A cleavable linker compound, its preparation and use

Abstract

The application discloses a cleavable linker compound for a prodrug administration system and pharmaceutically acceptable salts, solvates, polymorphs or isomers thereof. The application also discloses a synthesis method of the compound, a preparation method in an enzyme prodrug system. The application also discloses applications of the compound in preparation of an antitumor drug and in preparation of an antibody conjugated drug. The application innovatively connects a trigger fragment in the compound to a novel linker through a hydroxyl group, connects an active drug to the novel linker through a hydroxyl group of the active drug itself, and connects a modification fragment to the novel linker through an amino group. The application has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical technology and prodrug design for pharmaceutical formulations, and relates to a cleavable linker compound, its preparation, and its application. Background Technology

[0002] Many low-molecular-weight drugs used as chemotherapy agents face limitations in disease treatment due to their physicochemical properties. For example, they may be metabolized too quickly in the body or fail to accumulate accurately in diseased tissues, both of which can reduce therapeutic efficacy. To address these issues, research into drug delivery systems has been actively pursued. Currently, drug delivery systems have seen significant advancements. For instance, semaglutide binds to albumin via its side chain, extending its half-life in vivo to one week, thus greatly reducing the frequency of drug administration. Drug delivery technologies are even more widely used in the anti-tumor field, with the development of small molecule-drug conjugates (SMDCs), peptide-drug conjugates (PDCs), and antibody-drug conjugates (ADCs).

[0003] Antibody-drug conjugates (ADCs) are the most representative category of targeted anticancer agents in drug delivery systems. They aim to effectively release antibody-bound drugs into cancer cells through the selective binding between antigens and antibodies. Currently, the most popular ADC drug globally is trastuzumab deruxtecan, developed by Daiichi Sankyo. It is an antibody-drug conjugate targeting HER2, conjugated with the supplemental isomerase inhibitor Dxd. It received FDA approval in 2019 and 2022, respectively, and achieved sales of $2.5 billion in 2023. Currently, there are hundreds of ADCs in clinical trials.

[0004] In the field of modern pharmaceuticals, a considerable number of drugs contain hydroxyl groups. However, many hydroxyl compounds suffer from problems such as poor water solubility, rapid drug metabolism, and lack of targeting due to their inherent properties, leading to difficulties in administration, unsatisfactory efficacy, and significant side effects. Therefore, modifying these drugs to increase their targeting, solubility, stability, and improve their clinical suitability has become a major challenge in drug development. The novel linker of this invention, which simultaneously provides a trigger, a linker site for modifying side chains, and enables self-cleavage under specific conditions to release the active drug, represents one approach to solving this problem.

[0005] Glucuronate prodrugs are selectively activated by β-glucuronidase, an enzyme that is almost absent in the blood but highly expressed in the microenvironment of many solid tumors, including lung cancer, breast cancer, and gastrointestinal cancers. This enzyme has been used for drug delivery around tumors for the past two decades. Regarding conjugate-drugs containing β-glucuronide hydrolyzed by this enzyme, the drug is almost not released into the bloodstream but is selectively released into target cancer cells. In particular, β-glucuronide linkers are more hydrophilic than peptide linkers, thus having a significant advantage in improving the physical properties of conjugates and are widely used in the preparation of antibody-drug conjugates. Galactose prodrugs are selectively activated by β-galactosidase, an enzyme that hydrolyzes β-galactosidic bonds and exists as a protein in lysosomes within cells. Because this enzyme forms an active dimer only at low pH and exists as an inactive monomer at physiological pH 7.4, introducing novel β-galactosidase linkers can also significantly reduce the risk of drug release during in vivo circulation. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to propose a general formula compound for a cleavable linker used in prodrug delivery systems, and compounds synthesized by combining it with antibodies, albumins, proteins, peptides, and various small molecules with targeting effects, as well as its preparation method and applications, including its application in prodrug delivery systems. This invention proposes a novel approach to trigger drug release via β-glucuronidase or β-galactosidase. Specifically, the linker compound of this invention, applicable to prodrug delivery systems, releases the corresponding active drug through a self-closing ring process after glycosidic bond hydrolysis by β-glucuronidase or β-galactosidase to form a hydroxyl group.

[0007] This invention proposes a cleavable linker compound for use in prodrug delivery systems. This compound has one or more independently modifiable sites, allowing for the combination of different triggers, modified side chains, and / or active pharmaceutical ingredients to form a novel compound. The structure of the cleavable linker compound is shown in formula (1-1).

[0008]

[0009] R1 is selected from β-glucanide, β-galactoside, phosphate ester, sulfate ester, and pyrophosphate ester;

[0010] R2 is H or CH3(CH2) n n = 0-20;

[0011] D is a class of compounds containing hydroxyl groups, wherein compound D has a naked -OH group.

[0012] L is represented as W1-W2-W3,

[0013] W1 is selected from -O-, -N-; W2 is selected from C1-C12 straight-chain or branched carbon chains, C3-C8 carbon ring chains, C3-C8 heterocyclic chains, straight-chain or branched PEG chains, sugar chains, polyamino acid chains, and mixed chains of straight-chain or branched PEG chains and polysarcosine; W3 is selected from linking groups that can react with thiols, aminos, azides, alkynyls, maleimides, etc., and can be selected from:

[0014]

[0015] wait.

[0016] W1 is preferably -N-. When -W1- is -N-, the structure of the cleavable linker compound is as shown in formula (1-2):

[0017]

[0018] R1 is selected from β-glucanide, β-galactoside, phosphate ester, sulfate ester, and pyrophosphate ester;

[0019] R2 is selected from H or CH3(CH2). n n = 0-20;

[0020] D is selected from a class of compounds containing a hydroxyl group, wherein compound D has a naked -OH group.

[0021] R3 is selected from H, C1-C6 alkyl, or cycloalkyl;

[0022] L1 is selected from straight-chain or branched carbon chains of C1-C12, carbon ring chains of C3-C8, heterocyclic chains of C3-C8, straight-chain or branched PEG chains, sugar chains, polyamino acid chains, and mixed chains of straight-chain or branched PEG chains and polysarcosine.

[0023] R3 and L1 can be optionally connected together to form 3 to 6 loops. The looping of R3 and L1 can be selected from...

[0024] R4 is selected from one or more of H, C1-C6 alkyl or cycloalkyl;

[0025] The L2 is selected from straight-chain or branched carbon chains of C1-C12, carbon ring chains of C3-C8, heterocyclic chains of C3-C8, straight-chain or branched PEG chains, sugar chains, polyamino acid chains, and mixed chains of straight-chain or branched PEG chains and polysarcosine.

[0026] W3 is selected from linking groups that can react with thio, amino, azide, alkynyl, maleimide, etc., and can be chosen from: wait;

[0027] Furthermore,

[0028] When R1 is preferably a β-glucanide, the structure of the cleavable linker compound is shown in formulas (1-3):

[0029]

[0030] In equation (1-3), the definitions of R2, R3, L1, L2, and W3 are the same as in equation (1-2).

[0031] According to the formula (1-3), it includes the following compounds (1-4-1), (1-4-2), and (1-4-3), which contain maleimide in their structure (L2 is selected as lysine or glutamic acid as the branched backbone and / or the ligand linker W3 is selected as maleimide), and therefore have the ability to covalently bind to antibodies, albumin, proteins, peptides, and various small molecules with targeting effects.

[0032]

[0033] In the above formulas (1-4-1)-(1-4-3), n, m, and t represent the number of repeating units, where n is selected from 1 to 24 and m is selected from 0 to 12.

[0034] In equations (1-4-1) to (1-4-3), L is selected from: One or more of the following;

[0035] In equations (1-4-1) to (1-4-3), Q1 and Q2 are respectively selected from PEG fragments, polysarcosine fragments, etc.;

[0036] Wherein, D represents an active compound containing a hydroxyl group, and the active compound may be a drug, a toxin, an affinity ligand, a detection probe, or a combination thereof.

