CN107964018A - Substituted purin ketones derivant and its medical usage - Google Patents

Abstract

The present invention relates to formula (I) substituted purin ketones derivant and its in pharmaceutically acceptable salt, isomers, prodrug and drug composition, such compound is the stimulant of soluble guanylate cyclase, wherein R₁It is expressed as thiophene or substituted pyridinyl, R₂It is expressed as NH₂、‑NHCH₃, R₃Represent hydrogen or C₁~C₄Alkyl.Application the invention also discloses the preparation of the compound and its as medicine, especially as the application of the medicine for the treatment of angiocardiopathy.

Description

Substituted purin ketones derivant and its medical usage

Technical field

The present invention relates to the compound that can stimulate soluble guanylate cyclase and its in pharmaceutically acceptable salt, different Structure body, prodrug and drug composition, further relate to its preparation as well as the application of medicine, especially as the cardiovascular disease for the treatment of The application of the medicine of disease.

Background technology

Soluble guanylate cyclase (Soluble Guanylate Cyclasey, sGC) is that NO-sGC-cGMP signals turn Crucial signal transduction enzyme in guiding path, can be activated by nitric oxide (NO), so as to be catalyzed guanosine triphosphate (GTP) conversion For cyclic guanosine monophosphate (cGMP), cGMP adjusts the correlation effect device in Signal Transduction Pathways downstream as second messenger, including albumen swashs Enzyme, phosphodiesterase (PDE) and some ion channels etc., so that corresponding physiology course is adjusted, such as vasodilator, vascular smooth Myocyte's generation, hematoblastic aggegation and nerve conduction etc..

Soluble guanylate cyclase is made of a larger α subunit and the one smaller β subunits containing ferroheme Heterodimer, mankind sGC have 2 four kinds of subunits of α 1, α 2, β 1 and β, 2/ β 1 both heterodimers of α 1/ β 1 and α more allusion quotation Type.It is the part at Active Regulation position with β subunit combination ferrohemes position, it is particularly significant for activation mechanism.NO energy and blood The ferrous ion of red pigment combines, and so as to dramatically increase the activity of enzyme, still, NO cannot stimulate the enzyme without ferroheme.CO Also can be combined with the ferrous ion of ferroheme, but effect is poor compared with NO.

For sGC there are two kinds of kenels of oxidized form and reduced form, reduced form is its state of activation.The mistake produced under pathological conditions The NO of amount combines to form peroxynitrite with crossing negative oxygen ion, loses enzyme and other albumen by oxidation and nitrification It is living, cause cellular damage.Due to NO bioavailability reduce, sGC by the reduced form of activated state change into inactivation state oxidation Type, sGC reduce the susceptibility of endogenic NO and NO release medicines, cause NO-sGC-cGMP signal transduction pathways to be obstructed, It can cause such as hypertension, platelet activation, hyperplasia increase, endothelial dysfunction, atherosclerosis, angina pectoris, the heart Decline, thrombus, apoplexy, sex dysfunction and myocardial infarction, wherein more serious with pulmonary hypertension, it increases pulmonary vascular pressure It is big even dead so as to cause hypertrophy of right heart to ultimately result in right heart failure.Therefore, it is particularly important that reparation to the path just becomes, Mainly to improve the level of cGMP.A kind of therapy approach that sGC is directly activated independent of NO is a kind of more promising Method, because generally believing that this method is efficient and few side effects.According to whether it can be classified as dependent on ferroheme Two types：1st, ferroheme dependent form sGC stimulants (sGC stimulator)；2nd, non-heme dependent form sGC activator (sGC activator), some potential treatment methods further include：PPAR- gamma agonists, survivin antagonist, active t cell Nuclear factor inhibitor, statins, tyrosine kinase inhibitor, Rho kinase inhibitors, anti-inflammatory agent, dichloroacetate, on kidney Gland medullarin, elastatinal, thrombocytin plasma membrane-bound translocating protein inhibitor and 5-hydroxytryptamine receptor antagonist, blood vessel are lived Property intestines peptide, bone marrow derived stem cells etc..

In recent years, some document reports directly stimulate soluble guanylate cyclase independent of NO Compound is obtained, such as：3- (5 '-methylol -2 '-furyl) -1- benzylindoles (YC-1), aliphatic acid, diphenyl iodine hexafluoro phosphorus Hydrochlorate, isoliquiritigenin and various substituted pyrazole derivatives.In addition, WO98/16507, WO98/23619, WO00/ 06567、WO00/06568、WO00/06569、WO00/21954、WO02/42299、WO02/42300、WO02/42301、WO02/ 42302nd, WO02/092596, WO03/004503, US06/052397, US09/023717, WO2013/004785, in describe Pyrazolo pyridine derivatives as soluble guanylate cyclase stimulant.Wherein also particularly describe on 3 with phonetic The pyrazolo pyridine derivatives of pyridine residue, the compound of this type have very high in terms of soluble guanylate cyclase is stimulated External activity.

The content of the invention

Compound used in the present invention has the spy that activation soluble guanylate cyclase is directly acted on independent of NO Point, can be as the medicine for the treatment of angiocardiopathy.The compound has substituted purin ketone structure as described in claim 1, The characteristics of substituted purin ketones derivant of this new type is that 2, purinone ring is substituted by pyrido pyrazoles.

The present invention provides the compound shown in Formulas I and its pharmaceutically acceptable salt, isomers, solvate.

Wherein：

R₁For thiophene or substituted pyridinyl,

R₂For amino or substituted-amino,

R₃For hydrogen or C₁~C₄Alkyl.

Preferable compound has following formula I.

Wherein R1 is

R₂For-NH₂、-NHCH₃,

R₃For for hydrogen, methyl or ethyl.

Formula I can also be the form of its salt, the salt usually formed with organic or inorganic alkali or acid.

