WO2024140450A1 - Antibody-conjugated drug of n-alkoxyalkyl substituted camptothecin derivative - Google Patents

Abstract

An N-alkoxyalkyl-substituted camptothecin derivative is used as an antibody-drug conjugate represented by formula (I) and as a cytotoxic drug moiety, and the antibody-drug conjugate has a significant anti-tumor effect.

Description

Antibody-drug conjugates of N-alkoxyalkyl-substituted camptothecin derivatives Technical Field

The present invention relates to the field of medicine and specifically provides a series of N-alkoxyalkyl substituted camptothecin derivative antibody-drug conjugates, preparation methods thereof and applications thereof in the field of anti-tumor.

Background technique

Antibody-drug conjugate (ADC) combines the advantages of monoclonal antibody drugs with small molecule cytotoxins. Its structure includes three components: antibody, cytotoxin and linker. It achieves specific targeting of tumors through antibodies, and then releases cytotoxins to further kill tumor cells. ADC has overcome the drug resistance problem of monoclonal antibody drugs to a certain extent. Since the effectiveness of ADC mainly depends on the cytotoxins carried by antibodies, and the biological effects mediated by antibodies are not necessary, even if the antigen undergoes a certain degree of mutation, it will not affect the effectiveness of ADC. For example, the classic ADC drug Kadcyla, which treats HER2+ breast cancer that is resistant to naked anti-Herceptin, can further extend the overall survival by 6 months compared with standard therapy. Another clinical study showed that the incidence of grade 3 adverse reactions in the Kadcyla treatment group was reduced by 50% compared with the standard chemotherapy drug treatment group. ADC has greatly improved the therapeutic index compared with conventional chemotherapy drugs by reducing toxicity and increasing efficacy. As a new type of anti-tumor "weapon", ADC is expected to make breakthroughs in the treatment of tumor diseases.

The success of an ADC depends not only on the optimization of each component, but also on the reasonable combination and integration of the components. The three components of ADC play different roles, so ADC also has different requirements for its three components: the antibody of ADC needs to meet the requirements of specific tumor targeting, suitable affinity and internalization performance; the toxin of ADC needs to meet the requirements of high toxicity, clear mechanism of action, and conjugation. Camptothecin derivatives are a kind of broad spectrum anti-tumor drugs, which are widely used as toxins of ADC. However, there are few existing technologies on the structural design of alkoxyalkyl-modified camptothecin derivatives as toxins or their tumor inhibitory activity. The research and promotion of this topic is of great value to the development of camptothecin derivative ADC technology.

Summary of the invention

The object of the present invention is to provide an antibody-drug conjugate with an alkoxyalkyl-substituted camptothecin derivative as the drug toxic part, and these antibody-drug conjugates have significant anti-tumor effects.

In one aspect of the present invention, there is provided an antibody-drug conjugate represented by formula (I),

In the formula,

R ¹ and R ² are each independently selected from hydrogen, deuterium, and C ₁ -C ₆ alkyl;

L ¹ is selected from -L ¹¹ -L ¹² -L ¹³ -, wherein L ¹¹ , L ¹² , and L ¹³ are each independently selected from -O-, C ₁ -C ₃ alkylene, and phenyl;

L ^P is a peptide residue consisting of 2-7 amino acids;

Z is selected from ^-Lz - ^Lj- , wherein ^Lz is selected from -C(=O) _{-C1 -} C8 alkylene and -C(=O)-( _CH2CH2O ) _{2-6 - CH2CH2NH-} , _and ^Lj is a linker that can be coupled to an antibody;

n ₁ is selected from 0, 1, 2 or 3;

Ab is an antibody.

In the antibody-drug conjugate of formula (I), the drug-antibody ratio (DAR) is selected from 2-8, and can further be 2.1, 2.3, 4.3, 4.6, 6.2, 6.3, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5.

The antibody-drug conjugate represented by formula (I) is the antibody-drug conjugate represented by formula (I'),

n' is selected from 2-8, and can further be 2-4, 2-6 or 4-6, for example, it can be 2.1, 2.3, 4.3, 4.6, 6.2, 6.3, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5.

In some embodiments, R ¹ and R ² are each independently selected from hydrogen, deuterium and C ₁ -C ₃ alkyl.

In some embodiments, R ¹ and R ² are each independently selected from hydrogen, deuterium, methyl, ethyl and isopropyl.

In some embodiments, R ¹ is selected from hydrogen, deuterium, methyl, ethyl and isopropyl, and R ² is selected from hydrogen, deuterium, methyl and ethyl.

In some embodiments, R ¹ is methyl, and R ² is hydrogen or methyl.

In some specific embodiments, L ¹¹ , L ¹² , and L ¹³ are each independently selected from -O-, methylene, and phenyl.

In some specific embodiments, L ¹¹ -L ¹² -L ¹³ are selected from -O-CH ₂ -phenyl- and -CH ₂ -O-CH ₂ -.

In some specific embodiments, the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn).

In some specific embodiments, ^Lp is selected from a peptide residue consisting of 2-5 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn).

In some embodiments, ^Lp is selected from a peptide residue consisting of 2-4 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), and lysine (Lys).

In some specific embodiments, ^Lp is selected from -Val-Cit-, -Val-Ala-, -Gly-Val-Ala-, -Gly-Val-Ala-Gly-, -Gly-Lys-, -Gly-Gly-Lys-, -Gly-Gly-Lys-Gly-, -Val-Ala-, -Ala-Ala-Asn-, -Gly-Leu-, -Gly-Gly-Leu-, -Gly-Gly-Leu-Gly-, -Gly-Phe-, -Gly-Gly-Phe-, and -Gly-Gly-Phe-Gly-.

In some embodiments, L ^p is selected from Its carbonyl end is connected to -NH- and the other end is connected to Z.

In some embodiments, L ^p is selected from Its carbonyl end is connected to -NH- and the other end is connected to Z.

In some embodiments, L ^is selected from The positions shown indicate attachment to antibodies. The positions shown indicate attachment to the L ^Z group.

In some specific embodiments, L ^z is selected from —C(═O)—C ₁ -C ₈ alkylene and —C(═O)—(CH ₂ CH ₂ O) _2-6 —CH ₂ CH ₂ NH—.

In some specific embodiments, ^Lz is selected from -C( ₌ O) _{-C1 -C6} alkylene and -C(=O)-( _CH2CH2O ) _{2-4 - CH2CH2NH-} .

