RU2840789C2 - Antibody-drug conjugate intermediate comprising sn38, and method for preparation thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to an intermediate compound of an antibody-drug conjugate containing SN38, and a method for production thereof. Water-soluble group of PEG, having biological activity, is introduced into linker structure and structural modification of PEG is carried out. Method is simple and cheap and environmentally safe since heavy metal ions are not involved in the reaction.

EFFECT: as compared to antibody-drug conjugate intermediate compounds in which SN38 is a toxin in existing technology, use of the intermediate compound of the antibody-drug conjugate synthesized by said method provides higher stability and efficacy.

24 cl, 3 dwg, 6 tbl, 7 ex

Description

AREA OF TECHNOLOGY

[0001] The present disclosure relates to the field of antibody-drug conjugates and, in particular, to an antibody-drug conjugate intermediate and a method for producing the same.

LEVEL OF TECHNOLOGY

[0002] Antibody-drug conjugate (ADC), as a new biological targeted drug, realizes a powerful combination of the targeting effect of monoclonal antibodies with the cytotoxicity of small molecule drugs, and has become one of the fastest growing areas of targeted tumor therapy. The three components of ADC, antibody, cytotoxin and linker, together constitute a targeted drug delivery system, where the antibody provides the targeting effect, the linker ensures the stability of the ADC during transport in the blood, and the toxin exerts its killing effect on cancer cells after reaching the target. Currently, more than 60 ADC-based drugs are undergoing clinical trials for antitumor therapy. Among them, most of the toxins are tubulin inhibitors, and a small part are DNA inhibitors. DNA inhibitors have two advantages over tubulin inhibitors: 1) DNA inhibitors (picomolar IC50 values) are more active than tubulin inhibitors (subnanomolar IC50 values) and have better effects in treating tumors with few antigens; 2) they can kill cancer cells that are not in the division phase and have advantages in treating solid tumors.

[0003] Camptothecin compounds are clinically used topoisomerase 1 (TOP1) inhibitors and have good clinical effects on slow-growing solid tumors. However, the special structure of camptothecin results in its poor water solubility and lipid solubility, so it needs to be modified to ensure water solubility. ADC-based drugs with camptothecin as the target element offer a new solution to the above-mentioned shortcomings.

[0004] Currently, two camptothecin-derived ADCs are approved for sale: Enhertu (trastuzumab deruxtecan) and Trodelvy (sacituzumab govitecan). They fill a large clinical need in the treatment of tumors, especially malignancies. In Enhertu, developed by AstraZeneca/Daiichi Sankyo, the cathepsin B-activated tetrapeptide GGFG is used as a linker and a small self-cleaving structure is introduced to release Dxd, an exatecan derivative. In HER2-positive metastatic breast cancer, 99 patients treated with Enhertu had an objective remission rate of 54.5% and a disease control rate of 93.9%. Sacituzumab govitecan contains the Mcc-triazole spacer-PEG7-x-lysine-PABC linker, which is cleaved in lysosomes (pH approximately 5) in cells to release camptothecin (SN38). Phase II clinical results for sacituzumab govitecan in triple-negative breast cancer showed an efficacy of at least 30% and tumor shrinkage in 69.5% of patients, despite the fact that triple-negative breast cancer is virtually “incurable” clinically. Additionally, in small cell lung cancer, where multiple therapies have failed, sacituzumab govitecan can shrink tumors in up to 60% of patients. In non-small cell lung cancer, where chemotherapy, targeted therapy, and PD-1 treatment have failed, sacituzumab govitecan demonstrated a control rate of up to 43%. However, in clinical studies, despite the significant therapeutic effect, serious safety problems caused by ADC toxicity, such as hematological toxicity, neurotoxicity, pulmonary toxicity, skin toxicity, hepatotoxicity, ocular toxicity, metabolic disorders and cardiotoxicity, also arise. Therefore, improving the safety of ADC-based drugs is an issue that needs serious attention and solution in the ongoing drug development.

[0005] In addition, there is also a problem of difficult removal of metal ion reaction products during the synthesis of the linker and loading of some ADCs. For example, CL2A-SN38 used in sacituzumab govitecan involves a click chemistry reaction during the synthesis process (see Chinese Patent Application Publication No. CN102448494A, paragraph [0273] on page 50 of the description), which results in the presence of Cu ²⁺ in the reaction products, which is not easy to remove.

[0006] The linker plays a vital role in the structure of ADC and affects the pharmacokinetic parameters, therapeutic indices and efficacy of ADC. The linker can maintain the stability of the ADC complex in the blood. When designing the linker, stability should be primarily considered. Since the ADC-based drug has a long half-life, the stability of the linker is necessary to prevent the linker from being degraded in the blood with premature release of toxins. If the linker is not stable enough in the blood, it will lead to the decomposition of the ADC before it enters the tumor cells, in which case the drug will have a reduced effect on the tumor and even mistakenly kill other cells. Moreover, in the process of internalization of ADC by tumor cells, the linker should be able to quickly release cytotoxic drugs. As a result, the linker plays a significant decisive role in the safety and efficacy of the ADC-based drugs being developed. In addition, different linker structures also greatly affect the complexity factor, production cost and environmental friendliness of the ADC production process, and greatly affect the quality of ADC-based drugs, such as the stability of ADCs, which further affects the safety of ADC-based drugs.

ESSENCE INVENTIONS

[0007] The present invention provides an intermediate compound of an antibody-drug conjugate containing SN38 and a method for producing the same. The production method is simple in steps and economical, does not introduce heavy metal ions into reactions with increased environmental friendliness. Moreover, the intermediate compound of the antibody-drug conjugate synthesized by this production method has good stability, which significantly improves the safety of ADC-based drugs.

[0008] The present description relates to an intermediate antibody-drug conjugate represented by formula (I):

(I)

Where:

X ₁ is an alkane chain or a PEG chain,

X ₂ is H or -C(O)NR ¹ R ² , and

R ¹ is selected from the group consisting of hydrogen, halogen, hydroxyl, substituted or unsubstituted C _1-6 alkyl, substituted or unsubstituted C _1-6 hydroxyalkyl, substituted or unsubstituted C _1-6 aminoalkyl, substituted or unsubstituted C _1-6 alkoxyl, substituted or unsubstituted C _1-6 alkylacyl, and substituted or unsubstituted C _1-6 alkylaldehyde;

R ² is selected from the group consisting of hydrogen and substituted or unsubstituted C _1-6 alkyl; wherein each substituted C _1-6 alkyl is optionally substituted with 1-5 substituents independently selected from the group consisting of halogen, hydroxyl, amino, carbonyl, carboxyl, 5-10-membered heterocyclyl and C _1-6 haloalkyl, wherein the 5-10-membered heterocyclyl has 1-3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur.

[0009] Preferably, X ₁ is selected from the group consisting of -(CH ₂ ) _m - and -(CH ₂ CH ₂ O) _p -, where m is selected from 1, 2, 3, 4, 5 and 6, preferably m is 5; where p is selected from 1, 2, 3, 4, 5 and 6, preferably P is 2.