The preferably acceptable salt of physiology of the invention.The acceptable salt of physiology of the compounds of this invention can be the present invention The salt of material and inorganic acid, carboxylic acid or sulfonic acid, particularly preferably for example with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, high chlorine Acid, fumaric acid, acetic acid, propionic acid, butanedioic acid, hydroxyacetic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, flutter acid, the third two Acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, p-methyl benzenesulfonic acid, methanesulfonic acid, ethyl sulfonic acid, naphthalene- The salt that 2- sulfonic acid, benzene sulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, tannic acid are formed.Other acid, such as oxalic acid, although its Body is not pharmaceutically acceptable, but can be used for preparing the salt as intermediate, to obtain the compounds of this invention and its pharmacy Upper acceptable salt.

Physiologically acceptable salt equally can be the metal or ammonium salt of the compounds of this invention with free carboxy.It is special You Xuanshi not be such as sodium, potassium, magnesium or calcium salt and inorganic ammonia or organic amine such as ethamine, diethylamine, triethylamine, N, N '-dibenzyl Ethylenediamine, chloroprocaine, choline, N-METHYL-ALPHA-L-GLUCOSAMINE and procaine, diethanol amine, triethanolamine, dicyclohexylamine, Dimethylaminoethanol, arginine, the ammonium salt of lysine or ethylenediamine.

The compound of the present invention can exist with tautomeric form, and the present invention equally also contains such form.

The compound of the present invention can also be its possible solvate.

Halogen (halogen for the purpose of the present invention) is fluorine, chlorine, bromine and iodine.

The invention further relates to the synthetic method for preparing formula Compound I, including：

1), chloromethyl thiophene or substituted pyridines (II) are obtained with 3- cyano group -1H- pyrazolos [3,4-b] pyridines (III) reaction Intermediate 3- cyano group -1- thiophene/substituted pyridinyl methyl isophthalic acid H- pyrazolos [3,4-b] pyridine (IV).

2) 2- (1- thiophene or substituted pyridinyl methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl) -4,5,6- pyrimidine triamines (V) intermediate 4,6- diaminourea -2- (1- thiophene/substituted pyridinyl methyl isophthalic acid H- pyrazolos are obtained with chloro-formate (VI) reaction [3,4-b] pyridin-3-yl) -5- pyrimidinyl-aminos formic acid esters (VII).

Wherein：R₁、R₂Definition it is as claimed in claim 1.

3) 4,6- diaminourea -2- (1- thiophene/substituted pyridinyl methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl) -5- is phonetic Piperidinyl carbamate (VII) and R₃- X (VIII) reaction obtain 4,6- diaminourea -2- (1- thiophene/substituted pyridines ylmethyl - 1H- pyrazolos [3,4-b] pyridin-3-yl) -5- pyrimidine radicals (alkyl) carbamate (IX).

Wherein：R₁、R₂、R₃Definition it is as claimed in claim 1, X is leaving group.Such as halogen, preferably iodine, or first Sulphonic acid ester, is optional that what is carried out under cooling in organic solvent.

4) IX and potassium tert-butoxide or sodium hydroxide reacted in the tert-butyl alcohol 6- amino -2- (1- substituted pyridines ylmethyl - 1H- pyrazolos [3,4-b] pyridin-3-yl) -7- alkyl/H-7H- purine -8 (9H) -one.

Wherein：R₁、R₂、R₃Definition it is as claimed in claim 1.

The synthetic method for preparing the required formula of compound of formula I (V) compound defined in claim 2 is as follows：

Wherein, R₁Definition it is as claimed in claim 1.

The present invention also compound comprising at least one formula I and one or more organic nitrates or NO donors Combination.

Organic nitrate and NO donors for the purpose of the present invention shows it generally by release NO or NO materials The material of therapeutic effect.The preferred embodiment that can be mentioned that has：Sodium nitroprussiate, nitroglycerin, Isosorbide acid esters, the different sorb of single nitric acid Ester, Ma Duoming and SIN-1.

In addition, the group of compound of the present invention also comprising the decomposition that can inhibit cyclic guanylic acid (cGMP) with one or more Close.It is preferably phosphodiesterase 1,2 and 5；Beavo and Reifsnyder (1990) TiPS11, the name of page 150 to 155 Material inhibitor.PDE5 inhibitor, especially compound 'Xiduofeng ' (WO are particularly preferably in this respect 94/28902), one kind in Vardenafil (WO99/24433) or tadalafil (WO 95/19978).These inhibitor are strengthened The effect of the compounds of this invention, and add required pharmacotoxicological effect.

The invention further relates to a kind of medicine for including at least one the compounds of this invention, it is preferably also together comprising one kind Or the acceptable excipient of a variety of pharmacology or carrier, and further relate to its application for the above purpose.Here medicinal load Body includes but not limited to：Ion-exchanger, aluminium oxide, aluminum stearate, lecithin, haemocyanin such as human serum albumin, cushion Matter such as phosphate, glycerine, sorbic acid, potassium sorbate, the partial glyceride mixtures of saturated vegetable fatty acid, water, salt or electrolysis Matter, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, cabosil, magnesium trisilicate, polyethylene Pyrrolidones, cellulosic material, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, beeswax, lanolin.

The active ingredient can have whole body and/or local action, therefore, its can suitable approach be administered, institute The suitable route said such as oral, parenteral, lung, nose, sublingual, tongue, cheek, rectum, percutaneously, conjunctiva, local administration or with implant Form administration.

The form of medication that the active ingredient can be adapted to these methods of administration is administered.

There is the known administration shape that active ingredient can be transmitted rapidly and/or in a manner of change suitable for oral administration Formula, such as tablet (uncoated tablets or coating tablet, such as have enteric coating or the tablet not being coated), capsule, sugar coated tablet, particle, small medicine Ball, pulvis, emulsion, suspension and aerosol.

Using parenteral be able to may avoid absorption step (in intravenous, intra-arterial, intracardiac, intraspinal or waist marrow to Medicine) or include absorb (intramuscular, subcutaneous, intracutaneous, percutaneous or Intraperitoneal medication).It is special suitable for the form of medication of parenteral It is the preparation of solution, suspension, emulsion, freeze-drying thing and the aseptic powdery form for injecting and inputting.