In some specific embodiments, ^Lz is selected from -C(=O) _- ( _CH2 ) _5- and -C(=O)-( _CH2CH2O ) _{2 - CH2CH2NH-} .

In some embodiments, Z is selected from The positions shown indicate attachment to antibodies. The positions shown indicate attachment to the L ^p group.

In some embodiments, the antibody is an antibody against a tumor-associated antigen.

In some specific embodiments, the tumor-associated antigen antibody is selected from anti-Her2 antibody, anti-Trop2 antibody, anti-B7H3 antibody, anti-5T4, anti-Nectin-4 antibody, anti-CD20 antibody and anti-ROR1 antibody.

In some embodiments, the antibody-drug conjugate of formula (I) has a structure as shown in formula (I-1), formula (I-1'), formula (I-2'), or formula (I-2),

In the formula, R ¹ , R ² , n ₁ , L ^p , Z and Ab have the same definitions as those of the compound of formula (I), and n' has the same definition as above.

The present invention provides the following antibody-drug conjugates:

wherein Ab is defined as the compound of formula (I).

In some specific embodiments, Ab is selected from tumor-associated antigen antibodies, and is further selected from anti-Her2 antibodies, anti-Trop2 antibodies, anti-B7H3 antibodies, anti-5T4, anti-Nectin-4 antibodies, anti-CD20 antibodies, and anti-ROR1 antibodies.

n is selected from 2-8, and may further be 2-4, 2-6 or 4-6, for example, may be 2.1, 2.3, 4.3, 4.6, 6.2, 6.3, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5.

Furthermore, the present invention provides the following antibody-drug conjugates:

n is selected from 6.5-6.8.

n is selected from 7.3-7.6.

n is selected from 7.1-7.2.

n is selected from 7.3-7.5.

n is selected from 7.3-7.4.

n is selected from 2.1-2.3.

n is selected from 4.3-4.6.

wherein Ab is defined as the compound of formula (I).

In some embodiments, Ab is HS627 antibody or IP140B antibody, and the heavy chain amino acid sequence of HS627 is as shown in SEQ ID NO: 1 The amino acid sequence of its light chain is shown in SEQ ID NO:2; the amino acid sequence of the heavy chain of the IP140B antibody is shown in SEQ ID NO:3, and the amino acid sequence of its light chain is shown in SEQ ID NO:4.

Another aspect of the present invention provides a method for preparing the above-mentioned antibody-drug conjugate, which comprises the following steps:

The compound of formula (a) is subjected to a condensation reaction with HO- ^Lz - ^Lj ' to obtain the compound of formula (b), and the compound of formula (b) is linked to an antibody to obtain the compound of formula (I).

wherein n ₁ , R ¹ , R ² , L ¹ , L ^p , L ^z , and Ab each have the same meaning as the compound of formula (I).

L ^j 'selected

Another aspect of the present invention provides a pharmaceutical composition, which comprises the aforementioned antibody-drug conjugate or the antibody-drug conjugate prepared by the above method and a pharmaceutically acceptable carrier.

Another aspect of the present invention provides use of the aforementioned antibody-drug conjugate, the antibody-drug conjugate prepared by the aforementioned method, or the aforementioned pharmaceutical composition in the preparation of anti-tumor drugs.

Another aspect of the present invention provides a method for inhibiting or treating a tumor disease in a patient in need thereof, comprising administering the above-mentioned antibody-drug conjugate or pharmaceutical composition to the patient in need thereof.

In some embodiments, the amount of the antibody-drug conjugate or pharmaceutical composition administered is a therapeutically effective amount.

In some embodiments, the tumor is selected from a solid tumor.

In some embodiments, the tumor is selected from lung cancer, esophageal cancer, breast cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, or ovarian cancer.

In some embodiments, the lung cancer is non-small cell lung cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the tumor growth curve of the Calu-6 human non-small cell lung cancer CDX model in Test Example 2.

FIG2 is a photo of the tumor after dissection of the Calu-6 human non-small cell lung cancer CDX model in Test Example 2.

FIG3 is a graph showing the tumor growth curve of the KYSE-150 human esophageal squamous cell carcinoma CDX model in Test Example 2.

FIG. 4 is a tumor photograph of the KYSE-150 human esophageal squamous cell carcinoma CDX model after dissection in Test Example 2.

FIG5 is a graph showing the tumor growth curve of the ES-2 human ovarian cancer CDX model in Test Example 2.

FIG6 is a photo of the tumor after dissection of the ES-2 human ovarian cancer CDX model in Test Example 2.

Detailed ways

IDefinition:

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the relevant terms and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

The compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers include, for example, enantiomers and diastereomers. The stereoisomers include geometric isomers (such as cis and trans structures) and optical isomers (such as enantiomers), and therapeutic substances composed of monomers, racemates, racemic mixtures and pharmaceutically acceptable salts thereof. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure form or in racemic form. Optically pure forms can be separated from racemic mixtures or by using hand The invention relates to the synthesis of a novel compound using chiral raw materials or chiral reagents. Racemates, diastereomers, and enantiomers are all within the scope of the present disclosure.

Numeric ranges herein refer to each integer in the given range. For example, "C ₁ -C ₆ " means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.

When any variable (e.g., Rn) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 1-5 Rs, the group may be optionally substituted with up to 5 Rs, and each occurrence of R is an independent choice. In addition, combinations of substituents and/or variants thereof are permitted only if such combinations result in stable compounds.

The term "C _1- C ₆ alkyl" refers to a straight or branched alkyl group having 1 to 6 carbon atoms. Specific examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and various branched isomers thereof.

Refers to the chemical bond connection. It should be noted that the structural fragments described in the present invention (e.g., -L ¹¹ -L ¹² -L ¹² - or -L ^z -L ^j -), unless otherwise specified, represent the corresponding groups connected in sequence from left to right. For example, when -L ¹² - is C ₁ -C ₂ alkylene-O-, it means that the left side of C ₁ -C ₂ alkylene-O- is connected to -L ¹¹ -, and the right end is connected to -L ¹³ -.

It can be understood that in the antibody-drug conjugates of the present application, for example, in formula (I), (I-1), (I-2), the "-" between the drug-linker fragment and the antibody is intended to indicate the connection relationship between the antibody and the fragment, and is not intended to be limited to one antibody connected to one drug-linker fragment. It is well known in the art that an antibody can be connected to one or more drugs due to the presence of multiple inter-linking disulfide bonds.

