CN102485724B - Substituted thiophene base pyrazolo-pyridines and medicinal use thereof - Google Patents

Abstract

The present invention relates to new substituted thienyl pyrazolo-pyridines and medicinal use thereof.Specifically, the present invention relates to formula I, and isomer pharmacologically acceptable salts, solvate: wherein each substituent definition is as used in the description.The invention still further relates to the pharmaceutical composition that comprises described compound and described compound for the preparation for the treatment of and/or preventing and the purposes in the medicine of cardiovascular relevant disease or illness.Formula I can treat and/or prevent and cardiovascular relevant disease or illness valuably.

Description

Substituted thiophene base pyrazolo-pyridines and medicinal use thereof

Technical field

The present invention relates to 2-substituted thiophene base pyrazolo-pyridines and pharmaceutically acceptable salt thereof, the invention still further relates to the purposes that the method for preparation I compound, medicinal compositions containing above-claimed cpd and described compound are used for the treatment of cardiovascular disorder.

Background technology

Soluble guanylate cyclase (Soluble Guanylate Cyclasey, sGC) be the signal transduction enzyme of key in NO-sGC-cGMP signal transduction pathway, it can activate by nitrogen protoxide (NO), thus catalysis guanosine triphosphate (GTP) is converted into cyclic guanosine monophosphate (cGMP), cGMP can the correlation effect device in regulating rotary guiding path downstream as second messenger, comprise protein kinase, phosphodiesterase (PDE) and some ionic channel etc., thus regulate corresponding physiological process, as vasodilator, promote that vascular smooth muscle cell produces, regulate hematoblastic aggegation and nerve conduction etc.

The heterodimer that soluble guanylate cyclase is made up of a larger α subunit and a less β subunit being combined with protoheme, mankind sGC has α 1, α 2, β 1 and β 2 four kinds of subunits, these two kinds of heterodimers of α 1/ β 1 and α 2/ β 1 are comparatively typical.The part at Active Regulation position with β subunit in conjunction with protoheme position, very important for activation mechanism.NO can be combined with the iron atom of protoheme, thus significantly can increase the activity of enzyme, but NO can not stimulate not containing the enzyme of protoheme.CO also can be combined with the iron atom of protoheme, but the stimulation undertaken by CO is lower than the stimulation of NO.

There is oxidized form and reduced form two profiles state in sGC, reduced form is its active state.The excessive NO produced under pathological conditions is combined with mistake negative oxygen ion and forms peroxynitrite, makes enzyme and other protein inactivation, cause cell injury by oxidation and nitrification.Due to the oxidized form that the bioavailability of NO reduces, sGC to be changed into inactivation state by the reduced form of activated state, sGc reduces the susceptibility that endogenic NO and NO discharges medicine, NO-sGC-cGMP signal transduction pathway is caused to be obstructed, it can cause as hypertension, platelet activation, hyperplasia increase, endothelial dysfunction, atherosclerosis, stenocardia, heart failure, thrombus, apoplexy, sexual dysfunction and myocardial infarction, wherein comparatively serious with pulmonary hypertension, it makes pulmonary vascular pressure increase thus causes hypertrophy of right heart finally to cause right heart failure even dead.

Therefore, the reparation of this path is increased by path diseases, such as hypertension, platelet activation, the hyperplasia caused of being obstructed treatment, endothelial dysfunction, atherosclerosis, stenocardia, heart failure, thrombus, apoplexy, sexual dysfunction and myocardial infarction, particularly pulmonary hypertension, and cause pulmonary vascular pressure increase by pulmonary hypertension thus the right heart failure causing hypertrophy of right heart to cause even death is particularly important.

The method of repairing this passage mainly improves the level of cGMP at present.The level of cGMP can be improved by the approach activating sGC, reach the object of disease therapy.Therapy approach can be divided into two types according to whether depending on protoheme: the sGC stimulant (sGC stimulator) 1, depending on protoheme; 2, do not rely on the sGC activator (sGC activator) of protoheme, according to these two kinds classification, potential methods for the treatment of has: NO synthetic enzyme activator, inhaled NO and NO donor medicine, phosphodiesterase inhibitor etc.Up to the present, the compound based on NO, as organic nitrate can not be used for the stimulation therapy of soluble guanylate cyclase always, except side effect, forming tolerance is one of main drawback of this therapeutic modality.

Not relying on the therapy approach that NO directly activates sGC is an a kind of most promising method, generally believes that the efficiency of this method is high and is almost free from side effects.

Given this, the art need develop brand new do not rely on the activator that NO directly activates sGC.Novel 2-substituted thiophene base pyrazolo-pyridines is exactly the sGC activator for this shot design, and it significantly can strengthen the susceptibility of sGC to NO; Under the extremely low condition even lacked of NO level, directly activate sGC, thus there is treatment hypertension, platelet activation, hyperplasia increase, endothelial dysfunction, atherosclerosis, stenocardia, heart failure, thrombus, apoplexy, sexual dysfunction and myocardial infarction, particularly pulmonary hypertension, and cause pulmonary vascular pressure increase by pulmonary hypertension thus the right heart failure causing hypertrophy of right heart to cause the even effect of death.

Summary of the invention

What the object of the invention is searching brand new does not rely on the activator that NO directly activates sGC, by strengthening sGC to the susceptibility of NO; Under the extremely low condition even lacked of NO level, directly activate sGC, reach the object with Cardiovarscular, increase including, but not limited to hypertension, platelet activation, hyperplasia, endothelial dysfunction, atherosclerosis, stenocardia, heart failure, thrombus, apoplexy, sexual dysfunction and myocardial infarction, particularly pulmonary hypertension, and cause pulmonary vascular pressure increase by pulmonary hypertension thus the right heart failure causing hypertrophy of right heart to cause the even effect of death.The present inventor finds through research, and 2-substituted thiophene base pyrazolopyridines formula I provided by the invention has the effect of good anti-mycobacterium tuberculosis.The present invention is based on above-mentioned discovery and be accomplished.

For this reason, first aspect present invention provides formula I, and isomer pharmacologically acceptable salts, solvate:

Wherein:

R ₁for-NR ₂c (=O) OR ₃,

R ₂for hydrogen or C ₁-C ₄alkyl,

R ₃for C ₁-C ₆alkyl.

Preferred compound has following formula I a, Ib, Ic.

Preferred compound is wherein R ₁for-NR ₂c (=O) OR ₃, R ₂for hydrogen, methyl or ethyl, R ₃for methyl, ethyl and sec.-propyl.

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

The acceptable salt of the preferred physiology of the present invention.The acceptable salt of physiology of the compounds of this invention can be material of the present invention and mineral acid, the salt of carboxylic acid or sulfonic acid, particularly preferably be such as with hydrochloric acid, Hydrogen bromide, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succsinic acid, oxyacetic acid, formic acid, lactic acid, toxilic acid, tartrate, citric acid, flutter acid, propanedioic acid, hydroxymaleic acid, toluylic acid, L-glutamic acid, phenylformic acid, Whitfield's ointment, fumaric acid, tosic acid, methylsulfonic acid, ethyl sulfonic acid, naphthalene-2-sulfonic acid, Phenylsulfonic acid, hydroxynaphthoic acid, hydroiodic acid HI, oxysuccinic acid, steroic acid (steroic acid), the salt that tannic acid is formed.Other acid, as oxalic acid, although itself is not pharmaceutically acceptable, may be used for preparing the salt being used as intermediate, to obtain the compounds of this invention and pharmacy acceptable salt thereof.

On physiology, acceptable salt can be metal or the ammonium salt of the compounds of this invention with free carboxy equally.Particularly preferably be as sodium, potassium, magnesium or calcium salt and derive from ammonia or organic amine as the ammonium salt of ethamine, diethylamine, triethylamine, N, N '-dibenzyl-ethylenediamin, chloroprocaine, choline, N-METHYL-ALPHA-L-GLUCOSAMINE and PROCAINE HCL, PHARMA GRADE, diethanolamine, trolamine, dicyclohexylamine, dimethylaminoethanol, arginine, Methionin or quadrol.

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

Compound of the present invention can also be its possible solvate.

Alkyl normally has 1 to 6, preferably the side chain of 1 to 4 carbon atom, particularly preferably the side chain of 1 to 3 carbon atom or branched-chain alkyl, and its preferred example has methyl, ethyl, n-propyl, sec.-propyl, the tertiary butyl, n-pentyl and n-hexyl.

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

The invention still further relates to the synthetic method preparing formula I, comprising:

1) chloromethyl fluoro thiophene (II) and 3-cyano group-1H-pyrazolo [3,4-b] pyridine (III) are obtained by reacting intermediate 3-cyano group-1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridine (IV),

2) 2-(1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridin-3-yl)-4,5,6-pyrimidine triamine tri hydrochloride (V) and chloro-formic ester are obtained by reacting intermediate 4,6-diamino-2-(1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridin-3-yl)-5-pyrimidinyl-amino manthanoate

Wherein: R ₃definition as claimed in claim 1, optional is react in organic solvent.

3) 4,6-diamino-2-(1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridin-3-yl)-5-pyrimidinyl-amino manthanoate and R ₂-X is obtained by reacting 4,6-diamino-2-(1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridin-3-yl)-5-pyrimidyl (alkyl) carbamate,

Wherein: R ₂, R ₃definition as claimed in claim 1, X is leavings group, and as halogen, preferably iodine, or methanesulfonates, optional carries out in organic solvent under cooling.