[0037] Furthermore, the drug may be selected from, but is not necessarily limited to, the group consisting of topoisomerase I inhibitors (camptothecin derivatives), alkylating agents, dihydrofolate reductase inhibitors, thymidine synthase inhibitors, purine nucleoside synthase inhibitors, nucleoside reductase inhibitors, DNA polymerase inhibitors, RNA polymerase inhibitors, EGFR inhibitors, ALK inhibitors, MET inhibitors, CDK4 / 6 inhibitors; and pharmaceutically acceptable salts, solvates, acids, or derivatives thereof, and its structure is shown below:

[0038]

[0039] Furthermore, the cleavable linker compounds provided by the present invention include the following compounds (1-5-1), (1-5-2), (1-5-3) and (1-5-4), and their pharmaceutically acceptable salts, solvates, polymorphs or isomers:

[0040]

[0041] This invention also provides a method for preparing a cleavable linker compound of formula (1-2), the reaction process of which is shown in the following formula:

[0042]

[0043] The steps are as follows:

[0044] a) Substitution reaction: In the first solvent, under the action of silver oxide, compound 1 undergoes a nucleophilic substitution reaction with acetylated β-glucan or β-galactoside to generate compound 2;

[0045] The first solvent is acetone, acetonitrile, or N,N-dimethylformamide, etc.; preferably, it is acetonitrile.

[0046] The nucleophilic substitution reaction is carried out at a temperature of 5°C to 50°C; preferably, at 25°C.

[0047] The nucleophilic substitution reaction takes 4-24 hours; preferably, it takes 12 hours.

[0048] The molar ratio of compound 1, silver oxide, and acetylated β-glucoside or β-galactoside is 1:(1-2):(1-2); preferably, it is 1:1:1.2.

[0049] b) Condensation reaction: In the second solvent, compound 2 and amine undergo a condensation reaction under the action of a condensing agent to generate compound 3;

[0050] The condensing agent can be a commonly used condensing agent such as DCC, EEDQ, HATU, EDCI / HOBT, PyBOP; preferably, it is EDCI / HOBT.

[0051] The temperature of the condensation reaction is 5℃-50℃; preferably, it is 25℃.

[0052] The condensation reaction takes 2-48 hours; preferably, it takes 24 hours.

[0053] The molar ratio of compound 2 and condensing agent is 1:(1-2); preferably, it is 1:1.2.

[0054] The second solvent is one or more of DMF, CH3CN, DCM, etc.; preferably, it is DMF.

[0055] c) Reduction reaction: In the third solvent, compound 3 undergoes a reduction reaction under a hydrogen atmosphere by the action of platinum dioxide or Raney nickel to generate compound 4;

[0056] The temperature of the reduction reaction is 5℃-50℃; preferably, it is 25℃.

[0057] The reduction reaction takes 1-24 hours; preferably, it takes 12 hours.

[0058] The molar ratio of compound 3, platinum dioxide, or Raney nickel is 1:(0.01-0.5); preferably, it is 1:0.1.

[0059] The third solvent is one or more of methanol, ethanol, trifluoroethanol, tetrahydrofuran, tetradioxane, etc.; preferably, it is methanol.

[0060] d) Substitution reaction: In the fourth solvent, compound 4 undergoes a substitution reaction with a halogenated compound under basic conditions to generate compound 5;

[0061] The temperature for the substitution reaction is 5°C-50°C; preferably, it is 25°C.

[0062] The substitution reaction time is 1h-24h; preferably, it is 8h.

[0063] The molar ratio of compound 4, the base, and the halogenated compound is 1:(1-5):(1-2); preferably, it is 1:2:1.5.

[0064] e) Condensation reaction: Under nitrogen protection, compound 5 was dissolved in the fifth solvent, and di(trichloromethyl) carbonate and anhydrous organic base were added. After reacting for 1 hour, drug (D) and DMAP were added, and the reaction was carried out overnight at 20-60°C to obtain compounds 1-2.

[0065] The drug (D) must satisfy the requirement of having exposed -OH, -NH2, -NHR, -SH;

[0066] The fifth solvent is anhydrous tetrahydrofuran or dichloromethane, etc.

[0067] The anhydrous organic base is selected from triethylamine, DIPEA, pyridine, etc.;

[0068] The molar ratio of compound 5 to di(trichloromethyl) carbonate is 1:0.3-1; preferably, it is 1:0.5.

[0069] The molar ratio of compound 5 to anhydrous organic base is 1:3 to 5; preferably, it is 1:4.

[0070] The molar ratio of compound 5 to drug (D) derivative is 1:1 to 1.2; preferably, it is 1:1.1.

[0071] In one specific implementation, the following steps are included:

[0072] Substitution reaction: In the presence of silver oxide, compound 1 undergoes a nucleophilic substitution reaction with acetylated β-glucan bromide to generate compound 2. The solvent is acetonitrile, and the temperature is 25℃.

[0073] Condensation reaction: HATU was used as the condensing agent and DIPEA as the base. Compound 2 condensed with the corresponding amine to form compound 3. The solvent was DMF and the temperature was 25°C.

[0074] Reduction reaction: Compound 3 undergoes a reduction reaction in a hydrogen atmosphere via Raney nickel to produce compound 4. The solvent is methanol, and the temperature is 25°C.

[0075] Substitution reaction: In the fourth solvent, under basic conditions, compound 4 undergoes a substitution reaction with a halogenated compound to generate compound 5;

[0076] Condensation reaction: Under nitrogen protection, compound 5 is dissolved in an organic solvent, and di(trichloromethyl) carbonate and anhydrous organic base are added. The molar ratio of compound 5 to di(trichloromethyl) carbonate is 1:0.5, and the molar ratio of compound 5 to anhydrous organic base is 1:3-5. After reacting for 1 hour, drug (D) and DMAP are added, and the reaction is carried out overnight at 20-60°C to obtain compounds 1-2. The molar ratio of compound 5 to drug (D) derivative is 1:1-1.2. The solvent is anhydrous tetrahydrofuran or dichloromethane, etc.

[0077] This invention also provides a method for preparing a cleavable linker compound (1-4-1), the reaction process of which is shown in the following formula:

[0078]

[0079] The specific steps are as follows:

[0080] a) Condensation reaction: Under nitrogen protection, compound 1 was dissolved in an organic solvent, and di(trichloromethyl) carbonate and triethylamine were added. After reacting for 1 hour, a hydroxyl-containing drug was added, and the reaction was allowed to proceed overnight at room temperature to obtain compound 2.

[0081] The organic solvent is selected from tetrahydrofuran, dichloromethane, dichloroethane, etc.; preferably, it is dichloromethane.

[0082] b) Hydrolysis reaction: Compound 2 was dissolved in acetonitrile and water, an inorganic base was added, and after reacting for 2 hours, the pH was adjusted to acidic, and the solution was passed through a column to obtain compound 3.

[0083] The inorganic base is selected from lithium hydroxide, sodium hydroxide, etc.;

[0084] c) Condensation reaction: Compound 3 and Compound 4 are dissolved in an organic solvent and stirred at room temperature for 6-8 hours to generate the prodrug having the general formula (1-4-1);

[0085] The organic solvent is one or a mixture of methanol, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, and water.

[0086] In one specific implementation, the following steps are included:

[0087] Condensation reaction: Under nitrogen protection, compound 1 was dissolved in anhydrous dichloromethane, and 0.5 equivalents of di(trichloromethyl) carbonate and 2 equivalents of triethylamine were added. After reacting for 1 hour, 2 equivalents of DMAP and 1 equivalent of hydroxyl-containing drug were added, and the reaction was carried out overnight at room temperature to obtain compound 2.

[0088] Hydrolysis reaction: Compound 2 was dissolved in acetonitrile and water, 20 equivalents of lithium hydroxide were added, and after reacting for 2 hours, the pH was adjusted to acidic and then passed through a reverse-phase column to obtain compound 3.

