Antibody conjugated drugs of n-alkoxyalkyl-substituted camptothecin derivatives

Abstract

The present application relates to a kind of N-alkoxyalkyl-substituted camptothecin derivatives as the antibody-drug conjugate shown in formula (I) of drug toxicity part, wherein each group definition is described in the specification, the antibody-drug conjugate has significant antitumor effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine. Specifically, the present application provides a series of N-alkoxyalkyl-substituted camptothecin derivative antibody conjugate drugs, a preparation method thereof, and application thereof in the field of anti-tumor. BACKGROUND

[0002] An antibody-drug conjugate (ADC) realizes the combination of the advantages of monoclonal antibody drugs and small molecule cytotoxins. The ADC is structurally composed of three components, namely an antibody, a cytotoxin, and a linker. The ADC realizes specific targeting of tumors through the antibody, and then releases the cytotoxin to further kill tumor cells. The ADC overcomes the drug resistance problem of monoclonal antibody drugs to some extent. The effect of the ADC mainly depends on the cytotoxin carried by the antibody, and the biological effect mediated by the antibody is not necessary. Even if the antigen mutates to some extent, the ADC can still exert efficacy. For example, the classic ADC drug Kadcyla can treat HER2+ breast cancer that is resistant to naked Herceptin, and can further prolong the overall survival period by 6 months compared with standard therapy. Another clinical study shows that the incidence of grade 3 adverse reactions in the Kadcyla treatment group is reduced by 50% compared with the standard chemotherapy drug treatment group. The ADC can increase the therapeutic index by reducing toxicity and increasing efficacy compared with conventional chemotherapy drugs. As a new type of anti-tumor "weapon", the ADC is expected to make a breakthrough in the field of tumor disease treatment.

[0003] The success of an ADC depends not only on the optimization of each component, but also on the reasonable matching and integration of each component. The three components of the ADC play different roles, and therefore the ADC has different requirements for the three components. The antibody of the ADC needs to meet the requirements of specific targeting of tumors, appropriate affinity, and internalization performance. The toxin of the ADC needs to meet the requirements of high toxicity, clear mechanism of action, and conjugation. SUMMARY

[0004] The present application aims to provide an antibody conjugate drug using an alkoxylalkyl-substituted camptothecin derivative as a drug toxicity component. The antibody-drug conjugate has a significant anti-tumor effect. In one aspect of the present application, an antibody-drug conjugate represented by formula (I) is provided,

[0005]

[0006] in which

[0007] R 1 , R 2 are each independently selected from the group consisting of hydrogen, deuterium, C1-C6 alkyl;

[0008] L 1 is selected from the group consisting of -L 11 -L 12 -L 13 -, wherein L 11 , L 12 , L 13 are each independently selected from the group consisting of -O-, C1-C3 alkylene and phenyl;

[0009] L P is a peptide residue consisting of 2-7 amino acids;

[0010] Z is selected from the group consisting of -L z -L j -, wherein L z is selected from the group consisting of -C(=O)-C1-C8 alkylene and -C(=O)-(CH2CH2O) 2-6 -CH2CH2NH-, L j is a linker to a conjugatable antibody;

[0011] n is selected from the group consisting of an integer from 0 to 3;

[0012] Ab is an antibody.

[0013] In some embodiments, R 1 , R 2 are each independently selected from the group consisting of hydrogen, deuterium and C1-C3 alkyl;

[0014] Preferably, R 1 , R 2 are each independently selected from the group consisting of hydrogen, deuterium, methyl, ethyl and isopropyl;

[0015] Preferably, R 1 is selected from the group consisting of hydrogen, deuterium, methyl, ethyl and isopropyl, R 2 is selected from the group consisting of hydrogen, deuterium, methyl and ethyl;

[0016] Preferably, R 1 is methyl, R 2 is hydrogen or methyl.

[0017] In some embodiments, L 11 , L 12 , L 13 are each independently selected from the group consisting of -O-, methylene and phenyl;

[0018] Preferably, L 11 -L 12 -L 13 is selected from -O-CH2-phenyl- and -CH2-O-CH2-.

[0019] In some embodiments, the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and asparagine (Asn);

[0020] Preferably, L p 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 asparagine (Asn);

[0021] 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), and leucine (Leu), lysine (Lys);

[0022] Preferably, L p 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-;

[0023] Preferably, L p is selected from with its carbonyl end attached to -NH- and its other end attached to Z.

[0024] Preferably, L p is selected from with its carbonyl end attached to -NH- and its other end attached to Z.

[0025] In some embodiments, L j is selected from indicates the attachment to an antibody, the position indicated is attached to L Z the position indicated is attached to L

[0026] Preferably, L z is selected from -C(=O)-C1-C8alkylene and -C(=O)-(CH2CH2O) 2-6 -CH2CH2NH-;

[0027] Preferably, L z is selected from -C(=O)-C1-C6alkylene and -C(=O)-(CH2CH2O) 2-4 -CH2CH2NH-;

[0028] Preferably, L z is selected from -C(=O)-(CH2)5- and -C(=O)-(CH2CH2O)2-CH2CH2NH-;

[0029] Preferably, Z is selected from the position indicated is attached to L the position indicated is attached to L p the position indicated is attached to L.

[0030] In some embodiments, the antibody is a tumor-associated antigen antibody;

[0031] Preferably, the tumor-associated antigen antibody is selected from an anti-Her2 antibody, an anti-Trop2 antibody, an anti-B7H3 antibody, an anti-5T4 antibody, an anti-Nectin-4 antibody, an anti-CD20 antibody, and an anti-ROR1 antibody.

[0032] In some embodiments, the antibody-drug conjugate of formula (I) has a structure as shown in formula (I-1) or formula (I-2),

[0033]

[0034] wherein R 1 , R 2 , n, L p , Z, and Ab are each defined as in the compound of formula (I). The present application provides the following antibody-drug conjugates:

[0035]

[0036]

[0037] wherein Ab is defined as in the compound of formula (I).