Suitable for other administration route have for example the medicine of suction (particularly powder suction, spraying), nose drops/solution, Spray；For tongue, the tablet or capsule, suppository, preparation, vaginal capsule, water for ear and eyes of the administration of sublingual or cheek Property suspension (lotion, shaking mixture), lipophilicity suspension, ointment, emulsifiable paste, lotion, paste, dusting or implant, like that Rise special stent.

The active ingredient can be changed into the form of medication with method known per se.It can use inert non-toxic Suitable pharmaceutical excipient realize.It particularly includes carrier (such as microcrystalline cellulose), solvent (such as the poly- second two of liquid Alcohol), emulsifying agent (such as lauryl sodium sulfate), dispersant (such as polyvinylpyrrolidone), synthesis and natural biological polymerization Thing (such as protein), stabilizer (such as antioxidant and ascorbic acid), colouring agent (such as inorganic pigment such as iron oxide) are rectified Taste agent and/or odor mask.In the case of suitable, described active ingredient can be present in the form of microencapsulation it is a kind of or In a variety of above-mentioned carriers.

In addition to the compound of formula I, said medicine preparation can also include other drugs active ingredient.

Embodiment

See embodiment on preparing the more detailed data of compound of Formula I, but the following examples are only of the invention preferred Illustrative approach, the present invention is not limited in any way.

Embodiment 1：6- amino -2- [1- (5- picoline -3- bases) methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl] - 7H- purine -8 (9H) -one

A：Benzeneazo malononitrile

Weigh 4.66g (50mmol) aniline to be fitted into 250mL twoport flasks, in ice bath a moment, the dense salt of 8.5mL is slowly added dropwise Acid, maintains the temperature at 0~5 DEG C as far as possible；Drop finishes, and after temperature stabilization, is slowly added into 4.55g (66mmol) sodium nitrites and 8mL Aqueous solution, stirs 15min；Then 8.04g sodium acetates and 20mL aqueous solutions are slowly added into；Temperature stabilization is treated in stirring, and 5.19g is added dropwise The third two eyeball and 6mL ethanol solutions, produce a large amount of bright yellow solids, add 80mL water, the reaction was continued 1h.TLC is monitored, and is filtered, filter The appropriate water washing of cake, it is dry, obtain bright yellow solid 8.24g, yield 96.8%.¹H-NMR (400MHz, DMSO-d₆)δ13.00 (s, 1H), 7.41 (dt, J=14.9,8.1Hz, 4H), 7.18 (td, J=7.3,1.3Hz, 1H).

B：3- cyano group -1- (5- picoline -3- bases) methyl isophthalic acid H- pyrazolos [3,4-b] pyridine

Weigh 7.42g (41.629mmol) 3- (chloromethyl) -5- methyl pyridinium chlorides and 6g (41.629mmol) 3- cyanogen Base -1H- pyrazoles [3,4-b] pyridine is dissolved in 120mLDMF, stirs lower addition 14.36g (104.06mmol) K₂CO₃, stirring sheet Carve, overnight, TLC is monitored 40 DEG C or so reactions of oil bath heating, and fundamental reaction is complete.200mL water, ethyl acetate are added into reaction solution (120mL*3) is extracted, 80mL saturated common salt water washings, organic phase anhydrous Na₂SO₄Dry, column chromatography for separation, obtains white solid 8.77g, yield 84.5%.¹H-NMR (400MHz, DMSO-d₆) δ 8.81 (dd, J=4.5,1.5Hz, 1H), 8.51 (dd, J= 8.2,1.5Hz, 1H), 8.43 (d, J=1.8Hz, 1H), 8.36 (d, J=1.5Hz, 1H), 7.55 (dd, J=8.2,4.5Hz, 2H), 5.85 (s, 2H), 2.25 (s, 3H).ESI-MS m/z：250.1102[M+H]⁺。

C：1- (5- picoline -3- bases) methyl isophthalic acid H- pyrazolos [3,4-b] pyridine -3- carboximide acid methyl esters

Weigh 9.48g (38.03mmol) 3- cyano group -1- (5- picoline -3- bases) methyl isophthalic acid H- pyrazolos [3,4-b] pyrrole Pyridine is dissolved in 400mL methanol, stirs lower addition 8.77g (162.34mmol) sodium methoxide, reaction 2h is stirred at room temperature.TLC is monitored, instead Should be complete, product is single, does not carry out structural identification, is directly used in and reacts in next step.

D：1- (5- picoline -3- bases) methyl isophthalic acid H- pyrazolos [3,4-b] pyridine -3- carbonamidines

8.7mL (152.14mmol) glacial acetic acid, which is drawn, with syringe adds 1- (5- picoline -3- bases) methyl isophthalic acid H- pyrroles Azoles simultaneously in [3,4-b] pyridine -3- carboximides acid methyl esters reaction solution, then adds 3g (55.056mmol) NH₄Cl, oil bath add Heat, 64 DEG C or so back flow reactions are stayed overnight.TLC is monitored, and fundamental reaction is complete, removes solvent under reduced pressure, then adds 400mL water and 12g (86.96mmol)K₂CO₃, stirring, adds appropriate NaCl to water saturation, ethyl acetate (150mL*5) extraction, and organic phase is with anhydrous MgSO₄It is dry, filter, remove solvent under reduced pressure, it is dry, obtain white solid 8g, yield 79.0%.¹H-NMR (400MHz, DMSO- d₆) δ 8.71-8.59 (m, 2H), 8.36 (dd, J=20.8,1.7Hz, 2H), 7.50 (s, 1H), 7.35 (dd, J=8.0, 4.5Hz, 1H), 6.78 (s, 3H), 5.73 (s, 2H), 2.24 (s, 3H).ESI-MS m/z：267.1354[M+H]⁺。

E：2- [1- (5- picoline -3- bases) methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl] -5- [(E) phenyl two Nitrence base] -4,6- pyrimidinediamines

