CN107417669B

CN107417669B - 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound, preparation method and application thereof

Info

Publication number: CN107417669B
Application number: CN201610346543.8A
Authority: CN
Inventors: 方莲花; 焦晓臻; 杜冠华; 谢平; 袁天翊; 姚阳阳; 陈俞材; 张惠芳
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2016-05-24
Filing date: 2016-05-24
Publication date: 2021-07-06
Anticipated expiration: 2036-05-24
Also published as: CN107417669A

Abstract

The invention discloses a series of 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compounds shown in a general formula I, and a preparation method and medicinal application thereof. The novel 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound has the function of inhibiting Rho kinase activity; has effects in dilating aortic blood vessel, cerebral arteriole, mesenteric blood vessel, and protecting blood vessel; has pharmacological actions of promoting cell glucose consumption and the like, and is a novel compound which has important application in preparing medicaments for preventing and/or treating cardiovascular and cerebrovascular diseases such as hypertension, atherosclerosis, cerebral vasospasm, coronary vasospasm, myocardial infarction, heart failure, diabetes and the like, diabetes and complications thereof, including diabetic cardiovascular complications, diabetic cerebrovascular complications and the like.

Description

3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound, preparation method and application thereof

Technical Field

The invention relates to a 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound or a pharmaceutically acceptable salt, a preparation method and a pharmaceutical application thereof. In particular to a new compound, a new drug action and application thereof in preparing drugs and health products for preventing, relieving and/or treating cardiovascular and cerebrovascular diseases, diabetes or symptoms such as hypertension, atherosclerosis, cerebral vasospasm, coronary vasospasm, myocardial infarction, heart failure, diabetic complications and the like, belonging to the technical field of innovative drug research.

Technical Field

It is known that cardiovascular and cerebrovascular diseases are the first killers threatening human health, and the morbidity population is gradually younger, and the morbidity and the mortality are continuously increased. The main causes of these diseases are alterations in central or peripheral vascular structure and function, such as abnormal increases in vascular tone. The vasodilating active substance can be used for treating vascular diseases, diabetes and complications thereof caused by abnormal increase of vascular tension, such as hypertension, atherosclerosis, cerebral vasospasm, coronary vasospasm, myocardial infarction, heart failure and diabetic angiopathy.

Small molecule GTP-binding proteins are monomeric G-protein molecules that play a key role in the regulation of cellular function. Rho is a small G protein, Rho kinase is the earliest observed effector of Rho. At the molecular level, Rho kinase expresses various factors that promote inflammation, oxidative stress, thrombosis, and fibrosis, down-regulating endothelial nitric oxide synthase. At the cellular level, Rho kinase mediates vascular smooth muscle cell contraction, promotes proliferation and migration, and promotes inflammatory cell migration. Rho kinase directly participates in the development of many cardiovascular and cerebrovascular diseases, such as hypertension, atherosclerosis, cerebral vasospasm, coronary vasospasm, myocardial infarction, heart failure, diabetes and its complications, etc., through the interaction with vasoactive substances such as angiotensin II, endothelin-1, etc.

The Rho kinase inhibitor has important significance for treating various cardiovascular and cerebrovascular diseases, diabetes and complications thereof, such as hypertension, atherosclerosis, cerebral vasospasm, coronary vasospasm, myocardial infarction, heart failure, diabetic vasculopathy and the like, and has positive effects on preventing and treating the cardiovascular and cerebrovascular diseases, the diabetes and the complications thereof which seriously harm human health at present.

The 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound is a new compound, and no report related to application of the compound in preventing and treating cardiovascular and cerebrovascular diseases, diabetes and complications thereof exists.

Disclosure of Invention

The invention aims to provide a 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound shown as a general formula I, a preparation method thereof, a pharmaceutical composition and application thereof in preparing cardiovascular and cerebrovascular diseases, diabetes and complications thereof.

In order to solve the technical problem, the invention provides the following technical scheme:

the first aspect of the technical scheme of the invention provides a compound shown as a general formula (I) and pharmaceutically acceptable salts thereof,

R₁，R₂independently selected from hydrogen, halogen substituted C1-6 alkyl, cyano, carboxyl, C1-6 alkyl ester group, nitro, C1-6 alkyl, C1-6 alkoxy;

n is 0, 1, 2; the halogen substitution comprises single halogen substitution, double halogen substitution, trihalo substitution and polyhalo substitution.

Among them, preferred is R₁，R₂Independently selected from hydrogen, halogen, trihalo-substituted methyl, cyano, carboxyl, carbomethoxy, carboethoxy, propyl, isopropyl, nitro, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, methoxy, ethoxy, propoxy, isopropoxy; the halogen is selected from fluorine, chlorine, bromine and iodine; .

Most preferred compounds are selected from

In a second aspect of the present invention, there is provided a process for preparing a compound of the first aspect:

when n is 1 and 2: reacting 5-aminoindazole with methyl chlorobenzoate to obtain a compound 17, carrying out cyclization reaction on the compound 17 and 3-chloropropionic acid hydrochloride to obtain a compound 18, protecting the compound 18 with THP to obtain a compound 19, carrying out nucleophilic substitution reaction on the compound 18 and a compound of a formula (II) under the condition of NaH to obtain a compound of a formula (III), and removing THP to obtain a compound of a formula (I);

i amidation; ii a cyclization reaction; iii THP protection; iv substitution reaction; v de-THP protection, wherein R₁，R₂Is as defined for the first aspect of the invention;

when n is 0: when R is₁，R₂When halogen, halogen substituted C1-6 alkyl, cyano, carboxyl, C1-6 alkyl ester group and nitro electron-withdrawing group are adopted, the compound 19 is obtained in the same way as the above, and the compound 19 is applied to Xantphos and Pd (OAc)₂Under the catalysis of (3), carrying out coupling reaction with a compound of a formula (IV) to obtain a compound of a formula (V), and removing THP to obtain a compound of a formula (I);

when n is 0: when R is₁，R₂When the compound is hydrogen, C1-6 alkyl or C1-6 alkoxy, the compound 19 is obtained as above, the compound 19 and the compound of the formula (IV) are subjected to coupling reaction under the catalysis of cuprous iodide to obtain a compound of the formula (V), and then THP is removed to obtain the compound of the formula (I);

the term "halogen" refers to fluorine, chlorine, bromine, iodine;

i amidation; ii a cyclization reaction; iii THP protection; iv coupling reaction; v de-THP protection, wherein R₁，R₂Is as defined for the first aspect of the invention.

According to a third aspect of the technical scheme of the invention, a first pharmaceutical composition is provided, which comprises a prophylactically and/or therapeutically effective amount of the 3- (1H-indazole) -tetrahydropyrimidine-2-one compound, and optionally a pharmaceutically acceptable carrier and/or an auxiliary material.

In the present invention, the pharmaceutical composition of the 3- (1H-indazole) -tetrahydropyrimidin-2-one compound may be in a dosage form selected from the group consisting of: solutions, suspensions, emulsions, pills, capsules, powders, controlled release or sustained release formulations.

The pharmaceutical compositions of the 3- (1H-indazole) -tetrahydropyrimidin-2-one compounds of the present invention can be formulated in a known manner and administered to a subject using several routes including, but not limited to, parenteral, oral, topical, intradermal, intramuscular, intraperitoneal, subcutaneous, intravenous, intranasal routes.

The pharmaceutical compositions of the 3- (1H-indazole) -tetrahydropyrimidin-2-one compounds of the present invention optionally may be formulated by any conventional method with one or more pharmaceutically acceptable carriers and/or excipients. Thus, the 3- (1H-indazole) -tetrahydropyrimidin-2-one compounds and their pharmaceutically acceptable salts can be formulated, for example, for inhalation or insufflation (either through the mouth or the nose) or for oral, buccal, parenteral or rectal administration.

The pharmaceutical compositions of 3- (1H-indazole) -tetrahydropyrimidin-2-one compounds can also be in the form of solutions, suspensions, emulsions, pills, capsules, powders, controlled-release or sustained-release formulations. These formulations will contain a therapeutically effective amount of a 3- (1H-indazole) -tetrahydropyrimidin-2-one compound, preferably in purified form, and an appropriate amount of a carrier to provide a form suitable for administration to a patient.

