NO-donor type roxadustat derivatives, methods of preparation, compositions, and uses

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a class of NO donor type roxadustat derivatives, a preparation method, a composition and purposes. In the application, an ester group is used to connect an NO donor into a roxadustat structure to synthesize the NO donor type roxadustat. The NO donor type roxadustat can release NO rapidly under the action of enzymes in vivo, and exert the beneficial pharmacological activity of NO. Then, the released roxadustat can stabilize HIF-1 alpha, activate the downstream related genes of HIF-1, and exert the effects of inducing brain tissue hypoxia tolerance, promoting angiogenesis and promoting the survival of neurons. Thus, a new drug with the pharmacological activities of both nitric oxide molecules and roxadustat is obtained, and the new drug can exert better effects of preventing cerebral ischemia, treating and protecting brain tissues after cerebral ischemia, and improving the prognosis.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, and particularly relates to a class of NO donor-type roxadustat derivatives, their preparation methods, compositions, and uses. Background Technology

[0002] Ischemic stroke is a cerebrovascular disease primarily caused by a sudden blockage of cerebral blood vessels, leading to a sharp decrease in blood supply to the brain and resulting in widespread brain tissue damage and loss of brain function. It is characterized by high morbidity, high mortality, and high disability rates, seriously endangering human health. Currently, the primary clinical treatment is thrombolytic therapy, which aims to restore blood supply to the brain as quickly as possible to prevent the spread of ischemia. However, the effective time window for thrombolytic therapy is only 4 hours. Once this window is exceeded, irreversible damage to brain cells can occur, potentially leading to permanent loss of brain function. In addition, calcium channel antagonists, EAA antagonists, and free radical scavengers are also used clinically to combat cerebral ischemia-reperfusion injury, but their efficacy is limited.

[0003] Nitric oxide (NO) is an important gaseous messenger molecule involved in regulating normal blood flow, vasodilation, and the activity of neurons and glial cells. It is a key factor in the pathological changes and development of stroke. In ischemic brain injury or cerebral ischemia-reperfusion injury, NO has a dual role of neuroprotection and damage prevention. In the early stages of cerebral ischemia, NO can regulate vascular tone, maintain normal blood vessel and tissue perfusion, inhibit platelet aggregation and adhesion, and suppress the release of inflammatory factors, thereby exerting a protective effect against cerebral ischemia.

[0004] Roxadustat is the world's first oral hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI), originally discovered and developed by FibroGen in the United States. It was first launched in China in 2018 for the treatment of anemia in dialysis patients with chronic renal disease (CKD), including those on hemodialysis and peritoneal dialysis. Subsequently, a second indication was added: non-dialysis chronic renal anemia (NDD-CKD). Roxadustat works by stabilizing hypoxia-inducible factor 1α (HIF-1α), inhibiting its degradation, activating the transcription of related genes, increasing erythropoietin (EPO) concentration, increasing EPO receptor sensitivity, decreasing hepcidin levels, increasing transferrin receptor content and activity, thereby increasing oxygen levels and improving the body's tolerance to hypoxia. Furthermore, HIF-1α plays a crucial role in various ischemic-hypoxic diseases. Studies on animal models of focal cerebral ischemia have revealed that the expression of HIF-1α is upregulated in the ischemic penumbra, while the expression of vascular endothelial growth factor (VEGF) also increases, thereby promoting angiogenesis to combat ischemic-hypoxic injury. Furthermore, experimental intervention to reduce HIF-1α degradation induces the upregulation of EPO and VEGF expression, significantly improving the oxygen-carrying capacity of erythrocytes and alleviating post-ischemic cerebral edema and vascular leakage. The survival of neurons in ischemic brain tissue is also closely related to the stability of HIF-1α. HIF-1α can counteract apoptosis and promote neuronal survival under various stress conditions, thus playing a neuroprotective role.

[0005] Only a small percentage of stroke patients fully recover normal function; the vast majority suffer from sequelae such as hemiplegia and aphasia, placing a significant burden on society and families. The prevention and treatment of cerebrovascular diseases is a concern for the entire society, but currently there are no ideal treatments. Therefore, the search for and development of new, effective treatments is urgently needed. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a class of NO donor-type roxadustat derivatives, preparation methods, compositions and uses, with the aim of solving some of the problems in the prior art or at least alleviating some of the problems in the prior art.

[0007] This invention is achieved by developing a class of NO donor-type roxadustat derivatives. These compounds comprise a roxadustat core, a linker arm, and a NO donor structure, with the general structural formula shown in Formula I.

[0008]

[0009] Where X is the linker arm, selected from any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, n-hexyl or such alkyl structures, or selected from isosorbide; R is the NO donor structure, selected from nitrate esters.

[0010] Furthermore, when X is selected from any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, n-hexyl, or such alkyl structures, its structure is as shown by RN1, RN2, or RN3 in Formula II.

[0011]

[0012] When X is selected from isosorbide, its structure is as shown in RN4 of Formula III.

[0013]

[0014] This invention also provides a method for preparing a class of NO donor-type roxadustat derivatives. When X is selected from alkyl groups, the reaction route is as follows:

[0015]

[0016] Where X is selected from any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, n-hexyl or such alkyl structures;

[0017] When X is selected from isosorbide, the reaction route is as follows:

[0018]

[0019] The intermediates or target compounds involved in the above process can be purified using conventional separation techniques. If necessary, they can be separated into their monomers using conventional separation techniques and can be further converted into addition salts with pharmaceutically acceptable acids as needed.

[0020] The present invention also provides a pharmaceutical composition comprising, as described above, a NO donor-type roxadustat derivative or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a NO donor-type roxadustat derivative or a pharmaceutically acceptable salt thereof and pharmaceutical excipients in different dosage forms.

[0021] This invention also provides the use of the pharmaceutical composition described above in the preparation of a drug for treating or preventing ischemic stroke. The preventive effect against ischemic stroke is an antiplatelet aggregation effect, and the therapeutic effect against ischemic stroke is at least one of antiplatelet aggregation, angiogenesis, endothelial protection, anti-inflammatory, and neuroprotective effects.

