CN103450163B - Indazole compounds, its preparation method and its medicinal usage - Google Patents

Abstract

Present invention firstly discloses a series of indazole compounds or its physiologically acceptable derivative, its preparation method and its medicinal usage.The indazole compounds or its physiologically acceptable derivative have the effect for suppressing Rho kinase activities respectively；Effect with vasodilator, protection vascular function；With the reperfusion injury effect of protection cardiac muscle cell's anti anoxia；With the effect for promoting grape cell sugar consumption；Effect with protection vascular endothelial cell to anti-oxidative damage, is class noval chemical compound with important use in terms of preparation prevents and treats the medicines such as the cardiovascular and cerebrovascular diseases such as hypertension, atherosclerosis, cerebral angiospasm, coronarospasm, myocardial infarction, heart failure and diabetic complication, diabetes.

Description

Indazole compounds, its preparation method and its medicinal usage

Technical field

Used the present invention relates to indazole compounds or its physiologically acceptable derivative, its preparation method and its medicine On the way.Specifically related to noval chemical compound, new medicine act on and its are preparing prevention, alleviating and/or treatment hypertension, Atherosclerosis The cardiovascular and cerebrovascular diseases such as change, cerebral angiospasm, coronarospasm, myocardial infarction, heart failure and diabetic complication, diabetes Or the application in the medicine and health products of symptom, belong to innovation drug research technical field.

Technical background

It is well known that cardiac and cerebral vascular diseases are the number one killers for threatening human health, its morbidity colony is progressively young Change, morbidity and mortality are also constantly rising.The Etiological of these diseases is center or peripheral vascular 26S Proteasome Structure and Function Change, such as the abnormal increase of antiotasis.Diastolic blood vessel activity material may be used primarily for hypertension, atherosclerosis, brain blood Pipe spasm, coronarospasm, myocardial infarction, heart failure and diabetic angiopathy etc. increase induction with antiotasis extremely Vascular conditions, diabetes and its complication treatment.

Small GTP-binding proteins are a kind of monomer G-protein molecules played a crucial role during cell function regulation. Rho is a kind of small molecular G protein, and Rho kinases is the effector of the earliest Rho for finding.On molecular level, Rho kinase expressions The various factors for promoting inflammation, oxidative stress, thrombosis and fibrosis are adjusted, eNOS is lowered.In cell Level, the kinase mediated VSMCs of Rho shrink, and promote propagation and migrate, and promote inflammatory cell movement.Rho kinases leads to Cross and interacted with the vaso-active substance such as Angiotensin II, endothelin -1, directly participate in the hair of many cardiovascular and cerebrovascular diseases Hair tonic exhibition, such as hypertension, atherosclerosis, cerebral angiospasm, coronarospasm, myocardial infarction, heart failure and glycosuria Disease and its complication etc..

Using Rho kinase inhibitors, to hypertension, atherosclerosis, cerebral angiospasm, coronarospasm, cardiac muscle The treatment of various cardiovascular and cerebrovascular diseases such as infarct, heart failure and diabetic angiopathy, diabetes and its complication has weight Meaning is wanted, the prevention and treatment for the cardiovascular and cerebrovascular disease, diabetes and its complication of current serious harm human health are produced The positive effect of life.

Indazole compounds formula I of the invention and its derivative are noval chemical compound, not in prevention and treatment heart and brain blood The application relevant report of pipe disease, diabetes and its complication.

The content of the invention

One aspect of the present invention, be related to as shown in formula formula I indazole compounds (Chinese and English title, compound lead to Formulas I) and its physiologically acceptable derivative, belong to compound protection domain of the invention.

Another technical problem to be solved by the present invention is that providing the preparation method of this kind of compound.

The invention solves the problems that another technical problem be to provide the pharmaceutical composition containing this kind of compound.

The invention solves the problems that another technical problem be to provide this kind of compound and preparing cardiac and cerebral vascular diseases, diabetes And its application in complication.

To solve technical problem of the invention, adopt the following technical scheme that：

1st, formula (I) compound is led to

Wherein,

R1, R2, R3 independence selected from hydrogen, halogen, halo C1-6 alkyl, C1-6 alkyl, C1-6 alkoxies；

R4 may be selected from ketonic oxygen, 2 hydrogen atoms；

R5 may be selected from hydrogen, cyano group, acid amides；

n=0,1。

Preferably

R1, R2, R3 independence selected from hydrogen, halogen, three halogenated c1-4 alkyls, C1-4 alkyl, C1-4 alkoxies；

R4 may be selected from ketonic oxygen, 2 hydrogen atoms；

R5 may be selected from hydrogen, cyano group, acid amides；

n=0,1。

Preferred compound, including but not limited to

In the present invention, term " halogen " refers to fluorine, chlorine, bromine, iodine.

According to the present invention, formula (I) compound of the present invention can be prepared by following reaction scheme I-II：

When R4 is hydrogen, using route I：

I additions;Ii benzyl protections；Iii is reduced;Iv is hydrogenated;V amidatioons

R4 for carbonyl itch when, using route II：

I additions;V amidatioons

The third aspect of the present invention, is related to a kind of increase initiation extremely for preventing, alleviating and/or treat antiotasis The pharmaceutical composition of disease or symptom, its above-mentioned indazole compounds for containing prevention and/or therapeutically effective amount, and optionally Pharmaceutically acceptable carrier and/or auxiliary material.

In the present invention, according to route of administration, the pharmaceutical composition of the indazole compounds can be in selected from following formulation： Solution, suspension, emulsion, pill, capsule, powder, control release or extended release preparation.

Indazole compounds pharmaceutical composition of the invention can be prepared with known method, using several approach to tested Person applies, including but not limited to parenteral, oral, part, intracutaneous, intramuscular, intraperitoneal, subcutaneous, vein, intra-nasal route.

