A tetrahydropyrrolidine compound and its preparation method and application

Abstract

The invention belongs to the field of tumor drug technology, specifically to a tetrahydropyrrolidine compound, its preparation method, and application. The tetrahydropyrrolidine compound, as represented by formula (I), has excellent anti-tumor effects and low toxicity to normal cells, making it a safe, effective, and low-toxic candidate anti-tumor drug.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor drugs, and in particular relates to a tetrahydropyrrolidine compound and a preparation method and application thereof. Background Art

[0002] Malignant tumors are currently one of the leading causes of death worldwide, severely endangering human health and hindering socioeconomic development. Current treatments include chemotherapy, radiotherapy, and surgery, but chemotherapy remains the primary approach. Numerous chemical drugs, such as nitrogen mustards, pyrimidines, platinums, and porphyrins, are used to treat malignant tumors. However, the high toxicity and low bioavailability of most drugs limit their application. Therefore, the search for highly effective, low-toxic anti-tumor drugs has become a research hotspot in medicinal chemistry.

[0003] Tetrahydropyrrole rings containing chiral quaternary carbon centers are key structural units in numerous biologically active drug molecules and natural products. They have a wide range of applications in medicine, primarily in antibacterial, anti-tumor, neurological, and cardiovascular diseases, and are highly valued for their low toxicity and high efficacy. Because these tetrahydropyrrole rings contain chiral quaternary carbon centers, the introduction of different substituents into the rings allows for significant conformational variability, potentially improving existing physicochemical properties and pharmacological activity, playing a crucial role in clinical applications.

[0004] Therefore, it is necessary to explore a new tetrahydropyrrolidine compound containing a chiral quaternary carbon center. Summary of the Invention

[0005] To address the above issues, one objective of the present invention is to provide a tetrahydropyrrolidine compound containing a chiral quaternary carbon center. This compound is prepared by a 1,3-dipolar cycloaddition reaction of α-substituted acrylonitrile and polysubstituted nitroethylene as dipoleophiles with α-substituted or unsubstituted iminoesters in the presence of a bifunctionalized ligand in the presence of a metal, synergistically promoted by metal catalysis and organic catalysis. This compound and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs, and polymorphs exhibit excellent antitumor effects and low toxicity to normal cells, making it a safe, effective, and low-toxic candidate antitumor drug.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a tetrahydropyrrolidine compound of formula (I) and pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs thereof:

[0008]

[0009] Among them, R 1 Any one of:

[0010] R 2 and R 3 Independently selected from: R 2 and R 3 Same or different; R 4 Any one of:

[0011]

[0012] R 1 and R 4 Same or different;

[0013] R 5 Any one of:

[0014] R 2 、R 3 and R 5 Same or different.

[0015] Another aspect of the present invention provides a method for preparing the tetrahydropyrrolidine compound represented by the above formula (I), comprising: mixing a ligand L1, a molecular sieve, Cu(CH3CN)4BF4 and dichloromethane for reaction, and then cooling the mixture to 0°C±5°C to obtain a reaction solution; mixing a compound represented by formula (II), a compound represented by formula (III), triethylamine and dichloromethane, and then reacting the mixture with the reaction solution; after the reaction is completed, the compound represented by formula (I) is obtained;

[0016]

[0017] In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned compound and pharmaceutically acceptable salts, stereoisomers, tautomers, homologues, solvates, prodrugs or polymorphs thereof.

[0018] In another aspect, the present invention provides a preparation comprising the above-mentioned compound and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologues, solvates, prodrugs or polymorphs; or the above-mentioned pharmaceutical composition; and a pharmaceutically acceptable carrier.

[0019] In another aspect, the present invention provides the above-mentioned compound and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologues, solvates, prodrugs or polymorphs; or the use of the above-mentioned pharmaceutical composition as or in the preparation of a preparation with anticancer activity.

[0020] The beneficial effects of the present invention include: the tetrahydropyrrolidine compounds and pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs provided by the present invention have good anti-tumor effects and low toxicity to normal cells, and are safe, effective and low-toxic candidate anti-tumor drugs. DETAILED DESCRIPTION

[0021] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0022] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0023] The present invention provides a tetrahydropyrrolidine compound of formula (I) and pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs thereof:

[0024]

[0025] Among them, R 1 Any one of:

[0026] R 2 and R 3 Independently selected from: R 2 and R 3 Same or different;

[0027] R 4 Any one of:

[0028] R 1 and R 4 Same or different;

[0029] R 5 Any one of:

[0030] R 2 、R 3 and R 5 Same or different.