The synthetic method of the formula IIa compound required for formula Ia compound that preparation claim 2 defines is as shown below:

The synthetic method of the formula IIb compound required for formula Ib compound that preparation claim 2 defines is as shown below:

The synthetic method of the formula IIc compound required for formula Ic compound that preparation claim 2 defines is as shown below:

Formula III compound can be prepared by following reaction scheme:

Formula V compound can be prepared by following reaction scheme:

The present invention also comprises the compound of at least one formula I and the combination of one or more organic nitrates or NO donor.

It is generally the material being shown its therapeutic action by release NO or NO material for the organic nitrate of the object of the invention and NO donor.The preferred embodiment that can mention has: Sodium Nitroprusside, pannonit, Isosorbide acid esters, isosorbide mononitrate, Ma Duoming and SIN-1.

In addition, the present invention also comprises the combination of the compound of the decomposition that can suppress guanosine monophosphate (cGMP) with one or more.It is phosphodiesterase 1,2 and 5 preferably; Beavo and Reifsnyder (1990) TiPS11, the inhibitor of the material of the 150 to 155 page of name.Particularly preferably be the inhibitor of PDE5 in this respect, the one especially in compound 'Xiduofeng ' (WO 94/28902), Vardenafil (WO 99/24433) or Tadalafei (WO 95/19978).These inhibitor strengthen the effect of the compounds of this invention, and add required pharmacotoxicological effect.

The invention still further relates to a kind of medicine comprising at least one the compounds of this invention, it preferably also comprises the acceptable vehicle of one or more pharmacology or carrier together, and relates to its application for above-mentioned purpose.Here pharmaceutical carrier includes but not limited to: ion-exchanger, aluminum oxide, aluminum stearate, Yelkin TTS, serum protein is as human serum albumin, and buffer substance is as phosphoric acid salt, glycerine, Sorbic Acid, potassium sorbate, the partial glyceride mixtures of saturated vegetable fatty acid, water, salt or ionogen, as protamine sulfate, Sodium phosphate dibasic, potassium hydrogen phosphate, sodium-chlor, zinc salt, colloided silica, Magnesium Trisilicate, polyvinylpyrrolidone, cellulosic material, polyoxyethylene glycol, Xylo-Mucine, polyacrylic ester, beeswax, lanolin.

This activeconstituents can have whole body and/or local action, therefore, it can carry out administration with suitable approach, said suitable route as oral, parenteral, lung, nose, sublingual, tongue, cheek, rectum, through skin, conjunctiva, topical or the form administration with implant.

This activeconstituents can also carry out administration with the form of medication being suitable for these route of administration.

Being suitable for having of oral administration can rapidly and/or with the known form of medication of mode Transport Activities composition changed, as tablet (uncoated tablets or coating tablet, as having the tablet of enteric coating or not dressing), capsule, coated tablet, particle, piller, pulvis, emulsion, suspension and aerosol.

Adopt parenteral admin may can avoid absorption step (in intravenously, intra-arterial, intracardiac, backbone or administration in waist marrow) or comprise absorption (intramuscular, subcutaneous, intracutaneous, through skin or Intraperitoneal medication).Be suitable for the preparation of form of medication especially for the solution injected and input, suspension, emulsion, lyophilize thing and sterilized powder form of parenteral admin.

Be suitable for medicine, nose drops/solution, sprays that having of other administration route such as sucks (particularly powder sucks, sprays); For the tablet of tongue, sublingual or cheek administration or capsule, suppository, preparation for ear and eyes, vaginal capsule, aqueous suspension (lotion, jolting mixture), lipotropy suspension, ointment, emulsifiable paste, emulsion, paste, dusting or implant, as stent.

By known method itself, this activeconstituents can be changed into described form of medication.It can realize with the suitable pharmaceutical excipient of inert non-toxic.It particularly comprises carrier (such as Microcrystalline Cellulose), solvent (such as liquid macrogol), emulsifying agent (such as sodium lauryl sulphate), dispersion agent (such as polyvinylpyrrolidone), synthesis and natural biological copolymer (such as protein), stablizer (such as oxidation inhibitor and xitix), tinting material (such as mineral dye is as ferric oxide) or correctives and/or odor mask.When suitable, said activeconstituents can be present in one or more above-mentioned carriers with the form of microencapsulation.

Except the compound of formula I, said medicine preparation can also comprise other drug activeconstituents.

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Abbreviation

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CAN ceric ammonium nitrate

Con concentrates

DCM methylene dichloride

DMF DMF

DMSO methyl-sulphoxide

EA ethyl acetate

Et ₂o-HCl ethereal HCI

The gas chromatography/mass spectrometry of GC-MS series connection

H hour

HAc acetic acid

Two (trimethylsilyl) amination lithium of LiHMDS

MeOH methyl alcohol

Min minute

Mp fusing point

MS mass spectrum

NMR NMR (Nuclear Magnetic Resonance) spectrum

PE sherwood oil

Py pyridine

Rf retention index (in thin-layer chromatography)

RT room temperature

S second

TFA trifluoroacetic acid

TFAA trifluoroacetic anhydride

THF tetrahydrofuran (THF)

TLC thin-layer chromatography

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The data more detailed about preparation compound of Formula I is shown in embodiment.

Detailed Description Of The Invention

Be further described with feature to various aspects of the present invention below.

The various term that the present invention uses and phrase have and well known to a person skilled in the art general sense, nonetheless, the present invention still wishes to be described in more detail at this these terms and phrase and to explain, the term mentioned and phrase, if any inconsistent with common art-recognized meanings, are as the criterion with the implication that the present invention states.Here is the definition of the present invention's multiple term used, and these definition are applicable to term used in the whole specification sheets of the application, unless separately explained in particular case.

As described herein, term " significant quantity " refers to the dosage that can realize treating and/or preventing disease of the present invention or illness in experimenter.

As described herein, term " pharmaceutical composition ", it can also refer to " composition ", and it is used in experimenter and particularly realizes in Mammals treating and/or preventing disease of the present invention or illness.

As described herein, term " experimenter " can refer to patient or other accept formula I or its pharmaceutical composition to treat and/or prevent the animal of disease of the present invention or illness, particularly Mammals, such as people, dog, monkey, ox, horse etc.

As described herein, term " disease and/or illness " refers to a kind of physical state of described experimenter, and this physical state is relevant with disease of the present invention and/or illness.Such as, disease of the present invention and/or illness both can refer to a kind of physical state, such as, in the physical state compared with hyperglycemia, also can refer to a kind of morbid state, such as, show as the morbid state such as hyperglycemia, diabetes.Do not distinguish for physical state and morbid state in this article, or the two can refer to mutually, such as " hyperglycemia " and " hyperglycemia " can exchange use.

As described herein, as do not specialized, " % " refers to the per-cent of w/w, particularly when describing solid matter.Certainly, when describing liquid substance, being somebody's turn to do the per-cent (solid being dissolved in the situation of liquid) that " % " can refer to weight/volume, or volume/volume per-cent (liquid being dissolved in the situation of liquid) can be referred to.

As described herein, term " pharmacy is acceptable " or " pharmaceutically useful " that be used interchangeably with it, such as when describing " pharmacologically acceptable salts ", represent this salt its not still subject physiologic learn and can accept, but also the synthetic pharmaceutically having use value can be referred to, such as at the salt as intermediate for being formed when carrying out chiral separation, although the salt of this intermediate directly can not give experimenter, this salt can work for obtaining in end product of the present invention.

In one embodiment, the present invention relates to the compound of general formula I, its all possible isomer and pharmacologically acceptable salts, solvate or the hydrate purposes for the production of medicine, described medicine is used for the treatment of cardiovascular disorder.

In one embodiment, the present invention relates to the compound of general formula I, its all possible isomer and pharmacologically acceptable salts, solvate or hydrate for the production of can the purposes of Cardiovarscular medicine.

In one embodiment; the compound of general formula I of the present invention or its pharmaceutically useful salt can be used alone; or use with the form of pharmaceutical composition together with pharmaceutically useful carrier or vehicle; when using with the form of pharmaceutical composition; usually the general formula I compound or pharmaceutically acceptable salt thereof of the present invention of effective dose or hydrate and one or more pharmaceutically acceptable carrier or thinner are combined and make suitable administration form or dosage form, this program comprise by suitable mode, component is mixed, granulation, compression or dissolve.Therefore, the invention provides pharmaceutical composition, it comprises the compound of general formula I, its all possible isomer and pharmacologically acceptable salts, solvate or hydrate and the pharmaceutically useful carrier of at least one.

The pharmaceutical composition of the compounds of this invention, can the any-mode of following aspect grant: oral, spraying suction, rectal administration, intranasal administration, vagina administration, topical, parenterai administration are as in subcutaneous, vein, intramuscular, intraperitoneal, sheath, in ventricle, in breastbone or intracranial injection or input, or by a kind of reservoir medication of outer planting, wherein preferred oral, intramuscular injection, intraperitoneal or intravenous administration mode.

The compounds of this invention or the pharmaceutical composition containing it can administrations in a unit.Form of administration can be liquid dosage form, solid dosage.Liquid dosage form can be true solution class, colloidal type, particulate formulations, emulsion dosage form, mixed suspension form.Other formulations are tablet, capsule, dripping pill, aerosol, pill, pulvis, solution, suspensoid, emulsion, granule, suppository, lyophilized injectable powder, inclusion compound, implants, patch, liniment etc. such as.

