CN102485724A - Substituted thiazolyl pyrazolo pyridine compound and medical purpose thereof - Google Patents

Abstract

The invention relates to a novel substituted thiazolyl pyrazolo pyridine compound and medical purpose thereof. Specifically, the invention relates to a compound shown as a formula I, and isomer, pharmaceutically accepted salt and solvate thereof, wherein definitions of substituents are specified in the specification. The invention also relates to a pharmaceutical composition including the compound and purpose of the compound in preparation of medicaments for treating and / or preventing disease or illness related to cardiovascular. The compound shown as the formula I of the invention can beneficially treat and / or prevent disease or illness related to cardiovascular.

Description

Substituted thiazolyl pyrazolo-pyridines and medicinal use thereof

Technical field

The present invention relates to 2-substituted thiazolyl pyrazolo-pyridines and, the invention still further relates to the purposes that the method for preparation I compound, the medicinal compsns that contains above-claimed cpd and said compound are used to treat cardiovascular disorder at pharmacy acceptable salt.

Background technology

Soluble guanylate cyclase (Soluble Guanylate Cyclasey; SGC) be the signal transduction enzyme of the key in the NO-sGC-cGMP signal transduction pathway; It can be activated by nitrogen protoxide (NO); Thereby catalysis guanosine triphosphate (GTP) is converted into cyclic guanosine list phosphoric acid (cGMP), and cGMP comprises protein kinase, phosphodiesterase (PDE) and some ionic channel etc. as the correlation effect device that the second messenger can regulate the transduction passage downstream; Thereby regulate corresponding physiological process, produce, regulate hematoblastic aggegation and nerve conduction etc. like vasodilator, promotion VSMC.

Soluble guanylate cyclase is by a bigger α subunit and the heterodimer that the less β subunit that is combined with protoheme is formed; Human sGC has α 1; α 2, β 1 and 2 four kinds of subunits of β, and α 1/ β 1 is comparatively typical with α 2/ β 1 these two kinds of heterodimers.Combining the protoheme position with the β subunit is the part of active regulator site, very important for activation mechanism.NO can combine with the iron atom of protoheme, thereby can significantly increase the activity of enzyme, and still, NO can not stimulate the enzyme that does not contain protoheme.CO also can combine with the iron atom of protoheme, but is lower than the stimulation of NO by the stimulation that CO carries out.

SGC exists oxidized form and two kinds of kenels of reduced form, and reduced form is its active state.The excessive N O that produces under the pathological conditions combines to form peroxynitrite with the mistake negative oxygen ion, makes enzyme and other albumen inactivations through oxidation and nitrification, causes cell injury.Because the oxidized form that the bioavailability of NO reduces, sGC is changed into the inactivation attitude by the reduced form of activated state; SGC discharges the susceptibility reduction of medicine to endogenic NO and NO; Cause the NO-sGC-cGMP signal transduction pathway to be obstructed; It can cause increasing like hypertension, platelet activation, hyperplasia; Endothelial dysfunction, atherosclerosis, stenocardia, heart failure, thrombus, apoplexy, sexual dysfunction and myocardial infarction, wherein comparatively serious with pulmonary hypertension, increase causes that hypertrophy of right heart finally causes right heart failure even death thereby it makes pulmonary vascular pressure.

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

The method of repairing this passage mainly is to improve the level of cGMP at present.Can improve the level of cGMP through the approach that activates sGC, reach the purpose of treatment disease.Can the treatment approach be divided into two types according to whether depending on protoheme: the sGC stimulant (sGC stimulator) that 1, depends on protoheme; 2, do not rely on the sGC activator (sGC activator) of protoheme, according to these two kinds of classification, the potential treat-ment has: the NO synthetic enzyme activates agent, imbedibility NO and NO donor medicine, phosphodiesterase inhibitor etc.Up to the present, with NO the compound on basis, can not be used for the stimulation therapy of soluble guanylate cyclase like organic nitrate salt always, except spinoff, forming tolerance is one of main drawback of this therapeutic modality.

The treatment approach that a kind of NO of not relying on directly activates sGC is a kind of method the most likely, generally believes that the efficient of this method is high and almost is free from side effects.

Given this, the activator that NO directly activates sGC that do not rely on of brand new need be developed in the present technique field.Novel 2-substituted thiazolyl pyrazolo-pyridines is exactly the sGC activator to this target spot design, and it can significantly strengthen the susceptibility of sGC to NO; Under even the condition that lacks extremely low in the NO level; Directly activate sGC; Thereby have treatment hypertension, platelet activation, hyperplasia increase; Endothelial dysfunction, atherosclerosis, stenocardia, heart failure, thrombus, apoplexy, sexual dysfunction and myocardial infarction, particularly pulmonary hypertension, thus and cause pulmonary vascular pressure to increase by pulmonary hypertension to cause right heart failure even the dead effect that hypertrophy of right heart causes.

Summary of the invention

The objective of the invention is to seek the activator that NO directly activates sGC that do not rely on of brand new, through strengthening the susceptibility of sGC to NO; Under even the condition that lacks extremely low in the NO level; Directly activate sGC; Reach purpose, increase endothelial dysfunction, atherosclerosis, stenocardia, heart failure, thrombus, apoplexy, sexual dysfunction and myocardial infarction including, but not limited to hypertension, platelet activation, hyperplasia with treatment cardiovascular disorder; Pulmonary hypertension particularly, thus and cause pulmonary vascular pressure to increase by pulmonary hypertension to cause right heart failure even the dead effect that hypertrophy of right heart causes.The inventor is through discovering that 2-substituted thiazolyl Pyrazolopyridine class formula I compound 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 compound, and isomer pharmacologically acceptable salts, solvate:

Wherein:

R ₁For-NR ₂C (=O) OR ₃,

R ₂Be hydrogen or C ₁-C ₄Alkyl,

R ₃Be C ₁-C ₆Alkyl.

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

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

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

The acceptable salt of the preferred physiology of the present invention.The acceptable salt of the physiology of The compounds of this invention can be the salt of material of the present invention and mineral acid, carboxylic acid or sulfonic acid, for example particularly preferably is with hydrochloric acid, Hydrogen bromide, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetate, propionic acid, succsinic acid, oxyacetic acid, formic acid, lactic acid, toxilic acid, tartrate, Hydrocerol A, pounces on the salt that 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), tannic acid form.Other acid like oxalic acid, though itself be not pharmaceutically acceptable, can be used to prepare the salt as midbody, to obtain The compounds of this invention and pharmacy acceptable salt thereof.

Acceptable salt can be metal or the ammonium salt with The compounds of this invention of free carboxy equally on the physiology.Particularly preferably be like sodium, potassium, magnesium or calcium salt and derive from ammonia or organic amine such as ethamine, diethylamine, triethylamine, N, the ammonium salt of N '-dibenzyl-ethylenediamin, chloro PROCAINE HCL, PHARMA GRADE, choline, N-NMG and PROCAINE HCL, PHARMA GRADE, diethylolamine, trolamine, dicyclohexylamine, dimethylaminoethanol, l-arginine, Methionin or quadrol.

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

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

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

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

The invention still further relates to the compound method of preparation formula I compound of the present invention, comprising:

1) chloromethyl fluoro thiophene (II) obtains midbody 3-cyanic acid-1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridines (IV) with 3-cyanic acid-1H-pyrazolo [3,4-b] pyridines (III) reaction,

2) 2-(1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridin-3-yl)-4,5; 6-pyrimidine triamine tri hydrochloride (V) obtains midbody 4 with the chloro-formic ester reaction; 6-diamino--2-(1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridin-3-yl)-5-pyrimidinyl-amino manthanoate

Wherein: R ₃Definition according to claim 1, optional is in organic solvent, to react.

