Exatecan hydroxylamine derivative and use thereof

Abstract

The present invention relates to a method for preparing a series of exatecan derivatives containing a hydroxylamine structure substitution and pharmaceutically acceptable salts thereof, and the use thereof in the field of anti-tumor. These exatecan derivatives not only have a good activity of inhibiting the growth of tumor cells, but also have a good capability of permeating cell membranes or bystander effect, and can be used as monotherapy, combination therapy, or as toxin components of ADC for the treatment of tumors.

Description

Exatecan hydroxylamine derivatives and their applications

[0001] This application claims priority to Chinese patent application No. 2023111927049, filed on September 15, 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 exatecan hydroxylamine derivatives and applications thereof. Background Art

[0003] DNA topoisomerases are essential enzymes widely present in organisms. They influence DNA topology by regulating supercoiling, linkage, delinking, and nucleic acid dissociation. They are primarily divided into topoisomerase 1 (Top1) and topoisomerase 2 (Topo2). Compared to Top2 inhibitors, Top1 inhibitors have higher efficacy and a broader anti-tumor spectrum, making them important targets for the design of new anti-tumor drugs. Furthermore, Top1 levels in various tumor cells, such as colon cancer, cervical cancer, and ovarian cancer, are significantly higher than in normal tissues, and its activity is significantly enhanced in S-phase tumor cells. Therefore, Top1 inhibitors can selectively inhibit DNA replication in proliferating tumor cells, demonstrating excellent selectivity.

[0004] Camptothecin has Top1 inhibitory activity, particularly against Top1-DNA complexes. Topoisomerase 1 (Top1) is the primary target of camptothecin (CPT) and its analogs. Therefore, camptothecin has significant therapeutic effects on a variety of solid tumors, including gastric cancer, esophageal cancer, lung cancer, and bladder cancer, making it a broad-spectrum anti-tumor drug. Among them, irinotecan, topotecan, and belotecan have been approved for the treatment of various cancers in some countries. Furthermore, DX-8951 and SN38 have both been successfully used as payloads in antibody-drug conjugates (ADCs) for the targeted treatment of various solid tumors and have garnered significant attention for their significant efficacy. Currently, exatecan suffers from significant toxic side effects, poor cell penetration, and poor stability in the blood system.

[0005] Summary of the Invention

[0006] The present invention provides a series of novel exatecan derivatives, their pharmaceutically acceptable salts, stereoisomers, or prodrugs, and their applications in the anti-tumor field. Exatecan derivatives exhibit both potent tumor cell growth inhibition and excellent cell membrane permeability or bystander effects, and can be used for tumor treatment as single agents, in combination, or as toxin components of ADCs.

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

[0008] Wherein, R is selected from

[0009] wherein R1 is selected from C1-C6 alkyl, deuterated C1-C6 alkyl or C3-C6 cycloalkyl, wherein the C1-C6 alkyl or C3-C6 cycloalkyl is optionally substituted with one or more halogens;

[0010] Or, R1 is selected from C1-C6 alkyl, wherein two hydrogen atoms on the same carbon atom of the C1-C6 alkyl are simultaneously replaced by -(CH2) j -substituted to form a C3-C6 cycloalkyl group, wherein j is 1, 2, 3, 4 or 5;

[0011] R2 and R3 are each independently selected from hydrogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl or C3-C6 cycloalkyl,

[0012] X and Y are each independently selected from an O atom or are absent, provided that at least one of X and Y is O;

[0013] m and n are each independently selected from 0, 1, 2 or 3.

[0014] In one embodiment, R1 is selected from C1-C6 alkyl or deuterated C1-C6 alkyl;

[0015] Preferably, R1 is selected from -CH2-, -CD2-, -(CH2)2- or -CH(CH3)-; or,

[0016] R1 is selected from C1-C3 alkyl, wherein two hydrogen atoms on the same carbon atom of the C1-C3 alkyl are simultaneously replaced by -(CH2) j -substituted to form cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl;

[0017] Preferably, R1 is selected from

[0018] In one embodiment, R2 is selected from C1-C6 alkyl or deuterated C1-C6 alkyl;

[0019] Preferably, R2 is selected from methyl, ethyl or deuterated methyl.

[0020] In one embodiment, R3 is selected from hydrogen or C1-C6 alkyl.