[0038] Further, the present application provides the following antibody-drug conjugates:

[0039] Further, the present application provides the following antibody-drug conjugates:

[0040]

[0041] wherein, Ab is defined as the compound of formula (I).

[0042] Preferably, Ab is HS627 antibody or IP140B antibody, the heavy chain amino acid sequence of which is shown as SEQ ID NO: 1, and the light chain amino acid sequence of which is shown as SEQ ID NO: 2; the heavy chain amino acid sequence of which is shown as SEQ ID NO: 3, and the light chain amino acid sequence of which is shown as SEQ ID NO: 4.

[0043] In another aspect of the present application, a method for preparing the antibody-drug conjugate is provided, comprising the following steps:

[0044] The compound of formula (a) is condensed with HO-L z -L j to obtain the compound of formula (b), and the compound of formula (b) is linked with an antibody to obtain the compound of formula (I).

[0045]

[0046] wherein, n, R 1 , R 2 , L 1 , L p , L z , Ab are defined as the compound of formula (I) respectively;

[0047] L j is selected from

[0048] In another aspect of the present application, a pharmaceutical composition is provided, comprising the aforementioned antibody-drug conjugate or the antibody-drug conjugate prepared by the aforementioned method and a pharmaceutically acceptable carrier.

[0049] In another aspect of the present application, the 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 an antitumor drug is provided.

[0050] In another aspect of the present application, a method for inhibiting tumor diseases in a patient in need is provided, comprising administering the aforementioned antibody-drug conjugate or the pharmaceutical composition to the patient in need.

[0051] In some specific embodiments, the aforementioned antibody-drug conjugate or the pharmaceutical composition is administered in a therapeutically effective amount.

[0052] In some specific embodiments, the tumor is selected from solid tumors.

[0053] In some embodiments, the tumor is selected from lung cancer, esophageal cancer, breast cancer, non-small cell lung cancer, esophageal squamous cancer, or ovarian cancer. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 Tumor growth curve graph for the Calu-6 human non-small cell lung cancer CDX model in Test Example 2.

[0055] Figure 2 Post-dissection tumor photograph for the KYSE-150 human esophageal squamous cancer CDX model in Test Example 2.

[0056] Figure 3 Tumor growth curve graph for the KYSE-150 human esophageal squamous cancer CDX model in Test Example 2.

[0057] Figure 4 Post-dissection tumor photograph for the KYSE-150 human esophageal squamous cancer CDX model in Test Example 2.

[0058] Figure 5 Tumor growth curve graph for the ES-2 human ovarian cancer CDX model in Test Example 2.

[0059] Figure 6 Post-dissection tumor photograph for the ES-2 human ovarian cancer CDX model in Test Example 2. DETAILED DESCRIPTION

[0060] I Definitions:

[0061] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined. Also, the relevant terms and laboratory operation procedures used herein are the terms and conventional procedures widely used in the corresponding field. At the same time, in order to better understand the present application, the definitions and explanations of the relevant terms are provided as follows.

[0062] As used herein and unless otherwise indicated, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, are each generally understood either open- ended or non-limiting, e.g., not excluding additional unlisted elements or steps.

[0063] The compounds of the present disclosure can be asymmetric, for example, having one or more stereocenters. Unless otherwise indicated, all stereoisomers (e.g., enantiomers and diastereomers) are included within the scope of the present disclosure. The stereoisomers include geometric isomers (e.g., cis, trans isomers), and optical isomers (e.g., enantiomers), as well as racemates, racemic mixtures, and pharmaceutically acceptable salts thereof. Compounds of the present disclosure containing an asymmetric carbon atom can be isolated in optically active or racemic forms. The optically active forms can be obtained by separation from racemic mixtures, or by using chiral starting materials or chiral reagents in the synthetic sequence. Racemates, diastereomers, enantiomers are all included within the scope of the present disclosure.

[0064] The numerical ranges recited herein are inclusive of the integers within the given range. For example, "C1-C6" 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.

[0065] When any variable (e.g., Rn) occurs more than one time in a compound, its definition in each occurrence is independent of the definition of the other occurrences. Thus, for example, if a group is substituted with 1-5 R, then said group can optionally be substituted with up to 5 R, and each R is selected, independently, at each occurrence. Also, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0066] The term "C 1- C6alkyl" refers to a straight or branched chain alkyl group having from 1 to 6 carbon atoms. Specific examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-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 the like.

[0067] "-" refers to a chemical bond. It is noted that the structural fragments (e.g., -L 11 -L 12 -L 12 - or -L z -L j -) represent the order of attachment of the corresponding groups from left to right, unless otherwise indicated. For example, when -L 12- when -C1-C2alkylene-O- is C1-C2alkylene-O-, it represents the left side attachment -L 11 - the right end attachment -L 13 -.

[0068] pharmaceutical or pharmaceutical composition

[0069] The pharmaceutical or pharmaceutical composition 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 desired to administer the active agent orally. The active agent can be administered orally in the form of capsules, tablets, etc. (see Remington: The Science and Practice of Pharmacy, 20th Edition).

[0070] The term "treatment" includes inhibiting, ameliorating, preventing or eliminating one or more symptoms or side effects associated with the disease, disorder or malady being treated.

[0071] The use of the term "inhibition" is relative to a control. The skilled artisan 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 the response in a subject or cell not treated with the compound.

[0072] The term "pharmaceutical composition" means a composition comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically-acceptable ingredient selected from the group consisting of carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweetening agents, flavoring agents, flavoring adjuvants, antibacterial agents, antifungal agents, lubricating agents, dispersing agents, temperature-sensitive materials, temperature-regulating agents, adherents, stabilizers, suspending agents, and the like, as a function of the mode of administration and the nature of the dosage form.