Indazole compounds pharmaceutical composition of the invention is optional can be used one or more by any conventional method Pharmaceutically acceptable carrier and/or excipient are prepared.Therefore, indazole compounds and its pharmaceutically acceptable derivative can be special Be not formulated as example sucking or be blown into (by mouth or nose) or oral, containing changing, parenteral or rectally.

Indazole compounds pharmaceutical composition can also use solution, suspension, emulsion, pill, capsule, powder, controlled-release is released Put or extended release preparation.These preparations are by the indazole compounds containing therapeutically effective amount, preferably purified form, Yi Jishi The carrier of amount, in the form of providing to the appropriate administration of patient.

The fourth aspect of the invention, is related to indazole compounds formula I preparing prevention, alleviating and/or treatment heart and brain Application in the medicine of the symptom that vascular diseases trigger, as long as these indazole compounds and its derivative are applied to cardiovascular and cerebrovascular Property disease and diabetes and its complication, belong to compound protection domain of the invention.

Described cardiovascular and cerebrovascular disease is the vascular conditions caused by a variety of causes.Especially increased extremely by antiotasis Height induce vascular conditions, including hypertension, atherosclerosis, cerebral angiospasm, coronarospasm, myocardial infarction, Heart failure and diabetes and its complication.

In the present invention, the prevention, alleviation and/or treatment vascular conditions or vascular conditions trigger disease or disease Shape is selected from the activity for suppressing Rho kinases, and Myocytes Anoxia Reperfusion injury is protected in vasodilator, the effect of protection vascular function Wound, promotes grape cell sugar consumption, protects the medicine effect of Oxidation Induced Damages of Vascular Endothelial Cells.

The present invention is to realize by following technical solution：A series of indazole compounds are prepared by artificial synthesized And its derivative；Using Rho kinase protein activity evaluation systems, indazole class, phthalimide derivatives are evaluated to Rho The inhibitory activity of kinases；By extracorporeal blood vessel ring strain measurement system, indazole compounds are further looked to noradrenaline Element or potassium chloride cause vasoconstrictive effect, judge diastole effect of the indazole compounds to isolated rat blood vessel；Using original For cultured neonatal rat heart cells, protective effect of the indazole compounds to Myocytes Anoxia reperfusion injury is evaluated；With external Culture hepatocyte is facilitation of the model evaluation indazole compounds to grape cell sugar consumption；With in vitro culture blood vessel endothelium Cellular superoxide hydrogen damage model evaluation indazole compounds protect the effect of Oxidation Induced Damages of Vascular Endothelial Cells, and overall merit is sentenced Indazole compounds are determined to cardiac and cerebral vascular diseases and the preventive and therapeutic action of diabetes and its complication.

Indazole compounds formula I of the invention is respectively provided with prevention, alleviates and/or control when any formulation is made Treat the medicine effect of cardiac and cerebral vascular diseases or diabetes and its complication.Any medicament, if containing indazole class in its component Compound formula I only prepares patent medicine with indazole compounds formula I separate constituents, in the mark such as its packaging or specification As long as or indicated on other any propaganda materials or point out to have treatment cardiac and cerebral vascular diseases and diabetes and its complication or The effect of disease or symptom that vascular conditions trigger, falls within protection scope of the present invention.

In the present invention, the indazole compounds of the purified form refer to substantially pure, and especially purity is more than 80%, preferably greater than 85%, it is particularly preferred to be more than 90%, the structure indazole compounds even more preferably more than 95%.Institute The structure indazole compounds purity range for stating purified form can be such as 90-96%.

Advantageous Effects：

Indazole compounds formula I is monomer noval chemical compound, has the advantages that toxicity is relatively low, preparation process is simple；

With good applicating and exploitation prospect, be a kind of preferably prevention and treatment cardiovascular and cerebrovascular disease and diabetes and The noval chemical compound of its complication, can be applied to the preparation of medicine and health products.

Indazole compounds have the effect for suppressing Rho kinase activities, the work with vasodilator, protection vascular function With with protection Myocytes Anoxia reperfusion injury, with the medicine effect for promoting grape cell sugar to absorb, with protection blood The effect of endothelial cell oxidative damage, is one class preventing and control hypertension, atherosclerosis, cerebral angiospasm, coronary artery convulsion Contraction, hypertension, atherosclerosis, cerebral angiospasm, coronarospasm, myocardial infarction, heart failure and diabetes and its The cardiovascular and cerebrovascular diseases such as complication, diabetes aspect pole have the noval chemical compound of medical value, with good applicating and exploitation prospect.

Specific embodiment

With reference to the present invention, the medicine for further illustrating the preparation process of indazole compounds and its reducing blood pressure in vivo Thing acts on and increases extremely in antiotasises such as prevention and treatment hypertension the application of the disease of induction.Following embodiments are more detailed Ground illustrates the present invention, is not any limitation of the invention.

Prepare embodiment

Embodiment 1

Step A：Para-fluoroaniline (1.92mL, 20mmol), itaconic acid (3.12g, 24mmol), water are added in round-bottomed bottle 20mL, heating reflux reaction, 6 as a child, and TLC display raw materials disappear substantially, stop reaction, and most of water is removed under reduced pressure, and use afterwards The sodium hydrate aqueous solution of 1M is adjusted to alkalescence, is filtered to remove insoluble matter, then is adjusted to acidity with the hydrochloric acid of 1N, separates out solid, suction filtration, Wash with water 3 times, white solid 4.014g, yield 90% are obtained after drying.

¹H NMR(300M,CDCl₃, δ ppm) and 12.785 (s, 1H ,-COOH), 7.69-7.64 (m, 2H, Ar-H), 7.24- 7.18(m,2H,Ar-H),4.01(m,2H,-CH2-),3.34(m,1H,-CH-),2.72(m,2H,-CH2-).