[0022] The present invention also provides the use of the pharmaceutical composition described above in the preparation of a medicament having at least one of the following effects: antiplatelet aggregation, angiogenesis, vascular endothelial protection, anti-inflammatory, or neuroprotective effects.

[0023] Furthermore, the drug can be administered orally, via non-gastrointestinal routes, or via topical administration.

[0024] Furthermore, the non-gastrointestinal administration includes intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or transdermal administration.

[0025] The present invention also provides the application of a class of NO donor-type roxadustat derivatives as described above in the preparation of reagents for regulating the expression of cGMP and / or PKG2 in the NO / cGMP / PKG2 pathway.

[0026] The present invention also provides the use of a class of NO donor-type roxadustat derivatives as described above in the preparation of reagents for regulating the expression of VEGF, HIF-1α and / or p-VEGFR2 in the HIF-1α / VEGF / VEGFR2 pathway.

[0027] NO donors serve as storage and transport forms of NO both in vivo and in vitro. They can release NO in vivo through enzymatic or non-enzymatic processes, thereby increasing its stability after entering the body and prolonging its release half-life. Common NO donors include nitrates, nitrites, azomonium glycol salts, furazolidone N-oxides, and nitrosothiols. After ischemic stroke, the uncoupling of NO synthases leads to a decrease in NO synthesis and the generation of large amounts of reactive oxygen species and reactive nitrogen species (ROS / RNS), resulting in neurotoxicity. Therefore, after cerebral ischemia, exogenous use of NO donors to supplement NO and increase the NO concentration in the ischemic area can exert an anti-ischemic stroke effect.

[0028] Suitable lipid solubility is a key factor for drugs to cross the blood-brain barrier. Molecules with high lipid solubility can cross the blood-brain barrier via passive diffusion, while drugs with insufficient lipophilicity have difficulty crossing it. Considering that the ionic structure of carboxylic acids does not readily permeate biological membranes, esterified forms of roxadustat (methylated, ethylated, propylated, butylated, etc.) can increase the cell membrane permeability of roxadustat, promoting its accumulation within cells and resulting in intracellular concentrations 10-100 times higher than unmodified roxadustat. The slow and continuous deesterification process within cells also prolongs the effective duration of roxadustat and reduces its cytotoxicity.

[0029] In summary, the advantages and positive effects of this invention are as follows:

[0030] This application employs an ester group to link roxadustat to a NO donor, using linker arms of carbon chains of varying lengths to incorporate the NO donor into the roxadustat structure, thus synthesizing NO-donor roxadustat. This allows for the rapid release of NO under the action of enzymes in vivo, maximizing the beneficial pharmacological activity of NO. Subsequently, as the ester group is gradually hydrolyzed, the released roxadustat stabilizes HIF-1α, activates downstream HIF-1-related genes, and induces hypoxia tolerance in brain tissue, promotes angiogenesis, and enhances neuronal survival. This results in a novel drug possessing both nitric oxide molecule and roxadustat pharmacological activity, enabling better prevention of cerebral ischemia, or treatment and protection of brain tissue after cerebral ischemia, and improved prognosis. Attached Figure Description

[0031] Figure 1 This is the general structural formula of the compound provided by the present invention.

[0032] Figure 2 This invention relates to the effect of the representative compound RN1 on the survival and apoptosis of HUVEC cells under OGD / R. **p<0.01, ***p<0.001, vs control group; #p<0.05, ##p<0.01, ###p<0.001 vs OGD / R group.

[0033] Figure 3 This describes the effect of the representative compound RN1 of this invention on the NO content in HUVECs under OGD / R conditions. **p<0.01, vs. control group; #p<0.05, ##p<0.01, vs. OGD / R group.

[0034] Figure 4 This invention's representative compound RN1 demonstrates the tubule regeneration-promoting effect on HUVECs under OGD / R conditions. **p<0.01 vs control group; #p<0.05, ##p<0.01 vs OGD / R group; Δp<0.05, ΔΔp<0.01 vs OGD / R + 20 μM RN1 group.

[0035] Figure 5 This invention's representative compound RN1 affects the expression of cGMP and PKG2 proteins in HUVECs under OGD / R conditions. *p<0.05,**p<0.01 vs control group; #p<0.05,##p<0.01 vs OGD / R group.

[0036] Figure 6This invention describes the effect of the representative compound RN1 on the expression of HIF-1α, VEGFR, VEGFR2, and p-VEGFR2 in HUVECs under OGD / R conditions. *p<0.05,**p<0.01 vs control group; #p<0.05,##p<0.01 vs OGD / R group. Detailed Implementation

[0037] 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. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0038] Based on the information contained in this application, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0039] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "about". Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods. In this invention, "about" means within 10%, preferably within 5%, of a given value or range.

[0040] Unless otherwise specified, the following embodiments of the present invention are all under normal temperature conditions. Normal temperature refers to the natural room temperature conditions in all four seasons, without additional cooling or heating treatment. Generally, the normal temperature is controlled between 10 and 30°C, preferably between 15 and 25°C.

[0041] Unless otherwise stated, the following terms used in the specification and claims have the meanings discussed below:

[0042] "alkyl" includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, and n-hexyl.

[0043] The term "pharmaceutically acceptable salt" as used in this invention refers to salts that retain the bioavailability and properties of the parent compound. Such salts include:

[0044] (1) It forms salts with acids, which are obtained by reacting the free base of the parent compound with inorganic or organic acids. Inorganic acids include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, metaphosphoric acid, sulfuric acid, sulfurous acid and perchloric acid, etc. Organic acids include acetic acid, trifluoroacetic acid, propionic acid, acrylic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, tartaric acid, maleic acid, ascorbic acid, benzoic acid, hydroxybenzoic acid, γ-hydroxybutyric acid, methoxybenzoic acid, phthalic acid, methanesulfonic acid, camphoric acid, oxalic acid, ethanesulfonic acid, naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, cinnamic acid, dodecyl sulfate, gluconic acid, glutamic acid, aspartic acid, stearic acid, mandelic acid, succinic acid or malonic acid, etc.