[0073] The term "effective amount" or "therapeutically effective amount" refers to a nontoxic but sufficient amount of an agent to achieve a desired result. In the practice of the present application, the amount of a given agent to be administered to a patient in accordance with the present application will depend upon factors such as the particular dosing regimen chosen, the type and severity of the disease or condition, the identity of the subject or host to be treated, e.g., body weight, but, in accordance with a particular set of circumstances, including, for example, the particular agent employed, the route of administration, the condition being treated, and the subject or host being treated, the dosage administered can be routinely determined by methods known in the art. In general, in terms of the dosage ranges for adult human treatment, the dosage administered typically will be in the range of 0.02-5000 mg / day, e.g., about 1-1500 mg / day. The desired dose can conveniently be presented in a dosage unit or as divided doses (or within a short period of time) or at appropriate intervals, e.g., two, three, or four or more divided doses per day. It will be appreciated by those skilled in the art that the actual effective amount will depend on such factors as the particular condition being treated, the severity of the condition, the identity and age of the subject or host, and the particular agent employed, and adjustment of the dosage to these and other factors can be made by those skilled in the art. The term "antibody-drug conjugate" (ADC) is a small molecule drug with biological activity linked to a monoclonal antibody via a chemical linker, which serves as a carrier to target the small molecule drug to the target cell. The term "antibody conjugate drug" as used herein has the same meaning as antibody-drug conjugate.

[0074] Abbreviations:

[0075] Fmoc: 9-fluorenylmethyloxycarbonyl.

[0076] The amino acids constituting the peptide residue L P Val: valine, which has the structure Ala: alanine, which has the structure Gly: glycine, which has the structure; Phe: phenylalanine, which has the structure Leu: leucine, which has the structure Lys: lysine, which has the structure

[0077] L P The amino acids constituting the peptide residue L

[0078] PABC: ha:

[0079] In the present application, the antibody and the linker L j for linking the antibody can be linked by methods known in the art. For example, when L j has the structure (or ), it can be linked by (or ) with a reactive group such as thiol on the antibody to be connected, which is not particularly limited in the present application.

[0080] Example II:

[0081] The ADCs prepared in this example use, but are not limited to, HS627 antibody and IP140B antibody, the heavy chain amino acid sequence of HS627 antibody is as follows (SEQ ID NO: 1):

[0082]

[0083] The light chain amino acid sequence is as follows (SEQ ID NO: 2):

[0084]

[0085] The heavy chain amino acid sequence of IP140B antibody is as follows (SEQ ID NO: 3):

[0086]

[0087] The light chain amino acid sequence is as follows (SEQ ID NO: 4):

[0088]

[0089] Unless otherwise specified, the raw materials and equipment used in the specific embodiments of the present application are known products, which are obtained by purchasing commercially available products.

[0090] Intermediate HX-10a: N-(S-)Methoxyisopropylaminoethyl camptothecin

[0091]

[0092] To the reaction bottle, (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-methylenedioxy camptothecin (50 mg, 0.12 mmol) were added, and the mixture was stirred and heated to 120 °C for 50 minutes, then cooled to room temperature, methyl tert-butyl ether was added, and the solid was filtered to obtain the product title compound (36 mg, yield 61%, HPLC 99%); 1H NMR (500 MHz, DMSO-d6) δ 8.64 (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 = 4.7 Hz, 2H), 3.71 - 3.54 (m, 2H), 3.47 (dd, J = 10.1, 5.8 Hz, 6H), 3.25 (d, J = 13.6 Hz, 2H), 1.88 (dt, J = 14.2, 9.2 Hz, 2H), 1.24 (d, J = 6.4 Hz, 3H), 0.88 (dd, J = 9.4, 5.3 Hz, 3H); LC-MS (M+H)+508.37 (calcd 507.20).

[0093] Intermediate HX-14a: N-methoxyethylamine ethylcamptothecin

[0094]

[0095] DMSO (10 mL) and heated to 110 °C with stirring, 7-methyl-10,1 1 - methylenedioxycamptothecin (1.8 g, 4.4 mmol) was added and the temperature was increased to 120-130 °C for 1 h. The reaction was cooled to room temperature, methyl tert-butyl ether was added and filtered. The title compound was purified by silica gel column chromatography (739 mg, yield 34%, HPLC 97%). 1 H NMR (500 MHz, DMSO-d6) δ 8.64 (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 = 4.7 Hz, 2H), 3.71 - 3.54 (m, 2H), 3.47 (dd, J = 10.1, 5.8 Hz, 6H), 3.25 (d, J = 13.6 Hz, 2H), 1.88 (dt, J = 14.2, 9.2 Hz, 2H), 1.24 (d, J = 6.4 Hz, 3H), 0.88 (dd, J = 9.4, 5.3 Hz, 3H); LC-MS (M+H)+508.37 (calcd 507.20).

[0096] Example 1 : 7-[N-(HS627-mc-Gly-Gly-Phe-Gly-ha-Ac), N-(S)-methoxyisopropyl] amine ethyl-10,1 1 - methylenedioxycamptothecin (1)

[0097]

[0098] In a 250 ml single-mouth bottle, Fmoc-Gly-Gly-OH (5 g, 14.11 mmol) was dissolved in DMF (50 mL) and stirred to dissolve, and then 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 turn, and the reaction was warmed to 60°C for 20 min. The reaction solution was poured into ice water, and extracted with ethyl acetate to obtain a white solid product HX-9a (3.2 g, yield 66%); LCMS: (M+1) + 369.21 (theoretical value: 368.14).

[0099] HX-9a (3.2 g, 9.3 mmol) was dissolved in anhydrous DCM (30 mL), and benzyl glycolate (8.03 g, 93 mmol) was added, followed by the addition of PPTS (0.24 g, 0.93 mmol). 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 MgSO4, filtered and concentrated in vacuo. The crude residue was purified by column chromatography to obtain a white powder product HX-9b (4.05 g, yield 97%); LCMS: (M+1) + 475.03 (theoretical value: 474.18).