Drug or drug composition

The drugs or pharmaceutical compositions of the present disclosure can be administered orally, topically, parenterally or mucosally (e.g., buccally, by inhalation or rectally) in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers. It is generally desirable to use an oral route. The active agent can be administered orally in the form of capsules, tablets, etc.

The term "treating" includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with the disease, condition or disorder being treated.

The term "inhibit" is used relative to a control. One skilled in the art will readily determine the appropriate control for each experiment. For example, a reduced response in a subject or cell treated with a compound is compared to a response in a subject or cell not treated with the compound.

The term "patient" refers to an animal, preferably a mammal, more preferably a human.

The term "pharmaceutical composition" means a composition comprising the compound described in the present disclosure or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable ingredient selected from the following depending on the mode of administration and the nature of the dosage form, including but not limited to: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

The term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a non-toxic drug or medicament that can achieve the desired effect. In embodiments of the present invention, when treating a patient according to the present invention, the amount of a given drug depends on many factors, such as a specific dosing regimen, a disease or condition type and its severity, the uniqueness of the subject or host to be treated (e.g., body weight), but, according to specific surrounding conditions, including, for example, a specific drug, route of administration, condition to be treated, and the subject or host to be treated, the dosage can be routinely determined by methods known in the art. Usually, with respect to the dosage used for adult treatment, the dosage is typically in the range of 0.02-5000 mg/day, such as about 1-1500 mg/day. The desired dosage can be conveniently expressed as a single dose, or a dose administered simultaneously (or in a short period of time) or in a divided dose at appropriate intervals, such as two, three, four, or more divided doses per day. It will be appreciated by those skilled in the art that, although the above-mentioned dosage range is given, the specific effective amount can be appropriately adjusted according to the patient's condition and in conjunction with the physician's diagnosis.

The term "antibody-drug conjugate (ADC)" refers to a small molecule drug with biological activity connected to a monoclonal antibody through a chemical link. The monoclonal antibody acts as a carrier to transport the small molecule drug into the target cells. The "antibody-drug conjugate" mentioned in this article has the same meaning as antibody-drug conjugate.

The “antibody-drug conjugate” mentioned in this article has the same meaning as antibody-drug conjugate (ADC).

The term “drug-to-antibody ratio (DAR)” refers to the average number of payload molecules (anti-tumor compounds or drugs) attached to a single mAb.

abbreviation:

Fmoc: 9-fluorenylmethoxycarbonyl.

The amino acid constituting the peptide residue ^LP , Val: valine, has the structural formula Ala: Alanine, its structural formula is Gly: glycine, its structural formula is; Phe: phenylalanine, its structural formula is Leu: Leucine, its structural formula is Lys: lysine, its structural formula is

^LP can be obtained by an amino acid condensation method known in the art.

PABC: haha:

In the present invention, the antibody and the linker ^Lj used to connect the antibody can be connected by methods known in the art. For example, when the structure of ^Lj is (or ), which can be through (or ) is obtained by reacting with a thiol or other reactive group on an antibody, and the present invention is not particularly limited to this.

II Embodiment:

In this embodiment, ADCs prepared using, but not limited to, Pertuzumab and IP140B antibodies, Pertuzumab (HS627, PERTUZUMAB) heavy chain amino acid sequence is as follows (SEQ ID NO: 1):

The light chain amino acid sequence is as follows (SEQ ID NO: 2):

The heavy chain amino acid sequence of the IP140B antibody is as follows (SEQ ID NO: 3):

The light chain amino acid sequence is as follows (SEQ ID NO: 4):

Unless otherwise specified, the raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

Intermediate HX-10a: N-(S-)methoxyisopropylamine ethyl camptothecin

(S)-1-methoxy-2-propylamine (100 mg, 1.12 mmol), hydrochloric acid (0.7 mL, 0.8 mmol), DMSO (3 mL) and 7-methyl-10,11-methylenedioxycamptothecin (50 mg, 0.12 mmol) were added to the reaction flask, the temperature was raised to 120° for reaction for 50 minutes with stirring, and the mixture was cooled to room temperature. Methyl tert-butyl ether was added, the precipitated solid was filtered out, and the title compound was purified by silica gel column chromatography to obtain the product (36 mg, yield 61%, HPLC 99%); ¹ H NMR (500 MHz, DMSO-d ₆ )δ8.64(s,2H),7.69(s,1H),7.57(s,1H),7.27(s,1H),6.53(s,1H),6.33(s,2H),5.44(s,1H),5.33(d,J＝4.7Hz,2H),3.71-3.54(m,2H),3.47(dd,J＝10.1,5.8Hz,6H),3.25(d,J＝13.6Hz,2H),1.88(dt,J＝14.2,9.2Hz,2H),1.24(d,J＝6.4Hz,3H),0.88(dd,J＝9.4,5.3Hz,3H); LC-MS(M+H)+508.37(theoretical value 507.20).

Intermediate HX-14a: N-methoxyethylaminoethyl camptothecin

2-Methoxyethylamine (2.5 g, 0.03 mol) and hydrochloric acid (4 mL, 0.05 mol) were dissolved in DMSO (10 mL), heated to 110°C with stirring, 7-methyl-10,11-methylenedioxycamptothecin (1.8 g, 4.4 mmol) was added, and the temperature was continued to rise to 120-130°C for 1 h, cooled to room temperature, methyl tert-butyl ether was added, filtered, and purified by silica gel column chromatography to obtain the title compound (739 mg, yield 34%, HPLC 97%); ¹ H NMR (500 MHz, DMSO-d ₆ )δ7.55(s,1H),7.44(s,1H),7.20(s,1H),6.51(s,1H),6.27(d,J＝3.6Hz,2H),5.41(d,J＝4.6Hz,2H),5.13(q,J＝18.6Hz,2H),3.31-3.18(m,7H),2.94(t,J＝7.5Hz,2H),2.85(d,J＝4.9Hz,2H),1.86(dt,J＝19.4,7.0Hz,2H),0.88(t,J＝7.3Hz,3H); LC-MS(M+H)+494.12(theoretical value 493.18).

