阅读说明：本技术 一种no供体化合物及其制备方法和用途 (NO donor compound and preparation method and application thereof ) 是由 秦向阳 王玉琨 张星 姚团利 梁晶 王迎捷 刘珂珂 于 2021-09-09 设计创作，主要内容包括：本发明公开了一种NO供体化合物及其制备方法和用途,该化合物是通过5-单硝酸异山梨醇酯与NIT型氮氧自由基的拼合而形成的药物活性成分,结构如式I所示。所述化合物作为NO供体分子对缺血心肌具有显著的保护作用,可开发为高血压、缺血性心脏病治疗药物,且具有开发为无明显耐药性的治疗缺血性心脏病的NO供体药物的潜力。(The invention discloses an NO donor compound and a preparation method and application thereof, the compound is a pharmaceutical active ingredient formed by splicing 5-isosorbide mononitrate and NIT-type nitroxide free radicals, and the structure is shown as formula I. The compound has a remarkable protection effect on ischemic myocardium as NO donor molecules, can be developed into medicaments for treating hypertension and ischemic heart disease, and has the potential of being developed into NO donor medicaments for treating ischemic heart disease without obvious drug resistance.)

1. An NO donor compound characterized by: the NO donor compound is a compound with a structure shown in a formula I or any one of a tautomer, a stereoisomer and a pharmaceutically acceptable salt of the compound:

the connecting group comprises a functional structure for introducing a substituent, and the functional structure is a splicing body of any one or more than two of a benzene ring, a substituted benzene ring, acetic acid and a tetrahydropyrrole ring.

2. The NO donor compound of claim 1, wherein: the NO donor compound is a compound with a structure shown as a formula II, a formula III or a formula IV, or any one of a tautomer, a stereoisomer and a pharmaceutically acceptable salt of the compound:

wherein, R is H or a substituent group, and the substituent group is any one of halogen, halogenated hydrocarbon group, nitryl, hydroxyl and alkoxy.

3. The NO donor compound of claim 1, wherein: the NO donor compound is a compound with a structure shown as a formula II-1, a formula II-2, a formula II-3, a formula II-4, a formula II-5, a formula II-6, a formula II-7, a formula II-8, a formula II-9, a formula II-10 or a formula II-11, or any one of a tautomer, a stereoisomer and a pharmaceutically acceptable salt of the compound:

4. the NO donor compound of claim 1, wherein: the NO donor compound is a compound with a structure shown as a formula III-1, III-2, III-3, III-4, III-5, III-6, III-7, III-8, III-9, III-10 or III-11, or any one of a tautomer, a stereoisomer and a pharmaceutically acceptable salt of the compound:

5. a method of preparing a NO donor compound, comprising: the method comprises the following steps:

using 5-isosorbide mononitrate as a raw material for forming an NO donor part in the structure of the NO donor compound, and splicing the 5-isosorbide mononitrate and NIT-type nitroxide free radicals through connecting groups; the connecting group comprises a functional structure for introducing a substituent, and the functional structure is a splicing body of any one or more than two of a benzene ring, a substituted benzene ring, acetic acid and a tetrahydropyrrole ring.

6. The method of claim 5 for preparing a NO donor compound, wherein: the preparation method of the NO donor compound specifically comprises the following steps:

2.1) subjecting isosorbide 5-mononitrate to intermolecular condensation reaction with p-formylbenzoic acid or a p-formylbenzoic acid derivative in a solvent containing a condensing agent and a catalyst to obtain a first intermediate having the structure shown below:

wherein, R is H or a substituent group, and the substituent group is any one of halogen, halogenated hydrocarbon group, nitro, hydroxyl and alkoxy;

2.2) carrying out condensation reaction on the first intermediate and dihydroxylamine under the reflux condition to prepare a second intermediate with the structure shown as the following:

2.3) carrying out oxidation reaction on the second intermediate and an oxidant to prepare the NO donor compound.

7. The method of claim 5 for preparing a NO donor compound, wherein: the preparation method of the NO donor compound specifically comprises the following steps:

3.1) carrying out Williamson condensation reaction on p-hydroxybenzaldehyde or p-hydroxybenzaldehyde derivatives and chloroacetic acid or bromoacetic acid under alkaline conditions to prepare a third intermediate with the structure shown as the following formula:

wherein, R is H or a substituent group, and the substituent group is any one of halogen, halogenated hydrocarbon group, nitro, hydroxyl and alkoxy;

3.2) carrying out intermolecular condensation reaction on the third intermediate and 5-isosorbide mononitrate in a solvent containing a condensing agent and a catalyst to obtain a fourth intermediate with the structure shown as the following formula:

3.3) carrying out condensation reaction on the fourth intermediate and dihydroxylamine under the reflux condition to obtain a fifth intermediate with the structure shown as the following formula:

and 3.4) carrying out oxidation reaction on the fifth intermediate and an oxidant to prepare the NO donor compound.

8. The method of claim 5 for preparing a NO donor compound, wherein: the preparation method of the NO donor compound specifically comprises the following steps:

4.1) reacting p-formylbenzoic acid with an acyl chloride reagent contained in a solvent under the reflux condition to prepare a sixth intermediate;

4.2) carrying out intermolecular condensation reaction on the sixth intermediate and L-proline or D-proline in a solvent containing an acid-binding agent to prepare a seventh intermediate;

4.3) carrying out intermolecular condensation reaction on the seventh intermediate and 5-isosorbide mononitrate in a solvent containing a condensing agent and a catalyst to obtain an eighth intermediate;

4.4) carrying out condensation reaction on the eighth intermediate and dihydroxylamine under the reflux condition to prepare a ninth intermediate;

4.5) carrying out oxidation reaction on the ninth intermediate and an oxidant to prepare the NO donor compound.

