CN113493449A - NO donor coumarin furazan conjugate and pharmaceutical application thereof - Google Patents

Abstract

The invention belongs to the technical field of chemical pharmacy, relates to a coumarin furazan conjugate as an NO donor, and particularly relates to a furazan conjugate in which coumarin and open-loop derivatives thereof containing different substituents at the 3-position are combined with furazan nitric oxide through two-carbon linkage. Preliminary pharmacological experiments show that the compound can release high-concentration nitric oxide, does not influence the cell cycle, and has the characteristics of dependence on P-gp overexpression and the effect of inhibiting MDR tumor growth. The compound of the invention can be used for preparing a medicinal preparation for inhibiting sensitive or drug-resistant tumor cells.

Description

NO donor coumarin furazan conjugate and pharmaceutical application thereof

Technical Field

The invention belongs to the technical field of chemical pharmacy, relates to a coumarin furazan conjugate as an NO donor and pharmaceutical application thereof, and particularly relates to coumarin containing different substituents at the 3-position and a furazan conjugate as a ring-opening derivative thereof and pharmaceutical application thereof.

Background

The prior art discloses that Nitric Oxide (NO) is an important signaling molecule that can be involved in the regulation of various physiological functions and also can affect the growth, proliferation and metastasis processes of tumor cells. Research shows that low-concentration NO can promote angiogenesis of tumor to promote proliferation of tumor cells, and high-concentration NO can inhibit growth of tumor cells by means of DNA damage, cell apoptosis induction and the like.

In recent years, furazan derivatives have been studied more and more in the anti-tumor field, and furazan NO donors have become one of the enthusiasms for research. The literature reports a series of coupling compounds of furazan nitrogen oxides and antitumor drugs, such as: the toxicity of furazan-Tamibarotene conjugate (Bioorganic & Medicinal Chemistry letters.2011,21(23): 7025) -7029) and furazan-Thalidomide conjugate (Chemistry & Biodiversity,2009,6(4): 466-474) on tumor cells is increased, and simultaneously, the safety is improved. The Ling group designs that a synthetic farnesyl thiosalicylic acid (FTS, Ras antagonist) -furazan conjugate can inhibit the proliferation of triple negative breast cancer cells (MDA-MB-231) (Biorg Med Chem,2010,18(10):3448), and the compound also shows a relatively obvious effect of inhibiting the proliferation of liver cancer cells (J Med Chem,2011,54(9): 3251-9). And the Duan topic group designed and synthesized SAHA-furazan conjugate (journal of medicinal Chemistry,2015,58(10): 4325-4338) with broad-spectrum antitumor activity.

Based on the current situation of the prior art, the inventor of the application intends to provide a novel anti-tumor compound which has definite activity, improved water solubility and high selective inhibition effect on MDR tumor-resistant strains with over-expression of P-gp, and particularly relates to a NO donor coumarin furazan conjugate and pharmaceutical application thereof.

Disclosure of Invention

The invention aims to provide a novel antitumor compound which has definite activity, improved water solubility and high selective inhibition effect on MDR tumor drug-resistant strains with over-expression of P-gp based on the current situation of the prior art, relates to an NO donor coumarin furazan conjugate and pharmaceutical application thereof, and particularly relates to a compound 4- (2- ((3- (4-trifluoromethylbenzyl) -4-methyl-2-oxo-2H-chromene-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole 2-oxide (CY-16S-4A93, 7) and derivatives thereof and application thereof in preparing drugs with tumor growth inhibition activity.

In the invention, a lead compound CY-14S-4A83 is re-optimized to prepare 23 new 3-substituted coumarins and ring-opening derivative furazan conjugates, and the water solubility of the conjugates is increased by 70-820 times compared with that of CY-14S-4A 83; pharmacological experiments show that most of target compounds in the compound have obvious inhibition activity on A2780, A2780/CDDP, MCF-7/ADR and MDA-MB-231 tumor cell proliferation, and IC₅₀Values less than 100 nM; the antitumor activity of the drug-resistant strain MCF-7/ADR over-expressed to P-gp is obviously higher than that of the sensitive strain MCF-7; the release amount of nitric oxide of the compound in A2780 cells is equivalent to that of CY-14S-4A83, and the release of NO is closely related to the anti-tumor proliferation activity; can inhibit the formation of tumor cell colony and has no obvious influence on the cell cycle; the pharmacological mechanism of the high-selectivity inhibition effect on the drug-resistant strain MCF-7/ADR of P-gp over-expression is similar to that of CY-14S-4A 83.

Specifically, the invention designs and synthesizes a furazan conjugate which has obvious inhibition activity on tumor cell proliferation of A2780, A2780/CDDP, MCF-7/ADR and MDA-MB-231, contains different substituents at the 3-position of coumarin and replaces alpha, beta-unsaturated alicyclic ring of the coumarin with a hydrazide or hydrazone structure, as shown in figure 1, on the basis of an active compound CY-14S-4A 83.

In the invention, coumarin parent nucleus furazan derivatives with structures of I, II and III are preferably selected, wherein the coumarin furazan derivatives are products in type I formed by splicing 3-position of a coumarin parent nucleus with benzyl containing para-substituent, and 7-position with furazan nitrogen oxide through two carbon side chains; the 3-position of the coumarin parent nucleus is directly connected with chain hydrocarbon, naphthene base and heterocycle, and is linked and substituted by amide or piperazine ring to form a product in type II through two carbon side chains and furazan nitrogen oxide; and replacing the alpha, beta-unsaturated ester structure in the coumarin ring with hydrazide or hydrazone to form a ring-opening structural link to replace an aromatic ring, and then combining the ring-opening structural link with furazan oxynitride to form a product in type III (shown in figure 1),

wherein: r₁Is cyano, trifluoromethyl, nitro, methoxy,

R₂is ethyl, isopropyl, cyclopentyl methyl, cyclohexyl methyl, phenethyl, morpholine formyl ethyl,

R₃is a fluorine atom or a cyano group,

R₄is hydrazide-linked substituted aryl or hydrazone-linked substituted aryl,

R₅is a fluorine atom or a methoxy group,

the invention provides a preparation method of a furazan derivative of a coumarin parent nucleus, which comprises the following steps:

dissolving 1a and 60% sodium hydride in dry tetrahydrofuran, and reacting with chloromethyl methyl ether (MOMCl) to obtain a colorless oily product 1 b; reacting with methyl iodide in DMF under the catalysis of anhydrous potassium carbonate to obtain a compound 1 c; then 1N HCl is used for deprotection to obtain an intermediate 2 a; 1-2a, sodium iodide and anhydrous potassium carbonate are dissolved in DMF and are subjected to etherification reaction with chloroethanol to obtain products 1d and 2 b; further carrying out ammonolysis reaction with hydrazine hydrate to obtain hydrazide products 1e and 2 c; then reacts with 3a-b in ethanol under the catalytic amount of acetic acid to obtain compounds 1f, 2d and 3 c; finally, condensing the target compound with benzenesulfonyl furazan nitrogen oxide in anhydrous dichloromethane by using 1, 8-diazabicycloundec-7-ene (DBU) to obtain a hydrazide linked target compound 1-3; wherein the target compound 4 linked with hydrazide is obtained by reacting an intermediate 2c with 4-pyridylaldehyde (4a) under the catalysis of acetic acid to obtain an intermediate compound 4b and reacting the intermediate compound with furazan nitrogen oxide under the catalysis of DBU; the chemical reaction is carried out according to the following general formula I:

refluxing and etherifying 4-hydroxy-3-methoxy-acetophenone (5a), chloroethanol, potassium carbonate and sodium iodide in DMF to obtain 5 b; further hydrazine reacts to obtain a hydrazone product 5 c; condensing with 4-fluorobenzaldehyde in ethanol under the catalysis of acetic acid to obtain an intermediate 5 d; and finally, carrying out nucleophilic substitution with furazan nitrogen oxide under the catalysis of DBU to obtain a hydrazone-linked target compound 5, wherein the chemical reaction is as follows:

the preparation method comprises the following steps of (1) reacting commercially available p-cyanobenzyl bromide, p-trifluoromethyl benzyl bromide and p-nitro benzyl bromide (6-8a) with ethyl acetoacetate in NaH and Tetrahydrofuran (THF) to obtain an intermediate 6-8 b; further carrying out condensation reaction with resorcinol under 70% sulfuric acid to obtain 3-substituted coumarin parent nucleus products 6-8 c; wherein the 7-hydroxy group of 8c is protected with MOMCl to give 9a with SnCl₂Reducing nitro to form amino product 9b, further reacting with self-made morpholine sodium acetate to form amide 9c, then acidifying to remove MOM protecting group to obtain 9d intermediate, then carrying out reflux reaction on 6-8c, chloroethanol, potassium carbonate and sodium iodide in DMF to obtain 7-hydroxyethoxy 3-substituted coumarin intermediates 6-8d and 9e, and finally reacting with benzenesulfonyl furazan oxynitride in dichloromethane containing DBU at room temperature to synthesize 3-substituted coumarin skeleton target derivative 6-9; the chemical reaction is shown as the following general formula III:

adding 60% sodium hydride into dry THF, dropwise adding ethyl acetoacetate and 10-14a under ice bath, and carrying out nucleophilic substitution reaction to obtain ethyl acetoacetate derivative 10-14 b; further carrying out cyclization reaction with resorcinol under 70% sulfuric acid to obtain a 3-substituted coumarin parent nucleus product 10-14 c; then carrying out reflux reaction on the derivative, chloroethanol, potassium carbonate and sodium iodide in DMF to obtain a 7-hydroxyethoxy-coumarin derivative 10-14 d; finally reacting with benzenesulfonyl furazan nitrogen oxide in dichloromethane containing DBU at room temperature to synthesize a target derivative 10-14; the chemical reaction is shown as the following general formula IV:

synthesizing a coumarin derivative 15b from diethyl malonate and 2, 4-dihydroxybenzaldehyde (15a) in pyridine; diethyl acetylsuccinate (16a), diethyl acetylglutarate (20a) and resorcinol are cyclized to synthesize compounds 16b and 20b respectively under the catalysis of 75% sulfuric acid, and the compounds are hydrolyzed by 2N NaOH and then acidified by 2N HCl to obtain 15-16c and 20 c; further carrying out nucleophilic substitution reaction with 4-fluorobenzylamine, para-fluoroaniline, morpholine and a piperazine derivative 17-19b prepared from substituted bromobenzyl 17-19a to obtain amide products 15-16d,17-19c,20d and 21-23a, and then carrying out reflux reaction with chloroethanol, potassium carbonate and sodium iodide in DMF to obtain 15-16e,17-19d,20e and 21-23 b; finally, synthesizing the target coumarin parent nucleus derivative 15-23 with benzenesulfonyl furazan oxynitride in DBU-containing dichloromethane; the chemical reaction is shown as the following formula five:

the invention relates to a method for detecting the solubility of a furazan derivative (1-23) by replacing an open-loop derivative with an alpha, beta-unsaturated ester structure by a coumarin parent nucleus containing different substitutions at the 3-position and hydrazide or hydrazone thereof, and performing solubility detection and anti-tumor activity screening at a cellular level by the furazan derivative (1-23) coupled with a furazan nitrogen oxide through two carbon links; the experimental results of the solubility of the target compound in PBS (pH 7.4) show that the water solubility of this class of compounds is increased 70-820 times compared to the original active compound CY-14S-4a 83.

The invention carries out in vitro pharmacological experiments, tests the cytotoxicity of the prepared 23 target compounds on sensitive tumor cells A2780 (human ovarian cancer cells), KB (human oral epidermoid carcinoma cells), MCF-7 (human breast cancer cells), MDA-MB-231 (human triple negative breast cancer cells), drug-resistant tumor cells A2780/CDDP (cisplatin-resistant human ovarian cancer cells), KB-V (vincristine-resistant human oral epidermoid carcinoma cells), MCF-7/ADR (doxorubicin-resistant human breast cancer cells), normal cells HUVEC (human umbilical vein endothelial cells), T29 (immortalized human ovarian epithelial cells), WI-38 (human normal lung fibroblasts) and MCF-10A (human normal mammary epithelial cells) by taking CY-14S-4A83 and adriamycin, cisplatin, vincristine and gemcitabine as positive drug controls, the experimental result shows that most of the target compounds have obvious inhibition activity on A2780, A2780/CDDP, MCF-7/ADR and MDA-MB-231 tumor cell proliferation, and IC₅₀Small valueBetween 100 nM; the inhibitory activity to KB, KB-V and MCF-7 tumor cells is weaker; wherein the antitumor activity of the drug-resistant strain MCF-7/ADR over-expressed to P-gp is obviously higher than that of the sensitive strain MCF-7; particularly, the MCF-7/ADR inhibition activity of the compound 7 and the compound 8 is 499 and 276 times that of the sensitive cell strains, while the target compound has lower toxicity and increased safety to normal cells T29, HUVEC, WI-38 and MCF-10A;

the experimental result also shows that the 23 target compounds can effectively release NO in A2780 tumor cells, and the anti-tumor activity and the NO scavenger present a concentration-dependent negative correlation result, which shows that the anti-tumor activity of the compounds is related to the concentration of the released effective NO;

pharmacological experiments of the invention also show that the target compounds 6 and 7 can effectively inhibit the colony formation of A2780 and MCF-7/ADR cells; hardly affecting the cell cycle of A2780.

In the present invention, the solubility and biological activity of the compound were measured as follows:

1. preparing a standard curve, determining the relation between the area under the curve and the solubility, detecting the area under the curve of each compound by using HPLC (high performance liquid chromatography) and calculating the solubility;

2. using MTS method sensitive and drug-resistant solid tumor cell cytotoxicity determination, setting blank control group, target compound 1-23, compound CY-14S-4A83, and positive drug (adriamycin, cisplatin, vincristine and gemcitabine) control group, adding drug, culturing at 37 deg.C for 48 hr, performing MTS detection and calculating inhibition rate and IC₅₀A value; test tumor cell lines: a2780 and A2780/CDDP (human ovarian cancer cells, cis-platin resistant), KB and KB-V (human oral epidermoid cancer cells, vincristine resistant), MCF-7 and MCF-7/ADR (human breast cancer cells, adriamycin resistant) and MDA-MB-231 (human triple negative breast cancer cells);

3. MTS assay for IC of target Compounds on four non-neoplastic lines of HUVEC (human umbilical vein endothelial cells), T29 (immortalized human ovarian epithelial cells), WI-38 (human normal lung fibroblasts) and MCF-10A (human normal mammary epithelial cells)₅₀Verifying the toxicity of the target compound on normal cells, and further evaluating the safety of the target compound;

4. detecting the nitric oxide release level of a target compound in a tumor cell A2780 by using a total NO detection kit (Beyotime), wherein the detection principle is a classic Griess reagent principle;

5. compound CY-14S-4A83 was used as a control, and compounds 6 and 7 were tested for colony formation inhibition on two tumor cells, A2780 and MCF-7/ADR;

6. cell cycle experiments were performed using the cell cycle and apoptosis detection kit (Beyotime, C1052) and BD facscalibur flow cytometer, PI staining method.

The invention provides an NO donor coumarin furazan conjugate with a new structure, in particular to a 23 coumarin parent nucleus and an open-loop derivative furazan conjugate thereof, the compound has good solubility and has the function of inhibiting the proliferation activity of P-gp over-expression drug-resistant tumor cells with high selectivity, and the invention provides a new thought and direction for overcoming the multidrug resistance of the tumor cells caused by P-gp over-expression.

Drawings

FIG. 13-design of substituted coumarins and their ring-opened derivatives furazan conjugates.

Figure 2 effect of NO scavengers on antitumor activity.

FIG. 3 Effect of Compounds 6 and 7 on tumor cell colony formation.

Detailed Description

The following embodiments will help to understand the present invention, but are not limited to the contents of the present invention.

EXAMPLE 1 Synthesis of Compound 2-hydroxy-4-methoxymethyloxybenzoic acid methyl ester (1b)

According to the general formula one, 1a (10g, 60mmol, 1.0equiv) and 60% sodium hydride (3g, 75mmol, 1.2equiv) are added into a three-necked flask, dried tetrahydrofuran (60mL) is added, chloromethyl methyl ether (MOMCl, 5mL, 66mmol, 1.1equiv) is slowly added under ice bath, and after stirring for 30 minutes, the temperature is gradually increased to 80 ℃. The color of the reaction solution deepens, the reaction is monitored by TLC, and the reaction is completed in half an hour. Cooling to room temperature, evaporating most tetrahydrofuran, slowly pouring the reaction solution into ice water (50mL), extracting with ethyl acetate for three times, washing the organic phase with saturated saline, drying with anhydrous sodium sulfate, filtering, evaporating the filtrate to dryness to obtain colorless oily substance(1b)(96％)。ESI-MS m/z 213.2[M+H]⁺. The product was taken as starting material without purification and fed to the next step.

EXAMPLE 2 Synthesis of methyl 2-methoxy-4-methoxymethylbenzoate (1c), Compound

According to the general formula I, 1b (5.8g, 28mmol, 1.0equiv) and anhydrous potassium carbonate (15g, 109mmol, 4.0equiv) are added into DMF (40mL), the temperature is increased to 80 ℃, methyl iodide (3.5mL, 56mmol, 2.0equiv) is slowly added, after 20 minutes of reaction, the mixture is cooled to room temperature, the mixture is poured into ice water (200mL) after filtration, extraction is carried out by using ethyl acetate, an organic phase is rinsed by saturated saline water, and the mixture is dried by anhydrous sodium sulfate, filtered, and filtrate is evaporated to obtain a compound (1c) (92%); ESI-MS M/z 227.1[ M + H ]]⁺. The product was used as the starting material for the next reaction without purification.

Example 3

Synthesis of compound 2-hydroxy-4-hydroxyethoxy methyl benzoate (1d) in general formula I

1a (10mmol, 1.0equiv), sodium iodide (15mg, 0.1mmol, 0.01equiv) and anhydrous potassium carbonate (4.1g, 30mmol, 3.0equiv) were added to a three-necked flask, DMF (15mL) was added and the temperature was gradually raised to 80 ℃. After completion of the reflux reaction by adding chlorinated ethanol (1.3mL, 20mmol, 2.0equiv) in DMF (3mL), the mixture was cooled to room temperature and poured slowly into ice water (100mL), extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was evaporated to dryness to give compound (1d) (93%) as a white solid. ESI-MS M/z 213.2[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ10.98(s,1H,ArOH),7.75(d,J＝9.4Hz,1H,ArH),6.51–6.42(m,2H,ArH),4.13–4.07(m,2H,HOCH_{2 2}CHO-),3.98(m,2H,HO ₂CHCH₂-),3.92(s,3H,-OCH₃)。

Example 4

Synthesis of compound 2-hydroxy-4-hydroxyethoxy benzoyl hydrazine (1e) in general formula I

1d (11mmol, 1.0equiv) was dissolved in anhydrous ethanol (20mL), hydrazine hydrate (33mmol, 3.0equiv) was added, and the reaction was refluxed for 4 hours. The reaction solution was placed in an ice bath, and a solid precipitated, which was filtered off with suction and dried to give a white solid compound (1e) (98%)。ESI-MS m/z 213.2[M+H]⁺；¹H NMR(400MHz,CDCl₃)δ12.11(s,1H,-CONH-),7.36(d,J＝1.3Hz,1H,ArH),6.48(d,J＝1.2Hz,1H,ArH),6.44(dd,J＝10.4,1.7Hz,1H,ArH),4.10(d,J＝1.8Hz,2H,HOCH_{2 2}CHO-),3.85(d,J＝6.1Hz,2H,HO ₂CHCH₂-)。

