethylformamide (3 mL), add Mal-amido-PEG8-NHS ester (58 mg, 0.09 mmol) at room temperature, and react overnight at room temperature. After detecting the complete reaction of the raw materials by LC-MS, remove the solvent under reduced pressure with an oil pump and separate by preparative liquid phase to obtain 70 mg of a pale yellow oil, with a yield of 70%. 1 H NMR(400 MHz, DMSO-d 6) δ 9.97 (s, 1H), 8.30 (d, J = 8.1 Hz, 1H), 8.17 (t, J = 5.8Hz, 1H), 8.09 (d, J = 7.5 Hz, 1H), 7.99 (t, J = 5.3 Hz, 1H), 7.93 - 7.79 (m,2H), 7.57 (d, J = 8.3 Hz, 2H), 7.34 (s, 1H), 7.27 (d, J = 8.4 Hz, 2H), 6.99(s, 2H), 5.98 (t, J = 5.5 Hz, 1H), 5.90 (s, 1H), 5.64 - 5.34 (m, 6H), 4.95(d, J = 4.6 Hz, 3H), 4.77 (d, J = 5.8 Hz, 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),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 = 7.4 Hz, 3H). MS (ESI) m / z = 1347.6 (M + H + )。

[0171] 3. Synthesis of Compound Ⅲ-3

[0172] Referring to the synthesis method of Compound III-1, using Mal-amido-PEG12-NHS ester (CAS No.: 326003-46-7) instead of Mal-amido-PEG8-NHS ester, Compound Ⅲ-3 can be obtained. MS (ESI) m / z = 1609.8 (M + H + )。

[0173]

[0174] 4. Synthesis of Compound Ⅲ-4

[0175] Referring to the synthesis method of reference compound III-2, compound III-4 can be obtained by using Mal-amido-PEG12-NHS ester instead of Mal-amido-PEG8-NHS ester. MS (ESI) m / z = 1523.7 (M + H + ).

[0176]

[0177] Example 3: Preparation of III-5 and III-6

[0178] 1. Synthesis of compound III-5

[0179]

[0180] Step 1: Synthesis of compound 17

[0181] Under argon protection, Fmoc-Lys-OH (370 mg, 1.0 mmol) was dissolved in anhydrous dichloromethane (20 mL), and m-PEG17-NHS-ester (910 mg, 1.0 mmol) and N,N-diisopropylethylamine (650 mg, 1.0 mmol) were added at room temperature, followed by reaction at room temperature overnight. After LC-MS detection showed that the raw materials had completely reacted, the solvent was removed under reduced pressure with an oil pump and separated by a chromatographic column to obtain 1.0 g of a colorless liquid with a yield of 86%. 1 H NMR (400 MHz, Chloroform-d) δ 7.73–7.68 (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 + ).

[0182] Step 2: Synthesis of compound 18

[0183] 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 reaction was carried out at room temperature for 2 hours. After the raw material was completely reacted as detected by LC-MS, the solvent was removed under reduced pressure with an oil pump, and the product was separated by preparative liquid chromatography to obtain 420 mg of a colorless oil, with a yield of 90%. MS (ESI) m / z = 937.6 (M + H + )。

[0184] Step 3: Synthesis of compound 19

[0185] Under argon protection, compound 18 (240 mg, 0.25 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL), and N-succinimidyl 6-(maleimidocaproate) (105 mg, 0.5 mmol) and N,N-diisopropylethylamine (65 mg, 0.5 mmol) were added at room temperature. The reaction was carried out overnight at room temperature. After the raw material was completely reacted as detected by LC-MS, the solvent was removed under reduced pressure with an oil pump, and the product was separated by preparative liquid chromatography to obtain 220 mg of a colorless oil, with a yield of 80%. 1 H NMR (400 MHz, Chloroform-d) δ 6.86–6.83(m, 1H), 6.71 (s, 2H), 6.54–6.52 (m, 1H), 4.56–4.51 (m, 1H), 3.75–3.53 (m,68H), 3.40 (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 + )。

[0186] Step 4: Synthesis of compound III-5

[0187] Under argon protection, compound 19 (110 mg, 0.1 mmol) was dissolved in N,N-dimethylformamide (5 mL). At room temperature, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19 mg, 0.1 mmol) and N-hydroxysuccinimide (12 mg, 0.1 mmol) were added, and the reaction was continued at room temperature for 1 hour. After adding compound 13 (44 mg, 0.05 mmol) at room temperature, the reaction was continued at room temperature overnight. After the raw materials were completely reacted as detected by LC-MS, the solvent was removed under reduced pressure with an oil pump, and the product was separated by preparative liquid chromatography to obtain 50 mg of a pale yellow solid with a yield of 50%. MS (ESI) m / z = 1971.1 (M + H + ).