[0100] HX-9b (3 g, 6.32 mmol) was taken, 100 mL of acetonitrile was added, and then DBU (0.48 g, 3.16 mmol) was added. The reaction was carried out at room temperature for 2 h, and then 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) were added in turn. The reaction was carried out at room temperature for 15 h, and then 100 mL of DCM and 10 mL of isopropyl alcohol were added. The solution was washed with 100 mL*3 (0.5M) HCl, 100 mL*3 saturated sodium bicarbonate solution, 100 mL saturated sodium chloride solution, and dried over 20 g of anhydrous sodium sulfate. Filtration, rotary evaporation of the filtrate, and purification by silica gel column chromatography yielded a white powder solid product HX-9d (2.5 g, yield 63%); LCMS: (M+1) + 736.13 (theoretical value: 735.29).

[0101] HX-9d (2.5 g, 3.4 mmol) was dissolved in 60 ml of methanol and 30 ml of DCM mixed solvent in a 500 ml single-mouth bottle, 10% palladium-carbon (1200 mg) was added, and after hydrogen replacement, the reaction was carried out at room temperature and normal pressure for 2 h. After the reaction was completed, filtration and concentration were carried out to obtain the product HX-9e (2.01 g, yield 91%); LCMS: (M+1) + 645.42 (theoretical value: 645.67).

[0102] HX-9e (0.2 g, 0.31 mmol) was taken, 2 mL of DMF, HATU (1.17 g, 0.31 mmol), DIPEA (0.8 g. 0.62 mmol) and N-(S-) methoxyisopropylamine ethyl camptothecin (156 mg, 0.31 mmol) were added, and the reaction was carried out at room temperature for 15 h. 200 mL of DCM was added for dilution, and the saturated sodium chloride solution was washed. Anhydrous sodium sulfate was dried, and silica gel column chromatography was used for purification to obtain the yellow solid product HX-9f (201 mg, yield 56%); LCMS: (M+1) + 1135.36 (theoretical value: 1134.43).

[0103] HX-9f (201 mg, 0.177 mmol) was dissolved in 0.5 ml of DMF, then piperidine (138 mg, 1.77 mmol) was added, and 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).

[0104] HX-9g (121 mg, 0.131 mmol) was dissolved in 1 ml of anhydrous DMF, and 6-(maleimide) hexanoic acid succinimidyl ester (40 mg, 0.131 mmol) and DIPEA (17 mg, 0.131 mmol) were added in sequence. The reaction was carried out at room temperature for 30 min. The reaction solution was injected into a 25 g C18 pre-column (first balanced with acetonitrile, and then balanced with water containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 15% to 60% acetonitrile in water, time 50 min). Freeze-drying was carried out to obtain the yellow solid product HX-9 (35 mg, yield 24%); LCMS: (M+1) + 1106.16 (theoretical value: 1105.44).

[0105] Take the HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with a 1M Na2HPO4 solution, then add a 0.1M disodium ethylenediaminetetraacetate solution (25 μL), and add a prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and rotate the disc at room temperature 25°C for 90 min.

[0106] Compound HX-9 (0.89 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system, mixed, and rotated at room temperature for 2 h. After the reaction was completed, the buffer was replaced with a 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, 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).

[0107] UV-HPLC average value: n = 6.8.

[0108] Example 2: 7-[N-(IP140B-mc-Gly-Gly-Phe-Gly-ha-Ac), N-(S)-methoxyisopropyl] amine ethyl-10,11-methylenedioxy camptothecin (2)

[0109]

[0110] Take the IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with a 1M Na2HPO4 solution, then add a 0.1M disodium ethylenediaminetetraacetate solution (25 μL), and add a prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and rotate the disc at room temperature 25°C for 90 min.

[0111] Compound HX-9 (0.89 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system, mixed, and rotated at room temperature for 2 h. After the reaction was completed, the buffer was replaced with a 20 mM L-histidine acetate buffer, pH 6.0, 120 mM sucrose, 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).

[0112] UV-HPLC average value: n = 6.8.

[0113] Example 3: 7-[N-(HS627-Ac-PEG2-Val-Ala-PABC), N-(S)-methoxyisopropyl] amine ethyl-10,11-methylenedioxycamptothecin (3)

[0114]

[0115] In a 10 ml single-neck flask, N-(S-) methoxyisopropylamine ethyl camptothecin (50 mg, 0.098 mmol), 0.5 ml NMP, DIPEA (63 mg, 0.49 mmol), Fmoc-VA-PAB-PNP (67 mg, 0.098 mmol), HOBt (13.3 mg, 0.098 mmol) were added in sequence, and the reaction was stirred at room temperature for 0.5 h to obtain HX-10b, which was directly used in the next step; LCMS: (M+1) + 1049.15 (theoretical value: 1048.42).

[0116] In the above HX-10b reaction solution, 0.5 ml of piperidine (V:V = 10%) was added, and the reaction was carried out at room temperature for 30 min. Then 20 ml of methyl tert-butyl ether was added to the reaction solution, centrifuged, and the supernatant was removed. The solvent was removed under reduced pressure to obtain a solid product HX-10c (65 mg, yield 80%), which was directly used in the next step; LCMS: (M+1) + 826.35 (theoretical value: 825.35).

[0117] In a 10 ml single-neck flask, 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 (pre-equilibrated with acetonitrile, and then equilibrated with water containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min). After freeze-drying, a yellow solid product HX-10 (24 mg, yield 27%) was obtained; LCMS: (M+1) + 1106.02 (theoretical value: 1105.36).

[0118] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1M Na2HPO4solution, then add 0.1M disodium ethylenediaminetetraacetate solution (25 μL), and add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml). Stir at room temperature 25°C for 90 min.

[0119] Compound HX-10 (0.89 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system, mixed, and rotated 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, using a NAP-5 gel column (Cytiva) to obtain antibody drug conjugate ADC 3 (3.1 mg / ml, 2 ml).

[0120] UV-HPLC average value: n = 7.3.

[0121] Example 4: 7-[N-(IP140B-Ac-PEG2-Val-Ala-PABC), N-(S)-methoxyisopropyl] amine ethyl-10,11-methylenedioxy camptothecin (4)

[0122]

[0123] Take IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust pH to 7.2 with 1M Na2HPO4solution, then add 0.1M disodium ethylenediaminetetraacetate solution (25 μL), add prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and rotate at room temperature 25°C for 90 min.