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 reaction obtains 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 according to claim 1, X is a leavings group, like halogen, preferably iodine, or methanesulfonates, optional carries out under the refrigerative situation in organic solvent.

The compound method of the needed formula IIa compound of the preparation defined formula Ia compound of claim 2 is shown in the following figure:

The compound method of the needed formula IIb compound of the preparation defined formula Ib compound of claim 2 is shown in the following figure:

The compound method of the needed formula IIc compound of the preparation defined formula Ic compound of claim 2 is shown in the following figure:

The formula III compound can prepare through following reaction scheme:

Formula V compound can prepare through following reaction scheme:

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

The organic nitrate and the NO donor that are used for the object of the invention generally are to show the material of its therapeutic action through discharging NO or NO material.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 compounds that can suppress the decomposition of guanosine monophosphate (cGMP) with one or more.It is phosphodiesterase 1,2 and 5 preferably; Beavo and Reifsnyder (1990) TiPS11, the suppressor factor of the material of the 150th to 155 page of name.Particularly preferably be the suppressor factor of phosphodiesterase 5 in this respect, especially a kind of in compound 'Xiduofeng ' (WO 94/28902), Vardenafil (WO 99/24433) or the Tadalafei (WO 95/19978).These suppressor factor have been strengthened the effect of The compounds of this invention, and have increased required pharmacotoxicological effect.

The invention still further relates to a kind of medicine that comprises at least a The compounds of this invention, it preferably also comprises acceptable vehicle of one or more pharmacology or carrier together, and relates to the application that it is used for above-mentioned purpose.The pharmaceutical carrier here includes but not limited to: ionite, aluminum oxide, StAl, Yelkin TTS, serum proteins such as rHSA, buffer substance such as phosphoric acid salt; Glycerine, Sorbic Acid, POTASSIUM SORBATE GRANULAR WHITE, the partial glycerol ester mixture of saturated vegetable fatty acid, water; Salt or ionogen, like protamine sulfate, Sodium phosphate, dibasic, potassium hydrogen phosphate, sodium-chlor; Zinc salt, colloided silica, Magnesium Trisilicate, Vinylpyrrolidone polymer, cellulosic material; Polyoxyethylene glycol, Xylo-Mucine, polyacrylic ester, beeswax, yolk.

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

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

Being suitable for having of oral administration can be rapidly and/or transmit the known form of medication of activeconstituents with the mode that changes; Like tablet (uncoated tablets or coating tablet, as have the enteric coating or the tablet of dressing not), capsule, coated tablet, particle, piller, pulvis, emulsion, suspension and aerosol.

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

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

Can this activeconstituents be changed into described form of medication with known method itself.It can be realized with the suitable pharmaceutical excipient of inert non-toxic.It particularly comprises carrier (for example Microcrystalline Cellulose), solvent (for example liquid macrogol), emulsifying agent (for example sodium lauryl sulphate), dispersion agent (for example Vinylpyrrolidone polymer), synthetic and natural biological copolymer (for example protein), stablizer (for example oxidation inhibitor and xitix), tinting material (for example mineral dye such as red stone) or correctives and/or odor mask.In suitable situation, said activeconstituents can be present in the form of microencapsulation in one or more above-mentioned carriers.

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

Abbreviation

The CAN ceric ammonium nitrate

Con is spissated

The DCM methylene dichloride

DMF N, dinethylformamide

The DMSO methyl-sulphoxide

EA ETHYLE ACETATE

Et ₂O-HCl salt acid ether

The placed in-line gas chromatography/mass spectrometry of GC-MS

H hour

HAc acetate

Two (three silyls) the amination lithiums of LiHMDS

MeOH methyl alcohol

Min minute

The Mp fusing point

The MS mass spectrum

The NMR NMR spectrum

The PE sherwood oil

The Py pyridine

Rf retention index (in the thin-layer chromatography)

The RT room temperature

S second

The TFA trifluoroacetic acid

The TFAA trifluoroacetic anhydride

The THF THF

The TLC thin-layer chromatography

See embodiment about the more detailed data of preparation compound of Formula I.

Detailed Description Of The Invention

Do further to describe with characteristics to various aspects of the present invention below.

Various terms and phrase that the present invention uses have the general sense of well known to a person skilled in the art; Nonetheless; The present invention still hopes at this more detailed explanation and explanation to be done in these terms and phrase; Term of mentioning and phrase are as the criterion with the implication that the present invention was explained if any inconsistent with known implication.Be the definition of the used multiple term of the present invention below, these definition are applicable to used term in the whole specification sheets of the application, only if in particular case, explain in addition.

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

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

As described herein; Term " experimenter " can refer to that patient or other accept formula I compound of the present invention or its pharmaceutical composition to treat and/or prevent the animal of disease according to the invention or illness; Particularly Mammals, for example people, dog, monkey, ox, horse etc.

As described herein, term " disease and/or illness " is meant a kind of physical state of said experimenter, this physical state is relevant with disease according to the invention and/or illness.For example, disease according to the invention and/or illness both can refer to a kind of physical state, for example were the physical state than hyperglycemia, also can refer to a kind of morbid state, for example showed as morbid states such as hyperglycemia, mellitus.Do not distinguish for physical state and morbid state in this article, perhaps the two can refer to each other, and for example " hyperglycemia " can be exchanged use with " hyperglycemia ".

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

As described herein; " pharmaceutically useful " of term " pharmacy is acceptable " or use interchangeable with it, for example when describing " pharmacologically acceptable salts ", it still can not accepted on experimenter's physiology to represent this salt; But also can refer at the synthetic that use value is pharmaceutically arranged; For example being formed salt as midbody when carrying out chiral separation, though the salt of this midbody can not directly give the experimenter, this salt can work in the end product of the present invention for obtaining.

In one embodiment, the present invention relates to the purposes that compound, its all possible isomer and pharmacologically acceptable salts, solvate or the hydrate of general formula I are used to produce medicine, said medicine is used to treat cardiovascular disorder.

In one embodiment, the compound, its all possible isomer and pharmacologically acceptable salts, solvate or the hydrate that the present invention relates to general formula I are used to produce the purposes that can treat cardiovascular disease medicine.

In one embodiment; The compound of general formula I of the present invention or its pharmaceutically useful salt can use separately; Or use with the form of pharmaceutical composition with pharmaceutically useful carrier or vehicle; When using with the form of pharmaceutical composition; Usually the compound of Formula I of the present invention of effective dose or its pharmacologically acceptable salt or hydrate and one or more pharmaceutically acceptable carrier or thinner are combined to process suitable administration form or dosage form, this program comprises through suitable manner component mixing, granulation, compression or dissolving.Therefore, the invention provides pharmaceutical composition, it comprises compound, its all possible isomer and pharmacologically acceptable salts, solvate or hydrate and at least a pharmaceutically useful carrier of general formula I.

The pharmaceutical composition of The compounds of this invention; Any-mode that can following aspect is granted: in oral, spraying suction, rectal administration, intranasal administration, vagina administration, topical, parenterai administration such as subcutaneous, vein, intramuscular, intraperitoneal, the sheath, in the ventricle, in the breastbone or intracranial injection or input; Or by a kind of reservoir medication of outer planting, wherein preferred oral, intramuscular injection, intraperitoneal or intravenously application method.