Weigh 3.9g (22.92mmol) step D compounds and 6g (22.53mmol) embodiments 12A be dissolved in 120mLDMF, N₂12h is reacted under protection in 110 DEG C or so, TLC monitorings, fundamental reaction is complete, and cooling, decompression filters, the appropriate water washing of filter cake, Filtrate adds suitable water to solid is no longer produced, and filters, and filter cake is washed with 10% ethanol, dry, there are orange/yellow solid 7g, yield 71.2%.¹H-NMR (400MHz, DMSO-d₆) δ 9.20 (d, J=8.0Hz, 1H), 8.67 (d, J=4.5Hz, 1H), 8.54 (s, 2H), 8.39 (d, J=26.1Hz, 2H), 8.03 (d, J=8.2Hz, 2H), 7.93 (s, 2H), 7.44 (ddd, J= 20.5,13.8,7.4Hz, 5H), 5.81 (s, 2H), 2.25 (s, 3H).ESI-MS m/z：437.4[M+H]⁺。

F：2- [1- (5- picoline -3- bases) methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl] -4,5,6- pyrimidines three Amine

Weigh 6.88g (15.763mmol) step E compounds to be dissolved in 150mL DMF, it is (wet to add 3.89g Raney-Ni Weight), hydrogenation tank is put, the catalytic hydrogenation 12h under the conditions of 65 DEG C and 65Bar, TLC monitorings, remain a small amount of raw material unreacted, continue 12h is reacted, the reaction was complete for TLC monitorings, filters off Raney-Ni with diatomite, removes solvent under reduced pressure, column chromatography for separation, obtains yellow and consolidate Body 4.73g, yield 86.4%.¹H-NMR (400MHz, DMSO-d₆) δ 9.03 (d, J=7.8Hz, 1H), 8.59 (s, 1H), 8.35 (d, J=21.2Hz, 2H), 7.37 (d, J=56.7Hz, 2H), 6.34-5.11 (m, 6H), 4.07 (s, 2H), 2.23 (s, 3H). ESI-MS m/z：348.2[M+H]⁺。

G：4,6- diaminourea -2- [1- (5- picoline -3- bases) methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl] -5- Pyrimidinyl-amino isopropyl formate

Weigh 0.6g (1.73mmol) step F compounds to be dissolved in 35mL pyridines, ice bath stirring 15min, is inhaled with syringe Take the isopropyl chloroformate of 0.36g (2.94mmol) to be added dropwise in reaction solution, continue ice bath reaction 2h, remove ice bath, room temperature Reaction is overnight.TLC is monitored, and the reaction was complete, removes solvent, dry faint yellow solid 0.75g, yield about 100% under reduced pressure.mp 252~254 DEG C.¹H-NMR (400MHz, DMSO-d₆) δ 8.96 (dd, J=8.1,1.6Hz, 1H), 8.84-8.68 (m, 2H), 8.59 (s, 1H), 8.47 (s, 1H), 8.24 (dd, J=15.9,8.1Hz, 1H), 7.93-7.62 (m, 5H), 7.54 (dd, J= 8.1,4.5Hz, 1H), 5.92 (s, 2H), 4.89-4.76 (m, 1H), 2.31 (s, 3H), 1.20 (d, J=61.6Hz, 6H).ESI- MS m/z：434.3[M+H]⁺.Purity 99.1% (is analyzed, chromatographic column through HPLC：5 μ 250*4.6mm of C18, stream Dynamic phase：Acetonitrile/water=40: 60, flow velocity：1.0mL/min, wavelength：210nm).

H：6- amino -2- [1- (5- picoline 3- yls) methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl] -7H- is fast Purine -8 (9H) -one

Weigh 0.31g (0.7152mmol) step G compounds to be fitted into reaction bulb, add the 21mL tert-butyl alcohols, add in stirring Enter 0.06g (1.111mmol) sodium methoxide, 82 DEG C or so reaction 24h.TLC is monitored, and removes solvent under reduced pressure, column chromatography for separation, obtains 80mg white-yellowish solids, yield 30.0%.300 DEG C of mp ＞.¹H-NMR (400MHz, DMSO-d₆) δ 11.41 (s, 1H), 10.08 (s, 1H), 8.98 (dd, J=8.1,1.6Hz, 1H), 8.64 (dd, J=4.5,1.6Hz, 1H), 8.41 (d, J=1.8Hz, 1H), 8.34 (d, J=1.5Hz, 1H), 7.49 (s, 1H), 7.38 (dd, J=8.1,4.5Hz, 1H), 6.60 (s, 2H), 5.76 (d, J= 4.6Hz, 2H), 2.24 (s, 3H).ESI-MS m/z：372.13[M]^-.Purity 92.9% (is analyzed, chromatographic column through HPLC：5 μ 250*4.6mm of C18, mobile phase：Acetonitrile/water=30: 70, flow velocity：1.0mL/min), wavelength：210nm).

Embodiment 2：6- amino -2- [1- (2- fluorine pyridin-3-yl) methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl] -7- Methyl -7H- purine -8 (9H) -one

A：2- fluorine pyridin-3-yl-methanol

Weigh 10g (70.87mmol) 2- fluorine nicotinic acids to be dissolved in 330mL anhydrous tetrahydro furans, in ice bath stirring a moment, aliquot is slow It is slow to add 4.3g (113.07mmol) LiA₁H₄, a large amount of bubbles are produced, with addition LiA₁H₄The increase of amount, reaction solution is by white Muddy side yellow is muddy, finishes, the reaction was continued 20min, and TLC monitorings, the reaction was complete, is slowly added to 10.6mL water quenchings and goes out reaction, A large amount of bubbles and solid are produced, are filtered, solvent is evaporated off in filtrate decompression, obtains yellow liquid 7.78g, stand-by.