Step B：Compound 1 (526mg, 2.36mmol), 15mL dichloromethane, 5- Aminoindazoles are added in round-bottomed bottle (266mg, 2mmol), EDCI (575mg, 3mmol) room temperature reaction, as a child, TLC display reactions are complete, are concentrated under reduced pressure for reaction 3 Most of dichloromethane is removed, ethyl acetate dissolving is added, after adding the sodium hydrate aqueous solution of 0.5M to stir 5 minutes, point liquid, Organic phase is washed with water, anhydrous sodium sulfate drying, filtering, and solvent is evaporated off, and silica gel column chromatography obtains compound 472mg, yield 70%.

¹H NMR(300M,CDCl₃, δ ppm) and 12.97 (d, 1H ,-NH-), 10.17 (s, 1H ,-NH-), 8.12 (s, 1H, Ar- H),8.09(s,1H,-CH=N),7.72-7.67(m,2H,Ar-H),7.05-7.42(m,2H,Ar-H),7.21(m,2H,Ar- H),4.07(m,2H,-CH2-),3.46(m,1H,-CH-),2.84(m,2H,-CH2-).

Embodiment 2：

1- (3- trifluoromethyl-phenyls) -5- oxygen-pyrroles -3- carboxylic acids (644mg, 2.36mmol), 15mL is added in round-bottomed bottle Dichloromethane, 5- Aminoindazoles (266mg, 2mmol), EDCI (575mg, 3mmol) room temperature reaction, as a child, TLC shows for reaction 3 Show that reaction is complete, be concentrated under reduced pressure and remove most of dichloromethane, add ethyl acetate dissolving, add the NaOH of 0.5M water-soluble After liquid is stirred 5 minutes, point liquid, organic phase is washed with water, anhydrous sodium sulfate drying, and filtering is evaporated off solvent, and silica gel column chromatography is obtained Compound 504mg, yield 65%.

300M¹H-NMR(δppm,DMSO)12.97(s,1H);10.21(s,1H);8.21(s,1H);8.13(s,1H); 8.01(s,1H);7.85(d,1H,J=8.4Hz);7.62(t,1H,J=7.8Hz);7.46(m,3H);4.09(m,2H);3.46 (m,1H);2.86(m,2H).

Embodiment 3

1- (3- trifluoromethyl-phenyls) -5- oxygen-pyrroles -3- carboxylic acids (644mg, 2.36mmol), 15mL is added in round-bottomed bottle Dichloromethane, 5- Aminoindazoles -3- carbonyl acyls ammonia (352mg, 2mmol), EDCI (575mg, 3mmol) room temperature reaction reacts 3 hours Wait, TLC display reactions are complete, be concentrated under reduced pressure and remove most of dichloromethane, add ethyl acetate dissolving, add the hydrogen-oxygen of 0.5M After changing sodium water solution stirring 5 minutes, point liquid, organic phase is washed with water, anhydrous sodium sulfate drying, and filtering is evaporated off solvent, silica gel column layer Analysis, obtains compound 586mg, yield 68%.

300M¹H-NMR(δppm,DMSO)13.47(s,1H);10.28(s,1H);8.46(s,1H);8.21(s,1H); 7.86(d,1H,J=8.4Hz);7.65-7.47(m,5H);7.3(s,1H);4.08(m,2H);3.49(m,1H);2.85(m, 2H).

Embodiment 4

1- (4- fluoro-phenyls) -5- oxygen-pyrroles -3- carboxylic acids (526mg, 2.36mmol), 15mL dichloromethanes is added in round-bottomed bottle Alkane, 3- cyano group -5- Aminoindazoles (316mg, 2mmol), EDCI (575mg, 3mmol) room temperature reaction, as a child, TLC shows for reaction 3 Show that reaction is complete, be concentrated under reduced pressure and remove most of dichloromethane, add ethyl acetate dissolving, add the NaOH of 0.5M water-soluble After liquid is stirred 5 minutes, point liquid, organic phase is washed with water, anhydrous sodium sulfate drying, and filtering is evaporated off solvent, and silica gel column chromatography is obtained Compound 522mg, yield 72%.

300M¹H-NMR(δppm,DMSO)10.32(s,1H);8.47(s,1H);7.7(m,2H);7.62(s,2H);7.22 (t,2H,J=8.4Hz);4.07(m,2H);3.48(m,1H);2.78(m,2H).

Embodiment 5

1- (4- methylphenyls) -5- oxygen-pyrroles -3- carboxylic acids (516mg, 2.36mmol), 15mL dichloros is added in round-bottomed bottle Methane, 3- cyano group -5- Aminoindazoles (316mg, 2mmol), EDCI (575mg, 3mmol) room temperature reaction reacted 3 as a child, TLC Display reaction is complete, is concentrated under reduced pressure and removes most of dichloromethane, adds ethyl acetate dissolving, adds the NaOH water of 0.5M After solution is stirred 5 minutes, point liquid, organic phase is washed with water, anhydrous sodium sulfate drying, and filtering is evaporated off solvent, and silica gel column chromatography is obtained To compound 502mg, yield 70%.

300M¹H-NMR(δppm,DMSO)10.31(s,1H);8.47(s,1H);7.57(m,5H);7.18(d,2H,J= 8.4Hz);4.0(m,2H);3.5(m,1H);2.78(m,2H).

Embodiment 6

1- (the chloro- phenyl of 3-) -5- oxygen-pyrroles -3- carboxylic acids (564mg, 2.36mmol), 15mL dichloromethanes is added in round-bottomed bottle Alkane, 3- cyano group -5- Aminoindazoles (316mg, 2mmol), EDCI (575mg, 3mmol) room temperature reaction, as a child, TLC shows for reaction 3 Show that reaction is complete, be concentrated under reduced pressure and remove most of dichloromethane, add ethyl acetate dissolving, add the NaOH of 0.5M water-soluble After liquid is stirred 5 minutes, point liquid, organic phase is washed with water, anhydrous sodium sulfate drying, and filtering is evaporated off solvent, and silica gel column chromatography is obtained Compound 531mg, yield 70%.