9. Use of a NO donor compound as defined in claim 1 for the preparation of a medicament for the treatment of ischemic heart disease.

10. Use of a NO donor compound as defined in claim 1 for the preparation of a medicament for the treatment of hypertension.

Technical Field

The invention belongs to the technical field of medicines, and relates to an NO donor compound, and a preparation method and application thereof.

Background

Cardiovascular disease remains the leading cause of death worldwide, with up to 1730 million people dying from cardiovascular disease each year, and it is expected that by 2030, over 2360 million people will die from cardiovascular disease, with Ischemic Heart Disease (IHD) being the leading cause of death.

Nitrate drugs are first-line drugs widely used in clinical treatment of IHD, and mainly include the following four: nitroglycerin (GTN), isosorbide dinitrate (ISDN), isosorbide 5-mononitrate (5-ISMN), pentaerythrityl tetranitrate (PETN). The mechanism by which nitrate based drugs have pharmacological activity is their release of Nitric Oxide (NO) by interaction with thiol-containing enzymes in vascular smooth muscle and endothelial cells, which increases myocardial blood supply and reduces myocardial oxygen consumption. However, it not only causes a decrease in therapeutic effect due to its rapid development of drug resistance and cross-resistance, but also limits its clinical provision of continuous therapeutic effects. Therefore, the development of nitrate medicines without drug resistance has become a research hotspot in the cardiovascular field.

Research shows that nitrate medicine can promote the blood vessel to produce great amount of superoxide anion radicalWhileIs considered to be one of the causes of the drug resistance and cross resistance of nitrate drugs. Thus being scavenged by antioxidantsMay be an effective way to overcome the drug resistance of nitrate drugs. However, when the existing antioxidant and nitrate medicine are used together, the obvious defect that the synergistic effect of the medicine effect and the medicine metabolism in the same dosage form is difficult to realize exists, so that the drug combination is restricted.

The stable free-radical nitroxide is a new type of free-radical scavenger developed in recent years, and can be continuously decomposed in a catalytic modeThe rate constant of the catalytic reaction is as high as 10⁸M^-1·S^-1Is incomparable with stoichiometric radical scavengers. Furthermore, certain compounds containing NIT-type nitroxide radicals have the effect of treating ischemic heart diseases.

At present, reports of double-effect NO donor compounds formed by introducing NIT type nitroxide free radicals are not found.

Disclosure of Invention

The invention aims to provide an NO donor compound, a preparation method and application thereof, wherein the NO donor compound can generate NO and has better anti-oxidative stress capability, and can be used for treating cardiovascular diseases such as hypertension, ischemic heart disease and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

an NO donor compound, which is a compound with a structure shown in a formula I or any one of a tautomer, a stereoisomer and a pharmaceutically acceptable salt of the compound:

the connecting group comprises a functional structure for introducing a substituent, and the functional structure is a splicing body of any one or more than two of a benzene ring, a substituted benzene ring, acetic acid and a tetrahydropyrrole ring.

Preferably, the linking group is attached to the hydroxyl moiety of isosorbide 5-mononitrate (5-ISMN) and the carbon atom at position 2 of the NIT-type nitroxide radical, respectively.

Preferably, the NO donor compound is a compound having a structure shown in formula II, formula III or formula IV, or any one of a tautomer, a stereoisomer and a pharmaceutically acceptable salt of the compound:

wherein, R is H or a substituent, and the substituent is any one of various electron-donating groups or electron-withdrawing groups such as halogen, halogenated hydrocarbon group, nitro group, hydroxyl group, alkoxy group and the like (the C atom number of the substituent is not suitable to be too large, otherwise, the solubility of the NO donor compound is insufficient, for example, the C atom number of the alkoxy group can be selected to be 1-3).

Preferably, the NO donor compound is a compound having a structure shown in formula II-1 to formula II-11 or any one of a tautomer, a stereoisomer and a pharmaceutically acceptable salt of the compound:

preferably, the NO donor compound is a compound having a structure shown in formula III-1 to formula III-11 or any one of a tautomer, a stereoisomer and a pharmaceutically acceptable salt of the compound:

the preparation method of the NO donor compound comprises the following steps:

isosorbide 5-mononitrate (5-ISMN) is used as a raw material for forming an NO donor part in the structure of the NO donor compound, and the 5-ISMN and NIT type nitroxide free radical are spliced through the connecting group.

Preferably, the preparation method of the NO donor compound specifically comprises the following steps:

2.1) using 5-ISMN as a starting material, and carrying out intermolecular condensation reaction on the 5-ISMN and p-formylbenzoic acid or a derivative thereof in a solvent containing a condensing agent and a catalyst to prepare a first intermediate with the following structure:

wherein, R is H (compound 1 in the embodiment) or a substituent, the substituent is any one of various electron donating groups or electron withdrawing groups such as halogen, halogenated hydrocarbon group, nitro group, hydroxyl group, alkoxy group and the like, so as to investigate the influence of introducing different electric substituent groups into the connecting group on the biological activity of the NO donor compound;

2.2) condensation of the first intermediate with bis-hydroxylamine (2, 3-dimethyl-2, 3-dihydroxyaminobutane) under reflux conditions to produce a second intermediate (e.g. compound 2 in the example) having the structure shown below:

2.3) carrying out oxidation reaction on the second intermediate and an oxidant to prepare a compound (for example, the compound 3 in the embodiment) with the structure shown in the formula II.

