CN105272984B - Pyrazolo [3,4-d] pyrimidin-4-one-derivatives, preparation method and application - Google Patents

Abstract

A kind of pyrazolo [3,4-d] pyrimidin-4-one-derivatives or its stereoisomer or its salt have such as following formula (1) or formula (2) structure:

Description

Pyrazolo [3,4-d] pyrimidin-4-one-derivatives, preparation method and application

Technical field

The present invention relates to pyrazolo [3,4-d] pyrimidin-4-one-derivatives or pharmaceutical salt and preparation method thereof, Yi Jizhi Less include a kind of pharmaceutical formulations of compound or its salt as active constituent, and is preventing and treating hyperuricemia disease Application in disease, belongs to field of medicaments.

Background technique

Gout is one group of syndrome caused by human body purine metabolic disturbance, and hyperuricemia is one in its pathological development Stage.According to pathogenic factor, primary gout and secondary gout two types can be classified as.

Primary gout has apparent Family inherited inclination, is apt to occur in the middle-aged and the old, and onset peak is 30~50 years old, about 95% is male, and 5% women is often to fall ill after menopause.The cause of disease of primary gout mainly includes two aspects:

(1) inherent cause clinical findings, gout have apparent Family inherited inclination, and patient with gout relatives merge asymptomatic height The recall rate of uricacidemia is apparently higher than non-patient with gout.Gout and other metabolic diseases with genetic predisposition are (fat, high Blood pressure, hyperlipidemia, diabetes etc.) it is in close relations.Having found out causes uric acid to generate in excessive purine metabolism, causes the work of enzyme Sexually revise the hereditary basis of enzyme gene mutation.

(2) environmental factor overeating, to indulge in excessive drinking, eat rich in purine food be excessively urarthritis acute attack Common cause.The improvement of socioeconomic status, the metabolic diseases illness rate such as obesity, hypertension increase, and also make the illness rate of gout Increase.

Secondary gout onset caused by secondary gout is removed because of congenital Fanconi-de Toni syndrome and chronic renal failure Slowly outer, a lot of diseases are more anxious.The cause of disease of secondary gout is main including the following three aspects:

(1) after causing internal uric acid to generate the excessive cause of disease such as leukaemia, lymthoma progressive stage, especially chemotherapy, true property Red blood cell count(RBC) increase disease etc..After severe trauma, crush injury, major operation.

(2) causing kidney uric acid that the reduced cause of disease such as severe hypertension, eclampsia is discharged causes renal blood flow to reduce, and influences uric acid Filtration；Renal failure caused by any reason；Congenital Fanconi-de Toni syndrome, model can syndrome, Bartter syndromes etc.； The metabolic disorder of tubular secretion uric acid is influenced, such as ethylism, hungry excessive, ketoacidosis, lactic acidosis can draw It plays organic acid content in blood to increase, inhibits the secretion of renal tubule uric acid；Some drugs can cause hyperuricemia, such as ethamine fourth Alcohol.

(3) factor for influencing the variation of blood uric acid concentration uses the pre-renal dehydration of diuretic therapy, severe for a long time, keeps blood dense Contracting increases blood uric acid concentration.

Primary gout clinical manifestation has apparent Family inherited inclination, and according to disease progression feature, the gout course of disease can divide For following 4 phase: the asymptomatic hyperuricemia phase；Acute attack stage；The asymptomatic intermittent phase；Chronic phase.Main clinical manifestation has Following several respects:

(1) the generation Chang Shifen of asymptomatic hyperuricemia is hidden attacks, and it is gradually in duration that initial stage, which is that interruption occurs, more In physical examination or because other diseases are medical when be not intended to find.

(2) acute gouty arthritis this be gout most feature and common symptom, onset is hurried, a few hours it Interior affected joints may occur in which apparent red and swollen, heat pain, often in night-time attack, wake up because of joint severe pain, local joint is because of pain Cannot touch, in addition cannot lid sheet, limitation of activity.It is secondly other of brothers with foot the first plantar toe for most predilection site Minor articulus, ankle, knee, wrist, elbow, shoulder joint.Initial stage is mostly simple joint lesion, and two sides alternately occur, and the later period can be multi-joint lesion, Occur simultaneously or successively.Overeating is drunk beyond one's capacity, fatigue, infection, wound, operation, wound, periarticular compression, and shoes are carried out not Suitable wait can be risk factor.Acute attack symptom continues a Zhou Yu more, then gradually alleviates.After local joint redness subsides, There can be skin itch, peel, pigmentation.Stage of attack, constitutional symptom can have fever, out of strength, increased heart rate, headache etc..

(3) gout intermittent phase gout intermittent phase refers to the interphase of acute gouty arthritis breaking-out twice, short then several weeks, long Then many decades.Once in a while again without breaking-out after the 1st acute arthritis of some patients, majority is from long to short.The intermittent phase state of an illness is opposite Steadily, also known as stationary state.Some patientss can have discomfort after former affected joints activity, can be relieved after rest.Can still there be high urine Acidaemia is influenced by diet and treatment condition, and serum uric acid level is unstable.

(4) the chronic long-term hyperuricemia of gravel gout fails to correct, and it is soft that urate crystal can be deposited on extensively joint Bone, synovial membrane, ligament, subcutaneous, kidney, gradually form urate calculi, physiological function that is heavy then influencing deposition fabric.It is part of It after established calculus uric acid obtains control, can still melt, reduce, even disappear completely, this is more special for gouty calculus Lapse to.