[0045] (2) Salts formed by replacing acidic protons in the parent compound with metal ions or by coordinating with organic bases. Examples of metals include alkali metal ions, alkaline earth metal ions, or aluminum ions. Examples of organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucosamine, quinine, etc.

[0046] In this invention, "pharmaceutical composition" refers to a mixture of one or more of the compounds of this invention, or their pharmaceutically acceptable salts, solvates, hydrates, or prodrugs, with other chemical components, such as pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of drugs to the body.

[0047] In this invention, "pharmaceutical excipients" refers to inactive ingredients in pharmaceutical compositions that do not cause significant irritation to the organism and do not interfere with the biological activity and properties of the given compound. Examples include, but are not limited to: calcium carbonate, calcium phosphate, various sugars (e.g., lactose, mannitol, etc.), starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylic polymers or methacrylic polymers, gels, water, polyethylene glycol, propylene glycol, ethylene glycol, castor oil or hydrogenated castor oil or polyethoxylated hydrogenated castor oil, sesame oil, corn oil, peanut oil, etc.

[0048] This invention provides a pharmaceutical composition having the compound, isomer, or pharmaceutically acceptable salt thereof described in this invention as the active ingredient or the main active ingredient, supplemented with a pharmaceutically acceptable salt. The active ingredient of this pharmaceutical composition may be solely the compound of this invention, or it may be used in combination with other existing pharmaceutical products.

[0049] In this invention, when administering mammalian compounds of formula I and their pharmaceutically acceptable salts, as well as solvates of these compounds (collectively referred to herein as "therapeutic drugs"), they may be used alone or preferably in combination with a suitable pharmaceutical carrier or diluent according to standard pharmaceutical methods. Administration can be via various routes, including oral, non-gastrointestinal, or local administration. Non-gastrointestinal administration, as referred to herein, includes, but is not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and transdermal administration.

[0050] The compounds, isomers, or pharmaceutically acceptable salts thereof of the present invention may be used in the preparation of medicaments for the treatment or prevention of ischemic stroke, or in the preparation of medicaments for the treatment or prevention of ischemic-related diseases.

[0051] This invention discloses a class of NO donor-type roxadustat derivatives, their preparation methods, pharmaceutical compositions, and uses. The technical solution of this invention will be clearly and completely described below with reference to embodiments thereof.

[0052] Example 1

[0053] In this embodiment, a total of four compounds were synthesized, abbreviated as RN1, RN2, RN3 and RN4, with the following general structural formula: Figure 1 As shown.

[0054] Compound RN1 is named [[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-)carbonyl]-amino]-acetic acid-2-nitrooxyethanol ester, and its structural formula is as follows:

[0055]

[0056] Compound RN2 is named [[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-)carbonyl]-amino]-acetic acid-3-nitrooxypropanol ester, and its structural formula is as follows:

[0057]

[0058] Compound RN3 is named [[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-)carbonyl]-amino]-acetic acid-5-nitrooxypentanol ester, and its structural formula is as follows:

[0059]

[0060] Compound RN4 is named [[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-)carbonyl]-amino]-acetic acid-6-(nitrooxy)hexahydrofurano[3,2-b]furan-3-ol ester, and its structural formula is as follows:

[0061]

[0062] The reaction route involved in this embodiment of the invention is as follows:

[0063]

[0064] Step a: In a reaction flask, add methyl 4-hydroxy-1-methyl-7-phenoxy-3-isoquinoline carboxylate (309 mg, 1 mmol), dissolve in methanol (20 mL), add 2N sodium hydroxide solution (10 mL), heat to an internal temperature of 50–70 °C, and react for 3–10 h. After the reaction is complete, rotary evaporate most of the methanol, slowly add dilute hydrochloric acid (1 mol / L) to the residual liquid to adjust the pH to 4–5, slurry for 1 hour, filter, wash with water, collect the solid and dry to obtain yellow compound 1 (yield 89%).

[0065] Step b: In a reaction flask, glycine methyl ester hydrochloride (151 mg, 1.2 mmol), triethylamine (126 mg, 1.25 mmol), and dichloromethane (10 mL) were added, and the mixture was reacted at room temperature for 5 h. In another reaction flask, compound 1 (295 mg, 1 mmol), cyclohexylcarbodiimide (DCC, 248 mg, 1.2 mmol), a catalytic amount of 4-dimethylaminopyridine (DMAP), and dichloromethane (15 mL) were added sequentially, followed by dropwise addition to the reaction solution from the previous step. After the addition was complete, the mixture was reacted at room temperature for 8 h. After the reaction, the mixture was filtered, and silica gel was added to the filtrate for column chromatography. The filtrate was evaporated to dryness using a rotary evaporator, packed into a silica gel column (100–200 mesh), and subjected to column chromatography with PE:EA = 10:1 to obtain a pale yellow solid, which was compound 2 (yield 75.5%).

[0066] Step c: In a reaction flask, compound 2 (277 mg, 0.76 mmol) was added and dissolved in tetrahydrofuran (20 mL). Then, 0.5 mol / L sodium hydroxide solution (10 mL) was added, and the reaction was carried out at room temperature for 1.5 h. After the reaction was complete, dilute hydrochloric acid (1 mol / L) was slowly added to adjust the pH to approximately 5. The mixture was separated, and the aqueous phase was extracted three times with ethyl acetate until complete. The combined organic phases were dried over anhydrous sodium sulfate and rotary evaporated to obtain a white solid, which was compound 3 (yield 91%).

[0067] Step d: In a reaction flask, add 1.25 g (10 mmol) of 2-bromoethanol, 20 mL of acetonitrile, and 3.40 g (20 mmol) of silver nitrate. React at 70 °C for 3 h. After the reaction is complete, filter the solution, evaporate the filtrate to dryness using a rotary evaporator, add 20 mL of ethyl acetate, stir for 15 minutes, filter again, and evaporate the filtrate to dryness to give a pale yellow liquid, which is compound 4, with a yield of 80%.