B：3- chloromethyl -2- fluorine pyridines

Above-described embodiment 2- fluorine pyridin-3-yl-methanol is dissolved in 85mL dichloromethane, ice bath 10min, is slowly added dropwise 21.7mL (306.04mmol) thionyl chloride, reaction solution become brown by yellow, and drop finishes, and continue ice bath reaction 2h, TLC monitoring, instead Should be complete, continue ice bath a moment, 100mL water, N is slowly added dropwise₂Blow, lye absorbs exhaust gas, dichloromethane (100mL*3) extraction Take, 100mL saturated common salt water washings, anhydrous MgSO₄It is dry, remove solvent under reduced pressure, obtain 7.14g brown liquids, it is stand-by.¹H-NMR (400MHz, CHLOROFORM-d₁) δ 8.20 (d, J=4.8Hz, 1H), 7.89 (ddd, J=9.3,7.6,1.7Hz, 1H), 7.26-7.19 (m, 1H), 4.63 (s, 2H).

C：3- cyano group -1- (2- fluorine pyridin-3-yl) methyl isophthalic acid H- pyrazolos [3,4-b] pyridine

By 3- chloromethyls) -2- fluorine pyridine and 5.44g 3- cyano group -1H- pyrazolos [3,4-b] pyridine be dissolved in 110mL DMF In, add 10.42g K₂CO₃, N₂Protection, is stirred at room temperature 30min, then in 40 DEG C, N₂The lower reaction of protection is overnight.TLC is monitored, The reaction was complete, adds the stirring of 200mL water, ethyl acetate (110mL*3) extraction, 60mL saturated common salt water washings, anhydrous MgSO₄It is dry It is dry, filter, remove solvent under reduced pressure, column chromatography for separation, obtains white solid 7.54g, yield 78.9%.¹H-NMR (400MHz, DMSO-d₆) δ 8.81 (dd, J=4.5,1.5Hz, 1H), 8.53 (dd, J=8.2,1.5Hz, 1H), 8.27-8.19 (m, 1H), 7.92 (ddd, J=9.6,7.4,1.9Hz, 1H), 7.56 (dd, J=8.2,4.5Hz, 1H), 7.37 (ddd, J=7.1,4.9, 1.8Hz, 1H), 5.91 (s, 2H).

D：1- (2- fluorine pyridin-3-yl) methyl isophthalic acid H- pyrazolos [3,4-b] pyridine -3- carboximide acid methyl esters

It is molten to weigh 7g (27.64mmol) 3- cyano group -1- (2- fluorine pyridin-3-yl) methyl isophthalic acid H- pyrazolos [3,4-b] pyridine In 295mL methanol, 5.97g (110.51mmol) sodium methoxide is added, reaction 2h is stirred at room temperature.TLC is monitored, and the reaction was complete, production Thing is single, stand-by.

E：1- (2- fluorine pyridine 3- yls) methyl isophthalic acid H- pyrazolos [3,4-b] pyridine -3- carbonamidines

6.33mL (110.57mmol) and 2.96g (55.34mmol) is added into the reaction solution obtained by above-mentioned steps NH₄Cl, 64 DEG C of reaction 10h, TLC monitorings, has a small amount of raw material unreacted complete, removes solvent under reduced pressure, add 70mL acetone, stir number Minute, filter, a small amount of acetone washing, filter cake is added in 300mL water, and stirring is lower to add 9.9g (71.74mmol) K₂CO₃, acetic acid second Ester (200mL*4) extracts, and removes solvent under reduced pressure, is stirred with 15mL EtOAc-PE (2: 1) mixed solvent, stands, pours out solvent, It is dry, obtain white solid 6.28g, yield 84.0%.¹H-NMR (400MHz, DMSO-d₆) δ 8.69 (dd, J=8.0,1.6Hz, 1H), 8.63 (dd, J=4.5,1.6Hz, 1H), 8.19 (d, J=4.7Hz, 1H), 7.72 (ddd, J=9.6,7.5,1.8Hz, 1H), 7.40-7.28 (m, 2H), 6.60 (s, 3H), 5.79 (s, 2H).

F：2- [1- (2- fluorine pyridin-3-yl) methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl] -5- [(E) phenyl phenodiazines Alkenyl] -4,6- pyrimidinediamines

Weigh 5.5g (20.35mmol) E steps compound and 3.5g (20.5676mmol) benzeneazo malononitrile is dissolved in In 110mL DMF, as 110 DEG C of reactions 30min, power 50W in microwave reactor.TLC is monitored, and the reaction was complete, is cooled down, and is taken out Filter, the appropriate water washing of filter cake obtain bright yellow solid, and filtrate adds suitable quantity of water to there is no solid precipitation, filters, filter cake is used few The washing of 10% ethanol is measured, obtains khaki solid, it is dry, it there are product 7.96g, yield 88.8%.¹H-NMR (400MHz, DMSO- d₆) δ 9.21 (dd, J=8.1,1.6Hz, 1H), 8.66 (dd, J=4.5,1.6Hz, 1H), 8.53 (s, 2H), 8.21 (d, J= 4.9Hz, 1H), 8.03 (dd, J=8.2,1.0Hz, 2H), 7.91 (s, 2H), 7.75 (ddd, J=9.5,7.6,1.7Hz, 1H), 7.50 (t, J=7.7Hz, 2H), 7.46-7.32 (m, 3H), 5.86 (s, 2H).

G：2- [1- (2- fluorine pyridin-3-yl) methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl] -4,5,6- pyrimidine triamines

Weigh 4.81g (10.92mmol) embodiment F-step compound to be dissolved in 92mL DMF, add 2.3g Raney-Ni (weight in wet base), 65 DEG C, 65Bar catalytic hydrogenations 10h.TLC is monitored, and the reaction was complete, addition 400mL water, ethyl acetate (300mL*6), Column chromatography for separation, obtains faint yellow solid 2.85g, yield 75.0%.¹H-NMR (400MHz, DMSO-d₆) δ 9.05 (dd, J=8.1, 1.6Hz, 1H), 8.57 (dd, J=4.5,1.7Hz, 1H), 8.18 (d, J=4.5Hz, 1H), 7.79-7.62 (m, 1H), 7.32 (ddd, J=10.3,6.4,3.2Hz, 2H), 5.82 (s, 4H), 5.76 (s, 2H), 4.06 (s, 2H).