Example 1: 7-[N-(HS627-mc-Gly-Gly-Phe-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (1)

In a 250 ml single-necked bottle, Fmoc-Gly-Gly-OH (5 g, 14.11 mmol) was dissolved in DMF (50 mL), stirred to dissolve, copper acetate (768 mg, 4.23 mmol), acetic acid (1.69 g, 28.22 mmol), lead tetraacetate (6.872 g, 15.5 mmol) were added in sequence, the temperature was raised to 60 ° C, the reaction was carried out for 20 min, the reaction solution was poured into ice water, and extracted with ethyl acetate to obtain an off-white solid product HX-9a (3.2 g, yield 66%); LCMS: (M+1) ⁺ 369.21 (theoretical value: 368.14).

HX-9a (3.2 g, 9.3 mmol) was dissolved in anhydrous DCM (30 mL), benzyl glycolate (8.03 g, 93 mmol) was added, and then PPTS (0.24 g, 0.93 mmol) was added. The reaction solution was refluxed at 50 ° C overnight. After concentration, the reaction mixture was diluted with EtOAc (200 ml), washed with water (3x200 ml), dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo, and the crude residue was purified by column chromatography to give a white powder product HX-9b (4.05 g, yield 97%); LCMS: (M+1) ⁺ 475.03 (theoretical value: 474.18).

Take HX-9b (3 g, 6.32 mmol), add 100 mL of acetonitrile, then add DBU (0.48 g, 3.16 mmol), react at room temperature for 2 h, add PPTS (0.79 g, 3.16 mmol), HOBT (0.85 g, 6.32 mmol), Fmoc-Gly-Gly-Phe-OH (2.69 g, 5.36 mmol) and EDCI (1.21 g, 6.32 mmol) in sequence, react at room temperature for 15 h, spin dry, add 100 mL of DCM and 10 mL of isopropanol to dissolve, wash with 100 mL*3 (0.5 M) HCl, 100 mL*3 saturated sodium bicarbonate solution, 100 mL of saturated sodium chloride solution, and dry over 20 g of anhydrous sodium sulfate. The filtrate was filtered and dried in a rotary evaporator. The product was purified by silica gel column chromatography to obtain a white powdery solid product HX-9d (2.5 g, yield 63%); LCMS: (M+1) ⁺ 736.13 (theoretical value: 735.29).

In a 500ml single-necked bottle, HX-9d (2.5g, 3.4mmol) was dissolved in 60ml methanol and 30ml DCM mixed solvent, 10% palladium carbon (1200mg) was added, and the mixture was replaced with hydrogen and reacted at room temperature and normal pressure for 2h. After the reaction was completed, the product HX-9e (2.01g, yield 91%) was obtained by filtration and concentration; LCMS: (M+1) ⁺ 645.42 (theoretical value: 645.67).

Take HX-9e (0.2 g, 0.31 mmol), add 2 mL DMF, HATU (1.17 g, 0.31 mmol), DIPEA (0.8 g. 0.62 mmol) and N-(S-)methoxyisopropylamineethylcamptothecin (156 mg, 0.31 mmol), react at room temperature for 15 h, add 200 mL DCM to dilute, wash with saturated sodium chloride solution, dry over anhydrous sodium sulfate, and purify by silica gel column chromatography to obtain a yellow solid product HX-9f (201 mg, yield 56%); LCMS: (M+1) ⁺ 1135.36 (theoretical value: 1134.43).

HX-9f (201 mg, 0.177 mmol) was dissolved in 0.5 ml of DMF, and then piperidine (138 mg, 1.77 mmol) was added. The reaction was stirred at room temperature for 1 h. 30 ml of methyl tert-butyl ether was added to the reaction solution, centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure to obtain HX-9g solid (121 mg, yield 74%), which was directly used in the next step reaction; LCMS: (M+1) ⁺ 913.36 (theoretical value: 912.37).

HX-9g (121 mg, 0.131 mmol) was dissolved in 1 ml of anhydrous DMF, and 6-(maleimido)hexanoic acid succinimidyl ester (40 mg, 0.131 mmol) and DIPEA (17 mg, 0.131 mmol) were added in sequence. The reaction was allowed to react at room temperature for 30 min. The reaction solution was injected into a 25 g C18 pre-column (first equilibrated with acetonitrile and then with water, the aqueous phase containing 0.1% TFA), and then eluted by medium pressure reverse phase C18 chromatography (gradient: 15% to 60% acetonitrile in water, time 50 min), and freeze-dried to obtain a yellow solid product HX-9 (35 mg, yield 24%); LCMS: (M+1) ⁺ 1106.16 (theoretical value: 1105.44).

HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na ₂ HPO ₄ solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was carried out at room temperature 25°C with a rotating turntable for 90 min.

Compound HX-9 (0.89 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g/L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 1 (3.3 mg/ml, 2 ml).

UV-HPLC calculated average value: n=6.8.

Example 2: 7-[N-(IP140B-mc-Gly-Gly-Phe-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (2)

Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na ₂ HPO ₄ solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.

Compound HX-9 (0.89 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 2 (3.2 mg/ml, 2 ml).

UV-HPLC calculated average value: n=6.5.

Example 3: 7-[N-(HS627-Ac-PEG ₂ -Val-Ala-PABC), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (3)

In a 10 ml single-necked bottle, N-(S-)methoxyisopropylamineethylcamptothecin (50 mg, 0.098 mmol), 0.5 ml NMP, DIPEA (63 mg, 0.49 mmol), Fmoc-VA-PAB-PNP (67 mg, 0.098 mmol), and HOBt (13.3 mg, 0.098 mmol) were added in sequence. The reaction was stirred at room temperature for 0.5 h to obtain HX-10b, which was directly used for the next step; LCMS: (M+1) ⁺ 1049.15 (theoretical value: 1048.42).

To the above HX-10b reaction solution, add 0.5 ml of piperidine (V:V = 10%), react at room temperature for 30 min, add 20 ml of methyl tert-butyl ether to the reaction solution, centrifuge, remove the supernatant, and remove the solvent under reduced pressure to obtain a solid product HX-10c (65 mg, yield 80%), which is directly used for the next step reaction; LCMS: (M+1) ⁺ 826.35 (theoretical value: 825.35).

In a 10 ml single-mouth bottle, DCM (1 mL), HX-10c (65 mg, 0.078 mmol), bromoacetylaminoethoxyethoxypropionic acid (23 mg, 0.078 mmol) and DIC (10 mg, 0.078 mmol) were added in sequence. After stirring at room temperature for 90 min, the reaction solution was injected into a 25 g C18 pre-column (first balanced with acetonitrile and then balanced with water, the aqueous phase containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min), and freeze-dried to obtain a yellow solid product HX-10 (24 mg, yield 27%); LCMS: (M+1) ⁺ 1106.02 (theoretical value: 1105.36).

HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na ₂ HPO ₄ solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was carried out at room temperature 25°C with a rotating turntable for 90 min.

Compound HX-10 (0.89 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM Sucrose, 0.2 g/L polysorbate 20, to obtain antibody-drug conjugate ADC 3 (3.1 mg/ml, 2 ml).

UV-HPLC calculated average value: n=7.3.

Example 4: 7-[N-(IP140B-Ac-PEG ₂ -Val-Ala-PABC), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (4)

Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na ₂ HPO ₄ solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.

Compound HX-10 (0.89 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 4 (2.9 mg/ml, 2 ml).

UV-HPLC calculated average value: n=7.6.

Example 5: 7-[N-(HS627-Ac-PEG ₂ -Gly-Gly-Leu-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (5)

Take HX-9b (3 g, 6.32 mmol), add 100 mL of acetonitrile, then add DBU (0.48 g, 3.16 mmol), react at room temperature for 2 h, add PPTS (0.79 g, 3.16 mmol), HOBT (0.85 g, 6.32 mmol), Fmoc-Gly-Gly-Leu-OH (2.50 g, 5.36 mmol) and EDCI (1.21 g, 6.32 mmol), after the addition, react at room temperature for 15 h, spin dry the reaction solution, add 100 mL of DCM and 10 mL of isopropanol to dissolve, wash with 100 mL*3 (0.5 M) HCl, 100 mL*3 saturated sodium bicarbonate solution, 100 mL of saturated sodium chloride solution, and dry over 20 g of anhydrous sodium sulfate. The filtrate was filtered and dried in a rotary evaporator. The product was purified by silica gel column chromatography to obtain a white powdery solid product HX-11a (2.66 g, yield 61%); LCMS: (M+1) ⁺ 702.23 (theoretical value: 701.31).

In a 500ml single-necked bottle, HX-11a (2.66g, 3.4mmol) was dissolved in 50ml methanol and 25ml DCM mixed solvent, 1g of 10% palladium carbon was added, and after hydrogen substitution, the reaction was carried out at room temperature and normal pressure for 2h, and the product HX-11b (2.1g, 90%) was obtained by filtration and concentration; LCMS: (M+1) ⁺ 612.25 (theoretical value: 611.26).

Take N-(S-)methoxyisopropylamineethyl camptothecin (0.16 g, 0.31 mmol), add 2 mL DMF, HATU (1.17 g, 0.31 mmol), DIPEA (0.8 g. 0.62 mmol) and HX-11b (189 mg, 0.31 mmol), react at room temperature for 15 h, add 200 mL DCM to dilute, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, and purify by silica gel column chromatography to obtain a yellow solid product HX-11c (254 mg, yield 74%); LCMS: (M+1) ⁺ 1101.33 (theoretical value: 1100.45).

HX-11c (254 mg, 0.23 mmol) was dissolved in 0.5 ml of DMF, and then piperidine (179 mg, 2.3 mmol) was added. The reaction was stirred at room temperature for 1 h. 30 ml of methyl tert-butyl ether was added to the reaction solution, centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure to obtain a solid product HX-11d (150 mg, yield 73.4%), which was directly used in the next step reaction; LCMS: (M+1) ⁺ 879.36 (calculated value: 878.38).

In a 10 ml single-necked bottle, DCM (3 mL), HX-11d (150 mg, 0.17 mmol), HX-3b (49 mg, 0.17 mmol) and DIC (21 mg, 0.17 mmol) were added in sequence, and the reaction was stirred at room temperature for 90 min. The reaction solution was injected into a 25 g C18 pre-column (first balanced with acetonitrile and then balanced with water, the aqueous phase containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min), and freeze-dried to obtain a yellow solid product HX-11 (50 mg, yield 25%); LCMS: (M+1) ⁺ 1158.22 (theoretical value: 1157.39).

HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na ₂ HPO ₄ solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was carried out at room temperature 25°C with a rotating turntable for 90 min.

Compound HX-11 (0.93 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g/L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 5 (3.1 mg/ml, 2 ml).

UV-HPLC calculated average value: n=7.1.

Example 6: 7-[N-(IP140B-Ac-PEG ₂ -Gly-Gly-Leu-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (6)

Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na ₂ HPO ₄ solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.

Compound HX-11 (0.93 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 6 (3.3 mg/ml, 2 ml).

UV-HPLC calculated average value: n=7.2.

Example 7: 7-[N-(HS627-Ac-PEG ₂ -Val-Ala-PABC), N-methoxyethyl]aminoethyl-10,11-methylenedioxycamptothecin (7)

In a 10 ml single-necked bottle, N-methoxyethylamineethylcamptothecin (50 mg, 0.10 mmol), 0.5 ml NMP, DIPEA (65 mg, 0.50 mmol), Fmoc-VA-PAB-PNP (69 mg, 0.10 mmol), and HOBt (13.7 mg, 0.10 mmol) were added in sequence, and the reaction was stirred at room temperature for 0.5 h to obtain HX-14b. The reaction solution was directly put into the next step; LCMS: (M+1) ⁺ 1035.12 (theoretical value: 1034.41).

To the above HX-14b reaction solution, add 0.5 ml of piperidine (V:V = 10%), react at room temperature for 30 min, add 20 ml of methyl tert-butyl ether to the reaction solution, centrifuge, remove the supernatant, and remove the solvent under reduced pressure to obtain a solid product HX-14c (74 mg, yield 71%), which is directly used for the next step reaction; LCMS: (M+1) ⁺ 813.25 (theoretical value: 812.34).

In a 10 ml single-necked bottle, DCM (1 mL), HX-14c (74 mg, 0.091 mmol), HX-3b (26 mg, 0.091 mmol) and DIC (11.48 mg, 0.091 mmol) were added in sequence, and the reaction was stirred at room temperature for 90 min. The reaction solution was injected into a 25 g C18 pre-column (first balanced with acetonitrile and then balanced with water, the aqueous phase containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min), and freeze-dried to obtain a yellow solid product HX-14 (36 mg, yield 35%); LCMS: (M+1) ⁺ 1092.17 (theoretical value: 1091.35).

HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na ₂ HPO ₄ solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was carried out at room temperature 25°C with a rotating turntable for 90 min.

Compound HX-14 (0.88 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g/L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 7 (3.0 mg/ml, 2 ml).