[0068] Step e: In a reaction flask, add 3-bromopropanol (1.39 g, 10 mmol), acetonitrile (20 mL), and silver nitrate (3.40 g, 20 mmol). React at 70 °C for 3 h. After the reaction is complete, filter the solution, evaporate the filtrate to dryness using a rotary evaporator, add 20 mL of ethyl acetate, stir for 15 minutes, filter again, and evaporate the filtrate to dryness to give a pale yellow liquid, which is compound 5, with a yield of 78%.

[0069] Step f: In a reaction flask, add 5-bromopentanol (1.67 g, 10 mmol), acetonitrile (20 mL), and silver nitrate (3.40 g, 20 mmol). React at 70 °C for 3 h. After the reaction is complete, filter, evaporate the filtrate to dryness using a rotary evaporator, add 20 mL of ethyl acetate, stir for 15 minutes, filter again, and evaporate the filtrate to dryness to give a pale yellow liquid, which is compound 6, with a yield of 70%.

[0070] Step g: In a reaction flask, compound 3 (260 mg, 0.74 mmol), cyclohexylcarbodiimide (DCC, 183 mg, 0.89 mmol), catalytic amount of 4-dimethylaminopyridine (DMAP), compound 4 (95 mg, 0.89 mmol), or compound 5 (108 mg, 0.89 mmol), or compound 6 (132 mg, 0.89 mmol), or isosorbide mononitrate (170 mg, 0.89 mmol), and dichloromethane (15 mL) were added sequentially, and the reaction was carried out at room temperature for 4–6 h. After the reaction, the mixture was filtered, and silica gel for column chromatography was added to the filtrate. The mixture was then evaporated to dryness using a rotary evaporator and packed into a silica gel column (100-200 mesh). Column chromatography (PE:EA = 8:1) yielded a pale yellow solid, which was compound RN1 (yield 43%). Column chromatography (PE:EA = 7:1) yielded a yellow solid, which was compound RN2 (yield 34%). Column chromatography (PE:EA = 8:1) yielded a pale yellow solid, which was compound RN3 (yield 40%). Silica gel thin-layer chromatography (PE:EA = 4:1) yielded a white solid, which was compound RN4 (yield 29%).

[0071] RN1: 1 H NMR (600MHz, CDCl3) δ12.55 (s, 1H), 8.50 (s, 1H), 8.33 (d, J = 9.0Hz, 1H), 7.46

[0072] -7.38(m,4H),7.20(t,J=7.4Hz,1H),7.09(d,J=7.5Hz,2H),4.74-4.68(m,2H),4.52-4.46(m,2H),4.30(d,J=6.0Hz,2H),2.67(s,3H). 13C NMR(151MHz,CDCl3)δ170.25,169.31,158.49,155.97,153.77,147.06,132.17,130.11,125.61,124.44,124.17,122.29,119.65,111.77,77.21,77.00,76.79,70.04,61.13,40.62,21.67.HRMS(ESI):m / z:442.1250calc.for C 21 H 20 N3O8[M+H] + ,found 442.1254,ppm error 0.9.

[0073] RN2:1H NMR(600MHz,CDCl3)δ12.58(s,1H),8.49(s,1H),8.36(d,J=8.9Hz,1H),7.49

[0074] -7.39(m,4H),7.22(t,J=7.5Hz,1H),7.11(d,J=8.0Hz,2H),4.57(t,J=6.3Hz,2H),4.34(t,J=6.1Hz,2H),4.29(d,J=5.9Hz,2H),2.69(s,3H),2.16-2.11(m,2H). 13 C NMR(101MHz,CDCl3)δ170.33,169.43,158.48,156.04,153.76,147.07,132.23,130.14,125.64,124.44,124.22,122.28,119.67,119.47,111.82,77.34,77.23,77.02,76.71,69.57,61.45,40.80,29.70,21.78.MS(ESI):m / z:455.1calc.for C 22 H 21 N3O8[M+H] + ,found456.1.

[0075] RN3: 1 H NMR(600MHz,CDCl3)δ12.65(s,1H),8.52(s,1H),8.33(d,J=9.0Hz,1H),7.47

[0076] -7.38(m,4H),7.20(t,J=7.4Hz,1H),7.09(d,J=7.6Hz,2H),4.43(t,J=6.6Hz,2H),4.26(d,J=5.8Hz,2H),4.22(t,J=6.5Hz,2H),2.68(s,3H),1.78-1.67(m,4H),1.54-1.45(m,2H). 13 CNMR(151MHz,CDCl3)δ170.15,169.54,158.46,155.98,153.75,147.00,132.11,130.11,125.60,124.43,124.21,122.31,119.64,111.77,77.21,77.00,76.79,72.89,65.02,40.84,28.07,26.40,22.22,21.65.HRMS(ESI):m / z:484.1720calc.forC 24 H 26 N3O8[M+H] + ,found 484.1721,ppm error 0.2.

[0077] RN4: 1 H NMR(400MHz,CDCl3)δ12.53(s,1H),8.48(t,J=5.9Hz,1H),8.34(d,J=8.9Hz,

[0078] 1H),7.48-7.38(m,4H),7.22(t,J=7.4Hz,1H),7.11(d,J=7.5Hz,2H),5.36(dd,J=9.1,3.0Hz,2H),5.01(t,J=5.3Hz,1H),4.55(d,J=5.0Hz,1H),4.28(dd,J=11.9,5.9Hz,2H),4.04(dd,J=9.4,2.9Hz,2H),3.91(dd,J=11.3,5.6Hz,1H),2.67(s,3H). 13CNMR(101MHz,DMSO)δ170.63,169.34,158.33,156.01,153.36,147.54,131.96,130.86,125.76,125.04,123.93,122.95,119.96,119.80,112 .67,86.35,82.77,81.88,77.97,73.07,69.48,41.27,40.60,40.39,40.18,39.97,39.76,39.56,39.35,21.98.MS(ESI):m / z:525.1calc.for C 25 H 23 N3O 10 [M+H] + Found 526.1. Example 2: Antiplatelet aggregation pharmacological test and results of the representative compound of the present invention.

