Antibody conjugated drugs of n-azidoalkyl substituted camptothecin derivatives

Abstract

The present application relates to an antibody-drug conjugate of formula (I) with an azidoalkyl-substituted camptothecin derivative as a drug toxic moiety, wherein the groups are defined in the specification, the antibody-drug conjugate has a significant anti-tumor effect.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field. Specifically, this invention provides a series of antibody-drug conjugates of N-azidoalkyl-substituted camptothecin derivatives, their preparation methods, and their applications in the field of antitumor therapy. Background Technology

[0002] Camptothecin and its derivatives are inhibitors of top-1 topoisomerases and are broad-spectrum antitumor drugs. Irinotecan and topotecan have been approved in many countries for the treatment of various cancers, while belotecan has been approved in South Korea for the treatment of SCLC and ovarian cancer. Although in recent years, various camptothecin derivatives have been used as payloads for antibody-drug conjugates (ADCs) with very good antitumor effects, such as DS-8201 (Enhertu, Trastuzumab deruxtecan) using Dxd as its payload and IMMU-132 (Trodelvy, Sacituzumab govitecan) using SN-38 as its payload, the development of more drug conjugates with antitumor activity using camptothecin derivatives as payloads remains of great significance. Summary of the Invention

[0003] The purpose of this invention is to provide an antibody-drug conjugate in which a camptothecin derivative substituted with an azidoalkyl group is used as the toxic moiety of the drug, and the antibody-drug conjugate has a significant antitumor effect.

[0004] In one aspect, the present invention provides an antibody-drug conjugate of formula (I):

[0005]

[0006] In the formula,

[0007] Ab represents antibodies;

[0008] R 1 R 2 Each is independently selected from hydrogen, deuterium, hydroxyl, halogen, C1-C6 alkyl, and halogen-substituted C1-C6 alkyl; or, R 1 R 2 The carbon atoms on the benzene ring to which it is attached together form a 5- to 6-membered oxocyclic alkene;

[0009] R 3 Selected from hydrogen, deuterium, and C1-C6 alkyl groups;

[0010] L is selected from -L 1 -L 2 -L 3-, where L 1 L 2 L 3 Each is independently selected from -O-, C1-C3 alkylene and phenyl;

[0011] L P A peptide residue consisting of 2-7 amino acids;

[0012] Z is selected from -L z -L j -, where L z Selected from -C(=O)-C1-C8 alkylene or -C(=O)-(CH2CH2O) 2-6 -CH2CH2NH-, L j It serves as a linker for conjugable antibodies;

[0013] m and n are each independently selected from integers from 1 to 3.

[0014] In some specific implementations, R 1 R 2 Each is independently selected from hydrogen, deuterium, hydroxyl, halogen, and C1-C6 alkyl groups;

[0015] Preferably, R 1 R 2 Each is independently selected from hydrogen, deuterium, halogens, and C1-C6 alkyl groups;

[0016] Preferably, R 1 R 2 Each is independently selected from hydrogen, deuterium, F, and C1-C3 alkyl groups;

[0017] Preferably, R 1 R 2 Each is independently selected from hydrogen, F, and methyl;

[0018] Preferably, R 1 R 2 Each is independently selected from F and methyl;

[0019] Preferably, R 1 It is methyl, R 2 For F;

[0020] or,

[0021] R 1 R 2 The carbon atoms on the benzene ring to which it is attached together form a 5- to 6-membered oxocyclic alkene;

[0022] Preferably, R 1 R 2 The carbon atoms on the benzene ring to which it is attached together form dioxanepentene;

[0023] Preferably, R 1 R 2 The carbon atoms on the benzene ring to which it is attached together form meta-dioxane;

[0024] Preferably, R 1 R 2 The carbon atoms on the benzene ring it is attached to form

[0025] In some specific implementations, R 3 Selected from hydrogen, deuterium, and C1-C6 alkyl groups;

[0026] Preferably, R 3 Selected from hydrogen, deuterium, and C1-C4 alkyl groups;

[0027] Preferably, R 3 Selected from hydrogen, methyl, ethyl, and isopropyl;

[0028] Preferably, R 3 Selected from hydrogen and methyl;

[0029] Preferably, the Selected from

[0030] Preferably, the Selected from

[0031] Preferably, the Selected from

[0032] In some specific implementations, L 1 L 2 L 3 Each is independently selected from -O-, methylene, and phenyl;

[0033] Preferably, L is selected from -O-CH2-phenyl- and -CH2-O-CH2-.

