Halogen-substituted exatecan amide derivative, preparation method therefor, and use thereof

Abstract

The present invention relates to the field of biological medicines, and provides a halogen-substituted exatecan amide derivative, a preparation method therefor, and a use thereof; the halogen-substituted exatecan amide derivative or a pharmaceutically acceptable salt, stereoisomer, or prodrug thereof has good cancer cell inhibitory activity and activity across the cell membrane, and can be applied to the treatment of cancer as a single drug, a combined drug, or a toxin component of an ADC.

Description

Halogen-substituted amide derivatives of exatecan, preparation method and application thereof

[0001] This application claims priority to Chinese patent application No. 2023112892206, filed on September 28, 2023, and cites the full text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention relates to the field of biomedicine, and in particular to a halogen-substituted amide derivative of exatecan, a preparation method thereof, and an application thereof. Background Art

[0003] Exatecan, a derivative of the natural product camptothecin, exhibits inhibitory activity against topoisomerase Top1, particularly against the Top1-DNA complex. Camptothecin has significant therapeutic effects on gastric, esophageal, lung, and bladder cancers, and is a broad-spectrum anti-tumor drug. Irinotecan and topotecan have been approved in many countries for the treatment of various cancers.

[0004] Belotecan, a camptothecin derivative, has been approved in South Korea for the treatment of SCLC and ovarian cancer. The main drawbacks of camptothecin anti-tumor drugs are their toxicity, poor solubility and the development of drug resistance in tumor cells.

[0005] Summary of the Invention

[0006] To solve the above problems, the present invention provides a halogen-substituted amide derivative of exatecan, a preparation method thereof, and an application thereof, which has good tumor cell inhibitory activity and cell membrane permeability, and can be used in the treatment of cancer as a single drug, a combination drug, or as a toxin component of ADC.

[0007] In one aspect of the present invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof:

[0008] In the formula, R is selected from C1-C6 alkyl, halogen-substituted C1-C6 alkyl, Any of the following;

[0009] wherein R1 is selected from C1-C6 alkylene, said C1-C6 alkylene being optionally substituted with one or more substituents selected from halogen, C1-C6 alkyl or halogen-substituted C1-C6 alkyl;

[0010] R2 is selected from any one of halogen, C1-C6 alkyl or halogen-substituted C1-C6 alkyl; X is selected from any one of amino and imino.

[0011] In some embodiments of the present invention, R1 is selected from methylene, halogen-substituted methylene, or trifluoromethyl-substituted methylene.

[0012] In some embodiments of the present invention, R1 is selected from -CHF-, -CHF2-, -CH2-, or -CH(CF3)-.

[0013] In some embodiments of the present invention, R2 is selected from any one of halogen, methyl, methylene, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, and heptyl, preferably, any one of methylene, methyl, ethyl, and isopropyl.

[0014] The halogen is selected from any one of -F, -Cl, -Br, and -I, preferably -F.

[0015] In some embodiments of the present invention, X is selected from -NH- or -NH2.

[0016] In some embodiments of the present invention, Selected from

[0017] Wherein, R1 is selected from halogen; preferably F;

[0018] n is selected from 1, 2, 3 or 4, preferably, n is 1.

[0019] In some embodiments of the present invention, R is substituted with fluorine, which has better tumor cell inhibition activity and cell membrane permeability activity.

[0020] In some embodiments of the present invention, R is selected from

[0021] In some embodiments of the present invention, the above-mentioned compound or its pharmaceutically acceptable salt, stereoisomer or prodrug, wherein the compound of formula (I) is any one of the following compounds:

[0022] As another aspect of the present invention, a method for preparing the above-mentioned compound is provided, comprising: reacting a halogen-substituted carboxylic acid, a carboxylic anhydride, an acyl chloride and exatecan to prepare the above-mentioned compound; preferably, the halogen-substituted carboxylic acid, carboxylic anhydride, and acyl chloride are selected from any one of 2-fluoromalonic acid, 2-amino-3,3,3-trifluoropropionic acid, difluoroacetic anhydride, and 2,2-difluoropropionic acid.