The compounds of this invention or contain its pharmaceutical composition can the unit dosage form administration.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. for example.

Can also contain carrier commonly used in the pharmaceutical composition of the present invention, pharmaceutically acceptable carrier described here is including, but not limited to ionite, aluminum oxide, StAl, Yelkin TTS, serum proteins such as human serum protein; Buffer substance such as phosphoric acid salt, glycerine, Sorbic Acid, POTASSIUM SORBATE GRANULAR WHITE, the partial glycerol ester mixture of saturated vegetable fatty acid, water; Salt or ionogen, like protamine sulfate, Sodium phosphate, dibasic, potassium hydrogen phosphate, sodium-chlor; Zinc salt, colloided silica, Magnesium Trisilicate, Vinylpyrrolidone polymer, cellulosic material; Polyoxyethylene glycol, Xylo-Mucine, polyacrylic ester, beeswax, wool grease etc.The content of carrier in pharmaceutical composition can be 1 weight %-98 weight %, accounts for 80 weight % usually greatly.For simplicity, local anesthetic, sanitas, buffer reagents etc. can directly be dissolved in the carrier.

Oral tablet and capsule can contain vehicle such as tackiness agent, like syrup, and gum arabic, sorbyl alcohol, tragacanth, or Vinylpyrrolidone polymer; Weighting agent, like lactose, sucrose, W-Gum, calcium phosphate, sorbyl alcohol; Padil, lubricant, like Magnesium Stearate, talcum, polyoxyethylene glycol; Tripoli, disintegrating agent, like yam starch, or acceptable dibutyl phthalate, like bay sodium alkoxide vitriol.Tablet can be with known method dressing on the pharmacopedics.

Oral liquid can be processed the suspension-s of water and oil, solution, and emulsion, syrup or elixir also can be processed dry product, with preceding make up water or other suitable medium.This liquid preparation can comprise conventional additive, like suspension agent, and sorbyl alcohol, Walsroder MC 20000S, dextrose syrup; Gel, Natvosol, CMC 99.5, aluminium stearate gel, hydrogenant food oils; Emulsifying agent, like Yelkin TTS, sorb gathers candy list oleate, Sudan Gum-arabic; Or nonaqueous carrier (possibly comprise edible oil), like Prunus amygdalus oil, grease such as glycerine, terepthaloyl moietie, or ethanol; Sanitas is like methyl paraben or propyl ester, Sorbic Acid.Can add seasonings or tinting material like needs.

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

For the parenteral dispensing, liquid formulation is processed by compound and a kind of disinfectant carrier usually.The first-selected water of carrier.According to the different of selected carrier and drug level, compound had both dissolved in and also can be made into aaerosol solution in the carrier, and was earlier that compound is soluble in water when processing injection solution, packed into after the filter-sterilized and sealed in bottle or the ampoule.

Must recognize; The best dosage of compound of Formula I and be at interval by compound property with such as form, path and the position of administration and external conditionss such as the specific Mammals decision of being treated, and this best dosage can use conventional technology to confirm.Must recognize also simultaneously that the best course of treatment, promptly compound of Formula I is at the nominal dosage of every day in the time, available method well known in the art is confirmed.

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

Can change the actual dose level of each activeconstituents in the pharmaceutical composition of the present invention, obtain required therapeutic response so that the active compound amount of gained can effectively be directed against concrete patient, compsn and administering mode.The dosage level fibrous root is according to the severity of the activity of particular compound, route of administration, the patient's condition of treating and wait that the patient's condition and the medical history of treating the patient select.But the way of this area is, the dosage of compound begins from being lower than for obtaining the level that required result of treatment requires, and increases dosage gradually, up to obtaining required effect.

Generally speaking, formula I compound of the present invention is used for Mammals particularly people's dosage can be between the 0.001-1000mg/kg body weight/day, for example between the 0.01-100mg/kg body weight/day, for example between the 0.01-10mg/kg body weight/day.

For example,, can contain weight ratio 0.1% in the component according to the difference of administering mode, or the active ingredient of weight ratio 10-60% more suitably.But when comprising unitary dose in the component, each unit preferably comprises 5-500 milligram activeconstituents.For example, in one embodiment, different according to route of administration and administration frequency, the suitable therapeutic dose that is used to be grown up is 100-3000 milligram every day, like 1500 milligrams of every days.This dosage is corresponding to 1.5-50 milligram/kg/day, and proper dosage is 5-20 milligram/kg/day.

The inventor is surprised to find, and formula I compound provided by the invention is the activator that effectively activates sGC, can be through strengthening the susceptibility of sGC to NO; Under even the condition that lacks extremely low, directly activate sGC, have the effect of treatment cardiovascular disorder in the NO level.

Embodiment

Can further describe the present invention through following embodiment, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite that does not deviate from the spirit and scope of the present invention, can carry out various variations and modification to the present invention.

The present invention carries out generality and/or concrete description to the material and the TP that are used in the test.Though for realizing that employed many materials of the object of the invention and working method are well known in the art, the present invention still does to describe in detail as far as possible at this.

The fusing point of compound is measured by RY-1 fusing point appearance, and TM is without calibration.Mass spectrum is measured by Micromass ZabSpec HRMS (resolving power 1000). ¹H-NMR is measured by JNM-ECA-400 SUPERCONDUCTING NMR appearance, operating frequency ¹H-NMR 400MHz.

The universal synthesis method of initial compounds:

Embodiment 1A: to the methoxybenzyl hydrazine

Get 242g (4.11mol) 85% Hydrazine Hydrate 80 and be dissolved in the 350ml absolute ethyl alcohol, nitrogen protection is heated to backflow; Slowly drip the ethanol solution of 68g (0.434mol) to methoxyl group benzyl chloride and 200ml, 1h drips off, and continues backflow 6h; Stirring at room 18h removes solvent under reduced pressure.In resistates, add the 100ml methylene dichloride, 0 ℃ drips 100ml 5%NaOH solution, layering down; (4 * 80ml) extractions merge organic layer to water layer, and Anhydrous potassium carbonate is dry with methylene dichloride; After removing solvent under reduced pressure, it is for use to get light yellow oil 70g (GC-MS purity is 79%).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 is got the 350ml absolute ethyl alcohol in the 1000ml there-necked flask, places ice bath, gets 30.7g (1.334mol) sodium Metal 99.5; Carefully shred and add in the reaction solution, finish, reaction flask is placed oil bath, be heated to backflow; After undissolved sodium is dissolved entirely, reaction flask is placed ice bath again, temperature is reduced to about 0 ℃, drips 100.7g (0.689mol) oxalic acid diethyl ester and 35ml anhydrous ether solution; Drip and finish, continue to stir 20 minutes, drip 28.5g (0.694mol) acetonitrile and 35ml anhydrous ether solution then; In about 35 ℃, stir 24h, stopped reaction, reaction solution is centrifugal; The throw out baking is to half-dried, and the vacuum drier kept dry gets 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 the 4-dioxane, stir 20min, add 53g (0.465) TFA; Stir 20min, the dissolving of part solid adds in the reaction solution nitrogen protection with embodiment 1A to the methoxybenzyl hydrazine; Reflux 24h, suction filtration is removed solid, adds 300ml water, ETHYLE ACETATE (100ml * 4) extraction; Saturated aqueous common salt 50ml washing, anhydrous magnesium sulfate drying, suction filtration is removed siccative; Remove solvent under reduced pressure, PE: EA=2: 1 column chromatography for separation gets 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