[0034] In some specific embodiments, the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit), aspartic acid (Asn), and glutamic acid (Glu);

[0035] Preferably, the amino acid is selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn);

[0036] Preferably, L p Selected peptide residues consisting of 2-4 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), leucine (Leu), lysine (Lys), citrulline (Cit) and aspartic acid (Asn).

[0037] Preferably, L p Selected from -Val-Cit-, -Val-Ala-, -Gly-Val-Ala-, -Gly-Val-Ala-Gly-, -Gly-Lys-, -Gly-Gly-Lys-, -Gly-Gly-Lys-Gly-, -Val-A la-, -Ala-Ala-Asn-, -Gly-Leu-, -Gly-Gly-Leu-, -Gly-Gly-Leu-Gly-, -Gly-Phe-, -Gly-Gly-Phe-, and -Gly-Gly-Phe-Gly-;

[0038] Preferably, L p It is a peptide residue composed of 2 or 4 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), or alanine (Ala).

[0039] Preferably, L p Selected from Val-Ala and Gly-Gly-Phe-Gly;

[0040] Preferably, L p Selected from Its carbonyl end is connected to -NH-, and the other end is connected to Z.

[0041] Preferably, L p Selected from Its carbonyl end is connected to -NH-, and the other end is connected to Z.

[0042] In some specific implementations, L j Selected from The indicated location represents the connection to the antibody. The position shown indicates that it is related to L. Z Groups linked together;

[0043] Preferably, L z Selected from -C(=O)-C1-C8 alkylene groups and -C(=O)-(CH2CH2O) 2-6 -CH2CH2NH-;

[0044] Preferably, L z Selected from -C(=O)-C1-C6 alkylene groups and -C(=O)-(CH2CH2O)2-4 -CH2CH2NH-;

[0045] Preferably, L z Selected from -C(=O)-(CH2CH2O)2-CH2CH2NH- and -C(=O)-(CH2)5-;

[0046] Preferably, Z is selected from The indicated location represents the connection to the antibody; The position shown indicates that it is related to L. p Groups are linked together.

[0047] In some specific embodiments, the antibody is a tumor-associated antigen antibody;

[0048] 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.

[0049] In some specific embodiments, the antibody-drug conjugate structure shown in formula (I) is as shown in formula (II):

[0050]

[0051] In the formula, R 1 R 2 R 3 L p Each of Ab is a custom isoform (I) antibody-drug conjugate, where m and n are both 2;

[0052] Z is selected from -L z -L j -, where L z Selected from -C(=O)-(CH2CH2O) 2-6 -CH2CH2NH-, L j It serves as a linker for conjugable antibodies;

[0053] Preferably, L z Selected from -C(=O)-(CH2CH2O) 2-4 -CH2CH2NH-;

[0054] Preferably, L z It is -C(=O)-(CH2CH2O)2-CH2CH2NH-;

[0055] Preferably, Z is

[0056] In some specific embodiments, the compound structure represented by formula (I) is as shown in formula (III):

[0057]

[0058] In the formula, R 1 R 2 R 3 L p Each of Ab is a custom isoform (I) antibody-drug conjugate, where m and n are both 2;

[0059] Z is selected from -L z -L j -, where L z Selected from -C(=O)-C1-C8 alkylene groups, L j It serves as a linker for conjugable antibodies;

[0060] Preferably, L z Selected from -C(=O)-C1-C6 alkylene groups;

[0061] Preferably, L z It is -C(=O)-(CH2)5-;

[0062] Preferably, Z is

[0063] This invention also provides the following antibody-drug conjugates:

[0064]

[0065] Where Ab defines compounds of formula (I);

[0066] Preferably, the Ab is an HS627 antibody or an IP140B antibody, wherein the heavy chain amino acid sequence of the HS627 is shown in SEQ ID NO:1 and its light chain amino acid sequence is shown in SEQ ID NO:2; and the heavy chain amino acid sequence of the IP140B antibody is shown in SEQ ID NO:3 and its light chain amino acid sequence is shown in SEQ ID NO:4.