Preferably, in the step 2.1), the condensing agent is one or more of N, N' -Dicyclohexylcarbodiimide (DCC), Carbonyldiimidazole (CDI), Diisopropylcarbodiimide (DIC) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDCI), and the catalyst is one or more of 4-Dimethylaminopyridine (DMAP) and 1-hydroxybenzotriazole (HOBt); the solvent is one or more of tetrahydrofuran, acetonitrile, benzene, dichloromethane and N, N-dimethylformamide; the reaction temperature is 25-30 ℃, the reaction time is 15-20 h, the molar ratio of 5-ISMN to p-formylbenzoic acid or the molar ratio of 5-ISMN to formylbenzoic acid derivative is 1: 1-1: 2, the dosage of the catalyst is controlled to be 1-10% (molar ratio, relative to 5-ISMN), and the molar ratio of the condensing agent to 5-ISMN is 1: 1-1: 2; and after the reaction is finished, filtering, washing the filtrate by hydrochloric acid and saturated sodium bicarbonate in sequence, drying an organic phase by anhydrous magnesium sulfate, concentrating under reduced pressure, and carrying out column chromatography to obtain a first intermediate.

Preferably, in the step 2.2), the solvent used for the reaction is one or more of methanol, ethanol, N-dimethylformamide and tetrahydrofuran; the reaction temperature is 30-80 ℃, the reaction time is 12-24 hours, and the molar ratio of the first intermediate to the dihydroxylamine is 1: 1.1-1: 2; after the reaction is finished, the reaction solution is directly concentrated under reduced pressure to obtain a second intermediate (crude product).

Preferably, in the step 2.3), the solvent used for the reaction is one or more of water, methanol, tetrahydrofuran, dichloromethane or trichloromethane, and the oxidant is one or more of sodium periodate or lead dioxide; the reaction temperature is 0-25 ℃, the reaction time is 30-60 min, and the molar ratio of the first intermediate to the oxidant is 1: 1-1: 1.5; and after the reaction is finished, separating an organic phase, washing the organic phase by saturated sodium chloride, drying by anhydrous magnesium sulfate, concentrating under reduced pressure, and carrying out column chromatography to obtain the NO donor compound.

Preferably, in the step 2.1), the eluent for column chromatography is ethyl acetate and petroleum ether (in a volume ratio of 5: 1-1: 1), and in the step 2.3), the eluent for column chromatography is ethyl acetate and petroleum ether (in a volume ratio of 1: 5-1: 1).

Preferably, the preparation method of the NO donor compound specifically comprises the following steps:

3.1) Williamson condensation of p-hydroxybenzaldehyde or a derivative thereof (for example, an ortho-or meta-substituted p-hydroxybenzaldehyde derivative such as vanillin) with chloroacetic acid or bromoacetic acid under alkaline conditions to obtain a third intermediate having the following structure:

wherein, R is H or substituent, the substituent is any one of various electron-donating groups or electron-withdrawing groups such as halogen, halogenated hydrocarbon group, nitryl, hydroxyl, alkoxy (for example, compound 4 in the embodiment) and the like, so as to investigate the influence of introducing different electric substituent groups into the connecting group on the biological activity of the NO donor compound;

3.2) carrying out intermolecular condensation reaction on the third intermediate and 5-ISMN in a solvent containing a condensing agent and a catalyst to obtain a fourth intermediate (for example, a compound 5 in the example) with the structure shown as follows:

3.3) carrying out a condensation reaction of the fourth intermediate and dihydroxylamine (2, 3-dimethyl-2, 3-dihydroxyaminobutane) under reflux conditions to obtain a fifth intermediate (for example, compound 6 in the example) with the following structure:

and 3.4) carrying out oxidation reaction on the fifth intermediate and an oxidant to obtain a compound (for example, the compound 7 in the embodiment) with the structure shown in the formula III.

Preferably, in the step 3.1), the base used for the reaction is one or more of sodium hydroxide, potassium hydroxide, sodium hydride and potassium hydride, and the solvent used for the reaction is one or more of water, tetrahydrofuran and diethyl ether; the reaction temperature is 25-80 ℃, the reaction time is 5-13 h, the molar ratio of the p-hydroxybenzaldehyde (or the derivative thereof) to the chloroacetic acid or the molar ratio of the p-hydroxybenzaldehyde (or the derivative thereof) to the bromoacetic acid is 1: 1-1: 2, and the amount of the alkali is controlled to be 2.5-4 equivalents (the amount multiple of the substance relative to the p-hydroxybenzaldehyde or the derivative thereof); after the reaction is finished, adding water for dilution, washing by ethyl acetate, separating a water phase, acidifying by concentrated hydrochloric acid, extracting by ethyl acetate, separating a combined organic phase by saturated sodium bicarbonate, acidifying the water phase by the concentrated hydrochloric acid, and filtering to obtain a third intermediate.

Preferably, in the step 3.2), the condensing agent is one or more of N, N' -Dicyclohexylcarbodiimide (DCC), Carbonyldiimidazole (CDI), Diisopropylcarbodiimide (DIC) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDCI), and the catalyst is one or more of 4-Dimethylaminopyridine (DMAP) and 1-hydroxybenzotriazole (HOBt); the solvent is one or more of tetrahydrofuran, acetonitrile, benzene, dichloromethane and N, N-dimethylformamide; the reaction temperature is 25-30 ℃, the reaction time is 15-36 h, the molar ratio of the 5-ISMN to the third intermediate is 1: 1-1: 2, the dosage of the catalyst is controlled to be 1-10% (relative to the 5-ISMN), and the molar ratio of the 5-ISMN to the condensing agent is 1: 1-1: 2; and after the reaction is finished, filtering, washing the filtrate by hydrochloric acid and saturated sodium chloride in sequence, extracting the water phase by ethyl acetate, drying the extracted organic phase by anhydrous magnesium sulfate, concentrating under reduced pressure, and performing column chromatography to obtain a fourth intermediate.

