NO donor coumarin Mannich base compounds and preparation method and use thereof

Abstract

The present invention relates to NO donor type coumarin Mannich base compounds and preparation methods and uses thereof, and belongs to the field of pharmaceutical chemistry and pharmacotherapy. The method comprises using resorcinol as a raw material and reacting multiple people to obtain a lead compound 7-hydroxy-coumarin-3-ethyl formate; the obtained lead is then reacted with paraformaldehyde and an organic amine by Mannich reaction to obtain an intermediate 7-hydroxy-8-aminomethyl-coumarin-3-ethyl formate; then hydrolyzed to obtain 7-hydroxy-8-aminomethyl-coumarin-3-formic acid; 7-hydroxy-8-aminomethyl-coumarin-3-formic acid reacts with aminofurazan / hydroxyfurazan under the action of an ester condensation reagent to obtain an NO donor type coumarin Mannich base compound. The present invention introduces an aminomethyl Mannich base at the ortho position of the 7-hydroxyl group of coumarin, and introduces an NO donor at the C-3 position, thereby improving the anti-tumor activity of the lead compound, improving its water solubility, and giving it good drugability.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry and pharmacotherapy, and particularly relates to NO donor type coumarin Mannich base compounds and a preparation method and application thereof. Background Art

[0002] Cancer is one of the major diseases that threaten human life and health today. Although great progress has been made in the research of anticancer drugs, the poor selectivity and large toxic side effects of antitumor drugs, especially the existence of tumor chemotherapy resistance, affect the therapeutic effect of drugs. There is an urgent need to develop new drugs to meet the needs of cancer patients.

[0003] Natural products have long been considered an important source of effective therapeutic drugs. In addition to directly developing effective new therapeutic drugs from natural products, natural products are also an inexhaustible source of new lead compounds that new drug development relies on. Coumarin compounds are widely present in nature and have multiple physiological activities such as antibacterial, anticoagulant, antioxidant, anti-inflammatory and anti-tumor. Their unique molecular structure, good thermodynamic and photochemical stability, easy structural modification and convenient introduction of various functional groups have always attracted extensive attention from scholars at home and abroad. Studies have shown that some natural coumarin compounds can selectively act on tumor cells, while having low toxicity to normal cells, such as esculentol, daphnetolide, scopoletin, and neomycin (Journal of Chinese Materia Medica, 1998, 23(5): 303-305; Phytomedicine, 2014, 21: 970-977), which provides a good prospect for the development of highly effective and low-toxic anti-tumor drugs. In recent years, in order to obtain coumarin derivatives with stronger anti-tumor effects and explore their anti-tumor structure-activity relationship, several research groups have begun to structurally modify and transform coumarin, and have designed and synthesized many new coumarin derivatives.

[0004] The role of NO in the occurrence, development and death of tumors has become one of the hot topics in tumor biology research. A large number of studies have shown that high concentrations of NO have anti-tumor effects (Acta Pharmaceutica Sinica. 2019, 54(3): 514-521; Journal of China Pharmaceutical University, 2021, 52(1): 38-43; J Med Chem, 2014, 57(22): 9343–9356). Studies have also found that some tumor cells are more sensitive to NO-induced apoptosis than normal cells; the combination of NO donors and chemotherapeutic drugs can overcome tumor resistance and other advantages. Our research group used coumarin-3-carboxylic acid as a raw material to design and synthesize a series of NO-donor coumarin derivatives. In vitro antitumor activity studies have shown that the obtained derivatives have certain anti-proliferative activity against a variety of tumor cells, and some compounds have stronger effects than 5-FU, and have mild inhibitory effects on normal cells, showing good selectivity (Med Chem Res, 2018, 27(2): 1198–1205). Although this type of compound has good in vitro antitumor activity, its water solubility is poor and further structural modification and optimization are still needed to improve its shortcomings. Summary of the invention

[0005] In view of the above problems, the present invention provides NO donor coumarin Mannich base compounds and preparation methods and uses thereof. The NO donor coumarin Mannich base compounds use 7-hydroxy-coumarin-3-carboxylic acid ethyl ester as a lead compound, firstly introduce an aminomethyl structure at the 8-position thereof to obtain a corresponding coumarin Mannich base derivative, and then, after hydrolysis and acidification of the 3-position ester group, combine with an NO donor having an anti-tumor effect through different connecting chains to design and synthesize a new NO donor coumarin Mannich base derivative.

[0006] The first object of the present invention is to provide an NO donor coumarin Mannich base compound, wherein the chemical structural formula of the NO donor coumarin Mannich base compound is as shown in Formula I:

[0007]

[0008] In Formula I:

[0009] X represents -O- or -NH-;

[0010] R stands for -(CH2) n -, wherein n = 1 to 6, -CH(CH3)CH2-, -CH(CH3)(CH2)2-,

[0011] -(CH2)2O(CH2)2-, -Ph(CH2)2-, -(CH2)2NH(CH2)2-, -CH2CH=CHCH2- or -CH2C≡CCH2-;

[0012] R1 stands for NR 2 R 3 ;

[0013] R 2 and R 3 may be the same or different and independently represent a hydrogen atom, a C1-C6 alkyl group, a phenyl group, a benzyl group, or a phenethyl group;

[0014] or R 2 and R 3 Together with the nitrogen atom to which it is connected, it forms a five- to seven-membered aliphatic heterocycle or aromatic heterocycle, and the aliphatic heterocycle or aromatic heterocycle can be arbitrarily substituted by the following same or different substituents, wherein the substituents include: C1-C6 alkyl, C1-C6 alkoxy, hydroxyl or hydroxyl-(C1-C6) alkyl.

[0015] Further, X represents -O- or -NH-;

[0016] R represents -(CH2)n-, wherein n=2 to 6, -CH(CH3)CH2-, -CH(CH3)(CH2)2-,

[0017] -(CH2)2O(CH2)2-, -Ph(CH2)2-, -CH2CH=CHCH2- or -CH2C≡CCH2-;

[0018] R1 represents amino, dimethylamino, diethylamino, dipropylamino, di-n-butylamino, aniline, benzylamino, phenethylamino, pyrrolyl, piperidinyl, morpholinyl, imidazolyl, piperazinyl, 4-methylpiperidinyl, N-methylpiperazinyl, N-ethylpiperazinyl or 4-hydroxyethylpiperazinyl.