[0067] In another aspect, the present invention provides a method for preparing the above-mentioned antibody-drug conjugate, comprising the following steps:

[0068]

[0069] (1) The compound of formula (a) reacts with the compound of formula (b) to give the compound of formula (c);

[0070] (2) Compound of formula (d) and HO-L z -L j The condensation reaction yields compound (e); wherein compound (d) is obtained by removing the Fmoc protecting group from compound (c);

[0071] (3) Compound of formula (e) is linked with an antibody to obtain compound of formula (I);

[0072] Among them, R 1 R 2 R 3 L p L z Each of Ab, m, and n can define a compound of formula (I), formula (II), or formula (III);

[0073] L j Selected from

[0074] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned antibody-drug conjugate or an antibody-drug conjugate prepared by the aforementioned method and a pharmaceutically acceptable carrier. In another aspect, the present invention provides 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.

[0075] In another aspect, the present invention provides a method for inhibiting tumor disease in patients in need, comprising administering the above-mentioned antibody-drug conjugate or pharmaceutical composition to the patients in need.

[0076] In some specific implementations, the dosage of the above-mentioned antibody-drug conjugate or drug composition is a therapeutically effective amount.

[0077] In some specific embodiments, the tumor is selected from solid tumors.

[0078] In some specific embodiments, the tumor is esophageal cancer, lung cancer, or breast cancer. Detailed Implementation

[0079] I definition:

[0080] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the related terms and laboratory procedures used herein are all widely used terms and routine procedures in the respective fields. To better understand this invention, definitions and explanations of related terms are provided below.

[0081] As used herein and unless otherwise stated, the terms “comprising,” “including,” “having,” “containing,” and their grammatical equivalents, including their grammatical equivalents, should generally be understood as open-ended and non-restrictive, e.g., not excluding other unlisted elements or steps.

[0082] The compounds disclosed herein may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, for example, enantiomers and diastereomers. The stereoisomers include geometric isomers (e.g., cis, trans structures) and optical isomers (e.g., enantiomers), as therapeutic agents comprising monomers, racemates, racemic mixtures, and pharmaceutically acceptable salts thereof. The compounds containing asymmetric carbon atoms of this disclosure can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of this disclosure.

[0083] The numerical ranges used in this article refer to the integers within a given range. For example, "C1-C6" means that the group can have 1, 2, 3, 4, 5, or 6 carbon atoms.

[0084] When any variable (e.g., Rn) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by 1-5 Rs, the group can optionally be substituted by up to 5 Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0085] Term "C" 1- "C6 alkyl" refers to a straight-chain 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 their various branched isomers.

[0086] This refers to the chemical bond connection point. It should be noted that the structural segments described in this invention (e.g., -L) z -L j -), unless otherwise specified, indicates that the corresponding groups are connected in a left-to-right order. For example, when -L zWhen - is -C(=O)-(CH2CH2O)2-CH2CH2NH-, it represents the left-hand side connection of -L to -C(=O)-(CH2CH2O)2-CH2CH2NH-. p -, Right end connects -L j -

[0087] Drugs or drug compositions

[0088] The pharmaceutical products or pharmaceutical compositions disclosed herein can be administered orally, topically, parenterally, or via mucosal routes (e.g., sublingually, by inhalation, or rectally) in dosage units comprising a conventional, non-toxic, pharmaceutically acceptable carrier. Oral administration is generally preferred. The active agent can be administered orally in capsule, tablet, or other similar forms (see Remington: The Science and Practice of Pharmacy, 20th Edition).

[0089] The term "treatment" includes suppressing, alleviating, preventing, or eliminating one or more symptoms or side effects associated with the disease, condition, or disorder being treated.

[0090] The term "inhibition" is used relative to a control. Those 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 the compound is compared to a response in a subject or cell not treated with the compound.