The fourth aspect of the technical scheme of the invention provides application of the compound of the first aspect of the invention or pharmaceutically acceptable salts thereof in preparing products for preventing, relieving and/or treating cardiovascular and cerebrovascular diseases, diabetes and complications thereof. The cardiovascular and cerebrovascular diseases are selected from hypertension, atherosclerosis, cerebral vasospasm, coronary vasospasm, myocardial infarction and heart failure. The diabetic complications comprise diabetic cardiovascular complications and diabetic cerebrovascular complications. As long as the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compounds and the salts thereof are applied to cardiovascular and cerebrovascular diseases, diabetes and complications thereof, the compounds belong to the protection scope of the compounds.

The cardiovascular and cerebrovascular diseases are vascular diseases caused by various reasons. Especially vascular diseases induced by abnormally increased vascular tone, including hypertension, atherosclerosis, cerebral vasospasm, coronary vasospasm, myocardial infarction, heart failure and diabetes and its complications.

In the present invention, the prevention, alleviation and/or treatment of vascular diseases or conditions caused by vascular diseases is selected from the group consisting of inhibition of Rho kinase activity, vasodilation, protection of vascular function, promotion of cellular glucose consumption.

The invention is realized by the following technical scheme: a series of 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compounds and salts thereof are obtained by artificial synthesis; evaluating the inhibitory activity of 3- (1H-indazole) -tetrahydropyrimidine-2-one compounds on Rho kinase by using a Rho kinase protein activity evaluation system; the effect of 3- (1H-indazole) -tetrahydropyrimidine-2-one compounds on the contraction of isolated thoracic aorta blood vessels, cerebral basilar artery micro-vessels and mesenteric arterioles caused by norepinephrine or potassium chloride is further observed by an in vitro vascular ring tension measuring system, the effect of 3- (1H-indazole) -tetrahydropyrimidine-2-one compounds on promoting cell glucose consumption is observed by measuring the content of cell glucose, and the relaxation effect and the glucose consumption promoting effect of the 3- (1H-indazole) -tetrahydropyrimidine-2-one compounds on isolated large blood vessels and micro blood vessels of rats are judged. The comprehensive evaluation judges the prevention and treatment effects of the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compounds on cardiovascular and cerebrovascular diseases, diabetes and complications thereof.

The 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound has the pharmaceutical effect of preventing, relieving and/or treating cardiovascular and cerebrovascular diseases or diabetes and complications thereof when being prepared into any dosage form. Any medicament, if the components contain the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound shown in the general formula I or the medicament is prepared by only using the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound shown in the general formula I, the package, the instruction book and other labels or any other propaganda products only need to be noted or suggested to have the effect of treating cardiovascular and cerebrovascular diseases, diabetes and complications thereof or diseases or symptoms caused by vascular diseases, and the invention also belongs to the protection scope of the invention.

In the present invention, said purified form of the 3- (1H-indazole) -tetrahydropyrimidin-2-one compound refers to the structure 3- (1H-indazole) -tetrahydropyrimidin-2-one compound which is substantially pure, in particular with a purity of more than 80%, preferably more than 85%, particularly preferably more than 90%, even more preferably more than 95%. The purity of the purified form of the 3- (1H-indazol) -tetrahydropyrimidin-2-one compound can range, for example, from 90 to 96%.

The beneficial technical effects are as follows:

the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound is a new monomer compound, and has the advantages of low toxicity, simple preparation process and the like;

has good application and development prospect, is an ideal new compound for preventing and treating cardiovascular and cerebrovascular diseases, diabetes and complications thereof, and can be applied to the preparation of medicaments and health-care products.

The 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound has the effect of inhibiting Rho kinase activity, and has the effects of relaxing blood vessels, protecting blood vessels and promoting glucose consumption. The compound is a novel compound with great medicinal value in the aspects of preventing and treating cardiovascular and cerebrovascular diseases and diabetes, such as hypertension, atherosclerosis, cerebral vasospasm, coronary vasospasm, myocardial infarction, heart failure, diabetes and complications thereof, and the like, and has good application and development prospects.

Detailed Description

The following describes the preparation process of 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compounds, the effect of inhibiting Rho kinase activity, the effects of relaxing blood vessels, protecting blood vessel function and promoting glucose consumption, and the application of the compounds in preventing and treating cardiovascular and cerebrovascular diseases and diabetes caused by abnormal increase of blood vessel tension such as hypertension. The following examples illustrate the invention in more detail and are not intended to limit the invention in any way.

Examples

Preparation of intermediates of examples 1-16:

step A: a25 mL round bottom flask was charged with 5-aminoindazole (474mg, 3.56mmol), 6mL anhydrous pyridine in dichloromethane (6mL), and methyl chlorobenzoate (0.45mL, 3.56mmol) was slowly added dropwise at 80 ℃. The reaction was stirred at room temperature. The reaction was carried out for about 6 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped, and the solvent was removed under reduced pressure. Column chromatography on silica gel (dichloromethane: ethyl acetate 50:1) gave 812mg of the product in 90% yield.

¹H NMR(400MHz,DMSO,δppm)12.98(s,1H),10.17(s,1H),8.01(s,1H),7.92(s,1H),7.50(d,J＝8.8Hz,1H),7.42(t,J＝8.4Hz,3H),7.24(m,3H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₄H₁₂N₃O₂:254.09240；Found:254.09120.

And B: a10 mL round bottom flask was charged with Compound 17(100mg, 0.4mmol), 2mL anhydrous THF (6mL), cooled with stirring (ice bath), solid sodium hydroxide (47mg, 1.19mmol) was added, the reaction stirred below 10 deg.C, monitored by TLC, and compound 17 was consumed (about 2 hours) and tert-BuOK solution (112mg of tert-BuOK dissolved in 1mL THF) was added. The temperature is raised to 20 ℃ and the reaction is stirred. The reaction was carried out for about 18 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped, and the solvent was removed under reduced pressure. Diluting with water, adjusting to neutrality with concentrated hydrochloric acid, filtering, washing with ethyl acetate, and vacuum filtering to obtain 68mg of product with yield of 79%.

¹H NMR(400MHz,DMSO,δppm)12.98(s,1H),7.99(s,1H),7.54(s,1H),7.43(d,

J＝8.7Hz,1H),7.25(d,J＝8.7Hz,1H),6.49(s,1H),3.61(t,J＝5.4Hz,2H),3.24(m,2H),1.96(d,J＝5.4Hz,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₁H₁₃N₄O:217.10839；Found:217.10847.

And C: a10 mL round-bottom flask was charged with compound 18(5g, 0.023mol), p-toluenesulfonic acid (80mg, 0.46mmol), 50mL anhydrous DMF, 3, 4-dihydropyran (1.947g, 0.023mmol), and heated to 90 deg.C under reflux. The reaction was carried out for about 6 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped, and the solvent was removed under reduced pressure. The solution was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Filtration, concentration and silica gel column chromatography (dichloromethane: methanol ═ 50:1) gave 195.359 g of compound in 77% yield.

¹H NMR(400MHz,DMSO,δppm)8.03(s,1H),7.61(d,J＝8.9Hz,1H),7.56(s,1H),7.33(d,J＝8.9Hz,1H),6.51(s,1H),5.80(d,J＝9.5Hz,1H),3.86(m,1H),3.72(m,1H),3.62(m,2H),3.24(m,1H),2.39(m,1H),2.01(m,1H),1.96(m,2H),1.74(m,1H),1.57(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₆H₂₁N₄O₂:301.16590；Found:301.16528.

EXAMPLE 1 Synthesis of Compound 1

Step A: a25 mL round bottom flask was charged with compound 19(200mg, 0.67mmol), 4mL anhydrous 1, 4-dioxane, and NaH (44mg, 1.82mmol), benzyl bromide (124mg, 0.73mL) at 0 ℃. After stirring at room temperature for about 3 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped and the solvent was removed under reduced pressure. The solution was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Filtration, concentration and silica gel column chromatography (dichloromethane: methanol ═ 100:1) gave 223mg of the product in 86% yield.