(5) subcutaneous tophus tubercle is deposited on subcutaneous formation by urate crystal, is apt to occur in helix, (toe refers on joint periphery Between joint, knee joint, elbow joint, wrist joint etc.), tubercle differs in size, and sesame as low as 1~2 centimetre of major tubercle greatly, boundary is not advised Then, matter is hard, and table superficial part position is in yellow-white.With the tubercle of perienchyma clear-cut, no tenderness, but as merged bacterium infection can There are perienchyma's redness, tenderness, there is ulceration mouth more.Needle adopts out stone composition or ulceration secretion microscopy is urate crystal.

(6) joint tissue fibrosis and tophus caused by the repeated multiple times acute arthritis of gouty arthritis,chronic is broken out In articular cartilage, the deposition of synovial membrane, ligament, makes lesion joint gradually damaged deformation, lose motor function.Toe, interphalangeal joint, Ankle, knee, wrist joint are vulnerable to tired.

(7) the 2 kinds of forms that are deposited with of chronic gouty nephropathy and kidney stone urate crystal in kidney, including uric acid secretion Uric acid in renal tubule apurinic acid mineralization (uric acid concentration is normal in medullary interstitium, renal tubule) caused by excretion is insufficient and renal tubule Excessive concentration cannot be discharged in time and uric acid mineralization in the renal tubule that is detained.Chronic Uric-acid Nephropathy can be in both uric acid kidneys Occur on the basis of interior deposition pattern, for majority after the acute gouty arthritis of recurrent exerbation, minority can be only long-term Occur on the basis of hyperuricemia.

The clinical manifestation of secondary gout be before hyperuricemia occurs mostly secondary disease Clinical symptoms.Except because congenital Property Fanconi-de Toni syndrome and chronic renal failure caused by secondary gout onset it is slowly outer, a lot of diseases are more anxious.With high lithemia Mass formed by blood stasis and a large amount of lithates deposited in renal tubule cause acute renal failure be it is common, serum Uric Acid Concentration can > 1mmol/L, Urine uric acid increased significantly, visible a large amount of urate crystals in arena, under even visible mirror or gross hematuria.Patient can have urodynia, The symptoms such as lumbago, Nausea and vomiting, oliguresis or anuria.

The treatment of gout includes two aspects, i.e. two aspects of anti-inflammatory analgetic and reduction blood uric acid, the former is mark, Hou Zhewei This, symptomatic treatment in acute condition achievees the purpose that treating both manifestation and root cause of disease, and therefore, corresponding drug includes following two categories:

One, anti-inflammatory analgetic class drug

(1) colchicin is a kind of alkaloid extracted in colchici cormus, plays the role of preventing cell mitogen, can Inhibit the chemotactic of inflammatory cell and reduce inflammatory factor release, there is unique anti-inflammatory disappear for the gouty arthritis of acute attack Swollen effectiveness, can alleviate symptom within short a few hours.

(2) such types of drugs of non-steroidal anti-inflammatory drugs is more, not only orally available, local topical, it is possible to use suppository, mainly Local soft tissue redness, heat pain when by inhibiting tissue to mitigate the breaking-out of gout acute arthritis to the inflammatory reaction that uric acid deposits And general reaction, on serum uric acid level mostly without influence.

(3) Adrenal Glucocorticoid experimental study break out symptom especially severe, or to colchicin intolerant to Receptor can use small prednisone, dexamethasone, to mitigate the inflammatory reaction of tissue.

Two, anti-trioxypurine drugs

The generation of internal uric acid is related with purine metabolism, and in the final step of purine metabolism, hypoxanthine is in xanthine Xanthine is generated under the action of oxidoreducing enzyme (XOR), further generates uric acid, inhibits the activity of the enzyme that can effectively subtract The generation of oliguresis acid.Anti-trioxypurine drug, which mainly passes through, to be inhibited internal uric acid to generate and the discharge of uric acid in blood is promoted to play drop urine Sour effect, key agents have following several:

(1) the probenecid medicine can inhibit renal tubule to the re-absorption of lithate, to increase discharge of the uric acid from kidney, fit For blood uric acid height, the patient with gout of uric acid discharge capacity < 3.6mmol/d (< 600mg/d) is urinated.

(2) Benzbromarone (narcaricin) is benzofuran analog derivative, by inhibiting proximal tubular to the weight of uric acid The excretion of uric acid is absorbed and promoted, does not obstruct the metabolism of purine nucleotides.It is main that (intrahepatic metabolism, bile are discharged by gastrointestinal tract Discharge), suitable for urinating uric acid discharge capacity < 3.6mmol/d patient with gout, it can also be used to which slightly raised Renal function in early period is not or not inosine Full patient with gout.

(3) Allopurinol is xanthine oxidase inhibitor, can inhibit hypoxanthine and is changed into xanthine and switchs to uric acid again, from And reduce the synthesis of uric acid.Excessive primary or secondary patient with gout is generated suitable for itself uric acid.

It is the most commonly used with Allopurinol in common anti-trioxypurine drug, although novel anti-trioxypurine drug Febuxostat is Listing, but Allopurinol due to curative effect affirm, it is cheap, remain as the widest anti-trioxypurine drug of current clinical use.

However, the curative effect of Allopurinol is relatively on the weak side, and safety is poor, thus, development efficacy is more excellent, safety more preferably Allopurinol derivatives meet huge, urgent clinical demand and still have very important significance.