[0188] 2. The synthesis of compound III-6 was carried out with reference to the synthesis method of compound III-5. In step 4, compound 15 was used instead of compound 13 to obtain compound III-6. MS (ESI) m / z = 1885.0 (M + H + ).

[0189]

[0190] Example 4: Preparation of compound III-7

[0191]

[0192] Step 1: Synthesis of compound 21

[0193] Under argon protection, 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (100 mg, 0.37 mmol) was dissolved in dichloromethane (10 mL). At room temperature, 1-hydroxybenzotriazole (60 mg, 0.45 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (86 mg, 0.45 mmol) were added successively, and the reaction was continued at room temperature for 1 hour. At room temperature, amino-dodecaethylene glycol-tert-butyl propionate (250 mg, 0.375 mmol) was added, and the reaction was continued at room temperature overnight. After the raw material 20 was completely reacted as detected by TLC, the solvent was removed under reduced pressure and the product was separated by a chromatographic column to obtain 300 mg of a colorless liquid with a yield of 87%. 11H NMR (400 MHz, Chloroform-d) δ 9.39 (s, 2H), 3.73 (t, J = 7.9 Hz, 2H), 3.67 (t, J = 7.3 Hz, 2H), 3.52 – 3.40 (m, 44H), 3.28 (t, J = 7.3 Hz, 2H), 2.95 (s, 3H), 2.53 (t, J = 7.9 Hz, 2H), 2.34 (t, J = 5.4 Hz, 2H), 2.18 (t, J = 7.8 Hz, 2H), 1.95 – 1.85 - 3 (m, 2H), 1.42 (s, 9H). MS (ESI) m / z = 924.4 (M + H + )。

[0194] Step 2: Synthesis of Compound 22

[0195] Under argon protection, dissolve Compound 21 (300 mg, 0.32 mmol) in dichloromethane (10 mL), add trifluoroacetic acid (1 mL) at room temperature, and then continue the reaction at room temperature for 4 hours. After TLC detection shows that the raw material has completely reacted, remove the solvent under reduced pressure to obtain 270 mg of a pale yellow liquid with a yield of 96%. Without purification, it is directly used in the next step of the reaction. MS (ESI) m / z = 868.4 (M + H + )。

[0196] Step 3: Synthesis of Compound Ⅲ-7

[0197] Under argon protection, dissolve Compound 22 (100 mg, 0.12 mmol) in N,N-dimethylformamide (5 mL). At room temperature, successively add N-hydroxysuccinimide (14 mg, 0.12 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23 mg, 0.12 mmol), and then continue the reaction at room temperature for 1 hour. Add Compound 13 (50 mg, 0.06 mmol) at room temperature, and then react at room temperature overnight. After LC-MS detection shows that the raw material 13 has completely reacted, remove the solvent under reduced pressure using an oil pump and purify it through a C18 reverse-phase column to obtain Compound Ⅲ-7, 55 mg of a pale yellow solid with a yield of 55%. 1 1H NMR (400 MHz, DMSO-d 6) δ 9.97(s, 1H), 9.39 (s, 2H), 8.30 (d, J = 8.1 Hz, 1H), 8.17 (t, J = 5.8 Hz, 1H),8.09 (d, J = 7.5 Hz, 1H), 7.99 (t, J = 5.3 Hz, 1H), 7.93 - 7.79 (m, 2H), 7.57(d, J = 8.3 Hz, 2H), 7.34 (s, 1H), 7.27 (d, J = 8.4 Hz, 2H), 5.98 (t, J = 5.5Hz, 1H), 5.90 (s, 1H), 5.64 - 5.34 (m, 6H), 4.95 (d, J = 4.6 Hz, 2H), 4.77(d, J = 5.8 Hz, 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 = 7.8 Hz, 2H), 2.04 - 1.90 (m, 3H),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 = 7.4 Hz, 3H). MS (ESI) m / z =1709.8 (M + H + ).