[0010] Preferably, X ₁ is selected from the group consisting of:

[0011] Preferably, R ¹ or R ² is selected from the group consisting of: H, methyl, ethyl, propyl, butyl, pentyl, heptyl, Cl, Br, , , , And .

[0012] Preferably, X ₂ is selected from the group consisting of:

[0013] Preferably, the intermediate antibody-drug conjugate compound has a structure represented by formula (1) to (16):

(1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15),
And (16).

[0014] The present disclosure also provides a method for producing an antibody-drug conjugate intermediate.

[0015] In addition, the intermediate antibody-drug conjugate compound has the structure:

(4), or

(12), or

(I-1), or

(I-2).

[0016] R ¹ is selected from the group consisting of hydrogen, substituted or unsubstituted C _1-6 alkyl, substituted or unsubstituted C _1-6 hydroxyalkyl, substituted or unsubstituted C _1-6 aminoalkyl, substituted or unsubstituted C _1-6 alkoxyl, substituted or unsubstituted C _1-6 alkylacyl, and substituted or unsubstituted C _1-6 alkylaldehyde;

[0017] R ² is selected from the group consisting of hydrogen and substituted or unsubstituted C _1-6 alkyl; wherein each substituted C _1-6 alkyl is optionally substituted with 1-5 substituents independently selected from the group consisting of halogen, hydroxyl, amino, carbonyl, carboxyl, 5-10 membered heterocyclyl, and C _1-6 haloalkyl, wherein the 5-10 membered heterocyclyl has 1-3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur.

[0018] Preferably, R ¹ or R ² is selected from the group consisting of: H, methyl, ethyl, propyl, butyl, pentyl, heptyl, methoxyl, ethoxyl, Cl, Br, , , , , And .

[0019] In addition, the compound of formula (4) is obtained as follows:

Reaction A: dissolving compound a and compound b in a solvent, stirring at room temperature for an appropriate time, adding a reducing agent at a low temperature, stirring for an appropriate time, and then stirring at room temperature overnight to carry out the reaction; after completion of the reaction, drying the solvent by rotation, and then extracting, drying and purifying;

Reaction B: dissolving SN38 and DNPC in a solvent, adding an organic base and stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then triturating and filtering;

Reaction C: dissolving the product obtained by reaction B in a solvent, adding the product obtained by reaction A and an organic base, stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying;

Reaction D: dissolving the product obtained by reaction C in a solvent, adding an acid and stirring at a low temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation, dissolving Mc-VC-PAB-PNP in the solvent, stirring at a low temperature for an appropriate time, and adding an organic base to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying.

[0020] Reaction process 1:

[0021] In addition, the compound of formula (12) is obtained as follows:

Reaction A: dissolving compound a and compound b in a solvent, stirring at room temperature for an appropriate time, adding a reducing agent at a low temperature, stirring for an appropriate time, and then stirring at room temperature overnight to carry out the reaction; after completion of the reaction, drying the solvent by rotation, and then extracting, drying and purifying;

Reaction B: dissolving SN38 and DNPC in a solvent, adding an organic base and stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then triturating and filtering;

Reaction C: dissolving the product obtained by reaction B in a solvent, adding the product obtained by reaction A and an organic base, stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying;

Reaction D: dissolving the product obtained by reaction C in a solvent, adding an acid and stirring at a low temperature for an appropriate time to carry out the reaction; after completion of the reaction, spin-drying the solvent, dissolving MP2-VC-PAB-PNP in the solvent, stirring at a low temperature for an appropriate time, and adding an organic base to carry out the reaction; after completion of the reaction, spin-drying the solvent and then purifying.

[0022] Reaction process 2:

[0023] In addition, the compound of formula (I-1) is obtained as follows:

Reaction A: dissolving compound a and compound b in a solvent, stirring at room temperature for an appropriate time, adding a reducing agent at a low temperature, stirring for an appropriate time, and then stirring at room temperature overnight to carry out the reaction; after completion of the reaction, drying the solvent by rotation, and then extracting, drying and purifying;

Reaction B: dissolving SN38 and DNPC in a solvent, adding an organic base and stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then triturating and filtering;

Reaction C: dissolving the product obtained by reaction B in a solvent, adding the product obtained by reaction A and an organic base, and stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying;

Reaction D: dissolving the product obtained by reaction C in a solvent, adding bis(p-nitrophenyl)carbonate and an organic base, and stirring at a constant temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying;

Reaction E: dissolving the product obtained by reaction D and amine in a solvent, adding an organic base and stirring at a low temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying;

Reaction F: dissolving the product obtained by Reaction E in a solvent, adding an acid and stirring at a low temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation, dissolving Mc-VC-PAB-PNP in the solvent, stirring at a low temperature for an appropriate time, and adding an organic base to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying.

[0024] Reaction process 3:

[0025] In addition, the compound of formula (I-2) is obtained as follows:

Reaction A: dissolving compound a and compound b in a solvent, stirring at room temperature for an appropriate time, adding a reducing agent at a low temperature, stirring for an appropriate time, and then stirring at room temperature overnight to carry out the reaction; after completion of the reaction, drying the solvent by rotation, and then extracting, drying and purifying;

Reaction B: dissolving SN38 and DNPC in a solvent, adding an organic base and stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then triturating and filtering;

Reaction C: dissolving the product obtained by reaction B in a solvent, adding the product obtained by reaction A and an organic base, and stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying;

Reaction D: dissolving the product obtained by reaction C in a solvent, adding bis(p-nitrophenyl)carbonate and an organic base, and stirring at a constant temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying;

Reaction E: dissolving the product obtained by reaction D and amine in a solvent, adding an organic base and stirring at a low temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying;

Reaction F: dissolving the product obtained by Reaction E in a solvent, adding an acid and stirring at a low temperature for an appropriate time to carry out the reaction; after completion of the reaction, spin-drying the solvent, dissolving MP2-VC-PAB-PNP in the solvent, stirring at a low temperature for an appropriate time, and adding an organic base to carry out the reaction; after completion of the reaction, spin-drying the solvent and then purifying.

[0026] Reaction process 4:

[0027] Furthermore, compound (I-1) is selected from the group consisting of:

(1), (2), (3), (5), (6), (7)
And (8),

compound (I-2) is selected from the group consisting of:

(9), (10), (11), (13), (14), (15)
And (16).

[0028] Also, "at low temperature" in any of the above reaction processes refers to an ice water bath.

[0029] Furthermore, the solvent in any of the above reaction processes may independently be a polar solvent and/or a non-polar solvent, wherein the polar solvent is selected from the group consisting of THF, DMF, DMA, NMP and mixtures thereof; and the non-polar solvent is selected from the group consisting of dichloromethane, carbon tetrachloride and mixtures thereof.

[0030] Furthermore, the organic base in any of the above reaction processes may be independently selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine and a mixture thereof, preferably one or both of N,N-diisopropylethylamine and pyridine.

[0031] In addition, the acids are one or two acids selected from the group consisting of hydrochloric acid, trifluoroacetic acid and citric acid.

[0032] In addition, the amine is a primary amine or a secondary amine.

