A dual-responsive low-resistant nitric oxide donor and a preparation method and application thereof

Abstract

The application relates to the technical field of pharmaceutical chemistry, and discloses a double-response low-drug-resistance nitric oxide donor as well as a preparation method and application thereof, the low-drug-resistance nitric oxide donor is 5-mononitroisosorbide oxalate diester, has the following structural formula, and the preparation method adopts 5-mononitroisosorbide ester and oxalyl chloride or oxalic acid to react under the action of a catalyst to obtain the low-drug-resistance nitric oxide donor; the product prepared by the preparation method is high in purity and high in yield. The donor can realize double responses with different speeds in normal tissues and disease microenvironments, reduces side effects on normal tissues, reduces the generation of active nitrogen, reduces the nitration inactivation of related biological enzymes, avoids the drug resistance of a body to the nitric oxide donor. The donor is good in stability, long in half-life, can be used for preparing drugs for treating cardiovascular diseases and glaucoma-related diseases, and has good popularization and application values.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a dual-responsive, low-drug-resistance nitric oxide donor, its preparation method, and its applications. Background Technology

[0002] Nitric oxide (NO) is an important endogenous gaseous signaling molecule in the human body. Due to its high biological activity, it participates extensively in various physiological processes and plays a crucial regulatory role. Nitric oxide molecules are important in maintaining cardiovascular homeostasis, nerve signal transduction, and innate immune responses. Recent studies have also shown that nitric oxide is closely related to tumors, and certain concentrations of nitric oxide have significant effects on regulating tumor drug resistance, hypoxia, and apoptosis.

[0003] The research and development of exogenous nitric oxide donor drugs has received widespread attention due to the problem of low endogenous nitric oxide concentrations in the body caused by metabolic abnormalities in pathological conditions. The design and synthesis of nitric oxide donor drugs are of great significance in both basic research and clinical applications. For example, nitric oxide donors such as 5-mononitroisosorbide, nitroglycerin, and dinitroisosorbide are already being used clinically as first-line drugs for the treatment of cardiovascular diseases. Nitric oxide donor drugs release nitric oxide through cellular metabolism under physiological conditions to exert its physiological effects, and this has been one of the cutting-edge and hot topics in biomedical research in recent years.

[0004] For example, CN107459482A discloses a nitric oxide donor, its preparation and application, and discloses the following chemical formula. R2 is H, a C3-C8 cycloalkyl group, or optionally a C1-6 alkyl group substituted with 1-2 substituents selected from C1-4 alkoxy, -S(O)2-OH, and hydroxyl groups, wherein N is attached to the benzene ring of the fluorophore molecule. This compound can be used as a nitric oxide donor for hypertension and related diseases.

[0005] CN104119295A discloses a phenothiazine-based nitric oxide donor, its preparation method, and its uses. The compound's structural formula is as follows: n = 0, 1, or 2; R1 is: hydrogen, halogen, C1-C4 branched or straight-chain alkyl, halogenated C1-C4 branched or straight-chain alkyl; R2 is: hydrogen, C1-C4 branched or straight-chain alkyl. This compound is used in pharmaceutical compositions as an active ingredient, and in the preparation of drugs for treating breast cancer, lung cancer, and gastric cancer as an antitumor agent.

[0006] Nitric oxide itself is unstable and cannot be used directly in many medical settings. Nitric oxide donor drugs achieve efficient in vivo transport by loading nitric oxide and, to some extent, prolong its half-life. However, most NO donors currently have unsatisfactory stability, with half-lives typically ranging from seconds to hours under physiological conditions, often requiring cryogenic storage and transportation. This poses a significant challenge to the storage and biomedical applications of NO donors.

[0007] On the other hand, nitric oxide has a half-life of only 3–6 seconds and a diffusion radius of only 40–200 μm in vivo, which requires that the nitric oxide donor ideally be converted into NO at the lesion site to exert its effect. Furthermore, many nitric oxide donors easily induce drug resistance to NO after use, resulting in a significant reduction in the physiological efficacy of NO upon re-administration. This is mainly because high levels of reactive oxygen species in the disease microenvironment convert NO into reactive nitrogen species, oxidizing or nitrifying the enzymes involved in the metabolism of nitric oxide donors, causing them to become inactive and lose their ability to produce NO. Clinically, this problem is usually only addressed by discontinuing the medication, which increases the risk of developing related diseases. Even with such treatment, patients often develop stronger drug resistance after several cycles, frequently requiring longer withdrawal periods and higher doses, which increases drug-related toxicity. Therefore, the development of low-resistance nitric oxide donors is of great significance in disease treatment and has broad application prospects. Summary of the Invention

