A HNO donor compound and preparation method thereof

Abstract

The present invention provides a compound that can hydrolyze and release HNO under physiological conditions. This compound can release HNO under conditions similar to those of the human body, providing a new donor for HNO research. Furthermore, while releasing HNO, the compound provided by the present invention can monitor changes in fluorescence intensity following HNO release using fluorescence detection technology, enabling visualization of HNO release and combining HNO release with the application of fluorescent molecular probes.

Description

Technical Field

[0001] The present invention relates to a compound with a new structure and a preparation method thereof, in particular to a compound capable of releasing HNO by hydrolysis and a preparation method thereof. Background Art

[0002] Hydrogen nitrosyl (HNO) is the one-electron reduction product of nitric oxide (NO). As a reactive nitrogen and reactive oxygen species (RHOS), HNO has unique reactivity and pharmacology compared with its other redox counterparts, making it a hot topic for research and discussion by many scholars at home and abroad.

[0003] In the West, heart failure is the leading cause of hospitalization for many patients, and the mortality rate among hospitalized patients with heart failure is as high as 10%. Numerous studies have documented the widespread biomedical applications of HNO, including its reported positive inotropic effects and protective effects in experimental heart failure settings. HNO has been called a "miracle drug" capable of completely curing heart failure and cardiovascular and cerebrovascular diseases. Furthermore, there are documented reports that HNO can effectively inhibit the growth and reproduction of tumor cells. Given the high incidence of cancer and tumors in China, research on HNO is crucial and will continue to expand.

[0004] However, there are few known compounds in the prior art that can provide hydrogenated nitrosyl. As research on HNO increases, the currently known compounds can no longer fully meet people's research on HNO. Summary of the Invention

[0005] The object of the present invention is to address the deficiencies of the above-mentioned prior art and to provide a new HNO donor compound that can effectively release HNO and a preparation method thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is: an HNO donor compound, characterized in that the structural formula of the compound is as follows:

[0007]

[0008] Wherein, R is selected from the following groups:

[0009]

[0010] As a preferred embodiment of the HNO donor compound of the present invention, the compound can release hydrogenated nitrosyl at a pH of 7-10.

[0011] As a preferred embodiment of the HNO donor compound of the present invention, the compound can also release a fluorescent molecular probe.

[0012] As a preferred embodiment of the HNO donor compound of the present invention, the molar ratio of the hydrogenated nitrosyl released by the compound to the fluorescent molecular probe is 1:1.

[0013] In addition, another object of the present invention is to provide a method for synthesizing the above-mentioned HNO donor compound using coumarin-6-sulfonyl chloride as a raw material. To achieve this object, the technical solution adopted by the present invention is as follows:

[0014] When the R group in the structural formula of the HNO donor compound is H, the preparation method of the HNO donor compound comprises the following steps:

[0015] (1) Add NH2OH·HCl and triethylamine to a reaction flask, stir evenly, and then add tetrahydrofuran;

[0016] (2) adding coumarin-6-sulfonyl chloride to step (1) and reacting at room temperature to obtain a milky white reaction solution;

[0017] (4) Pour the milky white reaction solution obtained in step (2) into a separatory funnel, add ethyl acetate to the separated reaction solution, and then extract with water to obtain a colorless oily liquid in the upper organic phase;

[0018] (4) Anhydrous MgSO4 solid is added to the obtained colorless oily liquid and dried. After drying, the MgSO4 solid particles in the system are removed by suction filtration, and the solvent is removed to obtain white solid particles. The product is obtained after vacuum drying, that is, the HNO donor compound.

[0019] When, in the structural formula of the HNO donor compound, R is a group other than H, the preparation method of the HNO donor compound comprises the following steps:

[0020] (1) Add NH2OH·HCl and triethylamine to a reaction flask, stir evenly, and then add tetrahydrofuran;

[0021] (2) adding coumarin-6-sulfonyl chloride to step (1) and reacting at room temperature to obtain a milky white reaction solution;

[0022] (5) Pour the milky white reaction solution obtained in step (2) into a separatory funnel, add ethyl acetate to the separated reaction solution, and then extract with water to obtain a colorless oily liquid in the upper organic phase;

[0023] (4) adding anhydrous MgSO4 solid to the obtained colorless oily liquid and drying it. After drying, the MgSO4 solid particles in the system were removed by suction filtration, and the solvent was removed to obtain white solid particles, which were then vacuum dried to obtain the intermediate;

[0024] (5) Under N2 atmosphere and ice-water bath conditions, weigh the intermediate obtained in step (4) and pour it into a reaction flask, and add anhydrous THF to dissolve it;

[0025] (6) Add triethylamine to the reaction flask, then slowly add the acylating agent to the reaction flask. After 10 to 40 minutes, remove the ice-water bath and stir the reaction at room temperature for 5 to 10 hours.

[0026] (7) The solvent was dried using a rotary evaporator to obtain a white viscous solution, and then the crude product was dissolved in EtOAc, poured into a separatory funnel, and washed with water to remove water-soluble impurities. The obtained organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was again dried using a rotary evaporator to dry the solvent. The obtained preliminary product was dried in a vacuum drying oven at room temperature to obtain the HNO donor compound.