[0031] In some specific embodiments,

[0032] R 1 Any selection from: and / or

[0033] R 2 Any selection from: and / or

[0034] R 3 Any selection from: and / or

[0035] R 4 Any selection from: and / or

[0036] R 5 Any one of:

[0037] It should be noted that the tetrahydropyrrolidine compounds of formula (I) and their pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs, or polymorphs have the ability to kill cancer cells and have low cytotoxicity. Furthermore, stereoisomers, tautomers, homologs, solvates, prodrugs, and polymorphs are conventional technical terms in the art and have no other specific meanings.

[0038] In some specific embodiments, the tetrahydropyrrolidine compound of formula (I) and pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs thereof can be arbitrarily selected from:

[0039] (2S,3R,4S,5S)-2,4-dimethyl-4-nitro-3,5-diphenylpyrrolidine-2-carboxylic acid methyl ester;

[0040] (2S,3R,4S,5S)-5-(4-fluorophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0041] (2S,3R,4S,5S)-5-(4-chlorophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0042] (2S,3R,4S,5S)-5-(4-bromophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0043] (2S,3R,4S,5S)-5-(4-methoxyphenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0044] (2S,3R,4S,5S)-5-(benzo[d][1,3]dioxin-5-yl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0045] (2S,3R,4S,5S)-5-(4-Methylphenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0046] (2S,3R,4S,5S)-5-(4-trifluoromethylphenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0047] (2S,3R,4S,5S)-5-(4-nitrophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0048] (2S,3R,4S,5S)-5-(4-cyanophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0049] (2S,3R,4S,5S)-5-(2-fluorophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0050] (2S,3R,4S,5S)-5-(3-fluorophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0051] (2S,3R,4S,5S)-5-(3-bromophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0052] (2S,3R,4S,5S)-2,4-dimethyl-5-(naphthalen-1-yl)-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0053] (2S,3R,4S,5S)-2,4-dimethyl-5-(naphthalen-2-yl)-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0054] (2S,3R,4S,5R)-5-(thiophen-2-yl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0055] (2S,3R,4S,5R)-5-(furan-2-yl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0056] (2S,3R,4S,5S)-3-(4-bromophenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0057] (2S,3R,4S,5S)-3-(4-chlorophenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0058] (2S,3R,4S,5S)-3-(4-fluorophenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0059] (2S,3R,4S,5S)-3-(4-nitrophenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0060] (2S,3R,4S,5S)-3-(4-methoxyphenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0061] (2S,3R,4S,5S)-3-(furan-2-yl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0062] (2S,3R,4S,5S)-4-ethyl-2-methyl-4-nitro-3,5-diphenylpyrrolidine-2-carboxylic acid methyl ester;

[0063] (2S,3R,4S,5S)-2,4-Dimethyl-4-nitro-3,5-diphenylpyrrolidine-2-carboxylic acid ethyl ester;

[0064] tert-Butyl (2S,3R,4S,5S)-2,4-dimethyl-4-nitro-3,5-diphenylpyrrolidine-2-carboxylate;

[0065] (2S,3R,4S,5S)-2-ethyl-4-methyl-4-nitro-3,5-diphenylpyrrolidine-2-carboxylic acid methyl ester;

[0066] (2S,3R,4S,5S)-4-methyl-4-nitro-3,5-diphenylpyrrolidine-2-carboxylic acid methyl ester;

[0067] (2S,3S,4S,5S)-2-methyl-4-nitro-3,5-diphenylpyrrolidine-2-carboxylic acid methyl ester;

[0068] (2S,3R,4S,5S)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0069] (2S,3R,4S,5S)-5-(4-chlorophenyl)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitropyrrolidine-2-carboxylic acid methyl ester;

[0070] (2S,3R,4S,5S)-5-(4-methoxyphenyl)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitropyrrolidine-2-carboxylic acid methyl ester;

[0071] (2S,3R,4S,5S)-5-(4-trifluoromethylphenyl)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitropyrrolidine-2-carboxylic acid methyl ester;

[0072] (2S,3R,4S,5R)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitro-5-(thien-2-yl)pyrrolidine-2-carboxylic acid methyl ester;

[0073] (2S,3R,4S,5S)-3-(4-ethoxyphenyl)-4-methyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0074] (2S,3S,4S,5S)-3-(4-ethoxyphenyl)-2-methyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0075] and pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs thereof.