Example 5

Synthesis of compound 2-hydroxy-4-hydroxyethoxy-N' - ((E) - (4-fluorophenyl) methylene) benzoylhydrazone (1f) in the general formula I

1e (1.3mmol, 1.0equiv) was dissolved in ethanol (15mL), 3a (1.3mmol, 1.0equiv) and a catalytic amount of acetic acid (0.01equiv) were added to the reaction solution, and after a reaction for half an hour, suction filtration was performed, the cake was washed with ethyl acetate, and dried to obtain compound (1f) (86%) as a pale yellow solid. ESI-MS M/z319.1[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ8.46(s,1H,-N＝CH-),7.89(d,J＝9.0Hz,1H,ArH),7.82(d,J＝7.9Hz,2H,ArH),7.32(t,J＝8.8Hz,2H,ArH),6.57(dd,J＝8.9,2.1Hz,1H,ArH),6.49(d,J＝2.1Hz,1H,ArH),4.92(t,J＝5.4Hz,1H,-OH),4.03(t,J＝4.8Hz,2H,HOCH_{2 2}CHO-),3.72(dd,J＝9.6,5.0Hz,2H,HO ₂CHCH₂-)。

Example 6

Synthesis of compound 4- (2- (2-hydroxy-N' - ((E) - (4-fluorophenyl) methylene) benzoylhydrazone-4-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (1) in the general formula I

1f (0.7mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (380mg, 1mmol, 1.5equiv) were added to a single-necked flask, and anhydrous dichloromethane (8mL) was added; DBU (1, 8-diazabicycloundecen-7-ene, 1.1mL, 2.1mmol, 3.0equiv) was added, the reaction was stirred at room temperature for 2 hours, and extraction was performed with dichloromethane, the organic phase was rinsed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness, followed by column chromatography to separate and purify the title compound 1 (63%) as a white solid. ESI-MS M/z 543.1[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ12.52(s,1H,-CONH-),11.84(s,1H,-OH),8.48(s,1H,-N＝CH-),7.97(dd,J＝15.8,8.2Hz,2H,ArH),7.89–7.79(m,3H,ArH),7.68(t,J＝7.9Hz,2H,ArH),7.33(t,J＝8.7Hz,2H,ArH),6.63(d,J＝2.1Hz,1H,ArH),6.57(d,J＝2.1Hz,1H,ArH),4.81–4.69(m,2H,-O ₂CHCH₂O-),4.47–4.40(m,2H,-OCH_{2 2}CHO-).¹³C NMR(151MHz,DMSO-d₆)δ165.12,163.93,162.52,162.29,162.07,158.68,147.22,137.09,136.01,130.66,129.85,129.59,129.32,129.27,128.18,115.94,115.79,110.44,107.85,106.58,102.00,69.62,65.54。

Example 7

Synthesis of compound 4- (2- (2-methoxy-N' - ((E) - (4-fluorophenyl) methylene) benzoylhydrazone-4-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (2) in the general formula I

2d (0.7mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (380mg, 1mmol, 1.5equiv) were added to a single-necked flask, and anhydrous dichloromethane (8mL) was added; DBU (1, 8-diazabicycloundecen-7-ene, 1.1mL, 2.1mmol, 3.0equiv) was added, the reaction was stirred at room temperature for 2 hours and extracted with dichloromethane, the organic phase was rinsed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was evaporated to dryness, and the resulting product was purified by column chromatography to give the objective compound 2 (65%) as a white solid. ESI-MS M/z 557.5[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ10.74(s,1H,-CONH-),8.33(d,J＝8.8Hz,1H,ArH),8.24(s,1H,-N＝CH-),8.00(dd,J＝8.4,1.0Hz,2H,ArH),7.80(dd,J＝8.7,5.5Hz,2H,ArH),7.72(t,J＝7.5Hz,1H,ArH),7.54(t,J＝7.9Hz,2H,ArH),7.10(t,J＝8.6Hz,2H,ArH),6.69(dd,J＝8.8,2.2Hz,1H,ArH),6.61(d,J＝2.1Hz,1H,ArH),4.83–4.78(m,2H,-O ₂CHCH₂O-),4.49–4.45(m,2H,-O ₂CHCH₂O-),4.06(s,3H,-OCH₃).¹³C NMR(151MHz,CDCl₃)δ164.85,163.19,162.54,161.81,158.76,158.66,146.65,137.92,135.70,134.74,130.27,129.68,129.55,129.50,128.57,115.92,115.77,114.09,110.45,106.62,99.38,69.29,65.59,56.31。

Example 8

Synthesis of compound 4- (2- (2-methoxy-N' - ((E) - (4-methoxyphenyl) methylene) benzoylhydrazone-4-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (3) in the general formula I

3c (0.7mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (380mg, 1mmol, 1.5equiv) were added to a single-necked flask, and anhydrous dichloromethane (8mL) was added; DBU (1, 8-diazabicycloundecen-7-ene, 1.1mL, 2.1mmol, 3.0equiv) was added, the reaction was stirred at room temperature for 2 hours, and extraction was performed with dichloromethane, the organic phase was rinsed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness, followed by column chromatography to isolate and purify the objective compound 3 (62%) as a white solid. ESI-MS M/z 569.0[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ10.67(s,1H,-CONH-),8.33(d,J＝8.7Hz,1H,ArH),8.17(s,1H,-N＝CH-),8.00(d,J＝7.9Hz,2H,ArH),7.73(dd,J＝18.6,8.1Hz,3H,ArH),7.54(t,J＝7.9Hz,2H,ArH),6.94(d,J＝8.7Hz,2H,ArH),6.68(dd,J＝8.7,2.1Hz,1H,ArH),6.60(d,J＝1.9Hz,1H,ArH),4.84–4.78(m,2H,-O ₂CHCH₂O-),4.49–4.44(m,2H,-O ₂CHCH₂O-),4.05(s,3H,-OCH₃),3.85(s,3H,-OCH₃).¹³C NMR(151MHz,CDCl₃)δ162.38,161.60,161.43,158.75,158.61,147.72,137.92,135.70,134.65,129.67,129.30,128.55,126.65,114.28,114.14,110.44,106.55,99.33,69.31,65.57,56.27,55.38。

Example 9

Synthesis of compound 4- (2- (2-methoxy-N' - ((E) - (4-pyridine) methylene) benzoylhydrazone-4-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (4) in the general formula I

4b (100mg, 0.3mmol, 1.0equiv) and furazan nitroxide (130mg, 0.4mmol, 1.3equiv) were added to a single neck bottle and anhydrous dichloromethane (6mL) was added; DBU (0.13mL, 2.1mmol, 3.0equiv) was added, the reaction was stirred at room temperature for 2 hours, then ice water (20mL) was added, extraction was performed with dichloromethane, the organic phase was rinsed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was evaporated to dryness, and column chromatography (DCM/MeOH ═ 9/1) was performed to isolate and purify the objective compound 4 in 75% yield as a white solid, mp 193-. ESI-MS M/z 540.2[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ10.92(s,1H,-CONH-),8.67(d,J＝5.9Hz,2H,ArH),8.33(d,J＝8.5Hz,2H,overlapped),8.00(d,J＝8.5Hz,2H,ArH),7.73(t,J＝6.9Hz,1H,ArH),7.66(d,J＝5.9Hz,2H,ArH),7.55(t,J＝7.9Hz,2H,ArH),6.72(d,J＝2.1Hz,1H,ArH),6.63(d,J＝2.1Hz,1H,ArH),4.83–4.79(m,2H,-O ₂CHCH₂O-),4.51–4.46(m,2H,-O ₂CHCH₂O-),4.07(s,3H,-OCH₃).¹³C NMR(151MHz,CDCl₃)δ162.85,162.12,158.75,150.31,145.03,141.53,137.90,135.71,134.88,129.67,128.58,121.33,113.73,110.46,106.78,99.44,69.26,65.63,56.38.

Example 10

Synthesis of Compound 4- (2- (2-methoxy-4-hydroxyethoxy-N' - ((E) - (4-fluorophenyl)) phenethylhydrazone-4-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (5) in formula II

5d (120mg, 0.36mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (140mg, 0.38mmol, 1.1equiv) were added to a single-necked flask, and anhydrous dichloromethane (15mL) was added; DBU (0.13mL, 1.3mmol, 3.0equiv) was added, the reaction was stirred at room temperature for 2 hours, then water (30mL) was added, extraction was performed with dichloromethane, the organic phase was rinsed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was spin-dried under reduced pressure, and column chromatography (PE/EA ═ 2/1) was performed to isolate and purify the objective compound 5 at a yield of 65% as a white solid, mp 79-81 ℃. ESI-MS M/z 555.0[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ8.37(s,1H,-N＝CH-),8.06–8.02(m,2H,ArH),7.83(dd,J＝8.6,5.6Hz,2H,ArH),7.72(t,J＝7.5Hz,1H,ArH),7.58–7.49(m,3H,ArH),7.13(t,J＝8.8Hz,2H,ArH),6.55(s,1H,ArH),4.79(t,J＝6.0Hz,2H,-O ₂CHCH₂O-),4.44–4.40(m,2H,-O ₂CHCH₂O-),3.86(s,3H,-OCH₃),2.46(s,3H,-CH₃).¹³C NMR(151MHz,CDCl₃)δ167.03,165.16,160.54,159.18,158.81,156.72,138.04,135.62,130.56,130.19,130.14,129.67,128.59,123.05,115.95,115.80,110.43,105.42,99.58,69.41,65.52,55.61,19.16.