[0033] In addition, in reaction A, extraction is carried out with ethyl acetate, and purification is carried out by column chromatography using dichloromethane and methanol as an eluent.

[0034] In addition, in reaction B, trituration is carried out using ethyl acetate, hexane, dichloromethane, or a combination thereof.

[0035] In addition, in reaction C, purification is carried out by column chromatography using dichloromethane and methanol as eluent.

[0036] In addition, in reaction D, purification is carried out by column chromatography using dichloromethane and methanol as eluent.

[0037] In addition, in reaction E, purification is carried out by column chromatography using dichloromethane and methanol as eluent.

[0038] In addition, in reaction F, purification is carried out by preparative liquid chromatography using MeCN and 0.1% HCOOH as mobile phase A, and H ₂ O and 0.1% HCOOH as mobile phase B.

[0039] In addition, all reactions are carried out under nitrogen protection.

[0040] The method for producing an intermediate of an antibody-drug conjugate (particularly a covalent linker-SN38 conjugate) provided by the present disclosure, in addition to simple steps, reduces safety problems caused by heavy metal residues. Moreover, the antibody-drug conjugate obtained using the intermediate of the antibody-drug conjugate has higher stability in vivo.

[0041] Furthermore, the inventor unexpectedly discovered that an antibody-drug conjugate obtained by combining a linker and SN38 as described herein can exert a significant tumor inhibitory effect.

Brief description drawings

[0042] FIG. 1 shows the change in body weight of mice;

[0043] FIG. 2 shows the change in tumor volume;

[0044] FIG. 3 shows the relative tumor volume.

DETAILED DESCRIPTION

[Abbreviations]

[0045] Unless otherwise specified, all abbreviations used in this description have the same meaning as understood by those skilled in the art. In this description, commonly used abbreviations and their definitions are shown as follows:

Reduction Definition DCC N,N' -Dicyclohexylcarbodiimide EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride DICK N,N' -Diisopropylcarbodiimide GATU 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate GBTU Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate HBPIPU (Benzotrizol-1-yloxy)dipiperidinocarbenium hexafluorophosphate HBPyU O-(benzotriazol-1-yl)-N,N,N',N'-dipyrroluronium hexafluorophosphate GHTU 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate HDMA 1-[(Dimethylamino)(morpholino)methyl]-3-oxo-1H-[1,2,3]triazolo[4,5-b]pyridine-3-hexafluorophosphate TATTOO 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium tetrafluoroborate TBTU O-(benzotriazole)-N,N,N',N'-tetramethyluronium tetrafluoroborate TCTU O-(6-chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate TCFH N,N,N ', N' -tetramethylchloroformamidinium hexafluorophosphate TDBTU N,N,N',N'-tetramethyl-O-(4-carbonyl-3,4-dihydro-1,2,3-benzotriazin-3-yl)uronium tetrafluoroborate TFFH Fluoro-N,N,N',N'-tetramethyluronium hexafluorophosphate BTFFF N,N,N',N'-bis(tetramethylene)fluoroformamidinium hexafluorophosphate TSTU 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate PyBOP 1H-Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate PyCIOP Chlorotripyrrolidinophosphonium hexafluorophosphate PyAOP (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tri-1-pyrrolidinylphosphonium hexafluorophosphate PyCIU 1-(chloro-1-pyrrolidinylmethylene)pyrrolidinium hexafluorophosphate DEPBT 3-(Diethoxy- o -acyloxy)-1,2,3-benzotriazin-4-one EEDQ 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline Mc Maleimidohexanoyl SN38 Camptothecin DMF N,N-dimethylformamide DMA Dimethylacetamide DNPC Bis(p-nitrophenyl)carbonate DMSO Dimethyl sulfoxide THF Tetrahydrofuran DXM Dichloromethane DIPEA N,N-diisopropylethylamine TFC Trifluoroacetic acid

[Definitions]

[0046] The description and claims use various terms relating to various aspects of the description. Unless otherwise indicated, such terms are given their ordinary meaning in the art. Other specifically defined terms are to be understood in accordance with the definitions provided herein.

[0047] As used herein, the terms "a," "an," and "the" are used in accordance with standard practice and refer to one or more unless the context otherwise requires. Thus, for example, reference to "an antibody-drug conjugate" includes combinations of two or more antibody-drug conjugates, and so forth.

[0048] It should be understood that wherever the expression "comprising" is used herein to describe an aspect, similar aspects described as "consisting of......" and/or "consisting essentially of......" are also contemplated.

[0049] The term "antibody-drug conjugate" as used herein is a compound in which an antibody/functional antibody fragment, a linker, and a drug moiety are linked to each other by a chemical reaction, and its structure typically consists of three parts: an antibody or antibody ligand, a drug moiety, and a linker that links the antibody or antibody ligand and the drug. Currently, the preparation of antibody-drug conjugates typically involves two steps: in the first step, the linker and the drug moiety are chemically reacted to form a linker-drug conjugate, and in the second step, the linker portion of the linker-drug conjugate is covalently coupled to the antibody/functional antibody fragment via a sulfhydryl group or an amino group. The term "antibody-drug conjugate intermediate" as used herein refers to the "linker-drug" conjugate described above. In addition, the term "antibody-drug conjugate intermediate" as used herein refers to the covalent conjugate of the "linker" and SN38 as a whole.

[0050] The "antibody-drug conjugate" as used herein is prepared by a method conventional in the art. For example, the antibody-drug conjugate of the present disclosure is prepared by: preparing a 10 mg/mL solution of the antibody with PBS buffer at pH 7.4, adding an appropriate equivalent of TCEP, shaking for 1 hour to thoroughly mix, then adding 5.0 molar equivalents of the linker-toxin ( i.e., the compound shown in Formula 1 to Formula 16), shaking to thoroughly mix the reaction for 1 hour, removing residual small molecules by ultrafiltration after completion of the reaction, and loading into a hydrophobic interaction-high performance liquid chromatography (HIC-HPLC) system to determine the DAR, drug partitioning, and antibody ratio.

[0051] It is understood that the present disclosure is aimed at proposing a new intermediate compound of a drug conjugate, and a typical preparation of several ADCs with Heceptin is proposed to confirm the technical effect. The present disclosure has no limitation on the choice of antibody, and the structure of specific ADCs is provided as an example. ADC-1 has a linker and a toxin corresponding to the compound of formula (1), and is produced with Heceptin by the above-mentioned generally accepted method in the art for producing an "antibody-drug conjugate" (hereinafter the same: ADC-2 has a linker and a toxin corresponding to the compound of formula (2); ADC-3 has a linker and a toxin corresponding to the compound of formula (3); ADC-4 has a linker and a toxin corresponding to the compound of formula (4); ADC-5 has a linker and a toxin corresponding to the compound of formula (5); ADC-6 has a linker and a toxin corresponding to the compound of formula (6); ADC-7 has a linker and a toxin corresponding to the compound of formula (7); ADC-8 has a linker and a toxin corresponding to the compound of formula (8); ADC-9 has a linker and a toxin corresponding to the compound of formula (9); ADC-10 has a linker and a toxin corresponding to the compound of formula (10); ADC-11 has a linker and a toxin corresponding to a compound of formula (11); ADC-12 has a linker and a toxin corresponding to a compound of formula (12); ADC-13 has a linker and a toxin corresponding to a compound of formula (13); ADC-14 has a linker and a toxin corresponding to a compound of formula (14); ADC-15 has a linker and a toxin corresponding to a compound of formula (15); ADC-16 has a linker and a toxin corresponding to a compound of formula (16); and ADC-17 has a linker and a toxin corresponding to CL2A-SN38), wherein q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, ADC-6, ADC-7, ADC-8, ADC-9, ADC-10, ADC-11, ADC-12, ADC-13, ADC-14, ADC-15, ADC-16, ADC-17, CL2A-SN38.