[0027] The acylating agent is selected from acetyl chloride, 2,2,2-trifluoroacetyl chloride, propionyl chloride, 2-pyridinecarbonyl chloride, pivaloyl chloride, 4-chlorobenzoyl chloride, 4-fluorobenzoyl chloride, 4-methoxybenzoyl chloride, 4-(tert-butyl)benzoyl chloride, 4-cyanobenzoyl chloride, 2-naphthoyl chloride, 3-chlorobenzoyl chloride, 3-fluorobenzoyl chloride, quinoline-2-carbonyl chloride, 3-methoxybenzoyl chloride, 2,4,6-trimethylbenzoyl chloride, At least one of benzoyl chloride, 4-nitrobenzoyl chloride, 3,5-difluorobenzoyl chloride, 4-bromo-3-methylbenzoyl chloride, benzoyl chloride, 4-(trifluoromethyl)benzoyl chloride, 2,6-difluorobenzoyl chloride, methyl 4-chloroformylbenzoate, 3-(trifluoromethoxy)benzoyl chloride, diphenylcarbamoyl chloride, 2-(4-chlorophenyl)-3-methylbutanoyl chloride, and 2-phenoxybenzoyl chloride.

[0028] In a preferred embodiment of the method for preparing the HNO donor compound of the present invention, the molar ratio of NH2OH·HCl to coumarin-6-sulfonyl chloride in step (2) is: NH2OH·HCl: coumarin-6-sulfonyl chloride = 2.5 to 6:1. In a more preferred embodiment of the method for preparing the HNO donor compound of the present invention, the molar ratio of NH2OH·HCl to coumarin-6-sulfonyl chloride in step (2) is: NH2OH·HCl: coumarin-6-sulfonyl chloride = 5:1.

[0029] As a preferred embodiment of the method for preparing the HNO donor compound of the present invention, the molar volume ratio of NH2OH·HCl to triethylamine in step (1) is: NH2OH·HCl:triethylamine = 32-37 mmol / ml. As a preferred embodiment of the method for preparing the HNO donor compound of the present invention, the molar volume ratio of NH2OH·HCl to triethylamine in step (1) is: NH2OH·HCl:triethylamine = 20.5 mmol / ml.

[0030] In a preferred embodiment of the method for preparing the HNO donor compound of the present invention, the molar volume ratio of NH2OH·HCl to tetrahydrofuran in step (1) is: NH2OH·HCl:tetrahydrofuran = 1.01-1.1:1 mmol / ml. In a more preferred embodiment of the method for preparing the HNO donor compound of the present invention, the molar volume ratio of NH2OH·HCl to tetrahydrofuran in step (1) is: NH2OH·HCl:tetrahydrofuran = 20.5:20 mmol / ml.

[0031] In a preferred embodiment of the method for preparing the HNO donor compound of the present invention, the reaction in step (2) is carried out at room temperature for 12 to 20 hours. In a more preferred embodiment of the method for preparing the HNO donor compound of the present invention, the reaction in step (2) is carried out at room temperature for 14 to 18 hours. In a most preferred embodiment of the method for preparing the HNO donor compound of the present invention, the reaction in step (2) is carried out at room temperature for 16 hours.

[0032] As a preferred embodiment of the method for preparing the HNO donor compound of the present invention, step (1) is as follows: 1.425 g of NH2OH·HCl (M: 69.5N: 20.5 mmol) is weighed into a reaction flask, 0.583 ml of Et3N is pipetted into the reaction flask and stirring is started, and then 20 ml of tetrahydrofuran (THF) solution is pipetted into the reaction flask using a syringe.

[0033] As a preferred embodiment of the method for preparing the HNO donor compound of the present invention, step (2) is as follows: 1.0 g of coumarin-6-sulfonyl chloride (M: 243.96 N: 4.10 mmol) is weighed and slowly added to a reaction flask, and the reaction is carried out at room temperature for 16 h. The reaction liquid is milky white.

[0034] In a preferred embodiment of the method for preparing the HNO donor compound of the present invention, ethyl acetate is first added to the reaction solution after separation in step (3), and then extraction is performed with deionized water. In a more preferred embodiment of the method for preparing the HNO donor compound of the present invention, when deionized water is used for extraction in step (3), 60 ml of deionized water is used each time, and the washing and extraction are performed three times in total.

[0035] As a preferred embodiment of the method for preparing the HNO donor compound of the present invention, saturated brine is added during the extraction in step (3). Adding saturated brine during the extraction in step (3) can achieve a better separation effect. Preferably, the volume ratio of the saturated brine to ethyl acetate is: saturated brine:ethyl acetate = 15:80.

[0036] As a preferred embodiment of the method for preparing the HNO donor compound of the present invention, the vacuum drying time in step (4) is 24 to 48 hours.

[0037] In a preferred embodiment of the method for preparing the HNO donor compound of the present invention, the molar ratio of the acylating agent to the intermediate in step (6) is: acylating agent: intermediate = 1.1-1.3:1. In a more preferred embodiment of the method for preparing the HNO donor compound of the present invention, the molar ratio of the acylating agent to the intermediate in step (6) is: acylating agent: intermediate = 0.457:0.415.