The 1-that gets 55.1g (0.2mol) embodiment 3A is dissolved in 800ml 1 to methoxybenzyl-1H-pyrazolo [3,4-b] Nicotinicum Acidum ethyl ester, in the 4-dioxane; Add 19.8g dimethylamino acrolein and 27.4g TFA successively, reflux is 3 days under the nitrogen protection, removes solvent under reduced pressure; In resistates, add 300ml water, EA (150ml * 3) extraction, saturated aqueous common salt 50ml washing; Anhydrous magnesium sulfate drying, (PE: EA=8: 1) wash-out gets light yellow solid 21.8g (yield 35%) to column chromatography. 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 the ice bath, logical ammonia is to saturated in the 450ml anhydrous methanol, and the 1-that adds embodiment 4A is to methoxybenzyl-1H-pyrazolo [3; 4-b] Nicotinicum Acidum ethyl ester 21.8g (0.07mol), sealed reactor was in stirring at room 2 days; Revolve dried solvent, get 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-cyanic acid-1-is to methoxybenzyl-1H-pyrazolo [3,4-b] pyridine

The 1-that gets 33.8g (0.12mol) embodiment 5A is dissolved among the anhydrous THF of 300ml methoxybenzyl-1H-pyrazolo [3,4-b] pyridine-3-carboxamide, adds the 24.3g pyridine, places ice bath to stir; Slowly drip the 64.7g trifluoroacetic anhydride to reaction solution, drip and finish, spend the night in the stirring at room reaction; Add 600ml water, saturated sodium bicarbonate 250ml is used in EA (300ml * 3) extraction successively; 1M hydrochloric acid 150ml, saturated aqueous common salt 100ml washing, anhydrous magnesium sulfate drying; Suction filtration is removed siccative, revolve desolventize 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-cyanic acid-1H-pyrazolo [3,4-b] pyridine

3-cyanic acid-the 1-that gets 10.6g (0.04mol) embodiment 6A in the 450ml acetonitrile, is stirred to complete dissolving to methoxybenzyl-1H-pyrazolo [3,4-b] pyridine, adds 120ml water; Stir down, drip the 75g CAN and the 120ml aqueous solution, drip and finish; Add 300ml water after continuing to stir 2h, a shared 1500ml ethyl acetate extraction merges for several times; Revolve and desolventize, column chromatography for separation, 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

Get 29ml (0.318mol) aniline and add in the 1L there-necked flask, add an amount of ice cube, drip the 85ml concentrated hydrochloric acid under the mechanical stirring, drip and finish; Add few ice cubes, 0 ℃ of holding temperature, the aqueous solution of dropping 29g (0.42mol) Sodium Nitrite and 50ml, controlled temperature 0-5 ℃; Drip and finish, continue to be stirred to reaction solution in low temperature and make the variable color of starch-kalium iodide test paper, add 51g (0.622mol) sodium-acetate and the 100ml aqueous solution then, stirring moments later adds 33g (0.50mol) propane dinitrile and 25ml ethanolic soln; Produce a large amount of yellow solids, finish, continue stirring reaction 1h, suction filtration; Filter cake is used distilled water wash, and drying gets 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 the 125ml concentrated hydrochloric acid and be mixed with 6M hydrochloric acid 250ml, place ice bath, under the mechanical stirring, add 94g (0.598mol) 2-aminothiophene-3-methyl-formiate in batches; After room temperature continue to stir 30min, place ice bath once more, treat that temperature reduces to subzeroly, slowly drip the aqueous solution of 42g Sodium Nitrite and 100ml, controlled temperature is no more than 5 ℃; The reaction solution color burn drips and finishes, and continues to be stirred to the reaction fluid power and makes the variable color of starch-kalium iodide test paper, adds the 180ml fluoborate solution then; Produce a large amount of white solids, it is complete to deposition to continue to stir 15min, suction filtration; Use cold methyl alcohol and ether washing leaching cake successively, vacuum-drying gets white crystal 142g (yield 93%)

Embodiment 10A:3-fluoro-2-thiophenic acid

2-methoxycarbonyl thiophene-3-NITRODIAZONIUM FLUOROBORATE and the thick silica gel of 80g of getting 30g (0.117mol) embodiment 9A mix, and place the 1000ml round-bottomed flask, connect the Dewar formula condensing surface successively, cold-trap; Oil pump, decompression react in about 180 ℃ down, and question response is no longer violent; Temperature is increased to 200 ℃, and question response is no longer violent, stops decompression and heating; Wash out the product in reactor drum, condensing surface, the cold-trap with an amount of ethanol, merge, for use.

Get 9g NaOH and 200ml water and add in the above-mentioned ethanolic soln, behind the reflux 2h, revolve except that behind most of solvent; Add 150ml water, transfer to acidity, EA (75ml * 3) extraction with 10% hydrochloric acid; Column chromatography (PE: EA=2: 1) separate, get 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)。

Embodiment 11A:3-fluoro-2-thiophen(e)alcohol

The 3-fluoro-2-thiophenic acid of getting 9.0g (0.062mol) embodiment 10A is dissolved among the anhydrous THF of 90ml, slowly drips to the anhydrous THF of 3.9g (0.103mol) Peng Qinghuana and 90ml, and reaction produces great amount of bubbles; Drip and finish, continue to stir 30min, drip 11g (0.0433mol) iodine and 120ml anhydrous THF solution then to no longer producing bubble; In 50 ℃ of reaction 8h, water cancellation reaction, ether (150ml * 4) extraction; (yield 95%) for use revolved except that ether in saturated aqueous common salt 50ml washing.GC-MS(m/z)：M ⁺(132.0)

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

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

Embodiment 13A:2-cyanic acid-5-fluorine thiophene

Get 10.0g (64.9mmol) 2-cyanic acid-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 the 200ml tetramethylene sulfone, behind 180 ℃ of reaction 2h, is cooled to room temperature; Add 400ml water; Ether (100ml * 3) extraction, steaming desolventizes, and is for use.

Embodiment 14A:5-fluoro-2-thiophenic acid

Getting 5.0g (0.125mol) NaOH is dissolved in 60ml water and the 60ml ethanol; Add to then in 2-cyanic acid-5-fluorine thiophene of embodiment 13A, reflux 2h, steaming desolventizes; Add 120ml water; Transfer to acidity with concentrated hydrochloric acid, EA (40ml * 3) extraction, column chromatography for separation gets product 4.5g (two step yields 47.5%).

Embodiment 15A:5-fluoro-2-thiophen(e)alcohol

The 5-fluoro-2-thiophenic acid of getting 4.5g (0.031mol) embodiment 14A is dissolved among the anhydrous THF of 45ml, slowly drips to the anhydrous THF of 1.95g (0.052mol) Peng Qinghuana and 45ml, and reaction produces great amount of bubbles; Drip and finish, continue to stir 30min to no longer producing bubble, Dropwise 5 .5g (0.022mol) iodine and 60ml anhydrous THF solution then; In 50 ℃ of reaction 8h, water cancellation reaction, ether (75ml * 4) extraction; (yield 91%) for use revolved except that ether in saturated aqueous common salt 50ml washing.GC-MS(m/z)：M ⁺(132.0)。

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

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

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

Get 6.0g (0.0416mol) embodiment 7A and embodiment 12A and be dissolved among the 120ml DMF, add 16.3g (0.05mol) cesium carbonate, under the nitrogen protection; In about 40 ℃, react 15h; Add 250ml water, ETHYLE ACETATE (120ml * 3) extraction, the water washing of 50ml saturated common salt; Column chromatography for separation gets 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridine-3-formyl imidic acid methyl esters

3-cyanic acid-1-(3-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridine of getting 7.37g (0.0285mol) embodiment 17A is dissolved in the 300ml anhydrous methanol, adds 6.19g (0.115mol) sodium methylate, and stirring at room 2h, reaction solution directly do next step reaction.