Preferably, in the step 3.3), the solvent used for the reaction is one or more of methanol, ethanol, N-dimethylformamide and tetrahydrofuran; the reaction temperature is 30-80 ℃, the reaction time is 12-24 hours, the molar ratio of the fourth intermediate to the dihydroxylamine is 1: 1.1-1: 2, and after the reaction is finished, the fifth intermediate (crude product) is obtained by directly concentrating under reduced pressure.

Preferably, in the step 3.4), the solvent used for the reaction is one or more of water, methanol, tetrahydrofuran, dichloromethane and chloroform, and the oxidant is one or more of sodium periodate or lead dioxide; the reaction temperature is 0-25 ℃, the reaction time is 30-60 min, the molar ratio of the fourth intermediate to the oxidant is 1: 1-1: 1.5, the organic phase is separated after the reaction is finished, and the organic phase is dried, decompressed, concentrated and subjected to column chromatography sequentially through anhydrous magnesium sulfate to obtain the NO donor compound.

Preferably, in the above step 3.2) and 3.4), the eluent for column chromatography is ethyl acetate and petroleum ether (volume ratio is 1: 5-1: 1).

Preferably, the preparation method of the NO donor compound specifically comprises the following steps:

4.1) using p-formylbenzoic acid as a starting material, and reacting the p-formylbenzoic acid with an acyl chloride reagent contained in a solvent under reflux conditions to prepare a sixth intermediate (a compound 8 in the embodiment);

4.2) carrying out intermolecular condensation reaction on the sixth intermediate and L-proline or D-proline in a solvent containing an acid-binding agent to prepare a seventh intermediate (for example, a compound 9L in the embodiment);

4.3) carrying out intermolecular condensation reaction on the seventh intermediate and 5-ISMN in a solvent containing a condensing agent and a catalyst to prepare an eighth intermediate (for example, a compound 10L in the example);

4.4) carrying out a condensation reaction on the eighth intermediate and dihydroxylamine (2, 3-dimethyl-2, 3-dihydroxyaminobutane) under reflux conditions to obtain a ninth intermediate (for example, a compound 11L in the example);

4.5) carrying out oxidation reaction on the ninth intermediate and an oxidant to prepare a compound (for example, the compound 12L in the embodiment) with the structure shown in the formula IV.

Preferably, in the step 4.1), the acid chloride reagent is one or more of thionyl chloride and oxalyl chloride, and the solvent is one or more of dichloromethane and toluene; the reaction temperature is 80-90 ℃, the reaction time is 5-24 hours, and the molar ratio of the p-formylbenzoic acid to the acyl chlorination reagent is 1: 3-1: 10; after the reaction is finished, the reaction solution is concentrated under reduced pressure to obtain a sixth intermediate (crude product).

Preferably, in the step 4.2), the acid-binding agent is one or more of triethylamine and diisopropylethylamine, and the solvent is one or more of dichloromethane, trichloromethane and toluene; the reaction temperature is 25-80 ℃, the reaction time is 5-20 h, the molar ratio of the p-formylbenzoic acid to the L-proline or the molar ratio of the p-formylbenzoic acid to the D-proline is 1: 1-1: 2, and the dosage of the acid-binding agent is controlled to be 2.5-4.0 equivalent (relative to the multiple of the amount of the p-formylbenzoic acid); and after the reaction is finished, adding water for dilution, adjusting the pH value to 8-10, washing with dichloromethane, separating the water phase, adjusting the pH value to 2-4, extracting the organic phase with ethyl acetate, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and carrying out column chromatography to obtain a seventh intermediate.

Preferably, in the step 4.3), the condensing agent is one or more of N, N' -Dicyclohexylcarbodiimide (DCC), Carbonyldiimidazole (CDI), Diisopropylcarbodiimide (DIC), and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDCI), the catalyst is one or more of 4-Dimethylaminopyridine (DMAP) and 1-hydroxybenzotriazole (HOBt), and the solvent is one or more of tetrahydrofuran, acetonitrile, benzene, dichloromethane, and N, N-dimethylformamide; the reaction temperature is 25-30 ℃, the reaction time is 15-20 h, the molar ratio of the 5-ISMN to the seventh intermediate is 1: 1-1: 2, the dosage of the catalyst is controlled to be 1-10% (relative to the 5-ISMN), and the molar ratio of the 5-ISMN to the condensing agent is 1: 1-1: 2; and after the reaction is finished, filtering, washing the filtrate by hydrochloric acid and saturated sodium chloride in sequence, extracting the water phase by using ethyl acetate, drying the extracted organic phase by using anhydrous magnesium sulfate, concentrating under reduced pressure, and carrying out column chromatography to obtain an eighth intermediate.

Preferably, in the step 4.4), the solvent used for the reaction is one or more of methanol, ethanol, N-dimethylformamide and tetrahydrofuran; the reaction temperature is 30-85 ℃, the reaction time is 12-24 hours, the molar ratio of the eighth intermediate to the dihydroxylamine is 1: 1.1-1: 2, and after the reaction is finished, the pressure is directly reduced and the concentration is carried out to obtain a ninth intermediate (crude product).