[0008] This invention addresses the problems of poor stability, short half-life, and easy induction of drug resistance in existing nitric oxide donors by providing a dual-response, low-drug-resistance nitric oxide donor. This donor is stable, has a long half-life, and can be slowly hydrolyzed by esterases in normal tissues, avoiding burst release of nitric oxide and reducing the occurrence of drug resistance. Furthermore, under the stimulation of high concentrations of reactive oxygen species in the disease microenvironment, it rapidly releases the nitric oxide donor drug, reducing the nitrification inactivation of related biological enzymes and further preventing the body from developing drug resistance to the nitric oxide donor.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A dual-responsive nitric oxide donor with low drug resistance, wherein the low drug resistance nitric oxide donor is 5-mononitroisosorbide oxalate diester, having the following structural formula:

[0011]

[0012] This invention also provides a method for preparing the dual-response, low-drug-resistance nitric oxide donor, using either method one or method two:

[0013] Method 1 specifically includes the following steps: reacting 5-mononitroisosorbide (ISN) with oxaloyl chloride (OC) under the action of a catalyst to obtain the low-drug-resistance nitric oxide donor, namely 5-mononitroisosorbide oxalate diester (Di-ISN-Oxalate); the specific reaction formula can be represented as follows:

[0014]

[0015] Method two specifically includes the following steps: reacting 5-mononitroisosorbide (ISN) with oxalic acid (OA) under the action of a catalyst to obtain the low-drug-resistance nitric oxide donor, namely, 5-mononitroisosorbide oxalate diester (Di-ISN-Oxalate). The specific reaction formula can be represented as follows:

[0016]

[0017] In Method 1, the molar ratio of 5-mononitroisosorbide ester to oxaloyl chloride is 0.5:1-3:1, preferably 1.5-2.5:1, and more preferably 2:1.

[0018] In Method 2, the molar ratio of 5-mononitroisosorbide ester to oxalic acid is 0.5:1-1:3, preferably 1.5-2.5:1, and more preferably 2:1.

[0019] In Method 1 or Method 2, the solvent used includes one or more of dichloromethane, chloroform, acetonitrile, tetrahydrofuran, ethyl acetate, toluene, dioxane, or dimethyl sulfoxide. The stronger the hydrophilicity of the solvent, the lower its volatility, and the lower the product yield, while having less impact on purity. The stronger the Lewis basicity of the solvent, the lower the product yield and the lower the purity. Preferably, the solvent used is one or more of chloroform and dichloromethane.

[0020] In Method 1, the catalyst comprises any one or more of sodium carbonate, sodium bicarbonate, potassium carbonate, cesium carbonate, triethylamine, trimethylamine, benzimidazole, 4-dimethylaminopyridine, and pyridine; the molar ratio of the catalyst to oxalyl chloride is 0.1 to 5:1.

[0021] Preferably, the catalyst is any one or more of triethylamine, 4-dimethylaminopyridine, and pyridine. When oxalyl chloride is used as the reaction substrate, triethylamine has the best solubility as a catalyst in organic solvents. The resulting hydrochloride is more separable than other organic bases, and its efficiency is significantly higher than that of the inorganic catalyst K2CO3, which is insoluble in organic solutions. The yield and purity are also higher.

[0022] More preferably, the catalyst in Method 1 is triethylamine (TEA), and the molar ratio of the catalyst to oxalyl chloride is 0.5 to 2:1.

[0023] In Method 2, the catalyst comprises one or more of dicyclohexylcarbodiimide, 4-dimethylaminopyridine, 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NHS), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC); the molar ratio of the catalyst to oxalic acid is 0.1–5:1. Preferably, the molar ratio of the catalyst to oxalic acid is 0.5–2:1.

[0024] Preferably, in method two, the catalyst is a mixture of hydroxybenzotriazole (HOBt) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC).