[0019] The second object of the present invention is to provide a method for preparing an NO donor coumarin Mannich base compound, comprising:

[0020] Using resorcinol as a raw material, 2,4-dihydroxybenzaldehyde is prepared through Vilsmeier-Haack reaction under the action of an acylating agent and a first catalyst;

[0021] 2,4-dihydroxybenzaldehyde and diethyl malonate were reacted by Knoevenagel reaction to prepare 7-hydroxy-coumarin-3-carboxylic acid ethyl ester;

[0022] 7-hydroxy-coumarin-3-carboxylic acid ethyl ester, paraformaldehyde and organic amine are subjected to Mannich reaction to introduce different aminomethyl groups at the 8-position of 7-hydroxy-coumarin-3-carboxylic acid ethyl ester to obtain the intermediate 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid ethyl ester;

[0023] The intermediate 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid ethyl ester is hydrolyzed to obtain 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid;

[0024] 7-Hydroxy-8-aminomethyl-coumarin-3-carboxylic acid reacts with aminofurazan / hydroxyfurazan in the presence of an ester condensation reagent to obtain NO-donating coumarin Mannich base compounds;

[0025] The synthetic route is as follows:

[0026]

[0027] Wherein, the structural formula of the aminofurazan / hydroxyfurazan is as follows:

[0028]

[0029] X represents -O- or -NH-;

[0030] R stands for -(CH2) n -, wherein n = 1 to 6, -CH(CH3)CH2-, -CH(CH3)(CH2)2-,

[0031] -(CH2)2O(CH2)2-, -Ph(CH2)2-, -(CH2)2NH(CH2)2-, -CH2CH=CHCH2- or -CH2C≡CCH2-;

[0032] R1 stands for NR 2 R 3 ;

[0033] R 2 and R 3 may be the same or different and independently represent a hydrogen atom, a C1-C6 alkyl group, a phenyl group, a benzyl group, or a phenethyl group;

[0034] or R 2 and R 3 Together with the nitrogen atom to which it is connected, it forms a five- to seven-membered aliphatic heterocycle or aromatic heterocycle, and the aliphatic heterocycle or aromatic heterocycle can be arbitrarily substituted by the following same or different substituents, wherein the substituents include: C1-C6 alkyl, C1-C6 alkoxy, hydroxyl or hydroxyl-(C1-C6) alkyl.

[0035] Furthermore, in the preparation process of the 2,4-dihydroxybenzaldehyde, the molar ratio of resorcinol to the acylating agent is 1:1.1-1.5; the acylating agent is one of monosubstituted formamide and disubstituted formamide, and the first catalyst is one of phosphorus oxychloride, thionyl chloride, zinc chloride, and phosgene.

[0036] Furthermore, during the preparation of the 7-hydroxy-coumarin-3-carboxylic acid ethyl ester, a second catalyst is added, wherein the second catalyst is piperidine and glacial acetic acid; and the molar ratio of the 2,4-dihydroxybenzaldehyde to diethyl malonate is 1:1-1.5.

[0037] Furthermore, in the preparation process of the intermediate 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid ethyl ester, the molar ratio of 7-hydroxy-coumarin-3-carboxylic acid ethyl ester, paraformaldehyde and organic amine is 1:5-8:3-4.

[0038] Furthermore, the hydrolysis condition of the intermediate 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid ethyl ester is alkaline hydrolysis.

[0039] Furthermore, during the reaction of 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid with aminofurazan / hydroxyfurazan under the action of an ester condensation reagent, the molar ratio of 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid to aminofurazan / hydroxyfurazan is 1:1-1.5.

[0040] The third object of the present invention is to provide a pharmaceutical composition, comprising the NO donor coumarin Mannich base compound or a pharmaceutically acceptable salt thereof as described above;

[0041] The pharmaceutically acceptable salt is prepared by mixing an NO-donating coumarin Mannich base compound and a pharmaceutically acceptable acid;

[0042] The pharmaceutically acceptable acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, lactic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, tartaric acid, maleic acid, citric acid, ascorbic acid, methanesulfonic acid, camphoric acid, oxalic acid and the like.

[0043] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

[0044] The fourth object of the present invention is to provide the use of the above-mentioned pharmaceutical composition in the preparation of drugs for treating tumors, wherein the tumors include liver cancer, breast cancer, cervical cancer, lung cancer and colon cancer.

[0045] Beneficial effects of the present invention:

[0046] The NO donor type coumarin Mannich base compound provided by the present invention is obtained by introducing an amine methyl base (corresponding to CH2R1(NR 2 R 3 ))'s design improves the anti-tumor activity of the obtained NO donor coumarin Mannich base compounds, improves their water solubility, and gives them good drugability.

[0047] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1 A flow chart of a method for preparing a NO donor coumarin Mannich base compound according to an embodiment of the present invention is shown;

[0050] Figure 2 A graph showing the effect of the target product I7 according to an example of the present invention on the cell viability of normal human liver cells LO2 is shown. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] like Figure 1 As shown, the method for preparing the NO donor coumarin Mannich base compound according to an embodiment of the present invention comprises:

[0053] Step 1: using resorcinol as a raw material, preparing 2,4-dihydroxybenzaldehyde through a Vilsmeier-Haack reaction under the action of an acylating agent and a first catalyst;

[0054] Step 2: 2,4-dihydroxybenzaldehyde and diethyl malonate are reacted via Knoevenagel reaction to obtain 7-hydroxy-coumarin-3-carboxylic acid ethyl ester;

[0055] Step 3: 7-hydroxy-coumarin-3-carboxylic acid ethyl ester, paraformaldehyde and organic amine are subjected to Mannich reaction to introduce different aminomethyl groups at the 8-position of 7-hydroxy-coumarin-3-carboxylic acid ethyl ester to obtain the intermediate 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid ethyl ester;

[0056] Step 4: The intermediate 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid ethyl ester is hydrolyzed to obtain 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid;

[0057] Step 5: 7-Hydroxy-8-aminomethyl-coumarin-3-carboxylic acid reacts with aminofurazan / hydroxyfurazan in the presence of an ester condensation reagent to obtain a NO donor type coumarin Mannich base compound.