[0079] The IC50 of different compounds on arachidonic acid (AA) or adenosine 5'-diphosphate (ADP)-mediated platelet aggregation was determined by turbidimetric assay. 50 The specific implementation method is as follows:

[0080] Preparation of platelet-rich plasma (PRP) and anemic platelet-rich plasma (PPP): Rabbits fasted for 12-18 hours were anesthetized by intraperitoneal injection of 20% urethane solution. The common carotid artery was separated, and blood was collected through a polyethylene tube. The blood was injected into a siliconized centrifuge tube containing 1 / 10 of its volume of 3.8% sodium citrate solution. The blood was gently mixed with anticoagulant and centrifuged at 1000 rpm for 15 minutes. The upper light yellow suspension was aspirated, which is approximately platelet-rich plasma (PRP). The remaining plasma was centrifuged at 3000 rpm for 15 minutes, and the supernatant was aspirated to obtain anemic platelet-rich plasma (PPP). PPP was used to adjust the platelet count of PRP to 1×10⁻⁶. 8 / mL.

[0081] Platelet aggregation rate was determined by turbidimetric assay at 37°C. 260 μl of PRP was placed in a turbidimetric tube, followed by 10 μl of different concentrations of the test compound, positive control drug, or DMSO solution. The tubes were incubated at 37°C for 5 min, and then 30 μl of inducer was added sequentially. The final concentration of inducer ADP was 10 μM, and the final concentration of inducer AA was 1 mM. The maximum aggregation rate within 5 min was measured using a platelet aggregometer in both the control and test tubes. The inhibition rate of platelet aggregation was calculated as follows: Inhibition rate of platelet aggregation (IRPA) = (Platelet aggregation rate of control group - Platelet aggregation rate of experimental group) / Platelet aggregation rate of control group × 100%. The dose-response curve was plotted using GraphPad Prism software to obtain the IC50. 50 Data are expressed as mean ± SD. Statistical differences between groups were analyzed using one-way ANOVA and Tukey's test. A p-value less than 0.05 was considered statistically significant.

[0082] Table 1. Inhibitory effects of compounds on ADP / AA-induced platelet aggregation (Mean ± SD, n = 5, *P < 0.05, ***P < 0.001 vs Rox.)

[0083]

[0084]

[0085] As shown in Table 1, compound RN3 showed comparable inhibitory activity to Rox (roxadustat) only against AA-induced platelet aggregation, while its inhibitory activity against ADP-induced platelet aggregation was significantly lower than that of Rox. Compound RN1 exhibited some inhibitory effect on ADP-induced platelet aggregation, with an IC50 value of [missing information]. 50 The value was 0.8935±0.06, showing a significant difference compared to Rox; compounds RN1 and RN4 both exhibited good inhibitory effects on AA-induced platelet aggregation, with compound RN1 showing better efficacy, IC50... 50 The value was 0.6716±0.05, indicating stronger inhibitory activity compared to Rox, with a significant difference. This suggests that compound RN1 possesses certain antiplatelet aggregation activity, and its overall activity is superior to that of the original compound roxadustat.

[0086] Example 3: Pharmacological tests and results of the protective effect of the representative compound of the present invention on HUVECs under OGD / R.

[0087] HUVEC cells were passaged and cultured under F-12K medium containing penicillin (final concentration 100 U / mL), streptomycin (final concentration 100 μg / mL), 0.1 mg / mL heparin, 0.03 mg / mL ECGS, and 10% FBS. Experimental groups included a control group, an OGD / R group, an OGD / R + 10 μM RN1 group, an OGD / R + 20 μM RN1 group, an OGD / R + 40 μM RN1 group, and an OGD / R + 20 μM RN1 + 20 μM PTIO group. The control group was cultured under normal conditions. The OGD / R modeling conditions involved washing with deoxygenated glucose-free Hank's balanced salt solution, culturing in a hypoxic environment of 95% N2 and 5% CO2 for 3 hours, followed by incubation in normal medium containing the drug for another 24 hours under normal growth conditions. Cell viability was detected using the CCK8 assay 24 hours after drug treatment, and cell apoptosis was detected by flow cytometry in each group.

[0088] 1. CCK8 assay for cell viability: Cells from each group were collected by centrifugation and analyzed using a 1×10⁻⁶ assay. 4 The cells were seeded at the specified density into 96-well plates, with three replicates per group. Appropriate culture medium was added according to the grouping. After 24 hours, the CCK-8 assay was performed: the cells were washed three times with PBS, and 100 μl of culture medium containing 10% CCK-8 working solution was added to each well. The plates were gently shaken and incubated in a cell culture incubator. After 4 hours of incubation, the absorbance values ​​were measured at 450 nm using a microplate reader, and comparative analyses were performed. Results are as follows: Figure 2 As shown, all data are expressed as mean ± SD. Statistical differences between groups were analyzed using one-way ANOVA and Tukey's test. A p-value less than 0.05 was considered statistically significant.

[0089] 2. Flow cytometry detection of apoptosis: 1) Cells were treated sequentially according to the grouping method and then washed twice with PBS; 2) Cells were treated with EDTA-free trypsin until cell deformability and cell-cell junctions disappeared, at which point digestion was terminated with complete culture medium; 3) The cell suspension was transferred to sterile centrifuge tubes, centrifuged at 800 rpm for 5 min, and the supernatant was discarded; 4) 0.5 ml of staining buffer was added to each tube to resuspend the cells; 5) 5 μl of Annexin V-FITC staining solution was added to each tube, gently pipetted to mix, and then 5 μl of Propidium Iodide was added, mixed, and incubated at room temperature in the dark for 15 min; 6) Flow cytometry was used for detection. Results are as follows: Figure 2 As shown, all data are expressed as mean ± SD. Statistical differences between groups were analyzed using one-way ANOVA and Tukey's test. A p-value less than 0.05 was considered statistically significant.