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

Get 6.85g (0.114mol) Glacial acetic acid min. 99.5 and 2.22g (0.0415mol) ammonium chloride adds in the reaction solution of embodiment 18A, reflux 10h revolves and desolventizes; Resistates grinds with proper amount of acetone, and suction filtration, filter cake add in the 300ml water; Add 7.5g yellow soda ash and stir, ETHYLE ACETATE (200ml * 3) extraction, anhydrous magnesium sulfate drying; Revolve except that behind most of solvent, low temperature stirs down, and the dripping hydrochloric acid diethyl ether solution is to no longer producing solid in resistates; Revolve and desolventize, off-white color solid 6.77g (yield 76.1%).

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

1-(3-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridine-3-amitraz hydrochloride of getting 6.77g (0.0217mol) embodiment 19A adds among the 115ml DMF, stirs to add 1.29g (0.0239mol) sodium methylate down; Add 3.72g (0.0219mol) embodiment 8A then, behind 110 ℃ of reaction 12h, be cooled to room temperature under the nitrogen protection; To filtrate concentrate after; Suction filtration, filter cake washs with small amount of ethanol, gets the orange solid; In residual filtrate, add suitable quantity of water again to no longer producing deposition, suction filtration, filter cake use washing with alcohol, gets the red-brown solid, amount to 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-4,5,6-pyrimidine triamine tri hydrochloride

Get 2-[1-(3-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-[(E) phenyl diazenyl]-4 of 5.3g (11.9mmol) embodiment 20A, 6-pyrimidinediamine and 2.4g T-1 type Raney-Ni (weight in wet base) add among the 100ml DMF; In 65 ℃, hydrogenation is 12 hours under the 65bar pressure, removes catalyzer with the zeyssatite suction filtration; Revolve and desolventize the hydrochloric acid of adding 50ml 5N in resistates, stirring; Produce the tawny solid, in 90 ℃ of hot water baths, be incubated 45min, to be cooled to room temperature; Suction filtration gets tawny 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-cyanic acid-1-(5-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridine

Get 3.0g (0.0208mol) embodiment 7A and embodiment 16A and be dissolved among the 60ml DMF, add 8.2g (0.026mol) cesium carbonate, under the nitrogen protection; In about 40 ℃, react 15h; Add 125ml water, ETHYLE ACETATE (60ml * 3) extraction, the water washing of 30ml saturated common salt; Column chromatography for separation gets product 3.5g (yield 65.1%).

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

3-cyanic acid-1-(3-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridine of getting 3.5g (0.0135mol) embodiment 22A is dissolved in the 150ml anhydrous methanol, adds 3.1g (0.0576mol) sodium methylate, and stirring at room 2h, reaction solution directly do next step reaction.

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

Get 3.43g (0.0571mol) Glacial acetic acid min. 99.5 and 1.11g (0.0208mol) ammonium chloride adds in the reaction solution of embodiment 23A, reflux 10h revolves and desolventizes; Resistates grinds with proper amount of acetone, and suction filtration, filter cake add in the 150ml water; Add 3.8g yellow soda ash and stir, ETHYLE ACETATE (100ml * 3) extraction, anhydrous magnesium sulfate drying; Revolve except that behind most of solvent, low temperature stirs down, and the dripping hydrochloric acid diethyl ether solution is to no longer producing solid in resistates; Revolve and desolventize, off-white color solid 3.33g (yield 78.8%).

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

1-(5-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridine-3-amitraz hydrochloride of getting 3.33g (10.7mmol) embodiment 24A adds among the 55ml DMF, stirs to add 0.594g (11.0mmol) sodium methylate down; Add 1.85g (10.9mmol) embodiment 8A then, behind 110 ℃ of reaction 12h, be cooled to room temperature under the nitrogen protection; To filtrate concentrate after; Suction filtration, filter cake washs with small amount of ethanol, gets the orange solid; In residual filtrate, add suitable quantity of water again to no longer producing deposition, suction filtration, filter cake use washing with alcohol, gets the red-brown solid, amount to product 4.15g (yield 87.2%).

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

Get 2-[1-(5-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-[(E) phenyl diazenyl]-4 of 2.65g (5.95mmol) embodiment 25A, 6-pyrimidinediamine and 1.2g T-1 type Raney-Ni (weight in wet base) add among the 50ml DMF; In 65 ℃, hydrogenation is 12 hours under the 65bar pressure, removes catalyzer with the zeyssatite suction filtration; Revolve and desolventize the hydrochloric acid of adding 25ml 5N in resistates, stirring; Produce the tawny solid, in 90 ℃ of hot water baths, be incubated 45min, to be cooled to room temperature; Suction filtration gets tawny solid 2.1g (yield 75.8%).

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

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

Get the 2-[1-(3-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-4,5 of 0.35g (0.75mmol) embodiment 21A; 6-pyrimidine triamine tri hydrochloride is dissolved in the 15ml pyridine, and stirring at room moments later places ice bath to stir 15min, draws 0.12g (1.27mmol) methyl-chloroformate with syringe and adds in the reaction solution; Continue at and stir 2h in the ice bath, stirred overnight at room temperature is revolved dried solvent then; Adding 15ml water stirs; Generate pale brown look deposition, suction filtration, column chromatography for separation gets 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate

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

Method is with embodiment 1; 2-[1-(3-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3 with 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-yl) 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-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino methyl-formiate is in the anhydrous THF of 2ml; Place under the ice bath and to stir the THF solution that 30min gets 0.4ml LiHMDS (1.06M) and add to reaction solution, after continuing at ice bath and stirring 20min, get the 0.023ml methyl iodide and add in the reaction solution; Ice bath stirs 4h in the room temperature continuation after stirring 1h, and the aqueous ammonium chloride solution 2ml cancellation reaction with 5% is complete to the product extraction for several times with ETHYLE ACETATE and dichloromethane extraction; Merge; Revolve and desolventize, column chromatography for separation, product 100mg (yield 64.5%).