[0058] The above preparation route is as follows:

[0059]

[0060] Wherein, the structural formula of the aminofurazan / hydroxyfurazan is as follows:

[0061]

[0062] X represents -O- or -NH-;

[0063] R stands for -(CH2) n -, wherein n = 1 to 6, -CH(CH3)CH2-, -CH(CH3)(CH2)2-,

[0064] -(CH2)2O(CH2)2-, -Ph(CH2)2-, -(CH2)2NH(CH2)2-, -CH2CH=CHCH2- or -CH2C≡CCH2-;

[0065] R1 stands for NR 2 R 3 ;

[0066] R 2 and R 3 may be the same or different and independently represent a hydrogen atom, a C1-C6 alkyl group, a phenyl group, a benzyl group, or a phenethyl group;

[0067] or R 2 and R 3 Together with the nitrogen atom to which it is connected, it forms a five- to seven-membered aliphatic heterocycle or aromatic heterocycle, and the aliphatic heterocycle or aromatic heterocycle can be arbitrarily substituted by the following same or different substituents, wherein the substituents include: C1-C6 alkyl, C1-C6 alkoxy, hydroxyl or hydroxyl-(C1-C6) alkyl.

[0068] In step 1, during the preparation of 2,4-dihydroxybenzaldehyde, the molar ratio of resorcinol to acylating agent is 1:1.1-1.5; the acylating agent is one of monosubstituted formamide and disubstituted formamide, and the first catalyst is one of phosphorus oxychloride, thionyl chloride, zinc chloride, and phosgene.

[0069] In step 2, during the preparation of 7-hydroxy-coumarin-3-carboxylic acid ethyl ester, a second catalyst is added, wherein the second catalyst is piperidine and glacial acetic acid; the molar ratio of the 2,4-dihydroxybenzaldehyde to diethyl malonate is 1:1-1.5.

[0070] In step 3, during the preparation of the intermediate 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid ethyl ester, the molar ratio of 7-hydroxy-coumarin-3-carboxylic acid ethyl ester, paraformaldehyde, and organic amine is 1:5-8:3-4;

[0071] Different organic amines result in different R1 groups in the molecular structure formula I of the NO donor coumarin Mannich base compound. The organic amine is methylamine and its derivatives with hydrogen substitution on nitrogen. Its chemical structure is R1CH3, where R1 represents NR 2 R 3 ;

[0072] R 2 and R 3 may be the same or different and independently represent a hydrogen atom, a C1-C6 alkyl group, a phenyl group, a benzyl group, or a phenethyl group;

[0073] or R 2 and R 3 Together with the nitrogen atom to which it is attached, it forms a five- to seven-membered aliphatic heterocyclic ring or aromatic heterocyclic ring, wherein the aliphatic heterocyclic ring or aromatic heterocyclic ring may be arbitrarily substituted by the following identical or different substituents, wherein the substituents include: C1-C6 alkyl, C1-C6 alkoxy, hydroxyl or hydroxy-(C1-C6)alkyl;

[0074] In step 3, the reaction solvent of 7-hydroxy-coumarin-3-carboxylic acid ethyl ester, paraformaldehyde and organic amine is an alcohol solvent, such as methanol, ethanol, etc., preferably ethanol;

[0075] The amount of ethanol added is sufficient to dissolve 7-hydroxy-coumarin-3-carboxylic acid ethyl ester, paraformaldehyde, and the organic amine reactants, and is not specifically limited in the present invention.

[0076] In step 4, the hydrolysis condition of the intermediate 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid ethyl ester is alkaline hydrolysis;

[0077] In step 4, the hydrolysis reagent is preferably sodium hydroxide, and the molar ratio of the sodium hydroxide to 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid ethyl ester is 3-5:1.

[0078] In step 5, during the reaction of the 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid with aminofurazan / hydroxyfurazan under the action of an ester condensation reagent, the molar ratio of the 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid to aminofurazan / hydroxyfurazan is 1:1-1.5;

[0079] In step 5, the ester condensation reagent is preferably DIEA and HATU.

[0080] 1. Embodiment:

[0081] In certain embodiments of the present invention, the aminofurazan / hydroxyfurazan is specifically hydroxyfurazan, that is, X represents -O-, and its synthesis route is as follows:

[0082]

[0083] In the reaction of step iv, different compounds 4 will appear, resulting in different molecular structures of R. Specifically, R represents -(CH2) n -, wherein n = 1 to 6, -CH(CH3)CH2-, -CH(CH3)(CH2)2-, -(CH2)2O(CH2)2-, -Ph(CH2)2-, -(CH2)2NH(CH2)2-, -CH2CH=CHCH2- or -CH2C≡CCH2-;

[0084] Therefore, in the embodiment of the present invention, the synthesis route of the entire NO donor coumarin Mannich base compound is as follows:

[0085]

[0086] According to the above synthetic route, specifically: sodium benzenethiol is used as a raw material to react with bromoacetic acid to obtain benzenethiolacetic acid (1); 30% H2O2 is oxidized to obtain intermediate (2); fuming HNO3 is used to obtain intermediate (3); and then reacted with different diols to obtain intermediate (4). Then 1,3-benzenediol is used as a raw material to prepare 2,4-dihydroxybenzaldehyde (5) through Vilsmeier-Haack reaction, and then reacted with diethyl malonate through Knoevenagel reaction to obtain 7-hydroxycoumarin-3-carboxylic acid ethyl ester (6); Mannich reaction is performed to obtain intermediate (7); hydrolyzed under alkaline conditions to obtain intermediate (8); and then reacted with intermediate (4) to convert it into the target product;

[0087] Specific compounds I1, I2..., I22 for synthesizing target product I:

[0088] 1. Synthesis of Phenylthioacetic Acid

[0089] i. Add sodium thiophenolate (0.19 mol, 25 g) and 100 mL of water to a 500 mL round-bottom flask, and stir to dissolve at room temperature. Dissolve bromoacetic acid (0.20 mol, 27.8 g) in 50 mL of water, add anhydrous sodium carbonate (0.11 mol, 11.13 g) and stir until the solution is clear, then add the solution to the reaction solution, stir at room temperature for 4 h, condense and reflux for 2 h, and stop the reaction. Cool the reaction solution to room temperature, add 6 mol / L HCl to adjust pH = 2, freeze overnight, and filter to obtain 26.1 g of white crystals with a yield of 68.7%. mp 61.1-62.0℃.