[0089] Condensation reaction: Compound 3 and compound 4 were dissolved in DMF, and 2 equivalents of DIPEA were added. The mixture was stirred at room temperature for 6 hours to generate a cleavable linker compound with the general formula (1-4-1).

[0090] Another object of the present invention is to provide a possible drug release mechanism for a targeted drug linker formed by linking a ligand, an initiator and a drug to the cleavable linker compound under different conditions: the linker releases the corresponding active drug by hydrolyzing the glycosidic bond of the linker through β-glucuronidase or galactosidase to form a hydroxyl group, and then releasing the drug through a self-closing ring process.

[0091] The specific mechanism is as follows:

[0092]

[0093] The present invention also proposes a drug, a pharmaceutical composition comprising the said cleavable linker compound and its pharmaceutically acceptable salt, solvate, polymorph or isomer and pharmaceutically acceptable carrier.

[0094] The present invention also proposes the application of the cleavable linker compound (1-1), including but not limited to compounds of formula (1-5-1), formula (1-5-2), formula (1-5-3), and formula (1-5-4), in the preparation of antitumor drugs and in the preparation of antibody-drug conjugates.

[0095] The present invention also proposes the use of the drug / drug composition in the preparation of antitumor drugs, or in the preparation of antibody or albumin-bound antitumor drugs, or in the preparation of antibody-drug conjugates.

[0096] The advantages and beneficial effects of this invention include, but are not limited to: the cleavable linker compound formula (1-1) proposed in this invention, which can be used as a prodrug, has multiple independently modifiable sites, combining different triggers, modified side chains, and / or active drugs to form a new compound. This makes the active drug more stable, less toxic, and increases targeting while maintaining its activity. Figure 1 The retention times of the light and heavy chains of the conjugate compound were found to be similar to those of the naked antibody, indicating that the cleavable linker compound proposed in this invention has good water solubility. This invention provides an antibody-drug conjugate drug that undergoes β-glucuronidase cleavage to trigger the self-cleavage of the linker, its preparation method, and its applications.

[0097] In specific implementation schemes, experiments have demonstrated that the cleavable linker compound of formula (1-1), including but not limited to formulas (1-5-1) and (1-5-2), possesses excellent in vitro and in vivo stability and can rapidly release active drugs under simulated tumor microenvironment conditions. Upon binding with monoclonal antibodies, it forms antibody-drug conjugates, exhibiting significant antitumor activity in mouse models. Compound (1-5-1) significantly prolonged the survival time of mice and inhibited tumor growth in the NCI-N87 xenograft mouse model. This indicates that the novel linker and the compounds constructed based on it can achieve rapid drug release at the tumor site, improve drug-likeness, and enhance the anticancer effect.

[0098] This invention innovatively proposes a cleavable linker compound for use in prodrug delivery systems. This compound cleaves a hydroxyl group via a trigger, forming a self-closed ring structure in vivo and releasing the active drug. The cleavable linker compound of this invention has abundant modification sites, allowing for different modification methods to adapt to various drug delivery needs, thereby improving targeting, reducing the toxicity of the active drug, and improving drug-likeness. After administration, the cleavable linker compound of this invention is released at a specific target site in vivo via the trigger fragment, stabilized in vivo through long-term circulation via the modified fragment, and provides a therapeutic effect through the active drug portion. Ultimately, it achieves accumulation and targeted drug release at the target site, reduces the toxic side effects of the active drug, and improves drug-likeness. Attached Figure Description

[0099] Figure 1 The curves of compound 1-5-1 released by β-glucuronidase cleavage at different pH values ​​are shown.

[0100] Figure 2 The chromatogram of compound 1-5-1 after conjugation with trastuzumab;

[0101] Figure 3 The chromatogram is of compound 1-5-2 conjugated with trastuzumab.

[0102] Figure 4 The image shows the antitumor effects of compounds HER2-1-5-1 and HER2-1-5-2 on nude mice with NCI-N87 xenograft tumors. Detailed Implementation

[0103] The invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the invention are all common knowledge and general knowledge in the art, and the invention does not have any particular limitations.

[0104] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0105] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0106] Example 1: Synthesis of compound (1-5-1)

[0107]

[0108] Synthesis of Compound 2

[0109] Compound 1 (5 g, 27.30 mmol) was placed in a round-bottom flask, and 100 mL of anhydrous DMF was added. HATU (12.46 g, 32.77 mmol) and DIPEA (10.59 g, 81.91 mmol) were added sequentially. After stirring at room temperature for 30 min, 1-Boc-piperazine (6.10 g, 32.77 mmol) was added, and the reaction was allowed to proceed for 8 hours at room temperature. The reaction was confirmed to be complete by TLC. The pH was adjusted to 3-4 using 3M HCl solution. The mixture was extracted with EA / H2O. The EA phase was washed three times each with H2O and saturated NaCl solution, dried over anhydrous NaSO4, and concentrated under reduced pressure to obtain a yellow oily liquid. This liquid was purified by column chromatography (dichloromethane:methanol = 100:1) to give 7.5 g of a pale yellow solid, with a yield of 78.2%. 1H NMR (400MHz, CDCl3) δ10.71 (s, 1H), 8.22 (d, J = 2.2Hz, 1H), 7.68 (dd, J = 8.6, 2.3Hz, 1H), 7.23 (d, J = 8.6Hz, 1H), 3.55 (br, 8H), 1.48 (s, 9H). (ESI) m / z 352.1[M+H] +

[0110] Synthesis of Compound 3

[0111] Compound 2 (7 g, 19.92 mmol) was placed in a round-bottom flask, and 100 mL of anhydrous acetonitrile was added. Acetyl bromide-α-D-glucuronide methyl ester (8.70 g, 21.91 mmol) and activated silver oxide (4.62 g, 19.92 mmol) were added sequentially. Nitrogen gas was purged, and the reaction was carried out at room temperature for 8 hours. The reaction was confirmed to be complete by TLC. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain a yellow oily liquid. This liquid was purified by column chromatography (dichloromethane:methanol = 100:1) to give 12.7 g of a pale yellow solid, with a yield of 95.5%. 1 H NMR (400MHz, CDCl3) δ7.88(s,1H),7.62(d,J=8.6Hz,1H),7.42(d,J=8.6Hz,1H),5.43–5.29(m,4H),4. 25(d,J=8.6Hz,1H),3.74(s,3H),3.48(br,8H),2.13(s,3H),2.10–2.02(m,6H),1.48(s,9H).(ESI)m / z 668.2[M+H] +

[0112] Synthesis of Compound 4

[0113] Compound 3 (7.5 g, 11.23 mmol) was placed in a round-bottom flask, 100 mL of methanol was added, and Raney nickel (131 mg, 2.25 mmol) was added to displace hydrogen gas. The reaction was carried out at room temperature for 3 hours. The reaction was confirmed to be complete by TLC. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give 7.0 g of a pale yellow solid, with a yield of 97.8%. 1H NMR (400MHz, DMSO-d6) δ8.54(d,J=2.2Hz,1H),8.31(dd,J=8.5,2.2Hz,1H),7.53(d,J=8.6Hz,1H),5.31–5.16(m,3H),5.04(ddd,J=8.4,7.6 ,3.1Hz,1H),4.06(dt,J=8.4,2.7Hz,1H),3.64(s,3H),3.57–3.41(m,4H),3.37–3.31(m,4H),1.99(d,J=3.7Hz,9H),1.39(s,9H).(ESI)m / z 638.2[M+H] +