¹H NMR(400MHz,DMSO,δppm)8.05(s,1H),7.63(m,2H),7.35(m,3H),7.28(m,3H),5.81(m,1H),4.51(s,2H),3.85(m,1H),3.71(m,3H),3.28(m,2H),2.40(m,1H),1.97(m,4H),1.73(m,1H),1.57(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₃H₂₇N₄O₂:391.21285；Found:391.21249.

And B: a50 ml round-bottom flask was charged with Compound 20(163mg, 0.42mmol), 10ml of methanol, and about 9ml of 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed substantial disappearance of starting material, reaction was stopped, and the solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and purification on silica gel (dichloromethane: methanol ═ 50:1) gave 123mg of product in 96% yield.

¹H NMR(400MHz,DMSO,δppm)13.01(s,1H),8.02(s,1H),7.60(s,1H),7.46(d,J＝8.8Hz,1H),7.36(m,2H),7.28(m,4H),4.52(s,2H),3.69(t,J＝5.6Hz,2H),3.31(m,2H),2.04(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₈H₁₉N₄O:307.15534；Found:307.15445.

EXAMPLE 2 Synthesis of Compound 2

Step A: a25 mL round-bottom flask was charged with compound 19(80mg, 0.27mmol), 4mL anhydrous 1, 4-dioxane, and NaH (10mg,0.40mmol), p-methylbenzyl bromide (59mg, 0.32mmol) at 0 deg.C. After stirring at room temperature for about 3 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped and the solvent was removed under reduced pressure. The solution was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Filtration, concentration and silica gel column chromatography (dichloromethane: methanol ═ 100:1) gave 78mg of the product in about 72% yield.

¹H NMR(400MHz,DMSO,δppm)δ8.04(s,1H),7.64(d,J＝9.0Hz,1H),7.60(s,1H),7.35(d,J＝9.0Hz,1H),7.17(m,4H),5.82(d,J＝10.0Hz,1H),4.45(s,2H),4.07(q,J＝5.3Hz,1H),3.87(d,J＝11.4Hz,1H),3.73(q,J＝5.9,5.0Hz,1H),3.66(m,2H),3.27(t,J＝5.8Hz,2H),3.16(d,J＝5.2Hz,2H),2.39(m,1H),2.12–1.91(m,4H),1.74(m,1H),1.57(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₄H₂₉N₄O₂:405.22123；Found:405.22197。

And B: a25 mL round-bottom flask was charged with Compound 21(163mg, 0.42mmol), 3mL methanol, and about 3mL 6N HCl solution, stirred at 60 ℃ for about 3 hours, TLC showed the substantial disappearance of starting material, reaction was stopped, and solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and silica gel column purification (dichloromethane: methanol ═ 50:1) gave 40mg of product in 92% yield.

¹H NMR(400MHz,DMSO,δppm)13.00(s,1H),8.01(s,1H),7.58(s,1H),7.45(d,J＝8.9Hz,1H),7.28(d,J＝8.8Hz,1H),7.16(m,4H),4.45(s,2H),3.66(t,J＝5.5Hz,2H),3.26(t,J＝5.8Hz,2H),2.28(s,3H),2.01(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₉H₂₁N₄O:321.17099；Found:321.17117.

EXAMPLE 3 Synthesis of Compound 3

Step A: a25 mL round bottom flask was charged with compound 19(200mg, 0.67mmol), 4mL anhydrous 1, 4-dioxane, and NaH (44mg, 1.818mmol), p-methoxybenzyl bromide (146mg, 0.73mL) at 0 ℃. After stirring at room temperature for about 3 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped and the solvent was removed under reduced pressure. The solution was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Filtration, concentration and silica gel column chromatography (dichloromethane: methanol ═ 100:1) gave 222mg of the product in 87% yield.

¹H NMR(400MHz,DMSO)δ8.04(s,1H),7.64(d,J＝8.9Hz,1H),7.59(s,1H),7.34(d,J＝9.0Hz,1H),7.22(d,J＝8.0Hz,2H),6.90(d,J＝8.1Hz,2H),5.82(m,1H),4.43(s,2H),3.87(d,J＝11.4Hz,1H),3.73(s,3H),3.66(t,J＝5.7Hz,2H),3.26(t,J＝5.9Hz,2H),2.39(m,1H),1.99(m,4H),1.73(m,1H),1.58(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₄H₂₉N₄O₃:421.22342；Found:421.22311.

And B: a50 mL round-bottom flask was charged with Compound 22(182mg, 0.43mmol), 10mL methanol, and about 9mL 6N HCl solution, stirred at 60 ℃ for about 1 hour, TLC showed the substantial disappearance of starting material, the reaction was stopped, and the solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and silica gel column purification (dichloromethane: methanol ═ 50:1) gave 141mg of product in 97% yield.

¹H NMR(400MHz,DMSO,δppm)12.99(s,1H),8.00(s,1H),7.57(s,1H),7.45(d,J＝8.8Hz,1H),7.27(m,1H),7.22(d,J＝8.5Hz,2H),6.90(d,J＝8.6Hz,2H),4.42(s,2H),3.73(s,3H),3.65(m,2H),3.26(t,J＝6.0Hz,2H),2.01(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₉H₂₁N₄O₂:337.16590；Found:337.16510.

EXAMPLE 4 Synthesis of Compound 4

Step A: a25 mL round bottom flask was charged with compound 19(300mg, 0.10mmol), 6mL anhydrous 1, 4-dioxane, and NaH (65mg, 2.73mmol), p-fluorobenzyl bromide (206mg, 1.09mmol) at 0 deg.C. After stirring at room temperature for about 3 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped and the solvent was removed under reduced pressure. The solution was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Filtration, concentration and silica gel column chromatography (dichloromethane: methanol ═ 100:1) gave 367mg of the product in 99% yield.

¹H NMR(400MHz,DMSO)δ8.05(s,1H),7.64(d,J＝8.9Hz,1H),7.60(s,1H),7.34(m,3H),7.17(t,J＝8.5Hz,2H),5.81(dd,J＝9.6,2.5Hz,1H),4.48(s,2H),3.86(m,1H),3.70(m,3H),3.29(t,J＝5.9Hz,2H),2.40(m,1H),2.10–1.88(m,4H),1.75(m,1H),1.57(m,2H).

HR-MS(ESI)m/z(M+H)+calcd for C₂₃H₂₆N₄O₂F:409.20343；Found:409.20142.

And B: a50 mL round-bottom flask was charged with compound 23(378mg, 0.93mmol), 15mL methanol, and about 15mL 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed substantial disappearance of starting material, reaction was stopped, and solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and silica gel column purification (dichloromethane: methanol ═ 50:1) gave 228mg of product in 76% yield.

¹H NMR(400MHz,DMSO,δppm)13.00(s,1H),8.01(s,1H),7.58(s,1H),7.45(d,J＝8.8Hz,1H),7.33(m,1H),7.28(d,J＝8.8Hz,1H),7.17(t,J＝8.8,2H),4.48(s,2H),3.67(m,2H),3.29(m,2H),2.02(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₈H₁₈N₄OF:325.14592；Found:325.14584.

EXAMPLE 5 Synthesis of Compound 5

Step A: a25 mL round bottom flask was charged with compound 19(300mg, 0.10mmol), 6mL anhydrous 1, 4-dioxane, and NaH (65mg, 2.73mmol), p-cyanobenzyl bromide (214mg, 1.09mmol) at 0 deg.C. After stirring at room temperature for about 3 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped and the solvent was removed under reduced pressure. The solution was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Filtration, concentration and silica gel column chromatography (dichloromethane: methanol ═ 100:1) gave 355mg of the product in 94% yield.