[0090] 2. Synthesis of Benzenesulfonyl Methyl Ester (2) and 3,4-Diphenylsulfonyl Furazan (3)

[0091] ii+iii. Add compound 1 (0.16 mol, 26.1 g) and 100 mL of glacial acetic acid into a 500 mL three-necked flask, slowly drop 30% H2O2 (32 mmol, 33 mL) into it, and stir at room temperature for 3 h to generate benzenesulfonyl methyl ester (2). Slowly drop fuming nitric acid (65 mL, 1.72 mol) into the reaction solution while stirring under ice bath conditions to make the internal temperature of the reaction solution less than 40°C. After the dropwise addition, heat the solution to condensation reflux for 4 h to stop the reaction. Cool the reaction solution to room temperature, white crystals precipitate, freeze overnight, and filter to obtain 12.3 g of white crystals, with a yield of 42.8%. mp154.7-155.6°C.

[0092] 3. Synthesis of furazan diol (4)

[0093] iv. Add compound 3 (2.7mmol, 1.0g), diol (21mmol) and 10mL tetrahydrofuran (THF) into a 50mL reaction bottle, slowly drop 25% NaOH aqueous solution (0.5mL) under ice bath condition, monitor the reaction progress by TLC, and stop the reaction after 30min. Add the reaction solution into 100mL water, extract with dichloromethane (DCM) 3 times (50mL×3), combine the organic phases, extract the organic phases with saturated brine once, and then add anhydrous sodium sulfate to dry. Filter by suction, concentrate the filtrate under reduced pressure, separate by column chromatography [V (ethyl acetate): V (petroleum ether) = 1:4], and obtain the product.

[0094] 4. Synthesis of 2,4-dihydroxybenzaldehyde (5)

[0095] v. Add N,N-dimethylformamide (DMF) (0.26 mol, 20 mL) to a 500 mL three-necked flask under ice bath conditions, slowly add phosphorus oxychloride (POCl3) (0.22 mol, 20 mL) to the reaction solution while stirring, and stir in an ice bath for 1 h after the addition is complete.

[0096] Dissolve resorcinol (0.18 mol, 20 g) in 40 mL of acetonitrile and slowly drop it into the reaction solution. Stir in an ice bath for 8 h after the addition is complete. Filter with suction. Add the filter cake into a 250 mL round-bottom flask, add 100 mL of water, condense and reflux for 30 min, stir at room temperature for 1 h, filter with suction, and vacuum dry the filter cake to obtain 19.8 g of light pink needle-shaped crystals, with a yield of 78.8% and mp of 134.8-135.7 °C.

[0097] 5. Synthesis of 7-Hydroxycoumarin-3-carboxylic acid ethyl ester (6)

[0098] vi. Add compound 5 (10 g, 72 mmol) and 30 mL of anhydrous ethanol to a 250 mL round-bottom flask, stir thoroughly to dissolve, add diethyl malonate (13.1 mL, 86 mmol), hexahydropyridine (2.5 mL, 25 mmol), and glacial acetic acid (0.1 mL, 1.7 mmol), stir at room temperature, and monitor the reaction progress by TLC. After the reaction is complete, add 200 mL of ice water, freeze overnight, and filter to obtain 12.8 g of a yellow solid with a yield of 75.9%. The crude product was recrystallized from 95% ethanol to obtain 10.7 g of a light yellow solid with a yield of 63.5%, mp 172.2-172.9°C.

[0099] 6. Synthesis of intermediate 7-hydroxy-coumarin-8-aminomethyl-3-carboxylic acid ethyl ester (7)

[0100] vii. Add compound 6 (2.34 g, 10 mmol), paraformaldehyde (1.8 g, 60 mmol) and 20 mL of 95% ethanol to a 100 mL round-bottom flask and reflux for 1 h. Add an organic amine (30 mmol) dropwise to the reaction solution and continue the reaction. Monitor the reaction progress by TLC. The reaction is complete after 8 h. Cool the reaction solution to room temperature, concentrate under reduced pressure to remove ethanol, add the reaction solution to 100 mL of water, extract with dichloromethane (DCM) 3 times (50 mL × 3), combine the organic phases, back-extract the organic phase with saturated brine once, and then add anhydrous sodium sulfate to dry. Filter with suction, concentrate the filtrate under reduced pressure, and separate by column chromatography [V (methanol): V (dichloromethane) = 1:40] to obtain the product.

[0101] 7. Synthesis of 7-hydroxy-coumarin-8-aminomethyl-3-carboxylic acid (8)

[0102] viii. Add compound 7 (1 mmol), sodium hydroxide (3 mmol, 120 mg) and 20 mL of anhydrous ethanol to a 100 mL round-bottom flask, condense and reflux, monitor the reaction progress by TLC, and the reaction is complete after 30 min. Cool the reaction solution to room temperature, add 6 mol / L HCl dropwise to adjust the pH of the reaction solution to 3-4, filter with suction, and take the filter cake and directly put it into the next step of reaction.

[0103] 8. Synthesis of target compound I (I1-I22)

[0104] ix. Add compound 8 (1mmol), compound 4 (1.2mmol, 343mg), DIEA (2mmol, 0.35mL) and 5mL DMF into a 50mL round-bottom flask, add HATU (1.5mmol, 570mg) under ice bath, move the reaction solution to room temperature after addition, stir and monitor the reaction progress by TLC, the reaction is complete in 24h. Add the reaction solution into 100mL water, extract with dichloromethane (DCM) 3 times (50mL×3), combine the organic phases, extract the organic phases with saturated brine once, and then add anhydrous sodium sulfate to dry. Filter by suction, concentrate the filtrate under reduced pressure and separate by column chromatography to obtain the product.