[0025] In Method 1, oxalyl chloride is added dropwise. During the dropwise addition, the reaction temperature is -20 to 0°C, and the dropwise addition time is 0.5 to 3 hours. After the dropwise addition is completed, the temperature is raised to -5 to 25°C, and the reaction time is increased to more than 0.5 hours. Oxayl chloride is greatly affected by temperature; when the initial reaction temperature increases, the product yield decreases significantly. Therefore, a relatively low temperature should be maintained during the initial dropwise addition, and the reaction temperature can be appropriately increased during the subsequent reaction.

[0026] Preferably, in Method 1, the reaction time is more than 2 hours after the oxalyl chloride is added. The inventors found through experiments that the yield can reach more than 95% when the reaction time of Method 1 is more than 2 hours. After that, the yield increases slightly when the reaction time is extended. More preferably, the reaction time in Method 1 is 2 to 12 hours, including 4 hours, 6 hours, 8 hours, etc.

[0027] Method 2 includes the following steps: activating oxalic acid with a catalyst and then reacting it with 5-mononitroisosorbide to obtain the low-drug-resistance nitric oxide donor. The activated catalyst exhibits better catalytic performance and a higher reaction yield.

[0028] The activation temperature is below 25°C, and the reaction temperature after activation is 20-45°C, with a reaction time of 8 hours or more. Preferably, the reaction time in Method 2 is 12 hours or more, and the yield can reach 60% or more; more preferably, the reaction time in Method 2 is 12-48 hours, including 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, and 36 hours.

[0029] Preferably, the products obtained by method one or method two are purified by extraction and precipitation, and then dried. The desiccant is selected from one or more of phosphorus pentoxide, anhydrous magnesium sulfate, or anhydrous sodium sulfate. The drying method is selected from one or more of hot drying and freeze drying.

[0030] The present invention also provides the use of the aforementioned dual-responsive, low-drug-resistance nitric oxide donor in the preparation of drugs for the treatment or prevention of cardiovascular disease and / or pulmonary hypertension.

[0031] The inventors discovered that this donor can decompose under the stimulation of high concentrations of reactive oxygen species in inflamed tissues or esterases in normal tissues, releasing the nitric oxide donor 5-mononitroisosorbide ester. This ester can be used to treat cardiovascular diseases and glaucoma-related diseases. It can be used to prepare drugs of this type, such as intramuscular, intravenous, or subcutaneous injections, sublingual or swallowed tablets, inhalable formulations, and topical or ocular solutions or ointments, achieving good therapeutic effects in the treatment of cardiovascular diseases and glaucoma. It has broad application potential in drug development and has significant potential for widespread application.

[0032] The low-drug-resistance nitric oxide donor exhibits stable performance under physiological conditions and has a long half-life of over 45 days. Compared to the half-life of typical nitric oxide donors in the prior art, which ranges from a few seconds to several hours, this represents a significant improvement and advancement. It also achieves dual-response effects in different environments and has minimal side effects on normal tissues.

[0033] The rapid decomposition process of the dual-response nitric oxide donor of the present invention under high concentration of reactive oxygen species stimulation is as follows:

[0034]

[0035] The slow degradation process of the dual-responsive nitric oxide donor of the present invention under stimulation by normal tissue esterases is as follows:

[0036]

[0037] The dual-response nitric oxide donor in this invention can first effectively consume reactive oxygen species and then release nitric oxide in response, thereby reducing the generation of toxic reactive nitrogen species and the nitrification inactivation of related biological enzymes, reducing the occurrence of drug resistance, and overcoming the drug resistance problem of nitrate ester nitric oxide donors in the prior art.

[0038] The present invention also provides a medicament for treating or preventing cardiovascular disease, pulmonary hypertension or glaucoma, comprising the aforementioned dual-responsive low-drug-resistance nitric oxide donor, wherein the amount of the donor is approximately 10 nM-10 μM (4.36 μg-4.36 mg) per kilogram.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) The dual-response low-drug-resistant nitric oxide donor provided in this invention is a novel structure reported for the first time. Its preparation method is simple, and the yield can reach more than 60%, and up to more than 95%, which provides effective theoretical support for industrial mass production.

[0041] (2) The low-drug-resistant nitric oxide donor in this invention can achieve the effect of slow hydrolysis and release of nitric oxide donor drug 5-mononitroisosorbitol ester in normal tissues and rapid release of nitric oxide donor drug under the stimulation of reactive oxygen species in the disease microenvironment. It achieves dual response in different environments, which reduces the side effects on normal tissues, reduces the generation of reactive nitrogen, reduces the nitrification inactivation of related biological enzymes, and avoids the body from developing drug resistance to nitric oxide donor.