[0038] As a preferred embodiment of the method for preparing the HNO donor compound of the present invention, the molar ratio of triethylamine to the intermediate in step (6) is: triethylamine:intermediate=1.1-1.3:1. As a more preferred embodiment of the method for preparing the HNO donor compound of the present invention, the molar ratio of triethylamine to the intermediate in step (6) is: triethylamine:intermediate=0.457:0.415.

[0039] As a preferred embodiment of the method for preparing the HNO donor compound of the present invention, when water washing is used to remove water-soluble impurities in step (7), water washing is performed three times, with the amount of water used each time being 100 ml.

[0040] As a preferred embodiment of the method for preparing the HNO donor compound of the present invention, the initial product in step (7) is dried in a vacuum drying oven at room temperature for 24 hours.

[0041] Finally, the present invention also provides the use of the aforementioned HNO donor compounds in the preparation of drugs for treating heart failure, cardiovascular disease, and inhibiting tumor cells. The compounds described herein are capable of effectively releasing HNO and can serve as HNO donor compounds, providing new HNO donors for HNO research. HNO, as an endogenous signaling molecule, can effectively protect the cardiovascular system, improve ventricular diastolic and systolic function, reduce cardiac workload, and increase cardiac output, thereby achieving a therapeutic effect. Furthermore, HNO can effectively inhibit the growth and reproduction of tumor cells. Therefore, the HNO donor compounds described herein provide new pharmaceutical options for treating heart failure, cardiovascular disease, and tumors.

[0042] The HNO donor compound provided by the present invention can effectively release HNO, providing a new HNO donor for HNO research. The preparation method of the HNO donor compound of the present invention uses coumarin-6-sulfonyl chloride as a raw material, has simple process steps, and is easy to scale up. The HNO donor compound of the present invention, which releases HNO as a single-electron reduction product of NO, has been hailed as a "miracle drug" capable of completely treating heart failure and cardiovascular and cerebrovascular diseases due to its unique reactivity and pharmacological properties. It also inhibits the growth and proliferation of tumor cells, providing a new medical option for the treatment of heart failure, cardiovascular diseases, and tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The synthetic route of one embodiment of the HNO donor compound of the present invention is as follows;

[0044] Figure 2 A synthetic route diagram of another embodiment of the HNO donor compound of the present invention;

[0045] Figure 3 This is a hydrogen nuclear magnetic resonance spectrum of an embodiment of the HNO donor compound of the present invention;

[0046] Figure 4 This is a carbon NMR spectrum of an embodiment of the HNO donor compound of the present invention;

[0047] Figure 5 This is a mass spectrum of an embodiment of the HNO donor compound of the present invention;

[0048] Figure 6 This is a kinetic degradation test diagram of an embodiment of the HNO donor compound of the present invention;

[0049] Figure 7 This is a UV-visible spectrum of the reaction between the HNO donor compound and VB12 according to an embodiment of the present invention;

[0050] Figure 8 The reaction of the HNO donor compound and the phosphorus-containing compound according to one embodiment of the present invention 31 P NMR detection diagram.

[0051] Figure 9 This is a fluorescence data detection diagram of an embodiment of the HNO donor compound of the present invention.

[0052] Figure 10 FIG1 is another fluorescence data detection diagram of an embodiment of the HNO donor compound of the present invention.

[0053] Figure 11This is a fluorescence spectrum diagram of an embodiment of the HNO donor compound of the present invention.

[0054] Figure 12 This is a standard curve for estimating the released HNO concentration based on the fluorescence intensity of one embodiment of the HNO donor compound of the present invention. DETAILED DESCRIPTION

[0055] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present application will be further described below in conjunction with specific embodiments.

[0056] The present invention discloses an HNO donor compound, the structural formula of the compound is as follows:

[0057]

[0058] Wherein R is selected from the following groups

[0059]

[0060] When the above R selects H group, the synthetic route of the compound is as shown in the attached Figure 1 As shown, the preparation method comprises the following steps:

[0061] (1) Add NH2OH·HCl and triethylamine to a reaction flask, stir evenly, and then add tetrahydrofuran;

[0062] (2) adding coumarin-6-sulfonyl chloride to step (1) and reacting at room temperature to obtain a milky white reaction solution;

[0063] (3) Pour the milky white reaction solution obtained in step (2) into a separatory funnel, add ethyl acetate to the separated reaction solution, and then extract with water to obtain a colorless oily liquid in the upper organic phase;

[0064] (4) Anhydrous MgSO4 solid is added to the obtained colorless oily liquid and dried. After drying, the MgSO4 solid particles in the system are removed by suction filtration, and the solvent is removed to obtain white solid particles. The product is obtained after vacuum drying, that is, the HNO donor compound.