[0052] The term "linker" as used herein refers to a molecule having bifunctional or multifunctional groups that can react with a protein/antibody molecule and SN38, respectively, and thus act as a "bridge" linking the protein/antibody to SN38. A linker as used herein specifically refers to a group containing an acyl in the structure.

[Examples]

[0053] The present description is further illustrated below in conjunction with specific examples. It should be understood that these examples are used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not indicated, are generally carried out under conventional conditions or under conditions recommended by the manufacturer, and the reagents of unknown origin are conventional reagents that are commercially available. All percentages, rates, ratios, or parts are by weight unless otherwise indicated.

[0054] Unless otherwise specified, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present disclosure. The preferred examples and materials described herein are for illustrative purposes only.

[0055] Example 1 Preparation of a compound of formula (12)

[0056] 1) Synthesis of compound c (2-Boc-N-methylethylethyleneglycol)

[0057] 0.6 g of compound b (aminoethylene glycol) and 1.0 g of compound b (N-Boc-acetaldehyde) were dissolved in 20 ml of methanol and stirred at room temperature for 6 h. The reaction system was placed in an ice water bath, sodium triacetoxyborohydride was added, stirred in an ice water bath for 1 h, then returned to room temperature and stirred overnight. The product was detected by LC-MS and showed no UV absorption. The solvent was spin dried and the residue was extracted twice with ethyl acetate, dried over anhydrous Na ₂ SO ₄ , spin dried, purified by column chromatography using dichloromethane:methanol 20:1 as eluent, and rinsed to obtain 1.0 g of the product in 66% yield. LC-MS: (M+H) ⁺ 262.6.

[0058] 2) Synthesis of SN38-PNP (10-p-nitrophenyl carbonate-camptothecin)

[0059] 1.0 g of SN38 (camptothecin) and 1.6 g of DNPC (bis(p-nitrophenyl)carbonate) were dissolved in 100 ml of tetrahydrofuran, 2 ml of trimethylamine was added, and stirred at room temperature for 1.5 h. The completion of the reaction was monitored by LC-MS. After the completion of the reaction, the solvent was spin-dried, and the residue was triturated with ethyl acetate:n-hexane 20 ml:100 ml, and then filtered to obtain a solid. The solid was triturated with 20 ml of dichloromethane, filtered, and monitored by LC-MS to collect the product to obtain 700 mg of SN38-PNP solid in 49.3% yield. LC-MS: (M+H) ⁺ 557.4.

[0060] 3) Synthesis of compound d (Boc-N-methylethylglycol-camptothecin)

[0061] 250 mg SN38-PNP was dissolved in 30 ml N,N-dimethylformamide, 235 mg Boc-DMEDA-PEG and 233 μl N,N-diisopropylethylamine were added, and stirred at room temperature for 16 h. The completion of the reaction was detected by LC-MS. After completion of the reaction, the solvent was spin-dried, and the residue was purified by column chromatography using dichloromethane:methanol 20:1 as eluent, rinsed, and monitored by LC-MS to collect the product to obtain 213 mg Boc-DMEDA-PEG-SN38 solid in 70% yield. LC-MS: (M+H) ⁺ 680.5.

[0062] 4) Synthesis of compound g (10-(2-N-methylethylethyleneglycol)-trifluoroacetate camptothecin)

[0063] 73.0 mg of compound d was dissolved in 3 mL of dichloromethane and 3 mL of trifluoroacetic acid, placed in an ice water bath, and stirred at constant temperature for 1 h. The completion of the reaction was monitored by LC-MS. After completion of the reaction, the solvent was spin-dried. The residue was added to 2 mL of toluene and 2 mL of dichloromethane, spin-dried twice, and monitored by LC-MS to collect the product to give SN38-DMEDA⋅TFA, which was used directly in the next reaction. LC-MS: (M+H) ⁺ 580.6.

[0064] 5) Synthesis of a compound of formula (12) (maleimidediethoxy-L-valyl-L-citrulline-p-aminobenzyl-(2-N-methylethylethyleneglycol)-camptothecin)

[0065] 50 mg of MP2-VC-PAB-PNP was dissolved in 4 ml of N,N-dimethylformamide, placed in an ice water bath to maintain a constant temperature, and protected with nitrogen. 38 mg of compound g was added to the reaction system, followed by 42 μl of trimethylamine, stirred at a constant temperature for 0.5 h, and then returned to room temperature for 0.5 h of reaction. Completion of the reaction was detected by LC-MS. After completion of the reaction, the solvent was dried by spinning at low temperature, and the residue was purified by preparative liquid chromatography using a Sun Fire® Prep C18 OBD TM 5 μm, 19*250 mm preparative column, A MeCN mobile phase, and BH ₂ O mobile phase. LC-MS was performed to monitor and collect the pure product to obtain 18 mg of a solid of the compound of formula (12) in a yield of 22%. LC-MS: (M+H) ⁺ 1197.2.

[0066] Example 2 Preparation of a compound of formula (4)

[0067] Compound g (10-(2-N-methylethylethyleneglycol)-camptothecin trifluoroacetate) was prepared according to the synthesis procedure of Example 1: Compound d (73.0 mg) was dissolved in 3 mL dichloromethane and 3 mL trifluoroacetic acid, placed in an ice water bath, and stirred at constant temperature for 1 h. Completion of the reaction was monitored by LC-MS. After completion of the reaction, the solvent was spin-dried. The residue was added to 2 mL dichloromethane, spin-dried twice, and monitored by LC-MS to collect the product to afford compound g (70 mg), which was used directly in the next reaction. LC-MS: (M+H) ⁺ 580.6.

[0068] The compound of formula (4) (maleimidohexanoic acid-L-valyl-L-citrulline-p-aminobenzyl-(2-N-methylethylethyleneglycol)-camptothecin) was prepared according to the synthesis procedure of Example 1: Mc-VC-PAB-PNP (maleimidohexanoic acid-L-valyl-L-citrulline-p-aminobenzyl-p-nitrophenyl carbonate) (58 mg) and compound g (42 mg) were dissolved in 2.5 ml of N,N-dimethylformamide, stirred in an ice water bath for 30 min, DIPEA (42 mg) was added dropwise and stirred in an ice water bath for 1 h. The incomplete reaction was detected by LC-MS. Triethylamine (10 mg) was added to the reaction system again, and stirred at room temperature for 1 h. The reaction solution turned yellow, and the completion of the reaction was detected by LC-MS. After completion of the reaction, the solvent was dried by rotation at low temperature, and the residue was purified by preparative liquid chromatography using a Sun Fire® Prep C18 OBD TM 5 μm, 19*250 mm preparative column, mobile phase A MeCN and 0.1% HCOOH, and mobile phase BH ₂ O and 0.1% HCOOH. LC-MS was performed to monitor and collect the pure product, to obtain 34 mg of solid compound of formula (4) in 40.5% yield. LC-MS: (M +H) ⁺ 1179.2.