Can also containing conventional carrier in pharmaceutical composition of the present invention, pharmaceutically acceptable carrier described here is including, but not limited to ion-exchanger, aluminum oxide, aluminum stearate, Yelkin TTS, serum protein is as human serum protein, buffer substance is as phosphoric acid salt, glycerine, Sorbic Acid, potassium sorbate, the partial glyceride mixtures of saturated vegetable fatty acid, water, salt or ionogen, as protamine sulfate, Sodium phosphate dibasic, potassium hydrogen phosphate, sodium-chlor, zinc salt, colloided silica, Magnesium Trisilicate, polyvinylpyrrolidone, cellulosic material, polyoxyethylene glycol, Xylo-Mucine, polyacrylic ester, beeswax, wool grease etc.The content of carrier in pharmaceutical composition can be 1 % by weight-98 % by weight, usually accounts for greatly 80 % by weight.For simplicity, local anesthetic, sanitas, buffer reagent etc. can directly be dissolved in carrier.

Oral tablet and capsule can contain vehicle as tackiness agent, as syrup, and gum arabic, sorbyl alcohol, tragacanth, or polyvinylpyrrolidone, weighting agent, as lactose, sucrose, W-Gum, calcium phosphate, sorbyl alcohol, Padil, lubricant, as Magnesium Stearate, talcum, polyoxyethylene glycol, tripoli, disintegrating agent, as yam starch, or acceptable dibutyl phthalate, as bay sodium alkoxide vitriol.Tablet can with method dressing known in pharmacopedics.

Oral liquid can make the suspension of water and oil, and solution, emulsion, syrup or elixir, also can make dry product, with front make up water or other suitable medium.This liquid preparation can comprise conventional additive, as suspension agent, and sorbyl alcohol, Walsroder MC 20000S, dextrose syrup, gel, Natvosol, carboxymethyl cellulose, aluminium stearate gel, the food oils of hydrogenation, emulsifying agent, as Yelkin TTS, sorb gathers candy list oleate, Sudan Gum-arabic; Or nonaqueous carrier (may edible oil be comprised), as Prunus amygdalus oil, grease as glycerine, ethylene glycol, or ethanol; Sanitas, as methyl p-hydroxybenzoate or propyl ester, Sorbic Acid.As needs can add seasonings or tinting material.

Suppository can comprise conventional suppository base, as cocoa butter or other glyceryl ester.

For parenteral, liquid forms is made up of the carrier of compound and a kind of sterilization usually.The first-selected water of carrier.According to the difference of selected carrier and drug level, compound had both dissolved in carrier and also can be made into aaerosol solution, first that compound is soluble in water when making injection solution, loaded in sealed bottle or ampoule after filter-sterilized.

Must recognize, the best dosage of compound of Formula I and interval are determined by external conditionss such as the form of compound property and such as administration, path and position and the specific Mammalss treated, and this best dosage can be determined by the technology of routine.Also must recognize, the best course for the treatment of, i.e. the dosage of compound of Formula I every day within the specified time, available method well known in the art is determined simultaneously.

Formula I also comprises its isomer and solvate, and formula I and its isomer, solvate and ester can also form solvate, such as hydrate, alcohol adduct etc.Above-claimed cpd can also be prodrug or the form that can discharge described activeconstituents in vivo after metabotic change.Selecting and preparing suitable prodrug derivant is technology as well known to those skilled in the art.In general, for object of the present invention, as suitable with non solvate form in the solvate form thereof of water, ethanol etc. with the acceptable solvent of pharmacy.

The active compound amount of gained the actual dose level of each activeconstituents in pharmaceutical composition of the present invention can be changed, so that effectively can obtain required therapeutic response for concrete patient, composition and administering mode.Dosage level must according to the activity of particular compound, route of administration, treat the severity of the patient's condition and the patient's condition of patient to be treated and medical history and select.But the way of this area is, the dosage of compound, from lower than for obtaining level that required result for the treatment of requires, increases dosage, gradually until obtain required effect.

In general, formula I is used for the dosage of Mammals particularly people can between 0.001-1000mg/kg body weight/day, such as, between 0.01-100mg/kg body weight/day, such as, between 0.01-10mg/kg body weight/day.

Such as, according to the difference of administering mode, in component, can weight ratio 0.1% be contained, or the active ingredient of more suitably weight ratio 10-60%.But when comprising unitary dose in component, each unit preferably comprises 5-500 milligram activeconstituents.Such as, in one embodiment, the difference of foundation route of administration and administration frequency, the suitable therapeutic doses for being grown up is 100-3000 milligram every day, as every day 1500 milligrams.This dose corresponds to 1.5-50 mg/kg/day, and suitable dosage is 5-20 mg/kg/day.

The present inventor is surprised to find, and formula I provided by the invention is effectively activate the activator of sGC, can by strengthening sGC to the susceptibility of NO; Under the extremely low condition even lacked of NO level, directly activate sGC, there is the effect of Cardiovarscular.

Embodiment

Can be conducted further description the present invention by the following examples, but scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite not deviating from the spirit and scope of the present invention, can carry out various change and modification to the present invention.

The present invention carries out generality and/or concrete description to the material used in test and test method.Although for realizing many materials that the object of the invention uses and working method is well known in the art, the present invention still describes in detail as far as possible at this.

The fusing point of compound is measured by RY-1 melting point apparatus, and thermometer is without calibration.Mass spectrum is measured by Micromass ZabSpec high resolution mass spectrometer (resolving power 1000). ¹h-NMR is measured by JNM-ECA-400 SUPERCONDUCTING NMR instrument, operating frequency ¹h-NMR 400MHz.

The universal synthesis method of initial compounds:

Embodiment 1A: to methoxybenzyl hydrazine

Getting 242g (4.11mol) 85% hydrazine hydrate is dissolved in 350ml dehydrated alcohol; nitrogen protection; be heated to backflow; slow dropping 68g (0.434mol) is to the ethanol solution of methoxyl group benzyl chloride and 200ml; 1h drips off; continue backflow 6h, stirring at room temperature 18h, remove solvent under reduced pressure.100ml methylene dichloride is added in resistates, 100ml 5%NaOH solution is dripped at 0 DEG C, layering, water layer methylene dichloride (4 × 80ml) extracts, merge organic layer, Anhydrous potassium carbonate is dry, and after removing solvent under reduced pressure, give light yellow oil 70g (GC-MS purity is 79%) is stand-by.1H-NMR (D ₂o, 400MHz) (hydrochloride): δ 3.692 (s, 3H), 4.073 (s, 2H), 6.901 (d, 2H), 7.261 (d, 2H); GC-MS (m/z): 152.1 [M] ⁺.

Embodiment 2A: cyano propanone acetoacetic ester sodium salt

Dry instrument, get 350ml dehydrated alcohol in 1000ml there-necked flask, be placed in ice bath, get 30.7g (1.334mol) sodium Metal 99.5, carefully shred and add in reaction solution, finish, reaction flask is placed in oil bath, be heated to backflow, after making undissolved sodium entirely molten, reaction flask is placed in ice bath again, temperature is down to about 0 DEG C, drip 100.7g (0.689mol) oxalic acid diethyl ester and 35ml anhydrous ether solution, drip and finish, continue stirring 20 minutes, then 28.5g (0.694mol) acetonitrile and 35ml anhydrous ether solution is dripped, 24h is stirred in about 35 DEG C, stopped reaction, reaction solution is centrifugal, throw out is dried to half-dried, vacuum drier kept dry, obtain light yellow solid 100.7g (yield 89.6%). ¹H-NMR(d-DMSO，400MHz)：δ1.177(t，3H)，4.016(q，2H)，4.089(s，1H)；ES I-MS(m/z)：140.2[M-Na] ^-

Embodiment 3A:5-amino-1-is to methoxybenzyl-pyrazoles-3-ethyl formate

Get 51g (0.313mol) cyano propanone acetoacetic ester sodium salt and add to 900ml 1, in 4-dioxane, stir 20min, add 53g (0.465) TFA, stir 20min, fraction solids dissolves, embodiment 1A added in reaction solution to methoxybenzyl hydrazine, nitrogen protection, reflux 24h, suction filtration removing solid, add 300ml water, ethyl acetate (100ml × 4) extracts, saturated aqueous common salt 50ml washs, anhydrous magnesium sulfate drying, suction filtration removing siccative, remove solvent under reduced pressure, PE: EA=2: 1 column chromatography for separation, obtain light yellow solid 44.8g (yield 52.0%). ¹H-NMR(d-DMSO，400MHz)：δ1.238(t，3H)，3.718(s，3H)，4.179(q，2H)，5.113(s，2H)，5.539(br，2H)，5.712(s，1H)，6.889(dd，2H)，7.13(dd，2H)；GC-MS(m/z)：275.1[M] ⁺。

Embodiment 4A:1-is to methoxybenzyl-1H-pyrazolo [3,4-b] Nicotinicum Acidum ethyl ester

Get the 1-of 55.1g (0.2mol) embodiment 3A to methoxybenzyl-1H-pyrazolo [3; 4-b] Nicotinicum Acidum ethyl ester is dissolved in 800ml 1; in 4-dioxane; add 19.8g dimethylamino acrolein and 27.4g TFA successively; reflux 3 days under nitrogen protection; remove solvent under reduced pressure; 300ml water is added in resistates; EA (150ml × 3) extracts; saturated aqueous common salt 50ml washs; anhydrous magnesium sulfate drying, column chromatography (PE: EA=8: 1) wash-out, obtains light yellow solid 21.8g (yield 35%). ¹H-NMR(d-DMSO，400MHz)：δ1.376(t，3H)，3.702(s，3H)，4.404(q，2H)，5.728(s，2H)，6.881(d，2H)，7.273(d，2H)，7.468(dd，1H)，8.483(dd，1H)，8.713(dd，1H)；GC-MS(m/z)：311.1[M] ⁺。