[0042] (3) The low-drug-resistant nitric oxide donor in this invention has good stability and a long half-life, and can be used in the preparation of drugs for the treatment of cardiovascular diseases and glaucoma-related diseases, and has good application value. Attached Figure Description

[0043] Figure 1 The 1H NMR spectrum of 5-mononitroisosorbitol oxalate diester (Di-ISN-Oxalate) obtained in Example 1.

[0044] Figure 2 This is a high-resolution mass spectrometry of the 5-mononitroisosorbitol oxalate diester (Di-ISN-Oxalate) obtained in Example 1.

[0045] Figure 3 This is a comparison of the decomposition curves of the dual-response nitric oxide donor Di-ISN-Oxalate obtained in Example 1 in different environments.

[0046] Figure 4 The graph shows the reactive oxygen species scavenging ability of the dual-response nitric oxide donor Di-ISN-Oxalate obtained in Example 1.

[0047] Figure 5 This is a bar chart showing the ratio of the competitive activity of the dual-response nitric oxide donor Di-ISN-Oxalate obtained in Example 1 against endothelial cells relative to smooth muscle cells.

[0048] Figure 6 The bar chart shows the level of nitrosine (NT) protein after co-incubating the dual-response nitric oxide donor Di-ISN-Oxalate obtained in Example 1 with the clinically commonly used nitric oxide carrier drug isosorbide dinitrate (DISN) and H2O2-treated cells.

[0049] Figure 7 The study aimed to evaluate the vasodilatory effects of Di-ISN-Oxalate obtained in Example 1 and the clinically commonly used nitric oxide carrier drug isosorbide dinitrate (DISN) in in vitro perfusion and its drug resistance after repeated administration.

[0050] Figure 8The graph shows the stability curves of Di-ISN-Oxalate obtained in Example 1 in different environments. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.

[0052] All raw materials used in the following specific implementation methods were purchased from the market.

[0053] Example 1

[0054] 19 g of 5-mononitroisosorbide (ISN) was dissolved in 100 mL of chloroform, 12 g of oxaloyl chloride (OC) was added, followed by 20 μL of N-dimethylformamide (DMF). The mixture was stirred at 0 °C for 2 hours, then heated to 25 °C and 10 mL of triethylamine (TEA) was added. The mixture was stirred for another 10 hours. After removing most of the solvent by rotary evaporation under reduced pressure, the solid product was collected after washing with water and then freeze-dried under vacuum to obtain 5-mononitroisosorbide oxalate diester (Di-ISN-Oxalate) with a yield of 95% and a purity of 99%.

[0055] The obtained 1H NMR spectrum and high-resolution mass spectrum of Di-ISN-Oxalate are as follows: Figure 1 and Figure 2 As shown, 5-mononitroisosorbitol oxalate diester was successfully prepared.

[0056] Example 2

[0057] 19 g of 5-mononitroisosorbide (ISN) was dissolved in 100 mL of chloroform, 12 g of oxaloyl chloride (OC) was added, followed by 20 μL of N-dimethylformamide (DMF). The mixture was stirred at 0 °C for 2 hours, then heated to 25 °C and stirred for another 10 hours. After removing most of the solvent by rotary evaporation under reduced pressure, the solid product was washed with water and collected. The solid product was then freeze-dried under vacuum to obtain 5-mononitroisosorbide oxalate diester (Di-ISN-Oxalate), with a yield of 40% and a purity of 98%.

[0058] Example 3

[0059] 19 g of 5-mononitroisosorbide (ISN) was dissolved in 100 mL of chloroform, 12 g of oxaloyl chloride (OC) was added, followed by 20 μL of N-dimethylformamide (DMF). The mixture was stirred at 25 °C for 2 hours, then 10 mL of triethylamine (TEA) was added and stirring continued for 10 hours. After removing most of the solvent by rotary evaporation under reduced pressure, the solid product was collected after washing with water and then freeze-dried under vacuum to obtain 5-mononitroisosorbide oxalate diester (Di-ISN-Oxalate), with a yield of 30% and a purity of 95%.