[0091] The term "pharmaceutical composition" means a composition comprising the compounds described in this disclosure or their pharmaceutically acceptable salts, and at least one pharmaceutically acceptable ingredient selected from the following, depending on the manner of administration and the nature of the dosage form: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, thermosensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0092] The term "effective dose" or "therapeutic effective dose" refers to a sufficient amount of a drug or agent that is non-toxic but achieves the desired effect. In embodiments of the invention, when treating a patient according to the invention, the amount of a given drug depends on many factors, such as the specific dosing regimen, the type and severity of the disease or condition, and the unique characteristics of the patient or host requiring treatment (e.g., weight). However, depending on the specific surrounding circumstances, including, for example, the specific drug used, the route of administration, the condition being treated, and the patient or host being treated, the dosage can be conventionally determined by methods known in the art. Typically, for adult treatment, the dosage is typically in the range of 0.02-5000 mg / day, for example, about 1-1500 mg / day. This required dose can conveniently be expressed as a single dose, or concurrent (or over a short period of time) or fractions at appropriate intervals, such as two, three, four, or more doses per day. Those skilled in the art will understand that although the above dosage ranges are given, the specific effective dose can be appropriately adjusted according to the patient's condition and in conjunction with the physician's diagnosis.

[0093] The term "antibody-drug conjugate (ADC)" refers to a small molecule drug with biological activity linked to a monoclonal antibody via a chemical link. The monoclonal antibody then acts as a carrier to target and deliver the drug to specific cells. The term "antibody-drug conjugate" as used in this article has the same meaning as antibody-drug conjugate.

[0094] abbreviation:

[0095] Fmoc: 9-fluorenemethyloxycarbonyl.

[0096] The peptide residues L P The amino acid Val: valine, has the following structural formula: Ala: Alanine, its structural formula is... Gly: Glycine, its structural formula is as follows Phe: Phenylalanine, its structural formula is as follows

[0097] L P It can be obtained by amino acid condensation methods known to the public.

[0098] -PABC-: -ha-: mc-:

[0099] In this invention, the antibody and the adapter L for connecting the antibody... j It can be obtained by methods known in the art. For example, when L j The structure is (or When ), it can be through (or It is obtained by reacting with reactive groups such as thiols on the antibody, and the present invention does not make any particular limitation thereto.

[0100] Example II:

[0101] In this embodiment, the ADC was prepared using, but not limited to, pertuzumab and IP140B antibody. The heavy chain amino acid sequence of pertuzumab (HS627, PERTUZUMAB) is as follows (SEQ ID NO:1):

[0102]

[0103] The light chain amino acid sequence is as follows (SEQ ID NO:2):

[0104]

[0105] The heavy chain amino acid sequence of the IP140B antibody is as follows (SEQ ID NO:3):

[0106]

[0107] The light chain amino acid sequence is as follows (SEQ ID NO:4):

[0108]

[0109] Intermediate 1: 7-(azidoethyl)aminoethyl-10,11-methylenedioxycamptothecin

[0110]

[0111] 3,4-Methylenedioxy-6-aminoacetophenone (5 g, 0.028 mol) was dissolved in anhydrous toluene (10 mL), and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyranone-[3,4-f]indoleazine-3,6,10(4H)-trione (7.35 g, 0.028 mol) and pyridine p-toluenesulfonate (PPTS) (0.7 g, 0.0028 mol) were added. The reaction was heated to 115°C and stirred for 12 h. After cooling, the solvent was concentrated under reduced pressure, 20 mL of methanol was added, filtered, and dried to give 7-methyl-10,11-methylenedioxycamptothecin (11.0 g, 97% yield); LC-MS (M+H) + 407.15 (theoretical value 406.12).

[0112] 2-Azide ethylamine hydrochloride (63.56 mg, 0.74 mmol), DMSO (1 mL), and 7-methyl-10,11-methylenedioxycamptothecin (50 mg, 0.12 mmol) were reacted with stirring at 110-130 °C for 1 h. Isopropanol was added, the solid was filtered, and the mixture was purified by silica gel column chromatography to obtain the title compound (29 mg, yield 47.9%, HPLC 99%). 1 H NMR (500MHz, DMSO-d6) δ8.91(s,2H),7.61(s,1H),7.48(s,1H),7.20(s,1H),6.26(d,J=2.4Hz,1H),5.39(s,1H),5.22(s ,1H),3.75-3.69(m,2H),3.18(d,J=26.4Hz,4H),2.47-2.44(m,2H),1.83(dt,J=14.3,6.9Hz,2H),0.83(t,J=7.3Hz,3H); 13 C NMR (126MHz, DMSO) δ163.00,147.31,141.50,140.63,140.00,137.65,136.58,127.48,118.93,118.93,108. 67,96.12,93.28,89.64,86.54,62.88,55.70,40.28,37.46,36.36,36.36,30.86,20.71,16.74; LC-MS(M+H) + 505.17 (theoretical value 504.18).