Embodiment 5A:1-is to methoxybenzyl-1H-pyrazolo [3,4-b] pyridine-3-carboxamide

Under ice bath, in 450ml anhydrous methanol, logical ammonia is to saturated, add the 1-of embodiment 4A to methoxybenzyl-1H-pyrazolo [3,4-b] Nicotinicum Acidum ethyl ester 21.8g (0.07mol), sealed reactor, in stirring at room temperature 2 days, be spin-dried for solvent, obtain product and amount to 19.8g (yield 100%). ¹H-NMR(d-DMSO，400MHz)：δ3.701(s，3H)，5.673(s，2H)，6.876(d，2H)，7.258(d，2H)，7.376(dd，1H)，7.524(s，1H)，7.838(s，1H)，8.546(dd，1H)，8.651(dd，1H)；GC-MS(m/z)：M ⁺(282.1)

Embodiment 6A:3-cyano group-1-is to methoxybenzyl-1H-pyrazolo [3,4-b] pyridine

Get the 1-of 33.8g (0.12mol) embodiment 5A to methoxybenzyl-1H-pyrazolo [3, 4-b] pyridine-3-carboxamide is dissolved in the anhydrous THF of 300ml, add 24.3g pyridine, be placed in ice bath to stir, slow dropping 64.7g trifluoroacetic anhydride is in reaction solution, drip and finish, spend the night in stirring at room temperature reaction, add 600ml water, EA (300ml × 3) extracts, use saturated sodium bicarbonate 250ml successively, 1M hydrochloric acid 150ml, saturated aqueous common salt 100ml washs, anhydrous magnesium sulfate drying, suction filtration, removing siccative, revolve and desolventize to obtain light yellow crystal 30.7g (yield 96.8%). ¹H-NMR(d-DMSO，400MHz)：δ3.709(s，3H)，5.75(s，2H)，6.895(dd，2H)，7.314(dd，2H)，7.535(dd，1H)，8.494(dd，1H)，8.805(dd，1H)；GC-MS(m/z)：M ⁺(264.1)。

Embodiment 7A:3-cyano group-1H-pyrazolo [3,4-b] pyridine

3-cyano group-the 1-getting 10.6g (0.04mol) embodiment 6A in 450ml acetonitrile, is stirred to entirely molten to methoxybenzyl-1H-pyrazolo [3,4-b] pyridine, add 120ml water, under stirring, drip 75g CAN and the 120ml aqueous solution, drip and finish, 300ml water is added after continuing to stir 2h, one shares 1500ml extraction into ethyl acetate for several times, merges, revolves and desolventize, column chromatography for separation, obtains product 5.5g (yield 95.4%). ¹H-NMR(d-DMSO，400MHz)：δ7.472(dd，1H)，8.455(d，1H)，8.731(dd，1H)，15.013(s，1H)；GC-MS(m/z)：M ⁺(144.0)。

Embodiment 8A: benzeneazo propane dinitrile

Getting 29ml (0.318mol) aniline adds in 1L there-necked flask, add appropriate ice cube, 85ml concentrated hydrochloric acid is dripped under mechanical stirring, drip and finish, add a small amount of ice cube, holding temperature 0 DEG C, drip the aqueous solution of 29g (0.42mol) Sodium Nitrite and 50ml, control temperature 0-5 DEG C, drip and finish, continue to be stirred to reaction solution in low temperature and make the variable color of starch-kalium iodide test paper, then 51g (0.622mol) sodium-acetate and the 100ml aqueous solution is added, stir and add 33g (0.50mol) propane dinitrile and 25ml ethanolic soln in a moment, produce a large amount of yellow solid, finish, continue stirring reaction 1h, suction filtration, filter cake distilled water wash, dry, obtain yellow solid 42.2g (yield 78%)

¹H-NMR(CDCl ₃，400MHz)：δ7.254-7.273(m，1H)，7.291-7.339(m，2H)，7.425-7.465(m，2H)，9.775(s，1H)；GC-MS(m/z)：M ⁺(170.1)。

Embodiment 9A:2-methoxycarbonyl thiophene-3-NITRODIAZONIUM FLUOROBORATE

Get 125ml concentrated hydrochloric acid and be mixed with 6M hydrochloric acid 250ml, be placed in ice bath, under mechanical stirring, add 94g (0.598mol) 2-aminothiophene-3-methyl-formiate in batches, again ice bath is placed in after room temperature continues to stir 30min, treat that temperature is down to subzero, the aqueous solution of slow dropping 42g Sodium Nitrite and 100ml, control temperature is no more than 5 DEG C, reaction solution color burn, drip and finish, continue to be stirred to reaction solution and can make the variable color of starch-kalium iodide test paper, then 180ml fluoborate solution is added, produce a large amount of white solid, continue to stir 15min complete to precipitation, suction filtration, successively with cold methyl alcohol and washed with diethylether filter cake, vacuum-drying, obtain white crystal 142g (yield 93%)

The fluoro-2-thiophenic acid of embodiment 10A:3-

2-methoxycarbonyl thiophene-3-NITRODIAZONIUM FLUOROBORATE and the thick silica gel of 80g of getting 30g (0.117mol) embodiment 9A mix, and are placed in 1000ml round-bottomed flask, connect Dewar formula condenser successively, cold-trap, oil pump, under decompression, in about 180 DEG C reactions, question response is no longer violent, and temperature is increased to 200 DEG C, question response is no longer violent, stop decompression and heating, wash out the product in reactor, condenser, cold-trap by ethanol in proper amount, merge, stand-by.

Get 9g NaOH and 200ml water adds in above-mentioned ethanolic soln, after reflux 2h, revolve except after most of solvent, add 150ml water, acidity is adjusted to 10% hydrochloric acid, EA (75ml × 3) extracts, and column chromatography (PE: EA=2: 1) is separated, and obtains product 5.5g (two step yields 32%). ¹H-NMR(CDCl ₃，400MHz)：δ6.901(d，1H)，7.962(dd，1H)；GC-MS(m/z)：M ⁺(146.0)。

The fluoro-2-thiophen(e)alcohol of embodiment 11A:3-

The 3-fluoro-2-thiophenic acid getting 9.0g (0.062mol) embodiment 10A is dissolved in the anhydrous THF of 90ml, slowly drop in the anhydrous THF of 3.9g (0.103mol) sodium borohydride and 90ml, reaction produces a large amount of bubble, drip and finish, continue to stir 30min to no longer producing bubble, then 11g (0.0433mol) iodine and 120ml anhydrous THF solution is dripped, in 50 DEG C of reaction 8h, to go out reaction with shrend, ether (150ml × 4) extracts, saturated aqueous common salt 50ml washs, and revolves except ether, stand-by (yield 95%).GC-MS(m/z)：M ⁺(132.0)

Embodiment 12A:2-chloromethyl-3-fluorine thiophene

Get 30ml concentrated hydrochloric acid, add in the fluoro-2-thiophen(e)alcohol of 3-of embodiment 11A, stir 15min, add 70ml water, ether (50ml × 4) extracts, and washs with rare sodium hydrogen carbonate solution, then saturated common salt water washing is used, anhydrous magnesium sulfate drying, revolves except ether, stand-by (yield 96%).GC-MS(m/z)：150.0(100％)，152.0(36％)

Embodiment 13A:2-cyano group-5-fluorine thiophene

Get 10.0g (64.9mmol) 2-cyano group-5-nitrothiophene, 18.9g (323.6mmol) Potassium monofluoride, 2.5g (6.0mmol) 4-phenyl phosphonium bromide, 13.4g (66.0mmol) phthalyl chloride is dissolved in 200ml tetramethylene sulfone, after 180 DEG C of reaction 2h, is cooled to room temperature, add 400ml water, ether (100ml × 3) extracts, and steaming desolventizes, stand-by.

The fluoro-2-thiophenic acid of embodiment 14A:5-

Getting 5.0g (0.125mol) NaOH is dissolved in 60ml water and 60ml ethanol, then add in the 2-cyano group-5-fluorine thiophene of embodiment 13A, reflux 2h, steaming desolventizes, add 120ml water, be adjusted to acidity with concentrated hydrochloric acid, EA (40ml × 3) extracts, and column chromatography for separation obtains product 4.5g (two step yields 47.5%).

The fluoro-2-thiophen(e)alcohol of embodiment 15A:5-

The 5-fluoro-2-thiophenic acid getting 4.5g (0.031mol) embodiment 14A is dissolved in the anhydrous THF of 45ml, slowly drop in the anhydrous THF of 1.95g (0.052mol) sodium borohydride and 45ml, reaction produces a large amount of bubble, drip and finish, continue to stir 30min to no longer producing bubble, then 5.5g (0.022mol) iodine and 60ml anhydrous THF solution is dripped, in 50 DEG C of reaction 8h, to go out reaction with shrend, ether (75ml × 4) extracts, saturated aqueous common salt 50ml washs, and revolves except ether, stand-by (yield 91%).GC-MS(m/z)：M ⁺(132.0)。

Embodiment 16A:2-chloromethyl-5-fluorine thiophene

Get 15ml concentrated hydrochloric acid, add in the fluoro-2-thiophen(e)alcohol of 5-of embodiment 15A, stir 15min, add 35ml water, ether (30ml × 4) extracts, and washs with rare sodium hydrogen carbonate solution, then saturated common salt water washing is used, anhydrous magnesium sulfate drying, revolves except ether, stand-by (yield 95%).GC-MS(m/z)：150.0(100％)，152.0(36％)。

Embodiment 17A:3-cyano group-1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridine

Getting 6.0g (0.0416mol) embodiment 7A and embodiment 12A is dissolved in 120ml DMF; add 16.3g (0.05mol) cesium carbonate; under nitrogen protection; in about 40 DEG C reaction 15h; add 250ml water, ethyl acetate (120ml × 3) extracts, the water washing of 50ml saturated common salt; column chromatography for separation, obtains product 7.37g (yield 68.5%). ¹H-NMR(d-DMSO，400MHz)：δ5.935(s，2H)，7.001(d，1H)，7.573-7.542(m，2H)，8.511(dd，1H)， 8.831(dd，1H)；GC-MS(m/z)：M ⁺(258.1)。

Embodiment 18A:1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridine-3-carboximide acid methyl esters

Get 3-cyano group-1-(3-fluorine thiophene-2-base) the methyl isophthalic acid H-pyrazolo [3 of 7.37g (0.0285mol) embodiment 17A, 4-b] pyridine is dissolved in 300ml anhydrous methanol, add 6.19g (0.115mol) sodium methylate, stirring at room temperature 2h, reaction solution directly does next step reaction.