H：4,6- diaminourea -2- [1- (2- fluorine pyridin-3-yl) methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl] -5- is phonetic Piperidinyl methyl carbamate

Method weighs step reactant and reacts to obtain faint yellow solid with methyl chlorocarbonate with 1 step G of embodiment.mp 248~250 DEG C.¹H-NMR (400MHz, DMSO-d₆) δ 9.07 (dd, J=8.0,1.6Hz, 1H), 8.61 (dd, J=4.5, 1.6Hz, 1H), 8.18 (d, J=4.7Hz, 1H), 8.02 (s, 1H), 7.68 (t, J=8.0Hz, 1H), 6.25 (s, 4H), 5.81 (s, 2H), 3.62 (s, 3H).ESI-MS m/z：410.1[M+H]⁺, 432.3 [M+Na]⁺.Purity 97.5% (is analyzed, color through HPLC Compose column：5 μ 250*4.6mm of C18, mobile phase：Acetonitrile/water=40: 60, flow velocity 1.0mL/min, wavelength： 210nm)。

I：4,6- diaminourea -2- [1- (2- fluorine pyridin-3-yl) methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl] -5- is phonetic Piperidinyl (methyl) methyl carbamate

Weigh 0.148g (0.3213mmol) step H compounds to be fitted into reaction bulb, add the anhydrous THF of 2mL, put ice bath and stir 30min is mixed, the LiHMDS tetrahydrofuran solutions that 0.64mL 1.06mol/L are drawn with syringe are added in reaction solution, continue ice bath 20min is stirred, then with microsyringe by 20 μ L (0.3213mmol) CH₃I adds reaction solution, the reaction was continued 4.5h, ice cube Naturally melt.TLC is monitored, and the reaction was complete.The NH of 2mL5% is added into reaction solution₄Reaction, then another addition is quenched in Cl aqueous solutions 8mL water, ethyl acetate (25mL*3) extraction, removes solvent under reduced pressure, column chromatography for separation, obtains white solid.280~282 DEG C of mp.¹H-NMR (400MHz, DMSO-d₆) δ 9.07 (dd, J=8.0,1.5Hz, 1H), 8.60 (dd, J=4.4,1.7Hz, 1H), 8.18 (d, J=4.7Hz, 1H), 7.72-7.55 (m, 1H), 7.42-7.22 (m, 2H), 6.42 (s, 4H), 5.81 (s, 2H), 3.65 (s, 1H), 3.53 (s, 2H), 3.00 (s, 3H).ESI-MS m/z：424.19[M+H]⁺, 446.17 [M+Na]⁺.(the warp of purity 99.6% HPLC is analyzed, chromatographic column：5 μ 250*4.6mm of C18, mobile phase：Acetonitrile/water=40: 60, flow velocity 1.0mL/ Min, wavelength：210nm).

J：6- amino -2- [1- (2- fluorine pyridin-3-yl) methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl] -7- methyl - 7H- purine -8 (9H) -one

Weigh 0.11g (0.2598mmol) step Compound I to be fitted into reaction bulb, add the 4mL tert-butyl alcohols and 0.05g (0.3894mmol) potassium tert-butoxide, 82 DEG C of back flow reaction 11h.TLC is monitored, and the reaction was complete, adds 3mL water and 1.5mL ethanol, Gu Body dissolves, and in orange, 1h is stirred at room temperature, and adds 20mL water, ethyl acetate (25mL*4) extraction, anhydrous Na₂SO₄It is dry, column chromatography Separation, obtains white solid 20mg, yield 19.8%.300 DEG C of mp ＞.¹H-NMR (400MHz, DMSO-d₆) δ 11.63 (s, 1H), 9.06 (dd, J=8.1,1.6Hz, 1H), 8.64 (dd, J=4.5,1.6Hz, 1H), 8.20 (d, J=4.7Hz, 1H), 7.80- 7.69 (m, 1H), 7.42-7.29 (m, 2H), 6.77 (s, 2H), 5.81 (s, 2H), 3.48 (s, 3H).ESI-MS m/z：392.14 [M+H]⁺, 414.12 [M+Na]⁺.Purity 95.0% (is analyzed, chromatographic column through HPLC：5 μ 250*4.6mm of C18, Mobile phase：Acetonitrile/water=40: 60, flow velocity 1.0mL/min, wavelength：210nm).

Embodiment 3：6- amino -2- [1- (2- methoxypyridine -3- bases) methyl isophthalic acid H- pyrazolos [3,4-b] pyridine -3- Base] -8 (9H) -one of -7- methyl -7H- purine

Method is the same as 2,4,6- diaminourea -2- of embodiment [1- (2- methoxypyridine -3- bases) methyl isophthalic acid H- pyrazolos [3,4- B] pyridin-3-yl] -5- pyrimidine radicals (methyl) methyl carbamate and potassium tert-butoxide react, obtain white solid.300 DEG C of mp ＞.¹⁹F-NMR free-florides signal responds,¹H-NMR (400MHz, DMSO-d₆) δ 11.62 (s, 1H), 9.06 (dd, J=8.1,1.5Hz, 1H), 8.60 (dd, J=4.5,1.6Hz, 1H), 8.10 (dd, J=5.0,1.8Hz, 1H), 7.37 (dd, J=8.1,4.5Hz, 1H), 7.13 (d, J=5.7Hz, 1H), 6.91 (dd, J=7.3,5.0Hz, 1H), 6.76 (s, 2H), 5.70 (s, 2H), 3.89 (s, 3H), 3.48 (s, 3H).ESI-MS m/z：404.16[M+H]⁺, 426.14 [M+Na]⁺.Purity 92.5% (analyzed through HPLC, Chromatographic column：5 μ 250*4.6mm of C18, mobile phase：Acetonitrile/water=40: 60, flow velocity 1.0mL/min, wavelength： 210nm)。

Embodiment 4：Rna level up-regulation experiment：Embodiment compound is measured to human pulmonary artery smooth muscle cells SerpinB2 With level-off on the RNA of GREM1 genes

The culture and administration of cell：

People's arteria pulmonalis smooth muscle primary cell is at Smooth Muscle Cell base (ScienCell), 37 DEG C, 5%CO2 cultures, After being passaged to for 3~7 generations, the 0.5 μ L of given the test agent of 100mM are added into cell culture medium, are gently mixed, incubation at room temperature 8 is small When.