Summary of the invention

In order to solve the above technical problem, the present invention provides a kind of pyrazolo [3,4-d] pyrimidin-4-one-derivatives or it is vertical Body isomers or its salt, compared with Allopurinol, anti-trioxypurine activity is higher, safety is higher.

To achieve the goals above, the present invention adopts the following technical scheme:

A kind of pyrazolo [3,4-d] pyrimidin-4-one-derivatives or its stereoisomer or its salt, have such as following formula (1) or Formula (2) structure:

Wherein,

G be selected from aryl, alkyloxyaryl, alkyl carbonyl epoxide aryl, alkylcarbonyloxyalkyl aryl, heteroaryl, Alkyl oxy heteroaryl, alkyl carbonyl epoxide heteroaryl or alkylcarbonyloxyalkyl heteroaryl, the aryl or heteroaryl Or the H in the aryl or heteroaryl in functional group is optionally by one or more alkyl, aryl alkyl, xenyl alkyl, aryl acyl Base, heteroaryl alkyl, heteroaroyl, amino-sulfonyl, halogen replace, and wherein aryl, heteroaryl, amino, xenyl are optional Further replaced by one or more alkyl, halogen, tetrazole radical；

The heteroaryl is selected from pyridyl group, furyl, purine radicals, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidine radicals, pyrroles Base, quinazolyl, quinolyl, quinazinyl, imidazole radicals, indazolyl, indolinyl, indyl, diazosulfide base, different benzo Furyl, isochroman base, iso-dihydro-indole-group, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, phenodiazine Miscellaneous naphthalene, oxadiazoles, oxazolyl, phenazinyl, phenothiazinyl, phthalazinyl, pteridyl, quinoxalinyl, tetrahydro isoquinolyl, tetrahydro Quinolyl, tetrazole radical, thiadiazolyl group, thiazolyl, thio chromanyl, thienyl, triazolyl, different thiophene benzo dihydro pyrrole It mutters base, benzimidazolyl, benzodioxane base, benzo dioxepine base, benzodioxole group, benzo Furyl, benzofuraxan base, benzothiazolyl, benzoxadiazole, benzoxazinyl-, benzoxazolyl, benzimidazolyl, benzo Morpholinyl, the miscellaneous di azoly of benzo selenium, benzothienyl, carbazyl, chromanyl or imidazo [1,2-a] pyridyl group；

The halogen refers to fluorine, chlorine, bromine or iodine.

As the preferred solution of the invention, pyrazolo [3, the 4-d] pyrimidin-4-one-derivatives or its stereoisomer or its Salt:

Wherein,

G is selected from phenyl, C1-C5 alkyl oxy phenyl, C1-C5 alkyl carbonyl epoxide phenyl, C1-C5 alkyl carbonyl epoxide C1-C5 alkylaryl, heteroaryl, C1-C5 alkyl oxy heteroaryl, C1-C5 alkyl carbonyl epoxide heteroaryl or C1-C5 alkyl H in carbonyl oxygroup C1-C5 miscellaneous alkyl aryl, the phenyl or phenyl or heteroaryl in heteroaryl or functional group is optional By one or more C1-C5 alkyl, phenyl C1-C5 alkyl, xenyl C1-C5 alkyl, phenylacyl, heteroaryl alkyl, heteroaryl Base acyl group, amino-sulfonyl or halogen replace, wherein the phenyl in the phenyl C1-C5 alkyl, the heteroaryl in heteroaroyl The xenyl in amino, xenyl C1-C5 alkyl in base, amino-sulfonyl is optionally further by one or more alkyl, halogen Element, tetrazole radical replace；

The heteroaryl is selected from pyridyl group, furyl, purine radicals, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidine radicals, pyrroles Base, quinolyl, quinazinyl, imidazole radicals, indazolyl, indolinyl, indyl, isobenzofuran-base, isoindolyl, isoquinolin Base, isothiazolyl, isoxazolyl, oxazolyl, phenazinyl, tetrazole radical, thiadiazolyl group, thiazolyl, thienyl, triazolyl, benzo Imidazole radicals, benzofuranyl, benzothiazolyl, benzimidazolyl, benzo morpholinyl, the miscellaneous di azoly of benzo selenium, benzopyrrole base Or benzothienyl；

The halogen is selected from fluorine, chlorine or bromine.

As the further preferred scheme of the present invention, pyrazolo [3, the 4-d] pyrimidin-4-one-derivatives or its alloisomerism Body or its salt:

Wherein,

G is selected from phenyl, C1-C5 alkyl oxy phenyl, C1-C5 alkyl carbonyl epoxide phenyl or C1-C5 alkyl-carbonyl oxygen Phenyl in base C1-C5 miscellaneous alkyl aryl, the C1-C5 alkyl oxy phenyl or C1-C5 alkyl carbonyl epoxide phenyl, Huo Zhesuo H in the heteroaryl in C1-C5 alkyl carbonyl epoxide C1-C5 miscellaneous alkyl aryl is stated optionally by one or more methyl, ethyl, just Propyl, isopropyl, normal-butyl, phenyl formoxyl, heteroaroyl, diphenylmethyl or amino-sulfonyl replace, wherein phenyl, Xenyl, amino and heteroaryl are optionally further by one or more methyl, ethyl, n-propyl, isopropyl, halogen, tetrazole radical Replace；

The heteroaryl be selected from furyl, benzofuranyl, thienyl, benzothienyl, pyrrole radicals, benzopyrrole base, Imidazole radicals, benzimidazolyl, pyrazolyl, benzopyrene oxazolyl, pyridyl group or pyrimidine radicals；

The halogen is selected from fluorine, chlorine or bromine.