[0023] In some embodiments of the present invention, the halogen-substituted carboxylic acid, carboxylic anhydride, and acyl chloride further include halogen-substituted carboxylic acid, carboxylic anhydride, and acyl chloride with protected amino groups; exatecan further includes the corresponding salt of exatecan; and the reaction conditions are direct reaction or reaction under the action of a condensation reagent.

[0024] In some embodiments of the present invention, the amino protecting group in the amino protection method is selected from any one of benzyloxycarbonyl (Cbz-) and tert-butyloxycarbonyl (Boc-); the halogen-substituted carboxylic acid, carboxylic anhydride, and acyl chloride of the amino group being protected is selected from N-Cbz-N-trifluoroethylglycine N-Boc-4,4-difluoroproline any one of; the corresponding salt of exatecan is selected from exatecan mesylate; the condensation reagent is any one of N,N-diisopropylcarbodiimide (DIC) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

[0025] As another aspect of the present invention, an antibody-drug conjugate is provided, which comprises a small molecule drug, a linker and an antibody, wherein the small molecule drug comprises the above-mentioned compound or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof.

[0026] As another aspect of the present invention, a pharmaceutical composition is provided, comprising the above-mentioned compound or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof.

[0027] As another aspect of the present invention, provided is a use of the above-mentioned compound or its pharmaceutically acceptable salt, stereoisomer or prodrug, the compound obtained by the above-mentioned preparation method, the above-mentioned antibody-drug conjugate or the above-mentioned pharmaceutical composition in the preparation of a drug for treating cancer.

[0028] As another aspect of the present invention, a method for treating cancer is provided, comprising the step of administering the above-mentioned compound or its pharmaceutically acceptable salt, stereoisomer or prodrug, the compound obtained by the above-mentioned preparation method, the above-mentioned antibody-drug conjugate or the above-mentioned pharmaceutical composition to a patient in need.

[0029] Furthermore, the method comprises the step of administering a therapeutically effective amount of the above compound to a patient in need thereof.

[0030] In some embodiments of the present invention, the cancer includes any one of gastric cancer, esophageal cancer, esophageal cancer, breast cancer, mammary adenocarcinoma, and lung cancer.

[0031] In some embodiments of the present invention, the cancer cells are selected from any one of OE33 cells (human esophageal cancer cells), MDA-MB-231 (human breast cancer cells), NCI-N87 (human gastric cancer cells), NCI-H1975 (human lung cancer cells), and TE12 (human esophageal cancer cells). DETAILED DESCRIPTION

[0032] I. Definition

[0033] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the relevant terms and laboratory procedures used herein are those widely used in the relevant fields and routine procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0034] As used herein and unless otherwise specified, the terms "comprises," "includes," "has," "contains," and their grammatical equivalents should generally be understood as open-ended and non-limiting, e.g., not excluding other unlisted elements or steps.

[0035] The compounds of the present invention may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The stereoisomers include geometric isomers (such as cis, trans structures) and optical isomers (such as enantiomers), and are therapeutic substances consisting of monomers, racemates, racemic mixtures and pharmaceutically acceptable salts thereof. The compounds of the present invention containing asymmetric carbon atoms can be isolated in optically pure form or racemic form. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral raw materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of the present invention.

[0036] The compounds of the present invention also include tautomeric forms, which result from the exchange of a single bond with an adjacent double bond accompanied by the migration of a proton.

[0037] As used herein, "pharmaceutically acceptable salts" refer to salts formed between the corresponding amine compounds and inorganic or organic acids, or between the corresponding carboxylic acid compounds and alkali metals or alkaline earth metals, or between the corresponding carboxylic acid compounds and organic amines. Inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like; organic acids include, but are not limited to, acetic acid, propionic acid, butyric acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, succinic acid, lactic acid, citric acid, succinic acid, gluconic acid, maleic acid, fumaric acid, tartaric acid, and the like; alkali metal or alkaline earth metal salts include, but are not limited to, sodium, potassium, calcium, and magnesium salts; and organic amine salts include, but are not limited to, salts composed of ammonia, methylamine, ethylamine, propylamine, isopropylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, tert-butylamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, morpholine, piperidine, piperazine, and amino acids.