Preferably, in the step 4.5), the solvent used for the reaction is one or more of water, methanol, tetrahydrofuran, dichloromethane and chloroform, and the oxidant is one or more of sodium periodate or lead dioxide; the reaction temperature is 0-25 ℃, the reaction time is 30-60 min, the molar ratio of the eighth intermediate to the oxidant is 1: 1-1: 1.5, the organic phase is separated after the reaction is finished, and the organic phase is dried, decompressed, concentrated and subjected to column chromatography sequentially through anhydrous magnesium sulfate to obtain the NO donor compound.

Preferably, in the step 4.2), the column chromatography eluent is ethyl acetate and petroleum ether (in a volume ratio of 10: 1-1: 1), and in the steps 4.3) and 4.5), the column chromatography eluent is ethyl acetate and petroleum ether (in a volume ratio of 1: 5-1: 1).

The NO donor compound can be used as an active ingredient for preparing a medicament for treating ischemic heart disease, and particularly can be used for preparing a medicament for treating myocardial ischemia/reperfusion (MI/R) injury.

Preferably, the NO donor compound has activity in protecting ischemic myocardium.

The NO donor compound can be used as an active ingredient for preparing a medicament for treating hypertension.

Preferably, the dosage form of the medicament is a dosage form which can enable the active ingredients to effectively reach the body (specifically to the blood circulation system), and is specifically selected from common dosage forms such as tablets, capsules, powder, granules, syrup, solutions, suspensions, injections, tinctures, oral liquids, aerosols, buccal agents, medicinal granules, pills, powders and the like or sustained-release dosage forms such as nano-preparations and the like.

Preferably, the medicament may contain minor ingredients and/or pharmaceutically acceptable carriers which do not affect the effectiveness of the active ingredient in addition to the active ingredient, such as sweeteners to improve taste, antioxidants to prevent oxidation, and adjuvants necessary for various preparations.

The invention has the beneficial effects that:

the invention combines 5-ISMN and NIT type nitroxide free radicals to form a series of NO donor molecules (namely NO donor compounds with bifunctional groups) with the function of resisting oxidative stress, which can be used for preparing medicaments for treating hypertension and ischemic heart disease.

Drawings

FIG. 1 is a schematic diagram of the structure of a bifunctional NO donor compound formed by connecting NIT type nitroxide radicals to 5-ISMN.

FIG. 2 is a schematic diagram of a synthetic route of compound II-1 in one of its steps.

FIG. 3 is a second scheme showing the steps of the synthesis of compound II-1.

FIG. 4 is a single crystal structural view of compound II-1.

FIG. 5 is a schematic diagram of a synthetic route of compound III-5 in one of the steps.

FIG. 6 shows a second scheme of the synthesis of compound III-5 in two steps.

FIG. 7 is a third scheme showing the synthesis scheme of compound III-5 in steps.

FIG. 8 is a scheme showing one of the steps of the synthesis of compound 12L.

FIG. 9 is a second schematic step-by-step scheme of the synthesis of compound 12L.

Fig. 10 shows the results of the test of the activity of the objective compound against oxidative damage to cells (× 100%).

FIG. 11 shows the results of the activity test of Compound 12L in a mouse model of hypertension (in the figure, PE indicates epinephrine, Ach indicates acetylcholine, and Relaxation indicates the degree of vasodilation).

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention.

Referring to fig. 1, the invention combines NIT type nitroxide free radical and 5-ISMN by connecting group to obtain a series of novel NO donor compounds, the series of compounds simultaneously contain nitroxide free radical structural unit and 5-ISMN structural unit, and experimental results show that the molecular structure of the compounds can continuously and efficiently eliminate NO at NO release position accuratelyHas important function, thereby providing direct protection effect on ischemic myocardium and overcoming the drug resistance of nitrate drugs.

1. Preparation of Compound II-1

Referring to fig. 2, p-formylbenzoic acid (0.75g,5mmol), 5-ISMN (0.96g,5mmol), and DMAP (0.06g,0.5mmol) were added to 50mL of dried Tetrahydrofuran (THF), cooled to 0 ℃, DCC (1.03g,5mmol) was added, the mixture was reacted at room temperature for 16h, after disappearance of the starting material point was detected by TLC, dicyclohexylurea was removed by filtration, the filtrate was washed twice with 0.5mol/L hydrochloric acid, twice with a saturated aqueous sodium bicarbonate solution, the organic phase was dried over anhydrous magnesium sulfate, the solvent was evaporated in vacuo to give a white solid, and column chromatography was performed with an eluent of ethyl acetate: petroleum ether ═ 2:1(v/v) to obtain a white solid (compound 1). Compound 1 yield 66.7%; and Mp: 96.3-98 ℃;¹H NMR(400MHz,CDCl₃)δ10.11(s,1H),8.18(d,J＝7.2Hz,1H),7.96(d,J＝7.2Hz,1H),5.50(s,1H),5.40(s,1H),5.09(d,J＝4.8Hz,1H),4.65(d,J＝4.8Hz,1H),4.23–4.15(m,1H),4.15–4.01(m,1H),3.95(dd,J＝11.2,5.5Hz,1H)。

referring to FIG. 3, compound 1(0.5g,1.55mmol) is dissolved in 15mL of methanol, bis-hydroxylamine (0.28g,1.86mmol) is added and the temperature is raised to 80 ℃ for refluxing 24h, TLC detection of disappearance of starting material, solvent is evaporated in vacuo, residue is dissolved in 32mL of Dichloromethane (DCM), NaIO is added dropwise at 0 ℃₄Solution (NaIO)₄0.33g,1.55mmol), stirring for 30min, separating the organic layer, washing the aqueous phase with DCM, combining the organic phases and washing with saturated brine, separating the organic layer, anhydrous Na₂SO₄Drying and filtering, evaporating the solvent in vacuum, and carrying out column chromatography on the residue by using an eluant of ethyl acetate and petroleum ether (1: 2 (v/v)) to obtain a blue solid (a compound 3). The yield of the compound 3 (i.e. the compound II-1) is 73.1 percent; IR (KBr,400-4000 cm)^-1):3501，2928，2359，1722，1645，1450，1362，1281，1097，852，768，696，542，HRMS:450.1499。

Referring to FIG. 4, the solvent for single crystal growth of compound 3 is dichloromethane, ethyl acetate and petroleum ether (1:1: 5-1: 3: 5). The single crystal structure demonstrates the correctness of the structural formula.