[0090] like Figure 2 As shown in Figure A, compared with the Control group, the cell viability of the OGD / R group was significantly decreased; compared with the OGD / R group, the cell viability of the OGD / R+10μM RN1, OGD / R+20μM RN1, OGD / R+40μM RN1, and OGD / R+20μM RN1+20μM PTTIO groups was significantly increased in a dose-dependent manner; compared with the OGD / R+20μM RN1 group, the cell viability of the OGD / R+20μM RN1+20μM PTTIO group was decreased, indicating that the protective effect of compound RN1 on HUVECs under OGD / R was weakened after the application of NO scavenger, and the NO released by RN1 has a synergistic protective effect. Figure 2 As shown in B, compared with the Control group, the apoptosis rate of cells in the OGD / R group was significantly increased; compared with the OGD / R group, the apoptosis rate of cells in the OGD / R+10μMRN1, OGD / R+20μMRN1, OGD / R+40μMRN1, and OGD / R+20μMRN1+20μMPTIO groups decreased significantly in a dose-dependent manner.

[0091] Compared with the OGD / R+20μM RN1 group, the OGD / R+20μM RN1+20μM TIO group showed an increased apoptosis rate, indicating that the anti-apoptotic effect of compound RN1 on HUVECs under OGD / R was weakened after the application of NO scavenger, and the NO released by RN1 had a synergistic anti-apoptotic effect. These experimental results demonstrate that the compound has a significant protective effect on HUVECs cells under OGD / R.

[0092] Example 4: Tests and results of the representative compound of the present invention on the determination of NO content in HUVECs under OGD / R.

[0093] HUVEC cells were passaged and cultured under F-12K medium containing penicillin (final concentration 100 U / mL), streptomycin (final concentration 100 μg / mL), 0.1 mg / mL heparin, 0.03 mg / mL ECGS, and 10% FBS. Experimental groups included a control group, an OGD / R group, an OGD / R + 10 μM RN1 group, an OGD / R + 20 μM RN1 group, an OGD / R + 40 μM RN1 group, and an OGD / R + 20 μM RN1 + 20 μM PTIO group. The control group was cultured under normal conditions. The OGD / R modeling conditions involved washing with deoxygenated glucose-free Hank's balanced salt solution, culturing in a hypoxic environment of 95% N2 and 5% CO2 for 3 hours, followed by incubation in normal medium containing the drug for another 24 hours under normal growth conditions. NO content in cell supernatants was measured at 6 h, 12 h, and 24 h after drug treatment using the Griess method: 1) Griess Reagent I and II were removed and allowed to return to room temperature; 2) Standards were diluted to 1, 2, 5, 10, 20, 40, 60, 80, and 100 μM using cell culture medium; 3) 50 μL of standard and sample were added to each well; 4) 50 μL of Griess Reagent I was added to each well; 5) 50 μL of Griess Reagent II was added to each well; 6) The absorbance at 540 nm was measured, and a standard curve was plotted based on the standards to calculate the NO concentration in the samples. Results are as follows: Figure 3 As shown, all data are expressed as mean ± SD. Statistical differences between groups were analyzed using one-way ANOVA and Tukey's test. A p-value less than 0.05 was considered statistically significant.

[0094] like Figure 3 As shown in Figure A, after 24 hours of compound incubation, the NO content in cells of the OGD / R group was significantly lower than that of the Control group; compared with the OGD / R group, the NO content in cells of all experimental groups increased, with the OGD / R+20μM MRN1 and OGD / R+40μM MRN1 groups showing a significant increase in NO content; compared with the OGD / R+20μM MRN1 group, the NO content in cells of the OGD / R+20μM MRN1+20μM PTIO group decreased. Figure 3 As shown in Figure B, after 6, 12, and 24 hours of compound incubation, the NO content in cells increased in a time-dependent manner, while at the same time point, the NO content in cells increased in a dose-dependent manner. This indicates that compound RN1 releases NO in HUVECs, and the amount released is related to the concentration of the compound; the NO concentration increases with increasing compound concentration and also with increasing time.

[0095] Example 5: Pharmacological tests and results of the representative compounds of the present invention on the pro-angiogenic effect of HUVECs under OGD / R.

[0096] HUVEC cells were passaged and cultured under F-12K medium containing penicillin (final concentration 100 U / mL), streptomycin (final concentration 100 μg / mL), 0.1 mg / mL heparin, 0.03 mg / mL ECGS, and 10% FBS. Experimental groups included a control group, an OGD / R group, an OGD / R + 10 μM RN1 group, an OGD / R + 20 μM RN1 group, an OGD / R + 40 μM RN1 group, and an OGD / R + 20 μM RN1 + 20 μM PTIO group. The control group was cultured under normal conditions. The OGD / R modeling conditions involved washing with deoxygenated glucose-free Hank's balanced salt solution, culturing in a hypoxic environment of 95% N2 and 5% CO2 for 3 hours, followed by incubation in normal medium containing the drug for another 24 hours under normal growth conditions. After 24 hours of drug treatment, cells were transferred to Matrigel substrate and cultured for another 6 hours to observe the formation of tubules. After 24 hours of drug treatment, the expression levels of cGMP and VEGF were detected by ELISA, and the expression levels of PKG2, HIF-1α, VEGFR2, and p-VEGFR2 were detected by Western blotting.

[0097] 1. Tube Formation Assay: 1) Melting: Before the experiment, transfer the Matrigel stock solution from a -20℃ freezer to a 4℃ freezer and melt overnight. 2) Spreading: Add 50 μL of melted Matrigel to each well of a 96-well plate, gently shake the plate to allow the gel to spread evenly, and then incubate the 96-well plate at 37℃ for 30-60 min to allow the Matrigel to fully solidify. 3) Cell Implantation: Collect HUVEC cells after digestion and centrifugation according to the experimental grouping, resuspend them in serum-free medium, count them, and adjust the cell density to 2 × 10⁶ cells / well. 5 100 μL of single-cell suspension was gently added along the wall of a 96-well plate, with three replicates per well. The 96-well plate was then incubated at 37°C for 6 hours. 4) Microscopic examination: After 2 hours of incubation, the formation of tubule lumens was closely observed. After 6 hours of culture, when the lumens were fully formed, the 96-well plate was removed and microscopically examined. The total tube length and the number of branch points were calculated using the Image J plugin Angiogenesis Analyzer. Data are expressed as mean ± SD. Statistical differences between groups were analyzed using one-way ANOVA and Tukey's test. A p-value less than 0.05 was considered statistically significant.