[0105] The specific names of the obtained I1-I22 are as follows:

[0106] 7-Hydroxy-8-piperidinylmethyl-coumarin-3-carboxylic acid 1,2-ethylenediol benzenesulfonyl furazan (I1);

[0107] 7-Hydroxy-8-piperidinylmethyl-coumarin-3-carboxylic acid-1,3-propanediolbenzenesulfonylfuroxan (I2);

[0108] 7-Hydroxy-8-piperidinylmethyl-coumarin-3-carboxylic acid-1,4-butanediolbenzenesulfonylfuroxan (I3);

[0109] 7-Hydroxy-8-piperidinylmethyl-coumarin-3-carboxylic acid-1,5-pentanediolbenzenesulfonylfuroxan (I4);

[0110] 7-Hydroxy-8-piperidinylmethyl-coumarin-3-carboxylic acid-1,6-hexanediolbenzenesulfonylfuroxan (I5);

[0111] 7-Hydroxy-8-piperidinylmethyl-coumarin-3-carboxylic acid-ethylene glycol phenylsulfonyl furazan (I6);

[0112] 7-Hydroxy-8-piperidinylmethyl-coumarin-3-carboxylic acid-1,4-butynediol benzenesulfonylfuroxan (I7)

[0113] 7-Hydroxy-8-N-methylpiperazinylmethyl-coumarin-3-carboxylic acid 1,2-ethylenediol benzenesulfonylfuroxan (I8);

[0114] 7-Hydroxy-8-N-methylpiperazinylmethyl-coumarin-3-carboxylic acid-1,3-propanediolbenzenesulfonylfuroxan (I9);

[0115] 7-Hydroxy-8-N-methylpiperazinylmethyl-coumarin-3-carboxylic acid-1,4-butanediolbenzenesulfonylfuroxan (I10);

[0116] 7-Hydroxy-8-N-methylpiperazinylmethyl-coumarin-3-carboxylic acid-1,5-pentanediolbenzenesulfonylfuroxan (I11);

[0117] 7-Hydroxy-8-N-methylpiperazinylmethyl-coumarin-3-carboxylic acid-1,6-hexanediolbenzenesulfonylfuroxan (I12);

[0118] 7-Hydroxy-8-N-methylpiperazinylmethyl-coumarin-3-carboxylic acid monoethylene glycol benzenesulfonyl furazan (I13);

[0119] 7-Hydroxy-8-N-methylpiperazinylmethyl-coumarin-3-carboxylic acid-1,4-butynediol benzenesulfonylfurazan (I14)-I14;

[0120] 7-Hydroxy-8-N-ethylpiperazinylmethyl-coumarin-3-carboxylic acid 1,2-ethylenediol benzenesulfonylfuroxan (I15);

[0121] 7-Hydroxy-8-N-ethylpiperazinylmethyl-coumarin-3-carboxylic acid-1,3-propanediolbenzenesulfonylfuroxan (I16);

[0122] 7-Hydroxy-8-N-ethylpiperazinylmethyl-coumarin-3-carboxylic acid-1,4-butanediolbenzenesulfonylfuroxan (I17);

[0123] 7-Hydroxy-8-N-ethylpiperazinylmethyl-coumarin-3-carboxylic acid-1,5-pentanediolbenzenesulfonylfuroxan (I18);

[0124] 7-Hydroxy-8-N-ethylpiperazinylmethyl-coumarin-3-carboxylic acid-1,6-hexanediolbenzenesulfonylfuroxan (I19);

[0125] 7-Hydroxy-8-N-ethylpiperazinylmethyl-coumarin-3-carboxylic acid monoethylene glycol benzenesulfonyl furazan (I20);

[0126] 7-Hydroxy-8-N-ethylpiperazinylmethyl-coumarin-3-carboxylic acid-1,4-butynediol benzenesulfonylfuroxan (I21);

[0127] 7-Hydroxy-8-N-hydroxyethylpiperazinylmethyl-coumarin-3-carboxylic acid-1,4-butynediol benzenesulfonylfuroxan (I22);

[0128] In this embodiment, only some compounds in which the intermediate compound 4 is hydroxyfurazan are specifically listed, and the final product may also be any one of the following compounds:

[0129] 7-Hydroxy-8-morpholinylmethyl-coumarin-3-carboxylic acid 1,2-ethylenediol benzenesulfonylfuroxan;

[0130] 7-Hydroxy-8-morpholinylmethyl-coumarin-3-carboxylic acid-1,3-propanediolbenzenesulfonylfuroxan;

[0131] 7-Hydroxy-8-morpholinylmethyl-coumarin-3-carboxylic acid-1,4-butanediolbenzenesulfonylfuroxan;

[0132] 7-Hydroxy-8-morpholinylmethyl-coumarin-3-carboxylic acid-1,5-pentanediolbenzenesulfonylfuroxan;

[0133] 7-Hydroxy-8-morpholinylmethyl-coumarin-3-carboxylic acid-1,6-hexanediolbenzenesulfonylfuroxan;

[0134] 7-Hydroxy-8-morpholinylmethyl-coumarin-3-carboxylic acid monoethylene glycol benzenesulfonylfuroxan;

[0135] 7-Hydroxy-8-morpholinylmethyl-coumarin-3-carboxylic acid-1,4-butynediol benzenesulfonylfuroxan;

[0136] 7-Hydroxy-8-piperazinylmethyl-coumarin-3-carboxylic acid 1,2-ethylenediol benzenesulfonylfuroxan;

[0137] 7-Hydroxy-8-piperazinylmethyl-coumarin-3-carboxylic acid-1,3-propanediolbenzenesulfonylfuroxan;

[0138] 7-Hydroxy-8-piperazinylmethyl-coumarin-3-carboxylic acid-1,4-butanediolbenzenesulfonylfuroxan;

[0139] 7-Hydroxy-8-piperazinylmethyl-coumarin-3-carboxylic acid-1,5-pentanediolbenzenesulfonylfuroxan;

[0140] 7-Hydroxy-8-piperazinylmethyl-coumarin-3-carboxylic acid-1,6-hexanediolbenzenesulfonylfuroxan;

[0141] 7-Hydroxy-8-piperazinylmethyl-coumarin-3-carboxylic acid monoethylene glycol benzenesulfonylfuroxan;

[0142] 7-Hydroxy-8-piperazinylmethyl-coumarin-3-carboxylic acid-1,4-butynediol benzenesulfonylfuroxan;

[0143] 7-Hydroxy-8-N-hydroxyethylpiperazinylmethyl-coumarin-3-carboxylic acid 1,2-ethylenediol benzenesulfonylfuroxan;

[0144] 7-Hydroxy-8-N-hydroxyethylpiperazinylmethyl-coumarin-3-carboxylic acid-1,3-propanediolbenzenesulfonylfuroxan;

[0145] 7-Hydroxy-8-N-hydroxyethylpiperazinylmethyl-coumarin-3-carboxylic acid-1,4-butanediolbenzenesulfonylfuroxan;

[0146] 7-Hydroxy-8-N-hydroxyethylpiperazinylmethyl-coumarin-3-carboxylic acid-1,5-pentanediolbenzenesulfonylfuroxan;

[0147] 7-Hydroxy-8-N-hydroxyethylpiperazinylmethyl-coumarin-3-carboxylic acid-1,6-hexanediolbenzenesulfonylfuroxan;

[0148] 7-Hydroxy-8-N-hydroxyethylpiperazinylmethyl-coumarin-3-carboxylic acid monoacetate ethylene glycol benzenesulfonylfuroxan.