RNA is extracted：

Appropriate cell precipitation adds 1mL Trizol reagents, blows and beats repeatedly, makes fully to crack.Lysate is transferred at DEPC 5min is incubated on ice in the Eppendorf pipes managed, and is kept completely separate nucleoprotein complex.0.2mL chloroforms are added, are acutely rocked 15s, is incubated 3~5min on ice.12000rpm, 4 DEG C of centrifugation 15min, mixture are divided into three layers, upper strata RNA.By upper phase Move in the processed Eppendorf pipes of DEPC, add 0.5mL isopropanols, be incubated 20min after mixing on ice.4 DEG C, 12000rpm, centrifuges 10min.Supernatant is removed, is carefully washed with 75% ethanol (being prepared with the processed deionized waters of DEPC) Wash precipitation.7500rpm, 4 DEG C of centrifugation 3min, abandons supernatant.Drying at room temperature 10 minutes, adds water dissolving RNAs of the 20 μ L without RNase. Packing is saved backup after -80 DEG C of refrigerators.

Reverse transcription：

5 μ g cell total rna MMLV Reverse Transcriptase kits are respectively taken to complete the synthesis of the first chains of cDNA.5 μ g total serum IgEs, 1 μ L OligodT (0.5 μ g/ μ L) and 1 μ L of 10mM dNTPs mixtures are mixed, and add the processed deionized waters of DEPC to 10 μ L.65 DEG C effect is denatured RNA in 5 minutes, is then immediately transferred on ice, acts at least 1 minute.Sequentially add 10 × reverse transcription reaction 2 μ L of buffer solution (200mM Tris-HCl, pH 8.4,500mM KCl), 4 μ L of 25mM MgCl2,2 μ L of 0.1M DTT, RNase 1 μ L of inhibitor, mix, and 42 DEG C act on 2 minutes.1 μ L MMLV reverse transcriptases are added, 42 DEG C act on 50 minutes, and 72 DEG C go out for 15 minutes Reverse transcriptase living.1 μ L e. coli rna enzymes H (2 units/μ L) is added, 37 DEG C act on 15 minutes to digest single stranded RNA.Reaction production Thing is the first chains of cDNA.

Real-time PCR：

The reaction of Real-time PCR carries out sxemiquantitative, using ACTIN genes as internal reference, iQ5 according to SYBR-Green methods Multicolor Real-Time PCR detection systems (Bio-Rad) are detected.SerpinB2 gene primers for 5 '- TCCATTCATCCTTCCGCTCT-3 ' (upstream), 5 '-AAGTCTACTGCCTG GGGTTC-3 ' (downstream)；ACTIN gene primers For 5 '-GGCGGCACCACCATGTACCCT-3 ' (upstream), 5 '-AGGGGCCGGACTC GTCATACT-3 ' (downstream).Real- Time PCR reaction systems are as follows：2×SYBR-Green Premix EX Taq^TMReagent；50ng DNA；0.1 μM of gene primer, 20 μ l of final volume.Reaction condition for 95 DEG C 5 minutes, 95 DEG C 5 seconds, 60 DEG C 30 seconds, 40 circulation, by the solubility curve for analyzing product To determine the specificity of amplification, the relative expression quantity of SerpinB2 is calculated using CT values method, experiment every time repeats at least three times.

Data statistics and analysis：

Data acquired carries out statistical analysis using SPSS Statistics 20.0 and GraphPad Prism 5.01. The measurement data for meeting normal distribution is usedThe measurement data median and quartile table of normal distribution are not met Show, enumeration data is represented with rate.The comparison of measurement data is according to its characteristic distributions application independent samples t test or non-ginseng between two groups Number is examined.Baseline SerpinB2 is horizontal with 2^-ΔCT(Δ CT=CT_{Target gene}-CT_{Reference gene}) represent, wherein target gene is SerpinB2, reference gene ACTIN, calculate according to Real-time PCR results.The clear and definite baseline of application variables correlation analysis Whether SerpinB2 levels are related to the pulmonary hypertension order of severity, calculate Pearson correlation coefficient.P ＜ 0.05 are considered poor Exclusive or correlation has statistical significance.

Experimental result：

With the given the test agent of 100nM to level-off pair on the RNA of the SerpinB2 and GREM1 of human pulmonary artery smooth muscle cells Compound is screened.The results are shown in Table 1.

1. test-compound of table improves situation to the rna level of SerpinB2 and GREM1

Note：Blank group adds the DMSO of equivalent

Embodiment 5：Vascular circle diastole is tested, diastole effect of the measure embodiment compound to rat chest aorta blood vessel

1) preparation of in vitro aorta pectoralis vascular circle：

By rat sacrificed by decapitation, aorta pectoralis is taken out rapidly, is placed in and is filled ice-cold oxygen-saturated improvement Kreb ' s- (NaCl's Henseleit liquid 118 fulfills, NaHCO₃MM, KCl 4.7mM, MgCl₂1.2mM, KH₂PO₄, 1.2mM, CaCl₂2.5mM, Portugal Grape sugar 10mM, pH 7.4 ± 0.5) culture dish in, carefully wash away thrombus and remove the outer connective tissue of blood vessel.