[0069] Example 3 Preparation of a compound of formula (1)

[0070] 1) Synthesis of compound c (2-Boc-N-methylethylethyleneglycol)

[0071] 0.6 g of compound a (aminoethylene glycol) and 1.0 g of compound b (N-Boc-(methylamido)acetaldehyde) were dissolved in 20 ml of methanol and stirred at room temperature for 6 h. The reaction system was placed in an ice water bath, sodium triacetoxyborohydride was added, stirred in an ice water bath for 1 h, then returned to room temperature and stirred overnight. The product was detected by LC-MS and showed no UV absorption. The solvent was spin dried, and the residue was extracted twice with ethyl acetate and dried over anhydrous Na ₂ SO ₄ . After decanting the organic phase, the residue was spin dried, purified by column chromatography using dichloromethane:methanol 20:1 as eluent, and rinsed to obtain 1.0 g of the product in 66% yield. LC-MS: (M+H) ⁺ 262.6.

[0072] 2) Synthesis of SN38-PNP (10-p-nitrophenyl carbonate-camptothecin)

[0073] 1.0 g SN38 (camptothecin) and 1.6 g DNPC (bis(p-nitrophenyl)carbonate) were dissolved in 100 mL tetrahydrofuran, 2 mL trimethylamine was added and stirred at room temperature for 1.5 h. The completion of the reaction was monitored by LC-MS. After completion of the reaction, the solvent was spin-dried, the residue was triturated with ethyl acetate:n-hexane 20 mL:100 mL and filtered to obtain a solid. The solid was triturated with 20 mL dichloromethane, filtered and monitored by LC-MS to collect the product to obtain 700 mg of SN38-PNP solid in 49.3% yield. LC-MS: (M+H) ⁺ 557.4.

[0074] 3) Synthesis of compound d (Boc-N-methylethylglycol-camptothecin)

[0075] 500 mg of SN38-PNP was dissolved in 30 ml of N,N-dimethylformamide, 370 mg of compound c and 233 μl of N,N-diisopropylethylamine were added, and stirred at room temperature for 3 h. The completion of the reaction was detected by LC-MS. After completion of the reaction, the solvent was spin-dried, and the residue was purified by column chromatography using dichloromethane:methanol 20:1 as eluent, rinsed, and monitored by LC-MS to collect the product to give 250 mg of solid compound d in 45% yield. LC-MS: (M+H) ⁺ 680.5.

[0076] 4) Synthesis of compound h (Boc-N-methylethyldiethoxy p-nitroactive ester-camptothecin)

[0077] 220.0 mg of compound d were dissolved in 5 ml of dichloromethane, 150 mg of bis(p-nitrophenyl)carbonate and 86 mg of DIPEA were added and stirred at constant temperature for 16 h. The completion of the reaction was detected by LC-MS. After completion of the reaction, the solvent was spin-dried and the residue was purified by column chromatography using dichloromethane:methanol 50:1 as eluent. The product was collected to give 220 mg of solid compound h in 68.5% yield, which was then used directly in the next reaction. LC-MS: (M+H) ⁺ 845.6.

[0078] 5) Synthesis of compound i (Boc-N-methylethyldiethoxy-N,N,N-trimethylethylenediamine-camptothecin)

[0079] 220 mg of compound h (Boc-N-methylethyldiethoxy p-nitroactive ester-camptothecin) and 50 mg of N,N,N-trimethylethylenediamine were dissolved in 5 ml of N,N-dimethylformamide, DIPEA (65 mg) was added dropwise, and stirred in an ice water bath for 1 h. The completion of the reaction was detected by LC-MS. After completion of the reaction, the solvent was spin-dried and the residue was purified by column chromatography using dichloromethane:methanol 20:1 as eluent. The product was collected to give 157 mg of solid compound i in 75% yield, which was then used directly in the next reaction. LC-MS: (M+H) ⁺ 808.6.

[0080] 6) Synthesis of compound j (N-methylethyldiethoxy-N,N,N-trimethylethylenediamine-camptothecin trifluoroacetate)

[0081] 157.0 mg of compound i (Boc-N-methylethyldiethoxy-N,N,N-trimethylethylenediamine-camptothecin) were dissolved in 3 ml of dichloromethane and 1.3 ml of trifluoroacetic acid, placed in an ice water bath and stirred at a constant temperature for 1 h. The completion of the reaction was detected by LC-MS. After completion of the reaction, the solvent was spin-dried. The residue was added with 2 ml of dichloromethane and then spin-dried twice to obtain 120 mg of compound j, which was then directly used in the next reaction. LC-MS: (M+H) ⁺ 708.3.

[0082] 7) Synthesis of a compound of formula (1) (maleimidohexanoic acid-L-valyl-L-citrulline-p-aminobenzyl-(N-methylethyldiethoxy-N,N,N-trimethylethylenediamine)-camptothecin)

[0083] 52 mg of Mc-VC-PAB-PNP (maleimidohexanoic acid-L-valyl-L-citrulline-p-aminobenzyl-p-nitrophenyl carbonate) and 35 mg of compound j were dissolved in 2.5 ml of N,N-dimethylformamide, stirred in an ice water bath for 30 min, 129 mg of DIPEA were added dropwise, and stirred in an ice water bath for 0.5 h. The incomplete reaction was detected by LC-MS. Then 129 mg of DIPEA was added to the reaction system, and stirred at room temperature for 0.5 h. The reaction solution turned yellow, and the completion of the reaction was detected by LC-MS. After completion of the reaction, the solvent was dried by rotation at low temperature, and the residue was purified by preparative liquid chromatography using a Sun Fire® Prep C18 OBD TM 5 μm, 19*250 mm preparative column, mobile phase A MeCN and 0.1% HCOOH, and mobile phase BH ₂ O and 0.1% HCOOH. LC-MS was performed to monitor and collect the pure product to obtain 20 mg of solid compound of formula (1) with a yield of 20.5%. LC-MS: (M+H) ⁺ 1307.6.