It is different as still more preferably scheme, pyrazolo [3, the 4-d] pyrimidin-4-one-derivatives or its solid of the invention Structure body or its salt:

Wherein,

The G is

Pyrazolo [3,4-d] pyrimidin-4-one-derivatives of the present invention or its stereoisomer or its salt can be with three-dimensional different The form of structure body exists, including all geometric isomers, optical isomer and its mixture.The pyrazolo [3,4-d] is phonetic Pyridine -4- ketone derivatives or its stereoisomer or its salt can also be containing one or more asymmetric carbon atoms, and therefore may be used To show optical siomerism and/or diastereo-isomerism.Routine techniques, such as chromatography or fractional crystallization etc. can be used, point From enantiomter.Required optical isomer can not also will cause by suitable optically active starting material Racemic or difference to the reaction (i.e. ' chiral pond ' method) under conditions of (solid) isomerization, by suitable starting material and The reaction of ' chiral auxiliary ' (is split, including dynamic resolution) by derivatization, then such as chromatography point by way of conventional Mapping derivative is separated out, or by anti-with suitable chiral reagent or chiral catalyst under the conditions of to known to those skilled in the art It answers, obtains or isolate after reacting corresponding isomers, all stereoisomers and its mixture are all included in the present invention In the range of.

The compound of the present invention can also show tautomerism, all tautomeric forms and its mixture also by It is included within the scope of the invention.

Pyrazolo [3,4-d] pyrimidin-4-one-derivatives of the present invention or its stereoisomer or its salt are preferably changed as follows Close object:

The present invention also provides the systems of a kind of pyrazolo [3,4-d] pyrimidin-4-one-derivatives or its stereoisomer or its salt Preparation Method, in a suitable solvent, formula (3) compound or formula (4) compound and formula (5) compound are in the presence/absence of work Obtained from being reacted under conditions of agent,

Wherein G is as defined above.

The compound of formula (3) or (4) can refer to International Journal of Pharmaceutics, The synthesis of method described in Volume64, Issue1,1990, P75-87.

The present invention also provides another pyrazolo [3,4-d] pyrimidin-4-one-derivatives or its stereoisomers or its salt Preparation method, in a suitable solvent, formula (3 ') compound or formula (4 ') compound and formula (5 ') compound are in presence/do not deposit It is reacted under conditions of activator,

Wherein G ' is selected from:

The activator is selected from thionyl chloride, oxalyl chloride, dicyclohexylcarbodiimide, 1- (3- dimethylamino-propyl) -3- One or more of ethyl-carbodiimide hydrochloride, p-nitrophenol or allyl alcohol.

During the preparation process, the formula (5), formula (3 '), formula (4 ') compound can activator effect under will be carboxylic acid activated For acyl chlorides, activated amide or active ester.

Specifically, the formula (5), formula (3 '), the carboxylic acid in formula (4 ') compound are reacted with thionyl chloride or oxalyl chloride Generate acyl chlorides, or with active carbodiimide, such as dicyclohexylcarbodiimide, i.e. DCC or 1- (3- dimethylamino third Base) -3- ethyl-carbodiimide hydrochloride, i.e. EDC reaction activation, or form active ester with alcohol or phenol, for example, with p-nitrophenyl Phenol or allyl alcohol reaction.

It is partially the compound of commercialization in formula (5) and (5 ') compound, can directly uses, another part needs passes through The reagent being commercialized is generated into carboxylic acid by simple derivatization, carboxylic acid derivates are including but not limited to hydrolyzed to carboxylic Acid, or carboxylic acid is generated with reacting containing the organic acid anhydride there are two carboxyl.

The suitable solvent can be such as acetonitrile, acetone, tetrahydrofuran, methylene chloride, chloroform, four chlorinations Or mixtures thereof carbon, formamide, N,N-dimethylformamide, dimethyl sulfoxide, ethyl acetate, methyl acetate.

Preferred compound shown in table 1 is obtained using preparation method of the present invention.

The synthesis of the preferred compound of the present invention of table 1

The present invention also provides above-mentioned pyrazolo [3,4-d] pyrimidin-4-one-derivatives or its stereoisomer or its salt to exist Preparation prevents and treats the application in hyperuricemia disease medicament.

Pyrazolo [3,4-d] pyrimidin-4-one-derivatives of the present invention or its stereoisomer or its salt can be used for making The preparation of standby various administration routes, the form including emulsion, solution, suspension, aerosol and dry powder formulations carry out part and give Medicine (such as to skin or to lung and/or air flue)；Or whole body is carried out by oral administration such as with tablet, capsule, syrup, powder or particle and is given Medicine；Or parenteral administration is carried out in the form of solution or suspension；Or carry out subcutaneous administration；Or per rectum is given in the form of suppository Medicine；Or cutaneous penetration.

Pyrazolo [3,4-d] pyrimidin-4-one-derivatives of the present invention or its stereoisomer or its salt, with Allopurinol Compared to more preferably anti-trioxypurine effect and more preferably safety.

Specific embodiment

Further illustrating part with following non-limiting embodiment, this illustrates compound and preparation method thereof, while these The preparation process of certain raw materials used in embodiment is with subsequent preparation embodiment explanation.

For convenience of description, it is related to tautomer in the raw material in following embodiments, intermediate or product When, represented with the structural formula of one such tautomer or chemical name, and it is not construed as that there is only a certain The form of kind tautomer.