[0114] Synthesis of Compound 5

[0115] Compound 4 (6.7 g, 10.28 mmol) was placed in a round-bottom flask, and 50 mL of anhydrous DMF was added. NaHCO3 (1.73 g, 20.56 mmol) and iodomethane (2.92 g, 20.56 mmol) were added sequentially, and the reaction was carried out at room temperature for 8 hours. The reaction was confirmed to be complete by LC-MS. CH3I was quenched with saturated NaHCO3 solution, and the mixture was extracted with EA / H2O. The EA phase was washed three times each with H2O and saturated NaCl solution, dried over anhydrous NaSO4, and concentrated under reduced pressure to obtain a yellow oily liquid. This liquid was purified by column chromatography (dichloromethane:methanol = 75:1) to give 3.05 g of a pale yellow solid, with a yield of 46.5%. 1 H NMR (400MHz, DMSO) δ6.94(d,J=8.1Hz,1H),6.62(d,J=8.2Hz,1H),6.57(s,1H),5.61(d,J=7.8Hz,1H),5.49(t,J=9.6Hz,1H),5.13(t,J=8. 7Hz,1H),5.07(t,J=9.7Hz,1H),4.72(d,J=9.9Hz,1H),3.64(s,3H),3.45(br,8H),2.74(s,3H),2.06–1.96(m,9H),1.40(s,9H).(ESI)m / z 652.3[M+H] +

[0116] Synthesis of Compound 6

[0117] Compound 5 (1.26 g, 1.93 mmol) and triphosgene (286.8 mg, 0.967 mmol) were weighed into a three-necked flask, purged with nitrogen, and 40 mL of anhydrous dichloromethane was added. Under ice bath conditions, DIEA (750 mg, 5.10 mmol) was slowly added dropwise, and the reaction was carried out at room temperature for 1 hour. LC-MS was used to confirm the reaction was complete. DMAP (472 mg, 3.87 mmol) and camptothecin derivative (735 mg, 1.74 mmol) were added, and the reaction was carried out at room temperature for 8 hours. TLC was used to monitor the reaction completion. 40 mL of dichloromethane was added, and the mixture was washed twice with 1 M dilute hydrochloric acid and once with saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. Column chromatography (dichloromethane:methanol = 100:1 to 50:1) was used to purify the crude product, yielding 1.3 g of a yellow solid. The two-step yield was 61.0%. 1 H NMR (400MHz, DMSO-d6) δ8.04(d,J=2.2Hz,1H),7.76(dd,J=9.4,2.2Hz,1H),7.28(t,J=0.9Hz,1H),7.14(dd,J=12.1,8.7 Hz,2H),5.90(s,1H),5.40(dd,J=2.1,0.9Hz,2H),5.31–5.16(m,5H),5.04(ddd,J=8.4,7.6,3.1Hz,1H),4.06(dt,J=8.4 ,2.7Hz,1H),3.66–3.49(m,7H),3.46(ddd,J=12.3,6.1,3.7Hz,3H),3.34(dd,J=6.1,3.6Hz,4H),2.85–2.65(m,2H),2.2 4(tt,J=8.5,5.8Hz,2H),2.03–1.89(m,10H),1.72(dq,J=13.7,8.0Hz,1H),1.39(s,9H),0.86(t,J=8.0Hz,3H).(ESI)m / z 1101.1[M+H] +

[0118] Synthesis of Compound 7

[0119] Compound 6 (1.3 g, 1.30 mmol) was weighed into a flask, 10 mL of dichloromethane was added, followed by 2 mL of trifluoroacetic acid. The mixture was reacted at room temperature for 2 hours. The reaction was monitored by TLC until complete, and the solution was concentrated under reduced pressure to obtain a yellow oil. Under ice bath conditions, 65 mL of acetonitrile was added, followed by the slow addition of 65 mL of 0.4 M lithium hydroxide aqueous solution. The reaction was allowed to proceed for one hour at room temperature. The reaction was monitored by LC-MS until complete, and the solution was concentrated under reduced pressure and purified by reversed-phase column chromatography to give 850 mg of a yellow solid, with a yield of 75.9%. 1H NMR (400MHz, DMSO) δ12.95 (s, 1H), 8.82 (s, 2H), 7.90 (dd, J = 34.2, 11.1Hz, 1H), 7. 65(s,1H),7.58–7.46(m,1H),7.32(d,J=7.1Hz,2H),6.54(s,1H),5.56(s,1H),5. 44(s,2H),5.35–5.17(m,3H),3.70(s,4H),3.27(s,3H),3.18(d,J=7.5Hz,9H),2. 91(s,1H),2.03(s,2H),1.86(hept,J=6.9Hz,2H),0.87(t,J=7.1Hz,3H).(ESI)m / z 860.3 [M+H] +

[0120] Synthesis of Compound 1-5-1

[0121] Compound 7 (100 mg, 116.3 μmol) was placed in a round-bottom flask, and 2 mL of anhydrous DMF was added. Then, PEG4-maleimide OSU active ester (65.7 mg, 127.9 μmol) and DIPEA (26.8 mg, 232.6 μmol) were added sequentially. The reaction was carried out at room temperature for 2 hours, and the reaction was confirmed to be complete by LC-MS. The mixture was purified by reversed-phase column chromatography to give 60 mg of a pale yellow solid, with a yield of 41.1%. 1 H NMR (400MHz, DMSO-d6) δ10.17(s,1H),8.04(d,J=2.0Hz,1H),7.76(dd,J=9.3,2.0Hz,1H),7.71(t,J=5.1Hz,1H),7.28(t,J=0.9Hz,1H),7 .19–7.10(m,2H),6.61(s,2H),5.90(s,1H),5.40(dd,J=2.1,0.9Hz,2H),5.35(d,J=5.9Hz,1H),5.28(s,2H),5.17(d,J=6.1Hz,1H),5.11– 5.03(m,2H),4.72(t,J=6.2Hz,2H),3.86(dt,J=8.8,2.4Hz,1H),3.70–3.40(m,32H),2.85–2.65(m,2H),2.59(s,2H),2.35(t,J=6.7Hz,2 H),2.24(tt,J=8.5,5.8Hz,2H),1.96(dq,J=13.8,8.1Hz,1H),1.72(dq,J=13.7,8.0Hz,1H),0.86(t,J=8.0Hz,3H).(ESI)m / z1258.4[M+H] +

[0122] Example 2 Synthesis of compound (1-5-2)

[0123]

[0124] Synthesis of Compound 9

[0125] Compound 8 (50 mg, 93.18 μmol) was placed in a round-bottom flask, and 3 mL of anhydrous DMF was added. Compound 7 (67 mg, 77.65 μmol) and DIPEA (25.09 mg, 33.81 μmol) were then added sequentially. The reaction was carried out at room temperature for 2 hours, and LC-MS was used to confirm completion. The mixture was purified by reversed-phase column chromatography to give 65 mg of a pale yellow solid, with a yield of 65.32%. 1 H NMR (400MHz, DMSO-d6) δ10.34(s,1H),8.17(d,J=9.1Hz,1H),8.04(d,J=1.9Hz ,1H),7.76(dd,J=9.3,2.0Hz,1H),7.28(t,J=0.9Hz,1H),7.14(dd,J=12.1,8.7 Hz,2H),6.76(t,J=5.0Hz,1H),6.70(s,1H),5.90(s,1H),5.40(dd,J=2.1,0.9H z,2H),5.38–5.32(m,1H),5.28(s,1H),5.17(d,J=6.2Hz,1H),5.11–5.03(m,2H ),4.24(ddd,J=12.3,6.5,4.0Hz,2H),4.12(dt,J=9.1,6.6Hz,1H),3.86(dt,J= 8.7,2.4Hz,1H),3.63–3.41(m,12H),3.14–2.99(m,4H),2.85–2.65(m,2H),2.4 1–2.30(m,2H),2.24(tt,J=8.5,5.8Hz,2H),1.96(dq,J=13.8,8.1Hz,1H),1.82 –1.50(m,5H),1.37(s,9H),1.44–1.15(m,8H),0.86(t,J=8.0Hz,3H).(ESI)m / z 1282.3[M+H] +