[0060] Example 4

[0061] 19 g of 5-mononitroisosorbide (ISN) was dissolved in 100 mL of chloroform, 12 g of oxaloyl chloride (OC) was added, followed by 20 μL of N-dimethylformamide (DMF). The mixture was stirred at 0 °C for 2 hours, then heated to 25 °C and 10 mL of triethylamine (TEA) was added. The mixture was stirred for another 10 hours. The mixture was extracted with water, 0.1 M dilute hydrochloric acid, and saturated NaCl solution, respectively. The organic phases were collected and combined, dried with anhydrous magnesium sulfate, and most of the dichloromethane was removed by rotary evaporation under reduced pressure. The solid was collected and dried in a vacuum oven to obtain 5-mononitroisosorbide oxalate diester (Di-ISN-Oxalate), with a yield of 95% and a purity of 90%.

[0062] Example 5

[0063] 19 g of 5-mononitroisosorbide (ISN) was dissolved in 100 mL of chloroform, 12 g of oxaloyl chloride (OC) was added, followed by 20 μL of N-dimethylformamide (DMF). The mixture was stirred at 0 °C for 2 hours, then heated to 25 °C and 27 g of potassium carbonate (K₂CO₃) was added. The mixture was stirred for another 10 hours. After filtration, the organic phase was extracted with water, 0.1 M dilute hydrochloric acid, and saturated NaCl solution, respectively. The organic phases were collected and combined, dried over anhydrous magnesium sulfate, and most of the dichloromethane was removed by rotary evaporation under reduced pressure. The solid was collected and dried in a vacuum oven to obtain 5-mononitroisosorbide oxalate diester (Di-ISN-Oxalate), with a yield of 60% and a purity of 90%.

[0064] Example 6

[0065] 4.5 g of oxalic acid (OA), 13.5 g of 1-hydroxybenzotriazole (HOBt), and 19.2 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) were dissolved in 100 mL of DMF. After stirring at 25 °C for 2 hours, 8.5 g of 5-mononitroisosorbide (ISN) was added, and the mixture was stirred at 45 °C for 24 hours. After removing most of the solvent by rotary evaporation under reduced pressure, 100 mL of dichloromethane was added, and the mixture was extracted with water, 0.1 M dilute hydrochloric acid, and saturated NaCl solution, respectively. The organic phases were collected and combined, dried with anhydrous magnesium sulfate, and then removed most of the dichloromethane by rotary evaporation under reduced pressure. The solid was collected and dried in a vacuum oven to obtain 5-mononitroisosorbide oxalate diester (Di-ISN-Oxalate), with a yield of 35% and a purity of 95%.

[0066] Example 7

[0067] 4.5 g of oxalic acid (OA), 13.5 g of 1-hydroxybenzotriazole (HOBt), and 19.2 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) were dissolved in 100 mL of dichloromethane. After stirring at 25 °C for 2 hours, 8.5 g of 5-mononitroisosorbide (ISN) was added, and the mixture was stirred at 45 °C for 24 hours. The mixture was extracted with water, 0.1 M dilute hydrochloric acid, and saturated NaCl solution, respectively. The organic phases were collected and combined, dried with anhydrous magnesium sulfate, and most of the dichloromethane was removed by rotary evaporation under reduced pressure. The collected solid was dried in a vacuum oven, washed with water, and freeze-dried under vacuum to obtain 5-mononitroisosorbide oxalate diester (Di-ISN-Oxalate), with a yield of 85% and a purity of 98%.

[0068] Example 8

[0069] 4.5 g of oxalic acid (OA), 11.5 g of N-hydroxysuccinimide (NHS), and 19.2 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) were dissolved in 100 mL of dichloromethane. After stirring at 25 °C for 2 hours, 8.5 g of 5-mononitroisosorbide (ISN) was added, and the mixture was stirred at 45 °C for 24 hours. The mixture was extracted with water, 0.1 M dilute hydrochloric acid, and saturated NaCl solution, respectively. The organic phases were collected and combined, dried with anhydrous magnesium sulfate, and most of the dichloromethane was removed by rotary evaporation under reduced pressure. The collected solid was dried in a vacuum oven, washed with water, and freeze-dried under vacuum to obtain 5-mononitroisosorbide oxalate diester (Di-ISN-Oxalate), with a yield of 10% and a purity of 65%.