Embodiment 19A:1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridine-3-amitraz hydrochloride

Get 6.85g (0.114mol) Glacial acetic acid and 2.22g (0.0415mol) ammonium chloride adds in the reaction solution of embodiment 18A, reflux 10h, revolve and desolventize, resistates proper amount of acetone is ground, suction filtration, filter cake adds in 300ml water, add 7.5g sodium carbonate to stir, ethyl acetate (200ml × 3) extracts, and anhydrous magnesium sulfate drying, revolves except after most of solvent, under low temperature stirs, in resistates, dripping ethereal HCI solution to no longer producing solid, revolving and desolventizing, obtaining off-white color solid 6.77g (yield 76.1%).

Embodiment 20A:2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-[(E) phenyl-diazenyl]-4,6-pyrimidinediamines

Get 1-(3-fluorine thiophene-2-base) the methyl isophthalic acid H-pyrazolo [3 of 6.77g (0.0217mol) embodiment 19A, 4-b] pyridine-3-amitraz hydrochloride adds in 115ml DMF, 1.29g (0.0239mol) sodium methylate is added under stirring, then add 3.72g (0.0219mol) embodiment 8A, under nitrogen protection after 110 DEG C of reaction 12h, be cooled to room temperature, after filtrate is concentrated, suction filtration, a small amount of washing with alcohol of filter cake, obtains orange solid; In residual filtrate, add suitable quantity of water again to no longer producing precipitation, suction filtration, filter cake washing with alcohol, obtains brown-red solid, amounts to obtain product 8.52g (yield 88%). ¹H-NMR(d-DMSO，400MHz)：δ5.889(s，2H)，6.996(d，1H)，7.376-7.429(m，2H)，7.478-7.516(m，3H)，7.926(br，1H)，8.018-8.039(2H)，8.518(br，1H)，8.677(dd，1H)，9.190(dd，1H)；MS(m/z)：[M+H] ⁺(446.1)。

Embodiment 21A:2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-4,5,6-pyrimidine triamine tri hydrochlorides

Get 2-[1-(3-fluorine thiophene-2-base) the methyl isophthalic acid H-pyrazolo [3 of 5.3g (11.9mmol) embodiment 20A, 4-b] pyridin-3-yl]-5-[(E) phenyl-diazenyl]-4, 6-pyrimidinediamine and 2.4g T-1 type Raney-Ni (weight in wet base) add in 100ml DMF, in 65 DEG C, hydrogenation 12 hours under 65bar pressure, catalyzer is removed with suction filtered through kieselguhr, revolve and desolventize, the hydrochloric acid of 50ml 5N is added in resistates, stir, produce Tan solid, 45min is incubated in 90 DEG C of hot water baths, to be cooled to room temperature, suction filtration, obtain Tan solid 4.03g (yield 72.7%). ¹H-NMR(d-DMSO，400MHz)：δ3.449(br，4H)，4.931(br，1H)，5.925(s，2H)，6.996(dd，1H)，7.238-7.492(br，3H)，7.503-7.527(m，2H)，8.770(dd，1H)，8.929(dd，1H)，13.176(br，1H)；MS(m/z)：[M+H] ⁺(357.1)。

Embodiment 22A:3-cyano group-1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridine

Getting 3.0g (0.0208mol) embodiment 7A and embodiment 16A is dissolved in 60ml DMF; add 8.2g (0.026mol) cesium carbonate; under nitrogen protection; in about 40 DEG C reaction 15h; add 125ml water, ethyl acetate (60ml × 3) extracts, the water washing of 30ml saturated common salt; column chromatography for separation, obtains product 3.5g (yield 65.1%).

Embodiment 23A:1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridine-3-carboximide acid methyl esters

Get 3-cyano group-1-(3-fluorine thiophene-2-base) the methyl isophthalic acid H-pyrazolo [3 of 3.5g (0.0135mol) embodiment 22A, 4-b] pyridine is dissolved in 150ml anhydrous methanol, add 3.1g (0.0576mol) sodium methylate, stirring at room temperature 2h, reaction solution directly does next step reaction.

Embodiment 24A:1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridine-3-amitraz hydrochloride

Get 3.43g (0.0571mol) Glacial acetic acid and 1.11g (0.0208mol) ammonium chloride adds in the reaction solution of embodiment 23A, reflux 10h, revolve and desolventize, resistates proper amount of acetone is ground, suction filtration, filter cake adds in 150ml water, add 3.8g sodium carbonate to stir, ethyl acetate (100ml × 3) extracts, and anhydrous magnesium sulfate drying, revolves except after most of solvent, under low temperature stirs, in resistates, dripping ethereal HCI solution to no longer producing solid, revolving and desolventizing, obtaining off-white color solid 3.33g (yield 78.8%).

Embodiment 25A:2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-[(E) phenyl-diazenyl]-4,6-pyrimidinediamines

Get 1-(5-fluorine thiophene-2-base) the methyl isophthalic acid H-pyrazolo [3 of 3.33g (10.7mmol) embodiment 24A, 4-b] pyridine-3-amitraz hydrochloride adds in 55ml DMF, 0.594g (11.0mmol) sodium methylate is added under stirring, then add 1.85g (10.9mmol) embodiment 8A, under nitrogen protection after 110 DEG C of reaction 12h, be cooled to room temperature, after filtrate is concentrated, suction filtration, a small amount of washing with alcohol of filter cake, obtains orange solid; In residual filtrate, add suitable quantity of water again to no longer producing precipitation, suction filtration, filter cake washing with alcohol, obtains brown-red solid, amounts to obtain product 4.15g (yield 87.2%).

Embodiment 26A:2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-4,5,6-pyrimidine triamine tri hydrochlorides

Get 2-[1-(5-fluorine thiophene-2-base) the methyl isophthalic acid H-pyrazolo [3 of 2.65g (5.95mmol) embodiment 25A, 4-b] pyridin-3-yl]-5-[(E) phenyl-diazenyl]-4, 6-pyrimidinediamine and 1.2g T-1 type Raney-Ni (weight in wet base) add in 50ml DMF, in 65 DEG C, hydrogenation 12 hours under 65bar pressure, catalyzer is removed with suction filtered through kieselguhr, revolve and desolventize, the hydrochloric acid of 25ml 5N is added in resistates, stir, produce Tan solid, 45min is incubated in 90 DEG C of hot water baths, to be cooled to room temperature, suction filtration, obtain Tan solid 2.1g (yield 75.8%).

The specific embodiment of new texture 2-substituted thiophene base pyrazolo-pyridines:

Embodiment 1:4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino methyl-formiate

Get 2-[1-(3-fluorine thiophene-2-base) the methyl isophthalic acid H-pyrazolo [3 of 0.35g (0.75mmol) embodiment 21A, 4-b] pyridin-3-yl]-4, 5, 6-pyrimidine triamine tri hydrochloride is dissolved in 15ml pyridine, stirring at room temperature is placed in ice bath in a moment and stirs 15min, drawing 0.12g (1.27mmol) methyl-chloroformate with syringe adds in reaction solution, continue in ice bath and stir 2h, then stirred overnight at room temperature, be spin-dried for solvent, add 15ml water to stir, generation brown color precipitates, suction filtration, column chromatography for separation obtains product 0.3g (yield 96.5%). ¹H-NMR(d-DMSO，400MHz)：δ3.595(s，3H)，5.828(s，2H)，6.254(s，4H)，6.970(dd，1H)，7.340(dd，1H)，7.462(dd，1H)，8.123(br，1H)，8.617(dd，1H)，9.036(dd，1H)；MS(m/z)：[M+H] ⁺(415.2)，[M+Na] ⁺(437.1)。

Embodiment 2:4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate

Method is with embodiment 1, by 2-[1-(3-fluorine thiophene-2-base) the methyl isophthalic acid H-pyrazolo [3 of embodiment 21A, 4-b] pyridin-3-yl]-4,5,6-pyrimidine triamine tri hydrochloride and Vinyl chloroformate react to obtain light yellow solid 4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate.

Embodiment 3:4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino isopropyl formate

Method is with embodiment 1, by 2-[1-(3-fluorine thiophene-2-base) the methyl isophthalic acid H-pyrazolo [3 of embodiment 21A, 4-b] pyridin-3-yl]-4,5,6-pyrimidine triamine tri hydrochloride and isopropyl chlorocarbonate react light yellow solid 4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino isopropyl formate.MS(m/z)：[M+H] ⁺(443.1)，[M+Na] ⁺(465.1)。

Embodiment 4:4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) Urethylane

Get 4 of 0.15g (0.362mmol) embodiment 1, 6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3, 4-b] pyridin-3-yl]-5-pyrimidinyl-amino methyl-formiate is in the anhydrous THF of 2ml, stir the THF solution that 30min gets 0.4ml LiHMDS (1.06M) under being placed in ice bath and add to reaction solution, after continuing at ice bath stirring 20min, getting 0.023ml methyl iodide adds in reaction solution, continue to stir 4h in room temperature after ice bath stirs 1h, with the aqueous ammonium chloride solution 2ml cancellation reaction of 5%, complete to product extraction for several times with ethyl acetate and dichloromethane extraction, merge, revolve and desolventize, column chromatography for separation, obtain product 100mg (yield 64.5%).