The preparation of 1 A1-1 of embodiment

The preparation of 1.1 1- methylol -5- hydrogen-pyrazolo [3,4-d] pyrimidin-4-one

1,5- dihydro-pyrazol simultaneously [3,4-d] pyrimidin-4-one 20g is taken, the formalin of 1000ml0.2mol/l is added to In, it with sodium hydrate aqueous solution tune pH to 7, reacts at room temperature 24 hours, filters, 10ml × 10 time, vacuum 70 is washed with water in filter cake It is DEG C dry to constant weight, obtain white powdery solids 21.8g, yield 90%.

¹H-NMR (400MHz, d⁶DMSO): δ 12.41 (s, 1H), 8.18 (s, 1H), 8.14 (d, 1H), 6.37 (s, 2H), 2.32 (s, 1H).

The preparation of 1.2 2- methyl -2- (4- (4- chlorobenzene formacyl) phenoxy group) propionic acid

36g2- methyl -2- (4- (4- chlorobenzene formacyl) phenoxy group) isopropyl propionate is dissolved in 15ml methanol, then The sodium hydroxide solution 35ml of 3mol/L is added, after back flow reaction 2 hours, with the hydrochloric acid tune pH of 1mol/L for 2 under ice-water bath, Filtering water washing 3 times, obtains white solid 29g, yield 91.7% after dry.

¹H-NMR (400MHz, d⁶DMSO): δ 13.21 (s, 1H), δ 7.60-7.70 (m, 4H), 7.50-7.60 (d, 2H), 6.76-6.79 (d, 2H), 1.59 (s, 6H).

The preparation of 1.3 2- methyl -2- (4- (4- chlorobenzene formacyl) phenoxy group) propionyl chloride

5ml thionyl chloride and 1 drop DMF are added into the product acid of 1.2 steps of 15.6g, then reaction 2 is small at 70 DEG C Shi Hou, evaporated under reduced pressure obtain faint yellow solid 16.9g, are directly used in reaction in next step without further purification.

The preparation of 1.4 A1-1

100ml is added into 1- methylol -5- hydrogen-pyrazolo [3,4-d] pyrimidin-4-one three-necked flask for filling 8.3g Tetrahydrofuran, stirring after ten minutes, the previous step acyl chlorides of 16.8g are added in the case where ice-water bath is cooling, is then added dropwise to tri- second of 6ml Amine after removing ice-water bath, first reacts 1 hour at room temperature, stops reaction after reacting 1 hour at 50 DEG C.

¹H-NMR (400MHz, d⁶DMSO): δ 12.41 (s, 1H), 8.18 (s, 1H), 8.14 (d, 1H), δ 7.60-7.70 (m, 4H), 7.50-7.60 (d, 2H), 6.76-6.79 (d, 2H), 6.37 (s, 2H), 1.59 (s, 6H).

ESI-MS:m/z:467、468、469、470(M+H)。

The preparation of 2 compound A2-1 of embodiment

By p- [(dipropyl amino) sulfonyl] benzoic acid of 14.2g, 1- methylol -5- hydrogen-pyrazolo [3,4- of 8.3g D] pyrimidin-4-one and 10.3g N, be added in 100ml tetrahydrofuran after the mixing of N- dicyclohexylcarbodiimide, be stirred at room temperature 60 Minute, TLC monitors end of reaction, stops reaction, and filtering is evaporated to obtain crude product after filter cake washs 3 x10ml with tetrahydrofuran Then 23.0g recrystallizes to obtain 17g white solid, yield 78% with dehydrated alcohol.

¹H-NMR (400MHz, d⁶DMSO): δ 12.48 (s, 1H), 8.22-8.26 (d, 2H), 8.09-8.11 (d, 2H), 7.93-7.95 (d, 2H), 6.54 (s, 2H), 3.01-3.05 (t, 4H), 1.42-1.45 (m, 4H), 0.77-0.86 (m, 6H).

ESI-MS:m/z434,435,436 (M+1).

The preparation of 3 compound A-13-1 of embodiment

The preparation of 3.1 1- methylol -5- hydrogen-pyrazolo [3,4-d] pyrimidin-4-one succinate monoester

Succinic anhydride, the 1- methylol -5- hydrogen-pyrazolo [3,4-d] of 4.8g that 7.8g is added in 100ml reaction flask are phonetic It in pyridine -4- ketone and 60ml pyridine, is stirred at room temperature 96 hours, stops reaction, reaction solution is poured into the HCl of 150ml4mol/L, is stirred It mixes and a large amount of white solids is precipitated within 4 hours, filter, filter cake washing, dry white powdery solids 4.3g, yield 54%.

¹H-NMR (400MHz, CDCl₃): δ 12.36 (s, 1H), 11.48 (s, 1H), 8.04 (s, 1H), 7.97 (s, 1H), 6.28 (s, 2H), 2.64 (s, 2H), 2.53 (s, 2H).

The preparation of 3.2 compound A-13s -1

By the bromo- 4- hydroxyphenyl -2- ethyl -3- benzofuran of 3,5- bis- of the Intermediate carboxylic acids of 2.26g3.1 step, 2.12g 30ml DMF is added after the mixing of the N of base-ketone and 2.69g, N- dicyclohexylcarbodiimide, stops after reaction being stirred at room temperature 2 hours It only reacts, reaction system is filtered, 400ml ethyl acetate, after saturated common salt water washing 3 times, column are then added into filtrate Chromatograph 2.13g white object product.