¹H NMR(400MHz,DMSO)δ8.05(s,1H),7.81(d,J＝7.9Hz,2H),7.64(d,J＝8.9Hz,1H),7.61(s,1H),7.48(d,J＝7.9Hz,2H),7.35(dd,J＝8.9,1.9Hz,1H),5.81(dd,J＝9.7,2.4Hz,1H),4.58(s,2H),3.86(d,J＝11.3Hz,1H),3.72(m,3H),3.33(t,J＝6.4Hz,2H),2.40(m,1H),2.04(m,3H),1.95(m,1H),1.73(m,1H),1.57(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₄H₂₆N₅O₂:416.20810；Found:416.20599.

And B: a50 mL round-bottom flask was charged with Compound 24(355mg, 0.86mmol), 15mL methanol, and about 15mL 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed substantial disappearance of starting material, reaction was stopped, and solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and purification on silica gel (dichloromethane: methanol ═ 50:1) gave 218mg of product in 77% yield.

¹H NMR(400MHz,DMSO,δppm)13.01(s,1H),8.01(s,1H),7.82(d,J＝8.2Hz,2H),7.60(s,1H),7.47(m,3H),7.28(d,J＝8.8,1.8Hz,1H),4.58(s,2H),3.70(t,J＝5.6Hz,2H),3.33(m,2H),2.06(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₉H₁₈N₅O:332.15059；Found:332.15042.

EXAMPLE 6 Synthesis of Compound 6

Step A: a25 mL round bottom flask was charged with compound 19(300mg, 0.10mmol), 6mL anhydrous 1, 4-dioxane, and NaH (65mg, 2.73mmol), 2, 4-dimethylbenzyl bromide (217mg, 1.09mmol) at 0 deg.C. After stirring at room temperature for about 3 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped and the solvent was removed under reduced pressure. The solution was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Filtration, concentration and silica gel column chromatography (dichloromethane: methanol ═ 100:1) gave 369mg, 97% yield.

¹H NMR(400MHz,DMSO)δ8.04(s,1H),7.64(d,J＝8.9Hz,1H),7.59(d,J＝1.8Hz,1H),7.34(dd,J＝8.9,1.9Hz,1H),7.06(t,J＝7.7Hz,1H),6.99(m,2H),5.81(dd,J＝9.7,2.5Hz,1H),4.47(s,2H),4.07(q,J＝5.3Hz,1H),3.87(d,J＝11.6Hz,1H),3.81–3.57(m,4H),3.22(t,J＝6.0Hz,2H),3.16(d,J＝5.2Hz,3H),2.38(m,1H),2.31(s,1H),2.25(s,3H),2.22(s,3H),2.03(p,J＝5.5Hz,3H),1.96(m,1H),1.73(m,1H),1.57(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₅H₃₁N₄O₂:419.24415；Found:419.24295.

And B: a50 mL round-bottom flask was charged with Compound 25(360mg, 0.86mmol), 15mL methanol, and about 15mL 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed the substantial disappearance of starting material, reaction was stopped, and solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and silica gel column purification (dichloromethane: methanol ═ 50:1) gave 280mg of product in 97% yield.

¹H NMR(400MHz,DMSO,δppm)12.99(s,1H),8.00(s,1H),7.56(s,1H),7.44(d,J＝8.8Hz,1H),7.26(m,1H),7.02(m,3H),4.46(s,2H),3.66(t,J＝5.7Hz,1H),3.20(t,J＝5.9Hz,2H),2.48(m,1H),2.30(s,1H),2.24(s,3H),2.21(s,3H),2.01(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₀H₂₃N₄O:335.18664；Found:335.18591.

EXAMPLE 7 Synthesis of Compound 7

Step A: a25 mL round-bottom flask was charged with compound 19(300mg, 0.10mmol), 6mL anhydrous 1, 4-dioxane, and NaH (65mg, 2.73mmol), methyl p-bromomethylbenzoate (250mg, 1.09mmol) was added at 0 ℃. After stirring at room temperature for about 3 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped and the solvent was removed under reduced pressure. The solution was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Filtration, concentration and silica gel column chromatography (dichloromethane: methanol ═ 100:1) gave 330mg of product in 81% yield.

¹H NMR(400MHz,DMSO)δ8.05(s,1H),7.95(d,J＝7.8Hz,2H),7.70–7.59(m,2H),7.43(d,J＝7.9Hz,2H),7.36(d,J＝8.8Hz,1H),5.82(d,J＝9.3Hz,1H),4.58(s,2H),3.88(m,1H),3.84(s,3H),3.71(dt,J＝11.2,6.1Hz,3H),3.32(m,2H),2.39(m,1H),2.13–1.88(m,4H),1.73(m,1H),1.57(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₅H₂₉N₄O₄:449.21833；Found:449.21692.

And B: a50 mL round-bottom flask was charged with Compound 26(310mg, 0.69mmol), 15mL methanol, and about 15mL 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed the substantial disappearance of starting material, reaction was stopped, and solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and silica gel column purification (dichloromethane: methanol ═ 50:1) gave 200mg of product in 80% yield.

¹H NMR(400MHz,DMSO,δppm)13.00(s,1H),8.01(s,1H),7.95(d,J＝8.2Hz,2H),7.59(m,1H),7.44(t,J＝9.4Hz,3H),7.28(m,1H),4.58(s,2H),3.84(s,3H),3.69(t,J＝5.6Hz,2H),3.3(m,2H),2.05(m,2H).

HR-MS(ESI)m/z(M+H)+calcd for C₁₉H₂₁N₄O₃:365.16082；Found:365.16010.

EXAMPLE 8 Synthesis of Compound 8

Step A: a25 mL round-bottom flask was charged with compound 19(100mg, 0.33mmol), 2mL anhydrous 1, 4-dioxane, and NaH (39mg, 1.64mmol), p-bromomethylbenzoic acid (141mg, 0.66mmol) at 0 deg.C. After stirring at room temperature for about 3 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped and the solvent was removed under reduced pressure. The solution was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Filtration, concentration and silica gel column chromatography (dichloromethane: methanol ═ 100:1) gave 126mg of the product in 87% yield.

¹H NMR(400MHz,DMSO)δ12.86(s,1H),8.05(s,1H),7.93(d,J＝7.9Hz,2H),7.68–7.59(m,2H),7.40(d,J＝8.0Hz,2H),7.36(dd,J＝8.9,1.9Hz,1H),5.82(dd,J＝9.7,2.5Hz,1H),5.74(s,1H),4.58(s,2H),3.87(d,J＝11.5Hz,1H),3.72(dt,J＝11.3,5.9Hz,3H),2.39(m,2H),2.09–1.88(m,4H),1.76(m,2H),1.57(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₄H₂₇N₄O₄:435.20268；Found:435.20193.

And B: a25 mL round-bottom flask was charged with Compound 27(90mg, 0.21mmol), 5mL methanol, and about 5mL 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed the substantial disappearance of starting material, reaction was stopped, and solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and purification on silica gel column (dichloromethane: methanol ═ 50:1) gave 65mg of product in 89% yield.

¹H NMR(400MHz,DMSO,δppm)12.94(s,1H),8.01(s,1H),7.93(d,J＝8.1Hz,2H),7.60(s,1H),7.46(d,J＝8.8Hz,1H),7.40(d,J＝8.1Hz,2H),7.29(d,J＝8.8Hz,1H),4.57(s,2H),3.69(t,J＝5.5Hz,2H),3.32(t,J＝5.8Hz,2H),2.06(t,J＝5.5Hz,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₉H₁₉N₄O₃:351.14517；Found:351.14462.

EXAMPLE 9 Synthesis of Compound 9

Step A: a25 mL round bottom flask was charged with compound 19(100mg, 0.33mmol), 2mL anhydrous 1, 4-dioxane, and NaH (24mg,0.91mmol), p-bromobenzyl bromide (91mg, 0.36mmol) was added at 0 deg.C. After stirring at room temperature for about 3 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped and the solvent was removed under reduced pressure. The solution was diluted with dichloromethane, washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Filtration, concentration and silica gel column chromatography (dichloromethane: methanol ═ 100:1) gave 135mg of the product in 95% yield.