300M¹H-NMR(δppm,DMSO)10.37(s,1H);7.88(s,1H);7.82(s,1H);7.56(d,1H,J= 8.1Hz);7.37(m,3H);7.2(d,1H,J=7.8Hz);7.12(d,1H,J=8.1Hz);4.06(m,2H);3.44(m,1H); 2.8(m,2H).

Embodiment 7

1- (4- Methyl-benzvls) -5- oxygen-pyrroles -3- carboxylic acids (550mg, 2.36mmol), 15mL dichloros is added in round-bottomed bottle Methane, 5- Aminoindazoles (266mg, 2mmol), EDCI (575mg, 3mmol) room temperature reaction, as a child, TLC shows instead for reaction 3 Should be complete, it is concentrated under reduced pressure and removes most of dichloromethane, ethyl acetate dissolving is added, add the sodium hydrate aqueous solution of 0.5M to stir After mixing 5 minutes, point liquid, organic phase is washed with water, anhydrous sodium sulfate drying, and filtering is evaporated off solvent, and silica gel column chromatography obtains chemical combination Thing 542mg, yield 78%.

300M¹H-NMR(δppm,CD3OD)8.5(d,2H,J=11.7Hz);7.94(m,2H);7.69(s,3H);4.95 (s,2H);4.06(m,2H);3.82(m,1H);3.20(m,2H);2.80(s,3H).

Embodiment 8

1- (4- methyoxy-benzyls) -5- oxygen-pyrroles -3- carboxylic acids (587mg, 2.36mmol), 15mL bis- is added in round-bottomed bottle Chloromethanes, 5- Aminoindazoles (266mg, 2mmol), EDCI (575mg, 3mmol) room temperature reaction, as a child, TLC shows for reaction 3 Reaction is complete, is concentrated under reduced pressure and removes most of dichloromethane, adds ethyl acetate dissolving, adds the sodium hydrate aqueous solution of 0.5M After stirring 5 minutes, point liquid, organic phase is washed with water, anhydrous sodium sulfate drying, and filtering is evaporated off solvent, and silica gel column chromatography is changed Compound 553mg, yield 76%.

300M¹H-NMR(δppm,DMSO)12.96(s,1H);10.03(s,1H);8.07(s,1H);7.99(s,1H); 7.41(d,1H,J=9Hz);7.37(d,1H,J=10.8Hz);7.17(d,2H,J=8.7Hz);6.9(d,2H,J=8.4Hz); 4.32(s,2H);3.72(s,3H);3.47(m,1H);3.3(m,2H);2.58(d,2H).

Embodiment 9

1- (the fluoro- benzyls of 4-) -5- oxygen-pyrroles -3- carboxylic acids (559mg, 2.36mmol), 15mL dichloromethanes is added in round-bottomed bottle Alkane, 5- Aminoindazoles (266mg, 2mmol), EDCI (575mg, 3mmol) room temperature reaction, as a child, TLC displays are reacted for reaction 3 Completely, it is concentrated under reduced pressure and removes most of dichloromethane, add ethyl acetate dissolving, adds the sodium hydrate aqueous solution stirring of 0.5M After 5 minutes, point liquid, organic phase is washed with water, anhydrous sodium sulfate drying, and filtering is evaporated off solvent, and silica gel column chromatography obtains compound 549mg, yield 78%.

300M¹H-NMR(δppm,DMSO)12.96(s,1H);10.04(s,1H);8.08(s,1H);7.99(s,1H); 7.47-7.14(m,6H);4.38(s,2H);3.40(m,1H);3.30(m,2H);2.60(d,2H).

Embodiment 10

1- benzyls -5- oxygen-pyrroles -3- carboxylic acids (516mg, 2.36mmol), 15mL dichloromethane, 5- ammonia is added in round-bottomed bottle Base indazole (266mg, 2mmol), EDCI (575mg, 3mmol) room temperature reaction, as a child, TLC display reactions are complete, subtract for reaction 3 Pressure concentration removes most of dichloromethane, adds ethyl acetate dissolving, adds the sodium hydrate aqueous solution of 0.5M to stir 5 minutes Afterwards, divide liquid, organic phase is washed with water, anhydrous sodium sulfate drying, filter, solvent is evaporated off, silica gel column chromatography obtains compound 507mg, Yield 76%.

300M¹H-NMR(δppm,DMSO)12.95(s,1H);10.04(s,1H);8.07(s,1H);7.99(s,1H); 7.47-7.23(m,6H);4.39(s,2H);3.50(m,1H);3.37(m,2H);2.60(d,2H).

Embodiment 11

Step C：Compound 1 (1.784g8mmol) is added in round-bottomed bottle, after adding DMF20mL dissolvings, DBU is added (1.8mL, 12mmol), bromobenzyl (1.2mL, 9.6mmol), as a child, TLC display reactions are complete, add ether for room temperature reaction 3, Water, stirring divides liquid after 5 minutes, and organic phase is washed with water 2 times, and saturated sodium-chloride water solution is washed 1 time, anhydrous sodium sulfate drying, filtering, Solvent is evaporated off, (petroleum ether: ethyl acetate=4: 1), obtains white solid 2.26g, yield 90% to silica gel column chromatography.

¹H NMR(300M,CDCl₃, δ ppm) and 7.52-7.51 (m, 2H, Ar-H), 7.37 (s, 5H, Ar-H), 7.09-7.03 (m, 2H, Ar-H), 5.20 (s, 1H ,-OCH2Ph), 4.07 (m, 2H ,-CH2-), 3.40 (m, 1H ,-CH-), 2.92 (m, 2H ,- CH2-).