UV-HPLC calculated average value: n=7.3.

Example 8: 7-[N-(IP140B-Ac-PEG ₂ -Val-Ala-PABC), N-methoxyethyl]aminoethyl-10,11-methylenedioxycamptothecin (8)

Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na ₂ HPO ₄ solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.

Compound HX-14 (0.88 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 8 (3.3 mg/ml, 2 ml).

UV-HPLC calculated average value: n=7.5.

Example 9: 7-[N-(HS627-Ac-PEG ₂ -Gly-Gly-Leu-Gly-ha-Ac), N-methoxyethyl]aminoethyl-10,11-methylenedioxycamptothecin (9)

Take N-methoxyethylaminoethyl camptothecin (100 mg, 0.20 mmol), add 2 mL DMF, HATU (77 mg, 0.20 mmol), DIPEA (52 mg. 0.4 mmol) and HX-11b (123 mg, 0.20 mmol), react at room temperature for 15 h, add 200 mL DCM to dilute, wash with saturated sodium chloride solution, dry over anhydrous sodium sulfate, and purify by silica gel column chromatography to obtain a yellow solid HX-15a (180 mg, yield 81%); LCMS: (M+1) ⁺ 1087.15 (theoretical value: 1086.43).

HX-15a (180 mg, 0.17 mmol) was dissolved in 0.5 ml of DMF, and then piperidine (179 mg, 2.3 mmol) was added. The reaction was stirred at room temperature for 1 h. 30 ml of methyl tert-butyl ether was added to the reaction solution, centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure to obtain a solid product HX-15b (114 mg, yield 77%), which was directly used in the next step reaction; LCMS: (M+1) ⁺ 865.35 (theoretical value: 864.37).

In a 10 ml single-necked bottle, DCM (3 mL), HX-11d (114 mg, 0.13 mmol), HX-3b (38 mg, 0.13 mmol) and DIC (16.5 mg, 0.13 mmol) were added in sequence, and the reaction was stirred at room temperature for 90 min. The reaction solution was injected into a 25 g C18 pre-column (first equilibrated with acetonitrile and then with water, the aqueous phase containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min), and freeze-dried to obtain a yellow solid HX-15 (48 mg, yield 32%); LCMS: (M+1) ⁺ 1145.03 (theoretical value: 1143.38).

HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na ₂ HPO ₄ solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was carried out at room temperature 25°C with a rotating turntable for 90 min.

Compound HX-15 (0.92 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g/L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 9 (3.0 mg/ml, 2 ml).

UV-HPLC calculated average value: n=7.4.

Example 10: 7-[N-(IP140B-Ac-PEG ₂ -Gly-Gly-Leu-Gly-ha-Ac), N-methoxyethyl]aminoethyl-10,11-methylenedioxycamptothecin (10)

Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na ₂ HPO ₄ solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.

Compound HX-15 (0.92 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and rotated at room temperature. The reaction was allowed to proceed for 2 h. After the reaction was completed, the buffer was replaced with a 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 10 (3.2 mg/ml, 2 ml).

UV-HPLC calculated average value: n=7.3.

Example 11: 7-[N-(HS627-Ac-PEG ₂ -Gly-Gly-Lys-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (11)

Take HX-9b (2g, 4.21mmol) and dissolve it in 60mL acetonitrile, add DBU (0.31g, 2.08mmol), react at room temperature for 2h, add PPTS (0.52g, 2.08mmol), HOBT (0.56g, 4.2mmol), Fmoc-Gly-Gly-Lys-OH (3.29g, 4.2mmol) and EDCI (0.85g, 4.2mmol) in sequence, react at room temperature for 15h, concentrate the reaction solution under reduced pressure, add 100mL DCM and 10mL isopropanol to dissolve, wash with dilute HCl, saturated sodium bicarbonate solution, saturated sodium chloride solution, and dry over anhydrous sodium sulfate. Filter, spin dry the filtrate, and purify it by silica gel chromatography to obtain a white powder solid HX-34a (2.5g, yield 60%); LCMS: (M+1) ⁺ 959.11 (theoretical value: 958.43).

In a 500 ml single-necked bottle, HX-34a (2.5 g, 2.6 mmol) was dissolved in 40 ml of methanol and 20 ml of DCM mixed solvent, 10% palladium carbon (1.0 g) was added, and after hydrogen replacement, the reaction was carried out at room temperature and normal pressure for 2 h, and the solid product HX-34b (2.2 g, yield 97%) was obtained; LCMS: (M+1) ⁺ 869.12 (calculated value: 868.38).

HX-10a (0.1 g, 0.19 mmol) was dissolved in 2 mL DMF, HATU (0.15 g, 0.39 mmol), DIPEA (0.05 g. 0.39 mmol) and HX-34b (0.34 mg, 0.39 mmol) were added, and the mixture was reacted at room temperature for 15 h. 200 mL DCM was added to dilute the mixture, and the mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain a yellow solid product HX-34c (121 mg, yield 76%); LCMS: (M+1) ⁺ 1358.52 (theoretical value: 1357.57).

HX-11c (121 mg, 0.09 mmol) was dissolved in 0.2 mL of DMF, and then piperidine (71 mg, 0.9 mmol) was added. The reaction was stirred at room temperature for 1 h, and 10 mL of methyl tert-butyl ether was added. The mixture was centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure to obtain a solid product HX-34d (82 mg, yield 81%), which was directly used in the next step reaction; LCMS: (M+1) ⁺ 1136.27 (theoretical value: 1135.50).

In a 10 mL single-necked bottle, DCM (3 mL), HX-34d (82 mg, 0.07 mmol), bromoacetamide ethoxyethoxypropionic acid (21.5 mg, 0.07 mmol) and DIC (9.1 mg, 0.07 mmol) were added in sequence, and the reaction was stirred at room temperature for 90 min. 0.1 ml of dichloroacetic acid was added and the reaction was continued for 30 min. The reaction solution was injected into a 25 g C18 pre-column (first equilibrated with acetonitrile and then with water, the aqueous phase containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min) and freeze-dried to obtain a yellow solid HX-34 (43 mg, yield 51%); LCMS: (M+1) ⁺ 1173.36 (theoretical value: 1172.40).

HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na ₂ HPO ₄ solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the mixture was stirred at room temperature. The reaction was carried out at 25°C on a rotating turntable for 90 min.