[0084] Example 4 Preparation of a compound of formula (9)

[0085] The compound of formula (9) (maleimidiethoxy-L-valyl-L-citrulline-p-aminobenzyl-(N-methylethyldiethoxy-N,N,N-trimethylethylenediamine)-camptothecin) was prepared according to the synthesis procedure of Example 3: MP2-VC-PAB-PNP (maleimidiethoxy-L-valyl-L-citrulline-p-aminobenzyl-p-nitrophenyl carbonate) (52 mg) and compound j (34 mg) were dissolved in 2.5 ml of N,N-dimethylformamide, stirred for 30 min in an ice water bath, DIPEA (36 mg) was added dropwise, and stirred in an ice water bath for 0.5 h. The incomplete reaction was detected by LC-MS. DIPEA (8 mg) was then added to the reaction system, which was stirred at room temperature for 1.5 h. The reaction solution turned yellow, and the completion of the reaction was detected by LC-MS. After the completion of the reaction, the solvent was dried by rotation at low temperature, and the residue was purified by preparative liquid chromatography using a Sun Fire® Prep C18 OBD TM 5 μm, 19*250 mm preparative column, mobile phase A MeCN and 0.1% HCOOH, and mobile phase BH ₂ O and 0.1% HCOOH. LC-MS was performed to monitor and collect the pure product, to obtain 23 mg of the compound of formula (9) as a solid substance in a yield of 35.2%. LC-MS: (M+H) ⁺ 1325.4.

[0086] Example 5. Inhibitory effect of ADC-1 on NCI-N87 cells.

[0087] The compound of formula (1) and the intermediate CL2A-SN38 are subjected to sulfhydryl coupling with Heceptin by conventional methods in the art, respectively (for example, see Example 12 in Chinese Patent Application Publication No. CN102448494A) to obtain antibody-drug conjugates of ADC-1 and HER2-CL2A-SN38 (ADC-17) with an average DAR value of 8. NCI-N87 cells are digested with trypsin, then the cell density is adjusted to 50,000 cells/mL, added to a cell culture plate at 100 μL/well, and cultured in an incubator at 37°C, 5% _CO2 for 14 to 20 minutes. hour The samples and controls (for the composition of the test samples, see Table 1) were gradient diluted with basal medium, respectively, transferred to a cell culture plate inoculated with cells at a concentration of 100 μl/well, and cultured in an incubator at 37°C, 5% CO ₂ for 70-74 hours. CCK-8 was diluted 10-fold with the culture medium. The old medium from the 96-well plate was removed, and 100 μl of the diluted CCK-8 solution was added to each well. Color development was carried out under 5% CO ₂ for 2-4 hours. After centrifugation to remove air bubbles, the plates were detected with a microplate reader at a detection wavelength of 450 nm/655 nm for reading. The results are shown in Table 2.

[0088] Table 1. Composition of the tested samples

Number Test sample Average DAR Sample 1 ADC-1 8 Control group 1 ADC-17 8 Control group 2 HER2 antibody /

[0089] Table 2. Inhibitory effect on NCI-N87 cells

Number Test sample IC50 (ng/ml) Sample 1 ADC-1 251 Control group 1 ADC-17 210 Control group 2 HER2 antibody ND

[0090] The experimental results show that the ADC-1 group and the ADC-17 group have comparable inhibitory effects on NCI-N87 cells.

[0091] Example 6. Experimental studies of stability

[0092] 1) The compound of formula (1) (2.1 mg) and CL2A-SN38 (2.1 mg) are accurately weighed and then dissolved in 700 μL of DMSO, respectively.

[0093] 2) Prepare PB buffers (0.2 M) with pH 6, 7 and 8, respectively.

[0094] 3) 50 μl of a solution of the compound of formula (1) in DMSO are added to a centrifuge tube, and 950 μl of PB buffer (0.2 M) at pH 6, wherein the concentration of the compound is 60 μg/ml, are added. The system is placed in a system with a constant temperature of 37°C. 50 μl of CL2A-SN38 solution in DMSO are added to a centrifuge tube, and 950 μl of PB buffer (0.2 M) at pH 6, wherein the concentration of the compound is 60 μg/ml, are added. The system is placed in a system with a constant temperature of 37°C;

[0095] 4) 40 μl of sample are taken at 0 h, 2 h, 4 h, 6 h, 24 h and 48 h, respectively, 120 μl of acetonitrile are added and centrifuged. The supernatant is collected for LC-MS quantitative determination.

[0096] 5) Processing results.

[0097] The concentration at T=0 h in LC-MS is used as a standard, and the data are calculated as T/ _T0 *100%. The results are shown in Table 3 and Table 4, respectively.

[0098] Table 3. Compound of formula (1)

Time pH=6 pH=7 pH=8 0 1 1 1 2 0.988606 0.878866 0.56405 4 0.97612 0.801849 0.284131 6 0.92084 0.821934 0,123407 24 0.86821 0.256709 0.009607 48 0.78862 0.122173 0.012562

[0099] At pH 6, the compound of formula (1) decomposes by 21.2% after 48 h;

[00100] At pH 7, the compound of formula (1) is decomposed by 87.8% after 48 h;

[00101] At pH 8, the compound of formula (1) is decomposed by 98.7% after 48 h.

[00102] Table 4. CL2A-SN38

Time pH=6 pH=7 pH=8 0 1 1 1 2 0.747874 0.703812 0.628692 4 0.647065 0.585106 0.434695 6 0.412955 0.571543 0.38435 24 0.035931 0.022606 0.025316 48 0.020243 0,01906 0.019275

[00103] At pH 6, CL2A-SN38 is 98.0% degraded after 48 hours;

[00104] At pH 7, CL2A-SN38 is 98.1% degraded after 48 hours;

[00105] At pH 8, CL2A-SN38 is 98.1% degraded after 48 hours.

[00106] From the results of Table 3 and Table 4 it can be seen:

[00107] 1) Under weak acidic conditions, the compound of formula (1) is more stable than CL2A-SN38.

[00108] 2) Under neutral conditions, the compound of formula (1) is decomposed by 18% in 6 hours and 75% in 24 hours, and CL2A-SN38 is decomposed by 43% in 6 hours and 98% in 24 hours.

[00109] 3) Under alkaline conditions, the compound of formula (1) is decomposed by 97% in 24 hours, and CL2A-SN38 is decomposed by 98% in 24 hours.

[00110] Therefore, compared with CL2A-SN38, the compound of formula (1) obtained in the present invention has higher stability under weak acidic or neutral conditions in vivo, which is expected to significantly improve the safety of ADC-based drugs.

[00111] Example 7. Evaluation of the inhibitory effect of anti-Her2-drug conjugates on breast cancer development in situ

[00112] 1. Experimental methods

[00113] The BT474 cell line is thawed and passaged 1 to 2 times. After the cells grow in a stable state, they are cultured by expansion. A tumor cell suspension is prepared. Nude mice are inoculated with 0.2*10 ⁷ BT474 cells in the mammary fat pads, and a total of 60 female BALB/c nude mice are inoculated. When the average tumor volume reaches about 70 mm ³ , 42 mice are selected and randomly divided into 6 groups depending on the tumor volume: a negative control group as control group 1 (saline group), control group 2 (ADC-17), experimental group 1 (ADC-4), experimental group 2 (ADC-12), experimental group 3 (ADC-1), and experimental group 4 (ADC-9), with 7 mice in each group. After successfully establishing the model according to the above grouping, the control group 1 was intravenously administered with saline, and the other groups were intravenously administered with drugs at a dose of 5 mg/kg for the control group 2, 5 mg/kg for the experimental group 1, 5 mg/kg for the experimental group 2, 5 mg/kg for the experimental group 3, and 5 mg/kg for the experimental group 4 once a week for 21 weeks. The mice were weighed and tumor volumes were measured 3 times a week, and the mice were weighed and tumor volumes were measured on day 21.