Example 11

Synthesis of Compound 3- (4-cyano) benzyl-2-oxobutanoic acid ethyl ester (6b) in formula III

60% sodium hydride (3.79g, 94.8mmol, 1.1equiv), dried tetrahydrofuran (80mL), acetylAfter ethyl acetate (10.5mL, 79mmol, 1.0equiv), 6a (15.68g,80mmol, 1.0equiv), the ice bath was removed and the temperature was raised to 60 ℃ to continue the reaction for 3 hours, the reaction was filtered, most of the solvent was evaporated from the filtrate, water (100mL) was added, extraction was carried out three times with ethyl acetate, and the organic phase was washed three times with saturated brine and dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness to afford compound (6b) as a yellow oil (98%). The product was used as the next step without purification. ESI-MS (M/z)246.2[ M + H]⁺；

Example 12

Synthesis of compound 7-hydroxy-3- (4-cyanobenzyl) -4-methyl-2H-chromen-2-one (6c) in the formula III

Resorcinol (80mmol, 1.0equiv) was added to the reaction flask containing 6b, 70% sulfuric acid (30mL) was added under ice bath, the reaction was continued at room temperature for 3 hours, and then dropwise added to ice water (300mL), followed by suction filtration and drying to obtain a white solid compound (6c) (83%). ESI-MS M/z 292.3[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ10.51(s,1H,-OH),7.74(d,J＝7.9Hz,2H,ArH),7.66(d,J＝8.7Hz,1H,ArH),7.42(d,J＝7.8Hz,2H,ArH),6.82(dd,J＝8.5,1.0Hz,1H,ArH),6.72(s,1H,ArH),4.02(s,2H,-CH₂-),2.40(s,3H,-CH₃)。

Example 13

Synthesis of compound 7-hydroxy-3- (4-cyanobenzyl) -4-methyl-2H-chromen-2-one (6d) in the formula III

DMF (10mL) was added 6c (2.5mmol, 1.0equiv), sodium iodide (37.5mg, 0.25mmol, 0.1equiv) and anhydrous potassium carbonate (1.04g, 7.5mmol, 3.0equiv) and the temperature was gradually raised to 80 ℃. After completion of the reaction monitored by TLC, the reaction mixture was cooled to room temperature, slowly added to ice water (80mL), and then filtered with suction, to obtain a crude product, which was recrystallized from methanol to obtain a white solid compound (6d) (81%). ESI-MS M/z 336.3[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ7.56(t,J＝7.3Hz,3H,ArH),7.37(d,J＝8.0Hz,2H,ArH),6.91(d,J＝8.9Hz,1H,ArH),6.84(d,J＝1.6Hz,1H,ArH),4.18–4.13(m,2H,HOCH_{2 2}CHO-),4.08(m,2H,HO ₂CHCH₂-),4.02(s,2H,-CH₂-),2.42(s,3H,-CH₃)。

Example 14

Synthesis of Compound 4- (2- (3- (4-cyanobenzyl) -4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (6) in formula III

6d (1mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (550mg, 1.5mmol, 1.5equiv) were added to a single-necked flask, and anhydrous dichloromethane (10mL) was added; DBU (0.5mL, 3mmol, 3.0equiv) was added, stirred at room temperature for 2 hours, extracted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and isolated and purified by column chromatography to give the objective compound (6) (65%) as a white solid. ESI-MS M/z 560.1[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ8.04(d,J＝7.5Hz,2H,ArH),7.74(t,J＝8.0Hz,1H,ArH),7.63–7.55(m,5H,ArH),7.39(d,J＝7.0Hz,2H,ArH),6.95(dd,J＝9.0,1.9Hz,1H,ArH),6.89–6.85(m,1H,ArH),4.82(d,J＝2.4Hz,2H,-O ₂CHCH₂O-),4.47(d,J＝1.9Hz,2H,-O ₂CHCH₂O-),4.10(s,2H,-CH₂-),2.45(s,3H,-CH₃).¹³C NMR(151MHz,CDCl₃)δ161.88,160.70,158.71,153.95,148.25,144.67,137.97,135.70,132.40,129.67,129.09,128.59,126.13,121.23,118.88,114.55,112.55,110.40,110.33,101.72,69.09,65.74,33.13,15.55。

Example 15

Synthesis of Compound 4- (2- (3- (4-trifluoromethylbenzyl) -4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (phenylsulfonyl) -1,2, 5-oxadiazole-2-oxide (7) in formula III

7d (1mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (550mg, 1.5mmol, 1.5equiv) were added to a single-necked flask, and anhydrous dichloromethane (10mL) was added; DBU (0.5mL, 3mmol, 3.0equiv) was added, stirred at room temperature for 2 hours, extracted with dichloromethane, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and subjected to column chromatography to isolate and purify the objective compound (7) (62%). ESI-MS M/z 603.1[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ7.98(d,J＝8.0Hz,2H,ArH),7.83(dd,J＝14.5,7.8Hz,2H,ArH),7.66(dd,J＝14.4,7.6Hz,4H,ArH),7.46(d,J＝7.8Hz,2H,ArH),7.11(s,1H,ArH),7.04(dd,J＝8.9,1.0Hz,1H,ArH),4.77(d,J＝2.8Hz,2H,-O ₂CHCH₂O-),4.50(d,J＝1.7Hz,2H,-O ₂CHCH₂O-),4.06(s,2H,-CH₂-),2.47(s,3H,-CH₃).¹³C NMR(151MHz,DMSO)δ161.05,160.45,158.67,153.32,148.88,144.21,137.06,136.00,129.84,128.76,128.17,126.80,125.16,120.38,113.79,112.56,110.44,101.17,69.61,66.00,32.00,15.18。

Example 16

Synthesis of Compound 4- (2- (3- (4-nitrobenzyl) -4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (8) in formula III

Add 8d (1mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (550mg, 1.5mmol, 1.5equiv) to a single neck bottle and add anhydrous dichloromethane (10 mL); DBU (0.5mL, 3mmol, 3.0equiv) was added, and the mixture was stirred at room temperature for 2 hours and extracted with dichloromethane, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and subjected to column chromatography to isolate and purify the objective compound (8) (64%). ESI-MS M/z 580.1[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ8.15(d,J＝8.7Hz,2H,ArH),7.98(d,J＝7.5Hz,2H,ArH),7.87–7.78(m,2H,ArH),7.67(t,J＝7.9Hz,2H,ArH),7.52(d,J＝8.6Hz,2H,ArH),7.11(d,J＝2.4Hz,1H,ArH),7.05(dd,J＝8.9,2.4Hz,1H,ArH),4.77(d,J＝2.1Hz,2H,-O ₂CHCH₂O-),4.50(d,J＝4.4Hz,2H,-O ₂CHCH₂O-),4.11(s,2H,-CH₂-),2.47(s,3H,-CH₃).¹³C NMR(151MHz,CDCl₃)δ161.86,160.75,158.71,153.98,148.34,146.81,146.68,137.97,135.71,129.68,129.12,128.59,126.15,123.85,121.15,114.53,112.59,110.41,101.74,69.08,65.75,32.95,15.58。

Example 17

Synthesis of Compound 4- (2- (3- (4- (4-morpholinoacetamido) benzyl) -4-methyl-2-oxo-2H-chromen-7-yloxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (9) in formula III

9e (60mg, 0.14mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (60mg,0.16mmol, 1.1equiv) was added to a single-necked flask and anhydrous dichloromethane (4mL) was added; DBU (60 μ L, 0.42mmol, 3.0equiv) was added, and extraction with dichloromethane was performed for 2 hours under stirring at room temperature, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was subjected to column chromatography (DCM/MeOH ═ 9/1) to purify the objective compound 9 in 80% yield as a white solid. ESI-MS M/z 677.1[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ9.00(s,1H,-NHCO-),8.03(d,J＝7.4Hz,2H,ArH),7.73(t,J＝7.2Hz,1H,ArH),7.56(t,J＝9.4Hz,3H,ArH),7.47(d,J＝8.2Hz,2H,ArH),7.25(d,J＝9.1Hz,2H,ArH),6.92(dd,J＝8.7,1.7Hz,1H,ArH),6.85(d,J＝1.4Hz,1H,ArH),4.80(m,2H,-O ₂CHCH₂O-),4.44(m,2H,-O ₂CHCH₂O-),4.02(m,2H,-NCH_{2 2}CHO-),3.77(m,4H,overlapped),3.13(s,2H,-CH₂-),2.62(m,4H,overlapped),2.44(s,3H,-CH₃).¹³C NMR(151MHz,CDCl₃)δ167.81,162.09,160.36,158.71,153.82,147.51,138.00,135.70,135.15,129.67,128.92,128.57,125.97,122.66,119.83,114.87,112.26,110.38,101.63,69.11,66.99,65.67,62.41,53.78,32.37,15.45。

Example 18

Synthesis of Compound 3-phenethyl-2-oxobutanoic acid ethyl ester (10b) in formula IV

60% sodium hydride (300mg, 7.5mmol, 1.0equiv) was added to dry tetrahydrofuran (15mL), ethyl acetoacetate (0.94mL, 7.4mmol, 1.0equiv) and 10a (7.4mmol, 1.0equiv) were added dropwise, the temperature was raised to 60 ℃, after completion of the TLC monitoring reaction, the solid in the reaction solution was filtered off, most of the solvent was evaporated, water (30mL) was added, ethyl acetate was extracted three times, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to give compound 10b in 88% yield as a yellow oil. ESI-MS M/z 235.2[ M + H ]]⁺. The product was used as the next step without purification.