[0198] Example 5: Synthesis of Compounds HER2-ADC1 to HER2-ADC7

[0199]

[0200] At 37 °C, to the PBS buffer aqueous solution of antibody Trastuzumab (HER2 antibody, CAS: 180288-69-1, purchased from Hangzhou Haoyang Biotechnology Co., Ltd.) (0.05 M PBS buffer aqueous solution with pH = 6.5; 10.0 mg / mL, 1.0 mL, 67.6 nmol), an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 37.2 μL, 372 nmol) prepared was added. It was placed in a water bath oscillator and reacted with shaking at 37 °C for 3 hours, then the reaction was stopped. The reaction solution was cooled to 25 °C using a water bath. Compound III-1 (1.45 mg, 1014 nmol) was dissolved in 50 μL of DMSO and added to the above reaction solution. It was placed in a water bath oscillator and reacted with shaking at 25 °C for 3 hours, then the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (mobile phase: 0.05 M PBS buffer aqueous solution with pH 6.5, containing 0.001 M EDTA) to obtain the PBS buffer solution of conjugate HER2-ADC1 (1.1 mg / mL, 7.5 mL), which was stored at 4 °C. The purity was detected by SEC-HPLC: 96.68%, and the average drug loading calculated by RP-HPLC: p = 7.8.

[0201] Referring to the same synthesis method, III-1 was replaced with III-2, III-3, III-4, III-5, III-6, III-7, MC-GGFG-DXD (CAS No. 1599440-13-7, purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.) and conjugated with 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), HER2-GGFG-DXD (DS-8201, DAR = 7.9).

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208] Biological evaluation

[0209] Experimental Example 1. Determination of the inhibitory activity of compounds on the proliferation of tumor cells

[0210] The SRB method was used to test 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 the proliferation ability of tumor cells. Cells in the logarithmic growth phase were inoculated into 96-well culture plates at the corresponding concentrations, and each well was cultured overnight with 100 μL of complete medium. Different concentrations of the compounds were added, with three replicates for each concentration, and positive control wells without compound treatment and negative control wells without cells were set up. The cells were cultured at 37 °C and 5% CO2 for 72 h. After the drug treatment, the inhibitory effect of the compounds on cell proliferation was detected by the sulforhodamine B (SRB) protein staining method. The specific operation steps were as follows: Pour out the culture medium, fix the cells with 10% trichloroacetic acid, place them at 4 °C for 1 h, then wash them 5 times with distilled water, and dry them in an oven. Add 100 μL / well of an SRB solution (4 mg / ml) prepared with 1% glacial acetic acid, stain at room temperature for 15 min, remove the supernatant, wash 5 times with 1% glacial acetic acid, and dry in an oven. Finally, add 150 μL / well of Tris solution, shake well, and measure the OD value at 560 nm with a full-wavelength microplate reader SpectraMax190. The inhibition rate of the compounds on cell proliferation was calculated by the following formula:

[0211] Inhibition rate (%) = [1 - (OD of drug-treated wells - OD of negative control wells) / (OD of positive control wells - OD of negative control wells)] × 100%.

[0212] 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.

[0213] Table 1. Overview of cell lines used in this screening

[0214]

[0215] Table 2. Effects of compounds on the proliferation ability of tumor cells

[0216]

[0217] Experimental Example 2. Test methods for HER2-ADC1~ADC7 and HER2-GGFG-DXD on the proliferation ability of tumor cells:

[0218] Digest the NCI-N87 cells, resuspend the cells, count them, centrifuge to remove the supernatant, and adjust the cell density. Count the cells with a cell counter, aspirate 20 μL of the cell suspension, mix it with 20 μL of VisStain AOPI staining solution, take 20 μL of the liquid, and count the cells. Seed the cells into a 96-well cell culture plate, add 180 μL to each well, seed 1000 cells or 2000 cells per well. Gently tap to mix, and place the cell culture plate in a 37°C cell incubator for 16 hours. Dilute the compound with RPMI1640 medium / 10% FBS, add 20 μL to each well, with the maximum concentration being 100 nM, 3-fold dilution, 10 points. The vehicle control is DMSO, and add 20 μL of RPMI1640 medium / 10% FBS to the blank control. Gently tap to mix, and place the cell culture plate in a 37°C cell incubator for 7 days. After 7 days of culture, take the cell plate out of the incubator, aspirate the culture medium, add 20 μL of the culture medium, and then add 20 μL of CTG to each well. Oscillate on an oscillator for 5 min, let it stand at room temperature for 10 min, take 30 μL of the reaction solution into a Viewplate-96 white clear-bottom microplate, and perform data analysis on an EnVision microplate reader.

[0219] Calculation formula: Use the following formula to calculate the inhibition rate (IR) of the test antibody: IR (%) = (1 – (RLU antibody – RLU blank control) / (RLU vehicle control – RLU blank control)) * 100%. Calculate the inhibition rate of antibodies at different concentrations in Excel, and then use GraphPad Prism software to make an inhibition curve and calculate relevant parameters. The results are shown in Table 3.

[0220] Table 3. Effects of ADCs on the proliferative ability of NCl-N87 tumor cells

[0221]

[0222] Experimental Example 3. Antitumor efficacy test of HER2-ADCs on a xenograft NCI-N87 cell animal model in NOG mice:

[0223] In this experiment, the antitumor effect of HER2-ADCs was determined by subcutaneous inoculation of NCI-N87 cells into NOG mice.

[0224] NOG mice: Female NOG mice (6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were adaptively fed for 7 days after arrival and then the study began.

[0225] Cells: Human gastric cancer NCI-N87 cells (from ATCC, catalog number CRL-5822), were routinely passaged and cultured according to the instructions; the 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 an NCI-N87 tumor-bearing mouse model.

[0226] Drug administration:

[0227] On the 10th day after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with a tumor volume in the range of 130 mm 3 ~250 mm 3 were selected and evenly grouped according to tumor volume (5 mice in each group). Drugs were administered on the 11th day after inoculation, including 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.), positive control HER2-GGFG-DXd, HER2-ADC3, and HER2-ADC5. During drug administration, the tumor volume and body weight changes of each group of mice were monitored, and the monitoring frequency was 2 times / week for 3 consecutive weeks. Body weight and tumor volume were measured before each drug administration. On the 30th day after inoculation, the tumor volume inhibition rate (TGI%) was calculated. The calculation formula was as follows: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100; where Ti: the average tumor volume of the drug administration group, T0: the average tumor volume of the drug administration group on day D0, Vi: the average tumor volume of the isotype control group, V0: the average tumor volume of the isotype control group on day D0. Tumor volume measurement: The long diameter (a) and short diameter (b) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: TV = 1 / 2 × a × b 2 . Body weight was measured using an electronic balance.

[0228] The drug administration dose and method are shown in Table 4.

[0229] Table 4. Drug administration experimental design

[0230]

[0231] As shown in Table 5 and Figure 1 indicated, 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, and the tumor volume inhibition rates were 115.16% and 114.52%, respectively.

[0232] Table 5. ADC in vivo tumor growth inhibition ability on the 23rd day

[0233]

[0234] Note: The P value is compared with the negative control antibody

[0235] The above experiments showed that HER2-ADC3 and HER2-ADC5 significantly inhibited the growth of tumors in the animal model of subcutaneous transplantation of NCI-N87 cells in NOG mice, and the pharmacodynamic effect was also better than that of the control HER2-GGFG-DXD, and had no obvious 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 or , " ” indicates the connection position of the parallel ring; L is , where end a is connected to T and end b is connected to Q; Q is a single amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; X is , , , , , , or , where 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 or , where 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 , , , , , , , or , wherein the e-terminal is connected to -NH-, and the f-terminal 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 or , where 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 , , , , or ; (2) Q is or , wherein the e-terminal is connected to -NH-, and the f-terminal is connected to -C(=O)-; (3) X is , , or , where 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: 、 、 、 、 、 or , where 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 or , 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: 、 、 、 、 、 or 。

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