¹h-NMR (d-DMSO, 400MHz): δ 3.004 (s, 3H), 3.533 (main) and 3.657 (secondary) (s, 3H), 5.830 (s, 2H), 6.406 (s, 4H), 6.970 (dd, 1H), 7.340 (dd, 1H), 7.462 (dd, 1H), 8.617 (dd, 1H), 9.036 (dd, 1H); MS (m/z): [M+H] ⁺(429.1), [M+Na] ⁺(451.0).

Embodiment 5:4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) urethanum

Method is with embodiment 4, by 4 of embodiment 2,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate and iodomethane reaction must obtain light yellow solid 4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) urethanum. ¹H-NMR(d-DMSO，400MHz)：δ1.075-1.272(m，3H)，3.001(s，3H)，3.983-4.018(m，2H)，5.829(s，2H)，6.377(s，4H)，6.971(dd，1H)，7.341(dd，1H)，7.465(dd，1H)，8.616(dd，1H)，9.039(dd，1H)。

Embodiment 6:4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) carbamic acid isopropyl ester

Method is with embodiment 4, by 4 of embodiment 3,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino isopropyl formate and iodomethane reaction obtain light yellow solid 4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) carbamic acid isopropyl ester. ¹H-NMR(d-DMSO，400MHz)：δ1.089-1.279(m，6H)，2.992(s，3H)，4.805(m，1H)，5.829(s，2H)，6.343(s，4H)，6.971(dd，1H)，7.340(dd，1H)，7.466(dd，1H)，8.624(dd，1H)，9.052(dd，1H)。

Embodiment 7:4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) Urethylane

Method is with embodiment 4, by 4 of embodiment 1,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino methyl-formiate and iodoethane react to obtain light yellow solid 4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) Urethylane. ¹h-NMR (d-DMSO, 400MHz): δ 1.061 (t, 3H), 3.502 (q, 2H), 3.544 (main) and 3.673 (secondary) (s, 3H), 5.831 (s, 2H), 6.345 (s, 4H), 6.972 (dd, 1H), 7.339 (dd, 1H), 7.464 (dd, 1H), 8.623 (dd, 1H), 9.046 (dd, 1H).

Embodiment 8:4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) urethanum

Method is with embodiment 4, by 4 of embodiment 2,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate and iodoethane react to obtain light yellow solid 4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) urethanum.MS(m/z)：[M+H] ⁺(457.2)，[M+Na] ⁺(479.1)。

Embodiment 9:4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) carbamic acid isopropyl ester

Method is with embodiment 4, by 4 of embodiment 3,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino isopropyl formate and iodoethane react to obtain light yellow solid 4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) carbamic acid isopropyl ester. ¹H-NMR(d-DMSO，400MHz)：δ1.035-1.110(m，9H)，3.469-3.523(m，2H)，4.829(m，1H)，5.832(s，2H)，6.286(s，4H)，6.976(dd，1H)，7.337(dd，1H)，7.463(dd，1H)，8.625(dd，1H)，9.077(dd，1H)。

Embodiment 10:4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino methyl-formiate

Method is with embodiment 1, by 2-[1-(5-fluorine thiophene-2-base) the methyl isophthalic acid H-pyrazolo [3 of embodiment 26A, 4-b] pyridin-3-yl]-4,5,6-pyrimidine triamine tri hydrochloride and methyl-chloroformate react to obtain light yellow solid 4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino methyl-formiate.MS(m/z)：[M+H] ⁺(415.2)，[M+Na] ⁺(437.1)。

Embodiment 11:4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate

Method is with embodiment 1, by 2-[1-(5-fluorine thiophene-2-base) the methyl isophthalic acid H-pyrazolo [3 of embodiment 26A, 4-b] pyridin-3-yl]-4,5,6-pyrimidine triamine tri hydrochloride and Vinyl chloroformate react to obtain light yellow solid 4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate.MS(m/z)：[M+H] ⁺(429.2)，[M+Na] ⁺(451.1)。

Embodiment 12:4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino isopropyl formate

Method is with embodiment 1, by 2-[1-(5-fluorine thiophene-2-base) the methyl isophthalic acid H-pyrazolo [3 of embodiment 26A, 4-b] pyridin-3-yl]-4,5,6-pyrimidine triamine tri hydrochloride and isopropyl chlorocarbonate react to obtain light yellow solid 4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino isopropyl formate.MS(m/z)：[M+H] ⁺(443.1)，[M+Na] ⁺(465.1)。

Embodiment 13:4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) Urethylane

Method is with embodiment 4, by 4 of embodiment 10,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino methyl-formiate and iodomethane reaction obtain light yellow solid 4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) Urethylane.MS(m/z)：[M+H] ⁺(429.1)，[M+Na] ⁺(451.0)。

Embodiment 14:4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) urethanum

Method is with embodiment 4, by 4 of embodiment 11,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate and iodomethane reaction obtain light yellow solid 4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) urethanum. ¹H-NMR(d-DMSO，400MHz)：δ1.077-1.274(m，3H)，3.004(s，3H)，3.987-4.022(m，2H)，5.805(s，2H)，6.394(s，4H)，6.579(dd，1H)，6.888(dd，1H)，7.337(dd，1H)，8.622(dd，1H)，9.046(dd，1H)。

Embodiment 15:4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) carbamic acid isopropyl ester

Method is with embodiment 4, by 4 of embodiment 12,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino isopropyl formate and iodomethane reaction obtain light yellow solid 4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) carbamic acid isopropyl ester.MS(m/z)：[M+H] ⁺(457.2)，[M+Na] ⁺(479.1)。

Embodiment 16:4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) Urethylane

Method is with embodiment 4, by 4 of embodiment 10,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino methyl-formiate and iodoethane react to obtain light yellow solid 4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) Urethylane.MS(m/z)：[M+H] ⁺(443.2)，[M+Na] ⁺(465.0)。

Embodiment 17:4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) urethanum

Method is with embodiment 4, by 4 of embodiment 11,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate and iodoethane react to obtain light yellow solid 4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) urethanum.MS(m/z)：[M+H] ⁺(457.2)，[M+Na] ⁺(479.1)。

Embodiment 18:4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) carbamic acid isopropyl ester

Method is with embodiment 4, by 4 of embodiment 12,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino isopropyl formate and iodoethane react to obtain light yellow solid 4,6-diamino-2-[1-(5-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) carbamic acid isopropyl ester.MS(m/z)：[M+H] ⁺(471.2)，[M+Na] ⁺(493.0)。

Embodiment 19:N-{4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl } methane amide

By 2-[1-(3-fluorine thiophene-2-base) the methyl isophthalic acid H-pyrazolo [3 of embodiment 21A free state, 4-b] pyridin-3-yl]-4,5,6-pyrimidine triamine is dissolved in 10ml DMF, adds 20ml concentrated hydrochloric acid, stirring at room temperature 30min, suction filtration obtains yellow-brown solid, column chromatography () is separated to obtain product N-{4,6-diamino-2-[1-(3-fluorine thiophene-2-base) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl } methane amide. ¹H-NMR(d-DMSO，400MHz)：δ5.834(s，2H)，6.248(br，4H)，6.979(dd，1H)，7.350(dd，1H)，7.474(dd，1H)，8.126(s，1H)，8.630(dd，1H)，8.881(s，1H)，9.045(dd，1H)；MS(m/z)：[M+H] ⁺(385.1)，[M+Na] ⁺(407.0)。

Embodiment 20: vascular circle diastole is tested, measures embodiment compound to the diastole effect of rat chest aorta blood vessel

The preparation of in vitro thoracic aorta vascular circle:

By rat sacrificed by decapitation, take out thoracic aorta rapidly, be placed in and fill ice-cold oxygen-saturated improvement Kreb ' s-Henseleit liquid (NaCl 118mM, NaHCO ₃325mM, KCl 4.7mM, MgCl ₂1.2mM, KH ₂pO ₄1.2mM, CaCl ₂2.5mM, glucose 10mM, pH 7.4 ± 0.5) culture dish in, carefully wash away thrombus and remove the outer reticular tissue of blood vessel.

Blood vessel is cut into the vascular circle that 3mm is wide, be placed in 37 DEG C of thermostatic baths that 10ml contains Kreb ' s-Henseleit liquid, lower end is fixed, and upper end is connected in polygraph by tonotransducer.Organize in bath the mixed gas continuing to pass into 95% oxygen and 5% carbonic acid gas, every 15min changes nutritive medium once, after vascular circle hatches 20-30 minute, gives vascular circle 2g preload, stablizes 45-60 minute.With 3 × 10 ^-7molL ^-1norepinephrine (NE) brings out vascular circle and shrinks, and after contraction reaches maximum value and balances (about 8-10 minute), change nutritive medium, eccysis NE, reaches balance again.Tension value based on now tension value, starts to observe the impact of process factor on antiotasis.

Drug treating:

After blood vessel is hatched and arrived balance, by 3 × 10 ^-7molL ^-1nE injects preshrinking vascular circle in bath, until contraction reach maximum and stable after, give test-compound in the mode of increasing concen-trations, observe test-compound to the diastole effect of norepinephrine (NE) vasoconstriction ring; Calculate the EC50 value of diastole percentage and each compound.