Example 19

Synthesis of Compound 7-hydroxy-3-phenethyl-4-methyl-2H-chromen-2-one (10c) in formula IV

10b and Resorcinol (80mmol, 1.0equiv) in an ice bath70% sulfuric acid (30mL) was added, the reaction was continued at room temperature, TLC monitored the completion of the reaction, and slowly added dropwise to ice water (300mL), followed by suction filtration and drying to give compound 10c in 81% yield as a white solid, ESI-MS M/z 281.3[ M + H ] M/z 281.3]⁺；¹H NMR(400MHz,CDCl₃)δ7.44(d,J＝8.8Hz,1H,ArH),7.26(d,J＝3.1Hz,2H,ArH),7.24–7.17(m,3H,ArH),7.05(d,J＝0.6Hz,1H,ArH),6.85(dd,J＝9.3,1.2Hz,1H,ArH),2.93(t,J＝6.0Hz,2H,- ₂CHCH₂-),2.88–2.82(m,2H,-CH_{2 2}CH-),2.16(s,3H,-CH₃)。

Example 20

Synthesis of Compound 7-hydroxyethyl-3-phenethyl-4-methyl-2H-chromen-2-one (10d) in formula IV

10c (2.5mmol, 1.0equiv), sodium iodide (37.5mg, 0.25mmol, 0.1equiv) and anhydrous potassium carbonate (1.04g, 7.5mmol, 3.0equiv) DMF (10mL) was added, 80 ℃ DMF (3mL) containing chlorinated ethanol (330. mu.L, 5mmol, 2.0equiv) was added, after completion of the reflux reaction, cooling to room temperature, slowly adding ice water (80mL), suction filtration, drying to give the crude product, methanol recrystallization to give pure compound 10d, yield 82%, white solid, ESI-MS M/z 325.3[ M + H325.3 ]]⁺；¹H NMR(400MHz,CDCl₃)δ7.46(dd,J＝9.1,3.4Hz,1H,ArH),7.35–7.19(m,5H,ArH),6.87(dd,J＝4.9,3.6Hz,1H,ArH),6.84(d,J＝2.2Hz,1H,ArH),4.21–4.12(m,2H,HO ₂CHCH₂-),4.02(dd,J＝7.6,3.4Hz,2H,HOCH_{2 2}CHO-),2.95–2.92(m,2H,- ₂CHCH₂-),2.89–2.84(m,2H,-CH_{2 2}CH-),2.14(s,3H,-CH₃)。

Example 21

Synthesis of Compound 4- (2- (3-phenethyl-4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (10) in formula IV

10d (1mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (550mg, 1.5mmol, 1.5equiv) were added with anhydrous dichloromethane (10mL) and DBU (0.5mL, 3mmol, 3.0equiv), and after completion of the reaction monitored by TLC, a small amount of water was added and extracted with dichloromethane, and saturated common salt was addedWashing organic phase with water, drying with anhydrous sodium sulfate, filtering, purifying by column chromatography to obtain target compound 10 with yield of 63%, white solid ESI-MS M/z 549.0[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ7.97(d,J＝14.1Hz,2H,ArH),7.84(t,J＝7.4Hz,1H,ArH),7.74–7.62(m,3H,ArH),7.27(t,J＝7.3Hz,2H,ArH),7.20(dd,J＝12.7,7.0Hz,3H,ArH),7.08(d,J＝1.5Hz,1H,ArH),7.03–6.96(m,1H,ArH),4.80–4.72(m,2H,-O ₂CHCH₂O-),4.52–4.39(m,2H,-OCH_{2 2}CHO-),2.86–2.80(m,2H,- ₂CHCH₂-),2.80–2.70(m,2H,-CH_{2 2}CH-),2.21(s,3H,-CH₃).¹³C NMR(151MHz,DMSO-d₆)δ160.66,160.02,158.62,153.01,147.30,140.99,137.05,135.92,129.78,129.59,128.32,128.11,126.40,125.80,121.44,113.80,112.28,110.38,101.09,69.58,65.89,33.75,29.05,14.34.

Example 22

Synthesis of Compound 4- (2- (3-cyclohexyl-4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (11) in formula IV

11d (1mmol, 1.0equiv) and benzenesulfonylfurazan nitroxide (550mg, 1.5mmol, 1.5equiv) were added to dry dichloromethane (10mL) and DBU (0.5mL, 3mmol, 3.0equiv), and after completion of the TLC monitoring reaction, a small amount of water was added and extraction was performed with dichloromethane, the organic phase was rinsed with saturated brine, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography to give the title compound 11 in 66% yield as a white solid. ESI-MS M/z 541.5[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ7.96(d,J＝7.9Hz,2H,ArH),7.83(t,J＝7.5Hz,1H,ArH),7.73(d,J＝8.7Hz,1H,ArH),7.65(t,J＝7.7Hz,2H,ArH),7.04(d,J＝0.8Hz,1H,ArH),7.02–6.95(m,1H,ArH),4.85–4.66(m,2H,-O ₂CHCH₂O-),4.51–4.40(m,2H,-OCH_{2 2}CHO-),2.46(d,J＝7.2Hz,2H,- ₂CHCH＝),2.39(s,3H,-CH₃),1.61(dd,J＝15.0,7.2Hz,2H,-CH₂-),1.51(dd,J＝7.2,3.9Hz,1H,-CH₂ CH＝),1.26(d,J＝16.1Hz,2H,-CH₂-),1.16–1.08(m,2H,-CH₂-),1.06–0.93(m,2H,-CH₂-).¹³C NMR(151MHz,DMSO-d₆)δ160.95,159.92,158.61,152.94,147.14,137.04,135.92,129.78,128.10,126.39,121.39,113.93,112.22,110.38,101.02,69.59,65.88,37.29,34.06,32.54,30.99,29.68,25.81,25.66,15.12。

Example 23

Synthesis of Compound 4- (2- (3-cyclopentyl-4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (12) of formula IV

12d (1mmol, 1.0equiv) and benzenesulfonylfurazan nitroxide (550mg, 1.5mmol, 1.5equiv) were added anhydrous dichloromethane (10mL) and DBU (0.5mL, 3mmol, 3.0equiv), TLC monitored the completion of the reaction, a small amount of water was added, extraction was performed with dichloromethane, the organic phase was rinsed with saturated brine, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography to give the title compound 12 in 59% yield as a white solid. ESI-MS M/z 527.5[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ7.97(d,J＝7.6Hz,2H,ArH),7.84(t,J＝7.2Hz,1H,ArH),7.75(d,J＝9.1Hz,1H,ArH),7.67(t,J＝7.2Hz,2H,ArH),7.05(d,J＝1.3Hz,1H,ArH),7.01(dd,J＝9.0,1.5Hz,1H,ArH),4.82–4.68(m,2H,-O ₂CHCH₂O-),4.56–4.44(m,2H,-OCH_{2 2}CHO-),2.60(d,J＝7.4Hz,2H,- ₂CHCH＝),2.43(s,3H,-CH₃),2.06(dd,J＝12.4,5.9Hz,1H,-CH₂ CH＝),1.66–1.57(m,2H,-CH₂-),1.52–1.41(m,2H,-CH₂-),1.24(dd,J＝10.8,4.4Hz,2H,-CH₂-).¹³C NMR(151MHz,DMSO-d₆)δ161.03,159.92,158.62,152.92,146.77,137.03,135.93,129.78,128.10,126.40,122.22,113.96,112.25,110.38,101.00,69.58,65.88,54.74,39.91,31.84,31.61,24.14,14.99。

Example 24

Synthesis of the Compound 4- (2- (3-isopropyl-4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (13) in the formula VI

13d (1mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (550mg, 1.5mmol, 1.5equiv) Anhydrous dichloromethane (10mL) and DBU (N-butyl-N-methyl-N-ethyl-N-methyl-N-propyl-N-butyl-N-propyl-N-butyl-N-propyl-N-butyl-N) (DBU: (10 mL)) were added0.5mL, 3mmol, 3.0equiv), TLC monitoring the reaction completion, adding a small amount of water, extracting with dichloromethane, rinsing the organic phase with saturated saline, drying over anhydrous sodium sulfate, filtering, and purifying by column chromatography to obtain the target compound 13 with a yield of 67% as a white solid. ESI-MS M/z 487.4[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ7.98(d,J＝7.7Hz,2H,ArH),7.84(t,J＝7.5Hz,1H,ArH),7.78(d,J＝8.8Hz,1H,ArH),7.67(t,J＝7.8Hz,2H,ArH),7.03(d,J＝2.1Hz,1H,ArH),7.00(dd,J＝8.8,2.3Hz,1H,ArH),4.76(d,J＝2.4Hz,2H,-O ₂CHCH₂O-),4.48(d,J＝1.9Hz,2H,-OCH_{2 2}CHO-),3.34–3.21(m,1H,-CH＝),2.44(s,3H,-CH₃),1.27(d,J＝7.0Hz,6H,-(CH₃)₂).¹³C NMR(151MHz,DMSO-d₆)δ159.84,158.91,158.61,153.09,145.89,137.03,135.93,129.77,128.11,126.81,126.66,113.98,112.17,110.38,100.77,69.57,65.87,27.68,19.65,14.32。

Example 25

Synthesis of the Compound of formula IV 4- (2- (3-ethyl-4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (14)

14d (1mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (550mg, 1.5mmol, 1.5equiv) anhydrous dichloromethane (10mL) and DBU (0.5mL, 3mmol, 3.0equiv) were added, TLC monitored that after completion of the reaction, a small amount of water was added, extraction was performed with dichloromethane, the organic phase was rinsed with saturated saline, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography to give the title compound 14 in 66% yield, as a white solid, mp 155-. ESI-MS M/z 473.4[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ7.98(d,J＝8.0Hz,2H,ArH),7.84(t,J＝7.6Hz,1H,ArH),7.75(d,J＝8.4Hz,1H,ArH),7.67(t,J＝7.5Hz,2H,ArH),7.05(s,1H,ArH),7.01(dd,J＝9.3,0.8Hz,1H,ArH),4.77(m,2H,-O ₂CHCH₂O-),4.49(m,2H,-OCH_{2 2}CHO-),2.57(q,J＝7.5Hz,2H,- ₂CHCH₃),2.41(s,3H,-CH₃),1.05(t,J＝7.1Hz,3H,-CH_{2 3}CH).¹³C NMR(151MHz,DMSO-d₆)δ160.66,159.98,158.68,152.99,146.35,137.06,136.00,129.84,128.18,126.39,123.90,113.99,112.30,110.44,101.07,69.62,65.92,20.13,14.28,12.86。