[0065] When the above-mentioned R is selected from groups other than H, the synthetic route of the compound is as shown in the attached Figure 2 As shown, the preparation method comprises the following steps:

[0066] (1) Add NH2OH·HCl and triethylamine to a reaction flask, stir evenly, and then add tetrahydrofuran;

[0067] (2) adding coumarin-6-sulfonyl chloride to step (1) and reacting at room temperature to obtain a milky white reaction solution;

[0068] (3) Pour the milky white reaction solution obtained in step (2) into a separatory funnel, add ethyl acetate to the separated reaction solution, and then extract with water to obtain a colorless oily liquid in the upper organic phase;

[0069] (4) adding anhydrous MgSO4 solid to the obtained colorless oily liquid and drying it. After drying, the MgSO4 solid particles in the system were removed by suction filtration, and the solvent was removed to obtain white solid particles, which were then vacuum dried to obtain the intermediate;

[0070] (5) Under N2 atmosphere and ice-water bath conditions, weigh the intermediate obtained in step (4) and pour it into a reaction flask, and add anhydrous THF to dissolve it;

[0071] (6) Add triethylamine to the reaction flask, then slowly add the acylating agent to the reaction flask. After 10 to 40 minutes, remove the ice-water bath and stir the reaction at room temperature for 5 to 10 hours.

[0072] (7) The solvent was dried using a rotary evaporator to obtain a white viscous solution, and then the crude product was dissolved in EtOAc, poured into a separatory funnel, and washed with water to remove water-soluble impurities. The obtained organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was again dried using a rotary evaporator to dry the solvent. The obtained preliminary product was dried in a vacuum drying oven at room temperature to obtain the HNO donor compound.

[0073] The acylating agent is selected from acetyl chloride, 2,2,2-trifluoroacetyl chloride, propionyl chloride, 2-pyridinecarbonyl chloride, pivaloyl chloride, 4-chlorobenzoyl chloride, 4-fluorobenzoyl chloride, 4-methoxybenzoyl chloride, 4-(tert-butyl)benzoyl chloride, 4-cyanobenzoyl chloride, 2-naphthoyl chloride, 3-chlorobenzoyl chloride, 3-fluorobenzoyl chloride, quinoline-2-carbonyl chloride, 3-methoxybenzoyl chloride, 2,4,6-trimethylbenzoyl chloride, At least one of benzoyl chloride, 4-nitrobenzoyl chloride, 3,5-difluorobenzoyl chloride, 4-bromo-3-methylbenzoyl chloride, benzoyl chloride, 4-(trifluoromethyl)benzoyl chloride, 2,6-difluorobenzoyl chloride, methyl 4-chloroformylbenzoate, 3-(trifluoromethoxy)benzoyl chloride, diphenylcarbamoyl chloride, 2-(4-chlorophenyl)-3-methylbutanoyl chloride, and 2-phenoxybenzoyl chloride.

[0074] When the above R is an H group, the HNO donor compound (C1) can be directly and simply synthesized by using coumarin-6-sulfonyl chloride as a raw material; when the above R is a group other than an H group, the compound (C1) is further reacted with different acylating agents as an intermediate to finally obtain the compound in which the above R is a group other than H.

[0075] Example 1

[0076] An embodiment of the HNO donor compound of the present invention, the structure of the HNO donor compound in this embodiment is shown below:

[0077]

[0078] The synthetic route of the HNO donor compound described in this embodiment is shown in the attached diagram. Figure 1 As shown, the preparation method comprises the following steps:

[0079] (1) Weigh 1.425 g of NH2OH·HCl (M: 69.5 N: 20.5 mmol) into a reaction flask. Use a pipette to inject 0.583 ml of Et3N into the reaction flask and begin stirring. Then use a syringe to inject 20 ml of tetrahydrofuran (THF) solution into the reaction flask.

[0080] (2) Weigh 1.0 g of coumarin-6-sulfonyl chloride (M: 243.96 N: 4.10 mmol) and slowly add it to the reaction flask described in step (1). React at room temperature for 16 h to obtain a milky white reaction solution.

[0081] (3) The milky white reaction solution obtained in step (2) was poured into a separatory funnel, 80.0 ml of ethyl acetate was added to the separated reaction solution, and the mixture was extracted with deionized water (3 x 60.0 ml). 15.0 ml of saturated brine was added each time for better separation effect. The upper organic phase was a colorless oily liquid.

[0082] (4) The colorless oily liquid in the upper organic phase was poured into a beaker, and an appropriate amount of anhydrous MgSO4 solid was added for drying. After drying, the MgSO4 solid particles in the system were removed by filtration, and the mixture was poured into a round-bottom flask and rotary evaporated to remove the solvent. The white solid particles obtained after rotary evaporation adhered to the wall of the flask. The flask was placed in a vacuum drying oven and vacuum dried for 24 h to obtain 0.84 g of the product, which is the HNO donor compound described in this example.

[0083] Example 2

[0084] An embodiment of the HNO donor compound of the present invention, the structural formula of the HNO donor compound in this embodiment is as follows:

[0085]

[0086] Wherein R is the following group:

[0087]

[0088] The preparation method of the HNO donor compound described in this embodiment comprises the following steps:

[0089] (1) Weigh 1.425 g of NH2OH·HCl (M: 69.5 N: 20.5 mmol) into a reaction flask. Use a pipette to inject 0.583 ml of Et3N into the reaction flask and begin stirring. Then use a syringe to inject 20 ml of tetrahydrofuran (THF) solution into the reaction flask.

[0090] (2) Weigh 1.0 g of coumarin-6-sulfonyl chloride (M: 243.96 N: 4.10 mmol) and slowly add it to the reaction flask described in step (1). React at room temperature for 16 h to obtain a milky white reaction solution.