¹H-NMR (400MHz, CDCl₃): 11.51 (s, 1H), 8.20 (s, 1H), 8.06 (s, 1H), 8.01 (s, 2H), 7.50-7.52 (d, 1H), 7.38-7.40 (m, 1H), 7.30-7.34 (m, 1H), 723-7.27 (m, 1H), 6.39 (s, 2H), 3.09 (m, 2H), 2.88-2.90 (m, 4H), 1.34-1.36 (t, 3H).

ESI-MS:m/z:+Q1:671,673,675.

The preparation of 4 compound A4-1 of embodiment

By the Intermediate carboxylic acids of 2.26g3.1 step, the chloro- 1- of 2- butyl-the 4- [[2'- (1H-TETRAZOLE -5- base) of 4.62g [1,1 '-xenyl] -4- base] methyl]-H- imidazoles -5- methanol monopotassium salt and 2.69g N, the mixing of N- dicyclohexylcarbodiimide 30ml DMF is added afterwards, stops reaction after being stirred at room temperature reaction 2 hours, reaction system is filtered, is then added into filtrate 400ml ethyl acetate, after saturated common salt water washing 3 times, column chromatograph 1.09g white object product.

¹H-NMR (400MHz, CDCl₃): 11.0 (s, 1H), 8.04 (s, 1H), 7.97 (s, 1H), 7.92-7.94 (d, 1H), 7.52-7.65 (m, 2H), 7.43-7.47 (m, 1H), 7.15-7.17 (d, 2H), 6.87-6.89 (d, 2H), 6.28 (s, 2H), 5.08 (s, 2H), 4.91 (s, 2H), 2.42-2.68 (m, 6H), 1.22-1.34 (m, 2H), 0.82-0.86 (m, 3H).

ESI-MS:m/z:+Q1:405 (fragment peak), 671 (M-K+2), 693 (M-K+Na+1).

Embodiment 5 is directed to and gives exogenous uric acid while the medicine efficacy screening of uricolytic rat model being inhibited to test

1, materials and methods

1.1 experimental animal

SPF grades SD rat 110,150~180 grams of weight, male, Animal adaptability is raised 1 week, observes body surface sign, Free water and feeding.All cage tools pass through 121 DEG C of sterilization treatments, and feed is irradiated sterilization treatment, water sterilization treatment it is pure Water.

1.2 test drug

Screening compounds code name: A1-1, A2-1, A3-1, A4-1 (referred to as " A series compound " or " test medicine "),

Positive control drug: Allopurinol.

1.3 reagents and drug

Oteracil Potassium (Oxonic acid), uric acid (Uric), sodium carboxymethylcellulose (CMC), uric acid (UA) measure reagent Box.

1.4 laboratory apparatus

Uric acid (UA) detection uses U.S. MULTISKAN MK3 microplate reader, Italian BASA-18 full-automatic biochemical analysis Instrument.

1.5 groupings and administration

110 male SD rats are randomly divided into Normal group, model group, A1-115mg/kg group, A1-130mg/kg Group, A2-115mg/kg, A2-130mg/kg group, A3-130mg/kg group, A3-160mg/kg group, A4-130mg/kg group, A4- 160mg/kg group and positive drug Allopurinol 30mg/kg group.Every group animal 10, test medicine is given using stomach-filling.

2, experimental method and operating procedure:

Normal group does not give any drug, remaining each group takes orally give Oteracil Potassium 1.5g/kg+ uric acid 0.15g/ daily Kg (is dissolved in 0.5% sodium carboxymethylcellulose), gavages according to quantity on time 1 time daily, amounts to 10 days.Before experiment and 10 days eyes of modeling Socket of the eye takes blood 0.5ml, separates serum, measures serum uric acid.Oral administration gavage gives test medicine and Allopurinol 4 after modeling success It takes rat orbital vein blood 1ml, 3500RPM, 15min respectively 2 hours after administration the 4th day, and supernatant is taken to detect blood The content of clear uric acid.

3, statistical procedures

Data mean soil standard deviationIt indicates, it is soft using Excel7.0 and SPPS13.0for windows Part is analyzed, and comparison among groups are examined with q, is compared before and after medication and is examined with self pair t, and P < 0.001 indicates that difference has significantly Meaning.

4, experimental result

Influence of the 2 A series compound of table to uricacidemia rat blood serum uric acid content

^*P < 0.001 and model group ratio.

Influence of the 3 A series compound of table to uricacidemia rat body weight

Exogenous uric acid is given by foundation while inhibiting uricolytic rat uricacidemia model, to A series chemical combination Object carries out drug screening, and compared with the effect of the reduction serum uric acid of positive drug Allopurinol.

For table 2 the results show that compared with model group, Allopurinol, A1-1, A2-1, A3-1, A4-1 are significantly reduced rat The effect of middle serum uric acid, and A series compound reduces the effect of serum uric acid better than Allopurinol, and has dosage effect pass System.

For table 3 the results show that compared with model group, Allopurinol has a certain impact to the weight of rat, i.e., Allopurinol group is dynamic The increased weight of object is substantially less than model group, and the weight of animals increment of A series compound group it is suitable or slightly lower with model group but Difference is not significant.