[0098] 2. ELISA kit for VEGF expression detection: 1) Preparation of standards: Prepare 1000 pg / ml to 15.6 pg / ml standards: Prepare 7 Eppendorf tubes, add 500 μl of sample diluent to each tube, and label them as 1000 pg / ml, 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.3 pg / ml, and 15.6 pg / ml respectively. Take 500 μl of the 2000 pg / ml standard and add it to the tube labeled 1000 pg / ml, mix well, and then take 500 μl of the same and add it to the next tube. Repeat this process until the last sample tube. 2) Add 50 μL of detection diluent RD1W to each well; set up standard wells, control wells, and blank wells according to the experimental requirements, and add 200 μL of standard, control, or sample to each well. Seal the plate and incubate at room temperature for 2 hours. 3) Aspirate and wash each well, repeating this process twice for a total of three washes. 4) Add 200 μL of Human VEGF Conjugate to each well. Seal the plate and incubate at room temperature for 2 hours. 5) Aspirate and wash each well, repeating this process twice for a total of three washes. 6) Add 200 μL of substrate solution to each well and incubate at room temperature in the dark for 20 minutes. 7) Add 50 μL of stop solution to each well. 8) Measure the optical density of each well using a 450 nm microplate reader within 30 minutes. Data were analyzed and plotted using Graphpad Prism 9 (Version 9.4.0).

[0099] 3. ELISA kit for detecting cGMP levels: 1) Preparation of standards: Preparation of 30 pmol / ml → 0.23 pmol / ml standards: Prepare 8 Eppendorf tubes. Add 900 μl of diluent to the first tube, and 500 μl of diluent to each of the remaining tubes. Label them as 30 pmol / ml, 15 pmol / ml, 7.5 pmol / ml, 3.75 pmol / ml, 1.87 pmol / ml, 0.93 pmol / ml, 0.46 pmol / ml, and 0.23 pmol / ml, respectively. Take 100 μl of the 300 pmol / ml standard and add it to the tube labeled 30 pmol / ml. Mix well, and then take 500 μl of the mixture and add it to the next tube. 1) Continue in the same manner until the last sample tube; 2) Set up blank wells, maximum binding wells (B0), standard wells, and sample wells according to experimental requirements; 3) Add 100 μl of diluent to the blank well, 50 μl of diluent to the B0 well, 50 μl of diluted standard to the standard well, and 50 μl of the sample to be tested to the sample well; 4) Add 50 μl of GMP antibody to each well; 5) Add 50 μl of GMP antibody to each well except for the blank well; 6) Seal the plate and incubate at room temperature for 18 hours; 7) Empty the wells and wash 5 times with washing buffer; 8) Add 200 μl of Ellman reagent to each well; 9) Seal the plate and incubate at room temperature for 60-90 minutes; 10) Read the absorbance value at 405 nm. Data were analyzed and plotted using Graphpad Prism 9 (Version 9.4.0).

[0100] 4. Western Blot method for detecting the expression levels of PKG2, HIF-1α, VEGFR2, and p-VEGFR2: 1) Extraction of total cell protein: After drug treatment, cells were collected with trypsin, centrifuged, and the supernatant was discarded. The cell samples were washed twice with pre-cooled PBS. 1 ml of RIPA with added PMSF was added to every 100 μl compressed volume of cell sample. After complete lysis, the cells were centrifuged at 12000g for 5 min at 4℃. The supernatant was immediately aspirated into a pre-cooled Eppendorf tube, which is the extracted total cell protein. The extracted total cell protein was stored at -80℃ for later use. 2) Extraction of nuclear and plasma proteins: After processing cells according to the grouping method, collect cells with trypsin, centrifuge and discard the supernatant; add 200 μl of cytoplasmic extraction reagent A containing PMSF to every 20 μl of cell pellet; vortex for 5 seconds, incubate on ice for 10-15 minutes; add 10 μl of cytoplasmic protein extraction reagent B, vortex for 5 seconds, incubate on ice for 1 minute; vortex for 5 seconds, centrifuge at 12000g for 5 minutes at 4℃; transfer the supernatant to a pre-chilled EP tube, which contains the extracted plasma proteins; add 50 μl of nuclear protein extraction reagent containing PMSF to the pellet; vortex for 15-30 seconds, incubate on ice for 1-2 minutes, then vortex again for 15-30 seconds, for a total of 30 minutes; centrifuge at 12000g for 10 minutes at 4℃; transfer the supernatant to a pre-chilled EP tube, which contains the extracted nuclear proteins. 3) Gel electrophoresis and transfer: Prepare a 10% or 12% separating gel and a 5% compression gel, depending on the molecular weight of the protein to be measured, and pour the SDS-PAGE gel. After adding an appropriate amount of pre-chilled 1× electrophoresis buffer, add the previously biolabeled sample or total intracellular protein extract to the lanes (pre-stained protein marker and sample). Electrophoresis at a constant voltage of 80V for about 30 minutes. After the sample enters the separating gel, adjust the voltage to 120V and continue electrophoresis. When the target band reaches the appropriate position (refer to the position of the pre-stained protein marker), stop the electrophoresis. Activate the PVDF membrane in methanol for 1 minute, then soak it in transfer buffer. Soak the filter paper in transfer buffer for 15 minutes as well. Prepare the transfer "sandwich" according to the principle of PVDF membrane ≥ gel ≥ filter paper, ensuring that air bubbles are removed before starting constant voltage transfer. After transfer, stain the membrane with Ponceau S S for 5 minutes, then wash twice with TBST and observe the proteins on the membrane. 4) Development: After wetting the membrane with TBS from bottom to top, transfer it to a petri dish containing blocking solution (5% BSA or 5% skim milk powder TBST solution) and block it on a decolorizing shaker at room temperature for 1 hour. Wash off any residual liquid on the membrane with TBST, then seal three sides with a sealing machine. Add the primary antibody diluted with TBST to the appropriate concentration (PKG2, 1:500; HIF-1α, 1:1000; VEGFR2, 1:1000; p-VEGFR2, 1:1000; LaminB1, 1:1000; β-actin, 1:1000), seal the bag, and incubate overnight at 4°C.Cut open the resealable bag and wash the membrane three times with TBST, 10 min each time. Then place the membrane in a sealed bag, add appropriate amounts of secondary antibodies (Goat Anti-Rabbit IgG H&L (HRP), 1:10000; Rabbit Anti-Mouse IgG H&L (HRP), 1:10000), seal the bag, and incubate at room temperature for 1 h. Cut open the resealable bag and wash the membrane three times with TBST, 10 min each time. Mix equal volumes of chemiluminescence reagents A and B, and place the membrane protein side down in the mixture for thorough contact. After 5 min, detect the protein expression using a Tanon 6600 chemiluminescence imaging workstation. Analyze the optical density values ​​using ImageProPlus 6.0 software. The relative protein expression level is calculated as the gray value of the target protein / the gray value of the internal reference protein.