[0021] m and n are each independently selected from 0, 1 or 2;

[0022] Preferably, Selected from

[0023] In one embodiment, X is O and Y is absent or Y is O and X is absent.

[0024] In one embodiment, the compound of formula (I) is a compound represented by formula (IIa):

[0025] In formula (IIa), R1 and R2 are the same as those in formula (I);

[0026] Preferably, R1 is selected from -CH2-, -CD2-, -(CH2)2-, -CH(CH3)- or

[0027] Preferably, R2 is selected from methyl, ethyl or deuterated methyl.

[0028] In one embodiment, the compound of formula (I) is a compound represented by formula (IIb):

[0029] In formula (IIb), R1 and R2 are defined as the same as the compound of formula (I), preferably, R1 is selected from -CH2-, -CD2-, -(CH2)2-, -CH(CH3)- or

[0030] Preferably, R2 is selected from methyl, ethyl or deuterated methyl.

[0031] In one embodiment, the compound of formula (I) is a compound represented by formula (D1) to (D5):

[0032] Another aspect of the present invention provides a method for preparing any of the above compounds, comprising:

[0033] The 2'(S)-amino group of 7,9-cyclohexane in the exatecan structure is reacted with a corresponding hydroxylamine-containing substituted compound to prepare an exatecan hydroxylamine derivative;

[0034] in,

[0035] The amino group in the compound containing the hydroxylamine structure is protected by amino protection, and the amino protection method is to replace the hydrogen on the amino group with an amino protecting group; preferably, the amino protecting group is tert-butyloxycarbonyl;

[0036] The acyl group in the compound substituted with a hydroxylamine structure is selected from any one of hydroxylamine-substituted acetyl, hydroxylamine-substituted propionyl, hydroxylamine-substituted cyclobutanecarboxyl, hydroxylamine-substituted isoxazolidine-4'-carboxyl, and hydroxylamine-substituted deuterated acetyl.

[0037] In one embodiment, the compound containing a hydroxylamine structure is selected from Any one of .

[0038] In one embodiment, the preparation method comprises forming an amide from the -2'(S)-amino group of 7,9-cyclohexane and the acetyl group substituted with hydroxylamine.

[0039] In one embodiment, the preparation method comprises forming an amide from the -2'(S)-amino group of 7,9-cyclohexane and the propionyl group substituted with hydroxylamine.

[0040] In one embodiment, the preparation method comprises forming an amide from the -2'(S)-amino group of 7,9-cyclohexane and a cyclobutanecarbonyl group substituted with hydroxylamine.

[0041] In one embodiment, the preparation method comprises forming an amide from the 2'(S)-amino group of the 7,9-cyclohexane group of exatecan and the hydroxylamine-substituted isoxazolidine-4'-formyl group.

[0042] In one embodiment, the preparation method comprises forming an amide from the -2'(S)-amino group of 7,9-cyclohexane and the deuterated acetyl group substituted with hydroxylamine.

[0043] In another aspect of the present invention, an antibody-drug conjugate is provided, comprising 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.

[0044] In another aspect of the present invention, a pharmaceutical composition is provided, which comprises the above-mentioned compound or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof and a pharmaceutically acceptable excipient.

[0045] In another aspect of the present invention, there is provided use of the above-mentioned compound or its pharmaceutically acceptable salt, stereoisomer or prodrug, the antibody-drug conjugate or the pharmaceutical composition in the preparation of a drug for treating cancer.

[0046] In another aspect of the present invention, a method for treating cancer is provided, comprising the step of administering a therapeutically effective amount of the above-mentioned compound or a pharmaceutically acceptable salt, stereoisomer or prodrug, antibody-drug conjugate or pharmaceutical composition thereof to a patient in need.

[0047] The above-mentioned compounds or pharmaceutically acceptable salts, stereoisomers or prodrugs thereof can be used alone, in combination or in the form of antibody-drug conjugates for the treatment of cancer.

[0048] Preferably, the cancer includes any one or more of esophageal adenocarcinoma, gastric cancer, esophageal cancer, lung cancer, lung squamous cell carcinoma, breast cancer, mammary adenocarcinoma and bladder cancer cells. DETAILED DESCRIPTION

[0049] I. Definition

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] Numeric ranges herein 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.

[0057] 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.