Step D：After compound 2 (313mg, 1mmol), plus tetrahydrofuran 5mL dissolvings are added in round-bottomed bottle, it is added dropwise under ice bath Borane dimethylsulfide ethereal solution (0.2mL) adds rear room temperature reaction, and overnight rear TLC displays reaction is complete, is slowly added dropwise saturation chlorination Aqueous ammonium, after stirring 10 minutes, adds ethyl acetate and water, and point liquid, water is extracted 2 times with ethyl acetate, merges organic phase, Solution is set to wash 1 time with saturated sodium-chloride, anhydrous sodium sulfate drying, filtering is evaporated off solvent, silica gel column chromatography (petroleum ether: acetic acid second Ester=6: 1), obtain colorless oil 238mg, yield 79.5%.

¹H NMR(300M,CDCl₃, δ ppm) and 7.35-7.25 (d, 5H, Ar-H), 6.97 (m, 2H, Ar-H), 6.50-6.49 (m, 2H, Ar-H), 5.16 (s, 1H ,-OCH2Ph), 3.53 (d 2H ,-CH2-), 3.93-3.24 (m, 3H ,-CH- ,-CH2-), 2.30(m,2H,-CH2-).

Step E：Compound 3 (200mg, 0.73mmol) is added in round-bottomed bottle, methylene chloride/methanol (4: 1) adds palladium carbon (25mg), normal pressure hydrogenation reaction, TLC displays reaction is complete after 3 hours.Palladium carbon is filtered to remove, 150mg pale pinks are obtained after being evaporated Solid.Yield 98%.

¹H NMR(300M,CDCl₃, δ ppm) and 12.47 (s, 1H ,-COOH), 7.03-6.97 (m, 2H, Ar-H), 6.54- (m, the 2H ,-CH2-) of 6.50 (m, 2H, Ar-H), 3.40 (m, 2H ,-CH2-), 3.24-3.12 (m, 3H ,-CH- ,-CH2-), 2.15

Step F：Compound 4 (209mg, 1mmol), 5- Aminoindazoles (160mg, 1.2mmol) is added to add in round-bottomed bottle After DMF10mL dissolvings, EDCI (230mg, 1.2mmol), DMAP10mg are added, 80 DEG C are reacted, and 3 as a child, TLC displays have been reacted Entirely, after temperature is down to room temperature, ethyl acetate, the hydrochloric acid of 1N is added to stir 5 minutes, point liquid, water is extracted 2 times with ethyl acetate, After merging organic phase, successively with water, saturated sodium-chloride is washed, anhydrous sodium sulfate drying, filtering, and solvent, silica gel column chromatography (stone is evaporated off Oily ether: ethyl acetate=1: 1), obtains hazel-color solid 254mg, yield 78%

¹H NMR(300M,CDCl₃, δ ppm) and 12.97 (s, 1H ,-NH-), 10.08 (s, 1H ,-NH-), 8.14 (s, 1H, Ar- H),8.00(s,1H,-CH=N),7.47-7.45(m,2H,Ar-H),7.04-6.98(m,2H,Ar-H),6.54(m,2H,Ar- H),3.45(m,2H,-CH-),3.30(m,4H,-CH-,-CH2-),2.24(m,2H,-CH2-).

Embodiment 12

1- (3,5- difluorophenyl)-pyrroles -3- carboxylic acids (227mg, 1mmol), 5- Aminoindazoles is added in round-bottomed bottle (160mg, 1.2mmol), after adding DMF10mL dissolvings, adds EDCI (230mg, 1.2mmol), DMAP10mg, 80 DEG C of reactions, 3 As a child, TLC displays reaction was complete, after temperature is down to room temperature, added ethyl acetate, the hydrochloric acid of 1N to stir 5 minutes, point liquid, water Extracted 2 times with ethyl acetate, after merging organic phase, successively with water, saturated sodium-chloride is washed, anhydrous sodium sulfate drying, filtering is steamed Except solvent, silica gel column chromatography obtains solid 266mg, yield 78%

300M¹H-NMR(δppm,DMSO)12.97(s,1H);10.10(s,1H);8.14(s,1H);8.01(s,1H); 7.47(m,2H);6.28(m,3H);3.54(m,1H);3.42(m,2H);3.29(m,2H);2.28(m,2H).

Embodiment 13

1- (3,4- difluorophenyl)-pyrroles -3- carboxylic acids (227mg, 1mmol), 5- Aminoindazoles is added in round-bottomed bottle (160mg, 1.2mmol), after adding DMF10mL dissolvings, adds EDCI (230mg, 1.2mmol), DMAP10mg, 80 DEG C of reactions, 3 As a child, TLC displays reaction was complete, after temperature is down to room temperature, added ethyl acetate, the hydrochloric acid of 1N to stir 5 minutes, point liquid, water Extracted 2 times with ethyl acetate, after merging organic phase, successively with water, saturated sodium-chloride is washed, anhydrous sodium sulfate drying, filtering is steamed Except solvent, silica gel column chromatography obtains solid 266mg, yield 78%

300M¹H-NMR(δppm,DMSO)12.99(s,1H);10.12(s,1H);8.15(s,1H);8.01(s,1H); 7.45(m,2H);7.23(m,1H);6.56(m,1H);6.33(d,1H);3.52(m,1H);3.32(m,4H);2.26(m,2H).

Embodiment 14

1- (4- Methyl-benzvls)-pyrroles -3- carboxylic acids (219mg, 1mmol), 5- Aminoindazoles is added in round-bottomed bottle (160mg, 1.2mmol), after adding DMF10mL dissolvings, adds EDCI (230mg, 1.2mmol), DMAP10mg, 80 DEG C of reactions, 3 As a child, TLC displays reaction was complete, after temperature is down to room temperature, added ethyl acetate, the hydrochloric acid of 1N to stir 5 minutes, point liquid, water Extracted 2 times with ethyl acetate, after merging organic phase, successively with water, saturated sodium-chloride is washed, anhydrous sodium sulfate drying, filtering is steamed Except solvent, silica gel column chromatography obtains solid 267mg, yield 80%

300M¹H-NMR(δppm,DMSO)12.93(s,1H);9.82(s,1H);8.08(s,1H);7.98(s,1H); 7.40(m,2H);7.15(m,2H);3.53(s,2H);3.02(m,1H);2.85(m,1H);2.66(m,1H);2.35(m,2H); 2.04(s,3H);2.0(m,2H).