Data processing method:

3 × 10 ^-7the maximum tension value that mol/L NE induces=give 3 × 10 ^-7tension value-basal tension value after mol/L NE vasoconstriction balance

Compound diastole value=3 × 10 ^-7tension value after maximum tension value-the give test-compound that mol/L NE induces

Compound is to the diastole effect of rat chest aorta blood vessel:

With 3 × 10 ^-7the NE preshrinking rat chest aorta ring (1.50-2.2g of mol/L, P < 0.01), test-compound is given in increasing concen-trations mode after balance, observe the vasoconstrictive diastole effect that new compound is induced norepinephrine, group of solvents gives the Kreb ' s-Henseleit nutritive medium containing 1/1000 DMSO.

As a result 1: the maximum diastole effect under new compound certain concentration.

Initial onset and diastole effect is selected to be about two concentration (3*10 of 60-80% ^-8m, 1*10 ^-7m) carry out the evaluation of compound, measure the maximum diastole effect that each concentration of compound adds rear generation.From table 1: extend group of solvents vasoconstriction tension force in time and about reduce 8-10%; Medicine group can the remarkable diastole thoracic aorta of concentration dependent; Tested embodiment compound 4,5 and 7EC50 are all less than 100nM, demonstrate compound and have diastole effect to NE preshrinking blood vessel.

Table 1. compound causes norepinephrine and shrinks rat

The diastole effect of thoracic aortic ring

Blank solvent is made up of Kreb ' s-Henseleit damping fluid, the DMSO containing 1/1000 volume).Diastole value is mean value ± deviation n=3-4.

As a result 2: multiple doses adds up experimental result: reaction test-compound is diastole effect power during unit time (5 minutes)

Test-compound 5 concentration (1 × 10 ^-8m, 3 × 10 ^-8m, 1 × 10 ^-7m, 3 × 10 ^-7m, 10 ^-6m) add successively, each concentration incubation time is 5 minutes (previous concentration compound not yet reaches maximum diastole effect, adds next concentration when reaching 5 minutes).Extend in time, group of solvents shrink tension has the reduction of 8-15%; Tested embodiment compound 4,5,6,7 can the remarkable diastole thoracic aorta of concentration dependent, the IC of compound ₅₀be worth (maximum diastolic rate calculates with 100%) as shown in table 2:

Table 2. compound causes norepinephrine and shrinks rat

The diastole effect IC of thoracic aortic ring ₅₀value

Compound often plants concentration process rat chest aorta ring 5min.Blank solvent is made up of Kreb ' s-Henseleit damping fluid, the DMSO containing 1/1000 volume).Numerical value is mean value ± deviation n=2-4.

Embodiment 21:Langendoff Perfused isolated heart is tested

Prepared by Langendorff Isolated Heart perfusion Model:

Rat sacrificed by decapitation, opens rapidly thoracic cavity and exposes heart, insert heart canula, be fixed on the heart canula of perfusion device by aorta cotton thread immediately along aortic arch opening.Kreb ' s-Henseleit nutritive medium ((mM): NaCl 112, NaHCO of 1/10000DMSO is contained with 37 DEG C ₃25, KCl 5, MgSO ₄1.2, KH ₂pO ₄1, CaCl ₂1.2, glucose 11.5, pH 7.4 ± 0.5) perfusion retrogradely heart, Ppa pulmonary artery pressure is 85cm water column.So that the soft sacculus of water filling adjusted volume MedLab U/4CS System of organism signal (Mei Yi bio tech ltd, Nanjing) can be connected, sacculus is inserted left ventricle along left auricle of heart and adjusts Balloon size and make left indoor pressure/end-diastolic pressure minimum value at 4-6mmHg.Around whole filling system and heart, maintain about 37 DEG C with water bath with thermostatic control circulator, perfusion liquid with 95% O ₂with 5% CO ₂fully saturated, heart can recover independently to beat in such a case very soon.Pre-filled balance 30-50 minute, reaches steadily until every cardiac function and starts experiment.

Drug treating: isolated rat heart function indices reaches steadily, and first the record various heart function value of left ventricle and coronary flow are as basic value before administration.Adjustment three passes to 2# passage, give oxygenation in advance, heat after testing compound (with Kreb ' s-Henseleit nutritive medium diluted compounds mother liquor of oxygenation in advance to working concentration, DMSO final concentration 1/10000), record every heart function value, and the coronary flow after administration when the 3rd, 5,8,10,15,20 minute.The percentage of each time point coronary flow change after calculating administration.

Statistical treatment: experimental data is used represent.

Langendorff Perfused isolated heart experimental result: from after administration 1.5 minutes coronary flow start increase, to generally reaching maximum when 5 minutes, tested embodiment compound 4,5,6,7 all can concentration dependent diastole coronary artery, increase coronary flow, its IC ₅₀be worth as shown in table 3:

The IC of table 3. rat Langendorff Perfused isolated heart compound ₅₀value

Blank solvent is made up of Kreb ' s-Henseleit damping fluid, the DMSO containing 1/1000 volume).Numerical value is mean value ± deviation.

Embodiment 22: rat vivo pharmacokinetic is tested

Experimental animal: SD rat, male, body weight 200 ± 20g.20 rats are divided into 10 groups at random, and gavage and intravenous injection give embodiment compound respectively, and dosage is 2 mg/kg.

Test method: rat is all according to dosage intravenous injection or the gastric infusion respectively of 2mg/kg, intravenously administrable group before administration and after administration 2,5,15,30min and 1,2,4,6,8,12,24h, gastric infusion group before administration and after administration 5,15,30min and 1,2,4,6,8,12,24h, to be taken a blood sample 0.25mL by eye socket, the centrifugal 10min of 3000rpm, separated plasma 0.1mL, puts-20 DEG C of refrigerators and deposits pending.Get rat plasma 0.1mL, add 0.3mL methyl alcohol, the centrifugal 10min of vortex oscillation 1min, 14000r/min, transfer supernatant 100 μ L, in sample injection bottle, gets 10 μ L sample introduction LC/MS/MS and analyzes mensuration.Adopt DAS pharmacokinetic program to surveyed data analysis, calculate main pharmacokinetic parameter.

Detecting instrument and analysis condition: U.S. Finnigan company's T SQ Quantum type liquid chromatograph-mass spectrometer (LC/MS/MS), be made up of Finnigan Surveyor LC pump, Surveyor AS automatic sampler, electro-spray ionization ionizer (ESI) and thtee-stage shiplock mass spectrum.Control software design is Xcalibur1.4, and MASS SPECTRAL DATA ANALYSIS adopts Lcquan2.0 data handling system.

Chromatographic column is C18 post (2.1mm × 50mm, 5 μm), Thermo company; Moving phase is acetonitrile-water (0.1% formic acid), gradient elution, 0min 15% acetonitrile, 2.5min 95% acetonitrile, 2.51min 15% acetonitrile, 8.5min 15% acetonitrile; Flow velocity 0.2ml/min; Sample size 10 μ L; Column temperature is room temperature.

LC interface adopts Aeroassisted electro-spray ionization ionizer (ESI), spray voltage 4.8KV; Heated capillary temperature (TEM) 300 DEG C; Sheath gas N ₂, flow velocity 10psi; Assisted gas N ₂, flow velocity 1psi; Collision gas (CID) Ar, pressure 1.5 (mTorr); In-source collision induced dissociation (Source CID) energy is 8V; Positive ion mode detects; Scanning of the mass spectrum mode is Selective reaction monitoring (SRM), measures the characteristic reaction ion pair of compound respectively; Sweep time is 0.5s.

Result:

1, the kinetics of embodiment 4 compound in rat body and bioavailability

According to the dosage of 2mg/kg, prepare 2 kinds respectively to drug solns, press 0.2ml/200g intravenous administration according to the weight of animals, 2ml/200g gastric infusion.Fasting (freely drinking water) administration afterwards in 12 hours, every rat completes a drug-time curve.Rat is respectively after intravenous injection and gastric infusion, main metabolic kinetic parameter in table 4,5.

After Oral Administration in Rats administration, Tmax is 100.45ng/ml at about 0.5h, t1/2 at about 6 h, Cmax.

After table 4 rat vein administration embodiment compound 4 (2mg/kg)

Dominant dynamic parameters

After table 5 rat oral gavage administration embodiment compound 4 (2mg/kg)

Dominant dynamic parameters

2, the kinetics of embodiment 7 compound in rat body and bioavailability

According to the dosage of 2mg/kg, prepare 2 kinds respectively to drug solns, press 0.2ml/200g intravenous administration according to the weight of animals, 2ml/200g gastric infusion.Fasting (freely drinking water) administration afterwards in 12 hours, every rat completes a drug-time curve.After rat distinguishes intravenous injection and gastric infusion, main metabolic kinetic parameter is in Table 6-7.

After Oral Administration in Rats administration, Tmax is 12ng/ml at about 3h, t1/2 at about 10 h, Cmax.

After table 6 rat vein administration embodiment 7 compound (2mg/kg)

Dominant dynamic parameters

After table 7 rat oral gavage administration embodiment 7 compound (2mg/kg)

Dominant dynamic parameters

3, the kinetics of embodiment 5 compound in rat body and bioavailability

According to the dosage of 2mg/kg, prepare 2 kinds respectively to drug solns, press 0.2ml/200g intravenous administration according to the weight of animals, 2ml/200g gastric infusion.Fasting (freely drinking water) administration afterwards in 12 hours, every rat completes a drug-time curve.After rat distinguishes intravenous injection and gastric infusion, main metabolic kinetic parameter is in Table 8-9.

After Oral Administration in Rats administration, Tmax is 123ng/ml at about 0.5h, t1/2 at about 2 h, Cmax.