[0091] (3) The milky white reaction solution obtained in step (2) was poured into a separatory funnel, 80.0 ml of ethyl acetate was added to the separated reaction solution, and the mixture was extracted with deionized water (3 x 60.0 ml). 15.0 ml of saturated brine was added each time for better separation effect. The upper organic phase was a colorless oily liquid.

[0092] (4) Pour the colorless oily liquid of the upper organic phase into a beaker, add an appropriate amount of anhydrous MgSO4 solid and dry it. After drying, remove the MgSO4 solid particles in the system by suction filtration, pour it into a round-bottom flask and perform rotary evaporation to remove the solvent. The white solid particles obtained after rotary evaporation adhere to the wall of the flask. Place the flask in a vacuum drying oven and dry it under vacuum for 24 hours to obtain 0.84 g of the intermediate;

[0093] (5) Under N2 atmosphere and ice-water bath conditions, weigh the intermediate obtained in step (4) (100.0 mg, 0.415 mmol) and pour it into a reaction flask, and add anhydrous THF to dissolve;

[0094] (6) Et3N (0.063 ml, 0.457 mmol) was added to the reaction flask, and then trifluoroacetyl chloride (0.457 mmol) was slowly added to the reaction flask. After 10 to 40 minutes, the ice-water bath was removed and the reaction was stirred at room temperature for 5 to 10 hours.

[0095] (7) The solvent was dried using a rotary evaporator to obtain a white viscous solution. The crude product was then dissolved in EtOAc (50 ml) and poured into a separatory funnel. The water-soluble impurities were removed by washing with water (100 ml × 3). The obtained organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was again dried using a rotary evaporator to dry the solvent. The obtained initial product was dried in a vacuum drying oven at room temperature for 24 h to obtain the HNO donor compound described in this example.

[0096] Example 3

[0097] An embodiment of the HNO donor compound of the present invention, the structural formula of the HNO donor compound in this embodiment is as follows:

[0098]

[0099] Wherein R is the following group:

[0100]

[0101] The preparation method of the HNO donor compound described in this example is the same as that of Example 2, except for the different material selection of the acylating agent used. In the preparation method of the HNO donor compound described in this example, the acylating agent is pivaloyl chloride.

[0102] Example 4

[0103] An embodiment of the HNO donor compound of the present invention, the structural formula of the HNO donor compound in this embodiment is as follows:

[0104]

[0105] Wherein R is the following group:

[0106]

[0107] The preparation method of the HNO donor compound described in this example is the same as that of Example 2, except for the different material selection of the acylating agent used. In the preparation method of the HNO donor compound described in this example, the acylating agent is 4-methoxybenzoyl chloride.

[0108] Example 5

[0109] An embodiment of the HNO donor compound of the present invention, the structural formula of the HNO donor compound in this embodiment is as follows:

[0110]

[0111] Wherein R is the following group:

[0112]

[0113] The preparation method of the HNO donor compound described in this example is the same as that of Example 2, except for the different material selection of the acylating agent used. In the preparation method of the HNO donor compound described in this example, the acylating agent is p-tert-butylbenzoyl chloride.

[0114] Example 6

[0115] An embodiment of the HNO donor compound of the present invention, the structural formula of the HNO donor compound in this embodiment is as follows:

[0116]

[0117] Wherein R is the following group:

[0118]

[0119] The preparation method of the HNO donor compound described in this example is the same as that of Example 2, except for the different material selection of the acylating agent used. In the preparation method of the HNO donor compound described in this example, the acylating agent is 2-naphthoyl chloride.

[0120] Example 7

[0121] An embodiment of the HNO donor compound of the present invention, the structural formula of the HNO donor compound in this embodiment is as follows:

[0122]

[0123] Wherein R is the following group:

[0124]

[0125] The preparation method of the HNO donor compound described in this example is the same as that of Example 2, except for the different material selection of the acylating agent used. In the preparation method of the HNO donor compound described in this example, the acylating agent is m-fluorobenzoyl chloride.

[0126] Example 8

[0127] An embodiment of the HNO donor compound of the present invention, the structural formula of the HNO donor compound in this embodiment is as follows:

[0128]

[0129] Wherein R is the following group:

[0130]

[0131] The preparation method of the HNO donor compound described in this example is the same as that of Example 2, except for the different material selection of the acylating agent used. In the preparation method of the HNO donor compound described in this example, the acylating agent is quinoline-2-carbonyl chloride.

[0132] Example 9

[0133] An embodiment of the HNO donor compound of the present invention, the structural formula of the HNO donor compound in this embodiment is as follows:

[0134]

[0135] Wherein R is the following group:

[0136]

[0137] The preparation method of the HNO donor compound described in this example is the same as that of Example 2, except for the selection of the acylating agent used. In the preparation method of the HNO donor compound described in this example, the acylating agent is 2,4,6-trimethylbenzoyl chloride.