Compound HX-34 (0.94 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g/L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 11 (3.3 mg/ml, 2 ml).

UV-HPLC calculated average value: n=2.3.

Example 12: 7-[N-(IP140B-Ac-PEG ₂ -Gly-Gly-Lys-Gly-ha-Ac), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (12)

Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na ₂ HPO ₄ solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.

Compound HX-34 (0.94 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 12 (3.1 mg/ml, 2 ml).

UV-HPLC calculated average value: n=2.1.

Example 13: 7-[N-(HS627-Ac-PEG ₂ -Gly-Lys-PABC), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (13)

In a 10 ml single-necked bottle, HX-10a (101.5 mg, 0.2 mmol), 1 ml NMP, DIPEA (129 mg, 1 mmol), Fmoc-GK-PAB-PNP (187 mg, 0.2 mmol), and HOBt (27 mg, 0.2 mmol) were added in sequence. The reaction mixture was stirred at room temperature for 4 h to obtain HX-35a. The reaction solution was directly used in the next step; LCMS: (M+1) ⁺ 1306.48 (theoretical value: 1305.54).

To the above HX-32a reaction solution, add 0.1 ml of piperidine (V:V = 10%), react at room temperature for 0.5 h, add 20 ml of methyl tert-butyl ether to the reaction solution, centrifuge, remove the supernatant, and remove the solvent under reduced pressure to obtain a solid product HX-35b (130 mg, yield 50%), which is directly used for the next step reaction; LCMS: (M+1) ⁺ 1076.39 (theoretical value: 1075.43).

In a 10 ml single-mouth bottle, DCM (2 mL), HX-35b (130 mg, 0.12 mmol), HX-3b (35 mg, 0.12 mmol) and DIC (15 mg, 0.12 mmol) were added in sequence, and the reaction was stirred at room temperature for 90 min. 0.1 ml of TFA was added and the reaction was continued for 30 min. The reaction solution was injected into a 25 g C18 pre-column (first equilibrated with acetonitrile and then with water, the aqueous phase containing 0.1% TFA), and then eluted by medium-pressure reverse phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min) and freeze-dried to obtain a yellow solid product HX-35 (41 mg, yield 31%); LCMS: (M+1) ⁺ 1121.04 (theoretical value: 1120.38).

HS627 antibody (20.0 mg/mL, 10 mg, 0.066 mmol) was taken, and the pH was adjusted to 7.2 with 1 M Na ₂ HPO ₄ solution, and then 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was carried out at room temperature 25°C with a rotating turntable for 90 min.

Compound HX-35 (0.90 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, and 0.2 g/L polysorbate 20 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 14 (3.4 mg/ml, 2 ml).

UV-HPLC calculated average value: n=4.3.

Example 14: 7-[N-(IP140B-Ac-PEG2-Gly-Lys-PABC), N-(S)-methoxyisopropyl]aminoethyl-10,11-methylenedioxycamptothecin (14)

Take IP140B antibody (20.0 mg/mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na ₂ HPO ₄ solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C on a rotating turntable for 90 min.

Compound HX-35 (0.90 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA, added to the above solution system, mixed, and reacted on a rotating turntable at room temperature for 2 h. After the reaction was completed, the buffer was replaced with 20 mM histidine solution, 250 mM sorbitol, 0.02% Tween 80, pH 5.7 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 14 (3.2 mg/ml, 2 ml).

UV-HPLC calculated average value: n=4.6.

Test Example 1: In vitro inhibition of tumor cell growth activity by ADC

ADC in vitro inhibitory activity test method: Human esophageal cancer cell OE-33, lung cancer cell NCI-H1975 and breast cancer cell MDA-MB-231 cells used for activity detection were cultured in RPMI1640 (Cellmax), RPMI1640 (Cellmax) and DMEM (Cellmax) culture medium containing 10% fetal bovine serum (Cellmax) to the exponential growth phase, and after trypsin digestion, the supernatant was discarded by centrifugation and diluted with culture medium to 3×10 ⁴ cells/mL, 0.5×10 ⁴ cells/mL and 1.5×10 ⁴ cells/mL, respectively, and 100 μL per well was added to a 96-well cell culture plate and returned to a 37°C, 5% CO ₂ incubator for overnight culture. On the second day, the ADC to be tested was diluted to 2000nM, 400nM, 80nM, 16nM, 3.2nM, 0.64nM, 0.128nM, and 0.026nM using culture medium, and the diluted ADC was added to a 96-well cell culture plate at 100μL per well, with 3 replicates for each concentration, and 100μL of culture medium was added to each well of the negative control and blank control groups without ADC. After the addition of the sample, it was returned to the incubator at 37°C and 5% _CO2 for further incubation for 6 days. After the incubation was completed, the cell culture plate was removed, the culture medium in the culture plate was discarded with a pipette, 100μL of culture medium containing 10% CCK-8 was added to each well, and incubated at 37°C for 3h. After the incubation was completed, the culture plate was removed, protected from light, placed in an ELISA plate, and 630nm was selected as the reference wavelength and 450nm was selected as the measurement wavelength to measure the absorbance. According to the absorbance value, IC ₅₀ was calculated using four-parameter regression in GraphPad (Table 2). The corresponding ADC drug of Dxd was used as the positive control drug, which has the following structure:

Among them, Ab is IP140B antibody.

Table 1: IC ₅₀ (nM) of ADC drugs against NCI-H1975, MDA-MB-231 and OE-33 cells

For IC ₅₀ values, "++++" indicates IC ₅₀ ≤ 50 nM; "+++" indicates 50 nM < IC ₅₀ ≤ 200 nM; "++" indicates 100 < IC ₅₀ ≤ 500 nM; and "+" indicates IC ₅₀ > 500 nM.

The ADC compounds of the embodiments of the present invention have good inhibitory activity against NCI-H1975, MDA-MB-231 and OE-33 cells. The IC ₅₀ of the inhibitory activity of some ADC compounds such as ADC10 and 12 against these cancer cells is even lower than 50 nM.