Embodiment 6 adds uricolytic enzyme inhibitor to cause mouse hyperuricemia animal model for a large amount of purine substances Medicine efficacy screening experiment

1, materials and methods

1.1 experimental animal

SPF grades KM mouse 165,20~24 grams of weight, male, Animal adaptability is raised 3 days, observes body surface sign, from By drinking water and searching for food.All cage tools pass through 121 DEG C of sterilization treatments, and feed is irradiated sterilization treatment, the pure water of water sterilization treatment.

1.2 test drug

Screening compounds code name: A1-1, A2-1, A3-1, A4-1 (referred to as " A series compound " or " test medicine "),

Positive control drug: Allopurinol.

1.3 reagents and drug

Oteracil Potassium (Oxonic acid), hypoxanthine (Hypoxanthine), uric acid (UA) assay kit.

1.4 laboratory apparatus

Uric acid (UA) detection uses U.S. MULTISKAN MK3 microplate reader, Italian BASA-18 full-automatic biochemical analysis Instrument.

1.5 groupings and administration

165 male KM mouse are randomly divided into Normal group, model group, A1-140mg/kg group, A1-180mg/kg Group, A2-140mg/kg, A2-180mg/kg group, A3-140mg/kg group, A3-180mg/kg group, A4-140mg/kg group, A4- 180mg/kg group, positive drug Allopurinol 40mg/kg group.Every group animal 15.

2, experimental method and operating procedure:

Normal group does not give any drug, the daily Intraperitoneal injection of hypoxanthine 500mg/kg of remaining each group and subcutaneous injection Uricase inhibitor Oteracil Potassium 50mg/kg is used cooperatively, continuous 7d.Before experiment and after modeling 7d, every group takes 2-3 Mouse goes eyeball to take blood 0.5ml, separates serum, serum uric acid is measured, as mouse normal serum uric acid level and modeling Mice serum uric acid level.Test medicine and Allopurinol 4 days are given in stomach-filling after modeling success, are administered 2 hours at last 1 time respectively, It goes eyeball to take blood 0.5ml, 4000RPM, 20min, takes the content of supernatant detection serum uric acid.

3, statistical procedures

Data mean soil standard deviationIt indicates, unites using Excel7.0 and SPPS13.0for windows Meter software is analyzed, and comparison among groups are examined with q, is compared before and after medication and is examined with self pair t, and P < 0.001 indicates that difference has Significant meaning.

4, experimental result

Influence of the 4 A based compound of table to uricacidemia mouse uric acid content

^*P < 0.001 and model group ratio.

By establishing a large amount of purine uric acid while inhibiting uricolytic mouse uricacidemia model, to A series chemical combination Object carries out drug screening, and compared with the effect of the reduction serum uric acid of positive drug Allopurinol.

The results show that Allopurinol, A1-1, A2-1, A3-1, A4-1 are significantly reduced serum urine compared with model group The effect of acid, and low dose group is suitable with the anti-trioxypurine effect of Allopurinol, and the effect that high dose group reduces serum uric acid is better than Allopurinol, and have dose-effect relationship (being shown in Table 4).

Embodiment 7 is for the uricolytic rat model medicine efficacy screening experiment of inhibition

1, materials and methods

1.1 experimental animal

SPF grades SD rat 88,180~220 grams of weight, male, Animal adaptability is raised 3 days, observes body surface sign, from By drinking water and searching for food.All cage tools pass through 121 DEG C of sterilization treatments, and feed is irradiated sterilization treatment, the pure water of water sterilization treatment.

1.2 test drug

Screening compounds code name: A1-1, A2-1, A3-1, A4-1 (referred to as " A series compound " or " test medicine "),

Positive control drug: Allopurinol.

1.3 reagents and drug

Oteracil Potassium (Oxonic acid), uric acid (UA) assay kit.

1.4 laboratory apparatus

Uric acid (UA) detection uses U.S. MULTISKAN MK3 microplate reader, Italian BASA-18 full-automatic biochemical analysis Instrument.

1.5 groupings and administration

88 male SD rats are randomly divided into Normal group, model group, A1-115mg/kg group, A1-130mg/kg Group, A2-115mg/kg group, A2-130mg/kg group, A3-115mg/kg group, A3-130mg/kg group, A4-130mg/kg group, A4- 160mg/kg group, positive drug Allopurinol 20mg/kg group.Every group animal 8.

2, experimental method and operating procedure

Normal group does not give any drug, and stomach-filling is given after the intraperitoneal injection of remaining each group Oxonic Acid sylvite 300mg/kg, 1h Test medicine is given, 2h goes eyeball to take blood 1ml after administration, is centrifuged 4000RPM, 20min, and serum is taken to measure uric acid content.

3, statistical procedures

Data mean soil standard deviationIt indicates, unites using Excel7.0 and SPPS13.0for windows Meter software is analyzed, and comparison among groups are examined with q, is compared before and after medication and is examined with self pair t, and P < 0.001 indicates that difference has Significant meaning.

4, experimental result

Influence of the 5 A series compound of table to uricacidemia rat blood serum uric acid content

^*P < 0.001 and model group ratio.

Inhibit uricolytic large and small mouse uricacidemia model by establishing, drug screening carried out to A series compound, And compared with the effect of the reduction serum uric acid of positive drug Allopurinol.The results show that compared with model group, Allopurinol, A1-1, A2-1, A3-1, A4-1 are significantly reduced the effect of serum uric acid, and the anti-trioxypurine effect phase of low dose group and Allopurinol When, and the effect that high dose group reduces serum uric acid is better than positive control drug Allopurinol, and has dose-effect relationship (to be shown in Table 5)。

Other compounds of the present invention are detected using the above method, result is consistent with the above, the A series Compound all has the apparent effect for reducing serum uric acid.