[0138] Example 10

[0139] An embodiment of the HNO donor compound of the present invention, the structural formula of the HNO donor compound in this embodiment is as follows:

[0140]

[0141] Wherein R is the following group:

[0142]

[0143] The preparation method of the HNO donor compound described in this example is the same as that of Example 2, except for the selection of the acylating agent used. In the preparation method of the HNO donor compound described in this example, the acylating agent is 4-bromo-3-methylbenzoyl chloride.

[0144] Example 11

[0145] An embodiment of the HNO donor compound of the present invention, the structural formula of the HNO donor compound in this embodiment is as follows:

[0146]

[0147] Wherein R is the following group:

[0148]

[0149] The preparation method of the HNO donor compound described in this example is the same as that of Example 2, except for the different material selection of the acylating agent used. In the preparation method of the HNO donor compound described in this example, the acylating agent is 2,6-difluorobenzoyl chloride.

[0150] Example 12

[0151] An embodiment of the HNO donor compound of the present invention, the structural formula of the HNO donor compound in this embodiment is as follows:

[0152]

[0153] Wherein R is the following group:

[0154]

[0155] The preparation method of the HNO donor compound described in this example is the same as that of Example 2, except for the different material selection of the acylating agent used. In the preparation method of the HNO donor compound described in this example, the acylating agent is m-trifluoromethoxybenzoyl chloride.

[0156] Example 13

[0157] An embodiment of the HNO donor compound of the present invention, the structural formula of the HNO donor compound in this embodiment is as follows:

[0158]

[0159] Wherein R is the following group:

[0160]

[0161] The preparation method of the HNO donor compound described in this example is the same as that of Example 2, except for the different material selection of the acylating agent used. In the preparation method of the HNO donor compound described in this example, the acylating agent is diphenylcarbamoyl chloride.

[0162] Example 14

[0163] An embodiment of the HNO donor compound of the present invention, the structural formula of the HNO donor compound in this embodiment is as follows:

[0164]

[0165] Wherein R is the following group:

[0166]

[0167] The preparation method of the HNO donor compound described in this example is the same as that of Example 2, except for the different material selection of the acylating agent used. In the preparation method of the HNO donor compound described in this example, the acylating agent is 2-phenoxybenzoyl chloride.

[0168] Example 15

[0169] The H NMR spectrum, C NMR spectrum, mass spectrum and kinetic degradation data of the compound obtained in Example 1 and the H NMR spectrum and kinetic degradation data of the compound obtained in Example 2 were tested by the following method:

[0170] H NMR spectrum: 2.0 mg of the compound was dissolved in 0.7 mL of deuterated dimethyl sulfoxide, the resulting solution was added to an NMR tube, and sent to a NMR testing center for testing. The test results of the compound described in Example 1 are shown in the attached figure. Figure 3 As shown. Figure 3 It can be seen from the hydrogen spectrum that the expected target product was synthesized in this application with high purity.

[0171] Carbon NMR spectrum: Dissolve 20 mg of the compound in 0.7 mL of deuterated dimethyl sulfoxide, add the resulting solution into an NMR tube, and send it to the NMR detection center for detection. The detection results of the compound described in Example 1 are shown in the attached figure. Figure 4 As shown. Figure 4 It can be seen that the target compound synthesized in this application is Figure 1 The reaction structure is correct and has high purity.

[0172] Mass spectrometry: Dissolve 1 mg of the compound in 10 mL of anhydrous methanol, then take 0.1 ml of the resulting solution and dissolve it in 10 ml of anhydrous methanol for dilution, and send it to the mass spectrometry detection center for detection. The detection results of the compound described in Example 1 are shown in the attached figure. Figure 5 As shown. Figure 5 It can be seen that the molecular weight of the compound described in Example 1 of the present application detected by mass spectrometry is consistent with the molecular weight calculated by the inventors of the present application.

[0173] Kinetic degradation data: The test conditions are: the compound (40 μM) is added to a 5:95 volume ratio (MeCN: phosphate buffer solution 0.1M, pH 7.4) at 37°C. An appropriate amount of the solution is placed in a cuvette and quickly placed in the sample tank of a UV-visible spectrometer for detection. The parameters are set to measure once every 15 minutes. The reaction rate constant obtained for the compound described in the example is 0.00767±4.27688×10-5s-1, and the reaction half-period t1 / 2 is: 90 minutes. The test results are shown in the attached figure. Figure 6 The compound described in Example 2 has a degradation half-cycle t at pH 12.0. 1 / 2 The degradation half-cycle at pH 10.0 is t 1 / 2 It is 63 minutes.

[0174] In order to prove that the HNO donor compound described in this application can be degraded under physiological conditions (pH 7.4, 37°C), it was tested using a UV-visible spectrophotometer. Figure 6 It can be clearly seen that relatively clear isosbestic points are located at 298.5 nm, 253 nm and 227 nm. This indicates that the donor compound of the present application undergoes a single degradation reaction under physiological conditions. Figure 6It can also be seen that at 235 nm, the donor is uniformly degraded under physiological conditions and the degradation process is very slow. The spectral data at 235 nm are used for the scatter plot (see Appendix Figure 6 The inset of Figure 3 shows that a nonlinear fit clearly shows that the complete decomposition of the HNO donor compound of the present invention requires approximately 498 minutes. The degradation trend curve also shows a first-order reaction (K = 0.00767 ± 4.27688 × 10-5 s-1, t1 / 2 = 90 min).