[0038] As used herein, "precursor" refers to a compound that, after entering the human body via an appropriate administration route, undergoes metabolism or simple chemical changes within the patient's body to transform into the compound of Formula 1 of the present invention and its corresponding salt. Precursors of the compound include, but are not limited to, various carboxylic acid esters, carbonates, phosphates, sulfates, sulfonates, amino acid esters, gluconates, and various amides, acetals, hemiacetals, and carbonate amides.

[0039] As used herein, numerical ranges refer to the individual 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; "C3-C6" means that the group can have 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

[0040] 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 Rn, the group may be optionally substituted with up to 5 Rn, with each occurrence of Rn being an independent choice. Furthermore, combinations of substituents and / or their variants are permitted only if such combinations result in stable compounds.

[0041] The term "alkyl" refers to a saturated aliphatic hydrocarbon group containing 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms. Non-limiting examples include 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, and 2,3-dimethylbutyl. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include 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 the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available connection point. The substituent is preferably one or more of the following groups, which are independently selected from alkyl and halogen. Methyl, ethyl, isopropyl, tert-butyl and halogenated alkyl are preferred in the present invention.

[0042] The hydrogen atoms described in the present invention can all be replaced by their isotope deuterium.

[0043] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1-3 hydrogen atoms, in a group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0044] Refers to the chemical bond connection.

[0045] Drug or pharmaceutical composition

[0046] The medicaments or pharmaceutical compositions of the present invention 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. The oral route is generally desirable. The active agent can be administered orally in the form of capsules, tablets, and the like (see Remington: The Science and Practice of Pharmacy, 20th Edition).

[0047] For oral administration in the form of tablets or capsules, the active drug component can be mixed with non-toxic, pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, sucrose, glucose, mannitol, sorbitol and other reducing and non-reducing sugars, microcrystalline cellulose, calcium sulfate or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc or silica, stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate, etc.); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate), coloring and flavoring agents, gelatin, sweeteners, natural and synthetic gums (such as acacia, tragacanth or alginates), buffer salts, carboxymethylcellulose, polyethylene glycol, waxes, etc. For oral administration in liquid form, the drug component can be combined with a non-toxic, pharmaceutically acceptable inert carrier (e.g., ethanol, glycerol, water), an anti-settling agent (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), an emulsifier (e.g., lecithin or gum arabic), a non-aqueous carrier (e.g., almond oil, oily esters, ethanol or fractionated vegetable oils), a preservative (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid), etc. Stabilizers such as antioxidants (BHA, BHT, propyl citric acid, sodium ascorbate, citric acid) can also be added to stabilize the dosage form.

[0048] Tablets comprising the active compound can be coated by methods well known in the art. The compositions of the present invention comprising the compound of formula (I) as the active compound can also be introduced into beads, microspheres or microcapsules, for example, constructed from polyglycolic acid / lactic acid (PGLA). Liquid preparations for oral administration can take the form of, for example, solutions, syrups, emulsions or suspensions, or they can be presented as dry products reconstituted with water or other suitable adjuvants before use. Preparations for oral administration can be suitably formulated to release the active compound in a controlled or delayed manner.

[0049] 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.

[0050] 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.

[0051] The term "pharmaceutical composition" means a composition comprising the compound of the present invention 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.

[0052] The term "effective amount" or "therapeutically effective amount" refers to a non-toxic but sufficient amount of a drug or medicament that can achieve the desired effect. In embodiments of the present invention, when a patient is treated according to the present invention, the amount of a given drug depends on many factors, such as a specific dosage regimen, the type of disease or condition and its severity, the uniqueness (e.g., body weight) of the patient or host in need of treatment, but, according to specific surrounding circumstances, including, for example, the specific drug, route of administration, the condition to be treated, and the patient or host to be treated, the dosage can be conventionally determined by methods known in the art. Typically, with respect to the dosage used for adult treatment, the dosage is typically in the range of 0.02-5000 mg / day, for example, about 1-1500 mg / day. The desired dosage can be conveniently expressed as a single dose, or simultaneously administered (or in a short period of time) or in divided doses 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 has been given, the specific effective amount can be appropriately adjusted according to the patient's condition and in conjunction with the physician's diagnosis.