[0124] Compound HX-10 (0.89 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system, mixed, and rotated 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).

[0125] UV-HPLC average value: n = 7.6.

[0126] Example 5: 7-[N-(HS627-Ac-PEG2-Gly-Gly-Leu-Gly-ha-Ac), N-(S)-methoxyisopropyl] amine ethyl-10,11-methylenedioxy camptothecin (5)

[0127]

[0128] HX-9b (3 g, 6.32 mmol) was taken in 100 mL of acetonitrile, and DBU (0.48 g, 3.16 mmol) was added. The reaction was carried out at room temperature for 2 h. 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) were added. The reaction was carried out at room temperature for 15 h. The reaction mixture was evaporated. 100 mL of DCM and 10 mL of isopropyl alcohol were added to the reaction mixture. The reaction mixture was washed with 100 mL*3 (0.5 M) HCl, 100 mL*3 saturated sodium bicarbonate solution, and 100 mL saturated sodium chloride solution. The reaction mixture was dried over 20 g of anhydrous sodium sulfate. The reaction mixture was filtered, and the filtrate was evaporated. The product HX-11a (2.66 g, 61% yield) was obtained as a white powder. LCMS: (M+1) + 702.23 (theoretical value: 701.31).

[0129] HX-11a (2.66 g, 3.4 mmol) was dissolved in 50 mL of a mixture of methanol and DCM (25 mL), and 1 g of 10% palladium-carbon was added. After replacement with hydrogen, the reaction was carried out at room temperature for 2 h. The reaction mixture was filtered and concentrated to obtain the product HX-11b (2.1 g, 90%). LCMS: (M+1) + 612.25 (theoretical value: 611.26).

[0130] N-(S-)Methoxyisopropylamine ethyl camptothecin (0.16 g, 0.31 mmol) was taken in 2 mL of DMF, and HATU (1.17 g, 0.31 mmol), DIPEA (0.8 g. 0.62 mmol), and HX-11b (189 mg, 0.31 mmol) were added. The reaction was carried out at room temperature for 15 h. The reaction mixture was diluted with 200 mL of DCM and washed with a saturated sodium chloride solution. The reaction mixture was dried over anhydrous sodium sulfate. The product HX-11c (254 mg, 74% yield) was obtained as a yellow solid. LCMS: (M+1) + 1101.33 (theoretical value: 1100.45).

[0131] HX-11c (254 mg, 0.23 mmol) was dissolved in 0.5 mL of DMF, and piperidine (179 mg, 2.3 mmol) was added. The reaction was carried out at room temperature for 1 h. 30 mL of methyl tert-butyl ether was added to the reaction mixture, which was centrifuged. The supernatant was removed, and the solvent was removed under reduced pressure to obtain the product HX-11d (150 mg, 73.4% yield) as a solid, which was directly used in the next reaction. LCMS: (M+1) + 879.36 (calculated value: 878.38).

[0132] In a 10 ml single neck flask, 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 successively, 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 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 then lyophilized to obtain the product HX-11 (50 mg, yield 25%) as a yellow solid; LCMS: (M+1) + 1158.22 (theoretical value: 1157.39).

[0133] Take the HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with a 1M Na2HPO4 solution, then add a 0.1M disodium ethylenediaminetetraacetate solution (25 μL), and add a prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and rotate the turntable at room temperature 25°C for 90 min.

[0134] Compound HX-11 (0.93 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system, mixed well, and the reaction was carried out 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 using a NAP-5 gel column (Cytiva), to obtain antibody-drug conjugate ADC 5 (3.1 mg / ml, 2 ml).

[0135] UV-HPLC average value: n = 7.1.

[0136] Example 6: 7-[N-(IP140B-Ac-PEG2-Gly-Gly-Leu-Gly-ha-Ac), N-(S)-methoxyisopropyl] amine ethyl-10,11-methylenedioxy camptothecin (6)

[0137]

[0138] Take the IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with a 1M Na2HPO4 solution, then add a 0.1M disodium ethylenediaminetetraacetate solution (25 μL), and add a prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and rotate the turntable at room temperature 25°C for 90 min.

[0139] Compound HX-11 (0.93 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system, mixed, and rotated at room temperature 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 6 (3.3 mg / ml, 2 ml).

[0140] UV-HPLC average value: n = 7.2.

[0141] Example 7: 7-[N-(HS627-Ac-PEG2-Val-Ala-PABC), N-methoxyethyl] amine ethyl-10,11-methylenedioxy camptothecin (7)

[0142]

[0143] In a 10 ml single-necked flask, N-methoxyethyl camptothecin (50 mg, 0.10 mmol), 0.5 ml of NMP, DIPEA (65 mg, 0.50 mmol), Fmoc-VA-PAB-PNP (69 mg, 0.10 mmol), HOBt (13.7 mg, 0.10 mmol) were sequentially added, and the reaction was stirred at room temperature for 0.5 h to obtain a reaction solution of HX-14b, which was directly used in the next step; LCMS: (M+1) + 1035.12 (theoretical value: 1034.41).

[0144] In the above reaction solution of HX-14b, 0.5 ml of piperidine (V:V = 10%) was added, and the reaction was carried out at room temperature for 30 min. Then, 20 ml of methyl tert-butyl ether was added to the reaction solution, centrifuged, and the supernatant was removed. The solvent was removed under reduced pressure to obtain solid product HX-14c (74 mg, yield 71%), which was directly used in the next step; LCMS: (M+1) + 813.25 (theoretical value: 812.34).

[0145] HX-14 (0.88 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system, mixed well, and rotated 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 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 7 (3.0 mg / ml, 2 ml). + 1092.17 (theoretical value: 1091.35).

[0146] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust pH to 7.2 with 1M Na2HPO4solution, then add 0.1M disodium ethylenediaminetetraacetate solution (25 μL), add prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and rotate at room temperature 25°C for 90 min.