Embodiment 15

1- (4- methyoxy-benzyls)-pyrroles -3- carboxylic acids (235mg, 1mmol), 5- Aminoindazoles is added in round-bottomed bottle (160mg, 1.2mmol), after adding DMF10mL dissolvings, adds EDCI (230mg, 1.2mmol), DMAP10mg, 80 DEG C of reactions, 3 As a child, TLC displays reaction was complete, after temperature is down to room temperature, added ethyl acetate, the hydrochloric acid of 1N to stir 5 minutes, point liquid, water Extracted 2 times with ethyl acetate, after merging organic phase, successively with water, saturated sodium-chloride is washed, anhydrous sodium sulfate drying, filtering is steamed Except solvent, silica gel column chromatography obtains solid 287mg, yield 82%

300M¹H-NMR(δppm,DMSO)12.95(s,1H);9.83(s,1H);8.10(s,1H);7.99(s,1H); 7.42(m,2H);7.23(d,2H,J=8.7Hz);6.88(d,2H,J=8.7Hz);3.73(s,3H);3.05(m,1H);2.86 (m,1H);2.67(m,1H);2.50(s,2H);2.43(m,2H);1.99(m,2H).

Pharmacological evaluation

Experimental example 1：Inhibitory action evaluation of the indazole compounds to Rho kinase activities.

Rho kinase activities are detected using Enzyme-linked Immunosorbent Assay method (ELISA).Commodity in use ELISA reagents Box (Cyclex Rho-kinase Assay kit), wherein comprising having used coated 96 hole of Rho kinase substrates MBS (MYPT1) Plate, Rho kinases can make 696 threonine residues phosphorylations of substrate with substrate reactions in the presence of ATP.It is special according to antigen-antibody Different in nature association reaction principle, after the completion of kinase reaction, using the threonine phosphorylation monoclonal antibody specific conduct that HRP is marked Probe, specifically binds with the threonine residues of phosphorylation, finally with tetramethyl benzidine (Tetrabenzidine, TMB) with HRP reaction solutions, determine its absorbance, to react the activity of Rho kinases.

Enzyme storing liquid concentration is 100ng/ μ l, and it is 0.2ng/ μ l to be diluted to working concentration with kinase buffer liquid.Kinase reaction 20X ATP kinase buffer liquids are diluted to 1X by buffer solution.The μ l kinase reactions buffer solution of+10 μ l compounds of 10 μ l enzymes+80 is added To in ELISA Plate, 37 DEG C are reacted 30 minutes.Liquid is poured out, with elution buffer board-washing 5 times.HRP labelled antibodies are added, room temperature is anti- Answer 1 hour.Liquid is poured out, with elution buffer board-washing 5 times.Add TMB to develop the color about 5 minutes, reaction is added after colour developing is obvious Stop solution color development stopping, determines absorption value at 450nm.Judge that enzymatic activity is strong and weak according to OD values height, judge indazole class chemical combination Thing to the inhibitory activity of Rho kinases, inhibiting rate %=(OD standard-OD samples)/OD standards * 100%, and calculate to Rho kinases half Number inhibition concentration (IC₅₀) value.

The activity of the suppression Rho kinases of result display formula I indazole compounds energy dose dependents, part indazole class The IC of compound₅₀It is respectively 34.46,23.99 and 48.21 μ g/ml, judges that indazole compounds are lived in prevention and treatment Rho kinases Sexual abnormality increases the medicine effect of the cardiac and cerebral vascular diseases of induction and applies.

Inhibiting rate of the part indazole compounds of table 1. to Rho kinase activities.

Experimental example 2：The effect that indazole compounds shrink to norepinephrine pre-stimulation vascular circle.

Experimental animal selects male SD rat 6,250~300g of body weight.It is rapid to take out chest actively after rat sacrificed by decapitation Arteries and veins, is cut into 2-3mm vascular circles long, vascular circle is placed in fill K-H liquid 10mL (37 DEG C of constant temperature, and be continually fed into 95% oxygen and The mixed gas of 5% carbon dioxide) bath in, tension variation is transmitted and records in BL-420S biological functional systems In.Vascular circle changes K-H liquid 1 time in 1.2g tension stability 60min, period per 20min.Vascular circle is stimulated with 60mM KCl, makes blood Pipe ring shrinks, and is rinsed 2 times after reaching amplitude peak, vascular circle is returned to the state before stimulating, totally 2 times.Add 1 μM and remove first kidney Upper parathyrine, 10 μM of acetylcholine is given after reaching maximum shrinkage amplitude, and determines its diastole amplitude.If diastole amplitude is more than 80% represents that endothelium is complete, shows minimum to the loss of vascular circle endothelium in operating process, and endothelium integrality is good, to there is endothelium Group；If not diastole or diastole amplitude represent that endothelial loss is big less than 30%, endothelial function is imperfect, is without endothelium group.Detection endothelium After function, the state rinsed to before stimulating, then stable 30min, further influence of the detection medicine to vascular circle.

Norepinephrine (Norepinephrine, NE) is a kind of vasoconstrictor.The present embodiment is by reactant 1 μM of norepinephrine is added in system, makes blood vessel in contraction state, the indazole class of 1,2,5,10,25,50 μ g/ml cumulative concentrations To having obvious diastole to act on without endothelium and the contraction for having interior cutaneous vessel ring caused by norepinephrine, it relaxes compound Zhang Zuoyong strengthens with increasing for concentration, and in dose-effect relationship, the medium effective concentration (EC of part indazole compounds₅₀) table 2 It is shown, judge indazole compounds to the vasoconstrictive diastole effect caused by norepinephrine and in prevention and treatment blood Pipe tension force increases the medicine effect of the heart and brain disease of induction and applies extremely.