2. Preparation of Compound III-5

Referring to fig. 5, vanillin (3g,19.7mmol) is dissolved in 1mol/L NaOH, chloroacetic acid (2.05g, 21.7mmol) is added and stirred for 1h at room temperature (so that phenolic hydroxyl is converted into salt form, which is beneficial to subsequent reaction), then the temperature is raised to 80 ℃ and reflux is carried out for 12h, after the reaction is finished, the reaction solution is diluted by water, ethyl acetate is used for washing, the water phase is separated and acidified by concentrated hydrochloric acid (36% -38%), the mixed solution is extracted by ethyl acetate, saturated NaHCO is added₃The combined organic phases were separated by aqueous phase, the aqueous phase was acidified with concentrated hydrochloric acid and filtered to give a white solid (compound 4). Compound 4 yield was 38%; and Mp: 133.8-135.8 ℃.

Referring to FIG. 6, compound 4(0.98g,4.66mmol), 5-ISMN (0.89g,4.66mmol), and DMAP (0.057g,0.47mmol) were added to 27mL of dried DCM and DCC (0.96g,4.66mmol) was added at 0 ℃. The mixture is stirred for 30h at room temperature, after TLC monitoring the reaction is complete, the dicyclohexylurea precipitate is filtered, the filtrate is washed twice with 0.5mol/L hydrochloric acid and saturated aqueous NaCl solution, the organic phases are separated, the combined organic phases are saturated aqueous NaHCO₃Washing with water solution, mixing water phases, extracting with ethyl acetate twice, mixing organic phases, and adding anhydrous Na₂SO₄After drying, spin-drying under reduced pressure, and purification by column chromatography using an eluent of ethyl acetate to petroleum ether 1:1(v/v) gave a white solid (compound 5) with a yield of compound 5 of 88.97%, Mp: 122.8-113.6℃；¹H NMR(400MHz,CDCl₃)δ9.87(s,1H),7.42(dd,J＝11.2,3.1Hz,2H),6.87(d,J＝8.1Hz,1H),5.34(dd,J＝8.7,2.6Hz,2H),4.94(s,1H),4.81(s,2H),4.48(d,J＝5.0Hz,1H),4.11(d,J＝7.2Hz,1H),4.07–3.97(m,3H),3.95(s,3H),3.90(dd,J＝11.4,5.5Hz,1H),2.04(s,1H),1.25(d,J＝2.0Hz,1H)。

Referring to FIG. 7, compound 5(1.48g, 3.86mmol) and 2.3-dimethyl-2, 3-dihydroxyaminobutane (0.69g, 4.63mmol) were dissolved in 30mL of methanol, heated to 80 ℃ and refluxed for 22h, after completion of the reaction, the solvent was evaporated off, 75mL of DCM was added to the residue, after cooling to 0 ℃ under ice bath, NaIO was added₄(0.83g, 3.86mmol) was dissolved in 48mL of water and added thereto, stirred for 30min, the organic phase was separated, the aqueous phase was washed with DCM and the organic phases were combined, the organic phase was passed over anhydrous NaSO₄After drying, spin-drying, column chromatography using ethyl acetate-petroleum ether-1: 2(v/v) eluent, and purification gave a dark blue solid (compound 7). The yield of compound 7 (i.e., compound III-5) was 21.5%; IR (KBr,400-4000 cm)^-1):3452，2959，1705，1634，1568，1510，1440，1298，1207，1169，1016，922，912，843，758，735，671，609，515，488.HRMS:510.1600。

The single crystal culture solvent of the compound 7 adopts dichloromethane, ethyl acetate and petroleum ether (1:1: 5-1: 3:5), and the correctness of the structural formula is proved by a single crystal structure.

3. Preparation of Compounds III-3 and III-8

By replacing the starting material (vanillin) for the preparation of compound III-5 with 3-bromo-4-hydroxybenzaldehyde and 2-bromo-4-hydroxybenzaldehyde, III-3 and III-8 can be prepared, respectively, according to the same procedure.

4. Preparation of Compound 12L

Referring to FIG. 8, after refluxing p-formylbenzoic acid (2g,13.3mmol) and thionyl chloride (4.76g,40mmol, i.e., thionyl chloride) in 26mL of toluene at 90 ℃ for 5h, the solvent and excess thionyl chloride were evaporated under reduced pressure to give a pale yellow oily liquid, i.e., p-formylbenzoyl chloride (Compound 8). L-proline (1.53g, 13.3mmol) was reacted with 2mL triethylamine (Et) under ice-bath conditions₃N) was dissolved in dry 33mL of dichloromethane, stirred in ice bath, and the pale yellow oily liquid was extracted with 10Diluting with mL of DCM, adding the diluted DCM into a reaction system by using a constant-pressure funnel, reacting for 15 hours at room temperature, adding water for dilution, adjusting the pH to be alkaline (the pH is 8-10) by using 1mol/L NaOH, washing by using dichloromethane, separating a water phase, adjusting the pH to be 2-4, extracting for multiple times by using ethyl acetate, combining organic phases, and using anhydrous Na₂SO₄After drying, spin-drying and purification by column chromatography using an eluent of ethyl acetate-petroleum ether-10: 1(v/v) gave 2.98g of the product (compound 9L). Compound 9L yield 90.5%, light yellow oil;¹H NMR(500MHz,CDCl₃)δ10.02(s,1H),8.14(s,2H),7.91(d,J＝8.1Hz,2H),7.69(d,J＝8.1Hz,2H),4.67(dd,J＝8.4,5.3Hz,1H),3.56(dd,J＝6.8,3.3Hz,1H),3.50–3.39(m,1H),2.33(dd,J＝13.0,8.0Hz,1H),2.18–2.07(m,2H),2.01(dd,J＝13.1,6.6Hz,1H),1.90(dd,J＝12.9,6.5Hz,1H)。