[0058] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 8 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, and most preferably an alkyl group containing 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, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 2,2-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. 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 point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate groups. Methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuterated alkyl, alkoxy-substituted alkyl and hydroxy-substituted alkyl are preferred.

[0059] The hydrogen atoms described in the present invention can all be replaced by their isotope deuterium.

[0060] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 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 a person skilled in the art can determine (by experiment or theory) which substitutions are possible or impossible 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.

[0061] Refers to the chemical bond connection.

[0062] Drug or pharmaceutical composition

[0063] 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).

[0064] 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 (e.g., acacia, tragacanth or alginates), buffer salts, carboxymethylcellulose, polyethylene glycol, waxes, and the like. 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] The term "antibody-drug conjugate" or "antibody-drug conjugate" (ADC) refers to a small molecule drug with biological activity attached to a monoclonal antibody through a chemical link. The monoclonal antibody acts as a carrier to transport the small molecule drug into target cells.

[0071] 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.

[0072] 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.

[0073] Cells used

[0074] OE33: human esophageal cancer cells;

[0075] SK-BR-3: human breast cancer cells;

[0076] NCI-N87: human gastric cancer cells;

[0077] NCI-H1975: human lung cancer cells;

[0078] MDA-MB-231: human breast cancer cells;

[0079] NCI-H226: human lung squamous cell carcinoma cells;

[0080] TE12: human esophageal cancer cells.

[0081] II. Specific Examples

[0082] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below. The described embodiments should not be regarded as limiting the present invention. All other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0083] Before further explaining the embodiments of the present invention in detail, the nouns and terms involved in the embodiments of the present invention are explained. The nouns and terms involved in the embodiments of the present invention are subject to the following interpretations.

[0084] 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.

[0085] Example 1: N-(2'O-(N-trideuterated methylamino)oxyacetyl)exatecan (D1)

[0086] To a 100 mL flask containing 35 mL of H₂O was added CD₃NO₂ (2.24 g, 2.0 mL, 35 mmol) and NH₄Cl (1.50 g, 28 mmol). The mixture was stirred in an ice-water bath, and zinc powder (5.7 g, 87.5 mmol) was added proportionally over 1 hour. After stirring for an additional 2 hours, the mixture was filtered and rinsed with water. The filtrate was acidified to pH 3 with concentrated HCl(aq) and evaporated to dryness. CD₃NHOH was recrystallized from EtOH / Et₂O to afford D1-1, which was used without further purification.

[0087] At 25°C, in a 250 mL round-bottom flask, D1-1 (4.2 g, 50.29 mmol) was dissolved in THF (100 mL). Boc anhydride (11.03 g, 50.29 mmol, 11.59 mL) and K2CO3 (6.95 g, 50.29 mmol) were added sequentially and stirred magnetically until most of it dissolved. Under nitrogen protection, the mixture was reacted at 25°C for 24 hours. The reaction solution was added with ethyl acetate and extracted three times. The mixture was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated to obtain D1-2 (5.36 g, 36.17 mmol, 71.93% yield) as a light yellow liquid.

[0088] At 25°C, D1-2 (1 g, 6.61 mmol) was dissolved in H2O (15 mL) in a 250 mL round-bottom flask. Bromoacetic acid (1.16 g, 8.27 mmol) and NaOH (264.53 mg, 6.61 mmol) were added sequentially and stirred until most of the solution was dissolved. The mixture was reacted at 25°C under nitrogen for 24 hours. The reaction solution was extracted three times with ethyl acetate, washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated to obtain D1-3 (1.36 g, 5.52 mmol, 83.54% yield).

[0089] At 0°C, in a 250 mL round-bottom flask, D1-3 (500 mg, 2.44 mmol) was dissolved in DCM (10 mL). EDCI (467.1 mg, 2.44 mmol) and NHS (280.1 mg, 2.44 mmol) were added sequentially. The mixture was stirred until most of it was dissolved. Under nitrogen protection, the reaction was carried out at 25°C for 3 hours to obtain D1-4.

[0090] At 25°C, in a 25 mL round-bottom flask, exatecan (100 mg, 188.13 μmol) was dissolved in DMF (1 mL). TEA (30.5 mg, 301.00 μmol) and D1-4 (91 mg, 301.00 μmol) were added sequentially and stirred until most of the solution was dissolved. Under nitrogen protection, the mixture was reacted at 25°C for 2 hours. Silica gel was added and the sample was stirred. The mixture was passed through a silica gel column (DCM:MeOH=97:3) and concentrated to obtain a yellow solid D1-5 (85.3 mg, 72.8%); LCMS: [M+1] + 626.21 (calculated: 625.67).