¹H NMR(400MHz,DMSO)δ8.04(s,1H),7.71–7.57(m,2H),7.54(d,J＝7.9Hz,2H),7.34(d,J＝9.0Hz,1H),7.26(d,J＝8.0Hz,2H),5.81(dd,J＝9.7,2.5Hz,1H),4.47(s,2H),3.87(d,J＝11.2Hz,1H),3.71(dt,J＝23.1,6.2Hz,3H),3.20(m,1H),2.39(m,1H),2.03(p,J＝5.4Hz,3H),1.95(d,J＝13.0Hz,1H),1.73(m,1H),1.57(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₃H₂₆N₄O₂Br:469.12337；Found:469.12198.

And B: a25 mL round-bottom flask was charged with Compound 28(160mg, 0.34mmol), 8mL methanol, and about 8mL 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed substantial disappearance of starting material, reaction stopped, and solvent removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and silica gel column purification (dichloromethane: methanol ═ 50:1) gave 118mg of product in 90% yield.

¹H NMR(400MHz,DMSO,δppm)8.01(s,1H),7.58(s,1H),7.53(d,J＝8.2Hz,2H),7.45(d,J＝8.8Hz,1H),7.27(m,3H),4.46(s,2H),3.67(t,J＝5.7Hz,2H),3.29(t,J＝5.7Hz,2H),3.15(s,1H),2.03(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₈H₁₈N₄OBr:385.06585；Found:385.06534.

EXAMPLE 10 Synthesis of Compound 10

Step A: a25 mL round bottom flask was charged with compound 19(100mg, 0.30mmol), p-nitrobenzophenone (91mg, 0.46mmol), cesium carbonate (138mg, 0.24mmol), palladium (II) acetate (3mg, 0.012mmol), 5mL anhydrous 1, 4-dioxane, under argon, Xantphos (11mg, 0.018mmol) and stirred at 100 deg.C. The reaction was carried out for about 2 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped, and the solvent was removed under reduced pressure. Column chromatography on silica gel (dichloromethane: methanol ═ 20:1) gave 110mg of the product in 86% yield.

¹H NMR(400MHz,DMSO,δppm)8.17(m,2H),8.08(s,1H),7.74–7.67(m,2H),7.64(m,2H),7.40(dd,J＝8.9,1.9Hz,1H),5.83(dd,J＝9.6,2.3Hz,1H),3.98–3.82(m,3H),3.82–3.67(m,3H),2.40(m,1H),2.23(m,2H),2.12–1.89(m,2H),1.84–1.64(m,1H),1.58(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₂H₂₄N₅O₄:422.18228；Found:422.18201.

And B: a25 mL round-bottom flask was charged with Compound 29(100mg, 0.24mmol), 5mL methanol, and about 5mL 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed the substantial disappearance of starting material, reaction was stopped, and solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and silica gel column purification (dichloromethane: methanol ═ 50:1) gave 78mg of product in 98% yield.

¹H NMR(400MHz,DMSO,δppm)13.07(s,1H),8.17(d,J＝9.2Hz,2H),8.04(s,1H),7.69(s,1H),7.66–7.59(m,2H),7.50(d,J＝8.8Hz,1H),7.33(dd,J＝8.8,1.9Hz,1H),3.92(t,J＝5.8Hz,2H),3.77(t,J＝5.9Hz,2H),2.23(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₇H₁₆N₅O₃:338.12477；Found:338.12479.

EXAMPLE 11 Synthesis of Compound 11

Step A: a25 mL round-bottom flask was charged with compound 19(70mg, 0.23mmol), 4-fluorobromobenzene (48mg, 0.28mmol), cesium carbonate (97mg, 0.30mmol), palladium (II) acetate (2mg, 0.0085mmol), 4mL anhydrous 1, 4-dioxane, under argon, Xantphos (7mg, 0.013mmol) and stirred at 100 deg.C. The reaction was carried out for about 2 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped, and the solvent was removed under reduced pressure. Column chromatography on silica gel (dichloromethane: methanol ═ 20:1) gave 67mg of the product in 80% yield.

¹H NMR(400MHz,DMSO)δ8.06(s,1H),7.72–7.60(m,2H),7.44–7.27(m,4H),7.14(m,1H),5.82(dd,J＝9.6,2.5Hz,1H),3.87(dd,J＝11.5,3.9Hz,1H),3.75(m,5H),2.46–2.31(m,1H),2.20(p,J＝5.9Hz,2H),1.99(m,2H),1.75(m,1H),1.58(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₂H₂₄N₄O₂F:395.18788；Found:395.18661.

And B: a25 mL round-bottom flask was charged with compound 30(44mg, 0.11mmol), 3mL methanol, and about 3mL 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed the substantial disappearance of starting material, reaction was stopped, and solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and silica gel column purification (dichloromethane: methanol ═ 50:1) gave 34mg of the product in 98% yield.

¹H NM(500MHz,DMSO,δppm)13.01(s,1H),8.01(s,1H),7.64(s,1H),7.46(d,J＝8.8Hz,1H),7.37–7.27(m,3H),7.14(t,J＝8.7Hz,2H),3.76(m,4H),2.19(m,2H).HR-MS(ESI)m/z(M+H)⁺calcd for C₁₇H₁₆N₄OF:311.13027；Found:311.12912.

EXAMPLE 12 Synthesis of Compound 12

Step A: a25 mL round bottom flask was charged with compound 19(70mg, 0.23mmol), p-bromotrifluorotoluene (62mg, 0.28mmol), cesium carbonate (97mg, 0.30mmol), palladium (II) acetate (2mg, 0.0085mmol), 4mL anhydrous 1, 4-dioxane, under argon, and Xantphos (7mg, 0.013mmol) was added and the reaction stirred at 100 ℃. The reaction was carried out for about 2 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped, and the solvent was removed under reduced pressure. Column chromatography on silica gel (dichloromethane: methanol ═ 20:1) gave 72mg of the product in 77% yield.

¹H NMR(400MHz,DMSO)δ8.07(s,1H),7.67(m,4H),7.57(d,J＝8.4Hz,2H),7.39(d,J＝8.9Hz,1H),5.83(d,J＝9.4Hz,1H),3.87(m,3H),3.75(m,3H),2.39(m,1H),2.22(p,J＝5.8Hz,2H),1.99(m,2H),1.77(m,1H),1.57(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₃H₂₄N₄O₂F₃:445.18298；Found:445.18459.

And B: a25 mL round-bottom flask was charged with Compound 31(70mg, 0.16mmol), 5mL methanol, and about 5mL 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed the substantial disappearance of starting material, reaction was stopped, and solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and silica gel column purification (dichloromethane: methanol ═ 50:1) gave 45mg of the product in 80% yield.

¹H NMR(500MHz,DMSO,δppm)13.04(s,1H),8.03(s,1H),7.66(d,J＝10.2Hz,3H),7.57(d,J＝8.3Hz,2H),7.49(d,J＝8.8Hz,1H),7.32(d,J＝8.7Hz,1H),3.86(t,J＝5.3Hz,2H),3.77(t,J＝5.5Hz,2H),2.22(t,J＝5.2Hz,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₈H₁₆N₄OF₃:361.12707；Found:361.12582.

EXAMPLE 13 Synthesis of Compound 13

Step A: a25 mL round bottom flask was charged with compound 19(70mg, 0.23mmol), 4-bromoxynil (55mg, 0.30mmol), cesium carbonate (97mg, 0.30mmol), palladium (II) acetate (2mg, 0.0085mmol), 4mL anhydrous 1, 4-dioxane, under argon, Xantphos (7mg, 0.013mmol) and stirred at 100 deg.C. The reaction was carried out for about 2 hours, TLC showed that the starting material had substantially disappeared, the reaction was stopped, and the solvent was removed under reduced pressure. Column chromatography on silica gel (dichloromethane: methanol ═ 20:1) gave 81mg of the product in 87% yield.