The pressor diastole effect pre- to norepinephrine of the part indazole compounds of table 2..

Experimental example 3：The effect that indazole compounds shrink to high concentration potassium chloride pre-stimulation vascular circle.

Tension detection vascular circle preparation method is with embodiment 3.

High concentration potassium chloride can cause vessel retraction.The present embodiment is made by adding 60mM KCl in reaction system Blood vessel is in contraction state, the indazole compounds of 1,2,5,10,25,50 μ g/ml cumulative concentrations to caused by KCl without endothelium and The contraction for having interior cutaneous vessel ring has obvious inhibitory action, and its inhibitory action strengthens with increasing for concentration, and is closed in dose-effect System.Medium effective concentration (the EC of part indazole compounds₅₀) shown in table 3, judge indazole compounds to high concentration potassium chloride The medicine of the heart and brain disease of induction is increased in caused vasoconstrictive diastole effect and prevention and treatment antiotasis extremely Effect and application.

The pressor diastole effect pre- to high concentration potassium chloride of the part indazole compounds of table 3..

Experimental example 4：Protective effect of the indazole compounds to original cuiture neonatal rat myocardial cell Ischemic-reperfused.

Myocardial ischemia is coronary stenosis, the common disease caused by vascular sclerosis, and it can trigger angina pectoris, heart failure etc. serious Disease simultaneously easily causes sudden death.The pathogenesis of clinical ischemic heart disease is simulated using external suckling mouse primary myocardial cell culture, The supply of energy and oxygen is reduced, the ischemia of cardiac muscle cell can be produced.In recent years studies have found that, the variability heart twist Bitterly, in the disease such as TIA, heart failure, Rho kinases serves key effect.Therefore to swashing with Rho in the present invention The protective effect that the indazole compounds of enzyme inhibition carry out myocardial anoxia reperfusion injury is significant.

Cardiac muscle cell can produce damage when there is anoxic (filling again), with the rupture of cell membrane, intracellular lactic dehydrogenase (LDH) culture medium is released into, by detecting that LDH contents and mtt assay determine the damage that cell viability may know that cell in culture medium Degree, judges the medicine effect of the vascular conditions that indazole compounds induce in prevention and treatment ischemic, anoxia-induced apoptosis and answers With.

Suckling mouse primary myocardial cell culture starts experiment after 5 days in 96 orifice plates by following groups：Normal group：Normal oxygen, DMEM high glucose mediums；Model group：Anoxic 6h, sugar-free DMEM culture mediums；Administration group：Final concentration 10 μ g/ml, anoxic 6h, sugar-free DMEM culture mediums.Change cell culture medium into DMEM sugar-frees serum-free (logical nitrogen is with emptying oxygen in advance), at the same time add Compound, is put into anaerobic incubator 6 hours.Normal group same medium but it is positioned in common incubator as control.After 6 hours Cell conditioned medium is collected respectively.

LDH is detected：Turn into CytoTox-One reagents to adding 11ml detection buffer solutions to mix in 1 bottle of substrate mixture.To The orifice plate of black 384 adds 25 μ l cell supernatants, adds 25 μ l CytoTox-One reagents, and room temperature reaction 10 minutes is added 12.5 μ l reaction terminating liquids, shake 10 seconds, carry out fluoroscopic examination：Excitation wavelength Ex=560nm, launch wavelength Em=590nm.Lactic acid Dehydrogenase (LDH) discharges inhibiting rate %=(model-sample)/(model-normal) * 100%；MTT cytoprotection rate %=(samples-mould Type)/(normal-model) * 100%.

As shown in table 4, part indazole compounds in 10 μ g/ml concentration to Primary cultured myocardial cells anoxia-induced apoptosis Protective rate is respectively 48.38%, 37.59%, 49.60%, 58.48%, 27.89%, 35.48% and 49.19%, suppresses LDH release rates point It is not 82.37%, 68.92%, 61.81%, 54.24%, 67.92%, 50.31% and 63.42%.

The part indazole compounds of table 4. are to Primary cultured myocardial cells protective rate and LDH release inhibiting rates.

Experimental example 5：Indazole compounds promote the effect of hepatic glucose consumption.

Diabetes (diabetes mellitus, DM) are and the livers because insulin deficit or islet function are impaired The target tissues such as dirty, skeletal muscle, fat are reduced to the intake of glucose, and a series of metabolism being characterized with hyperglycaemia for triggering Disorderly syndrome.New antidiabetic drug is researched and developed, promotes islet cell function and target organ to absorb glucose, into It is the important topic of diabetes medicament research.

Liver is the center of metabolism in animal body, is one of target organ of insulin action, and it is both to utilize grape Sugar is again the major organs for producing glucose.HepG2(human hepatocellular liver carcinoma cell Line) cell derived, in liver cell, is a kind of liver embryonic germ strain very much like with liver cell of phenotype, remains liver cell Many biological characteristicses, glucokinase and insulin receptor, IGF etc. can be expressed, be that research is external One of usual cell model of blood sugar reducing function.This model selection HepG2 cells, research compound disappears to HepG2 grape cell sugar The influence of consumption.

After cultured cells digestion, by every milliliter 5 × 10⁴Individual density is accessed in 96 orifice plates, per the μ L of hole 200, after 24 hours The good cell of growth conditions is chosen, the PRMI-1640 nutrient solutions for changing serum-free synchronize 12h.Random packet, respectively just Normal control group, positive drug insulin group, the compound group of various concentrations.Change to serum-free containing compound or without compound PRMI-1640 nutrient solutions, the glucose content in determination of glucose oxidase nutrient solution is used after cultivating 24 hours (GC).It is control with each group blank well average, calculates the changing value of glucose content in each hole nutrient solution of the group.Disappear in glucose Consumption experiment is incubated after terminating and removes nutrient solution to be measured for 24 hours, and the MTT serum-free mediums containing 0.5mg/mL of substituting are incubated Original fluid is abandoned in 4h, suction, and the μ L of DMSO 150 are added per hole, is mixed and is surveyed OD values (MTT) on rearmounted ELIASA at 540nm, with OD Value reflection cytoactive and quantity number；The sugar consumption amount of unit cell, indazole compounds shown in table 5 are represented with GC/MTT Promote the effect of hepatic glucose consumption, judge indazole compounds in prevention and treatment diabetes and its medicine of complication Effect and application.