[0076] In some specific embodiments, the tetrahydropyrrolidine compound of formula (I) and pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs thereof can be arbitrarily selected from:

[0077] (2S,3R,4S,5S)-3-(4-methoxyphenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0078] (2S,3R,4S,5S)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0079] (2S,3R,4S,5S)-5-(4-chlorophenyl)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitropyrrolidine-2-carboxylic acid methyl ester;

[0080] (2S,3R,4S,5R)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitro-5-(thien-2-yl)pyrrolidine-2-carboxylic acid methyl ester;

[0081] (2S,3R,4S,5S)-3-(4-ethoxyphenyl)-4-methyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester;

[0082] and pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs thereof.

[0083] It should be noted that pharmaceutically acceptable salts of the compound of formula (I) include, but are not limited to, various inorganic or organic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, citrate, lactate, tartrate, maleate, fumarate, mandelate and oxalate; various inorganic or organic base salts such as sodium hydroxide, trishydroxymethylaminomethane and N-methyl-glucamine.

[0084] It should also be noted that the above-mentioned preferred compound of formula (I) and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs have stronger killing ability against cancer cells than other compounds; and have low cytotoxicity.

[0085] Another embodiment of the present invention provides a method for preparing the tetrahydropyrrolidine compound represented by the above formula (I), comprising: mixing a ligand L1, a molecular sieve, Cu(CH3CN)4BF4 and dichloromethane for reaction, and then cooling the mixture to 0°C±5°C to obtain a reaction solution; mixing a compound represented by formula (II), a compound represented by formula (III), triethylamine and dichloromethane, and then reacting the mixture with the reaction solution; after the reaction is completed, the compound represented by formula (I) is obtained;

[0086]

[0087] It should be noted that in the above preparation method, the R 1 、R 2 、R 3 、R 4 and R 5 and R in the tetrahydropyrrolidine compound represented by the above formula (I) 1 、R 2 、R 3 、R 4 and R 5 consistent.

[0088] Yet another embodiment of the present invention provides a pharmaceutical composition comprising the compound of formula (I) above and pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs thereof.

[0089] It should be noted that the above-mentioned compound of formula (I) and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologues, solvates, prodrugs or polymorphs can be used in combination with other active drugs to form a compound to enhance the killing ability of cancer cells or supplement other therapeutic effects to achieve a synergistic effect.

[0090] It should also be noted that the pharmaceutical composition of the present invention contains 0.1%-99.9% by weight of the above-mentioned compound of formula (I) and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologues, solvates, prodrugs or polymorphs as active ingredients.

[0091] In another aspect, the present invention provides a preparation comprising the compound of formula (I) above and pharmaceutically acceptable salts, stereoisomers, tautomers, homologues, solvates, prodrugs or polymorphs thereof; or the pharmaceutical composition above; and a pharmaceutically acceptable carrier.

[0092] It should be noted that in the above-mentioned preparations, pharmaceutically acceptable carriers include, but are not limited to: ion exchange materials, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-tocopherol polyethylene glycol 1000 succinate, Tween or other similar polymeric media, surfactants for pharmaceutical preparations, serum proteins such as human serum albumin, buffer substances such as phosphates, aminoacetic acid, sorbic acid, potassium sorbate, a mixture of partial glycerides of saturated vegetable fatty acids, water, salts, electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium silicate, etc. Polyvinyl pyrrolidone, cellulosic materials, polyvinyl alcohol, sodium carboxymethyl cellulose, polyacrylates, ethylene-polyoxyethylene-block polymers and lanolin, cyclodextrins such as α-, β-, γ-cyclodextrin or their chemically modified derivatives such as 2- and 3-hydroxypropyl-β-cyclodextrin and other hydroxyalkyl cyclodextrins or other soluble derivatives can all be used to promote the drug delivery of the compound represented by the above formula (I) and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs.