[00114] 2. Experimental results and analysis

[00115] 1) Changes in body weight of mice:

[00116] Table 5 Body weight of mice

Group Quantity Weight (g) D1 D21 D1 D21 Control group 1 Saline solution 7 7 18.40±
1.41 19.94±
1.22 Control group 2 ADC-17 7 7 18.73±
0.78 20.36±
0.90 Experimental group 1 ADC-4 7 7 18.43±
0.95 20.42±
0.86 Experimental group 2 ADC-12 7 7 18.43±
0.95 19.94±
0.72 Experimental group 3 ADC-1 7 7 17.88±
1.01 18.64±
1.31 Experimental group 4 ADC-9 7 7 18.13±
1.14 18.61±
1.04

[00117] No mice died during the experiment. At the end of the experiment, each group of control group 2 (ADC-17 group), experimental group 1 (ADC-4), experimental group 2 (ADC-12), experimental group 3 (ADC-1), and experimental group 4 (ADC-9) showed no significant difference in body weight compared to control group 1. See Table 5 and FIG. 1 for details.

[00118] 2) Tumor volume in mice:

[00119] Table 6. Tumor volume in mice

Group Tumor volume ( ^mm3 ) RTV T/C (%) D1 D21 Control group 1 Saline solution 75.60±
24.66 107.07±
19.30 ^## 1.52±
0.39 ^## - Control group 2 ADC-17 67.95±
15.73 49.17±
13.29 ^** 0.75±
0.19** 49.30 Experimental group 1 ADC-4 68.24±
13,13 57.12±
20.28 ^** 0.83±
0.27** 55,00 Experimental group 2 ADC-12 70.73±
16.36 66.26±
21.37 ^** 0.95±
0.26* 62.58 Experimental group 3 ADC-1 66.47±
14.09 61.32±
12.82 ^** 0.95±
0.23** 62.48 Experimental group 4 ADC-9 70.73±
17.99 75.76±
46.18 ^* 1.02±
0.35* 67.19

Note: Each group is compared with the control group 1 of the saline group, where “*” represents P < 0.05, and “**” represents P < 0.01; and each group is compared with the control group 2, where “#” represents P < 0.05, and “##” represents P < 0.01.

[00120] The tumor volume of the control group 1 reached 107±19.30 mm ³ on the 21st day after the administration, and the tumor volume of the control group 2, experimental group 1, experimental group 2, experimental group 3 and experimental group 4 were 49.17±13.29, 57.12±20.28, 66.26±21.37, 61.32±12.82, 75.76±46.18 and 75.19±25.99 mm ³ , respectively (see Table 6). Among them, the control group 2, experimental group 1, experimental group 2 and experimental group 3 are statistically significantly different (P<0.01) from the control group 1 (saline group), and the experimental group 4 is statistically significantly different (P<0.05) from the control group 1. The tumor volumes of the experimental group 1, experimental group 2 and experimental group 3 are not statistically significantly different (P>0.05) from those of the control group 2, and their relative tumor volumes are also not significantly different (P>0.05), i.e., the effects are comparable. Among them, the relative tumor proliferation rate (T/C) in the control group 2, experimental group 1, experimental group 2, experimental group 3 and experimental group 4 were 49.30%, 55.00%, 62.58%, 62.48%, 67.19% and 63.20%, respectively. From the above data, it can be learned that all four antibody-drug conjugates, corresponding to the control group 2, experimental group 1, experimental group 2 and experimental group 3, tested in this experiment significantly inhibited the growth of BT474 tumor with comparable effects, and the experimental group 4 also inhibited the growth of BT474 tumor. The changes in tumor volume and relative tumor volume in each group during the experiment are shown in FIG. 2 and FIG. 3, respectively.

[00121] Thus, ADCs constructed using the drug conjugate intermediates of the present disclosure exhibit significant antitumor activity against the BT474 breast cancer cell model. Preferred ADC-1, ADC-4, and ADC-12 have significant advantages and are expected to significantly improve in vivo safety while providing tumor inhibitory effects comparable to those of the ADC-17 group.

[00122] The present description is illustrated by various specific examples. However, a person skilled in the art can understand that the present description is not limited to each specific embodiment, and a person skilled in the art can make various changes or modifications within the scope of the present description, and each technical feature mentioned in various places in this description can be combined with each other without departing from the spirit and scope of the present invention. Such changes and modifications are within the scope of the present invention.

Claims (72)