¹H-NMR (d-DMSO, 400MHz): δ 3.004 (s, 3H), 3.533 (main) and 3.657 (accessory) (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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) urethanum

Method is with embodiment 4; With 4 of embodiment 2; 6-diamino--2-[1-(3-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3; 4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate and iodomethane reaction must get light yellow solid 4,6-diamino--2-[1-(3-fluorine thiophene-2-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) carbamic acid isopropyl ester

Method is with embodiment 4; With 4 of embodiment 3; 6-diamino--2-[1-(3-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3; 4-b] pyridin-3-yl]-5-pyrimidinyl-amino isopropyl formate and iodomethane reaction get light yellow solid 4,6-diamino--2-[1-(3-fluorine thiophene-2-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) Urethylane

Method is with embodiment 4; With 4 of embodiment 1; 6-diamino--2-[1-(3-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3; 4-b] pyridin-3-yl]-5-pyrimidinyl-amino methyl-formiate and iodoethane react light yellow solid 4,6-diamino--2-[1-(3-fluorine thiophene-2-yl) 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 (accessory) (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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) urethanum

Method is with embodiment 4; With 4 of embodiment 2; 6-diamino--2-[1-(3-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3; 4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate and iodoethane react light yellow solid 4,6-diamino--2-[1-(3-fluorine thiophene-2-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) carbamic acid isopropyl ester

Method is with embodiment 4; With 4 of embodiment 3; 6-diamino--2-[1-(3-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3; 4-b] pyridin-3-yl]-5-pyrimidinyl-amino isopropyl formate and iodoethane react light yellow solid 4,6-diamino--2-[1-(3-fluorine thiophene-2-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino methyl-formiate

Method is with embodiment 1; 2-[1-(5-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3 with embodiment 26A; 4-b] pyridin-3-yl]-4,5,6-pyrimidine triamine tri hydrochloride and methyl-chloroformate react light yellow solid 4; 6-diamino--2-[1-(5-fluorine thiophene-2-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate

Method is with embodiment 1; 2-[1-(5-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3 with embodiment 26A; 4-b] pyridin-3-yl]-4,5,6-pyrimidine triamine tri hydrochloride and Vinyl chloroformate react light yellow solid 4; 6-diamino--2-[1-(5-fluorine thiophene-2-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidinyl-amino isopropyl formate

Method is with embodiment 1; 2-[1-(5-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3 with embodiment 26A; 4-b] pyridin-3-yl]-4,5,6-pyrimidine triamine tri hydrochloride and isopropyl chlorocarbonate react light yellow solid 4; 6-diamino--2-[1-(5-fluorine thiophene-2-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) Urethylane

Method is with embodiment 4; With 4 of embodiment 10; 6-diamino--2-[1-(5-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3; 4-b] pyridin-3-yl]-5-pyrimidinyl-amino methyl-formiate and iodomethane reaction get light yellow solid 4,6-diamino--2-[1-(5-fluorine thiophene-2-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) urethanum

Method is with embodiment 4; With 4 of embodiment 11; 6-diamino--2-[1-(5-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3; 4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate and iodomethane reaction get light yellow solid 4,6-diamino--2-[1-(5-fluorine thiophene-2-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (methyl) carbamic acid isopropyl ester

Method is with embodiment 4; With 4 of embodiment 12; 6-diamino--2-[1-(5-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3; 4-b] pyridin-3-yl]-5-pyrimidinyl-amino isopropyl formate and iodomethane reaction get light yellow solid 4,6-diamino--2-[1-(5-fluorine thiophene-2-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) Urethylane

Method is with embodiment 4; With 4 of embodiment 10; 6-diamino--2-[1-(5-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3; 4-b] pyridin-3-yl]-5-pyrimidinyl-amino methyl-formiate and iodoethane react light yellow solid 4,6-diamino--2-[1-(5-fluorine thiophene-2-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) urethanum

Method is with embodiment 4; With 4 of embodiment 11; 6-diamino--2-[1-(5-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3; 4-b] pyridin-3-yl]-5-pyrimidinyl-amino ethyl formate and iodoethane react light yellow solid 4,6-diamino--2-[1-(5-fluorine thiophene-2-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl (ethyl) carbamic acid isopropyl ester

Method is with embodiment 4; With 4 of embodiment 12; 6-diamino--2-[1-(5-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3; 4-b] pyridin-3-yl]-5-pyrimidinyl-amino isopropyl formate and iodoethane react light yellow solid 4,6-diamino--2-[1-(5-fluorine thiophene-2-yl) 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-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-5-pyrimidyl } methane amide

2-[1-(3-fluorine thiophene-2-yl) methyl isophthalic acid H-pyrazolo [3,4-b] pyridin-3-yl]-4,5 with embodiment 21A free state; 6-pyrimidine triamine is dissolved among the 10ml DMF, adds the 20ml concentrated hydrochloric acid, stirring at room 30min; Suction filtration gets pale brown look solid; Column chromatography () separate product N-{4,6-diamino--2-[1-(3-fluorine thiophene-2-yl) 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 experiment, measure of the diastole effect of embodiment compound to the rat chest aorta blood vessel

The preparation of stripped thoracic aorta vascular circle:

With the rat sacrificed by decapitation, take out thoracic aorta rapidly, place to 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) petridish in, careful flush away thrombus is also removed the outer reticular tissue of blood vessel.

Blood vessel is cut into the wide vascular circle of 3mm, places 10ml to contain 37 ℃ of thermostatic baths of Kreb ' s-Henseleit liquid, the lower end is fixed, and the upper end is connected in polygraph through tonotransducer.Organize the mixed gas that continues to feed 95% oxygen and 5% carbonic acid gas in the bath, every 15min changes nutritive medium once, treat that vascular circle is hatched 20-30 minute after, give vascular circle 2g preload, stablized 45-60 minute.With 3 * 10 ^-7MolL ^-1Sympathin (NE) brings out vascular circle and shrinks, waits to shrink to reach (about 8-10 minute) after peak and the balance, and the replacing nutritive medium, eccysis NE reaches balance again.With this moment tension value as basic tension value, begin to observe of the influence of processing factor to antiotasis.

Drug-treated:

After blood vessel is hatched and is arrived balance, with 3 * 10 ^-7MolL ^-1NE injects preshrinking vascular circle in the bath, wait to shrink reach maximum and stable after, the mode that increases progressively with concentration gives test-compound, observes the diastole effect of test-compound to sympathin (NE) vasoconstriction ring; Calculate the EC50 value of diastole percentage and each compound.

Data processing method:

3 * 10 ^-7Mol/L NE inductive maximum tension value=give 3 * 10 ^-7Tension value-basic tension value after the mol/L NE vasoconstriction balance

Compound diastole value=3 * 10 ^-7Tension value behind mol/L NE inductive maximum tension value-the give test-compound

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); Give test-compound with the concentration incremental manner after the balance; Observe new compound to the vasoconstrictive diastole effect of sympathin inductive, group of solvents contains the Kreb ' s-Henseleit nutritive medium of 1/1000 DMSO.

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

Select two concentration (3*10 of initial onset and the about 60-80% of diastole effect ^-8M, 1*10 ^-7M) carry out the evaluation of compound, measure each concentration of compound and add the maximum diastole effect that the back produces.Visible by table 1: as to prolong group of solvents vasoconstriction tension force in time and reduce 8-10% approximately; But the remarkable diastole thoracic aorta of drug group concentration dependent; Tried embodiment compound 4,5 and 7EC50 all less than 100nM, demonstrate compound NE preshrinking blood vessel is had the diastole effect.

Table 1. compound causes sympathin and shrinks rat

The diastole effect of thoracic aortic ring

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

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

5 concentration (1 * 10 of test-compound ^-8M, 3 * 10 ^-8M, 1 * 10 ^-7M, 3 * 10 ^-7M, 10 ^-6M) add successively, each concentration incubation time is 5 minutes (previous concentration compound does not reach maximum diastole effect as yet, adds next concentration when reaching 5 minutes).Prolong in time, the group of solvents shrink tension has the reduction of 8-15%; Tried embodiment compound 4,5,6, but the remarkable diastole thoracic aorta of 7 concentration dependents, the IC of compound ₅₀Be worth (maximum diastolic rate is calculated with 100%) as shown in table 2:

Table 2. compound causes sympathin and shrinks rat

The diastole effect IC of thoracic aortic ring ₅₀Value

Every kind of concentration of compound is handled rat chest aorta ring 5min.Blank solvent is made up of Kreb ' s-Henseleit damping fluid, contains the DMSO of 1/1000 volume).Numerical value is MV ± deviation n=2-4.