[0091] D1-5 (53 mg, 84.57 μmol) was dissolved in EA (0.5 mL), and 4 M HCl / EA (0.5 mL) was added. The mixture was stirred until most of it was dissolved. Under nitrogen protection, the reaction was carried out at 25°C for 2 hours, and the mixture was concentrated to obtain a yellow solid D1 (30.2 mg, 45.80 μmol, yield 54.16%). 1 H NMR (600MHz, DMSO-d6) δ8.53(d,J=8.8Hz,1H),7.81(d,J=10.9Hz,1H),7.31(s,1H),5.66-5.57(m,1H),5.42(s,2H),5.26(s,1H),4.28 (s,2H),3.18(t,J=6.6Hz,2H),2.41(s,3H),2.18(dt,J=36.9,7.2Hz,2H),1.86(ddd,J=21.4,14.2,7.0Hz,2H),0.87(t,J=7.3Hz,3H).

[0092] Example 2: N-(1'-O-(N-methylamino)oxycyclobutane-1'-formyl)exatecan (D2)

[0093] In a 10 mL vial, NaOH (555.46 mg, 13.89 mmol), H2O (1 mL), and tert-butyl N-hydroxy-N-methylcarbamate (82.31 mg, 555.50 μmol) were added. Finally, a 0.5 mL aqueous solution of 1-bromocyclobutanecarboxylic acid (100 mg, 555.50 μmol) was added dropwise to the reaction flask. The mixture was stirred at 25°C for 1 h, extracted with DCM, and concentrated to give D2-1 (100 mg, yield 67.98%, 93% HPLC); LCMS: [M+1] + 245.83 (calculated: 245.13).

[0094] Exatecan (159.50 mg, 365.44 μmol) was dissolved in DMF (1 mL), and D2-1 (100 mg, 406.04 μmol), EDCI (77.84 mg, 406.04 μmol), NHS (46.73 mg, 406.04 μmol), and DCC (83.78 mg, 406.04 μmol) were added in sequence. The mixture was stirred at 25°C for 2 h and purified by silica gel column chromatography to give D2-2 (130 mg, 168.45 μmol, yield 41.48%, HPLC 94%); LCMS: [M+1] + 663.27 (calculated: 662.28).

[0095] D2-2 (100 mg, 150.67 μmol) was dissolved in DCM (5 mL), and TFA (34.36 mg, 301.34 μmol, 23.06 μL) was added. The mixture was stirred at room temperature for 1 h. The DCM was concentrated and purified using a C18 Spherical 20-35 μm 100A 40 g reverse phase column with acetonitrile and 0.05% TFA aqueous solution as mobile phases B2 and A2, respectively. HPLC preparation method (254 nm and 214 nm wavelength monitoring) was used to obtain D2 (50 mg, 86.94 μmol, yield 57.70%, HPLC 98%). 1 H NMR(600MHz,DMSO-d6)δ8.29(d,J=8.5Hz,1H),7.76(d,J=10.7Hz,1H),7.31(s,1H),5 .57(q,J=7.3,6.9Hz,1H),5.41(s,2H),5.15(s,1H),5.11(s,1H),4.05(s,2H),3.23-3 .07(m,2H),2.71(s,3H),2.38(s,3H),2.16(q,J=6.5Hz,2H),1.86(dtt,J=21.5,14.4, 6.6Hz, 2H), 1.24 (tp, J=12.7, 7.1, 6.4Hz, 2H), 0.88 (q, J=7.4, 6.4Hz, 6H); LCMS: [M+1] + 563.26 (calculated: 562.22).

[0096] Example 3: N-(2'-methyl-2'-O-(N-methylamino)oxyacetyl)exatecan (D3)

[0097] To the reaction flask was added a solution of NaOH (64.94 mg, 1.62 mmol) in H2O (1 mL), followed by (2S)-2-bromopropionic acid (100 mg, 649.43 μmol) and tert-butyl N-hydroxy-N-methylcarbamate (96.23 mg, 649.43 μmol). The mixture was stirred at room temperature for 2 h, extracted with DCM, and concentrated to give the product D3-1 (80 mg, 290.59 μmol, yield 44.75%, HPLC 94%). LCMS: [M+1] + 220.23 (calculated: 219.24).