referring to FIG. 9, compound 9L (2.5g,10.3mmol), 5-ISMN (2.0g,10.3mmol) and DMAP (0.13g,0.1mmol) were dissolved in 70mL of dry THF, DCC (2.83g,13.7mmol) was added at 0 deg.C, the mixture was reacted at room temperature for 18h, after completion of the TLC monitoring reaction, dicyclohexylurea was precipitated by filtration, the filtrate was washed twice with 0.5mol/L hydrochloric acid and saturated aqueous NaCl solution, respectively, the organic phase was separated and the organic phase was washed with saturated aqueous NaHCO solution₃Washing once, combining water phases, extracting the water phases twice by ethyl acetate, combining organic phases and passing through anhydrous Na₂SO₄After drying, the solvent was removed in vacuo and column chromatography was performed with an eluent of petroleum ether and ethyl acetate 1:1(v/v) to give a white solid (compound 10L). Compound 10L yield 65%;¹H NMR(400MHz,CDCl3)δ9.99(s,1H),7.83(s,2H),7.70(s,2H),4.60(s,1H),4.11(d,J＝7.1Hz,1H),3.53(dd,J＝94.2,54.8Hz,4H),2.14(s,1H),2.04(s,1H),1.83(d,J＝78.1Hz,2H)。

refluxing compound 10L (1.76g, 4.2mmol) and 2.3-dimethyl-2, 3-dihydroxyaminobutane (1.0g, 7.14mmol) in 80mL methanol at 85 deg.C for 24h, after TLC detection of reaction completion, vacuum drying, adding 80mL DCM to the residue (oily residue), cooling to 0 deg.C in ice bath, and adding NaIO₄(0.9g, 4.2mmol) was dissolved in 40mL of water and added thereto, stirred for 30min and the organic phase separated, the aqueous phase washed with DCM and the organic phases combined, over anhydrous Na₂SO₄After drying, spin-drying and column chromatography with eluent of petroleum ether ethyl acetate ═ 2:1(v/v), purification gave a dark blue solid (compound 12L). Compound 12L yield 83.5%; IR (KBr,400-4000 cm)^-1):3445，2955，2923，1745，1632，1414，1389，1364，1306，1281，1202，1167，1132，1082，1020，968，851，766，669，542。

The compound 12L single crystal culture solvent adopts dichloromethane, ethyl acetate and petroleum ether (1:1: 5-1: 3:5), and the single crystal structure proves the correctness of the structural formula.

The enantiomer of compound 12L prepared above was compound 12D, the preparation process was essentially the same except that L-proline was replaced by D-proline:

5. preparation of Compound V-2

In a 25mL two-necked round bottom flask equipped with a reflux condenser were placed 3-bromo-4-hydroxybenzaldehyde (201.02mg,1mmol), bishydroxylamine (177.85mg,1.2mmol) and 8mL of methanol, and the mixture was reacted at 80 ℃ for 60 hours. After the reaction, the cooled reaction product was transferred to a 50mL round bottom flask, methanol was removed under reduced pressure, the spin-dried residue was dissolved in 8mL dichloromethane, stirred at 0 ℃ under nitrogen protection, 12.4mL sodium periodate (213.89mg,1mmol) was slowly added dropwise, the reaction was carried out at 0 ℃ for about 30min, the organic phase was separated, the aqueous phase was washed with DCM, the organic phases were combined, and anhydrous Na was added₂SO₄After drying, spin-drying and column chromatography with petroleum ether, ethyl acetate 2:1(v/v) eluent, purification yielded 125.5mg of a dark blue solid (compound v-2). Yield of compound V-2 38.2%, Mp: 208.1-210.2 ℃; IR (KBr,400-4000 cm)^-1):2922,1718,1653,1558,1456,1273,1126,1078,964,827；HRMS(ESI)calculated for C₁₃H₁₆B_rN₂O₃[M+H]⁺:328.04171,found:328.04060；EPR(CH₂Cl₂):g factor,2.0083；a_N，6.5G。

The starting material 3-bromo-4-hydroxybenzaldehyde was replaced with p-formylbenzoic acid, 2-bromo-4-hydroxybenzaldehyde and vanillin, and V-1, V-3 and V-4 were prepared according to the same method. These resulting compounds containing NIT-type nitroxide radicals were prepared for activity control experiments.

Preparation example of NO Donor drug tablets

After 10g of each of compounds II-1, III-3, III-8, III-5 and 12L was mixed with 87.5g of an auxiliary material (Baihu Jing: lactose 7:3, mass ratio), 95% ethanol was added to granulate, dried, sized (sieved), 2.5g of sodium stearate was added thereto and mixed uniformly and then tableted to obtain a tablet having a weight of 100mg per tablet and a content of compound II-1, III-3, III-8, III-5 or 12L of 10 mg.