[0053] The term "antibody-drug conjugate (ADC)" refers to the connection of a biologically active small molecule drug to a monoclonal antibody through a chemical link. The monoclonal antibody acts as a carrier to transport the small molecule drug into target cells.

[0054] As used herein, the terms "reduce," "inhibit," "mitigate," or "reduce" are 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.

[0055] 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 commercial products.

[0056] abbreviation

[0057] DIPEA: diisopropylethylamine

[0058] DMF: N',N'-dimethylformamide

[0059] DIC: N,N-diisopropylcarbodiimide

[0060] DCM: dichloromethane

[0061] HATU: 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0062] N-Cbz-: N-benzyloxycarbonyl

[0063] TFA: trifluoroacetic acid

[0064] EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0065] EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide

[0066] NHS: N-hydroxysuccinimide

[0067] TEA: triethylamine

[0068] II. Specific Examples

[0069] Example 1: N-(2'-Fluoro-3'-amino-3'-oxy-propionyl)exatecan (1)

[0070] In a 50 mL single-necked bottle, exatecan mesylate (265.8 mg, 0.5 mmol) and DIPEA (125.3 mg, 1.0 mmol) were dissolved in DMF (5 mL), 2-fluoromalonic acid (183.1 mg, 1.5 mmol) and DIC (63.1 mg, 0.5 mmol) were dissolved in DCM (2.5 mL) and added to the single-necked bottle. After stirring at room temperature for 3 h, the product 1a was purified by reverse phase column chromatography to obtain a yellow solid product 1a, 2-fluoromalonyl exatecan (155 mg, yield 57%); LCMS: (M+1) + 540.22 (calculated: 539.49).

[0071] In a 10 mL single-necked bottle, the intermediate 1a (50 mg, 92.68 μmol) was dissolved in DMF (2 mL) and stirred until clear. HATU (70.5 mg, 185.5 μmol), DIPEA (48 mg, 371 μmol), and NH4Cl (14.9 mg, 278 μmol) were added sequentially. The reaction was stirred at room temperature for 1 h. The product was purified by reverse phase column chromatography to obtain a yellow solid product, 1, N-(2-fluoro-3-amino-3-oxo-propionyl)exatecan (30.2 mg, 60.5% yield). 1 H NMR(500MHz,DMSO-d6)δ9.05(d,J=8.5Hz,1H),7.81(dt,J=11.2,5.5Hz,2H),7.69(d,J=4.1 Hz,1H),7.32(d,J=1.5Hz,1H),6.52(s,1H),5.60(dt,J=8.3,4.2Hz,1H),5.43(s,2H),5.38 -5.24(m,2H),5.21-5.05(m,2H),3.18(ddd,J=18.0,11.1,5.4Hz,2H),2.47-2.35(s,3H),2 .30-2.19(m,1H),2.18-2.07(m,1H),1.95-1.80(m,2H),0.87(t,J=7.3Hz,3H); LCMS:(M+1) + 539.28 (calculated: 538.51).

[0072] Example 2: N-(N-(2'-trifluoroethyl)aminoacetyl)exatecan (2)

[0073] Under ice-bath cooling, benzyl chloroformate (8.84 mL, 62.74 mmol), trifluoroethylamine (7.5 g, 57.03 mmol), Na2CO3 (9.07 g, 85.55 mmol) and dioxane:H2O (1:1, 150 mL) were added to the reaction flask. The reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure, and the mixture was extracted with methyl tert-butyl ether. After concentration, the crude product 2a, N-Cbz-trifluoroethylamine (9 g, crude yield 68%) was obtained, which was used directly in the next step without purification.