[0070] Example 9

[0071] 4.5 g of oxalic acid (OA) was dissolved in 100 mL of dichloromethane and stirred at 25 °C for 2 hours. Then, 8.5 g of 5-mononitroisosorbide (ISN) was added, and the mixture was stirred at 45 °C for 24 hours. The mixture was extracted with water, 0.1 M dilute hydrochloric acid, and saturated NaCl solution, respectively. The organic phases were collected and combined, dried with anhydrous magnesium sulfate, and most of the dichloromethane was removed by rotary evaporation under reduced pressure. The collected solid was dried in a vacuum oven, washed with water, and freeze-dried under vacuum to obtain 5-mononitroisosorbide oxalate diester (Di-ISN-Oxalate), with a yield of 1% and a purity of 60%.

[0072] Based on the product results of the above embodiments, the yield of Di-ISN-Oxalate in the above embodiments is approximately 1≈4≥7≥5≥6≥3≥8≥1, and the product purity is approximately 1≈2≈3≈7≥4≈5≈6≥8≥9. Considering all factors, Example 1 is the optimal one. In the preparation of Di-ISN-Oxalate, oxalyl chloride has significantly higher activity than oxalic acid, and the required catalyst formulation is simpler. Di-ISN-Oxalate can still be prepared under catalyst-free conditions, although the yield is lower but the purity is not affected.

[0073] When oxalyl chloride is used as the reaction substrate, triethylamine, as a catalyst, exhibits better solubility in organic solvents and is significantly more efficient than the inorganic catalyst K₂CO₃, which is insoluble in organic solutions, resulting in higher yields and purity. Oxayl chloride is highly sensitive to temperature; as the initial reaction temperature increases, the product yield decreases significantly.

[0074] When oxalic acid is used as the reaction substrate, the HOBt and EDC catalyst systems exhibit higher yields and purity, with significantly higher efficiency than the NHS and EDC catalyst systems, and also higher than the system without a catalyst. DMF is miscible with water and has a higher boiling point, making it more difficult to remove than dichloromethane. Therefore, using DMF as a solvent affects product precipitation, resulting in a lower yield, but it does not affect product purity (the yield is higher when dichloromethane is used as the solvent than when DMF is miscible with water). During the preparation process, the impurity exchange efficiency during extraction is lower than that during water washing, leading to lower product purity. Therefore, low-boiling-point solvents such as chloroform and dichloromethane are preferred.

[0075] Example 10

[0076] Following the 10-hour reaction conditions of Example 1, the reaction time for preparing Di-ISN-Oxalate after heating was replaced with 0.5 hours, 1 hour, 3 hours, and 24 hours, respectively. The yields of Di-ISN-Oxalate obtained were 87%, 90%, ≥95%, and ≥95%, respectively, and the purities were 98%, 98%, ≥98%, and ≥98%, respectively.

[0077] It is evident that the reaction time in this step has a significant impact on the results. When the reaction time is within 3 hours, the reaction yield gradually increases with time. However, when the reaction time is more than 3 hours, the yield can reach more than 95%. Subsequently, further extending the reaction time does not result in a qualitative change in the yield.

[0078] Example 11

[0079] Following the reaction conditions of Example 7, only the reaction time for preparing Di-ISN-Oxalate was replaced with 6 hours, 12 hours, 36 hours, and 48 hours, resulting in PBAP-ISN yields of 15%, 50%, 88%, and 90%, with purities of 10%, 33%, ≥98%, and ≥98%, respectively.

[0080] Similarly, it can be seen that in this step, when the reaction time is within 12 hours or even within 36 hours, the yield gradually increases with the increase of the reaction time. However, when the reaction time is more than 24 hours, the yield increase slows down and the increase is smaller when the reaction time is extended further.

[0081] Application Example 1

[0082] To investigate the stability of the antioxidant nitric oxide donor, stability testing experiments were conducted:

[0083] Hydrogen peroxide was used to simulate the high concentration of reactive oxygen species in the disease microenvironment. The Di-ISN-Oxalate prepared in Example 1 was added to different solutions (A: phosphate-buffered saline solution PBS; B: 5 nM esterase solution; C: 5 mM hydrogen peroxide solution). The final concentration of Di-ISN-Oxalate in the solution was 1 mM. The decomposition rate of Di-ISN-Oxalate was then measured.