[0126] Synthesis of Compound 10

[0127] Compound 9 (65 mg, 50.73 mmol) was weighed into a bottle, 10 mL of dichloromethane was added, followed by 2 mL of trifluoroacetic acid. The mixture was reacted at room temperature for 2 hours, and the reaction was confirmed to be complete by LC-MS. The mixture was purified by reversed-phase column chromatography to give 40 mg of a pale yellow solid, with a yield of 66.76%.1 H NMR (400MHz, DMSO-d6) δ10.67(s,1H),8.17(d,J=9.0Hz,1H),8.04(d,J=2.0Hz,1H ),7.76(dd,J=9.3,2.0Hz,1H),7.28(t,J=0.9Hz,1H),7.19–7.10(m,2H),6.70(s, 2H),5.90(s,1H),5.40(dd,J=2.1,0.9Hz,2H),5.38–5.32(m,1H),5.28(s,2H),5. 17(d,J=6.2Hz,1H),5.11–5.03(m,2H),4.24(ddd,J=12.3,6.5,4.0Hz,2H),4.12( dt,J=9.1,6.6Hz,1H),3.86(dt,J=8.8,2.4Hz,1H),3.63–3.40(m,13H),3.04(ddd ,J=12.5,6.5,4.0Hz,2H),2.85–2.66(m,2H),2.61(tt,J=6.5,5.0Hz,2H),2.41–2 .30(m,2H),2.24(tt,J=8.5,5.8Hz,2H),1.96(dq,J=13.8,8.1Hz,1H),1.82–1.50 (m,5H),1.38–1.15(m,8H),1.05(t,J=6.5Hz,2H),0.86(t,J=8.0Hz,3H).(ESI)m / z 1182.2[M+H] +

[0128] Synthesis of compound 1-5-2

[0129] Compound 10 (40 mg, 33.86 μmol) was placed in a round-bottom flask, and 2 mL of anhydrous DMF was added. Then, methyl-terminated PEG12 active ester (30 mg, 40.64 μmol) and DIPEA (11 mg, 84.66 μmol) were added sequentially. The reaction was carried out at room temperature for 2 hours, and LC-MS was used to confirm completion. The mixture was purified by reversed-phase column chromatography to give 36 mg of a pale yellow solid, with a yield of 59.2%. 1H NMR (400MHz, DMSO) δ12.76(s,1H),9.13(s,1H),8.78–8.43(m,1H),8.40–8.23(m,2H),8.23–8.02(m,1H),7.93(t,J=5.7Hz,1H),7.84(d,J=8 .1Hz,1H),7.77(dd,J=13.2,7.6Hz,2H),7.50(d,J=9.4Hz,2H),7.32(s,1H),7.26–7.09(m,2H),6.99(s,2H),5.55–5.28(m,6H),4.92(d,J=7 .5Hz,1H),4.82–3.64(m,26H),3.56(t,J=6.5Hz,23H),3.47–3.26(m,17H),3.23(s,3H),3.01(dq,J=40.3,7.0Hz,4H),2.58(d,J=7.1Hz,2H) ,2.27(t,J=6.5Hz,2H),2.07(h,J=7.1Hz,2H),1.99–1.78(m,J=7.3Hz,2H),1.62–1.41(m,6H),1.22(m,10H),0.88(t,J=7.2Hz,3H).(ESI)m / z 1796.9[M+H] +

[0130] Example 3 Synthesis of compound (1-5-3)

[0131]

[0132] Synthesis of Compound 12

[0133] Compound 11 (1.20 g, 2.82 mmol) was placed in a round-bottom flask, and 20 mL of anhydrous DMF was added. HATU (1.29 g, 3.38 mmol) and DIPEA (1.09 g, 8.46 mmol) were added sequentially. After stirring at room temperature for 30 min, aminooctaglycol monomethyl ether (1.08 g, 2.82 mmol) was added, and the reaction was allowed to proceed for 8 hours at room temperature. The reaction was confirmed to be complete by TLC. The pH was adjusted to 3-4 using 3M HCl solution. The mixture was extracted with EA / H2O. The EA phase was washed three times each with H2O and saturated NaCl solution, dried over anhydrous NaSO4, and concentrated under reduced pressure to obtain a yellow oily liquid. This liquid was purified by column chromatography (dichloromethane:methanol = 100:1) to give 1.8 g of a pale yellow solid, with a yield of 80.7%. 1H NMR (400MHz, DMSO) δ8.09–7.83(m,4H),7.74(t,J=6.2Hz,2H),7.50(d,J=8.3Hz,1H ),7.42(t,J=7.5Hz,2H),7.33(t,J=7.4Hz,2H),4.37–4.15(m,3H),3.98(td,J=8.4 ,5.3Hz,1H),3.50(s,28H),3.45–3.38(m,4H),3.23(s,3H),2.22(t,J=8.0Hz,2H), 1.87(dt,J=14.2,6.4Hz,1H),1.74(dt,J=13.9,7.7Hz,1H),1.39(s,9H).(ESI)m / z 791.4[M+H] +

[0134] Synthesis of Compound 13

[0135] Compound 12 (1.80 g, 2.28 mmol) was weighed into a round-bottom flask, 10 mL of dichloromethane was added, followed by 5 mL of trifluoroacetic acid. The mixture was reacted at room temperature for 6 hours. The reaction was confirmed to be complete by LC-MS. The solution was concentrated under reduced pressure to obtain a yellow oil. The solution was purified by reversed-phase column chromatography to give 1.04 g of a colorless bv oil, with a yield of 62.3%. 1 H NMR (400MHz, DMSO-d6) δ8.03(t,J=5.0Hz,1H),7.78(dd,J=7.6,1.5Hz,2H),7.61(dd,J=7.8,1.3Hz,2H),7.49(td,J =7.7,1.3Hz,2H),7.41(td,J=7.8,1.5Hz,2H),7.32(d,J=8.5Hz,1H),4.44(d,J=4.8Hz,2H),4.22(t,J=4.9Hz,1H),4 .17–4.07(m,1H),3.89(dq,J=14.0,4.8Hz,1H),3.65(s,3H),3.65(d,J=3.8Hz,1H),3.67–3.57(m,1H),3.59(s,3H), 3.61–3.52(m,5H),3.53(d,J=3.8Hz,1H),3.55–3.50(m,1H),3.48(s,2H),2.57–2.31(m,4H),1.37(s,6H).(ESI)m / z 735.4[M+H] +

[0136] Synthesis of Compound 14

[0137] Compound 13 (1.04 g, 1.42 mmol) was placed in a round-bottom flask, and 10 mL of anhydrous DCM was added. EDCI (406.97 mg, 2.12 mmol) and 2,3,5,6-tetrafluorophenol (470.08 mg, 2.83 mmol) were added sequentially, and the reaction was carried out at room temperature for 8 hours. LC-MS confirmed the reaction was complete. The solution was concentrated under reduced pressure to obtain a yellow oily liquid, which was purified by column chromatography (dichloromethane:methanol = 100:1) to give 0.83 g of a white oily substance, with a yield of 66.4%. 1 H NMR (400MHz, DMSO) δ8.14–7.92(m,3H),7.90(d,J=7.5Hz,2H),7.74(t,J=6.5Hz,2H),7.61(d,J =8.1Hz,1H),7.42(t,J=7.4Hz,2H),7.33(t,J=7.4Hz,2H),4.28(ddt,J=32.4,13.3,6.6Hz,3H) ,4.08(td,J=8.4,5.3Hz,1H),3.54(d,J=28.2Hz,29H),3.41(dd,J=5.9,2.8Hz,4H),3.23(s,3H ),2.80(t,J=7.9Hz,2H),2.06(dt,J=14.6,6.9Hz,1H),1.93(dt,J=14.8,8.1Hz,1H).(ESI)m / z 883.4[M+H] +