[0093] It should also be noted that in the above-mentioned preparations, pharmaceutically acceptable carriers also include pharmaceutically acceptable excipients, such as fillers (such as anhydrous lactose, starch, lactose beads and glucose), binders (such as microcrystalline cellulose), disintegrants (such as cross-linked sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose and cross-linked PVP), lubricants (such as magnesium stearate), absorption promoters, flavoring agents, sweeteners, diluents, excipients, wetting agents, solvents, solubilizers and colorants, etc., which can also be used as pharmaceutically acceptable carriers for preparing the above-mentioned compounds and pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs thereof into preparations.

[0094] In some specific embodiments, in the above preparations, the pharmaceutically acceptable carrier is suitable for liquid dosage form, solid dosage form or paste dosage form.

[0095] It should be noted that pharmaceutically acceptable carriers suitable for different dosage forms can be selected according to the administration method of the compound represented by the above formula (I) and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologues, solvates, prodrugs or polymorphs. It should also be noted that liquid dosage forms, solid dosage forms, paste dosage forms or emulsion dosage forms are known in the art. For example, liquid dosage forms include but are not limited to: injections, sprays or oral solutions; solid dosage forms include but are not limited to: tablets, powders, granules or capsules; paste dosage forms include but are not limited to: creams or ointments. In some specific embodiments, the compound represented by the above formula (I) and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs can be administered by enteral or parenteral routes; parenteral preparations include injections, creams, ointments, patches, sprays, etc.; administration routes include subcutaneous, intradermal, intraarterial, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial injection or infusion, or oral, topical, rectal, nasal, buccal, vaginal, sublingual, intradermal, mucosal, tracheal, urethral administration, or administration by inhalation of aerosol or implant accumulation or acupuncture.

[0096] In another aspect, the present invention provides a compound of formula (I) and pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs thereof; or the use of the above-mentioned pharmaceutical composition as or in the preparation of a preparation with anticancer activity.

[0097] In some specific embodiments, in the above applications, the cancer cells are cancer cells known in the art, preferably melanoma cells, lung adenocarcinoma cells, colon cancer cells, breast cancer cells, liver cancer cells or cervical cancer cells.

[0098] It should be noted that the therapeutically effective amount of the compound represented by the above formula (I) and its pharmaceutically acceptable salts, stereoisomers, tautomers, homologs, solvates, prodrugs or polymorphs is between 0.001 mg / kg / d and 100 mg / kg / d, which can be used for single drug or combination therapy for related diseases, which is within the range understood by those skilled in the art.

[0099] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0100] In the following examples, tetrahydropyrrolidine compounds containing a chiral quaternary carbon center were synthesized according to the following synthetic route:

[0101]

[0102] In the following examples, the method for preparing tetrahydropyrrolidine compounds containing a chiral quaternary carbon center according to the above synthetic route includes:

[0103] (1) Preparation of Compound 3

[0104] Weigh compound 2 (1.5 eq) and anhydrous magnesium sulfate (1.5 eq) into a dry round-bottom flask, add a stirrer, protect with nitrogen, add dry CH2Cl2 as solvent, add Et3N (1.5 eq), stir at room temperature for 1 h, then add compound 1 (1 eq), stir at room temperature for 24 h. After the reaction is complete, filter the reaction solution, wash the filtrate with a saturated sodium chloride solution prepared with deionized water and a saturated sodium bicarbonate solution twice in sequence, and finally wash with pure water twice. Combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under vacuum to obtain a crude product of compound 3. The crude product does not need to be purified and is directly used in the next reaction.

[0105] (2) Preparation of Compound 6

[0106] Compound 4 (1 eq), compound 5 (used as solvent), and ammonium acetate (1.3 eq) were weighed into a round-bottom flask and refluxed at 120°C for 2 hours. After the reaction was complete, water was added to quench the reaction, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed once with brine, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by silica gel column chromatography to obtain the product, compound 6.