Blood vessel is cut into the vascular circle of 3mm wide, is placed in 37 DEG C of thermostatic baths that 10ml contains Kreb ' s-Henseleit liquid In, lower end is fixed, and upper end is connected in polygraph by tonotransducer.Tissue bath in be continually fed into 95% oxygen and The mixed gas of 5% carbon dioxide, replaces nutrient solution once per 5min, after vascular circle is incubated 20-30 minutes, gives vascular circle 2g preloads, stablize 45-60 minutes.With 3 × 10^-7mol.L^-1Norepinephrine (NE) induces vascular circle and shrinks, and treats that contraction reaches After to maximum and balance (about 8-10 minutes), nutrient solution is replaced, resistance is removed, reaches balance again.Using tension value at this time as Basal tension value, starts to observe influence of the processing factor to antiotasis.

2) drug-treated：

After blood vessel is incubated arrival balance, by 3 × 10^-7mol.L^-1It is pre- in NE injection baths to shrink vascular circle, wait to shrink up to most It is big and after stablizing, test-compound is given in a manner of increasing concen-trations, observation test-compound receives norepinephrine (NE) The diastole effect of contracting vascular circle：Calculate diastole percentage and the EC of each compound₅₀Value.

3) data processing method：

3×10^-7mol.L^-1The maximum tension value of NE inductions=give 3 × 10^-7mol.L^-1.NE after vessel retraction balance Tension value-basal tension value

Compound diastole value=3 × 10^-7mol.L^-1The maximum tension value of NE inductions-give the tension force after test-compound Value

Diastole percentage=diastole value/maximum collapse value × 100%

4) diastole effect of the compound to rat chest aorta blood vessel：

With 3 × 10^-7mol.L^-1NE shrink rat chest aorta ring (1.50-2.2g, P ＜ 0.01) in advance, with dense after balance Degree incremental manner gives test-compound, and the vasoconstrictive diastole that observation noval chemical compound induces norepinephrine acts on, Solvent group gives the Kreb ' s-Henseleit nutrient solutions containing 1/1000DMSO.

5) result 1：Maximum diastole effect under noval chemical compound certain concentration.

Select concentration (3*10^-6M the evaluation of compound, the maximum diastole effect that measure compound produces after adding) are carried out. Measurement result is shown in Table 2.

2. compound of table causes norepinephrine oneself to shrink the diastole effect of rat chest aorta ring

Blank solvent is made of Kreb ' s-Henseleit buffer solutions, the DMSO containing 1/1000 volume).Diastole value is flat Mean value ± deviation, n=3-4.

Claims (11)

1. compound and its pharmaceutically acceptable salt, isomers, solvate shown in Formulas I.

Wherein：

R₁For thiophene or substituted pyridinyl,

R₂For amino or substituted-amino,

R₃For hydrogen or C₁~C₄Alkyl.

2. compound as claimed in claim 1 and its pharmaceutically acceptable salt, isomers, solvate.

Wherein：

R₁For

R₂For-NH₂、-NHCH₃,

R₃For hydrogen, methyl or ethyl.

3. compound as claimed in claim 2 and its pharmaceutically acceptable salt, isomers, solvate, wherein the chemical combination Thing preferably has following structure.

4. preparing the method for any one of the claims 1 to 3 compound, it includes：

1) chloromethyl thiophene or chloromethyl substituted pyridines (II) are reacted with 3- cyano group -1H- pyrazolos [3,4-b] pyridines (III) To intermediate 3- cyano group -1- thenyls/pyridylmethyl substitution -1H- pyrazolos [3,4-b] pyridine (IV).

Wherein：R₁Definition it is as claimed in claim 1.

2) 2- (1- thenyls or substituted pyridinyl methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl) -4,5,6- pyrimidine triamines (V) intermediate 4,6- diaminourea -2- (1- thenyls/substituted pyridinyl methyl isophthalic acid H- pyrroles are obtained with chloro-formate (VI) reaction Azoles simultaneously [3,4-b] pyridin-3-yl) -5- pyrimidinyl-aminos formic acid esters (VII).

Wherein：R₁、R₂Definition it is as claimed in claim 1.

Formula V compound is prepared by following reaction scheme：

3) 4,6- diaminourea -2- (1- thenyls or substituted pyridinyl methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl) -5- Pyrimidinyl-amino formic acid esters (VII) and R₄- X (VIII) reactions obtain 4,6- diaminourea -2- (1- thenyls/substituted pyridinyl Methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl) -5- pyrimidine radicals (alkyl) carbamate (I).

Wherein：R₁、R₂、R₃Definition it is as claimed in claim 1, X is leaving group.

4) IX reacts to obtain 6- amino -2- (1- thenyls or substituted pyridinyl with potassium tert-butoxide or sodium hydroxide in the tert-butyl alcohol Methyl isophthalic acid H- pyrazolos [3,4-b] pyridin-3-yl) -7- alkyl/H-7H- purine -8 (9H) -one.

Wherein：R₁、R₂、R₃Definition it is as claimed in claim 1.

5. a kind of medicine, it includes at least one compound of formula I as defined in claim 1 or its pharmaceutically acceptable salt, Isomers, solvate and at least one other excipient.

6. a kind of medicine, it includes at least one compound of formula I as defined in claim 1 or its pharmaceutically acceptable salt, Isomers, solvate and at least one organic nitrate or NO donor in connection.

7. a kind of medicine, it includes at least one compound of formula I as defined in claim 1 or its pharmaceutically acceptable salt, Isomers, solvate and at least one compound that can suppress cGMP decomposition in connection.

8. compound of formula I or its pharmaceutically acceptable salt, isomers, solvate are used to prepare as defined in claim 1 Treat the application in terms of the medicine of angiocardiopathy.

9. compound of formula I or its pharmaceutically acceptable salt, isomers, solvate are used to prepare as defined in claim 1 Treat the application in terms of the medicine of hypertension.

10. compound of formula I or its pharmaceutically acceptable salt, isomers, solvate are used to prepare as defined in claim 1 Application in terms for the treatment of thrombotic disease and the medicine of ischaemic.

11. the application as described in any one in claim 9~10, wherein using Formulas I chemical combination as defined in claim 1 Thing or its pharmaceutically acceptable salt, isomers, solvate and at least one organic nitrate or NO donor in connection or At least one compound that can suppress cGMP decomposition in connection.