[0138] Synthesis of Compound 15

[0139] Compound 7 (100 mg, 113.3 μmol) was placed in a round-bottom flask, and 2 mL of anhydrous DMF was added. Compound 14 (95.9 mg, 98.5 μmol) and DIPEA (31.8 mg, 246.2 μmol) were then added sequentially. The reaction was carried out at room temperature for 6 hours, and LC-MS was used to confirm completion. The mixture was purified by reversed-phase column chromatography to give 68 mg of a pale yellow solid, with a yield of 43.8%. 1H NMR (400MHz, DMSO) δ12.84(s,1H),8.85(s,1H),7.92(d,J=7.7Hz,2H),7.83(s,2H),7.68(s,2H),7 .57(d,J=26.1Hz,2H),7.53–7.43(m,2H),7.44–6.92(m,6H),6.60(d,J=65.2Hz,1H),5.43(s,3H), 5.31(s,1H),5.28–5.05(m,2H),4.21(d,J=32.2Hz,3H),4.11–3.84(m,3H),3.46–3.28(m,21H),3. 24(d,J=17.6Hz,12H),2.05(d,J=33.6Hz,2H),1.93–1.72(m,4H),0.88(t,J=7.3Hz,3H).(ESI)m / z 1576.6[M+H] +

[0140] Synthesis of Compound 16

[0141] Compound 15 (60 mg, 38.1 μmol) was placed in a round-bottom flask, and 10 mL of THF and 2 mL of diethylamine were added. The mixture was reacted at room temperature for 4 hours. The reaction was confirmed to be complete by LC-MS. The product was purified by reversed-phase column chromatography and lyophilized to give 41 mg of a yellow solid, with a yield of 79.5%. 1 H NMR (400MHz, CD3OD_SPE) δ7.64(s,1H),7.51(d,J=7.5Hz,1H),7.49–7.35(m,3H),5.50(t,J=16.1 Hz,1H),5.39–5.20(m,2H),5.14(d,J=7.0Hz,1H),4.99(s,1H),4.12–3.99(m,1H),3.94(d,J=6.4H z,1H),3.74(s,4H),3.45(d,J=22.8Hz,5H),3.34–3.25(m,11H),3.06–2.84(m,4H),2.59(s,2H), 2.26–2.05(m,3H),2.05–1.92(m,2H),1.90(d,J=6.2Hz,2H),0.95(q,J=8.2,7.6Hz,3H).(ESI)m / z 1354.6[M+H] +

[0142] Synthesis of compound 1-5-3

[0143] Compound 16 (30 mg, 22.2 μmol) was placed in a round-bottom flask, and 2 mL of anhydrous DMF was added. Then, PEG4-maleimide OSU active ester (13.1 mg, 25.5 μmol) and DIPEA (7.16 mg, 55.38 μmol) were added sequentially. The reaction was carried out at room temperature for 2 hours, and the reaction was confirmed to be complete by LC-MS. The product was purified by reversed-phase column chromatography to give 27 mg of a pale yellow solid, with a yield of 69.6%. 1 H NMR (400MHz, DMSO-d6) δ10.17(s,1H),8.17(d,J=8.4Hz,1H),8.06–8.00(m,2H),7.76(dd,J=9.3,2.0Hz,1H),7.71(t,J=5.1Hz,1H),7.28( t,J=1.0Hz,1H),7.19–7.10(m,2H),6.61(s,2H),5.90(s,1H),5.40(dd,J=2.1,0.9Hz,2H),5.35(d,J=5.9Hz,1H),5.28(s,2H),5.17(d,J=6 .2Hz,1H),5.11–5.03(m,2H),4.72(t,J=6.2Hz,2H),4.04–3.95(m,1H),3.95–3.82(m,2H),3.70–3.40(m,67H),3.25(s,3H),2.85–2.65(m, (ESI)m / z 1753.8[M+H] +

[0144] Example 4 Synthesis of compound (1-5-4)

[0145]

[0146] Synthesis of Compound 17

[0147] Compound 5 (1.5 g, 2.30 mmol) and triphosgene (307.35 mg, 1.04 mmol) were weighed into a three-necked flask, purged with nitrogen, and 20 mL of anhydrous dichloromethane was added. DIEA (892.5 mg, 6.91 mmol) was slowly added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 1 hour. LC-MS was used to confirm the reaction was complete. DMAP (562.4 mg, 4.60 mmol) and Dxd (1.14 g, 2.30 mmol) were added, and the reaction was carried out at room temperature for 8 hours. TLC was used to monitor the reaction completion. 40 mL of dichloromethane was added, and the mixture was washed twice with 1 M dilute hydrochloric acid and once with saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. Column chromatography (dichloromethane:methanol = 100:1 to 50:1) was used to purify the crude product, yielding 1.3 g of a yellow solid. The two-step yield was 48.1%. 1 H NMR (400MHz, DMSO-d6) δ8.31(d,J=9.1Hz,1H),8.04(d,J=1.9Hz,1H),7.76(dd,J=9.3,2. 0Hz,1H),7.32(d,J=7.9Hz,1H),7.28(t,J=0.9Hz,1H),7.13(d,J=9.3Hz,1H),5.90(s,1H) ,5.40(dd,J=2.1,0.9Hz,2H),5.31–5.23(m,1H),5.26–5.19(m,1H),5.23–5.16(m,1H),5 .04(ddd,J=8.4,7.6,3.1Hz,1H),4.90(ddd,J=9.1,6.5,3.7Hz,1H),4.55(d,J=15.0Hz,1H ),4.35(d,J=15.0Hz,1H),4.22(s,1H),4.06(dt,J=8.4,2.7Hz,1H),3.64(s,2H),3.57–3 .41(m,4H),3.34(dd,J=6.1,3.6Hz,4H),2.70(ddd,J=12.3,8.5,5.9Hz,1H),2.56(ddd,J= 12.3,8.5,5.9Hz,1H),2.37–2.25(m,1H),2.12–2.02(m,1H),1.99(d,J=3.8Hz,9H),2.00 –1.89(m,1H),1.72(dq,J=13.7,8.0Hz,1H),1.39(s,6H),0.86(t,J=8.0Hz,3H).(ESI)m / z 1172.2[M+H] + .

[0148] Synthesis of Compound 18

[0149] Compound 17 (300 mg, 1.30 mmol) was weighed into a bottle, 10 mL of dichloromethane was added, followed by 2 mL of trifluoroacetic acid. The mixture was reacted at room temperature for 2 hours. The reaction was confirmed to be complete by TLC. The solution was concentrated under reduced pressure to obtain a yellow oil. Under ice bath conditions, 25 mL of acetonitrile was added, followed by the slow addition of 25 mL of 0.2 M lithium hydroxide aqueous solution. The mixture was reacted at room temperature for one hour. The reaction was confirmed to be complete by LC-MS. The solution was concentrated under reduced pressure and purified by reversed-phase column chromatography to obtain 120 mg of a yellow solid, with a yield of 50.3%. 1 H NMR (400MHz, DMSO) δ12.97(s,1H),8.84(s,2H),8.64(d,J=42.2Hz,1H),7.84(d,J=10.9Hz,1H),7.55(s,1H),7. 39(d,J=23.0Hz,2H),7.18(d,J=55.4Hz,1H),6.58(s,1H),5.76(d,J=4.0Hz,1H),5.54(d,J=37.0Hz,2H),5.45(d ,J=4.9Hz,2H),5.31(d,J=19.4Hz,1H),5.09(d,J=56.4Hz,2H),4.80–4.15(m,2H),3.73(s,6H),3.21(d,J=25.9H z,10H),3.11(s,3H),2.42(s,3H),2.21–1.99(m,2H),1.89(hept,J=7.8Hz,2H),0.88(d,J=7.3Hz,3H).(ESI)m / z 931.9[M+H] +