[0113] Intermediate 2: N-(azidoethyl)aminoethyl-10-methyl-11-fluorocamptothecin

[0114]

[0115] 2-Azide ethylamine hydrochloride (218.30 mg, 2.54 mmol), 7,10-dimethyl-11-fluorocamptothecin (500 mg, 1.27 mmol), and DMSO (10 mL) were added to the reaction flask. The mixture was stirred and heated to 120-135 °C for 50 minutes. After cooling to room temperature, methyl tert-butyl ether was added, and the mixture was filtered and purified by silica gel column chromatography to obtain the title compound (294 mg, yield 47%, HPLC 95%). 1H NMR (600MHz, DMSO-d6) δ8.33 (d, J = 8.1 Hz, 1H), 8.24 (s, 2H), 7.90 (d, J = 10. 6Hz,1H),7.30(s,1H),6.55(s,1H),5.41(s,2H),5.31(s,2H),3.66(dd,J=1 2.9,4.7Hz,2H),3.54(dd,J=12.9,6.6Hz,2H),2.47(d,J=2.2Hz,2H),2.30 (s,2H),1.90-1.86(m,2H),1.20(d,J=6.7Hz,3H),0.88(s,3H); LC-MS(M+H) + 493.21 (492.19).

[0116] Example 1: 7-[N-(HS627-mc-Gly-Gly-Phe-Gly-ha-Ac),N-(azidoethyl)]aminoethyl-10,11-methylenedioxycamptothecin (1)

[0117]

[0118]

[0119] 7-(azidoethyl)aminoethyl-10,11-methylenedioxycamptothecin (50 mg, 0.099 mmol) was mixed with 1 mL of LDMF, HATU (1.17 g, 0.198 mmol), DIPEA (0.8 g, 0.198 mmol), and Fmoc-Gly-Gly-Phe-Gly-ha-AcOH (127 mg, 0.198 mmol). The mixture was reacted at room temperature for 15 h. After dilution with 200 mL of DCM, 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 HX-12b (90 mg, 80% yield). LCMS: (M+1) + 1132.16 (Theoretical value: 1131.41).

[0120] HX-12b (90 mg, 0.079 mmol) was dissolved in 0.9 mL of DMF, and piperidine (62 mg, 0.79 mmol) was added. The mixture was stirred at room temperature for 1 h, then 20 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 solid product HX-12c (52 mg, 70% yield), which was directly used in the next reaction. LCMS: (M+1) + 910.23 (Theoretical value: 909.34).

[0121] HX-12c (52 mg, 0.055 mmol) was dissolved in 1 mL of anhydrous DMF, and 6-(maleimide)hexanoic acid succinimide ester (16.9 mg, 0.055 mmol) and DIPEA (7 mg, 0.055 mmol) were added sequentially. The mixture was reacted at room temperature for 30 min. The reaction solution was injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water; the aqueous phase contained 0.1% TFA). Elution was performed by medium-pressure reversed-phase C18 chromatography (gradient: 15% to 60% acetonitrile in water, time 50 min). The product was lyophilized to obtain a yellow solid product HX-12 (15 mg, yield 26%); LCMS: (M+1). + 1103.12 (Theoretical value: 1102.41).

[0122] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate 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) by rotating the disc for 90 min.

[0123] Compound HX-12 (0.88 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc 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 (3.4 mg / ml, 2 ml).

[0124] UV-HPLC calculated average: n = 6.5.

[0125] Example 2: 7-[N-(IP140B-mc-Gly-Gly-Phe-Gly-ha-Ac),N-(azidoethyl)]aminoethyl-10,11-methylenedioxycamptothecin (2)

[0126]

[0127] Take IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) solution (10 mM, 0.04 mL), and react with a rotating disc at room temperature (25 °C) for 90 min.

[0128] Compound HX-12 (0.88 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc 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 (3.3 mg / ml, 2 ml).

[0129] UV-HPLC calculated average: n = 6.6.