Preparation example of NO Donor drug powder injection

1g of each of the compounds II-1, III-3, III-8, III-5 and 12L is dissolved in 170mL of mannitol respectively, after primary mixing, the volume is determined to be 200mL, the obtained solution is filtered and filled into penicillin bottles with 1mL of solution in each bottle, and freeze-drying, sealing and sterilizing are carried out to obtain freeze-dried powder injection containing 5mg of the compounds II-1, III-3, III-8, III-5 or 12L in each bottle.

Preparation example of NO Donor drug Capsule

15g of each of the compounds II-1, III-3, III-8, III-5 and 12L was mixed with 135g of an adjuvant (white lake essence: lactose: 7:3, mass ratio), and then granulated with 95% ethanol, dried, granulated (sieved) and encapsulated, wherein the weight of each granule is about 150mg, and the content of the compound II-1, III-3, III-8, III-5 or 12L is about 15 mg.

9. Endothelial cell oxidative damage protection (CCK-8 measuring free radical) experiment and in-vitro vasodilation function detection

9.1. Experimental Material

Experimental reagents and drugs: fetal Bovine Serum (FBS), 0.25% pancreatin-0.02% EDTA, penicillin-streptomycin, low sugar DMEM basal medium (LD-DMEM), all purchased from Gibco; tert-butyl hydroperoxide solution (TBHP), CCK-8.

Experimental cells: human Umbilical Vein Endothelial Cell (HUVEC)

9.2. Cell culture

Preparing a culture solution: 50mL of LD-DMEM medium was removed, followed by 50mL of FBS, and 5mL of penicillin-streptomycin.

Cell recovery: dissolving the cryovial containing HUVEC cells in 37 deg.C water bath by gentle shaking, slowly adding cell suspension into 15mL centrifuge tube, centrifuging for 5min in centrifuge, sucking out supernatant, resuspending with prepared 10mL culture solution, adding the resuspension solution into 10cm cell culture dish, placing into 37 deg.C 5% CO₂The cell culture box of (2) and the liquid is changed the next day. When the growth reaches more than 80%, digesting and passaging by using a 0.25% pancreatin-0.02% EDTA solution. After three passages, CCK-8 experiment is carried out.

9.3 Effect of bifunctional NO Donor Compounds on TBHP-injured HUVEC

Taking cells with good cell activity in logarithmic growth phase, preparing into cell suspension with DMEM culture solution containing fetal calf serum, and making into 6 × 10 cells per well⁴Cell concentration per mL, 100. mu.L per well of 96-well plate, and then the plate was incubated at 37 ℃ with 5% CO₂Culturing in a cell culture box until the cells adhere to the wall overnight. Absorbing the culture medium, adding DMEM culture medium into a blank control group (Ctrl group), adding prepared DMEM culture medium containing different drugs (Tempol, prepared NO donor compounds with multiple bifunctional groups such as II-1, III-5, III-3, III-8, 12L and the like and control compounds V-1, V-2, V-3, V-4) into an administration group and an experimental group (TBHP is added into the experimental group before administration), wherein the concentration is 5 mu mol/mL, and adding DMEM culture medium containing TBHP into the TBHP group. Each set was prepared in duplicate wells of 100. mu.L each. After 6h, absorbing the supernatant in the pore plate, adding 100 mu L of CCK-8 into each pore in a light-proof ultra-clean workbench, wrapping a 96-pore plate by using tinfoil paper, placing the 96-pore plate in a shaking table at 37 ℃, shaking for 2h at a low speed, and measuring the absorbance value at the position of 450nm of wavelength by using an enzyme labeling instrument after 2 h. Experimental results show that the NO donor compounds 12L, III-8 and III-3 (figure 10) with the bifunctional groups have better protection on endothelial cellsEffect (Compounds with endothelial cell protective Effect also include II-1, III-5, 12D), and NO donor molecules III-8, III-3, etc. can overcome the disadvantage of oxidative stress caused by existing NO donor molecules (e.g. 5-ISMN).

The experimental results show that the NO donor compound with the bifunctional group provided by the invention has a remarkable protective effect on an antioxidant stress model of Human Umbilical Vein Endothelial Cells (HUVEC). Therefore, the compound can be used as an active ingredient for preparing a medicament for treating ischemic heart disease and has obvious clinical application potential.

In order to further prove the application value of the prepared NO donor compound with the bifunctional group in treating cardiovascular diseases such as ischemic heart disease, the in vitro vasodilation function detection is carried out. Experimental results show that the NO donor compounds 12L, III-8, III-3, II-1, III-5 and 12D of the bifunctional groups have vasodilator activity.

10. Extended experiment of pharmacological action of bifunctional NO Donor Compound (experiment takes 12L as an example)

A mouse hypertension model is constructed by taking continuous infusion of angiotensin II (Ang II) micro-pump as a means, 12L (20mg/kg, once a day for 28 days) is injected into the abdominal cavity of a mouse while the Ang II is infused, and the blood pressure and the vascular function of the mouse are detected in the period. Experimental results as shown in fig. 11, it can be seen that the 12L treatment significantly reduced blood pressure and improved vascular function in the hypertensive model mice (the degree of vasodilation shown in the figure is the experimental result on day 28 during the experiment). These results not only validate the vasodilatory activity of the above bifunctional NO donor compounds, but also indicate their potential for the treatment of hypertension.

In a word, the NO donor compound with the bifunctional group can be used for preparing NO donor medicaments, and has a protection effect on hypertension, ischemic myocardium and the like (and has the potential of being developed into NO donor medicaments for treating ischemic heart diseases without obvious drug resistance).