[0150] Synthesis of Compound 19

[0151] Compound 18 (100 mg, 113.3 μmol) was placed in a round-bottom flask, and 2 mL of anhydrous DMF was added. Compound 10 (102.9 mg, 98.5 μmol) and DIPEA (31.8 mg, 246.2 μmol) were then added sequentially. The reaction was carried out at room temperature for 6 hours, and LC-MS was used to confirm completion. The mixture was purified by reversed-phase column chromatography to give 81 mg of a pale yellow solid, with a yield of 49.9%. 1H NMR(400MHz, DMSO-d6)δ10.17(s,1H),8.31(d,J=9.1Hz,1H),8.06–8.00(m,2H),7.81–7 .72(m,3H),7.61(dd,J=7.9,1.3Hz,2H),7.49(td,J=7.7,1.3Hz,2H),7.41(td,J=7.8,1. 5Hz,2H),7.35–7.25(m,3H),7.13(d,J=9.3Hz,1H),5.90(s,1H),5.40(dd,J=2.1,0.9Hz, 2H),5.35(d,J=5.9Hz,1H),5.17(d,J=6.2Hz,1H),5.11–5.03(m,2H),4.90(ddd,J=9.1,6 .5,3.7Hz,1H),4.55(d,J=15.0Hz,1H),4.44(d,J=4.8Hz,2H),4.35(d,J=15.1Hz,1H),4 .22(s,2H),4.12(dt,J=8.7,6.2Hz,1H),3.95–3.82(m,2H),3.70–3.62(m,4H),3.65–3.4 0(m,41H),2.70(ddd,J=12.3,8.5,5.9Hz,1H),2.56(ddd,J=12.3,8.5,5.9Hz,1H),2.29( s,3H),2.44–1.89(m,7H),1.72(dq,J=13.6,8.0Hz,1H),0.86(t,J=8.0Hz,3H).(ESI)m / z 1648.7[M+H] +

[0152] Synthesis of Compound 20

[0153] Compound 19 (81 mg, 49.16 μmol) was placed in a round-bottom flask, and 10 mL of THF and 2 mL of diethylamine were added. The mixture was reacted at room temperature for 4 hours. The reaction was confirmed to be complete by LC-MS. The product was purified by reversed-phase column chromatography and lyophilized to give 51 mg of a yellow solid, with a yield of 72.8%. 1H NMR(400MHz, DMSO-d6)δ10.17(s,1H),8.31(d,J=9.1Hz,1H),8.07–7.99(m,2H), 7.76(dd,J=9.3,2.0Hz,1H),7.35–7.25(m,2H),7.13(d,J=9.3Hz,1H),5.90(s,1 H),5.40(dd,J=2.1,0.9Hz,2H),5.35(d,J=5.9Hz,1H),5.17(d,J=6.2Hz,1H),5. 11–4.99(m,4H),4.90(ddd,J=9.1,6.5,3.7Hz,1H),4.55(d,J=15.0Hz,1H),4.35( d,J=15.0Hz,1H),4.22(s,2H),3.95–3.85(m,1H),3.89–3.82(m,1H),3.70–3.63 (m,4H),3.67–3.53(m,28H),3.56–3.51(m,4H),3.53–3.40(m,12H),3.25(s,2H), 2.70(ddd,J=12.3,8.5,5.9Hz,1H),2.56(ddd,J=12.3,8.5,5.9Hz,1H),2.37–2. 25(m,1H),2.25–1.89(m,5H),1.88–1.65(m,2H),0.86(t,J=8.0Hz,3H).(ESI)m / z 1425.6[M+H] +

[0154] Synthesis of compound 1-5-4

[0155] Compound 18 (30 mg, 21.1 μmol) was placed in a round-bottom flask, and 2 mL of anhydrous DMF was added. Then, PEG4-maleimide OSU active ester (12.4 mg, 24.2 μmol) and DIPEA (6.8 mg, 52.6 μmol) were added sequentially. The reaction was carried out at room temperature for 2 hours, and the reaction was confirmed to be complete by LC-MS. The product was purified by reversed-phase column chromatography to give 25 mg of a pale yellow solid, with a yield of 65.1%. 1H NMR (400MHz, DMSO) δ8.82–8.08(m,1H),7.78(d,J=6.1Hz,2H),7.67(s,1H),7.55(d,J=11.0H z,1H),7.21(t,J=21.5Hz,1H),7.15–6.83(m,3H),6.75(s,2H),5.32(s,1H),5.19(s,2H),5. 08–4.71(m,3H),4.51–3.87(m,2H),3.81–3.30(m,29H),3.04–2.52(m,19H),2.27(s,5H),2. 22–1.77(m,12H),1.74–1.39(m,5H),1.29(d,J=7.0Hz,1H),0.64(d,J=7.6Hz,3H).(ESI)m / z 1824.9[M+H] +

[0156] Example 5: Preparation of ADC Compounds

[0157] Weigh out two 10 mg portions of trastuzumab, add 10 equivalents of TCEP, react at 37°C for 1 hour, and detect complete ring opening of the monoclonal antibody by RPLC. Add 10 equivalents of compounds 1-5-1 and 1-5-2 prepared in this invention, react at 25°C for 4 hours, and detect complete coupling by RPLC. Use a 0.2 μm filter membrane to remove aggregates, and use an ultrafiltration tube to ultrafilter 6 times to remove excess small molecules, to obtain the target ADC compounds HER2-1-5-1 and HER2-1-5-2.

[0158] Example 6 Stability Experiment

[0159] The solutions of the corresponding ADC compounds (HER2-1-5-1 or HER2-1-5-2) prepared in Example 5 of this invention were added to plasma to achieve an effector molecule concentration of 25 μmol / L, and incubated on a shaker at 37°C. On days 1, 3, 5, and 7, 60 μL of the corresponding sample (n=3) was taken, precipitated with 240 μL of cold methanol, centrifuged at 4°C for 25 minutes, and 80 μL of the supernatant was taken. The content of camptothecin derivatives was detected by HPLC.

[0160] The experimental results are shown in the table below. The results indicate that the ADC compounds HER2-1-5-1 and HER2-1-5-2 can be stably present in plasma within 7 days.

[0161] Percentage of HER2-1-5-1 effector molecules released in plasma

[0162]

[0163] Percentage of HER2-1-5-2 effector molecules released in plasma

[0164]

[0165] Example 7 Enzyme release experiment

[0166] A solution (100 μM, pH 7.4 or pH 5.0) of the compound (1-5-1) synthesized in this invention was added to N-acetylcysteine ​​to quench maleimide, and then mixed with β-glucuronidase (300 U) and incubated in a shaker at 37°C. Samples (n=3) were collected at 5 min, 10 min, 15 min, 30 min, 60 min, 120 min, 240 min, and 360 min, precipitated with cold acetonitrile, centrifuged at 4°C for 25 min, and the supernatant was collected. The content of camptothecin derivatives was determined by HPLC.

[0167] The experimental results are shown in Figure 1 The results showed that within 4 hours, under pH 7.4 or pH 5.0 conditions, the camptothecin derivative could be released 100% under the triggering of β-glucuronidase, indicating that the compound (1-5-1) of the present invention can rapidly release the drug, converting the prodrug into camptothecin molecules with antitumor activity.

[0168] Example 8: Conjugation of Compound 1-5-1 with Her2 Antibody

[0169] The compound 1-5-1 synthesized in this invention was conjugated with the Her2 antibody, and the results are shown in the figure. Figure 2 This indicates that compound 1-5-1 and Her2 antibody can be successfully conjugated, with a DAR value of 7.9.

[0170] Example 9: Antitumor effect in nude mice with NCI-N87 xenograft tumors

[0171] The ADC compounds HER2-1-5-1 and HER2-1-5-2 prepared in Example 5 of this invention were subjected to anti-tumor experiments in a mouse NCI-N87 tumor model. By day 10, the tumor volume had grown to 120 mm. 3 The medication was administered once, at a dose of 3 mg / kg. Results were as follows: Figure 3 The ADC compounds HER2-1-5-1 and HER2-1-5-2 prepared in Example 5 of this invention both have significant anti-tumor effects and are superior to the positive control HER2-GGFG-Dxd.