Embodiment 21:Langendoff isolated heart perfusion experiment

The preparation of Langendorff isolated heart perfusion model:

The rat sacrificed by decapitation is opened the thoracic cavity rapidly and is exposed heart, inserts heart canula along the aortic arch opening, immediately aorta is fixed on the heart canula of perfusion device with cotton thread.With 37 ℃ of Kreb ' s-Henseleit nutritive medium ((mM): NaCl 112, NaHCO that contain 1/10000DMSO ₃25, KCl 5, MgSO ₄1.2, KH ₂PO ₄1, CaCl ₂1.2, glucose 11.5, pH 7.4 ± 0.5) the perfusion heart that drives in the wrong direction, Ppa pulmonary artery pressure is the 85cm water column.But the soft sacculus with the water filling adjusted volume connects MedLab U/4CS bio signal acquisition processing system (the beautiful bio tech ltd that is prone in Nanjing), sacculus is inserted left ventricle along left auricle of heart and adjusts the sacculus size make left indoor pressure/diastole end press minimum value at 4-6mmHg.Around whole filling system and the heart, maintain about 37 ℃ with the water bath with thermostatic control circulator, perfusion liquid is with 95% O ₂With 5% CO ₂Fully saturated, heart can recover independently to beat under this environment very soon.The perfusion balance is 30-50 minute in advance, treats to begin experiment after each item heart function index reaches steadily.

Drug-treated: after isolated rat heart function each item index reaches steadily, earlier record various heart function values of left ventricle and coronary artery flow as administration before basic value.Adjustment three passes to the 2# passage; Give oxygenation in advance, the testing compound after heating is (with the Kreb ' s-Henseleit nutritive medium diluted compounds mother liquor of oxygenation in advance to working concentration; DMSO final concentration 1/10000); Record each item heart function value, and the coronary flow the 3rd, 5,8,10,15,20 minute time the after the administration.The percentage that each time point coronary flow changes after the calculating administration.

Statistical treatment: experimental data is expression with .

Langendorff isolated heart perfusion experiment result: 1.5 minutes coronary flows begin to increase after the administration, generally reach maximum during to 5 minutes, are tried embodiment compound 4,5,6, but the diastole coronary artery of 7 equal concentration dependents, increase coronary flow, its IC ₅₀Be worth as shown in table 3:

The IC of table 3. rat Langendorff isolated heart perfusion compound ₅₀Value

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

Embodiment 22: the experiment of rat drug disposition dynamic metabolism

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

TP: rat is all distinguished intravenous injection or gastric infusion according to the dosage of 2mg/kg; The intravenously administrable group before administration and after the administration 2,5,15,30min and 1,2,4,6,8,12,24h; The gastric infusion group before administration and after the administration 5,15,30min and 1,2,4,6,8,12,24h, by eye socket blood sampling 0.25mL, the centrifugal 10min of 3000rpm; Separated plasma 0.1mL puts-20 ℃ of refrigerators and deposits pending.Get rat plasma 0.1mL, add 0.3mL methyl alcohol, vortex oscillation 1min, the centrifugal 10min of 14000r/min shifts supernatant 100 μ L in the sample introduction bottle, gets 10 μ L sample introduction LC/MS/MS assay determinations.Adopt DAS pharmacokinetics program that the survey data are analyzed, calculate main pharmacokinetic parameter.

Detecting instrument and analysis condition: U.S. Finnigan company's T SQ Quantum type liquid chromatographmass spectrometer (LC/MS/MS), form by Finnigan Surveyor LC pump, Surveyor AS automatic sampler, electro-spray ionization ionizer (ESI) and three grades of MSMSs.Control software is Xcalibur1.4, and MASS SPECTRAL DATA ANALYSIS adopts the Lcquan2.0 DPS.

Chromatographic column is C18 post (2.1mm * 50mm, 5 μ m), 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 a room temperature.

The LC interface adopts pneumatic auxiliary electro-spray ionization ionizer (ESI), spray voltage 4.8KV; 300 ℃ of heated capillary temperature (TEM); Sheath gas N ₂, flow velocity 10psi; Auxiliary gas N ₂, flow velocity 1psi; Collision gas (CID) Ar, pressure 1.5 (mTorr); Collision induced dissociation in the source (Source CID) energy is 8V; The positive ion mode detects; The scanning of the mass spectrum mode is selective reaction monitoring (SRM), measures the characteristic reaction pair ion of compound respectively; Be 0.5s sweep time.

The result:

1, embodiment 4 compounds are in intravital kinetics of rat and bioavailability

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

Tmax is about 0.5h behind the rat oral administration, and t1/2 is at 6 left and right sides h, and Cmax is 100.45ng/ml.

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

The major impetus mathematic(al) parameter

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

The major impetus mathematic(al) parameter

2, embodiment 7 compounds are in intravital kinetics of rat and bioavailability

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

Tmax is about 3h behind the rat oral administration, and t1/2 is at 10 left and right sides h, and Cmax is 12ng/ml.

Behind table 6 rat vein administration embodiment 7 compounds (2mg/kg)

The major impetus mathematic(al) parameter

Behind table 7 rat oral gavage administration embodiment 7 compounds (2mg/kg)

The major impetus mathematic(al) parameter

3, embodiment 5 compounds are in intravital kinetics of rat and bioavailability

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

Tmax is about 0.5h behind the rat oral administration, and t1/2 is at 2 left and right sides h, and Cmax is 123ng/ml.

Behind table 8 rat vein administration embodiment 7 compounds (2mg/kg)

The major impetus mathematic(al) parameter

Behind table 9 rat oral gavage administration embodiment 7 compounds (2mg/kg)

The major impetus mathematic(al) parameter

4, embodiment 6 compounds are in intravital kinetics of rat and bioavailability

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

Tmax is about 0.25h behind the rat oral administration, and t1/2 is at 2 left and right sides h, and Cmax is 20.8ng/ml.

Behind table 10 rat vein administration embodiment 6 compounds (2mg/kg)

The major impetus mathematic(al) parameter

Behind table 11 rat oral gavage administration embodiment 6 compounds (2mg/kg)

The major impetus mathematic(al) parameter

Embodiment 23: the embodiment compound suppresses the CYP enzyme and inducibility research

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

TP:

1, enzyme inhibition test

People's hepatomicrosome incubation reaction system is the 0.05molL of 200 μ L ^-1K ₂HPO ₄Damping fluid (pH=7.4) system contains people's hepatomicrosome (protein concentration 0.2g/L), the positive suppressor factor of series concentration or receive the reagent thing; The substrate of each CYP enzyme; Every kind parallel 3 parts, Incubating Solution adds behind 37 ℃ of water-bath preincubate 5min equally and starts reaction at the NADPH of 37 ℃ of preincubate 5min solution (final concentration 1mmol/L), hatch add behind the 30min 600 μ L contain in target stop buffer termination reaction; Vortex 2min; 4 ℃, the centrifugal 10min of 19000g gets supernatant 10 μ L sample introductions detect each substrate utilization product to LC-MS generation.The corresponding meta-bolites of each probe is following:

CYP substrate utilization product MS detects ion

1A2 Phenacetin PARACETAMOL BP98 m/z 152

2C9 tolbutamide 4-hydroxyl-tolbutamide m/z 285

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

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

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

The LC-MS testing conditions:

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

ESI source positive ion detection mode: 300 ℃ of capillary temperatures, collision energy 120, capillary voltage+4000V, in be designated as the hydrochloric acid propranolol; 4-hydroxyl-tolbutamide ESI source negative ion detection mode: 300 ℃ of capillary temperatures, collision energy 120, capillary voltage-3000V, in be designated as chlorzoxazone.