[0074] Under ice-cooling, N-Cbz-trifluoroethylamine 2a (3.5 g, 14.94 mmol), DMF (30 mL), NaH (60% dispersion in oil, 597.74 mg, 14.94 mmol), and tert-butyl 2-bromoacetate (2.20 mL, 14.94 mmol) were added to the reaction flask. The reaction was stirred at room temperature for 3 hours. Aqueous ammonium chloride was added, and the mixture was extracted with methyl tert-butyl ether. The product was purified by silica gel column chromatography to obtain 2b, N-Cbz-N-trifluoroethylglycine tert-butyl ester (3.6 g, 69% yield). 1 H NMR (600MHz, CDCl3) δ7.35 (dq, J = 18.0, 7.2, 6.8Hz, 5H), 5.18 (d, J = 10.1Hz, 2H), 4.07-3.89 (m, 4H), 1.42 (d, J = 44.5Hz, 9H).

[0075] Under ice-cooling, 2b (3.6 g, 10.33 mmol), DCM (30 mL), and TFA (6 mL) were added to the reaction flask and stirred at room temperature overnight. The solvent was removed under reduced pressure, and the mixture was extracted with methyl tert-butyl ether. The solvent was removed to give 2c as a colorless oil, N-Cbz-N-trifluoroethylglycine (3.4 g, 81% yield). 1 H NMR (600MHz, DMSO-d6) δ7.47-7.20 (m, 2H), 5.13 (d, J = 17.7Hz, 1H), 4.17 (dq, J = 18.7, 9.2Hz, 1H), 4.03 (d, J = 33.5Hz, 1H).

[0076] 2c (267.52 mg, 915.43 μmol), NHS (105.72 mg, 910.62 μmol), EDCI (176.10 mg, 918.60 μmol) and DCM (10 mL) were added to the reaction flask and stirred at room temperature for 1 hour. A solution of exatecan mesylate (0.25 g, 574.12 μmol) in DCM (5 mL) was added to the reaction solution and stirred at room temperature for 2 hours. The mixture was poured into water and extracted with DCM. The solvent was removed under reduced pressure and purified by silica gel column chromatography to give the white solid product 2d, N-trifluoromethyl-N-Cbz-glycine acyl exatecan (300 mg, yield 73.5%). 1H NMR (600MHz, CDCl3) δ7.62(d,J=10.4Hz,1H),7.53(s,1H),7.18(d,J=75.9Hz,6H),6.73( s,1H),5.65(d,J=16.2Hz,1H),5.56(s,1H),5.16(dt,J=19.9,14.0Hz,4H),5.08-4.99(m, 1H),4.33-4.13(m,2H),4.05(d,J=16.4Hz,2H),3.96(s,1H),3.04(d,J=61.6Hz,2H),2.40 (s,3H),2.26(d,J=27.8Hz,2H),1.96-1.84(m,2H),1.05(t,J=7.3Hz,3H).LCMS:m / z[M+H] + 709.10 (theoretical value 708.22).

[0077] 2d (0.25 g, 352.28 μmol), THF (5 mL) and Pd-C (5%, 37.49 mg) were added to the reaction flask and reacted under a hydrogen balloon. The mixture was stirred overnight, filtered, and the filtrate was concentrated to obtain a solid product 2, N-trifluoromethylglycine acyl exatecan (156 mg, 72% yield); 1 H NMR(600MHz,DMSO-d6)δ8.42(d,J=8.8Hz,1H),7.70(d,J=10.8Hz,1H),7.26( s,1H),6.53(s,1H),5.56(dt,J=9.5,5.0Hz,1H),5.40(s,2H),5.19-5.02(m, 2H),3.46-3.23(m,8H),3.13(dq,J=11.7,5.7Hz,2H),2.33(s,3H),2.17(tq, J=13.7,7.7,6.2Hz,2H),1.85(m,J=21.5,7.3Hz,2H),0.86(t,J=7.3Hz,3H); 13C NMR(151MHz,DMSO-d6)δ172.90,171.24,162.85,161.20,157.11,152.75,150.32,148.34,148 .24,145.61,140.43,136.61,136.57,127.57,126.10,125.72,124.23,124.10,121.78,119.60 ,110.37,110.22,97.10,72.79,65.73,51.97,50.02,49.96,49.81,49.61,44.47,40.39,40.2 5,40.11,39.97,39.83,39.69,39.56,30.85,28.03,23.41,11.36,11.33,8.19; LCMS:m / z[M+H] + 575.26 (theoretical value 574.18).