[0098] Exatecan (142.68 mg, 326.91 μmol) was dissolved in DMF (2.04 mL), and TEA (110.27 mg, 1.09 mmol, 151.88 μL), D3-1 (80 mg, 363.24 μmol), EDCI (69.63 mg, 363.24 μmol), and NHS (41.80 mg, 363.24 μmol) were added in sequence. The mixture was stirred at 25° C. for 1 h, extracted with DCM, and purified by silica gel column chromatography to give D3-2 (130 mg, 177.36 μmol, yield 48.83%, HPLC 97%); LCMS: [M+1] + 637.54 (calculated: 636.68).

[0099] D3-2 (90 mg, 141.14 μmol) was dissolved in DCM (5 mL), and TFA (2.98 g, 26.14 mmol, 2 mL) was added. The mixture was stirred at 25°C for 1 h and purified using a C18 Spherical 20-35 μm 100A 40 g reverse phase column with acetonitrile and 0.05% TFA aqueous solution as mobile phases B2 and A2, respectively. The product D3 (40 mg, 72.92 μmol, yield 51.67%, HPLC 98%) was obtained by HPLC (254 nm and 214 nm wavelength monitoring). 1H NMR (600MHz, DMSO-d6) δ8.49(d,J=8.7Hz,1H),7.74(t,J=9.3Hz,1H),7.29(s,1H),6.50(s,1H),5.57(q,J=6.6,6.2Hz,1H) ,5.41(s,2H),5.24(s,0H),5.23-5.14(m,1H),5.12(s,0H),4.39-4.35(m,0H),4.32(q,J=6.8Hz,1H),3.18(td,J=15.7,15. 0,6.8Hz,1H),3.12(s,0H),2.50(s,2H),2.37(s,2H),2.34(d,J=20.3Hz,1H),2.16(q,J=6.3Hz,2H),2.13(s,0H),1.87(td, J=13.9,7.2Hz,2H),1.82(d,J=7.0Hz,0H),1.42(t,J=7.4Hz,1H),1.34(d,J=6.8Hz,2H),0.86(t,J=7.3Hz,3H); LCMS:[M+1] + 537.80 (calculated: 536.56).

[0100] Example 4: N-(2',2'-dideutero-2'-O-(N-trideuteromethylamino)oxyacetyl)exatecan (D4)

[0101] At room temperature and under nitrogen protection, exatecan (4 g, 7.77 mmol) and DMF (10 mL) were added to a 50 mL round-bottom flask and stirred to dissolve. 2-Bromo-2,2-dideuteroacetic anhydride (2.06 g, 7.77 mmol) and DIEA (1.00 g, 7.77 mmol) were added in sequence and stirred to dissolve. The mixture was reacted at room temperature for 24 hours, and then extracted three times with ethyl acetate. The mixture was washed with saturated brine and dried over anhydrous magnesium sulfate. After concentration, a light yellow solid D4-1 (4.05 g, yield 88.47%) was obtained. LCMS: [M+1] + 559.37 (calculated: 558.40).

[0102] At room temperature under nitrogen, D4-1 (2 g, 3.58 mmol) was dissolved in DMF (10 mL). D1-2 (0.753 g, 5.01 mmol) and DBU (0.818 g, 5.37 mmol) were added sequentially. The mixture was stirred magnetically until most of it dissolved. The mixture was stirred at room temperature overnight and extracted three times with ethyl acetate. The mixture was washed with saturated brine and dried over anhydrous magnesium sulfate. The mixture was concentrated to obtain a light yellow solid D4-2 (1.05 g, 47% yield). LCMS: [M+1] +628.58 (calculated: 527.68).