After table 8 rat vein administration embodiment 7 compound (2mg/kg)

Dominant dynamic parameters

After table 9 rat oral gavage administration embodiment 7 compound (2mg/kg)

Dominant dynamic parameters

4, the kinetics of embodiment 6 compound in rat body and bioavailability

According to the dosage of 2mg/kg, prepare 2 kinds respectively to drug solns, press 0.2ml/200g intravenous administration according to the weight of animals, 2ml/200g gastric infusion.Fasting (freely drinking water) administration afterwards in 12 hours, every rat completes a drug-time curve.After rat distinguishes intravenous injection and gastric infusion, main metabolic kinetic parameter is in Table 10-11.

After Oral Administration in Rats administration, Tmax is 20.8ng/ml at about 0.25h, t1/2 at about 2 h, Cmax.

After table 10 rat vein administration embodiment 6 compound (2mg/kg)

Dominant dynamic parameters

After table 11 rat oral gavage administration embodiment 6 compound (2mg/kg)

Dominant dynamic parameters

Embodiment 23: embodiment compound is to CYP enzyme level and inducibility research

Test-compound: cytotoxicity > 300 μMs, effective concentration are 3 μMs.

Test method:

1, enzyme level test

People's liver microsomes incubation reaction system is the 0.05molL of 200 μ L ^-1k ₂hPO ₄damping fluid (pH=7.4) system, containing people's hepatomicrosome (protein concentration 0.2g/L), the positive inhibitor of series concentration or test medicine, the substrate of each CYP enzyme, parallel 3 parts of every sample, Incubating Solution adds equally in NADPH solution (final concentration 1mmol/L) the startup reaction of 37 DEG C of preincubate 5min after 37 DEG C of water-bath preincubate 5min, 600 μ L are added containing interior target stop buffer termination reaction after hatching 30min, vortex 2min, 4 DEG C, the centrifugal 10min of 19000g, gets supernatant 10 μ L sample introduction detects each substrate metabolite generation to LC-MS.The corresponding meta-bolites of each probe is as follows:

CYP substrate metabolite MS detects ion

____________________________________________________________

1A2 Phenacetin paracetamol m/z 152

2C9 tolbutamide 4-hydroxy-toluene sulphur butyl urea m/z 285

2C19 omeprazole 5-hydroxy-omeprazole m/z 362

2D6 S-right romilar Levorphanol d-form m/z 258

3A4 midazolam 1 '-hydroxyl-midazolam m/z 342

LC-MS testing conditions:

Chromatographic column is Capcell PAK C18 MGII S5 column (2.0 × 100mm ID, 5 μm, Shiseido, Japan); Moving phase is the 5mM ammonium formiate aqueous solution: acetonitrile: formic acid=72: 28: 0.1 (positive ion detection) or the 5mM ammonium formiate aqueous solution: acetonitrile: formic acid=55: 45; 0.1 (anionic textiles), flow velocity is 0.2mlmin ^-1.

ESI source positive ion detection mode: capillary temperature 300 DEG C, collision energy 120, capillary voltage+4000V, is inside designated as hydrochloric acid propranolol; 4-hydroxy-toluene sulphur butyl urea ESI source anionic textiles mode: capillary temperature 300 DEG C, collision energy 120, capillary voltage-3000V, is inside designated as chlorzoxazone.

Data processing:

The relative inhibition Irel of each inhibitor is calculated by following formula:

Irel(％)＝1-Ci(n)/Ci(0)×100％

In formula Ci (n) for Incubating Solution add different concns positive inhibitor after the substrate metabolite relative quantity that obtains; Ci (0) is the substrate metabolite relative quantity that the blank group not adding positive inhibitor obtains.The logarithmic value GraphPad Prism 5 software mapping of the relative inhibition Irel tried to achieve through above formula to the Log10 of respective concentration is calculated IC ₅₀value.

2, enzyme induction test

Positive controls:

Primary rat hepatocyte, adopts sandwich glue culture method to be inoculated in 12 porocyte culture plates.Take sodium phenobarbital as the positive inductor of CYP3A2, be diluted to 2mmolL respectively with liver cell culture liquid ^-1with 50 μm of olL ^-1administration, in 37 DEG C, 5%CO ₂, hatch 72h under saturated humidity condition, every 24h changes liquid once (1ml hole ^-1), often group establishes 2 multiple holes.Blank group adds the cell culture fluid containing 0.2% methyl alcohol.After 72h, every hole adds the Phenacetin (PH that 1ml contains probe; 50 μm of olL ^-1) and midazolam (MDZ; 10 μm of olL ^-1) cell culture fluid continue to hatch 1h.Hatch terminal absorption Incubating Solution 200 μ l and add 600 μ L reaction terminating liquid termination reactions, the centrifugal 10min of vortex 2min, 19000g, 10 μ L sample introduction LC-MS instrument detect the relative growing amount of meta-bolites.

Experimental group:

By each tested material (0.3,3,30 μm of olL of 3 concentration levels ^-1) replace positive inductor, each concentration group establishes 2 multiple hole administrations to carry out cell incubation, and all the other are with positive inductor group.

Data processing:

Evaluate positive inductor with fold induction or be subject to reagent to the inducing action of CYP3A.

With the growing amount of LC-MS method detection probes meta-bolites, the growing amount of hatching the product after 60min with probe substrate calculates fold induction according to the following formula:

The growing amount of the growing amount/negative control group product of positive fold induction=inductor group product

By the per-cent=by reagent group product formation/positive group growing amount × 100% of reagent corresponding positive group

Result:

1, enzyme level merit rating

The experimental model positive inhibitor of CYP enzyme level is verified, the positive inhibitor of application different concns is hatched with probe substrate in people's hepatomicrosome simultaneously, the IC of each positive inhibitor obtained ₅₀value, all within the scope of bibliographical information, shows that experiment incubation system used may be used for the evaluation of tested material to CYP enzyme level ability, the IC of each tested material obtained ₅₀value is in table 12.

This tests all test-compounds to the equal unrestraint effect of 2C19 and 2D6.

Table 12. embodiment compound is to the rejection ability evaluation of CYP isozyme

"-": fail to obtain IC in tested concentration range ₅₀value, compound is to enzyme unrestraint effect.

2, the evaluation of CYP3A enzyme induction ability

CYP3A is the main CYP isozyme participating in drug metabolism, take sodium phenobarbital as the positive inductor of CYP3A, Rat Primary Hepatocytes is evaluated the inducibility of series compound to CYP3A2.

The positive group of phenylethyl barbituric acid is all more than 2 times of solvent control group or close to 2 times to the induced activity of enzyme, shows that cell incubation system used may be used for the evaluation of tested material to CYP enzyme induction ability.The induction experiment of tested material to CYP3A2 the results are shown in Table 13.

Table 13. embodiment compound is to the inducibility evaluation of rat CYP3A2

*: cell state is poor, result is not counted.

Each tested material to CYP3A enzyme all without remarkable inducing action; Because CYP3A and 2C is regulated and controled by same nuclear receptor, above-claimed cpd to the main CYP enzyme of C also without obvious inducing action.

Claims (9)

1. formula I, and pharmacologically acceptable salts:

Wherein:

R ₁for-NR ₂c (=O) OR ₃,

R ₂for hydrogen or C ₁-C ₄alkyl,

R ₃for C ₁-C ₆alkyl.

2. formula I as claimed in claim 1, it has following formula I a, Ib, Ic,

Wherein:

R ₁for-NR ₂c (=O) OR ₃,

R ₂for hydrogen or C ₁-C ₄alkyl,

R ₃for C ₁-C ₆alkyl.

3. formula I as claimed in claim 2 and pharmacologically acceptable salts thereof,

Wherein:

R ₁for-NR ₂c (=O) OR ₃,

R ₂for hydrogen, methyl or ethyl,

R ₃for methyl, ethyl or sec.-propyl.

4. formula I as claimed in claim 2 and pharmacologically acceptable salts thereof, wherein said compound has structure below:

5. prepare the method for the formula I described in any one of claim 1-4, it comprises:

1) chloromethyl fluoro thiophene (II) and 3-cyano group-1H-pyrazolo [3,4-b] pyridine (III) are obtained by reacting intermediate 3-cyano group-1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridine (IV),

2) 2-(1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridin-3-yl)-4,5,6-pyrimidine triamine tri hydrochloride (V) and chloro-formic ester (VI) are obtained by reacting intermediate 4,6-diamino-2-(1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridin-3-yl)-5-pyrimidinyl-amino manthanoate (Ii)

Wherein: R ₃definition as claimed in claim 1,

Formula V compound is prepared by following reaction scheme:

3) 4,6-diamino-2-(1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridin-3-yl)-5-pyrimidinyl-amino manthanoate (Ii) and R ₂-X (VII) is obtained by reacting 4,6-diamino-2-(1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridin-3-yl)-5-pyrimidyl (alkyl) carbamate (Iii),

Wherein: R ₂, R ₃definition as claimed in claim 1, X is leavings group.

6. a pharmaceutical composition, it comprises the formula I described in any one of Claims 1-4 or its pharmacologically acceptable salts that treat and/or prevent significant quantity, and one or more optional pharmaceutically acceptable carriers or vehicle.

7. as defined in claim 1 formula I or its pharmacologically acceptable salts for the preparation of the application of the medicine aspect of Cardiovarscular.

8. as defined in claim 1 formula I or its pharmacologically acceptable salts for the preparation of the application of the hypertensive medicine aspect for the treatment of.

9. as defined in claim 1 formula I or its pharmacologically acceptable salts for the preparation of the application for the treatment of thrombotic disease and ischemic medicine aspect.