[0084] See results Figure 3 The results indicate that the dual-responsive, low-drug-resistance nitric oxide donor Di-ISN-Oxalate hardly decomposes in phosphate-buffered saline (PBS) simulating a physiological environment, demonstrating its excellent physiological stability. In the presence of 5 mM hydrogen peroxide, simulating the highly reactive oxygen species environment of inflamed tissue, Di-ISN-Oxalate rapidly decomposes, releasing carbon dioxide and the nitric oxide donor 5-mononitroisosorbide (ISN). However, under the action of esterases, Di-ISN-Oxalate decomposes slowly, with the ISN release rate being significantly lower than that under hydrogen peroxide stimulation.

[0085] Application Example 2

[0086] To investigate the antioxidant capacity of the nitric oxide donor Di-ISN-Oxalate, the reactive oxygen species scavenging capacity was tested: Di-ISN-Oxalate prepared in Example 1 was added to a 5 mM hydrogen peroxide solution, and the final concentration of Di-ISN-Oxalate in the solution was 10 mM. The concentration of hydrogen peroxide in the solution was measured at regular intervals using the xylenol orange method.

[0087] See results Figure 4 This indicates that the dual-response, low-drug-resistance nitric oxide donor Di-ISN-Oxalate can effectively remove hydrogen peroxide from the solution, thus playing an antioxidant role.

[0088] Application Example 3

[0089] To investigate the effects of a dual-response, low-drug-resistance nitric oxide donor on the growth of endothelial cells and smooth muscle cells, a cell proliferation behavior test was conducted. When endothelial cells (HUVECs) and smooth muscle cells (HUVSMCs) reached 80%–90% confluence, they were digested, centrifuged, and dispersed in culture medium. After being labeled with different colors using live cell staining agents, they were seeded into 24-well cell culture plates at a density of 2500 cells per well. After adhesion for 4 hours, 1 mL of cell culture medium containing 0 μM, 5 μM, 10 μM, and 15 μM of Di-ISN-Oxalate prepared in Example 1 was added every 12 hours. The cell culture medium containing 0 μM nitric oxide donor was the PBS control group. After 2 days of culture, cell density was statistically analyzed and proportions were calculated.

[0090] See results Figure 5 This indicates that Di-ISN-Oxalate can effectively enhance the proliferative advantage of endothelial cells over smooth muscle cells.

[0091] Application Example 4

[0092] The nitrosation and inactivation of intracellular enzymes are the main reasons for the development of drug resistance in nitrate nitric oxide carriers. To investigate the effect of a dual-response, low-resistance nitric oxide donor on the nitrosation of intracellular enzymes, a nitrosation protein assay was conducted: When HUVEC endothelial cells reached 80%–90% confluence, they were digested, centrifuged, and dispersed in culture medium. Cells were seeded at a density of 20,000 cells per well into 6-well cell culture plates. After adhesion for 4 hours, the cells were treated with 1 mM H2O2 for 12 hours. Then, 3 mL of cell culture medium containing 15 μM Di-ISN-Oxalate prepared in Example 1 was added. Cell culture medium containing PBS and the clinically commonly used nitric oxide carrier drug isosorbide dinitrate (DISN) were used as control groups. After 2 days of culture, the nitrosation protein levels and ratios were measured and calculated.

[0093] See results Figure 6This indicates that Di-ISN-Oxalate can effectively reduce the level of intracellular nitrosamines under high H2O2 stimulation in a simulated pathological microenvironment. In contrast, the traditional drug DISN significantly increased the level of intracellular nitrosamines under the same stimulation.

[0094] Application Example 5

[0095] To investigate the drug resistance of a dual-response, low-drug-resistance nitric oxide donor under simulated physiological conditions, an in vitro blood vessel perfusion dilation test was conducted: Isolated rat carotid arteries were connected to an extracorporeal circulation apparatus and perfused at 37°C with a high-glucose culture medium. At 10, 20, 30, 40, and 60 minutes, 10 μM of Di-ISN-Oxalate prepared in Example 1 was administered. Isosorbide dinitrate (DISN), a clinically commonly used nitric oxide carrier drug, was used as a control group. Changes in the diameter of the isolated blood vessels were measured.

[0096] See results Figure 7 The vasodilatory response of DISN-treated blood vessels weakened after repeated administration, while the vasodilatory response of Di-ISN-Oxalate-treated blood vessels remained good after repeated administration. This shows that the nitric oxide carrier drug Di-ISN-Oxalate of the present invention induces lower drug resistance.