Test Example 2: In vivo tumor growth inhibition activity of ADC

ADC in vitro inhibitory activity test method: Human non-small cell lung cancer Calu-6, human esophageal squamous cell carcinoma cell KYSE-150 and human ovarian cancer cell ES-2 were cultured in vitro monolayers. When the cell saturation was 80%-90%, they were digested with trypsin-EDTA, centrifuged and discarded the supernatant, and the cells were resuspended in PBS. The cell suspension was adjusted to an appropriate concentration. Calu-6 and KYSE-150 cells (2-10×10 ⁶ cells/0.1ml) were subcutaneously inoculated into BALB/c nude mice, and ES-2 cells were subcutaneously inoculated into NOD-SCID mice. The animals and transplanted tumor growth were observed regularly. When the tumor volume grew to about 100-200mm ³ , they were randomly divided into vehicle control group (normal saline) and ADC administration group (dissolved in normal saline), with 6 animals in each group. The drugs were administered by intravenous injection, and the administration frequency was Q4D, with a total of 2 administrations (the time of the first administration was recorded as Day 1, and the second administration was recorded on Day 5). The experimental grouping and administration settings are shown in Table 2. The long diameter a (mm), short diameter b (mm) of the tumor and the weight of the mice were measured with a vernier caliper twice a week, and the tumor volume (V) was calculated according to the following formula: V = 1/2 × a × b ² (mm ³ ), where a and b represent the length and width of the tumor, respectively. A growth curve was drawn, and the tumor was finally removed and weighed. Statistical analysis The tumor inhibition results were obtained by analyzing the data of tumor volume and weight of tumor-bearing mice at the end of the experiment using GraphPad Prism software. The control group "\" in the figure means that there is no result in this group and the tumor condition was not measured. There is no corresponding tumor in the experimental group, and "tumor reduction to 0" means that no tumor residue was found in the corresponding dissected animal.

Table 2: Mouse grouping and drug administration settings

The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims (11)

1. The antibody-drug conjugate represented by formula (I)
   

   In the formula,

   R ¹ and R ² are each independently selected from hydrogen, deuterium, and C ₁ -C ₆ alkyl;

   L ¹ is selected from -L ¹¹ -L ¹² -L ¹³ -, wherein L ¹¹ , L ¹² , and L ¹³ are each independently selected from -O-, C ₁ -C ₃ alkylene, and phenyl;

   L ^P is a peptide residue consisting of 2-7 amino acids;

   Z is selected from ^-Lz - ^Lj- , wherein ^Lz is selected from -C(=O) _{-C1 -} C8 alkylene and -C(=O)-( _CH2CH2O ) _{2-6 - CH2CH2NH-} , _and ^Lj is a linker that can be coupled to an antibody;

   n ₁ is an integer selected from 0-3;

   Ab is an antibody.
2. The antibody-drug conjugate according to claim 1, wherein R ¹ and R ² are each independently selected from hydrogen, deuterium and C ₁ -C ₃ alkyl;

   Preferably, R ¹ and R ² are each independently selected from hydrogen, deuterium, methyl, ethyl and isopropyl;

   Preferably, R ¹ is selected from hydrogen, deuterium, methyl, ethyl and isopropyl, and R ² is selected from hydrogen, deuterium, methyl and ethyl;

   Preferably, ^R1 is methyl and ^R2 is hydrogen or methyl.
3. The antibody-drug conjugate according to claim 1 or 2, wherein L ¹¹ , L ¹² , and L ¹³ are each independently selected from -O-, methylene, and phenyl;

   Preferably, L ¹¹ -L ¹² -L ¹³ are selected from -O-CH ₂ -phenyl- and -CH ₂ -O-CH ₂ -.
4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn);

   Preferably, ^Lp is selected from a peptide residue consisting of 2-5 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn);

   Preferably, L ^p is selected from a peptide residue consisting of 2-4 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys);

   Preferably, ^Lp is selected from -Val-Cit-, -Val-Ala-, -Gly-Val-Ala-, -Gly-Val-Ala-Gly-, -Gly-Lys-, -Gly-Gly-Lys-, -Gly-Gly-Lys-Gly-, -Val-Ala-, -Ala-Ala-Asn-, -Gly-Leu-, -Gly-Gly-Leu-, -Gly-Gly-Leu-Gly-, -Gly-Phe-, -Gly-Gly-Phe- and -Gly-Gly-Phe-Gly-;

   Preferably, L ^p is selected from Its carbonyl end is connected to -NH- and the other end is connected to Z.
5. The antibody-drug conjugate according to any one of claims 1 to 4, wherein L ^is selected from The positions shown indicate attachment to antibodies. The positions shown indicate attachment to the L ^Z group;

   Preferably, L ^z is selected from —C(═O)—C ₁ -C ₈ alkylene and —C(═O)—(CH ₂ CH ₂ O) _2-6 -CH ₂ CH ₂ NH—;

   Preferably, L ^z is selected from —C(═O)—C ₁ -C ₆ alkylene and —C(═O)—(CH ₂ CH ₂ O) _2-4 —CH ₂ CH ₂ NH—;

   Preferably, L ^z is selected from -C(=O)-(CH ₂ ) ₅ - and -C(=O)-(CH ₂ CH ₂ O) ₂ -CH ₂ CH ₂ NH-;

   Preferably, Z is selected from The positions shown indicate attachment to antibodies; The positions shown indicate attachment to the L ^p group.
6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein the antibody is a tumor-associated antigen antibody;

   Preferably, the tumor-associated antigen antibody is selected from anti-Her2 antibody, anti-Trop2 antibody, anti-B7H3 antibody, anti-5T4, anti-Nectin-4 antibody, anti-CD20 antibody and anti-ROR1 antibody.
7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein the antibody-drug conjugate represented by formula (I) has a structure as represented by formula (I-1) or formula (I-2),
   

   In the formula, R ¹ , R ² , n ₁ , L ^p , Z and Ab have the same meanings as the compound of formula (I).
8. The following antibody-drug conjugates:
   
   

   wherein Ab is defined as the compound of formula (I);

   Preferably, Ab is HS627 antibody or IP140B antibody, the heavy chain amino acid sequence of HS627 is shown in SEQ ID NO:1, and the light chain amino acid sequence thereof is shown in SEQ ID NO:2; the heavy chain amino acid sequence of IP140B antibody is shown in SEQ ID NO:3, and the light chain amino acid sequence thereof is shown in SEQ ID NO:4.
9. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.
10. Use of the antibody-drug conjugate according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 in the preparation of an anti-tumor drug;

    Preferably, the tumor is selected from solid tumors, more preferably lung cancer, esophageal cancer, breast cancer, non-small cell lung cancer, esophageal squamous cell carcinoma or ovarian cancer.
11. A method for treating tumor diseases, comprising the step of administering the antibody-drug conjugate according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 to a patient in need thereof.