[0147] HX-14 (0.88 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system, mixed well, and rotated 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 using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 7 (3.0 mg / ml, 2 ml).

[0148] UV-HPLC average value: n = 7.3.

[0149] Example 8: 7-[N-(IP140B-Ac-PEG2-Val-Ala-PABC), N-methoxyethyl] amine ethyl-10,11-methylenedioxy camptothecin (8)

[0150]

[0151] Take IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust pH to 7.2 with 1M Na2HPO4solution, then add 0.1M disodium ethylenediaminetetraacetate solution (25 μL), add prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and rotate at room temperature 25°C for 90 min.

[0152] Compound HX-14 (0.88 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system, mixed, and rotated on a rotary plate at room temperature 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 an NAP-5 gel column (Cytiva), to obtain antibody-drug conjugate ADC 8 (3.3 mg / ml, 2 ml).

[0153] UV-HPLC average value: n = 7.5.

[0154] Example 9: 7-[N-(HS627-Ac-PEG2-Gly-Gly-Leu-Gly-ha-Ac), N-methoxyethyl] amine ethyl-10,11-methylenedioxy camptothecin (9)

[0155]

[0156] Take N-methoxyethyl camptothecin (100 mg, 0.20 mmol), add 2 mL of 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, dilute with 200 mL of DCM, wash with saturated sodium chloride solution, dry over anhydrous sodium sulfate, and purify by silica gel column chromatography to obtain yellow solid HX-15a (180 mg, yield 81%); LCMS: (M+1) + 1087.15 (theoretical value: 1086.43).

[0157] HX-15a (180 mg, 0.17 mmol) was dissolved in 0.5 ml of DMF, then piperidine (179 mg, 2.3 mmol) was added, and the reaction was stirred at room temperature for 1 h. To the reaction solution was added 30 ml of methyl tert-butyl ether, centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure to obtain solid product HX-15b (114 mg, yield 77%) which was directly used in the next reaction; LCMS: (M+1) + 865.35 (theoretical value: 864.37).

[0158] HX-15 (48 mg, yield 32%) was obtained as a yellow solid by LCMS: (M+1) 1145.03 (theoretical value: 1143.38). + 1145.03 (theoretical value: 1143.38).

[0159] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust pH to 7.2 with 1M Na2HPO4solution, then add 0.1M disodium ethylenediaminetetraacetate solution (25 μL), add prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and rotate the turntable at room temperature 25°C for 90 min.

[0160] Compound HX-15 (0.92 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system, mixed, and reacted 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 using a NAP-5 gel column (Cytiva) to obtain antibody drug conjugate ADC 9 (3.0 mg / ml, 2 ml).

[0161] UV-HPLC average value: n = 7.4.

[0162] Example 10: 7-[N-(IP140B-Ac-PEG2-Gly-Gly-Leu-Gly-ha-Ac), N-methoxyethyl] amine ethyl-10,11-methylenedioxy camptothecin (10)

[0163]

[0164] Take IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust pH to 7.2 with 1M Na2HPO4solution, then add 0.1M disodium ethylenediaminetetraacetate solution (25 μL), add prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and rotate the turntable at room temperature 25°C for 90 min.

[0165] Compound HX-15 (0.92 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system, mixed well, and rotated at room temperature 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 an NAP-5 gel column (Cytiva) to obtain antibody drug conjugate ADC 10 (3.2 mg / ml, 2 ml).

[0166] UV-HPLC average value: n = 7.3.

[0167] Example 11: 7-[N-(HS627-Ac-PEG2-Gly-Gly-Lys-Gly-ha-Ac), N-(S)-methoxyisopropyl] amine ethyl-10,11-methylenedioxy camptothecin (11)

[0168]

[0169] HX-9b (2 g, 4.21 mmol) was dissolved in 60 mL of acetonitrile, and DBU (0.31 g, 2.08 mmol) was added and reacted at room temperature for 2 h. PPTS (0.52 g, 2.08 mmol), HOBT (0.56 g, 4.2 mmol), Fmoc-Gly-Gly-Lys-OH (3.29 g, 4.2 mmol), and EDCI (0.85 g, 4.2 mmol) were sequentially added, and after the addition was completed, the reaction was carried out at room temperature for 15 h. The reaction solution was concentrated under reduced pressure, 100 mL of DCM and 10 mL of isopropanol were added, and the solution was washed with HCl, saturated sodium bicarbonate solution, and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated and purified by silica gel chromatography to obtain white powder solid HX-34a (2.5 g, yield 60%); LCMS: (M+1) + 959.11 (theoretical value: 958.43).

[0170] HX-34a (2.5 g, 2.6 mmol) was dissolved in 40 mL of a mixture of methanol and 20 mL of DCM in a 500 mL single-neck flask, and 10% palladium-carbon (1.0 g) was added. After replacement with hydrogen gas, the reaction was carried out at room temperature under normal pressure for 2 h. After filtration and concentration, solid product HX-34b (2.2 g, yield 97%) was obtained; LCMS: (M+1) + 869.12 (calculated value: 868.38).

[0171] 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 reaction was allowed to proceed at room temperature for 15 h. The reaction was diluted with 200 mL DCM, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain the product HX-34c (121 mg, yield 76%) as a yellow solid; LCMS: (M+1) + 1358.52 (theoretical value: 1357.57).

[0172] 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, 10 mL methyl tert-butyl ether was added, centrifuged, the supernatant was removed, and the solvent was removed under reduced pressure to obtain the product HX-34d (82 mg, yield 81%) as a solid, which was directly used in the next reaction; LCMS: (M+1) + 1136.27 (theoretical value: 1135.50).

[0173] HX-34d (82 mg, 0.07 mmol), bromoacetamide ethoxy ethoxy propionic acid (21.5 mg, 0.07 mmol) and DIC (9.1 mg, 0.07 mmol) were sequentially added to a 10 mL single-neck flask containing DCM (3 mL), 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 (pre-equilibrated with acetonitrile, and then equilibrated with water containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min). After freeze-drying, HX-34 (43 mg, yield 51%) was obtained as a yellow solid; LCMS: (M+1) + 1173.36 (theoretical value: 1172.40).