Example 26

Synthesis of Compound 7-hydroxy-2H-chromen-2-one-3-carboxylic acid Ethyl ester (15b) in formula V

2, 4-dihydroxybenzaldehyde (15a, 3.0g, 21mmol, 1.0equiv), diethyl malonate (6.0mL, 38mmol, 1.8equiv) and pyridine (3mL) were stirred at room temperature for 1 hour, then 1N HCl (20mL) was added to the reaction solution, stirred for half an hour, filtered with suction, and dried to obtain compound 15b in 98% yield as a pale yellow solid. ESI-MS M/z 235.2[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ11.10(s,1H,-OH),8.69(s,1H,ArH),7.76(d,J＝8.6Hz,1H,ArH),6.84(dd,J＝8.6,2.1Hz,1H,ArH),6.73(d,J＝2.0Hz,1H,ArH),4.26(q,J＝7.1Hz,2H,-O ₂CHCH₃),1.29(t,J＝7.1Hz,3H,-OCH_{2 3}CH)。

Example 27

Synthesis of Compound 7-hydroxy-2H-chromen-2-one-3-carboxylic acid (15c) in formula V

15b (4.0g, 17.1mmol) was added 2N NaOH solution (20mL) and stirred at room temperature overnight before 2N HCl (23mL) was added and stirred at room temperature before suction filtration and drying to give compound 15c in 94% yield as a pale green solid. ESI-MS M/z 205.1[ M-H ]]^-；¹H NMR(400MHz,DMSO-d₆)δ10.10(s,1H,-OH),8.53(s,1H,ArH),7.56(d,J＝7.5Hz,1H,ArH),6.74(dd,J＝7.6,2.0Hz,1H,ArH),6.68(d,J＝2.1Hz,1H,ArH)。

Example 28

Synthesis of compound N- (4-fluorobenzyl) -7-hydroxy-2H-chromen-2-one-3-carboxamide (15d) in general formula V

15c (500mg, 2.4mmol, 1.0equiv) and CDI (1, 1-carbonyldiimidazole, 2.0g, 12.3mmol, 5.0equiv) tetrahydrofuran (10mL) was added and stirred at room temperature for 2 hours. 4-fluorobenzylamine (1mL, 7.7mmol, 3.3equiv) was added, and after completion of the reaction was monitored by room temperature TLC, poured into ice water (30mL), filtered with suction, dried, and purified by column chromatography (PE/EA ═ 8/1) to give compound 15d in 64% yield as a white solid. ESI-MS M/z 314.0[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ11.08(s,1H,-OH),9.08(t,J＝6.0Hz,1H,-CONH-),8.80(s,1H,ArH),7.82(d,J＝8.6Hz,1H,ArH),7.38(dd,J＝8.4,5.7Hz,2H,ArH),7.28(dd,J＝8.4,5.8Hz,2H,ArH),6.88(dd,J＝8.6,2.2Hz,1H,ArH),6.81(d,J＝1.9Hz,1H,ArH),4.51(d,J＝6.1Hz,2H,-NH ₂CH-)。

Example 29

Synthesis of compound N- (4-fluorobenzyl) -7-hydroxyethoxy-2H-chromen-2-one-3-carboxamide (15e) in general formula V

15d (200mg, 0.64mmol, 1.0equiv), sodium iodide (10mg, 0.06mmol, 0.1equiv) and anhydrous potassium carbonate (300mg, 2.2mmol, 3.0equiv) DMF (8mL) was added, chlorinated ethanol (130uL, 1.9mmol, 3.0equiv) DMF (1mL) was added at 80 deg.C, reflux TLC was used to monitor the completion of the reaction, ice water (30mL) was added at room temperature followed by suction filtration, and after drying column chromatography (PE/EA: 5/1), compound 15e was purified as a white solid in 84% yield. ESI-MS M/z 358.3[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ9.10(t,J＝6.4Hz,1H,-CONH-),8.85(s,1H,ArH),7.91(d,J＝8.7Hz,1H,ArH),7.39(dd,J＝7.4,6.3Hz,2H,ArH),7.16(t,J＝8.9Hz,2H,ArH),7.12(d,J＝1.0Hz,1H,ArH),7.05(dd,J＝8.4,1.1Hz,1H,ArH),4.98(brs,1H,-OH),4.51(d,J＝5.8Hz,2H,-NH ₂CH-),4.15(t,J＝4.5Hz,2H,-CH_{2 2}CHO-),3.79–3.68(m,2H,-HO ₂CHCH₂O-)。

Example 30

Synthesis of the Compound of formula V4- (2- (3- (N- (4-fluorobenzyl) carboxamide) -2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (15)

15f (30mg, 0.08mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (35mg, 0.09mmol, 1.1equiv) were added and then reacted for 2 hours with anhydrous dichloromethane (4mL) and DBU (40 μ L, 0.26mmol, 3.2equiv), a small amount of water was added and extracted with dichloromethane, the organic phase was rinsed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated and then subjected to column chromatography (PE/EA ═ 2/1) to isolate and purify the objective compound 15 in a yield of 57% as a white solid. ESI-MS M/z 582.5[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ9.10(t,J＝6.2Hz,1H,-CONH-),8.87(s,1H,ArH),7.96(dd,J＝8.4,2.9Hz,3H,ArH),7.82(t,J＝7.6Hz,1H,ArH),7.65(t,J＝7.8Hz,2H,ArH),7.39(dd,J＝8.1,5.8Hz,2H,ArH),7.21(d,J＝0.8Hz,1H,ArH),7.16(t,J＝8.7Hz,2H,ArH),7.10(dd,J＝8.7,1.5Hz,1H,ArH),4.80–4.76(m,2H,-O ₂CHCH₂O-),4.53(s,2H,-OCH_{2 2}CHO-),4.51(d,J＝6.1Hz,2H,-NH ₂CH-).¹³C NMR(151MHz,DMSO-d₆)δ162.37,160.40,160.29,158.76,158.61,153.20,148.24,146.88,137.02,135.94,134.25,129.78,129.70,129.65,128.13,126.66,121.12,115.10,114.86,114.74,113.90,112.44,110.38,101.10,68.56,65.93,32.21,15.04。

Example 31

Synthesis of the Compound of formula V4- (2- (3- (N- (4-fluorophenyl) acetamide) -4-methyl-2-oxo-2H-chromene-7-oxo) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (16)

16e (150mg, 0.4mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (180mg, 0.49mmol, 1.2equiv) anhydrous dichloromethane (10mL) and DBU (200 μ L, 1.34mmol, 3.0equiv) were added and stirred at room temperature for 2 hours, a small amount of water was added and extracted with dichloromethane, the organic phase was rinsed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography (DCM/MeOH ═ 8/1) to give the title compound 16 in 54% yield as a white solid. ESI-MS M/z 596.5[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ10.22(s,1H,-NHCO-),7.98(d,J＝7.5Hz,2H,ArH),7.83(dd,J＝14.1,8.1Hz,2H,ArH),7.67(t,J＝7.8Hz,2H,ArH),7.61(dd,J＝8.7,5.0Hz,2H,ArH),7.15(t,J＝10.3Hz,2H,ArH),7.10(d,J＝2.3Hz,1H,ArH),7.05(dd,J＝8.9,2.3Hz,1H,ArH),4.78(d,J＝4.6Hz,2H,-OCH_{2 2}CHO-),4.51(d,J＝2.8Hz,2H,-O ₂CHCH₂O-),3.73(s,2H,-CH₂CO-),2.44(s,3H,-CH₃).¹³C NMR(151MHz,DMSO)δ167.76,160.97,160.38,158.67,158.56,156.98,153.24,149.48,137.06,136.00,135.50,129.84,128.17,126.70,126.53,120.68,120.63,117.21,115.22,115.07,113.84,112.48,110.44,101.16,69.60,65.98,34.62,15.21。

Example 32

Synthesis of the Compound of formula V4- (2- (3- (N- (N' -benzylpiperazine) acetamide) -4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (17)

17d (0.4mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (0.49mmol, 1.2equiv) were added to anhydrous dichloromethane (8mL) and DBU (1.35mmol, 3.0equiv), followed by stirring at room temperature for 2 hours, addition of a small amount of water, extraction with dichloromethane, rinsing of the organic phase with saturated brine, drying over anhydrous sodium sulfate, filtration, and purification by column chromatography to give the title compound 17 in 55% yield as a white solid. ESI-MS M/z 661.6[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ8.03(d,J＝7.4Hz,2H,ArH),7.73(t,J＝6.9Hz,1H,ArH),7.63–7.51(m,3H,ArH),7.34(d,J＝3.9Hz,4H,ArH),7.29(dd,J＝8.8,4.7Hz,1H,ArH),6.91(dd,J＝8.9,2.0Hz,1H,ArH),6.83(d,J＝2.0Hz,1H,ArH),4.84–4.77(m,2H,-OCH_{2 2}CHO-),4.47–4.39(m,2H,-O ₂CHCH₂O-),3.72(s,2H,-CH₂CO-),3.72–3.68(m,2H,-CON ₂CHCH₂N＝),3.67–3.63(m,2H,-CON ₂CHCH₂N＝),3.54(s,2H,＝NCH₂-),2.56–2.50(m,2H,-CONCH_{2 2}CHN＝),2.45(s,3H,-CH₃),2.42(d,J＝4.2Hz,2H,-CONCH_{2 2}CHN＝).¹³C NMR(151MHz,CDCl₃)δ167.81,161.99,160.42,158.70,153.85,149.59,138.01,135.72,129.68,129.19,128.56,128.37,127.33,126.16,118.19,114.96,112.23,110.37,101.58,69.09,65.65,62.85,53.17,52.74,45.95,42.08,31.39,15.81。

Example 33

Synthesis of the Compound of formula V4- (2- (3- (N- (N' - (4-fluorobenzyl) piperazine) acetamide) -4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (18)