The experiment of 8 given the test agent Oral Acute Toxicity of embodiment

Given the test agent:

Given the test agent (Allopurinol, A1-1, A2-1, A3-1 and A4-1)

Preparation method: suspension is made into the grinding of 0.2% compound

Animal subject:

ICR mouse.

Weight: 18-22g.

Number of animals: 300.

Dosage setting:

Trial test shows that Allopurinol, A1-1, A2-1, A3-1 and A4-1 have certain toxicity, 1A1-1, A2-1, and A3-1 exists 600mg/kg dosage can cause 4/4 dead mouse, and Allopurinol and A4-1 cause 4/ under 200 and 400mg/kg dosage respectively 4 dead mouses.On the basis of preliminary experiment, each tested material formal test dosage setting is as follows:

The setting of each tested material formal test dosage of table 6

Administration route:

Gastric infusion (ig).

Test method:

Test room environmental: 24 ± 2 DEG C of room temperature, relative humidity 60~70%.

Observation index: compound is made into the drug of respective concentration by above-mentioned dosage according to administration volume by proportional diluted method Solution etc. holds ig and is administered once, and the various poisoning symptoms of record mouse and death condition, dead animal perform an autopsy on sb..

Observation period: 14 days.

Test result:

1, abnormal response:

Mouse ig Allopurinol, A1-1, A2-1, after five compounds of A3-1 and A4-1 within 12h, only Allopurinol group high dose Dosed portions animal appearance activity is reduced, other group of no abnormality seen；Administration 24 in each dosage group of A1-1, A2-1, A3-1, A4-1 not See animal dead, Allopurinol part high dose group animal dead；Subsequent each group has animal to occur death successively, is administered the 6th day Each group surviving animals have no dead afterwards.Dead animal and the rarely seen activity reduction of surviving animals and syntexis, have no that other are obvious abnormal.

2. autopsy findings:

The visible bilateral renal lighter of dead animal postmortem, the retention of urine, white content (compound) remains in stomach, Other organs are shown no obvious abnormalities, but A1-1, A2-1, and the ratio and degree of the internal organs exception of A3-1 and A4-1 group are significantly lower than Allopurinol group；Surviving animals postmortem shows that only the still visible kidney lighter of Allopurinol group Some Animals, the retention of urine, caecum increase Greatly, the symptoms such as intestinal tympanites, and A1-1, A2-1, it is abnormal that A3-1 and A4-1 compound group surviving animals postmortem is showed no obvious internal organs Change.

The death condition and LD of mouse ig compound₅₀Value.

The LD of 7 mouse ig compound of table₅₀Value (uses Bliss method^[1]It calculates)

The Allopurinol used in this test of # is to the LD50 of mouse compared with the LD50 value for the Allopurinol that standard literature is reported It is close, Allopurinol LD50 value reported in the literature be 78mg/kg (I.M.Ovcharova, I.M.Zasosova, L.N.Gerchikov,M.E.Shuvalova,E.S.Golovchinskaya,and S.S.Liberman.ALLOPURINOL, ITS SYNTHESIS AND PHARMACOLOGICAL ACTIVITY, Pharmaceutical Chemistry Journal (English translation) .Translation of KHFZAN.1973,7 (11), 735-736).

Brief summary: the safety of compound A1-1~A4-1 is significantly better than Allopurinol.

Other A series compounds of the present invention are detected using identical method, the results show that other A systems The safety of column compound is also significantly better than Allopurinol.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Industrial applicibility

Pyrazolo [3,4-d] pyrimidin-4-one-derivatives of the present invention or its stereoisomer or its salt, with Allopurinol Compared to more preferably anti-trioxypurine effect and more preferably safety.

Claims (2)

1. the preparation method of pyrazolo [3,4-d] pyrimidin-4-one-derivatives shown in a kind of formula (1) or formula (2) or its salt:

It is characterized in that, in a suitable solvent, formula (3) compound or formula (4) compound and formula (5) compound presence/ There is no reacting to obtain under conditions of activator,

G in formula (1), formula (2) and formula (5) is selected from:

The activator is selected from thionyl chloride, oxalyl chloride, dicyclohexylcarbodiimide or 1- (3- dimethylamino-propyl) -3- ethyl One or more of carbodiimide hydrochloride；

The suitable solvent is selected from acetonitrile, acetone, tetrahydrofuran, methylene chloride, chloroform, carbon tetrachloride, formamide, N, Or mixtures thereof dinethylformamide, dimethyl sulfoxide, ethyl acetate, methyl acetate.

2. the preparation method of pyrazolo [3,4-d] pyrimidin-4-one-derivatives or its salt shown in a kind of formula (1 ') or formula (2 '):

It is characterized in that, in a suitable solvent, formula (3 ') compound or formula (4 ') compound are being deposited with formula (5 ') compound / activator is not present under conditions of react and obtain,

G ' in formula (1 '), formula (2 ') and formula (5 ') is selected from:

The activator is selected from thionyl chloride, oxalyl chloride, dicyclohexylcarbodiimide or 1- (3- dimethylamino-propyl) -3- ethyl One or more of carbodiimide hydrochloride；

The suitable solvent is selected from acetonitrile, acetone, tetrahydrofuran, methylene chloride, chloroform, carbon tetrachloride, formamide, N, Or mixtures thereof dinethylformamide, dimethyl sulfoxide, ethyl acetate, methyl acetate.