The facilitation that the part class compound of table 5. is consumed to HepG2 hepatic glucoses.

Experimental example 6：Protective effect of the indazole compounds to Oxidation Induced Damages of Vascular Endothelial Cells.

Vascular endothelial cell produces and expresses various Molecular regulators, so as to vascular function under physiology and pathological state Regulation plays a crucial role.Vascular endothelial cell damage is not only the initiating agent of atherosclerosis, and to coronary heart disease, blood high The occurrence and development of the diseases such as pressure play an important role.Therefore protection of the research to vascular endothelial cell damage is preventing and treating heart and brain The new way of vascular diseases.

Hydrogen peroxide can be done directly on membrane lipid as a member in active oxygen family, form MDA, from And MDA generations increase, plasma membrane permeability increases, and extrinsic AGB stars increase, and LDH spills and also increase, while SOD activity decreases；Peroxide Change hydrogen can coup injury endoplasmic reticulum, and mitochondria dysfunction can be aggravated, make oxidative phosphorylation disorderly, cause endothelial cell damage Even activation regulated genes, inspire apoptosis.

CRL-1730 Human umbilical vein endothelial cells are counted, and are incubated at 96 hole flat-bottomed plates.After 24 hours, cell first uses sample Product process 4h, then use H₂O₂Treatment 20h causes cell oxidative damage model, and determining vascular endothelial cell mitochondria with mtt assay lives Property, to judge indazole compounds to H₂O₂The protective effect of caused Oxidation Induced Damages of Vascular Endothelial Cells, part indazole shown in table 6 The protective rate of class compound on vascular endothelium oxidative damage.

The protective effect of the part class compound on vascular endothelium oxidative damage of table 6.

In sum, indazole compounds or its physiologically acceptable derivative have suppression Rho kinase activities respectively Effect；Effect with vasodilator, protection vascular function；With the reperfusion injury effect of protection cardiac muscle cell's anti anoxia； With the effect for promoting grape cell sugar consumption；Effect with protection vascular endothelial cell to anti-oxidative damage, is that a class exists Preparation prevents and treats hypertension, atherosclerosis, cerebral angiospasm, coronarospasm, myocardial infarction, heart failure and diabetes The medicines such as the cardiovascular and cerebrovascular diseases such as complication, diabetes aspect has the noval chemical compound of important use.

Claims (10)

1. formula (I) compound or its isomers and its esters are led to

R₁, R₂, R₃It is independent selected from hydrogen, halogen, halo C1-6 alkyl, C1-6 alkyl, C1-6 alkoxies；

R₄May be selected from ketonic oxygen, 2 hydrogen atoms；

R₅May be selected from hydrogen, cyano group, acid amides；

N=0,1.

2. compound according to claim 1 or its isomers and its esters, it is characterised in that

R₁, R₂, R₃It is independent selected from hydrogen, halogen, three halogenated c1-4 alkyls, C1-4 alkyl, C1-4 alkoxies；

R₄May be selected from ketonic oxygen, 2 hydrogen atoms；

R₅May be selected from hydrogen, cyano group, acid amides；

N=0,1.

3. according to the compound or its isomers and its esters of claim any one of 1-2, it is characterised in that the compound choosing From

4. the preparation method of compound any one of claim 1-3, it is characterised in that comprise the following steps：

R₄During=ketonic oxygen, using following route：

I additions；V amidatioons

1) formula (III) compound and itaconic acid Michael additions, intramolecular amideization reaction obtain formula (IV) compound, R₄=carbonyl Base oxygen,

2) formula (IV) compound obtains formula (I) compound, R with 5- Aminoindazoles generation amidation process₄=ketonic oxygen；

R₄During=two hydrogen atoms, using following route：

I additions；Ii benzyl protections；Iii is reduced；Iv is hydrogenated；V amidatioons

1) formula (III) compound, with itaconic acid Michael additions, intramolecular amideization reaction obtains formula (IV) compound, R₄=carbonyl Base oxygen,

2) formula (IV) compound obtains formula (V) compound with bromobenzyl reaction,

3) formula (V) compound borane reduction obtains formula (VI) compound,

4) formula (VI) compound sloughs benzyl protection and obtains formula (VII) compound,

5) formula (VII) compound obtains formula (I) compound with 5- Aminoindazoles generation amidation process；

Wherein R₁, R₂, R₃, R₄, R₅As any one of claim 1-3 is defined.

5. a kind of pharmaceutical composition, it is characterised in that in compound and pharmaceutical field containing any one of claim 1-3 Available carrier.

6. pharmaceutical composition according to claim 5, it is characterised in that described pharmaceutical composition is selected from following formulation：Piece Agent, capsule, pill, injection.

7. pharmaceutical composition according to claim 5, it is characterised in that described pharmaceutical composition is selected from following formulation：It is molten Liquid, suspension, emulsion, powder, control release or extended release preparation.

8. the compound of any one of claim 1-3 is preparing prevention, is alleviating and/or treatment cardiovascular and cerebrovascular diseases, diabetes Application in product.

9. application according to claim 8, it is characterised in that described cardiovascular and cerebrovascular diseases be selected from hypertension, atherosclerosis, Cerebral angiospasm, coronarospasm, myocardial infarction, heart failure and diabetic complication.

10. application according to claim 8, it is characterised in that described product includes medicine, health products.