[0078] Example 3: 2'-amino-3',3',3'-trifluoropropionyl exatecan (3)

[0079] Under ice bath cooling, 2-amino-3,3,3-trifluoropropionic acid hydrochloride (1.08 g, 6.02 mmol), EDC hydrochloride (1.15 g, 6.02 mmol) and DMF (5 mL) were added to the reaction flask, and the reaction solution was stirred at 0°C for 30 minutes, and then stirred at room temperature for 1 hour. A solution of exatecan mesylate (2.0 g, 3.76 mmol) in DMF (80 mL) and triethylamine (609.17 mg, 6.02 mmol) were added to the reaction solution. The reaction solution was stirred at room temperature overnight, water was added, extracted with DCM, dried, and purified by silica gel column chromatography to obtain compound 3 as a brown solid, 2-amino-3,3,3-trifluoropropionic acid exatecan (1.2 g, yield 44.92%, HPLC 96%). 1 H NMR(600MHz,DMSO-d6)δ9.41(t,J=9.2Hz,1H),7.86-7.76(m,1H),7.30(s,1H),6 .53(s,1H),5.61(dt,J=8.3,4.2Hz,1H),5.47-5.28(m,4H),4.93(d,J=18.8Hz,1H ),4.58(dd,J=15.0,7.4Hz,1H),3.32-3.03(m,3H),2.39(s,3H),2.35-2.25(m,1H ),2.21-2.12(m,1H),1.86(dh,J=20.8,6.9Hz,2H),0.87(dt,J=13.8,7.3Hz,3H);13 C NMR(151MHz,DMSO-d6)δ172.84,163.02,162.64,161.37,157.15,152.86,150.44,148.54 ,148.44,145.56,138.57,136.44,126.55,124.62,124.49,121.68,121.50,119.81,110.6 1,110.45,97.33,72.80,65.71,54.62,49.49,45.24,44.30,40.39,40.25,40.11,39.97,3 9.83,39.69,39.55,30.81,27.24,27.06,22.61,11.43,11.40,8.23,8.13; LCMS:m / z[M+H] + 561.45 (theoretical value 560.17).

[0080] Example 4: 4',4'-difluoroproline acetatecan (4)

[0081] DCM (30 mL), N-Boc-4,4-difluoroproline (1.78 g, 7.05 mmol), EDC hydrochloride (1.35 g, 7.05 mmol) and NHS (811.92 mg, 7.05 mmol) were added to the reaction flask under ice cooling. The reaction solution was stirred under ice bath for 5 minutes and then at room temperature for 2 hours. A solution of exatecan mesylate (2.5 g, 4.70 mmol) in DMF (30 mL) and triethylamine (713.87 mg, 7.05 mmol) was slowly added. The mixture was stirred at room temperature overnight. Water was added, the mixture was extracted with DCM, and the mixture was purified by silica gel column chromatography to obtain compound 4a as a white solid, N-Boc-4,4-difluoroproline acyl exatecan (2.8 g, yield 87%, HPLC 98%); LCMS: (M+1) + 669.17 (calculated: 668.25).

[0082] The above-mentioned Boc-compound 4a (2.8 g, 4.10 mmol) was dissolved in DCM (25 mL), and TFA (8.94 g, 78.41 mmol) was added and stirred at room temperature overnight. The solvent was removed under reduced pressure, and acetonitrile was added to precipitate compound 4 as a yellow solid, 4,4-difluoroproline acetyl exatecan (2.3 g, yield 81%, HPLC 98.5%). 1H NMR(600MHz,DMSO-d6)δ9.15(d,J=8.7Hz,3H),7.78(d,J=10.8Hz,1H),7.29(s,1H),6.55(s,1H), 5.62(dt,J=8.8,4.4Hz,1H),5.42(s,2H),5.28-5.07(m,2H),4.52(t,J=8.6Hz,1H),3.76(td,J=12 .2,7.6Hz,3H),3.18(qt,J=17.1,8.4Hz,2H),2.82(tt,J=14.6,8.8Hz,1H),2.61(qd,J=14.7,8.8H z,1H),2.43-2.30(m,3H),2.29-2.13(m,2H),1.85(dh,J=21.6,7.2Hz,2H),0.87(t,J=7.3Hz,3H); 13 C NMR(151MHz,DMSO-d6)δ172.84,163.02,162.64,161.37,157.15,152.86,1 50.44,148.54,145.56,138.57,136.44,126.55,124.49,121.68,119.81,1 10.61,97.33,72.80,65.71,49.49,45.24,40.39,40.25,40.11,39.97,39. 83,39.69,39.55,30.81,27.24,22.61,11.43,11.40,8.23; LCMS:m / z[M+H] + 569.24 (theoretical value 568.19).