18d (0.4mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (0.49mmol, 1.2equiv) after addition of anhydrous dichloromethane (8mL) and DBU (1.35mmol, 3.0equiv), stirring at room temperature for 2 hours, addition of a small amount of water, extraction with dichloromethane, washing of the organic phase with saturated brine, drying over anhydrous sodium sulfate, and passing throughFiltering, and purifying by column chromatography to obtain the target compound 18 with the yield of 65 percent and white solid. ESI-MS M/z 679.0[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ8.03(d,J＝7.7Hz,2H,ArH),7.73(t,J＝6.9Hz,1H,ArH),7.64–7.52(m,3H,ArH),7.33–7.27(m,2H,ArH),7.02(t,J＝8.6Hz,2H,ArH),6.92(dd,J＝8.8,2.5Hz,1H,ArH),6.83(d,J＝2.3Hz,1H,ArH),4.86–4.77(m,2H,-OCH_{2 2}CHO-),4.47–4.39(m,2H,-O ₂CHCH₂O-),3.72(s,2H,-CH₂CO-),3.71(d,J＝1.1Hz,2H,-CON ₂CHCH₂N＝),3.67–3.63(m,2H,-CON ₂CHCH₂N＝),3.51(s,2H,＝NCH₂-),2.56–2.50(m,2H,-CONCH_{2 2}CHN＝),2.45(s,3H,-CH₃),2.44–2.42(m,2H,-CONCH_{2 2}CHN＝).¹³C NMR(151MHz,CDCl₃)δ167.85,162.97,162.00,161.34,160.44,158.70,153.84,149.64,138.01,135.72,130.65,129.68,128.56,126.17,118.16,115.27,115.13,114.95,112.25,110.37,101.59,69.09,65.65,61.99,53.07,52.64,45.93,42.03,31.36,15.82。

Example 34

Synthesis of the Compound of formula V4- (2- (3- (N- (N' - (4-cyanobenzyl) piperazine) acetamide) -4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (19)

After anhydrous dichloromethane (8mL) and DBU (1.35mmol, 3.0equiv) were added to 19d (0.4mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (0.49mmol, 1.2equiv), a small amount of water was added thereto under stirring at room temperature for 2 hours, extraction was performed with dichloromethane, the organic phase was rinsed with saturated brine, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography to give the objective compound 19 in 54% yield as a white solid. ESI-MS M/z 686.1[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ8.03(d,J＝7.7Hz,2H,ArH),7.73(t,J＝7.4Hz,1H,ArH),7.63(d,J＝8.4Hz,2H,ArH),7.57(dd,J＝14.9,7.1Hz,3H,ArH),7.47(d,J＝7.8Hz,2H,ArH),6.92(dd,J＝8.7,0.9Hz,1H,ArH),6.84(d,J＝1.8Hz,1H,ArH),4.84–4.78(m,2H,-OCH_{2 2}CHO-),4.39(d,J＝5.5Hz,2H,-O ₂CHCH₂O-),3.72(m,4H,-CON( ₂CHCH₂)₂N-),3.68–3.64(s,2H-CH₂CO-),3.59(s,2H,＝NCH₂-),2.55–2.51(m,2H,-CONCH_{2 2}CHN＝),2.46(s,3H,-CH₃),2.45–2.40(m,2H,-CONCH_{2 2}CHN＝).¹³C NMR(151MHz,CDCl₃)δ167.94,162.01,160.48,158.71,153.85,149.71,143.65,138.01,135.72,132.25,129.68,129.49,128.57,126.20,118.87,118.10,114.94,112.30,111.16,110.38,101.60,69.09,65.66,62.24,53.26,52.81,45.99,42.07,31.33,15.84。

Example 35

Synthesis of the Compound of formula V4- (2- (3- (N- (4-fluorobenzyl) propionamide) -4-methyl-2-oxo-2H-chromen-7-oxo) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (20)

After addition of anhydrous dichloromethane (8mL) and DBU (200 μ L, 1.34mmol, 3.0equiv) to 20e (170mg, 0.43mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (180mg, 0.49mmol, 1.1equiv), stirring was carried out at room temperature for 2 hours, a small amount of water was added, extraction was carried out with dichloromethane, the organic phase was rinsed with saturated brine, dried over anhydrous sodium sulfate, filtered, and column chromatography (DCM/MeOH ═ 12/1) was carried out to obtain the objective compound 20 in 58% yield as a white solid. ESI-MS M/z 624.0[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ8.41(t,J＝5.7Hz,1H,ArH),7.98(d,J＝7.7Hz,2H,ArH),7.83(t,J＝7.4Hz,1H,ArH),7.74(d,J＝8.9Hz,1H,ArH),7.66(t,J＝7.8Hz,2H,ArH),7.19(dd,J＝8.0,6.0Hz,2H,ArH),7.06(d,J＝2.2Hz,1H,ArH),7.03(d,J＝2.6Hz,1H,ArH),7.00(t,J＝5.5Hz,1H,ArH),4.81–4.73(m,2H,-O ₂CHCH₂O-),4.52–4.45(m,2H,-OCH_{2 2}CHO-),4.20(d,J＝5.8Hz,2H,-NH ₂CH-),2.81(t,J＝7.3Hz,2H,-CH_{2 2}CHCO-),2.37(s,3H,-CH₃),2.38–2.32(m,2H,- ₂CHCH₂CO-).¹³C NMR(151MHz,DMSO)δ171.10,161.72,160.60,160.09,158.68,153.08,147.49,137.07,136.00,135.63,129.84,128.98,128.93,128.17,126.59,121.45,114.80,114.66,113.92,112.30,110.43,101.09,69.59,65.96,41.23,33.74,23.45,14.61。

Example 36

Synthesis of the Compound of formula V4- (2- (3- (N- (4-fluorophenyl) propionamide) -4-methyl-2-oxo-2H-chromene-7-oxo) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (21)

21b (180mg, 0.46mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (200mg, 0.54mmol, 1.2equiv) in anhydrous dichloromethane (10mL) and DBU (200 μ L, 1.34mmol, 3.0equiv), stirred at room temperature for 2 hours, a small amount of water was added, extraction was performed with dichloromethane, the organic phase was rinsed with saturated brine, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography (PE/EA ═ 1/1) to give the title compound 21 in 51% yield as a white solid. ESI-MS M/z 610.0[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)δ10.00(s,1H,-CONH-),7.98(d,J＝8.1Hz,2H,ArH),7.83(t,J＝7.7Hz,1H,ArH),7.75(d,J＝8.7Hz,1H,ArH),7.66(t,J＝7.5Hz,2H,ArH),7.57(dd,J＝8.0,5.2Hz,2H,ArH),7.12(t,J＝8.6Hz,2H,ArH),7.07(s,1H,ArH),7.00(d,J＝8.7Hz,1H,ArH),4.77(d,J＝1.9Hz,2H,-OCH_{2 2}CHO-),4.49(d,J＝3.7Hz,2H,-O ₂CHCH₂O-),2.88(t,J＝7.1Hz,2H,-CH_{2 2}CHCO-),2.52(t,J＝7.4Hz,2H,- ₂CHCH₂CO-),2.42(s,3H,-CH₃).¹³C NMR(151MHz,DMSO-d₆)δ170.08,160.70,160.12,158.68,158.54,156.95,153.09,147.53,137.07,135.99,135.48,129.83,128.17,126.57,121.33,120.74,120.69,115.17,115.03,113.90,112.35,110.43,101.11,69.60,65.95,34.78,23.25,14.66。

Example 37

Synthesis of the Compound 4- (2- (3- (N-morpholinepropionamide) -4-methyl-2-oxo-2H-chromene-7-oxo) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (22) in general formula V

22b (90mg, 0.25mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (110mg, 0.29mmol, 1.3equiv) in dry dichloromethane (7mL) and DBU (120 μ L, 0.76mmol, 3.0equiv), stirring at room temperature for 2 hours, adding water (20mL), extracting with dichloromethane, rinsing the organic phase with saturated brine, drying over anhydrous sodium sulfate, filtering, and purifying by column chromatography (PE/EA ═ 2/1) to obtain the title compound 22, yield 57%, white solid. ESI-MS M/z 586.2[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ8.04(d,J＝7.9Hz,2H,ArH),7.74(t,J＝6.2Hz,1H,ArH),7.58(dd,J＝14.0,7.1Hz,3H,ArH),6.93(dd,J＝8.1,2.7Hz,1H,ArH),6.85(d,J＝1.1Hz,1H,ArH),4.82(d,J＝2.5Hz,2H,-OCH_{2 2}CHO-),4.46(d,J＝2.5Hz,2H,-O ₂CHCH₂O-),3.67(m,4H,-N(CH_{2 2}CH)₂O-),3.64–3.61(m,2H,-N ₂CHCH₂O-),3.57–3.54(m,2H,-N ₂CHCH₂O-),2.98(t,J＝7.4Hz,2H,-CH_{2 2}CHCO-),2.63(t,J＝7.3Hz,2H,- ₂CHCH₂CO-),2.50(s,3H,-CH₃).¹³C NMR(151MHz,CDCl₃)δ170.83,161.88,160.27,158.72,153.67,147.58,138.00,135.69,129.67,128.59,126.15,122.27,114.88,112.32,110.39,101.59,69.10,66.88,66.73,65.68,46.10,41.98,31.58,23.90,15.06。

Example 38

Synthesis of the Compound of formula V4- (2- (3- (N- (N' -benzylpiperazine) propionamide) -4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (23)