¹H NMR(400MHz,DMSO)δ8.07(s,1H),7.76(d,J＝8.4Hz,2H),7.68(d,J＝9.0Hz,2H),7.56(d,J＝8.5Hz,2H),7.39(dd,J＝8.9,1.9Hz,1H),5.83(dd,J＝9.7,2.5Hz,1H),3.86(m,3H),3.76(t,J＝6.2Hz,2H),3.72(m,1H),2.46–2.31(m,1H),2.21(p,J＝5.8Hz,2H),1.99(m,2H),1.73(m,1H),1.57(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₃H₂₄N₅O₂:402.19245；Found:402.19086.

And B: a25 mL round-bottom flask was charged with Compound 32(40mg, 0.10mmol), 3mL methanol, and about 3mL 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed the substantial disappearance of starting material, reaction was stopped, and solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and purification on silica gel column (dichloromethane: methanol ═ 50:1) gave 30mg of product in 95% yield.

¹H NMR(400MHz,DMSO,δppm)13.05(s,1H),8.03(s,1H),7.75(d,J＝8.7Hz,2H),7.67(s,1H),7.56(d,J＝8.7Hz,2H),7.49(d,J＝8.9Hz,1H),7.31(dd,J＝8.8,1.8Hz,1H),3.87(t,J＝5.7Hz,2H),3.76(t,J＝5.9Hz,2H),2.21(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₈H₁₆N₅O:318.13494；Found:318.13354.

EXAMPLE 14 Synthesis of Compound 14

Step A: a50 mL round bottom flask was charged with compound 19(100mg, 0.30mmol), p-bromoanisole (56. mu.L, 0.46mmol), 10mL dry toluene, under argon, and copper iodide (462mg, 2.42mmol), N, N-dimethyl-1, 2-ethylenediamine (0.28mL, 3.03mmol) was added rapidly, stirred at 100 ℃ for about 36 hours, TLC showed substantial disappearance of starting material, the reaction was stopped, filtered through celite, the filter cake was washed with ethyl acetate, the organic phases combined, and the solvent was removed under reduced pressure. Column chromatography on silica gel (dichloromethane: methanol ═ 100:1) gave 70mg of the product in 57% yield.

¹H NMR(400MHz,DMSO,δppm)8.04(s,1H),7.64(m,2H),7.37(d,J＝8.9Hz,1H),7.22(d,J＝7.1Hz,2H),6.87(d,J＝7.2Hz,2H),5.81(d,J＝9.5Hz,1H),3.86(m,1H),3.76-3.71(m,6H)3.74(s,3H),2.39(m,1H),2.18(m,2H),2.09–1.86(m,2H),1.74(m,1H),1.57(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₃H₂₇N₄O₃:407.20777；Found:407.20630.

And B: a25 mL round-bottom flask was charged with compound 33(68mg, 0.17mmol), 3mL methanol, and about 3mL 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed the substantial disappearance of starting material, reaction was stopped, and solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and silica gel column purification gave 50mg of product in 94% yield.

¹H NMR(500MHz,DMSO,δppm)13.02(s,1H),8.01(s,1H),7.62(s,1H),7.45(m,1H),7.29(m,1H),7.22(m,2H),6.97(m,2H),3.76(m,2H),3.73(s,3H),3.71(m,2H),2.18(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₈H₁₉N₄O₂:323.15025；Found:323.14969.

EXAMPLE 15 Synthesis of Compound 15

Step A: a50 mL round bottom flask was charged with compound 19(300mg, 0.10mmol), bromobenzene (214mg, 1.36mmol), 30mL dry toluene, under argon, copper iodide (1523mg, 8.00mmol), N, N-dimethyl-1, 2-ethylenediamine (882mg, 10.00mmol) was added rapidly and stirred at 100 deg.C for about 36 hours, TLC showed substantial disappearance of starting material, the reaction was stopped, celite was filtered, the filter cake was washed with ethyl acetate, the organic phases were combined and the solvent was removed under reduced pressure. Chromatography on silica gel (dichloromethane: methanol ═ 100:1) gave 220mg of the product in 59% yield.

¹H NMR(400MHz,DMSO)δ8.06(m,1H),7.65(m,2H),7.50–7.24(m,4H),7.13(m,1H),5.82(d,J＝9.3Hz,1H),3.87(d,J＝11.4Hz,1H),3.83–3.62(m,5H),2.39(m,1H),2.20(m,2H),2.13–1.89(m,2H),1.74(m,1H),1.57(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₂H₂₅N₄O₂:377.19720；Found:377.19711.

And B: a25 mL round-bottom flask was charged with compound 34(200mg, 0.53mmol), 10mL methanol, and about 10mL 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed the substantial disappearance of starting material, reaction was stopped, and solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and silica gel column purification gave 149mg of product in 96% yield.

¹H NMR(400MHz,DMSO,δppm)13.02(s,1H),8.02(s,1H),7.64(s,1H),7.46(d,J＝8.9Hz,1H),7.39–7.24(m,5H),7.13(m,1H),3.77(m,4H),2.20(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₇H₁₇N₄O:293.13969；Found:293.13892.

EXAMPLE 16 Synthesis of Compound 16

Step A: a50 mL round bottom flask was charged with compound 19(300mg, 0.10mmol), p-bromotoluene (257mg, 1.500mmol), 30mL dry toluene, under argon, and copper iodide (1523mg, 8.00mmol), N, N-dimethyl-1, 2-ethylenediamine (882mg, 10.00mmol) was added rapidly and stirred at 100 deg.C for about 36 hours, TLC showed substantial disappearance of starting material, the reaction was stopped, celite was filtered, the filter cake was washed with ethyl acetate, the organic phases were combined and the solvent was removed under reduced pressure. Column chromatography on silica gel (dichloromethane: methanol ═ 100:1) gave 297mg of the product in 76% yield.

¹H NMR(500MHz,DMSO)δ8.05(s,1H),7.65(m,2H),7.37(d,J＝9.0Hz,1H),7.20(d,J＝7.5Hz,2H),7.12(d,J＝8.0Hz,2H),5.82(d,J＝9.5Hz,1H),3.86(d,J＝11.0Hz,1H),3.75(m,5H),2.39(m,1H),2.26(s,3H),2.18(m,2H),2.10–1.90(m,2H),1.74(m,1H),1.57(s,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₂₃H₂₇N₄O₂:391.21285；Found:391.21140.

And B: a25 mL round-bottom flask was charged with Compound 34(288mg, 0.74mmol), 12mL methanol, and about 12mL 6N HCl solution, stirred at 60 ℃ for about 1.5 hours, TLC showed the substantial disappearance of starting material, reaction was stopped, and solvent was removed under reduced pressure. Diluting with water, adjusting pH to 10 with 2N NaOH solution, extracting with dichloromethane, mixing organic phases, washing with saturated sodium chloride solution, and drying with anhydrous sodium sulfate. Filtration, concentration and silica gel column purification gave 200mg of product in 89% yield.

¹H NMR(500MHz,DMSO,δppm)13.03(s,1H),8.01(s,1H),7.63(s,1H),7.46(d,J＝8.8Hz,1H),7.29(d,J＝8.8Hz,1H),7.20(m,2H),7.11(m,2H),3.75(m,4H),2.26(3,3H),2.18(m,2H).

HR-MS(ESI)m/z(M+H)⁺calcd for C₁₈H₁₉N₄O:307.15534；Found:307.15421.

Pharmacological experiments

Experimental example 1: evaluation of Rho kinase Activity inhibition by 3- (1H-indazole) -tetrahydropyrimidin-2-one compounds.

The Rho kinase activity was detected by enzyme-linked immunosorbent assay (ELISA). A commercially available ELISA kit (Cyclex Rho-kinase Assay kit) containing a 96-well plate coated with the Rho kinase substrate MBS (MYPT1) was used to phosphorylate the threonine residue at position 696 of the substrate in the presence of ATP. According to the principle of antigen-antibody specific binding reaction, after the kinase reaction is completed, using threonine phosphorylation specific monoclonal antibody marked by HRP as a probe to be specifically bound with phosphorylated threonine residues, finally using Tetramethylbenzidine (TMB) to react with HRP for color development, and measuring the absorbance so as to reflect the activity of Rho kinase.