[0175] By the attached Figure 3 It can be seen that 1H NMR (400 MHz, DMSO-d6) δ9.72 (d, J = 3.2 Hz, 1H), 9.69 (d, J = 3.2 Hz, 1H), 8.26 (dd, J = 7.6, 4.0 Hz, 2H), 8.00 (dd, J = 8.8, 2.0 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 6.65 (d, J = 9.6 Hz, 1H).

[0176] The hydrogen nuclear magnetic resonance spectrum of the compound described in Example 2 is: 1H NMR (400 MHz, DMSO-d6) δ10.21 (s, 1H), 8.29 (dd, J=7.6, 4.0 Hz, 2H), 8.10 (dd, J=8.8, 2.0 Hz, 1H), 7.62 (d, J=8.8 Hz, 1H), 6.85 (d, J=9.6 Hz, 1H), 3.23 (s, 3H).

[0177] The detection methods and results of the compounds described in Examples 3 to 14, such as H NMR, C NMR, mass spectrometry and kinetic degradation data, are similar to those of the compounds described in Examples 1 and 2 and are not described in detail here.

[0178] Example 16

[0179] Detection of HNO production standards

[0180] 1. HNO donor and VB 12 The reaction was detected by UV-visible spectrometry to detect HNO production.

[0181] UV-visible spectra of the reaction of the compound obtained in Example 1 (100 μM) and HOCbl (50 μM) in an anaerobic phosphate buffer solution (pH 7.4, 0.1 M) mixed with anhydrous acetonitrile (95.5, v / v) were used, with each scan interval being 220 seconds. K = 0.12642 ± 0.00438, t 1 / 2 =33, the results are as follows Figure 7 shown.

[0182] By the attached Figure 7It is known that cobalamin (NOCbl) can be used to detect HNO production. We will utilize the reaction of cobalamin (NOCbl) with an HNO donor to detect HNO production. Within a short period of time, the pink solution of HOCbl can be directly observed to change to the characteristic orange color of nitroxylcobalamin (NOCbl). Clear isosbestic points at wavelengths of 493 and 341 nm are visible in the spectral data, indicating the single degradation of the compound in the mixed solution. A nonlinear fit of the equation using the data at 525 nm in the spectral data is also evident in the inset, showing that the overall trend indicates a first-order reaction and that the reaction of HNO with VB12 is relatively rapid (K = 0.12642 ± 0.00438, t½ = 5.5 minutes). All isosbestic points in the spectral data and the overall degradation trend are consistent with those reported in the literature, confirming the production of HNO.

[0183] 2. Use phosphorus-containing compounds to react with the compound of Example 1 31 P NMR detection

[0184] The phosphorus-containing compound (methyl-2-diphenylphosphine terephthalate) was reacted with a pH 10.2 buffer solution, the compound of Example 1, and a mixture of deuterated acetonitrile (CD3CN). 31 P NMR confirmed the formation of HNO. The results are shown in the attached Figure 8 shown.

[0185] By the attached Figure 8 It can be seen that HNO reacts with the organophosphorus compound to form phosphorus oxide 2 (δ 33.88 ppm) and phosphoramidite 3 (δ 34.52 ppm), and their integrated ratio is about 1:1, indicating the ability of the HNO donor to release HNO in a buffer solution of pH 10.2.

[0186] The compounds described in Examples 2 to 14 were tested using the same method, and all of them were able to demonstrate the generation of HNO, which will not be described in detail here.

[0187] Example 17

[0188] Fluorescence performance detection of the HNO donor compound of the present invention

[0189] The compound described in Example 1 of the present invention was used as the test object. The compound of Example 1 (50 μM) was mixed with a volume ratio of 5:95 (MeCN: phosphate buffer solution 0.1M, pH 7.4). The fluorescence performance was tested at a temperature of 25 degrees. The degraded compound was prepared in advance and placed in a 50-degree oven overnight. The test results are shown in the attached figure. Figures 9-11 shown.

[0190] Attachment Figure 9 and attached Figure 10Comparison of fluorescence intensity of HNO donor compounds before and after degradation at different excitation wavelengths is shown.

[0191] The HNO donor compound (50 micromolar) described in Example 1 of the present application was mixed in a phosphate buffer solution (pH 7.4, 0.1 M) and the excitation wavelength of the test was 365 nm. Figure 11 As shown in the figure, the fluorescence intensity of the HNO donor compound after degradation in phosphate buffer solution (pH 7.4, 0.1M) varies at different times. It can be seen that when the HNO donor is not degraded, its fluorescence intensity is very weak. Over time, the donor slowly degrades and its fluorescence intensity gradually increases. After 900 minutes, the donor compound is completely degraded in the phosphate buffer solution, and the fluorescence intensity also reaches the peak of the strongest fluorescence emission at 473nm.

[0192] The standard curve for estimating the released HNO concentration based on fluorescence intensity is shown in the attached figure. Figure 12 To further confirm the relationship between the concentration of the donor after complete degradation and the fluorescence intensity at the corresponding concentration, the fluorescence intensity at different concentrations (50M, 25M, 12.5M, 6.25M, 3.125M) was detected, and the minimum HNO release concentration was determined using the lowest fluorescence intensity corresponding to the concentration. Figure 12 A linear standard curve can be clearly observed (y = 18.08x-13.09, R2 = 0.999). According to the standard curve equation, the detection limit of the HNO concentration-releasing fluorescent probe reaction concentration is calculated to be approximately 0.72M.