23b (100mg, 0.22mmol, 1.0equiv) and benzenesulfonyl furazan nitroxide (100mg, 0.27mmol, 1.2equiv) anhydrous dichloromethane (7mL) and DBU (100 μ L, 0.66mmol, 3.0equiv) were stirred at room temperature for 2 hours, a small amount of water was added, extraction was performed with dichloromethane, the organic phase was rinsed with saturated brine, dried over anhydrous sodium sulfate, filtered, and column chromatography (PE/EA ═ 2/1) was performed to obtain the objective compound 23 in 58% yield as a white solid. ESI-MS M/z 675.0[ M + H ]]⁺；¹H NMR(400MHz,CDCl₃)δ8.03(dd,J＝8.3,1.9Hz,2H,ArH),7.73(dd,J＝9.5,5.8Hz,1H,ArH),7.62–7.52(m,3H,ArH),7.33–7.26(m,5H,ArH),6.92(dd,J＝11.6,2.9Hz,1H,ArH),6.84(d,J＝2.6Hz,1H,ArH),4.80(d,J＝4.2Hz,2H,HOCH_{2 2}CHO-),4.44(d,J＝2.4Hz,2H,HO ₂CHCH₂O-),3.66–3.58(m,2H,-CONCH_{2 2}CHN-),3.56–3.50(m,2H,-CONCH_{2 2}CHN-),3.48(s,2H,＝NCH₂-),2.96(t,J＝8.9Hz,2H,-CH_{2 2}CHCO-),2.62(t,J＝6.4Hz,2H,- ₂CHCH₂CO-),2.48(s,3H,-CH₃),2.40(d,J＝1.4Hz,4H,- _{2 2}CHCHCO-).¹³C NMR(151MHz,CDCl₃)δ170.56,161.86,160.22,158.72,153.66,147.51,138.00,135.69,129.67,129.19,128.58,128.36,127.35,126.14,122.47,114.95,112.25,110.39,101.58,69.10,65.67,62.87,53.14,52.74,45.58,41.58,31.68,23.96,15.06。

Example 39 cell level antitumor Activity assay

Detecting the activity of the compound of the invention for inhibiting the proliferation of tumor cells by adopting an MTS method, inoculating cancer cells in logarithmic growth phase into a 96-well plate, dividing the cancer cells into a blank control group, a positive control group and a compound or positive drug treatment group with different concentrations, culturing the cancer cells at 37 ℃ for 48 hours, then performing MTT detection, and calculating the inhibition rate of cell proliferation and IC₅₀(ii) a The results show that most of the compounds of the present invention have IC activity for inhibiting the growth of four tumor cells, A2780/CDDP, MCF-7/ADR and MDA-MB-231₅₀The value is below 100nM, which is obviously higher than the proliferation inhibition activity to KB, KB-V and MCF-7 tumor cells; wherein the compounds 4- (2- (3- (4-cyanobenzyl) -4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (6), 4- (2- (3- (4-trifluoromethylbenzyl) -4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) -3- (benzenesulfonyl) -1,2, 5-oxadiazole-2-oxide (7) and 4- (2- (3- (4-nitrobenzyl) -4-methyl-2-oxo-2H-chromen-7-oxy) ethoxy) - The antiproliferative activity of 3- (phenylsulfonyl) -1,2, 5-oxadiazole-2-oxide (8) on A2780, A2780/CDDP, MCF-7/ADR and MDA-MB-231 was comparable or superior to that of the compound CY-14S-4A83, IC₅₀Values were between 5.0-44.2nM (see Table 1). The antiproliferative activity of the compound of the invention on a drug-resistant strain MCF-7/ADR is 10 to 500 times higher than that of a sensitive strain MCF-7, and the compound behaves similarly to the original active compound CY-14S-4A 83. No significant selective inhibitory activity was shown between sensitive A2780 and drug-resistant A2780/CDDP and between sensitive KB and drug-resistant KB-V; wherein the compound 7 (the 3-position contains trifluoromethyl benzyl coumarin furazan conjugate) and the compound 8 (the 3-position contains nitrobenzyl coumarin furazan conjugate)Conjugate), IC for MCF-7/AD₅₀The values were 5.0 and 8.5nM, respectively, for the specific sensitive cell line (IC for MCF-7)₅₀Values of 2495 and 2351nM, respectively) were 499 and 276 fold higher in antiproliferative activity (as shown in table 2).

The cytotoxicity of the compounds 1-23 of the invention on three normal cells was at the same level as that of the compound CY-14S-4A83 (shown in Table 3); IC of all Compounds on HUVEC₅₀The value is between 1.9 and 17.0. mu.M.

Table 1 shows the antiproliferative activity of the target compounds 1 to 23 on seven tumor cells.

Table 2 shows the IC of three groups of sensitive solid tumor cells and drug-resistant cells₅₀A ratio.

TABLE 3 antiproliferative activity of compounds of interest 1-23 on three normal cells.

TABLE 1

TABLE 2

TABLE 3

Example 40 intracellular NO Release assay and Effect of NO scavengers on antitumor Activity

Detection of intracellular NO Release levels of Compounds of the invention Using classical Griess reagent, test A2780 tumor cells (1)×10⁷One in a 10cm dish) is incubated with the compound of the invention with a concentration of 100 μ M for 150 minutes, the collected cells are lysed with RIPA lysate for 30 minutes on ice, the cell lysate is centrifuged at 4 ℃ and 12000rpm for 15 minutes, NADPH, FAD and Nitrate Reductase are added to the supernatant, and the incubation is carried out for 30 minutes at 37 ℃; adding LDH and LDH Buffer, and continuing incubation at 37 ℃ for 30 min; then mixing with Griess reagent for 30 minutes, detecting absorbance at 540nm, treating cells without drug effect under the same condition as background, treating with sodium nitrite of different concentrations by the same method to prepare standard curve, and using original active compound CY-14S-4A83 as control; as a result, as shown in Table 4, the compounds of the present invention released a considerable concentration of NO.

The test cells were seeded in a 96-well plate, a zero-set without cells was set, and the NO scavenger Carboxy-PITO (Beyotime) was prepared as a 10mM solution. Setting 8 concentrations, placing in an incubator at 37 ℃, adding the test compound at a concentration of 100nM or 200nM after 24 hours, and detecting the OD value at 490nM with a microplate reader after incubation for 48 hours in a control incubator, and calculating the inhibition ratio according to the following formula:

inhibition rate [ (OD)_Control-OD_{Administration of drugs})/(OD_Control-OD_{Zero setting})]×100％

The results show (see fig. 2) that the inhibition rate of compounds 6 and 7 on tumor cells A2780 gradually decreases with the increase of the concentration of NO scavenger C-PITO, and the concentration dependence is shown. Indirectly shows that the activity of the compound of the invention has close relation with the NO release amount.

Table 4 shows the concentrations of NO released by the compounds 1 to 23 according to the invention in tumor cells.

TABLE 4

TABLE 4

Example 41 colony formation inhibition experiment

The test cells are A2780 and MCF-7/ADR cells, which are inoculated on a 6-well plate and placed in an incubator for 24 hours. Test compounds were set at 5 concentrations and blanks. After 48 hours of incubation, the solution was changed according to the cell growth state. When the number of colonies was large, the culture was stopped, the original medium was aspirated, and the cells were rinsed twice with PBS solution. 1mL of methanol was added to each well and fixed at room temperature for 20 minutes. Methanol was aspirated, 1mL of crystal violet staining solution (C0121, Beyotime) was added to each well, and the staining was performed for 30 minutes at room temperature. The staining solution was aspirated off, rinsed twice with PBS, dried at room temperature, and photographed. The results show that both compounds 6 and 7 are effective in inhibiting tumor cell colony formation (as shown in FIG. 3).

Example 42 solubility testing experiment

Conditions for detection using HPLC 1mg of test compound was placed in a stoppered tube and 2mL of PBS solution (WISENT, China) and 20. mu.L of DMSO solution were added and the tube sealed until use. After the test, the solubility is calculated by comparing with the prepared standard curve. The results show that the water solubility of all the target compounds of the invention is significantly increased (70-820 times) compared to compound CY-14S-4a83, with 730 times increase in the water solubility of compound 6 (see table 5). Indicating that introduction of cyano and amide bonds favors increased water solubility.

Table 5 solubility of the compounds of the invention.

TABLE 5

Claims (6)

1. NO donor coumarin furazan conjugate is characterized in that it is a 3-substituted coumarin furazan derivative, and the derivative is a substituted coumarin parent nucleus and ring-opening thereof at the 3-position Derivatives (hydrazide or hydrazone replacing α,β-unsaturated ester structure in coumarin core), furazan derivatives bound to furazan nitroxide through a two-carbon linkage, the compounds have formulae I, II and the structure of III,

Wherein: R ₁ is cyano, trifluoromethyl, nitro, methoxy, R ₂ is ethyl, isopropyl, cyclopentylmethyl, cyclohexylmethyl, phenethyl, morpholineformylethyl, R ₃ is a fluorine atom, a cyano group, R ₄ is a hydrazide-linked substituted aryl group, a hydrazone-linked substituted aryl group, R ₅ is a fluorine atom or a methoxy group. 2. the purposes of the NO donor coumarin furazan conjugate of claim 1 in the preparation of the pharmaceutical preparation that suppresses sensitive or drug-resistant tumor cells; Described NO donor coumarin furoxan conjugate is formula I , II and III compounds. 3. according to the purposes described in claim 2, it is characterized in that, described sensitive tumor cell is sensitive tumor cell line human triple negative breast cancer cell (MDA-MB-231), human ovarian cancer cell (A2780), breast cancer cell line Cancer cells (MCF-7) or oral epidermal carcinoma cells (KB). 4. The purposes according to claim 2, wherein the drug-resistant tumor cells are cisplatin-resistant A2780/CDDP, vincristine-resistant KB-V and doxorubicin-resistant MCF-7/ADR . 5. The use according to claim 2, wherein the compounds of formula I, II and III inhibit tumor cell proliferation by releasing high concentrations of nitric oxide. 6. The use according to claim 2, wherein the compounds of formula I, II and III have the effect of selectively inhibiting the proliferation of MDR tumor cells overexpressing P-gp by releasing high concentrations of nitric oxide .

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