[0101] like Figure 4 As shown in Figure A, compared with the Control group, the OGD / R group showed significantly reduced neovascularization; compared with the OGD / R group, the OGD / R+20μM RN1 and OGD / R+20μM RN1+20μM PTIO groups showed significantly increased neovascularization. Figure 4 As shown in B and 4C, compared with the Control group, the total tubule length and number of branching points in the OGD / R group were significantly decreased; compared with the OGD / R group, the total tubule length and number of branching points in the OGD / R+20μM RN1 and OGD / R+20μM RN1+20μM PTIO groups were significantly increased. This indicates that after using compound RN1, the tubule regeneration activity under OGD / R was significantly restored, and it had a significant promoting effect on angiogenesis in HUVECs cells after oxygen-glucose stripping. Compared with the OGD / R+20μM RN1 group, the total tubule length and number of branching points in the OGD / R+20μM RN1+20μM PTIO group were significantly decreased, indicating that the pro-angiogenic activity of compound RN1 was partially inhibited after using the NO scavenger, suggesting that the NO released by compound RN1 has a synergistic pro-angiogenic activity.

[0102] like Figure 5As shown, compared with the Control group, the expression levels of cGMP and PKG2 in the OGD / R group decreased; compared with the OGD / R group, the expression levels of cGMP and PKG2 in the OGD / R+10μM RN1, OGD / R+20μM RN1, OGD / R+40μM RN1, and OGD / R+20μM RN1+20μM RN1 groups increased significantly in a dose-dependent manner; compared with the OGD / R+20μM RN1 group, the expression levels of cGMP and PKG2 in the OGD / R+20μM RN1+20μM RN1 group decreased, indicating that compound RN1 has a promoting effect on the expression of cGMP and PKG2 in the NO / cGMP / PKG2 pathway, and this promoting effect can be partially inhibited by NO scavengers, indicating that the NO released by compound RN1 activates the NO / cGMP / PKG2 pathway and promotes angiogenesis in HUVECs cells after oxygen-glucose desquamation.

[0103] like Figure 6 As shown, compared with the Control group, the expression levels of VEGF, HIF-1α, and p-VEGFR2 in cells of the OGD / R group increased; compared with the OGD / R group, the expression levels of VEGF, HIF-1α, and p-VEGFR2 in cells of the OGD / R+10μM RN1, OGD / R+20μM RN1, OGD / R+40μM RN1, and OGD / R+20μM RN1+20μM RN1 groups increased significantly; the expression level of VEGFR2 did not change significantly in any group. This indicates that compound RN1 has a significant promoting effect on the expression of the HIF-1α / VEGF / VEGFR2 pathway, suggesting that compound RN1 promotes the upregulation of HIF-1α / VEGF / VEGFR2 pathway expression and significantly exerts a pro-angiogenic effect.

[0104] In summary, the representative compound RN1 of this invention has a significant promoting effect on angiogenesis of HUVECs under OGD / R. This promoting effect is related to the upregulation of the expression of the NO / cGMP / PKG2 pathway and the HIF-1α / VEGF / VEGFR2 pathway, and leads to the upregulation of the expression of related proteins.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A class of NO donor-type roxadustat derivatives, characterized in that, This class of compounds includes a roxadustat core, a linker arm, and a NO donor structure, and its general structural formula is shown in Formula I. ， Wherein X is the linker arm, selected from any one of methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, n-pentylene, and n-hexylene, or selected from... R is a NO donor structure, selected from nitrate ester groups.

2. The NO donor-type roxadustat derivative according to claim 1, characterized in that: When X is selected from any one of ethylidene, n-propylidene, and n-pentylidene, its structure is as shown by RN1, RN2, or RN3 in Formula II. ； When X is selected At that time, its structure is as shown in RN4 of Equation III. 。 3. The method for preparing a type of NO donor-type roxadustat derivative as described in claim 1, characterized in that: When X is selected from any one of methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, n-pentylene, and n-hexylene, the reaction route is as follows: ； When X is selected At that time, its reaction route is as follows: 。 4. A pharmaceutical composition, characterized in that: Includes NO donor-type roxadustat derivatives as described in claim 1 or 2, or pharmaceutically acceptable salts thereof.

5. The use of the pharmaceutical composition as described in claim 4 in the preparation of a medicament for the treatment or prevention of ischemic stroke.

6. Use of the pharmaceutical composition of claim 4 in the preparation of a medicament having at least one of the following effects: antiplatelet aggregation, angiogenesis, endothelial protection, anti-inflammatory, or neuroprotective effects.

7. The application according to claim 5 or claim 6, characterized in that: The drug can be administered orally, via non-gastrointestinal routes, or via topical administration.

8. The application according to claim 7, characterized in that: The non-gastrointestinal administration includes intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or transdermal administration.

9. The use of a NO donor-type roxadustat derivative as described in claim 1 or 2 in the preparation of reagents for regulating the expression of cGMP and / or PKG2 in the NO / cGMP / PKG2 pathway.

10. The use of a NO donor-type roxadustat derivative as described in claim 1 or 2 in the preparation of reagents for regulating the expression of VEGF, HIF-1α and / or p-VEGFR2 in the HIF-1α / VEGF / VEGFR2 pathway.

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