[0103] Under nitrogen protection at 25°C, D4-2 (0.7 g, 1.12 mmol) was dissolved in DCM (10 mL), TFA (1 mL) was added, and the mixture was stirred for 0.5 h. The mixture was concentrated to obtain a light yellow solid D4 (0.536 g, yield 91%). 1 H NMR(500MHz,DMSO-d6)δ8.64(dd,J=8.9,5.5Hz,1H),7.78(dd,J=15.1,10.8Hz,1 H),7.30(d,J=7.8Hz,1H),6.55(s,1H),5.61(dt,J=9.2,4.7Hz,1H),5.42(d,J=4. 0Hz,2H),5.24(d,J=8.5Hz,2H),3.18(t,J=6.4Hz,3H),2.39(d,J=7.9Hz,3H),2. 28-2.10(m,2H),1.86(dp,J=21.4,7.1Hz,2H),0.87(t,J=7.4Hz,3H); LCMS:[M+1] + 528.46 (calculated: 527.56).

[0104] Example 5: N-(isoxazolidine-4'-formyl)exatecan (D5)

[0105] Concentrated sulfuric acid (0.5 mL) was added to 3-bromo-(2-bromomethyl)propionic acid (3 g, 12.15 mmol) in ethanol (30 mL), stirred and heated under reflux for 18 h, concentrated, extracted with methyl tert-butyl ether, washed with brine, and concentrated to obtain an oily liquid 3-bromo-(2-bromomethyl)propionic acid ethyl ester, which was used directly in the next reaction without purification.

[0106] N-Boc hydroxylamine (3 g, 22.36 mmol), DMF (10 mL) and 60% NaH (1.79 g, 44.72 mmol) were added to the reaction flask and stirred at 0°C for 10 min. Ethyl 3-bromo-(2-bromomethyl)propionate (6.15 g, 22.36 mmol) was added and reacted at room temperature for 3 h. The mixture was poured into ice water and extracted with methyl tert-butyl ether. The mixture was purified by silica gel column chromatography to give D5-1 (1.16 g, yield 21%), which was dissolved in 1:1 THF / H2O (10 mL). LiOH (252.82 mg, 10.56 mmol) was added and stirred at room temperature for 3 h. The reaction solution was concentrated, 1N HCl aqueous solution was added, extracted with DCM, and concentrated to give D5-2 (0.9 g, yield 78%).

[0107] D5-3 (1.46 g, 3.35 mmol), D5-2 (0.73 g, 3.35 mmol), DMF (10 mL), EDCI (961.90 mg, 5.02 mmol), and triethylamine (507.74 mg, 5.02 mmol, 699.37 μL) were added to the reaction flask and stirred at room temperature for 3 h. The mixture was concentrated and purified by silica gel column chromatography to obtain a white solid product D5-4 (700 mg, 19% yield). The obtained D5-4 was dissolved in a 4M HCl solution in EtOAc (5 mL) and stirred at room temperature for 3 h. The reaction solution was concentrated to obtain a white solid product D5 (170 mg, 82% yield). 1 H NMR(500MHz,DMSO-d6)δ7.81(dd,J=11.1,4.2Hz,1H),7.31(d,J=3.5Hz,1H),5.82(dd,J=1 9.1,13.1Hz,1H),5.49-5.31(m,4H),5.16(d,J=14.4Hz,2H),3.74(dt,J=12.5,6.2Hz,1H) ,3.69-3.31(m,5H),3.23(ddd,J=18.3,12.8,5.5Hz,1H),2.85(d,J=14.7Hz,1H),2.37(d, J=5.2Hz,4H),2.22(t,J=13.1Hz,1H),1.87(tt,J=14.4,6.5Hz,3H),0.87(t,J=7.2Hz,3H); 13 C NMR(126MHz,DMSO-d6)δ172.85,171.98,171.86,163.20,161.21,157.13,152.91,150.43,148.34,1 48.23,145.49,135.92,133.07,129.22,129.11,125.44,125.29,121.25,121.14,120.00,119.62,11 0.75,110.57,97.44,72.81,65.80,53.04,52.82,50.93,49.69,49.25,44.71,44.62,40.47,40.30,4 0.14,39.97,39.80,39.63,39.47,38.00,37.83,30.92,22.96,22.59,21.74,11.54,8.29.LCMS[M+H] + 535.20 (calculated value 534.19).

[0108] Effect Example 1: Inhibition of tumor cell growth activity

[0109] Tumor cells OE33, SK-BR-3, NCI-N87, NCI-H1975, MDA-MB-231, NCI-H226, and TE12 were cultured in RPMI1640 (Cellmax) containing 10% fetal bovine serum (Cellmax). Tumor cells in the exponential growth phase were diluted 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 using culture medium. 2 μL of the diluted compound was added to each well of the 96-well cell culture plate, with three replicates 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 and incubated for an additional 72 hours. After incubation, the cell culture 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).