[0083] Example 5: 2',2'-difluoroacetylexatecan (5)

[0084] To 10 mL of DMF, add exatecan mesylate (500 mg, 0.94 mmol) and TEA (237.96 mg, 2.3 mmol) and stir until dissolved. Add difluoroacetic anhydride (163.72 mg, 0.94 mmol) dropwise and stir at room temperature for 60 min. Remove the solvent under reduced pressure and purify by silica gel column chromatography to obtain 2',2'-difluoroacetatecan (420 mg, 87% yield, HPLC 99%). 1H NMR(600MHz,DMSO-d6)δ9.43(d,J=8.4Hz,1H),7.82(d,J=10.8Hz,1H),7.32(s,1H),6.5 3(s,1H),6.25(s,0H),5.62(dt,J=9.1,4.9Hz,1H),5.43(s,2H),5.27(s,1H),5.17(s,1H ),3.18(p,J=6.0Hz,2H),2.41(s,3H),2.25(dq,J=15.3,5.2Hz,1H),2.18(ddt,J=14.0, 9.9, 5.0Hz, 1H), 1.87 (dtt, J=21.4, 14.4, 7.3Hz, 2H), 0.87 (t, J=7.3Hz, 3H); LCMS: (M+1) + 514.25 (theoretical value: 513.47).

[0085] Example 6: 2',2'-difluoropropionyl exatecan (6)

[0086] 2,2-difluoropropionic acid (50 mg, 0.45 mmol) was dissolved in DMF (2 mL) and DIPEA (59 mg, 0.46 mmol) was added to dissolve it and clarify it. DIC (58 mg, 0.45 mmol) and HATU (171 mg, 0.45 mmol) were added in sequence. The mixture was stirred at room temperature for 30 minutes. Exatecan mesylate (239 mg, 0.45 mmol) was added and stirring was continued at room temperature for 3 hours. The mixture was purified by C18 Spherical`20-35um`100A`40g reverse column chromatography. The mobile phases used acetonitrile and 0.05% TFA aqueous solution as B2 and A2 mobile phases, respectively (the mobile phase ratio was from A2:B2=95:5 to A2:B2=61:39). Then, the acetonitrile ratio was maintained at 39% and eluted for 30 minutes to obtain product 6 as a yellow solid, 2',2'-difluoropropionyl exatecan (135 mg, yield 57%). 1H`NMR(600MHz,DMSO-d6)δ9.40(d,J=8.6Hz,1H),7.78(d,J=10.9Hz,1H),7.30(s, 1H),6.52(s,1H),5.60(dt,J=8.5,5.9Hz,1H),5.41(s,2H),5.15(s,1H),5.09(s, 1H),3.22(dt,J=16.8,6.3Hz,1H),3.15(dt,J=16.7,6.3Hz,1H),2.39(d,J=1.7Hz ,3H),2.21(q,J=6.4Hz,2H),1.92-1.79(m,5H),0.87(t,J=7.3Hz,3H); LCMS:[M+1] + 528.50 (theoretical value: 527.17).