[0193] The compounds described in Examples 2 to 14 were tested using the same method and had similar effects to the compound in Example 1, which will not be described in detail here.

[0194] By the above attachment Figures 3 to 12 It can be seen that the compounds described in the present application can release HNO with a half-life of several minutes to several hours under physiological and alkaline conditions, and the production of HNO under physiological conditions was successfully captured by fluorescence detection.

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An HNO donor compound, characterized in that The structural formula of the compound is as follows: Where R is H or .

2. The HNO donor compound according to claim 1, wherein The compound can release hydrogenated nitrosyl at a pH of 7-10.

3. The HNO donor compound according to claim 1 or 2, characterized in that The compounds are also capable of releasing fluorescent molecular probes.

4. The HNO donor compound according to claim 3, wherein The molar ratio of the hydronitrosyl released by the compound to the fluorescent molecular probe is 1:

1.

5. The method for preparing the HNO donor compound according to claim 1, wherein: The steps include: (1) Add NH2OH·HCl and triethylamine into the reaction flask, stir evenly, and then add tetrahydrofuran; (2) Add coumarin-6-sulfonyl chloride to step (1) and react at room temperature to obtain a milky white reaction solution; (3) Pour the milky white reaction solution obtained in step (2) into a separatory funnel, add ethyl acetate to the separated reaction solution, and then extract with water to obtain a colorless oily liquid in the upper organic phase; (4) adding anhydrous MgSO4 solid to the obtained colorless oily liquid and drying the mixture. After drying, the MgSO4 solid particles in the system are removed by filtration, and the solvent is removed to obtain white solid particles. The product is obtained by vacuum drying, i.e., the HNO donor compound. or (1) Add NH2OH·HCl and triethylamine into the reaction flask, stir evenly, and then add tetrahydrofuran; (2) Add coumarin-6-sulfonyl chloride to step (1) and react at room temperature to obtain a milky white reaction solution; (3) Pour the milky white reaction solution obtained in step (2) into a separatory funnel, add ethyl acetate to the separated reaction solution, and then extract with water to obtain a colorless oily liquid in the upper organic phase; (4) Add anhydrous MgSO4 solid to the obtained colorless oily liquid and dry it. After drying, remove the MgSO4 solid particles in the system by filtration, remove the solvent, and obtain white solid particles. After vacuum drying, obtain the intermediate; (5) Under N2 atmosphere and ice-water bath conditions, weigh the intermediate obtained in step (4) and pour it into a reaction flask, and add anhydrous THF to dissolve it; (6) Add triethylamine to the reaction flask, then slowly add the acylating agent to the reaction flask. After 10-40 minutes, remove the ice-water bath and stir the reaction at room temperature for 5-10 hours. (7) The solvent was dried using a rotary evaporator to obtain a white viscous solution, and then the crude product was dissolved in EtOAc, poured into a separatory funnel, and washed with water to remove water-soluble impurities. The obtained organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was again dried using a rotary evaporator to dry the solvent. The obtained initial product was dried in a vacuum drying oven at room temperature to obtain the HNO donor compound. The acylating agent is 2,2,2-trifluoroacetyl chloride.

6. The method for preparing the HNO donor compound according to claim 5, wherein: The molar ratio of NH2OH·HCl to coumarin-6-sulfonyl chloride in step (2) is: NH2OH·HCl: coumarin-6-sulfonyl chloride = 2.5-6:

1.

7. The method for preparing the HNO donor compound according to claim 5, wherein: At least one of the following conditions is met in step (1): (a) The molar volume ratio of NH2OH·HCl to triethylamine is: NH2OH·HCl:triethylamine = 32-37:1 mmol / ml; (b) The molar volume ratio of NH2OH·HCl to tetrahydrofuran is: NH2OH·HCl:tetrahydrofuran = 1.01-1.1:1 mmol / ml.

8. The method for preparing the HNO donor compound according to claim 5, wherein: At least one of the following conditions is met in step (6): (a) The molar ratio of the acylating agent to the intermediate is: acylating agent: intermediate = 1.1-1.3:1; (b) The molar ratio of triethylamine to the intermediate is: triethylamine:intermediate=1.1-1.3:

1.

9. The method for preparing the HNO donor compound according to claim 7 or 8, wherein: At least one of the following must be met: (1) In step (1), the molar volume ratio of NH2OH·HCl and triethylamine is: NH2OH·HCl:triethylamine = 20.5:0.583 mmol / ml; (2) The molar volume ratio of NH2OH·HCl to tetrahydrofuran in step (1) is: NH2OH·HCl:tetrahydrofuran = 20.5:20 mmol / ml; (3) The molar ratio of the acylating agent to the intermediate in step (6) is: acylating agent: intermediate = 0.457:0.415; (4) The molar ratio of triethylamine to the intermediate in step (6) is: triethylamine:intermediate=0.457:0.415.

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