[0172] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0173] As used in this invention, the term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention but does not exclude other aspects.

[0174] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0175] Although embodiments of the present description have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present description, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A cleavable linker compound for use in prodrug delivery systems, and its pharmaceutically acceptable salts, solvates, polymorphs, or isomers, characterized in that, The structure of the cleavable linker compound is shown in formula (1-1): R1 is selected from β-glucanide, β-galactoside, phosphate ester, sulfate ester, and pyrophosphate ester; Where R2 is H or CH3(CH2) n n = 0-20; Among them, compound D contains hydroxyl groups, and compound D has exposed -OH groups; Where L represents W1-W2-W3; W1 is selected from -O-, -N-; W2 is selected from C1-C12 straight-chain or branched carbon chains, C3-C8 carbon ring chains, C3-C8 heterocyclic chains, straight-chain or branched PEG chains, sugar chains, polyamino acid chains, and mixed chains of straight-chain or branched PEG chains and polysarcosine; W3 is selected from linking groups that react with thio, amino, azide, alkynyl, or maleimide groups, and is selected from...

2. The pyrolytic linker compound according to claim 1, characterized in that, When W1 in L is selected as -N-, the structure of the cleavable linker compound is shown in formula (1-2): R1 is selected from β-glucanide, β-galactoside, phosphate ester, sulfate ester, and pyrophosphate ester; R2 is selected from H or CH3(CH2). n n = 0-20; D is selected from compounds of class D containing a hydroxyl group, wherein compound D has a naked -OH group. R3 is selected from H, C1-C6 alkyl, or cycloalkyl; L1 is selected from straight or branched carbon chains of C1-C12, carbon ring chains of C3-C8, heterocyclic chains of C3-C8, straight or branched PEG chains, sugar chains, polyamino acid chains, and mixed chains of straight or branched PEG chains and polysarcosine. R3 and L1 can be optionally connected together to form 3 to 6 loops. The looping of R3 and L1 can be selected from... The L2 is selected from straight-chain or branched carbon chains of C1-C12, carbon ring chains of C3-C8, heterocyclic chains of C3-C8, straight-chain or branched PEG chains, sugar chains, polyamino acid chains, and mixed chains of straight-chain or branched PEG chains and polysarcosine. W3 is selected from the linking group that reacts with thio, amino, azide, alkynyl, or maleimide. R4 is selected from one or more of H, C1-C6 alkyl or cycloalkyl.

3. The linker compound according to claim 2, characterized in that, When R1 is selected from β-glucan, the structure of the cleavable linker compound is shown in formula (1-3): Where R2 is H or CH3(CH2). n n = 0-20; Wherein, D is a class of compounds containing hydroxyl groups, and the compound D has exposed -OH groups; R3 is selected from H, C1-C6 alkyl or cycloalkyl; L1 is selected from straight or branched carbon chains of C1-C12, carbon ring chains of C3-C8, heterocyclic chains of C3-C8, straight or branched PEG chains, sugar chains, polyamino acid chains, and mixed chains of straight or branched PEG chains and polysarcosine. R3 and L1 can be optionally connected together to form 3 to 6 loops. The looping of R3 and L1 can be selected from... R4 is selected from one or more of H, C1-C6 alkyl or cycloalkyl; The L2 is selected from straight-chain or branched carbon chains of C1-C12, carbon ring chains of C3-C8, heterocyclic chains of C3-C8, straight-chain or branched PEG chains, sugar chains, polyamino acid chains, and mixed chains of straight-chain or branched PEG chains and polysarcosine. L2 is selected from straight-chain or branched carbon chains of C1-C12, carbon ring chains of C3-C8, heterocyclic chains of C3-C8, straight-chain or branched PEG chains, sugar chains, polyamino acid chains, and mixed chains of straight-chain or branched PEG chains and polysarcosine. W3 is selected from the linking group that reacts with thio, amino, azide, alkynyl, or maleimide.

4. The linker compound according to claim 3, characterized in that, The cleavable linker compounds include compounds represented by formulas (1-4-1), (1-4-2), and (1-4-3) as follows: In the above equations (1-4-1)-(1-4-3), n, m, and t represent the number of repeating units, where n is selected from 1 to 24 and m is selected from 0 to 12. In equations (1-4-1) to (1-4-3), L is selected from: One or more of the following; In equations (1-4-1) to (1-4-3), Q1 and Q2 are respectively selected from PEG fragments and polysarcosine fragments; In the formula, D represents an active compound containing a hydroxyl group, and the active compound is a drug, toxin, affinity ligand, detection probe or a combination thereof; The drug is selected from the group consisting of topoisomerase I inhibitors (camptothecin derivatives), immune agonists, alkylating agents, dihydrofolate reductase inhibitors, thymidine synthase inhibitors, purine nucleoside synthase inhibitors, nucleoside reductase inhibitors, DNA polymerase inhibitors, RNA polymerase inhibitors, EGFR inhibitors, ALK inhibitors, MET inhibitors, CDK4 / 6 inhibitors, EG5 inhibitors; and pharmaceutically acceptable salts, solvates, acids, or derivatives thereof, with the structure shown below:

5. The pyrolytic linker compound according to claim 4, characterized in that, The compounds of formulas (1-4-1), (1-4-2), and (1-4-3) include the following compounds (1-5-1), (1-5-2), (1-5-3), and (1-5-4):

6. A method for preparing compound formula (1-2) as described in claim 2, characterized in that, The preparation reaction process of the method is shown in the following formula: The steps are as follows: a) Substitution reaction: In the first solvent, under the action of silver oxide, compound 1 undergoes a nucleophilic substitution reaction with acetylated β-glucan or β-galactoside to generate compound 2; b) Condensation reaction: In the second solvent, compound 2 and amine undergo a condensation reaction under the action of a condensing agent to generate compound 3; c) Reduction reaction: In the third solvent, compound 3 undergoes a reduction reaction under a hydrogen atmosphere by the action of platinum dioxide or Raney nickel to generate compound 4; d) Substitution reaction: In the fourth solvent, compound 4 undergoes a substitution reaction with a halogenated compound under basic conditions to generate compound 5; e) Condensation reaction: Under nitrogen protection, compound 5 was dissolved in a fourth solvent, and di(trichloromethyl) carbonate and anhydrous organic base were added. After reacting for 1 hour, drug (D) and DMAP were added, and the reaction was carried out overnight at 20-60°C to obtain compounds 1-2.

7. A method for preparing a compound of formula (1-4-1), characterized in that, The reaction process of the preparation method is shown in the following formula: The steps are as follows: a) Condensation reaction: Under nitrogen protection, compound 1 was dissolved in an organic solvent, and di(trichloromethyl) carbonate and triethylamine were added. After reacting for 1 hour, a hydroxyl-containing drug was added, and the reaction was carried out overnight at room temperature to obtain compound 2. b) Hydrolysis reaction: Compound 2 was dissolved in acetonitrile and water, an inorganic base was added, and after reacting for 2 hours, the pH was adjusted to acidic, and the solution was passed through a column to obtain compound 3; c) Condensation reaction: Compound 3 and Compound 4 are dissolved in an organic solvent and stirred at room temperature for 6-8 hours to generate a compound with the general formula (1-4-1).

8. A drug and / or a drug composition, characterized in that, It includes the compounds of any one of claims 1-5 and their pharmaceutically acceptable salts, solvates, polymorphs or isomers and pharmaceutically acceptable carriers.

9. The use of the linker compound as described in any one of claims 1-5 and its pharmaceutically acceptable salt, solvate, polymorph or isomer, or the drug and / or pharmaceutical composition as described in claim 8 in the preparation of an antitumor drug, or in the preparation of an antibody or albumin-bound antitumor drug, or in the preparation of an antibody-drug conjugate.

10. The application as described in claim 9, characterized in that, The applications include the preparation of antibody-coupled β-glucuronidase prodrugs for tumor treatment.

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