[0107] (3) Preparation of Compound I

[0108] Weigh ligand L1 (0.011 mmol, 0.055 eq) and Cu(CH3CN)4BF4 (0.01 mmol, 0.05 eq) in a dry reaction tube and add Molecular sieves (200 mg) were added with a stirring bar and nitrogen protection. 2.5 mL of dry dichloromethane was added and the mixture was stirred at room temperature for 30 min. The mixture was moved to 0°C. Compound 3 (0.22 mmol, 1.1 eq) prepared in (2), compound 6 (0.2 mmol, 1 eq) prepared in (2) and triethylamine (0.1 mmol, 0.5 eq) were dissolved in 0.5 mL of dry dichloromethane and added to the reaction solution. The mixture was reacted at 0°C and the reaction progress was monitored by TLC. After the reaction was complete, the molecular sieves were filtered off and the filtrate was washed with saturated sodium bicarbonate solution, saturated sodium chloride solution, and the organic phase was dried with anhydrous sodium sulfate. The mixture was concentrated under vacuum and the crude product was purified by silica gel column chromatography to obtain the target product, compound I.

[0109] 1. Preparation and Characterization of Compound I

[0110] (1) Preparation of Compound I

[0111] In Examples 1 to 36, Compound I was prepared using the different raw materials shown in Table 1 below according to the above preparation method.

[0112] Table 1 Raw materials used in Examples 1 to 36

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] (2) Characterization of Compound I

[0119] The names, structural formulas, yields, and H NMR data of compounds I prepared in Examples 1 to 36 are shown in Table 2 below.

[0120] Table 2 Characterization data of compound I prepared in Examples 1 to 36

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] II. In vitro antitumor activity and cytotoxicity assays of compound I

[0127] The MTT [3-(4,5)-dimethyl-2-thiazole-(2,5)-phenyl tetrazolium blue bromide] method was used to determine the drug concentration (half maximal inhibitory concentration, IC) at which the inhibition rate of compound I prepared in Examples 1 to 36 on human melanoma cell line (A375), human lung adenocarcinoma cell line (A549) and human colon cancer cell line (HCT116) reached 50%. 50 ); and cytotoxicity to human hepatocytes (L02), the toxicity of each compound was measured by the concentration CC at which the viability of L02 cells was inhibited by 50% 50 To express.

[0128] The specific steps are as follows:

[0129] (1) Preparation of culture medium: DMEM (basal medium) 89%, fetal bovine serum 10%, penicillin-streptomycin solution (10,000 IU / mL, 10,000 μg / mL) 1%;

[0130] (2) Cultivation of four cell lines: Using the culture medium prepared in (a) (the volume of the culture medium is approximately 1 / 10 of the volume of the culture flask), culture human melanoma cell line (A375), human lung adenocarcinoma cell line (A549), human colon carcinoma cell line (HCT116), and human hepatocytes (L02) in a 37°C, 5% CO2 incubator. The subculture time was determined based on the growth status of the cells.

[0131] (3) Preparation of different drug concentrations: Prepare the stock solution using DMEM (with a small amount of DMSO for solubilization). The final DMSO concentration in each well of the cell suspension after drug addition generally does not exceed 0.05%-0.1%. Dilute the stock solution to six concentration gradients (100 μM, 50 μM, 10 μM, 1 μM, 0.1 μM, and 0.01 μM) using DMEM and store in a -20°C refrigerator until use.

[0132] (4) Cell incubation: Take tumor cells in the logarithmic growth phase and adjust the cell suspension concentration to 5×10 4 / mL, mix well and add to 96-well culture plate (100 μL / well), and culture in a 37°C, 5% CO2 incubator for 24 h;

[0133] (5) Drug addition: The diluted drugs at different concentration gradients were added to 96-well culture plates, with three replicates for each concentration gradient. The culture was continued for 72 h. The experiment was divided into an experimental group (culture medium, cells, and compound I), a control group (culture medium and cells), and a blank group (culture medium only).

[0134] (6) Survival cell detection: After 72 h of culture, add 20 μL / well of MTT (5 mg / mL) to a 96-well plate; after incubation at 37°C for 4 h, remove the supernatant, add 200 μL / well of DMSO, and shake until the formazan crystals are completely dissolved; use an automatic microplate reader to measure the optical density (OD value) of each well at a wavelength of 570 nm.

[0135] (7) Calculation of inhibition rate: The killing ability of compound I prepared in Examples 1 to 36 on cancer cells (A375, A549 and HCT116) and toxicity on normal cells (L02) were calculated using the following formulas.