[0149] In some other embodiments of the present invention, the aminofurazan / hydroxyfurazan is specifically aminofurazan, that is, X represents -NH-, corresponding to the above-mentioned compound 4 where -OH is replaced by -NH2. Aminofurazan is easily available in the technical field and will not be described in detail here.

[0150] 2. Performance test:

[0151] 2.1 Yield, melting point and structural characterization:

[0152] In certain embodiments of the present invention, some of the target products and intermediates prepared in the above embodiments were selected to characterize the yield, melting point and hydrogen mass spectrum. The specific results are as follows:

[0153] Intermediate product of target product I1 (Synthesis of ethyl 7-hydroxy-coumarin-8-(piperidin-1-ylmethyl)-3-carboxylate (7a)):

[0154] Yield: 58.1%;

[0155] Melting point mp120.4-121.3℃;

[0156] Spectral data analysis:

[0157] 1 HNMR(400MHz,DMSO-d6)δ8.51(s,1H,ArH),7.54(d,J=8.7Hz,1H,ArH),6.54(d,J=8.7Hz,1H,ArH),4.25-4.19(m,2H,-OCH2-),4.03(s,2H,-NCH2-),2 .80(t,J=5.5Hz,4H,Piperidine),1.65-1.58(m,4H,Piperidine),1.52-1.44(m,2H,Piperidine),1.28(t,J=7.1Hz,3H,-CH2CH3).HRMS(m / z):calcd for C 18 H 22 NO5[M+H] + :332.1498,found.332.1480.

[0158] Target product I1 (4-(2-((7-hydroxy-8-(piperidin-1-ylmethyl)-2-oxo-2H-benzopyran-3-carbonyl)oxy)ethoxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide):

[0159] Yield: 32.1%;

[0160] Melting point mp89.2-90.1℃;

[0161] Spectral data analysis: 1 H NMR (600MHz, DMSO-d6) δ8.48(s,1H,ArH),7.99(d,J=7.9Hz,2H,ArH),7.77(t,J=7.5Hz,1H,ArH ),7.63(t,J=7.6Hz,2H,ArH),7.47(d,J=8.8Hz,1H,ArH),6.52(d,J=8.7Hz,1H,ArH),4.74(t,J= 4.4Hz,2H,-OCH2-),4.55(t,J=4.4Hz,2H,-OCH2-),4.08(s,2H,-NCH2-),2.87(t,J=5.5Hz,4H,P iperidine),1.66-1.61(m,4H,Piperidine),1.52-1.46(m,2H,Piperidine).HRMS(m / z):calcd for C 26 H 25 N3O 10 SNa[M+Na] + :594.1158,found.594.1153.

[0162] Intermediate product of target product I8 (Synthesis of ethyl 7-hydroxy-coumarin-8-(methylpiperazine-1-ylmethyl)-3-carboxylate (7b)):

[0163] Yield: 45.7%;

[0164] mp129.8-130.7℃;

[0165] Spectral data analysis: 1HNMR(400MHz,DMSO-d6)δ8.59(s,1H,ArH),7.62(d,J=8.7Hz,1H,ArH),6.70(d,J=8.7Hz,1H,ArH),4.27-4.21(m,2H,-OCH2-),3.92(s,2H,-NCH2-),2.6 9-2.59(m,4H,N-Methylpiperazine),2.46-2.26(m,4H,N-Methylpiperazine),2.18(s,3H,-NCH3),1.29(t,J=7.1Hz,3H,-CH2CH3).HRMS(m / z):calcd for C 18 H 23 N2O5[M+H] + :347.1607,found.347.1580.

[0166] Target product I8 (4-(2-((7-hydroxy-8-(methylpiperazin-1-ylmethyl)-2-oxo-2H-benzopyran-3-carbonyl)oxy)ethoxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide):

[0167] Yield: 32.6%;

[0168] mp57.6-59.2℃;

[0169] Spectral data analysis: 1 H NMR (400MHz, DMSO-d6) δ8.60 (s, 1H, ArH), 7.98 (d, J = 8.4Hz, 2H, ArH), 7.77 (t, J = 7 .5Hz,1H,ArH),7.64(t,J=7.8Hz,2H,ArH),7.53(d,J=8.7Hz,1H,ArH),6.71(d,J= 8.7Hz,1H,ArH),4.75(t,J=7.1Hz,2H,-OCH2-),4.60(t,J=7.1Hz,2H,-OCH2-),4. 14(s,2H,-NCH2-),2.72-2.34(m,8H,N-Methylpiperazine),2.19(s,3H,-NCH3). 13C NMR(151MHz,DMSO-d6)δ162.79,158.90,156.25,155.39,150.17,137.20,135.98,131.02,129.88,128.3 2,115.25,110.56,109.08,108.56,107.09,69.27,61.98,53.88,51.53,51.35,45.23.HRMS(m / z):calcd for C 26 H 27 N4O 10 S[M+H] + :587.1448,found.587.1440.

[0170] Intermediate product of target product I18 (Synthesis of ethyl 7-hydroxy-coumarin-8-(piperidin-1-ylmethyl)-3-carboxylate (7c)):

[0171] Yield: 50.8%;

[0172] mp122.3-123.1℃;

[0173] Spectral data analysis: 1 HNMR(400MHz,DMSO-d6)δ8.60(s,1H,ArH),7.64(d,J=8.7Hz,1H,ArH),6.71(d, J=8.6Hz,1H,ArH),4.30-4.18(m,2H,-OCH2-),3.95(s,2H,-NCH2-),2.73-2.58 (m,4H,N-Ethylpiperazine),2.46-2.32(m,4H,N-Ethylpiperazine,-NCH2-),1.28(t,J=7.1,3H,-CH2CH3),0.98(t,J=7.2Hz,3H,-CH2CH3).HRMS(m / z):calcd for C 19 H 25 N2O5[M+H] + :361.1764,found.361.1746.