[0097] Application Example 6

[0098] To investigate the half-life of the dual-response, low-drug-resistance nitric oxide donor under simulated physiological conditions, drug stability tests were conducted. Di-ISN-Oxalate prepared in Example 1 was dissolved in DMSO and mixed 1:100 with PBS (phosphate-buffered saline), PBS containing 10% BSA (bovine serum albumin), and PBS containing 10% FBS (fetal bovine serum albumin). The UV absorbance of Di-ISN-Oxalate at 273 nm was measured at different time points (6 hours, 12 hours, 24 hours, 2 days, 3 days, 6 days, 12 days, 20 days, and 30 days). The residual concentration (C0) of Di-ISN-Oxalate was calculated using a standard curve. 剩余 mg·L -1 ), calculate its concentration compared to the original concentration (C). 原始 The percentage of ) is the remaining percentage R. 剩余 :

[0099] R 剩余 =(C 剩余 ÷C 原始 )×100%

[0100] See results Figure 8The remaining percentage of Di-ISN-Oxalate in different solutions at different time points shows that after incubation in various solutions simulating physiological environments for 30 days, ≥90% of the Di-ISN-Oxalate of the present invention remains stable.

Claims

1. A dual-response nitric oxide donor with low drug resistance, characterized in that, The low-drug-resistance nitric oxide donor is 5-mononitroisosorbitan oxalate diester, which has the following structural formula:

2. The method for preparing a dual-response, low-drug-resistance nitric oxide donor according to claim 1, characterized in that, Use either method one or method two: The method specifically includes the following steps: reacting 5-mononitroisosorbide ester with oxaloyl chloride under the action of a catalyst to obtain the low-drug-resistant nitric oxide donor; The second method specifically includes the following steps: reacting 5-mononitroisosorbide ester with oxalic acid under the action of a catalyst to obtain the low-drug-resistant nitric oxide donor.

3. The method for preparing a dual-response, low-drug-resistance nitric oxide donor according to claim 2, characterized in that, The molar ratio of 5-mononitroisosorbide ester to oxaloyl chloride in Method 1 is 0.5:1-3:1; In Method 2, the molar ratio of 5-mononitroisosorbide ester to oxalic acid is 0.5:1-3:

1.

4. The method for preparing a dual-response, low-drug-resistance nitric oxide donor according to claim 2, characterized in that, In either method one or method two, the solvent used is selected from one or more of dichloromethane, chloroform, acetonitrile, tetrahydrofuran, ethyl acetate, toluene, dioxane, or dimethyl sulfoxide.

5. The method for preparing a dual-response, low-drug-resistance nitric oxide donor according to claim 2, characterized in that, In Method 1, the catalyst is selected from any one or more of sodium carbonate, sodium bicarbonate, potassium carbonate, cesium carbonate, triethylamine, trimethylamine, benzimidazole, 4-dimethylaminopyridine, and pyridine; the molar ratio of the catalyst to oxalyl chloride is 0.1 to 5:

1. And / or, in Method 2, the catalyst is selected from one or more of dicyclohexylcarbodiimide, 4-dimethylaminopyridine, 1-hydroxybenzotriazole, N-hydroxysuccinimide, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; the molar ratio of the catalyst to oxalic acid is 0.1 to 5:

1.

6. The method for preparing a dual-response, low-drug-resistance nitric oxide donor according to claim 2, characterized in that, In Method 1, oxalyl chloride is added dropwise. During the dropwise addition, the reaction temperature is -20 to 0°C and the dropwise addition time is 0.5 to 3 hours. After the dropwise addition is completed, the temperature is raised to -5 to 25°C and the reaction is carried out for more than 0.5 hours.

7. The method for preparing a dual-response, low-drug-resistance nitric oxide donor according to claim 2, characterized in that, Method 2 includes the following steps: activating oxalic acid with a catalyst and then adding 5-mononitroisosorbide ester to react and obtain the low-drug-resistant nitric oxide donor.

8. The method for preparing a dual-response, low-drug-resistance nitric oxide donor according to claim 7, characterized in that, The activation temperature is below 25℃, and the reaction temperature after activation is 20-45℃, with a reaction time of more than 8 hours.

9. The use of the dual-responsive, low-drug-resistance nitric oxide donor according to claim 1 in the preparation of medicaments for the treatment or prevention of cardiovascular diseases.

10. A drug for treating or preventing cardiovascular diseases, characterized in that, It includes the dual-response, low-drug-resistance nitric oxide donor as described in claim 1.

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