[0174] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was adjusted to pH 7.2 with 1M Na2HPO4solution, and then 0.1M disodium ethylenediaminetetraacetate solution (25 μL) was added. A prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml) was added, and the reaction was allowed to proceed at room temperature for 90 min.

[0175] Compound HX-34 (0.94 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA and added to the above solution system, mixed, and reacted at room temperature for 2 h on a rotary plate. 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, using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 11 (3.3 mg / ml, 2 ml).

[0176] UV-HPLC average value: n = 2.3.

[0177] Example 12: 7-[N-(IP140B-Ac-PEG2-Gly-Gly-Lys-Gly-ha-Ac), N-(S)-methoxyisopropyl] amine ethyl-10,11-methylenedioxy camptothecin (12)

[0178]

[0179] Take IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust pH to 7.2 with 1M Na2HPO4solution, then add 0.1M disodium ethylenediaminetetraacetate solution (25 μL), and add prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 ml), and react at room temperature 25°C for 90 min on a rotary plate.

[0180] Compound HX-34 (0.94 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA and added to the above solution system, mixed, and reacted at room temperature for 2 h on a rotary plate. 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).

[0181] UV-HPLC average value: n = 2.1.

[0182] Example 13: 7-[N-(HS627-Ac-PEG2-Gly-Lys-PABC), N-(S)-methoxyisopropyl] amine ethyl-10,11-methylenedioxy camptothecin (13)

[0183]

[0184] 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), HOBt (27 mg, 0.2 mmol) were added in sequence in a 10 ml single-neck flask. The reaction was stirred at room temperature for 4 h, and the reaction solution of HX-35a was directly used in the next step. LCMS: (M+1) + 1306.48 (theoretical value: 1305.54).

[0185] HX-32a reaction solution was added with 0.1 ml of piperidine (V:V=10%) and reacted at room temperature for 0.5 h. Then 20 ml of methyl tert-butyl ether was added to the reaction solution, centrifuged, and the supernatant was removed. The solvent was removed under reduced pressure to obtain solid product HX-35b (130 mg, yield 50%) which was directly used in the next step. LCMS: (M+1) + 1076.39 (theoretical value: 1075.43).

[0186] 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 in a 10 ml single-neck flask. The reaction was stirred at room temperature for 90 min, and then 0.1 ml of TFA was added. The reaction was continued for 30 min. The reaction solution was injected into a 25 g C18 pre-column (pre-equilibrated with acetonitrile, and then equilibrated with water containing 0.1% TFA), and then eluted by medium-pressure reverse-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min). After freeze-drying, yellow solid product HX-35 (41 mg, yield 31%) was obtained. LCMS: (M+1) + 1121.04 (theoretical value: 1120.38).

[0187] HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol) was adjusted to pH 7.2 with 1M Na2HPO4solution, and then 0.1M disodium ethylenediaminetetraacetate solution (25 μL) was added. Then 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 for 90 min on a rotary disc.

[0188] Compound HX-35 (0.90 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA and added to the above solution system, mixed, and rotated 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, using a NAP-5 gel column (Cytiva) to obtain antibody-drug conjugate ADC 14 (3.4 mg / ml, 2 ml).

[0189] UV-HPLC average value: n = 4.3.

[0190] Example 14: 7-[N-(IP140B-Ac-PEG2-Gly-Lys-PABC), N-(S)-methoxyisopropyl] amine ethyl-10,11-methylenedioxy camptothecin (14)

[0191]

[0192] Take IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust pH to 7.2 with 1M Na2HPO4solution, then add 0.1M disodium ethylenediaminetetraacetate solution (25 μL), and add prepared TCEP·HCl (tris(2-carboxyethyl) phosphine hydrochloride) solution (10 mM, 0.04 ml), and rotate at room temperature 25°C for 90 min.

[0193] Compound HX-35 (0.90 mg, 0.8 mmol) was dissolved in 0.09 mL of DMA and added to the above solution system, mixed, and rotated 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).

[0194] UV-HPLC average value: n = 4.6.

[0195] Test Example 1: In vitro tumor cell growth inhibition activity of ADC

[0196] In vitro ADC inhibition activity test method: Human esophageal cancer cells OE-33, lung cancer cells NCI-H1975 and breast cancer cells MDA-MB-231 cells for activity detection were cultured in RPMI1640 (Cellmax) containing 10% fetal bovine serum (Cellmax), RPMI1640 (Cellmax) and DMEM (Cellmax) medium to the exponential growth phase, trypsinized, centrifuged, and discarded the supernatant, diluted with medium to 3x104 cells / mL, 0.5 x 10 4 cells / mL and 1.5 x 10 4 cells / mL, 100 μL per well into 96-well cell culture plates, and placed back into the incubator at 37°C, 5% CO2 overnight. The next day, the ADCs to be tested were diluted to 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.128 nM, 0.026 nM using the culture medium, and 100 μL of the diluted ADCs were added to the 96-well cell culture plates, with 3 replicates for each concentration, and 100 μL of culture medium was added to the negative control and blank control groups without the addition of ADCs. After the addition of the samples was completed, the plates were placed back into the incubator at 37°C, 5% CO2 for 6 days of incubation. After the incubation was completed, the cell culture plates were removed, and the culture medium in the plates was aspirated using a pipette, 100 μL of culture medium containing 10% CCK-8 was added to each well, and the plates were incubated at 37°C for 3 h. After the incubation was completed, the plates were removed, protected from light, and placed into an enzyme-labeled plate, and the absorbance was measured at a reference wavelength of 630 nm and a measurement wavelength of 450 nm. According to the absorbance values, the IC50 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:

[0197] wherein Ab is the IP140B antibody.