Data processing:

Relative inhibition Irel by each suppressor factor of computes:

Irel(％)＝1-Ci(n)/Ci(0)×100％

Ci in the formula (n) adds the substrate utilization product relative quantity that obtains behind the positive suppressor factor of different concns for Incubating Solution; The substrate utilization product relative quantity that Ci (0) obtains for the blank group that does not add positive suppressor factor.To calculate IC to the logarithmic value of the Log10 of respective concentration with the mapping of GraphPad Prism 5 softwares through the relative inhibition Irel that following formula is tried to achieve ₅₀Value.

2, enzyme induction test

Positive controls:

Primary rat hepatocyte adopts sandwich glue culture method to be inoculated in the 12 porocyte culture plates.With the Soluble phenobarbitone is the positive inductor of CYP3A2, is diluted to 2mmolL respectively with liver cell culture liquid ^-1With 50 μ molL ^-1Administration is in 37 ℃, 5%CO ₂, hatch 72h under the saturated humidity condition, every 24h changes once (1ml hole of liquid ^-1), establish 2 multiple holes for every group.The blank group adds the cell culture fluid that contains 0.2% methyl alcohol.Every hole adds the Phenacetin (PH that 1ml contains probe behind the 72h; 50 μ molL ^-1) and midazolam (MDZ; 10 μ molL ^-1) cell culture fluid continue to hatch 1h.Hatch terminal point absorption Incubating Solution 200 μ l and add 600 μ L reaction terminating liquid termination reactions, vortex 2min, the centrifugal 10min of 19000g, 10 μ L sample introduction LC-MS appearance detect the relative growing amount of meta-bolites.

Experimental group:

3 concentration levels are respectively tried thing (0.3,3,30 μ molL ^-1) replace positive inductor, each concentration group is established 2 multiple hole administrations and is carried out cell and hatch, and all the other are with positive inductor group.

Data processing:

With inducing multiple to estimate positive inductor or receiving the inducing action of reagent to CYP3A.

With the growing amount of LC-MS method detection probes meta-bolites, the growing amount of hatching the product behind the 60min with probe substrate is induced multiple according to computes:

The positive is induced the growing amount of the growing amount/negative control group product of multiple=inductor group product

Receive the per-cent of the positive relatively group of reagent=receive reagent group product growing amount/positive to organize growing amount * 100%

The result:

1, enzyme suppresses merit rating

The experimental model that the CYP enzyme suppresses verifies that with positive suppressor factor the positive suppressor factor of using different concns is hatched the IC of the positive suppressor factor of each that obtains simultaneously with probe substrate in people's hepatomicrosome ₅₀Value all within the scope of bibliographical information, shows that the used incubation system of experiment can be used to tried thing the CYP enzyme is suppressed ability assessment, the IC that is respectively tried thing that obtains ₅₀Value is seen table 12.

The all test-compound of this test is to 2C19 and the equal unrestraint effect of 2D6.

Table 12. embodiment compound is to the inhibition merit rating of CYP isozyme

"-": in the concentration range of being tested, fail to obtain IC ₅₀Value, compound is to enzyme unrestraint effect.

2, CYP3A enzyme induction ability assessment

CYP3A is the main CYP isozyme of participating in drug metabolism, is the positive inductor of CYP3A with the Soluble phenobarbitone, on the rat primary hepatocyte, estimates the inducibility of series compound to CYP3A2.

The positive group of phenylethyl barbituric acid all is more than 2 times or near 2 times of solvent control group to the induced activity of enzyme, shows that used cell incubation system can be used to tried thing to CYP enzyme induction ability assessment.Tried thing the induction experiment result of CYP3A2 is seen table 13.

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

*: cell state is poor, does not count the result.

Respectively tried thing the CYP3A enzyme is not all had remarkable inducing action; Because CYP3A and 2C are regulated and control by same nuclear receptor, above-claimed cpd does not have tangible inducing action to the main CYP enzyme of C yet.

Claims (11)

1. formula I compound, and isomer, pharmacologically acceptable salts, solvate:

Wherein:

R ₁For-NR ₂C (=O) OR ₃,

R ₂Be hydrogen or C ₁-C ₄Alkyl,

R ₃Be C ₁-C ₆Alkyl.

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

Wherein:

R ₁For-NR ₂C (=O) OR ₃,

R ₂Be hydrogen or C ₁-C ₄Alkyl,

R ₃Be C ₁-C ₆Alkyl.

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

Wherein:

R ₁For-NR ₂C (=O) OR ₃,

R ₂Be hydrogen, methyl or ethyl,

R ₃Be methyl, ethyl or sec.-propyl.

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

5. prepare the method for each described formula I compound of claim 1-4, it comprises:

1) chloromethyl fluoro thiophene (II) obtains midbody 3-cyanic acid-1-fluoro thienyl methyl-1H-pyrazolo [3,4-b] pyridines (IV) with 3-cyanic acid-1H-pyrazolo [3,4-b] pyridines (III) reaction,

2) 2-(1-fluoro thienyl methyl-1H-pyrazolo [3; 4-b] pyridin-3-yl)-4; 5,6-pyrimidine triamine tri hydrochloride (V) obtains midbody 4,6-diamino--2-(1-fluoro thienyl methyl-1H-pyrazolo [3 with chloro-formic ester (VI) reaction; 4-b] pyridin-3-yl)-5-pyrimidinyl-amino manthanoate (Ii)

Wherein: R ₃Definition according to claim 1,

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) reaction obtains 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 according to claim 1, X is a leavings group.

6. pharmaceutical composition; It comprises each described formula I compound of claim 1 to 4 of treating and/or preventing significant quantity or its isomer, pharmacologically acceptable salts, solvate, and optional one or more pharmaceutically acceptable carriers or vehicle.

7. the application that is used to prepare the medicine aspect of treating cardiovascular disorder like the defined formula I compound of claim 1 or its pharmacologically acceptable salts, isomer, solvate.

8. be used to prepare the application of the hypertensive medicine of treatment aspect like the defined formula I compound of claim 1 or its pharmacologically acceptable salts, isomer, solvate.

9. be used to prepare the application of treatment thrombotic disease and ischemic medicine aspect like the defined formula I compound of claim 1 or its pharmacologically acceptable salts, isomer, solvate.

10. in the experimenter who needs is arranged, treat and/or prevent the method with cGMP diseases associated or illness, this method comprises to experimenter's administering therapeutic that needs are arranged and/or prevents each the described formula I compound of claim 1 to 4 or the described pharmaceutical composition of its isomer, pharmacologically acceptable salts, solvate or claim 6 of significant quantity.

11. be used to treat and/or prevent and each described formula I compound of claim 1 to 4 of cardiovascular disorder diseases associated or illness or its isomer, pharmacologically acceptable salts, solvate.