[0174] Target product I18 (4-(2-((7-hydroxy-8-(ethylpiperazin-1-ylmethyl)-2-oxo-2H-benzopyran-3-carbonyl)oxy)pentyloxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide):

[0175] Yield: 28.3%;

[0176] mp94.5-96.2;

[0177] Spectral data analysis: 1 H NMR (400MHz, DMSO-d6) δ8.55(s,1H,ArH),7.99(d,J=7.9Hz,2H,ArH),7.78(t,J=7.5Hz,1H,ArH),7.64( t,J=7.8Hz,2H,ArH),7.57(d,J=8.8Hz,1H,ArH),6.65(d,J=8.7Hz,1H,ArH),4.74(t,J=6.4Hz,2H,-OCH2 -),4.56(t,J=6.4Hz,2H,-OCH2-),4.15(s,2H,-NCH2-),3.05-2.77(m,10H,N-Ethylpiperazine),1.71- 1.61(m,4H,-CH2CH2-),1.56-1.46(m,2H,-CH2CH2-),1.24(t,J=7.5Hz,3H,-CH2CH3).HRMS(m / z):calcd forC 30 H 35 N4O 10 S[M+H] + :643.2074,found.643.2062.

[0178] Intermediate product of target product I22 (Synthesis of ethyl 7-hydroxy-coumarin-8-(hydroxyethylpiperazine-1-ylmethyl)-3-carboxylate (7d)):

[0179] Yield: 52.7%;

[0180] mp122.3-123.1℃;

[0181] Spectral data analysis: 1 HNMR(400MHz,DMSO-d6)δ8.54(s,1H,ArH),7.57(d,J=8.7Hz,1H,ArH),6.65(d,J=8.7Hz,1H,ArH),4 .29-4.15(m,2H,-OCH2-),3.87(s,2H,-NCH2-),3.47(t,J=6.2Hz,2H,-CH2OH),2.69-2.55(m,4H,N- Hydroxyethylpiperazine ),2.47-2.32(m,6H,N- Hydroxyethylpiperazine, -NCH2-),1.28(t,J=7.0Hz,3H,-CH2CH3).HRMS(m / z):calcd forC 19 H 26N2O6[M+H] + :377.1713,found.377.1687.

[0182] Target product I22 (4-(2-((7-hydroxy-8-(hydroxyethylpiperazin-1-ylmethyl)-2-oxo-2H-benzopyran-3-carbonyl)oxy)but-2-yn-1-yl)oxy)-3-(benzenesulfonyl)-1,2,5-oxadiazole 2-oxide):

[0183] Yield: 32.6%;

[0184] mp95.7-97.1℃;

[0185] Spectral data analysis: 1 H NMR(600MHz,Chloroform-d)δ8.54(s,1H,ArH),8.08(d,J=7.9Hz,2H,ArH),7.77(t,J=7 .5Hz,1H,ArH),7.65(t,J=7.6Hz,2H,ArH),7.43(d,J=8.6Hz,1H,ArH),6.79(d,J=8.6Hz ,1H,ArH),5.12(s,2H,-OCH2-),4.98(s,2H,-OCH2-),4.09(s,2H,-NCH2-),3.65(t,J=5 .4Hz,2H,-CH2OH),3.05-2.34(m,10H,N-Hydroxyethylpiperazine).HRMS(m / z):calcd for C 29 H 29 N4O 11 S[M+H] + :641.1554,found.641.1548.

[0186] 2.2 In vitro anticancer activity test:

[0187] The MTT method was used to evaluate the antiproliferative activity of the compound of the present invention on four human cancer cell lines. The MTT method has been widely used in large-scale anti-tumor drug screening, cytotoxicity tests, and tumor radiosensitivity assays.

[0188] Cell lines: human liver cancer cell line HepG-2, human colon cancer cell line HCT-29, human breast cancer cell line MCF-7, human lung cancer cell line A549 and human normal liver cell line LO2.

[0189] Experimental method: Compounds (I1-I21) and positive control drug 5-FU were dissolved in DMSO and diluted to the required concentration with PBS. A bottle of cells in the exponential growth phase and in good growth condition was taken, and 0.25% trypsin was added to digest the cells to make them fall off. The mixture was made into a solution containing 2×10 4 -4×10 4 Take the cell suspension and inoculate it on a 96-well plate, 180 μL per well, and culture it in a constant temperature CO2 incubator for 24 hours. Change the liquid, add the test solution, 20 μL per well, and culture it for 48 hours. Add tetramethyl blue nitrate to the 96-well plate, 20 μL per well, and react in the incubator for 4 hours. Aspirate the supernatant, add DMSO, 150 μL per well, and shake on a plate shaker for 5 minutes. Use an enzyme-linked immunosorbent assay to measure the absorbance of each well at a wavelength of 490 nm, and calculate the cell inhibition rate according to formula (1).

[0190]

[0191]

[0192] IC was calculated using SPSS (Staffstical Package for the Social Science) 17.0 using the probability unit weighted regression method (Bliss method). 50 Some experimental results are shown in Table 1.

[0193] Table 1 Inhibitory activity of the compounds of the present invention on tumor cell proliferation (IC 50 , μM)

[0194]

[0195]

[0196] a Data represent the mean ± SD of data from three independent experiments.

[0197] The data in Table 1 show that I1-I21 in the examples of the present invention can inhibit the proliferation of tumor cells to varying degrees, and most of the compounds have a strong effect of inhibiting the proliferation of tumor cells.

[0198] Effect test on normal hepatocyte cell viability:

[0199] The target product I7 was incubated for 24 h at six concentrations (2 μM, 5 μM, 10 μM, 20 μM, 40 μM and 50 μM) and the cell viability was tested by MTT assay. The results of three separate experiments are the mean ± standard deviation. Figure 2 shown. Figure 2**p<0.01, ****p<0.0001, from Figure 2 It can be seen that the target product I7 has little cytotoxicity to normal liver cells LO2.

[0200] 2.3 Determination of water solubility of target compounds

[0201] Some target products with good proliferation inhibition activity against four types of tumor cells were selected for water solubility testing. The experimental results are shown in Table 2

[0202] Table 2 Solubility of target product in water

[0203]

[0204] a represents the mean ± standard deviation of the data from three independent experiments, Compd.6 is compound 6, unmodified 7-hydroxycoumarin-3-carboxylic acid ethyl ester.