1. An intermediate compound of an antibody-drug conjugate represented by formula (I) where X ₁ is selected from the group consisting of -(CH ₂ ) _m - and -(CH ₂ CH ₂ O) _p -, where m is selected from 1, 2, 3, 4, 5 and 6 and p is selected from 1, 2, 3, 4, 5 and 6; X ₂ is H or -C(O)NR ¹ R ² ; and R ¹ is selected from the group consisting of hydrogen, substituted or unsubstituted C _1-6 alkyl and R ² is selected from the group consisting of hydrogen, substituted or unsubstituted C _1-6 alkyl and wherein each substituted C _1-6 alkyl is optionally substituted with 1-5 substituents independently selected from the group consisting of halogen, hydroxyl, amino, carboxyl, and 5-10 membered heterocyclyl, wherein 5-10 membered heterocyclyl has 1-3 heteroatoms selected from the group consisting of oxygen and nitrogen. 2. An intermediate compound of the antibody-drug conjugate according to claim 1, characterized in that m is equal to 5 and p is equal to 2. 3. An intermediate compound of the antibody-drug conjugate according to claim 1, characterized in that X ₁ is selected from the group consisting of: 4. An intermediate compound of the antibody-drug conjugate according to claim 1, characterized in that R ¹ or R ² is independently selected from the group consisting of: H, methyl, ethyl, propyl, butyl, pentyl, And 5. An intermediate compound of an antibody-drug conjugate according to any one of claims 1-4, characterized in that X ₂ is selected from the group consisting of: 6. An intermediate compound of an antibody-drug conjugate, characterized in that the intermediate compound of the antibody-drug conjugate has a structure represented by formulas (1) - (16): 7. A method for producing an intermediate antibody-drug conjugate compound, wherein the intermediate antibody-drug conjugate compound is: where R ¹ is selected from the group consisting of hydrogen, substituted or unsubstituted C _1-6 alkyl and R ² is selected from the group consisting of hydrogen and substituted or unsubstituted C _1-6 alkyl and wherein each substituted C _1-6 alkyl is optionally substituted with 1-5 substituents independently selected from the group consisting of halogen, hydroxyl, amino, carboxyl, and 5-10 membered heterocyclyl, wherein 5-10 membered heterocyclyl has 1-3 heteroatoms selected from the group consisting of oxygen and nitrogen; preferably R ¹ or R ² is selected from the group consisting of: H, methyl, ethyl, propyl, butyl, pentyl, where the method is selected from the following reaction processes: reaction process 1: 8. The method according to item 7, characterized in that the compound (1-1) is selected from the group consisting of: 9. The method according to item 7, characterized in that the reaction process 1 includes the following conditions: Reaction A: dissolving compound a and compound b in a solvent, stirring at room temperature for an appropriate time, adding a reducing agent at a low temperature, stirring for an appropriate time, and then stirring at room temperature overnight to carry out the reaction; after completion of the reaction, drying the solvent by rotation, and then extracting, drying and purifying; Reaction B: dissolving SN38 and bis(p-nitrophenyl)carbonate (DNPC) in a solvent, adding an organic base and stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then triturating and filtering; Reaction C: dissolving the product obtained by reaction B in a solvent, adding the product obtained by reaction A and an organic base, stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying; Reaction D: dissolving the product obtained by reaction C in a solvent, adding an acid and stirring at a low temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation, dissolving maleimidohexanoic acid-L-valyl-L-citrulline-p-aminobenzyl-p-nitrophenyl carbonate (Mc-VC-PAB-PNP) in the solvent, stirring at a low temperature for an appropriate time, and adding an organic base to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying. 10. The method according to item 7, characterized in that the reaction process 2 includes the following conditions: Reaction A: dissolving compound a and compound b in a solvent, stirring at room temperature for an appropriate time, adding a reducing agent at a low temperature, stirring for an appropriate time, and then stirring at room temperature overnight to carry out the reaction; after completion of the reaction, drying the solvent by rotation, and then extracting, drying and purifying; Reaction B: dissolving SN38 and bis(p-nitrophenyl)carbonate (DNPC) in a solvent, adding an organic base and stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then triturating and filtering; Reaction C: dissolving the product obtained by reaction B in a solvent, adding the product obtained by reaction A and an organic base, stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying; Reaction D: dissolving the product obtained by Reaction C in a solvent, adding an acid and stirring at a low temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation, dissolving maleimidiethoxy-L-valyl-L-citrulline-p-aminobenzyl-p-nitrophenyl carbonate (MP2-VC-PAB-PNP) in the solvent, stirring at a low temperature for an appropriate time, and adding an organic base to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying. 11. The method according to item 7, characterized in that the reaction process 3 includes the following operating conditions: Reaction A: dissolving compound a and compound b in a solvent, stirring at room temperature for an appropriate time, adding a reducing agent at a low temperature, stirring for an appropriate time, and then stirring at room temperature overnight to carry out the reaction; after completion of the reaction, drying the solvent by rotation, and then extracting, drying and purifying; Reaction B: dissolving SN38 and bis(p-nitrophenyl)carbonate (DNPC) in a solvent, adding an organic base and stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then triturating and filtering; Reaction C: dissolving the product obtained by reaction B in a solvent, adding the product obtained by reaction A and an organic base, and stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying; Reaction D: dissolving the product obtained by reaction C in a solvent, adding bis(p-nitrophenyl)carbonate and an organic base, and stirring at a constant temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying; Reaction E: dissolving the product obtained by reaction D and amine in a solvent, adding an organic base and stirring at a low temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying; Reaction F: dissolving the product obtained by Reaction E in a solvent, adding an acid and stirring at a low temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation, dissolving maleimidohexanoic acid-L-valyl-L-citrulline-p-aminobenzyl-p-nitrophenyl carbonate (Mc-VC-PAB-PNP) in the solvent, stirring at a low temperature for an appropriate time, and adding an organic base to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying. 12. The method according to claim 7, characterized in that the reaction process 4 includes the following operating conditions: Reaction A: dissolving compound a and compound b in a solvent, stirring at room temperature for an appropriate time, adding a reducing agent at a low temperature, stirring for an appropriate time, and then stirring at room temperature overnight to carry out the reaction; after completion of the reaction, drying the solvent by rotation, and then extracting, drying and purifying; Reaction B: dissolving SN38 and bis(p-nitrophenyl)carbonate (DNPC) in a solvent, adding an organic base and stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then triturating and filtering; Reaction C: dissolving the product obtained by reaction B in a solvent, adding the product obtained by reaction A and an organic base, and stirring at room temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying; Reaction D: dissolving the product obtained by reaction C in a solvent, adding bis(p-nitrophenyl)carbonate and an organic base, and stirring at a constant temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying; Reaction E: dissolving the product obtained by reaction D and amine in a solvent, adding an organic base and stirring at a low temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying; Reaction F: dissolving the product obtained by Reaction E in a solvent, adding an acid and stirring at a low temperature for an appropriate time to carry out the reaction; after completion of the reaction, drying the solvent by rotation, dissolving maleimidiethoxy-L-valyl-L-citrulline-p-aminobenzyl-p-nitrophenyl carbonate (MP2-VC-PAB-PNP) in the solvent, stirring at a low temperature for an appropriate time, and adding an organic base to carry out the reaction; after completion of the reaction, drying the solvent by rotation and then purifying. 13. The method according to any of paragraphs 7-12, characterized in that “at a low temperature” refers to a bath with ice water. 14. The method according to any one of claims 7-12, characterized in that the solvent is a polar solvent and/or a non-polar solvent, wherein the polar solvent is selected from the group consisting of THF, DMF, DMA, NMP and mixtures thereof; and the non-polar solvent is selected from the group consisting of dichloromethane, carbon tetrachloride and mixtures thereof. 15. The method according to any one of claims 7 to 12, characterized in that the organic base is selected from the group consisting of N,N-diisopropylethylamine, triethylamine, pyridine and mixtures thereof, preferably one or both of N,N-diisopropylethylamine and pyridine. 16. The method according to any one of paragraphs 7-12, characterized in that the acid is one or two acids selected from the group consisting of hydrochloric acid, trifluoroacetic acid and citric acid. 17. The method according to any one of paragraphs 7-12, characterized in that the amine is a primary amine or a secondary amine. 18. The method according to any one of paragraphs 7-12, characterized in that in reaction A the extraction is carried out using ethyl acetate and the purification is carried out by column chromatography using dichloromethane and methanol as the eluent. 19. The method according to any one of paragraphs 7-12, characterized in that in reaction B, trituration is carried out using ethyl acetate, hexane, dichloromethane or a combination thereof. 20. The method according to any one of paragraphs 7-12, characterized in that in reaction C the purification is carried out by column chromatography using dichloromethane and methanol as an eluent. 21. The method according to any one of paragraphs 7-12, characterized in that in reaction D the purification is carried out by column chromatography using dichloromethane and methanol as the eluent. 22. The method according to any one of paragraphs 7-12, characterized in that in reaction E the purification is carried out by column chromatography using dichloromethane and methanol as the eluent. 23. The method according to any one of paragraphs 7-12, characterized in that in reaction F the purification is carried out by preparative liquid chromatography using MeCN and 0.1% HCOOH as mobile phase A and _H2O and 0.1% HCOOH as mobile phase B. 24. The method according to any of paragraphs 7-12, characterized in that all reactions are carried out under the protection of nitrogen.

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