The concentration of the enzyme stock solution was 100 ng/. mu.l, and the solution was diluted with the kinase buffer solution to a working concentration of 0.2 ng/. mu.l. Kinase reaction buffer 20X ATP was diluted 1X with kinase buffer. Mu.l enzyme + 10. mu.l compound + 80. mu.l kinase reaction buffer was added to the microplate and reacted at 37 ℃ for 30 minutes. The liquid was decanted and the plate washed 5 times with elution buffer. Adding HRP-labeled antibodyThe reaction was carried out at room temperature for 1 hour. The liquid was decanted and the plate washed 5 times with elution buffer. Adding TMB for developing for about 5-10 min, adding reaction stopping solution to stop developing when the color is obvious, and measuring the absorption value at 450 nm. Judging the activity of the enzyme according to the OD value, judging the inhibitory activity of the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound on Rho kinase, and calculating the half Inhibitory Concentration (IC) of Rho kinase, wherein the inhibition rate is (OD standard-OD sample)/OD standard is 100 percent₅₀) The value is obtained.

The results show that the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound in the general formula I can inhibit the activity of Rho kinase in a dose-dependent manner, and the IC of partial 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound₅₀6.01 and 9.46 mu M respectively, and the pharmaceutical effect and the application of the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound in preventing and treating cardiovascular and cerebrovascular diseases induced by abnormal increase of Rho kinase activity are judged.

TABLE 1 inhibition of Rho kinase activity by part of 3- (1H-indazole) -tetrahydropyrimidin-2-one compounds.

Experimental example 2: the effect of 3- (1H-indazole) -tetrahydropyrimidine-2-ones on the pre-stimulation of vasoconstriction by norepinephrine.

6 male SD rats with the weight of 250-300 g are selected as experimental animals. After the rat is dead at the broken end, the thoracic aorta is rapidly taken out, cut into a blood vessel ring with the length of 2-3mm, the blood vessel ring is placed in a bath containing 10mL of K-H liquid (constant temperature of 37 ℃, and mixed gas of 95% oxygen and 5% carbon dioxide is continuously introduced), and the tension change is transmitted and recorded in a BL-420S biological function experimental system. The vascular ring was stabilized at 1.2g tension for 60min, during which time K-H solution was changed 1 time every 20 min. The vascular ring is stimulated by 60mM KCl to contract, and the vascular ring is flushed 2 times after reaching the maximum amplitude, so that the vascular ring is restored to the state before stimulation for 2 times. mu.M Norepinephrine (Norependerine, NE) was added, 10. mu.M acetylcholine was administered after the maximum amplitude of contraction was reached, and the amplitude of relaxation was measured. If the diastolic amplitude is greater than 80%, the endothelium is complete, which indicates that the loss to the vascular endothelial ring is very small in the operation process, the endothelium integrity is good, and the vascular endothelial ring is a group with endothelium; if the relaxation is not performed or the relaxation amplitude is less than 30%, the loss of endothelium is large, the function of endothelium is incomplete, and the group is the non-endothelium group. Experiments were performed using an endothelial intact vascular ring in this example. After detecting the endothelial function, the vessel is washed to the state before stimulation and then stabilized for 30min, and the influence of the drug on the vascular ring is further detected.

Norepinephrine is a vasoconstrictor. In this example, blood vessels are contracted by adding 1 μ M norepinephrine into the reaction system, the cumulative addition of 3- (1H-indazole) -tetrahydropyrimidine-2-one compounds at concentrations of 0.3, 1, 3, 10, 30, and 100 μ M has significant relaxation effect on the contraction of endothelial vascular rings caused by norepinephrine, the relaxation effect is enhanced with the increase of the concentration, and the half Effective Concentration (EC) of part of 3- (1H-indazole) -tetrahydropyrimidine-2-one compounds is in dose-effect relationship₅₀) Shown in Table 2, the relaxation effect of the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound on the vasoconstriction caused by norepinephrine and the drug effect and the application of the compound in preventing and treating the cardiovascular and cerebrovascular diseases induced by abnormal increase of vascular tone are judged.

TABLE 2 relaxation of noradrenaline pre-systolic blood vessels by some 3- (1H-indazole) -tetrahydropyrimidin-2-ones.

Experimental example 3: the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound has the function of pre-stimulating vasoconstriction by high-concentration potassium chloride.

The method for preparing the vascular ring for tonometry was the same as in example 2.

High concentrations of potassium chloride can cause vasoconstriction. In the embodiment, the blood vessel is in a contraction state by adding 60mM KCl into a reaction system, the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compounds added in cumulative concentrations of 0.3, 1, 3, 10, 30 and 100 mu M have obvious inhibition effect on the contraction of endothelial vascular rings caused by KCl, the inhibition effect is enhanced along with the increase of the concentration,and are in dose-effect relationship. Half the Effective Concentration (EC) of a fraction of 3- (1H-indazol) -tetrahydropyrimidin-2-ones₅₀) Table 3 shows the effect of 3- (1H-indazole) -tetrahydropyrimidine-2-one compounds on vasodilation of vasoconstriction caused by high concentration of potassium chloride, and the pharmaceutical effect and use thereof for preventing and treating cardiovascular and cerebrovascular diseases induced by abnormal increase in vascular tone.

TABLE 3 vasodilation of part of 3- (1H-indazole) -tetrahydropyrimidin-2-ones on pre-vasoconstriction of potassium chloride at high concentrations.

Experimental example 4: the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound has the effect of pre-stimulating cerebral basilar arterioconstriction by high-concentration potassium chloride.

The reduction of blood and oxygen supply to tissues by cerebrovascular contraction is an important cause of ischemic stroke diseases. The isolated blood vessel tension measurement is one of the classical methods for measuring the vasodilatation activity of a compound, and the experiment adopts a DMT 620M four-channel micro-vascular tension measurement system to measure the isolated cerebral basilar artery blood vessel tension of a rat, and the high K is⁺The contraction is caused by the depolarization of the vascular smooth muscle cell membrane, activating the voltage-dependent channel Ca²⁺Open to promote Ca²⁺Internal flow through high K⁺To stimulate vasoconstriction, compounds are added to allow vasodilation activity.

A100 mL beaker was taken, a small amount of prepared pre-cooled PSS buffer was poured in, and 95% O was passed through₂And 5% CO₂The air mixture was pre-saturated and the dish was pre-cooled on ice. The rats are sacrificed by decapitation, the brains are taken out, fixed, the meninges are stripped, the cerebral vessels are separated, and the vascular strips are cut into vascular rings with the length of about 2 mm.

The microvascular tensiometer switch was turned on and the instrument was heated to 37 ℃. Take off the measuring instrumentOne channel of the blood vessel ring is arranged under a dissecting mirror, a section of tungsten wire with the diameter of 40 mu M is cut, one end of the tungsten wire is arranged at the right end of a fixed frame in a bath and is tightly fixed by an upper end screw, the blood vessel ring is sleeved at the other end of the tungsten wire, the end is also fixed at the right end of the fixed frame and is fixed by a lower end screw. And a section of tungsten wire is cut and inserted into the fixed vascular ring, two ends of the tungsten wire are fixed at the left end of the fixing frame, and the upper screw and the lower screw are tightened to tighten the tungsten wire. And finally, adjusting the tungsten filaments at the left end and the right end to enable the two tungsten filaments to be in a horizontal state. And adjusting the optimal initial tension of 4 channels after the tension of each channel on the display of the microvessel instrument is zeroed. Stabilize for 1h, during which time the pre-incubated PSS buffer was changed every 20 min. After the vasoconstriction was stimulated to reach plateau with KCl-PSS solution (60mM), 0.1-100. mu.M of 3- (1H-indazole) -tetrahydropyrimidin-2-one compound was administered cumulatively and it was observed whether it could cause vasodilation. The diastolic rate was calculated as (maximum tension-after-tension given to the sample)/(maximum systolic tension-base tension) × 100%. Half the Effective Concentration (EC) of a fraction of 3- (1H-indazol) -tetrahydropyrimidin-2-ones₅₀) Table 4 shows the effect of 3- (1H-indazole) -tetrahydropyrimidine-2-one compounds on the relaxation of cerebral basolate microvascular contraction induced by high-concentration potassium chloride, and the pharmaceutical effect and application thereof in preventing and treating cardiovascular and cerebrovascular diseases induced by abnormal increase in vascular tone.