[0130] Example 3: 7-[N-(HS627-Ac-Val-Ala-PABC),N-(azidoethyl)]aminoethyl-10,11-methylenedioxycamptothecin (3)

[0131]

[0132] In a 10 ml single-necked flask, 7-(azidoethyl)aminoethyl-10,11-methylenedioxycamptothecin (50 mg, 0.098 mmol), 0.5 ml NMP, DIPEA (63 mg, 0.49 mmol), Fmoc-Val-Ala-PAB-PNP (67 mg, 0.098 mmol), and HOBt (13.3 mg, 0.098 mmol) were added sequentially. The mixture was stirred at room temperature for 0.5 h to obtain HX-13a reaction solution, which was then directly added to the next step. LCMS: (M+1) + 1046.11 (Theoretical value: 1045.40).

[0133] To the above HX-13a 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, centrifuge, remove the supernatant, remove the solvent under reduced pressure, and obtain solid product HX-13b (55 mg, yield 67%), which can be directly used for the next reaction; LCMS: (M+1) + 824.21 (Theoretical value: 823.33).

[0134] In a 10 mL single-necked flask, DCM (1 mL), HX-13b (55 mg, 0.066 mmol), bromoacetaminophen ethoxyethoxypropionic acid (19 mg, 0.066 mmol), and DIC (8.42 mg, 0.066 mmol) were added sequentially. The mixture was stirred at room temperature for 90 min. The reaction solution was injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water; the aqueous phase contained 0.1% TFA). Elution was performed by medium-pressure reversed-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min). The lyophilized product yielded a yellow solid, HX-13 (22 mg, yield 30%). LCMS: (M+1) + 1103.98 (Theoretical value: 1102.24).

[0135] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate 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) by rotating the disc for 90 min.

[0136] Compound HX-13 (0.88 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc 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 3 (3.2 mg / ml, 2 ml).

[0137] UV-HPLC calculated average: n = 6.6.

[0138] Example 4: 7-[N-(IP140B-Ac-Val-Ala-PABC),N-(azidoethyl)]aminoethyl-10,11-methylenedioxycamptothecin (4)

[0139]

[0140] Take IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) solution (10 mM, 0.04 mL), and react with a rotating disc at room temperature (25 °C) for 90 min.

[0141] Compound HX-13 (0.88 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc 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 (3.3 mg / ml, 2 ml).

[0142] UV-HPLC calculated average: n = 6.7.

[0143] Example 5: 7-[N-(HS627-Ac-Val-Ala-PABC),N-(azidoethyl)]aminoethyl-10-methyl-11-fluorocamptothecin (5)

[0144]

[0145] In a 10 ml single-necked flask, N-(azidoethyl)aminoethyl-10-methyl-11-fluorocamptothecin (60 mg, 0.121 mmol), 0.5 ml NMP, DIPEA (78.65 mg, 0.609 mmol), Fmoc-Val-Ala-PAB-PNP (82.9 mg, 0.121 mmol), and HOBt (16.46 mg, 0.098 mmol) were added sequentially. The mixture was stirred at room temperature for 0.5 h to obtain the HX-30b reaction solution, which was then directly added to the next step. LCMS: (M+1) + 1034.12 (Theoretical value: 1033.41).

[0146] To the above HX-30b 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, centrifuge, remove the supernatant, remove the solvent under reduced pressure, and obtain solid product HX-30c (63 mg, yield 63.6%), which can be directly used for the next reaction; LCMS: (M+1) + 812.16 (Theoretical value: 811.35).

[0147] In a 10 mL single-necked flask, DCM (1 mL), HX-30c (63.6 mg, 0.078 mmol), HX-30 (23 mg, 0.078 mmol), and DIC (10 mg, 0.078 mmol) were added sequentially. The mixture was stirred at room temperature for 90 min. The reaction solution was injected into a 25 g C18 pre-column (equilibrated first with acetonitrile, then with water; the aqueous phase contained 0.1% TFA). Elution was performed by medium-pressure reversed-phase C18 chromatography (gradient: 5% to 40% acetonitrile in water, time 30 min). The lyophilized product yielded a yellow solid, HX-30 (27 mg, yield 31.2%). LCMS: (M+1) + 1091.25 (Theoretical value: 1090.36).

[0148] Take HS627 antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate 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) by rotating the disc for 90 min.