[0136] Growth inhibition rate = (1 - survival rate) × 100% = [1 - (ODexperimental - ODblank) / (ODcontrol - ODblank)] × 100%, where ODexperimental is the average optical density of the experimental group, ODcontrol is the average optical density of the control group, and ODblank is the average optical density of the blank group.

[0137] (8) Based on the growth inhibition rate of different cancer cells (A375, A549 and HCT116) based on the concentration of compound I, calculate its IC 50 , unit μM; according to the concentration of compound I - normal cell (L02) growth inhibition rate, calculate its CC 50 , unit μM; the results are shown in Table 3 below.

[0138] Table 3 Cancer cell killing ability and normal cell toxicity data of compound I prepared in Examples 1 to 36

[0139]

[0140]

[0141] Note: In Table 3, IC 50 : Half inhibitory concentration, the ability of the compound to kill cancer cells; CC 50 : Half toxic concentration, the toxicity of the compound to normal cells.

[0142] The above results indicate that the tetrahydropyrrolidine compounds containing a chiral quaternary carbon center (Compound I) prepared in Examples 1 to 36 all have anti-tumor effects and low toxicity to normal cells.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A tetrahydropyrrolidine compound and a pharmaceutically acceptable salt thereof, characterized in that: Tetrahydropyrrolidine compounds are arbitrarily selected from: (2S,3R,4S,5S)-2,4-dimethyl-4-nitro-3,5-diphenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(4-fluorophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(4-chlorophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(4-bromophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(4-methoxyphenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(benzo[d][1,3]dioxin-5-yl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(4-methylphenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(4-trifluoromethylphenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(4-nitrophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(4-cyanophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(2-fluorophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(3-fluorophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(3-bromophenyl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-2,4-dimethyl-5-(naphthalen-1-yl)-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-2,4-dimethyl-5-(naphthalen-2-yl)-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5R)-5-(thiophen-2-yl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5R)-5-(furan-2-yl)-2,4-dimethyl-4-nitro-3-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-3-(4-bromophenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-3-(4-chlorophenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-3-(4-fluorophenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-3-(4-nitrophenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-3-(4-methoxyphenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-3-(furan-2-yl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-4-ethyl-2-methyl-4-nitro-3,5-diphenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-2,4-dimethyl-4-nitro-3,5-diphenylpyrrolidine-2-carboxylic acid ethyl ester; tert-Butyl (2S,3R,4S,5S)-2,4-dimethyl-4-nitro-3,5-diphenylpyrrolidine-2-carboxylate; (2S,3R,4S,5S)-2-ethyl-4-methyl-4-nitro-3,5-diphenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(4-chlorophenyl)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitropyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(4-methoxyphenyl)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitropyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(4-trifluoromethylphenyl)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitropyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5R)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitro-5-(thien-2-yl)pyrrolidine-2-carboxylic acid methyl ester; Methyl (2S,3R,4S,5S)-3-(4-ethoxyphenyl)-4-methyl-4-nitro-5-phenylpyrrolidine-2-carboxylate.

2. The compound according to claim 1, characterized in that Any one of: (2S,3R,4S,5S)-3-(4-methoxyphenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitro-5-phenylpyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5S)-5-(4-chlorophenyl)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitropyrrolidine-2-carboxylic acid methyl ester; (2S,3R,4S,5R)-3-(4-ethoxyphenyl)-2,4-dimethyl-4-nitro-5-(thien-2-yl)pyrrolidine-2-carboxylic acid methyl ester; Methyl (2S,3R,4S,5S)-3-(4-ethoxyphenyl)-4-methyl-4-nitro-5-phenylpyrrolidine-2-carboxylate.

3. A pharmaceutical composition, characterized in that The invention comprises the compound according to claim 1 or 2 and a pharmaceutically acceptable salt thereof.

4. A preparation, characterized in that The invention comprises the compound according to claim 1 or 2 and a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 3; and a pharmaceutically acceptable carrier.

5. The preparation according to claim 4, characterized in that The pharmaceutically acceptable carrier is suitable for liquid dosage forms, solid dosage forms or paste dosage forms.

6. Use of the compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 3, in the preparation of a preparation having anticancer activity, wherein the cancer cells are melanoma cells, human lung adenocarcinoma cells, or human colon cancer cells.

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