TABLE 4 relaxation of a portion of 3- (1H-indazole) -tetrahydropyrimidin-2-one compounds on pre-systolic cerebral basal microvasculature with high concentrations of potassium chloride.

Experimental example 5: the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound has the function of pre-stimulating mesenteric arteriolar constriction by high-concentration potassium chloride.

Mesenteric microvessels are a readily available class of blood vessels whose systolic or diastolic function represents the systemic peripheral vascular resistance. The compound with the relaxation effect on mesenteric microvessels may have a certain treatment effect on various hypertension diseases. The experiment in this example was also performed using a DMT 620M four channel microvascular tonometry system.

A100 mL beaker was taken, a small amount of prepared PSS buffer was poured in, and 95% O was passed through₂And 5% CO₂The air mixture was pre-saturated and the dish was pre-cooled on ice. Killing rat by cutting head, taking mesentery, fixing, separating mesentery micro-motion vein blood vessel, carefully cutting off fat, vein and connective tissue connected on the blood vessel, cutting the blood vessel into blood vessel ring with length of about 2mm, and passing tungsten wire through the blood vessel.

The method and procedure for measuring vascular tone were the same as in example 4. Half the Effective Concentration (EC) of a fraction of 3- (1H-indazol) -tetrahydropyrimidin-2-ones₅₀) Table 5 shows that the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compound has the relaxation effect on cerebral fundus microvascular contraction caused by high-concentration potassium chloride and the pharmaceutical effect and the application of the pharmaceutical composition for preventing and treating cardiovascular and cerebrovascular diseases induced by abnormal increase of vascular tone are judged.

TABLE 5 relaxation of part of 3- (1H-indazole) -tetrahydropyrimidin-2-ones on pre-systolic mesenteric microvessels with high concentrations of potassium chloride.

Experimental example 6: 3- (1H-indazole) -tetrahydropyrimidine-2-one compounds have the effect of promoting glucose consumption of liver cells.

Diabetes Mellitus (DM) is a series of metabolic disorder syndromes characterized by hyperglycemia due to insulin deficiency or impaired pancreatic islet function, and decreased glucose uptake by target tissues such as liver, skeletal muscle, and fat. Research and development of novel hypoglycemic drugs, promotion of islet cell functions and absorption and utilization of glucose by target organs become important subjects of diabetes drug research.

The liver is the center of metabolism of substances in animals, is one of the target organs of insulin action, and is the main organ for utilizing and producing glucose. HepG2(human hepatocellular liver cancer cell line) cell is derived from a hepatocyte, is a hepatoblastoma cell line with a phenotype very similar to that of the hepatocyte, retains a plurality of biological characteristics of the hepatocyte, can express glucokinase, insulin receptor, insulin-like growth factor and the like, and is one of common cell models for researching the in-vitro hypoglycemic action. HepG2 cells are selected for the model, and the influence of the compound on the glucose consumption of HepG2 cells is researched.

After digestion of the cultured cells, 5X 10 cells/ml⁴The density of each cell was inoculated into a 96-well plate, 200. mu.L per well, and cells in good growth state were selected 24 hours later and synchronized for 12 hours by replacing the serum-free PRMI-1640 culture medium. The groups were randomly divided into normal control group, positive drug insulin group and compound group with different concentrations. The culture medium was replaced with a serum-free PRMI-1640 medium containing or not containing the compound, and the Glucose Content (GC) in the medium was measured by the glucose oxidase method after 24 hours of culture. And calculating the change value of the glucose content in the culture solution of each group of blank wells by taking the average value of each group of blank wells as a control. Removing the culture solution to be detected after the 24-hour incubation of the glucose consumption test is finished, replacing a human with MTT serum-free culture solution containing 0.5mg/mL for incubation for 4 hours, removing the original culture solution, adding 150 mu L of DMSO into each hole, uniformly mixing, measuring an OD value (MTT) at 540nm on an enzyme labeling instrument, and reflecting the activity and the quantity of cells by the OD value; GC/MTT represents the sugar consumption of a unit cell, 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compounds shown in Table 5 have the effect of promoting the glucose consumption of liver cells, and the pharmaceutical effect and the application of the 3- (1H-indazole) -tetrahydropyrimidine-2-ketone compounds in preventing and treating diabetes and complications thereof are judged.

TABLE 6 promoting effect of part of 3- (1H-indazole) -tetrahydropyrimidin-2-ones on glucose consumption by HepG2 hepatocytes.

In summary, the 3- (1H-indazole) -tetrahydropyrimidine-2-one compounds or physiologically acceptable salts thereof respectively have the effect of inhibiting Rho kinase activity; has effects in dilating thoracic aorta blood vessel, protecting blood vessel, and lowering blood pressure; has effects in dilating cerebral basilar arterioles and preventing and treating cerebral ischemia; has the functions of relaxing mesenteric arteriole and reducing peripheral vascular resistance of an organism, has the function of promoting glucose consumption, and is a novel compound which has important application in the aspect of preparing medicaments for preventing and/or treating cardiovascular and cerebrovascular diseases such as hypertension, atherosclerosis, cerebral vasospasm, coronary vasospasm, myocardial infarction, heart failure, diabetes and the like, diabetes and complications thereof and the like.

Claims

1. The compound shown as the general formula (I) and the pharmaceutically acceptable salt thereof,

n＝0、1、2；

the halogen substitution comprises single halogen substitution, double halogen substitution, trihalo substitution and polyhalo substitution.

2. The compound according to claim 1 and pharmaceutically acceptable salts thereof, wherein R is₁，R₂Independently selected from hydrogen, halogen, trihalo-substituted methyl, cyano, carboxyl, carbomethoxy, carboethoxy, propyl, isopropyl, nitro, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, methoxy, ethoxy, propoxy, isopropoxy; the halogen is selected from fluorine, chlorine, bromine and iodine.

3. A compound according to any one of claims 1-2, characterized in that said compound is selected from the group consisting of

4. A process for the preparation of a compound according to any one of claims 1 to 3, comprising the steps of:

when n is 1 and 2: reacting 5-aminoindazole with methyl chlorobenzoate to obtain a compound 17, carrying out cyclization reaction on the compound 17 and 3-chloropropylamine hydrochloride to obtain a compound 18, protecting the compound 18 with THP to obtain a compound 19, carrying out nucleophilic substitution reaction on the compound 18 and a compound of a formula (II) under the condition of NaH to obtain a compound of a formula (III), and removing THP to obtain a compound of a formula (I);

i amidation; ii a cyclization reaction; iii THP protection; iv substitution reaction; v de-THP protection, wherein R₁，R₂Is as defined in any one of claims 1 to 3;

the term "halogen" refers to fluorine, chlorine, bromine, iodine;

i amidation; ii a cyclization reaction; iii THP protection; iv coupling reaction; v de-THP protection, wherein R₁，R₂As defined in any one of claims 1 to 3,

in the compounds of the formulas II and IV, X is selected from bromine.

5. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition comprises a tablet, capsule, pill, or injection.

7. The pharmaceutical composition of claim 5, wherein said pharmaceutical composition is selected from the group consisting of a solution, a suspension, an emulsion, a powder, a controlled release formulation, and a sustained release formulation.

8. Use of a compound according to any one of claims 1 to 3, and pharmaceutically acceptable salts thereof, for the manufacture of a product for the prevention, alleviation and/or treatment of cardiovascular and cerebrovascular diseases, diabetes and complications thereof.

9. The use according to claim 8, wherein said cardiovascular or cerebrovascular disease is selected from the group consisting of hypertension, atherosclerosis, cerebral vasospasm, coronary vasospasm, myocardial infarction, and heart failure.

10. The use of claim 8, wherein the diabetic complications comprise diabetic cardiovascular complications and diabetic cerebrovascular complications.

11. The use according to claim 8, wherein said product comprises a pharmaceutical or nutraceutical product.