[0205] The data in Table 2 show that the water solubility of the measured target products is higher than that of the lead compound 7-hydroxycoumarin-3-carboxylic acid ethyl ester (Compod.6, 0.03 mg / mL), ranging from 0.06 to 1.51 mg / mL, which is 2 to 50 times the water solubility of the lead compound. The water solubility of the target product with the introduction of methylpiperazine / ethylpiperazine group at the C-8 position is stronger than that of the target product with the introduction of piperidine group. When the link arm of the target product is 1,4-butynediol, the water solubility of the target product is better than that of the target compound with the link arm being alkyl. The water solubility of the target product I21 is the strongest at 1.51 mg / mL.

[0206] 2.4 Prediction of ester-water distribution coefficient of target product

[0207] The dissolution, absorption, distribution and transport of drugs in the body are related to the lipid-water partition coefficient of the drug. The clogP of some target compounds with good activity was calculated to predict their absorption in the body. The results are shown in Table 3:

[0208] Table 3 Lipid-water partition coefficients of compounds

[0209]

[0210] In Table 3, Compd.6 is compound 6, unmodified 7-hydroxycoumarin-3-carboxylic acid ethyl ester.

[0211] It can be seen from Table 3 that the cLogP values ​​of the measured target products are all between 0 and 5 (the data are not included in the table), among which the cLogP values ​​of the target products I5, I11, I12, I14, I19 and I21 are between 0 and 3, indicating that the target products have good absorption performance and drugability.

[0212] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. NO donor coumarin Mannich base compound, characterized in that: The chemical structural formula of the NO donor coumarin Mannich base compound is shown in Formula I: ， In Formula I: X represents -O- or -NH-; R stands for -(CH2) n -, where n = 1 to 6, -CH(CH3)CH2-, -CH(CH3)(CH2)2-, -(CH2)2O(CH2)2-, -Ph(CH2)2-, -(CH2)2NH(CH2)2-, -CH2CH=CHCH2- or -CH2C≡CCH2-; R1 stands for NR 2 R 3 ; R 2 and R 3 Together with the nitrogen atom to which it is connected, it forms a five- to seven-membered aliphatic heterocyclic ring, which can be arbitrarily substituted by the following substituents: C1-C6 alkyl, hydroxy-(C1-C6) alkyl.

2. The method for preparing the NO donor coumarin Mannich base compound according to claim 1, characterized in that: include: Using resorcinol as a raw material, 2,4-dihydroxybenzaldehyde is prepared through Vilsmeier-Haack reaction under the action of an acylating agent and a first catalyst; 2,4-dihydroxybenzaldehyde and diethyl malonate were reacted by Knoevenagel reaction to prepare 7-hydroxy-coumarin-3-carboxylic acid ethyl ester; 7-hydroxy-coumarin-3-carboxylic acid ethyl ester, paraformaldehyde and organic amine are subjected to Mannich reaction to introduce different aminomethyl groups at the 8-position of 7-hydroxy-coumarin-3-carboxylic acid ethyl ester to obtain the intermediate 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid ethyl ester; The intermediate 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid ethyl ester is hydrolyzed to obtain 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid; 7-Hydroxy-8-aminomethyl-coumarin-3-carboxylic acid reacts with aminofurazan / hydroxyfurazan in the presence of an ester condensation reagent to obtain NO-donating coumarin Mannich base compounds; The synthetic route is as follows: Wherein, the structural formula of the aminofurazan / hydroxyfurazan is as follows: ； X represents -O- or -NH-; R stands for -(CH2) n -, where n = 1 to 6, -CH(CH3)CH2-, -CH(CH3)(CH2)2-, -(CH2)2O(CH2)2-, -Ph(CH2)2-, -(CH2)2NH(CH2)2-, -CH2CH=CHCH2- or -CH2C≡CCH2-; R1 stands for NR 2 R 3 ; R 2 and R 3 Together with the nitrogen atom to which it is connected, it forms a five- to seven-membered aliphatic heterocyclic ring, which can be arbitrarily substituted by the following substituents: C1-C6 alkyl, hydroxy-(C1-C6) alkyl.

3. The method for preparing the NO donor coumarin Mannich base compound according to claim 2, characterized in that: In the preparation process of the 2,4-dihydroxybenzaldehyde, the molar ratio of resorcinol to the acylating agent is 1:1.1-1.5; the acylating agent is one of monosubstituted formamide and disubstituted formamide, and the first catalyst is one of phosphorus oxychloride, thionyl chloride, zinc chloride, and phosgene.

4. The method for preparing the NO donor coumarin Mannich base compound according to claim 2, characterized in that: During the preparation of the 7-hydroxy-coumarin-3-carboxylic acid ethyl ester, a second catalyst is added, wherein the second catalyst is piperidine and glacial acetic acid; the molar ratio of the 2,4-dihydroxybenzaldehyde to diethyl malonate is 1:1-1.

5.

5. The method for preparing the NO donor coumarin Mannich base compound according to claim 2, characterized in that: In the preparation process of the intermediate 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid ethyl ester, the molar ratio of 7-hydroxy-coumarin-3-carboxylic acid ethyl ester, paraformaldehyde and organic amine is 1:5-8:3-4.

6. The method for preparing the NO donor coumarin Mannich base compound according to claim 2, characterized in that: The hydrolysis condition of the intermediate 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid ethyl ester is alkaline hydrolysis.

7. The method for preparing the NO donor coumarin Mannich base compound according to any one of claims 2 to 6, characterized in that: In the process of the reaction of the 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid with aminofurazan / hydroxyfurazan under the action of an ester condensation reagent, the molar ratio of the 7-hydroxy-8-aminomethyl-coumarin-3-carboxylic acid to aminofurazan / hydroxyfurazan is 1:1-1.

5.

8. A pharmaceutical composition, characterized in that The invention comprises the NO donor coumarin Mannich base compound or a pharmaceutically acceptable salt thereof as claimed in claim 1.

9. Use of the pharmaceutical composition according to claim 8 in the preparation of a drug for treating tumors, wherein the tumors include liver cancer, breast cancer, cervical cancer, lung cancer and colon cancer.

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