[0149] Compound HX-30 (0.87 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc 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 (3.2 mg / ml, 2 ml).

[0150] UV-HPLC calculated average: n = 7.5.

[0151] Example 6: 7-[N-(IP140B-Ac-Val-Ala-PABC),N-(azidoethyl)]aminoethyl-10-methyl-11-fluorocamptothecin (6)

[0152]

[0153] Take IP140B antibody (20.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetate solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) solution (10 mM, 0.04 mL), and react with a rotating disc at room temperature (25 °C) for 90 min.

[0154] Compound HX-30 (0.87 mg, 0.8 mmol) was dissolved in 0.09 ml of DMA and added to the above solution system. The mixture was stirred and reacted at room temperature by rotating the disc 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).

[0155] UV-HPLC calculated average: n = 7.4.

[0156] Test Example 1: ADC Inhibits Tumor Cell Growth Activity

[0157] In vitro ADC inhibitory activity assay: Human esophageal cancer cells OE-33, lung cancer cells NCI-H1975, and breast cancer cells MDA-MB-231, used as activity assays, were cultured in RPMI 1640 (Cellmax), RPMI 1640 (Cellmax), and DMEM (Cellmax) media containing 10% fetal bovine serum (Cellmax), respectively, until the exponential growth phase. After trypsin digestion, the supernatant was discarded by centrifugation, and the cells were diluted with culture medium to a concentration of 3 × 10⁻⁶. 4 cells / mL, 0.5×10 4 cells / mL and 1.5×10 4 Cells / mL were added at 100 μL per well to a 96-well cell culture plate and incubated overnight at 37°C with 5% CO2. The next day, the ADC was diluted to 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.128 nM, and 0.026 nM using culture medium. 100 μL of the diluted ADC was added to each well of the 96-well cell culture plate, with three replicates for each concentration. 100 μL of culture medium was added to each well for the negative control and blank control (without ADC). After addition, the plates were incubated at 37°C with 5% CO2 for 6 days. After incubation, the cell culture plates were removed, the culture medium was aspirated from the plates, and 100 μL of medium containing 10% CCK-8 was added to each well. The plates were incubated at 37°C for 3 hours. After incubation, remove the culture plate, protect it from light, and place it in a microplate. Select 630 nm as the reference wavelength and 450 nm as the measurement wavelength to measure the absorbance. Based on the absorbance values, calculate the IC50 using four-parameter regression in GraphPad. 50 (Table 1). Using the corresponding ADC drug of Dxd as a positive control, it has the following structure:

[0158] Ab is the IP140B antibody.

[0159] Table 1: Inhibitory activity IC50 of ADC drugs against NCI-H1975, MDA-MB-231 and OE-33 cells 50 (nM)

[0160]

[0161] For IC 50 The value, where "++++" represents IC. 50 <50nM; "++" indicates IC 50 Between 50nM and 100nM; "++" indicates IC 50 Between 100-250nM; "+" indicates IC 50 >250nM.

[0162] The ADC compounds of the present invention exhibit good inhibitory activity against NCI-H1975, MDA-MB-231, and OE-33 cells, with an IC50 value of [missing information]. 50 All were below 250 nM, and some ADC compounds, such as ADC4, showed inhibitory activity IC50 against these cancer cells. 50 Even those are all below 50 nM.

Claims

1. The following antibody-drug conjugates: 、 、 ； in, Ab stands for antibody.

2. The antibody-drug conjugate according to claim 1, wherein, The Ab is either HS627 antibody or IP140B antibody; the heavy chain amino acid sequence of the HS627 is shown in SEQ ID NO: 1, and its light chain amino acid sequence is shown in SEQ ID NO: 2; the heavy chain amino acid sequence of the IP140B antibody is shown in SEQ ID NO: 3, and its light chain amino acid sequence is shown in SEQ ID NO:

4.

3. A pharmaceutical composition comprising the antibody-drug conjugate of claim 1 or 2 and a pharmaceutically acceptable carrier.

4. Use of the antibody-drug conjugate of claim 1 or 2 or the pharmaceutical composition of claim 3 in the preparation of an antitumor drug.

5. The use according to claim 4, wherein, The tumor is a solid tumor.

6. The use according to claim 4, wherein, The tumor is esophageal cancer, lung cancer, or breast cancer.

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