[0110] Table 1. IC values ​​of compounds for inhibiting tumor cell growth 50 (nM) value

[0111] "Dxd" is a positive control drug, and its structural formula is

[0112] From the results in the table, it can be seen that the exemplary compounds in the examples of the present invention have a good inhibitory effect on the growth of tumor cells, and their active inhibitory IC 50 The values ​​were all lower than 500 nM, or lower than 100 nM or 50 nM, and even lower than 10 nM.

[0113] 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 represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof: In the formula, R is selected from in, R1 is selected from C1-C6 alkyl, deuterated C1-C6 alkyl or C3-C6 cycloalkyl, wherein the C1-C6 alkyl or C3-C6 cycloalkyl is optionally substituted with one or more halogens; Or, R1 is selected from C1-C6 alkyl, wherein two hydrogen atoms on the same carbon atom of the C1-C6 alkyl are simultaneously replaced by -(CH2) j -substituted to form a C3-C6 cycloalkyl group, wherein j is 1, 2, 3, 4 or 5; R2 and R3 are each independently selected from hydrogen, C1-C6 alkyl, deuterated C1-C6 alkyl, halogenated C1-C6 alkyl or C3-C6 cycloalkyl, X and Y are each independently selected from an O atom or are absent, provided that at least one of X and Y is O; m and n are each independently selected from 0, 1, 2 or 3.

2. The compound according to claim 1, wherein R1 is selected from C1-C6 alkyl or deuterated C1-C6 alkyl; Preferably, R1 is selected from -CH2-, -CD2-, -(CH2)2- or -CH(CH3)-; or, R1 is selected from C1-C3 alkyl, wherein two hydrogen atoms on the same carbon atom of the C1-C3 alkyl are simultaneously replaced by -(CH2) j -substituted to form cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; Preferably, R1 is selected from 3. The compound according to claim 1 or 2, wherein R2 is selected from C1-C6 alkyl or deuterated C1-C6 alkyl; Preferably, R2 is selected from methyl, ethyl or deuterated methyl.

4. The compound according to any one of claims 1 to 3, wherein R3 is selected from hydrogen or C1-C6 alkyl; m and n are each independently selected from 0, 1 or 2; Preferably, Selected from 5. The compound according to any one of claims 1 to 4, wherein X is O and Y does not exist, or Y is O and X does not exist.

6. The compound according to any one of claims 1 to 5, wherein The compound of formula (I) is a compound represented by formula (IIa) or formula (IIb): In formula (IIa), R1 and R2 are respectively defined as the compound of formula (I); Preferably, R1 is selected from -CH2-, -CD2-, -(CH2)2-, -CH(CH3)- or Preferably, R2 is selected from methyl, ethyl or deuterated methyl; In formula (IIb), R1 and R2 are the same as those in formula (I), preferably, R1 is selected from -CH2-, -CD2-, -(CH2)2-, -CH(CH3)- or Preferably, R2 is selected from methyl, ethyl or deuterated methyl.

7. The compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein the compound of formula (I) is a compound represented by formula (D1) to (D5):

8. An antibody-drug conjugate comprising a small molecule drug, a linker and an antibody, characterized in that: The small molecule drug comprises the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof.

9. A pharmaceutical composition, characterized in that The invention comprises the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof and a pharmaceutically acceptable excipient or the antibody-drug conjugate according to claim 8.

10. Use of the compound according to any one of claims 1 to 7 or its pharmaceutically acceptable salt, stereoisomer or prodrug, 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 or more of esophageal adenocarcinoma, gastric cancer, esophageal cancer, lung cancer, lung squamous cell carcinoma, breast cancer, breast adenocarcinoma and bladder cancer cells.

11. A method for treating cancer, comprising the step of administering to a patient in need thereof a therapeutically effective amount of the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, the antibody-drug conjugate according to claim 8 or the pharmaceutical composition according to claim 9; Preferably, the cancer includes any one or more of esophageal adenocarcinoma, gastric cancer, esophageal cancer, lung cancer, lung squamous cell carcinoma, breast cancer, breast adenocarcinoma and bladder cancer cells.

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