[0087] Example 7: Tumor cell growth inhibition activity

[0088] Human esophageal cancer cells OE33, human breast cancer cells MDA-MB-231, human gastric cancer cells NCI-N87, human lung cancer cells NCI-H1975, human esophageal cancer cells TE12, and human breast cancer cells SK-BR-3 were cultured in RPMI1640 (Cellmax) containing 10% fetal bovine serum (Cellmax). Tumor cells in the exponential growth phase were diluted with culture medium to 1×10 5 cells / mL, 100 μL was added to each well of a 96-well cell culture plate and returned to a 37°C, 5% CO2 incubator for overnight incubation. The next day, the compound was diluted to 10,000 nM, 2,000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, and 0.13 nM in culture medium, and 2 μL of the diluted compound was added to each well of the 96-well cell culture plate. Three replicates were set for each concentration. For the negative control and blank control groups without compound addition, 2 μL of the dilution was added to each well. After addition, the plates were returned to a 37°C, 5% CO2 incubator for an additional 72 hours. After incubation, the plates were removed, the culture medium was aspirated, and 100 μL of culture 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 from light, and place it in an ELISA plate. Select 630 nm as the reference wavelength and 450 nm as the measurement wavelength to measure the absorbance. Based on the absorbance value, use the four-parameter regression in GraphPad to calculate the IC 50 Values ​​(Table 1).

[0089] Table 1. IC values ​​of compounds for inhibiting tumor cell growth 50 (nM) value

[0090] "Dxd" is a positive control drug, and its structural formula is

[0091] The compounds of the embodiments provided by the present invention have good inhibitory effects on the growth of tumor cells. Some compounds have an IC value of 0. 50 The value can reach below 10nM, or even below 1nM, with significant anticancer activity.

[0092] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof: In the formula, R is selected from C1-C6 alkyl, halogen-substituted C1-C6 alkyl, Any of the following: in, R1 is selected from C1-C6 alkylene, wherein the C1-C6 alkylene is optionally substituted by one or more substituents selected from halogen, C1-C6 alkyl or halogen-substituted C1-C6 alkyl; R2 is selected from any one of halogen, C1-C6 alkyl or halogen-substituted C1-C6 alkyl; X is selected from any one of amino and imino.

2. The compound according to claim 1 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein: R1 is selected from methylene, halogen-substituted methylene or trifluoromethyl-substituted methylene; Preferably, R1 is selected from -CHF-, -CHF2-, -CH2- or -CH(CF3)-.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein: R2 is selected from any one of halogen, methyl, methylene, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, and heptyl, preferably any one of methylene, methyl, ethyl, and isopropyl; The halogen is selected from any one of -F, -Cl, -Br, and -I, preferably -F.

4. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein: X is selected from -NH- or -NH2.

5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein: Selected from Wherein, R1 is selected from halogen; preferably F; n is selected from 1, 2, 3 or 4, preferably, n is 1; Preferably, R is selected from 6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein: The compound of formula (I) is any one of the following compounds:

7. A method for preparing the compound according to any one of claims 1 to 6, comprising: The compound according to any one of claims 1 to 6 is prepared by reacting a halogen-substituted carboxylic acid, a carboxylic anhydride, an acyl chloride and exatecan; Preferably, the halogen-substituted carboxylic acid, carboxylic anhydride, or acyl chloride is selected from 2-fluoromalonic acid, 2-amino-3,3,3-trifluoropropionic acid, Any one of difluoroacetic anhydride and 2,2-difluoropropionic acid.

8. An antibody-drug conjugate comprising a small molecule drug, a linker and an antibody, wherein: The small molecule drug comprises the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof.

9. A pharmaceutical composition comprising the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof or the antibody-drug conjugate according to claim 8.

10. Use of the compound according to any one of claims 1 to 6 or its pharmaceutically acceptable salt, stereoisomer or prodrug, the compound obtained by the preparation method according to claim 7, the antibody-drug conjugate according to claim 8 or the pharmaceutical composition according to claim 9 in the preparation of a drug for treating cancer; preferably, the cancer includes any one of gastric cancer, esophageal cancer, esophageal cancer, breast cancer, breast adenocarcinoma and lung cancer.

11. A method for treating cancer, comprising the step of administering to a patient in need thereof a compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, a compound obtained by the preparation method according to claim 7, an antibody-drug conjugate according to claim 8 or a pharmaceutical composition of the pharmaceutical composition according to claim 9; Preferably, the cancer includes any one of gastric cancer, esophageal cancer, esophageal cancer, breast cancer, breast adenocarcinoma, and lung cancer.

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