[0198] Table 1: Inhibitory activity IC50 of ADC drugs on NCI-H1975, MDA-MB-231 and OE-33 cells 50 (nM)

[0199]

[0200]

[0201] For the IC 50 values, wherein “++++” indicates that the IC 50 ≤ 50 nM; “+++” indicates that 50 nM < IC 50 ≤ 200 nM; “++” indicates that 100 < IC 50 ≤ 500 nM; “+” indicates that IC 50 > 500 nM.

[0202] The ADC compounds of the embodiments of the present application have good inhibitory activity on NCI-H1975, MDA-MB-231 and OE-33 cells, and the inhibitory activity IC 50 of some ADC compounds, such as ADC10, 12, on these cancer cells is much lower than 50 nM.

[0203] Test Example 2: In vivo anti-tumor activity of ADCs

[0204] In vitro anti-tumor activity test method: Human non-small cell lung cancer Calu-6, human esophageal squamous cell carcinoma KYSE-150 and human ovarian cancer ES-2 cells were cultured in monolayer in vitro, and when the cell saturation was 80%-90%, they were digested with trypsin-EDTA, centrifuged to discard the supernatant, resuspended with PBS, and the cell suspension was adjusted to the appropriate concentration. Calu-6 and KYSE-150 cells (2-10 x 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 the growth of the transplanted tumors were observed regularly. When the tumor volume reached 100-200 mm 3 left and right, the animals were randomly divided according to the tumor volume and body weight, i.e. the vehicle control group (normal saline) and the ADC administration group (dissolved in normal saline), with 6 animals in each group. Intravenous administration was performed, with a Q4D administration frequency for a total of 2 administrations (the first administration time was recorded as Day 1, Day 5 for the second administration). The experimental grouping and administration settings are shown in Table 2. The tumor long diameter a (mm) and short diameter b (mm) and the body weight of the mice were measured twice a week using a vernier caliper, the tumor volume (V) was calculated according to the following formula: V = 1 / 2 x a x b 2 (mm 3 ), where a and b represent the tumor length and width, respectively, and the growth curve was plotted. Finally, the tumor was peeled off and weighed. Statistical analysis was performed based on the tumor volume and tumor-bearing mouse body weight data at the end of the experiment using GraphPad Prism software to obtain the tumor inhibition results. In the figure, the control group "\ " indicates that there was no result, the tumor was not measured, and the experimental group had no corresponding tumor, "tumor shrinkage to 0" indicates that no tumor residue was found in the corresponding dissected animal.

[0205] Table 2: Mouse grouping and administration settings

[0206]

Claims

1. An antibody-drug conjugate represented by Formula (I), Ab-(L)n-D (I), wherein, Ab is an antibody; L is a linker; n is an integer selected from 0-3; and D is a drug.

2. The antibody-drug conjugate of claim 1, wherein, Ab is an antibody. R 1 , R 2 each independently is selected from hydrogen, deuterium, C1-C6alkyl; L 1 selected from -O-CH2-phenyl- and -CH2-O-CH2-; L P -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-; Z is selected from the position indicated means attached to the antibody; the position indicated means attached to the L p group; 3. The antibody-drug conjugate of claim 1 or 2, wherein, Ab is a tumor-associated antigen antibody.

4. The antibody-drug conjugate of claim 3, wherein, the tumor-associated antigen antibody is selected from the group consisting of an anti-Her2 antibody, an anti-Trop2 antibody, an anti-B7H3 antibody, an anti-5T4 antibody, an anti-Nectin-4 antibody, an anti-CD20 antibody, and an anti-ROR1 antibody.

2. The antibody-drug conjugate of claim 1, wherein, R 1 , R 2 each independently is selected from the group consisting of hydrogen, deuterium and C1-C3alkyl.

3. The antibody-drug conjugate of claim 1, wherein, R 1 , R 2 are each independently selected from the group consisting of hydrogen, deuterium, methyl, ethyl and isopropyl.

4. The antibody-drug conjugate of claim 1, wherein, R 1 selected from hydrogen, deuterium, methyl, ethyl and isopropyl, R 2 selected from hydrogen, deuterium, methyl and ethyl.

5. The antibody-drug conjugate of claim 1, wherein, R 1 is methyl, R 2 is hydrogen or methyl.

5. The antibody-drug conjugate of claim 4, wherein, the tumor-associated antigen antibody is an anti-Her2 antibody. L p selected from with its carbonyl end attached to -NH- and the other end to Z.

7. The antibody-drug conjugate of claims 1-6, wherein, 6. The antibody-drug conjugate of claim 1, wherein, Ab is an antibody.

8. The antibody-drug conjugate of claim 7, wherein, 7. The antibody-drug conjugate of claim 1, wherein, Ab is an antibody.

8. The antibody-drug conjugate of claim 1, represented by Formula (I) has a structure as shown in Formula (I-1) or Formula (I-2), Ab-(L)n-D (I-1) Ab-(L)n-D (I-2). wherein R 1 , R 2 , n, L p , and Z are each as defined for the compound of formula (I).

9. The antibody-drug conjugate of claim 8, represented by Formula (I) has a structure as shown in Formula (I-1) or Formula (I-2). wherein 10. The antibody-drug conjugate of claim 9, selected from the group consisting of:

11. The antibody-drug conjugate of claim 10, wherein, Ab is defined as the compound of Formula (I).

11. The antibody-drug conjugate of claim 10, wherein, Ab is an IP140B antibody, the heavy chain amino acid sequence of which is set forth in SEQ ID NO: 3, and the light chain amino acid sequence of which is set forth in SEQ ID NO:

4.

12. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1-11 and a pharmaceutically acceptable carrier.

14. The use according to claim 13, wherein, 13. Use of the antibody-drug conjugate of any one of claims 1-11 or the pharmaceutical composition of claim 12 in the manufacture of a medicament for treating a solid tumor.

15. The use according to claim 13, wherein, 14. The use of claim 13, wherein, the solid tumor is selected from the group consisting of lung cancer, esophageal cancer, breast cancer, esophageal squamous cell carcinoma, and ovarian cancer.

15. The use of claim 13, wherein, the solid tumor is non-small cell lung cancer.

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