CN1177960A - 6-Substituted pyrazolo[3,4-d]pyrimidin-4-ones, compositions and methods of use thereof - Google Patents

Abstract

6-Substituted pyrazolo[3, 4-d] pyrimidin-4-one derivatives, pharmaceutical compositions containing them and methods for a) effecting c-GMP-phosphodiesterase inhibition, b) treating heart failure and hypertension, c) reversing or reducing nitrate-induced tolerance and d) treating angina pectoris, congestive heart disease and myocardial infarction utilizing them.

Description

6-Substituted pyrazolo[3,4-d]pyrimidin-4-ones and compositions and methods of use thereof

Background of the Invention

(a) Field of Invention

The present invention relates to 6-substituted pyrazolo[3,4-d]pyrimidin-4-ones, to pharmaceutical compositions containing them and to their use for a) inhibition of c-GMP-phosphodiesterase, b) treatment of heart failure and/or hypertension, c) reversing or alleviating nitrate-induced drug resistance and d) methods of treating angina, congestive heart disease and myocardial infarction.

(b) Publication status of references

其中：US Patent No. 3,165,520 issued January 12, 1965 by Schmidt et al. discloses pyrazolo[3,4-d]pyrimidines as coronary vasodilators with the general formula:

in:

^R represents a hydrogen atom or an alkyl, hydroxyalkyl, haloalkyl or oxaalkyl or cycloalkyl, cycloalkylalkyl, aralkyl or heterocyclylalkyl group or in most cases a dinuclear aryl group or heterocyclyl;

R ³ represents a hydrogen atom or a lower alkyl group;

^R represents an aliphatic group, a cycloaliphatic group, a cycloaliphatic group-aliphatic group, an arylaliphatic group or a heterocyclic aliphatic group; and

R ⁶ represents an aliphatic group or an aralkyl group or a heterocyclylalkyl group which may be substituted.

This patent more specifically discloses wherein R ¹ represents a hydrogen atom or a lower alkyl or cycloalkyl. Hydroxy-lower alkyl or halogenated lower alkyl, oxa-lower alkyl, or mono-, which may be replaced by halogen, alkoxy, alkyl, methylenedioxy, trifluoromethyl, nitro, amino or pyridyl, Di-, or tri-substituted aryl; R ³ represents a hydrogen atom or a lower alkyl; R ⁵ represents a lower alkyl or a lower alkylamino; and R ⁶ represents a special value of a compound of a lower alkyl or aralkyl.

It further discloses a series of 1-R ¹ -3-R ³ -4-hydroxy-6-R ⁶ -pyrazolo[3,4-d]pyrimidines as intermediates for the synthesis of the final product. Specifically disclosed among these intermediates are 1-cyclopentyl-4-hydroxy-6-benzyl-pyrazolo[3,4-d]pyrimidine and 1-isopropyl-4-hydroxy-6-m-methanol Oxybenzylpyrazolo[3,4-d]pyrimidine.

US Patent No. 3,211,731 issued October 12, 1965 by Schmidt et al. discloses pyrazolo[3,4-d]pyrimidines as coronary vasodilators with the general formula: in;

^R represents hydrogen, alkyl, hydroxyalkyl, haloalkyl or oxaalkyl, cycloalkyl, cycloalkylalkyl, aralkyl, heterocyclylalkyl or in most cases dinuclear aryl or heteroalkyl ring group;

^R3 represents hydrogen, or secondly, lower alkyl; and

R ⁶ represents aralkyl or heterocyclylalkyl which may be substituted.

The patent more specifically discloses wherein R ¹ represents a hydrogen atom or a lower alkyl, cycloalkyl, hydroxy lower alkyl, halogenated lower alkyl, oxa lower alkyl, or aryl; R ³ represents a hydrogen atom or a lower Alkyl and ^R6 is a special value for compounds of substituted or unsubstituted aralkyl. Among these compounds, 1-isopropyl-4-hydroxy-6-(3'-methoxyphenylmethyl)pyrazolo[3,4-d]pyrimidine, 1-cyclopentyl-4 -Hydroxy-6-benzylpyrazolo[3,4-d]pyrimidine, 1-isopropyl-4-hydroxy-6(β-phenethyl)pyrazolo[3,4-d]pyrimidine and 1 -Isopropyl-4-hydroxy-6-(4-aminobenzyl)pyrazolo[3,4-d]pyrimidine.

US Patent 3,211,732 issued October 12, 1965 to Schmidt et al. discloses 1-R ¹ -3-R ³ -6-R ⁶ -4-hydroxy-pyrazolo[3,4 -d] pyrimidine, where:

R ¹ represents a hydrogen atom, a lower alkyl unsubstituted or substituted by hydroxyl or lower alkyloxy, or a cyclopentyl or cyclohexyl or phenyl or phenyl lower alkyl;

R ³ represents a hydrogen atom or a lower alkyl group; and

R ⁶ represents substituted or unsubstituted phenyl lower alkyl.

In particular 1-isopropyl-4-hydroxy-6-benzylpyrazolo[3,4-d]pyrimidine is disclosed.

Also disclosed as an intermediate is 1-R ¹ -3-R ³ -6-R ⁶ -4-hydroxypyrazolo[3,4-d]pyrimidine, wherein:

R ¹ represents a hydrogen atom, lower alkoxy lower alkyl or hydroxy lower alkyl, cyclopentyl or cyclohexyl or phenyl or phenyl lower alkyl which may be substituted;

^R3 is as defined above; and

R ⁶ represents a phenyl group which may be substituted. In particular 1-isopropyl-4-hydroxy-6-phenylpyrazolo[3,4-d]pyrimidine is disclosed.

其中：US Patent No. 3,732,225, Breuer et al., issued May 8, 1973, discloses pyrazolo[3,4-d]pyrimidines of the general formula as hypoglycemic agents and anti-inflammatory agents:

in:

R is hydrogen or lower alkyl; ^R is lower alkyl, cycloalkyl, phenyl or substituted phenyl; R is ^phenyl , substituted phenyl or cycloalkyl; and ^R is hydrogen, lower alkyl, Cycloalkyl, phenyl or substituted phenyl. In particular 1-methyl-6-phenyl and 1-methyl-6-(4-chlorophenyl)pyrazolo[3,4-d]pyrimidin-4-one are disclosed.

US Patent No. 3,350,397, Burch, issued October 31, 1967, discloses pyrazolo[3,4-d]pyrimidines of the general formula as antibacterial agents: in:

R represents hydroxy, chloro or -N(X)(Y), where X represents hydrogen, (lower) alkyl, hydroxy(lower) alkyl, (lower) alkoxy(lower) alkyl or amino; Y represents hydrogen , hydroxy(lower)alkyl, (lower)alkoxy(lower)alkyl or morpholinopropyl; X and Y together with N represent pyrrolidinyl; and ^R represents (lower)alkyl or methoxyethyl base.

The patent further discloses 4-amino and 4-hydroxy-1-R ¹ -6-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidine as intermediates. Burch in J. Med. Chem. 1968, 11, 79 further exemplifies the preparation of intermediates and the preparation and biological tests of final products.

British Patent 937,722, published on September 25, 1963, belongs to CIBALIMITED and discloses 1-isopropyl-4-hydroxy-6-benzyl-pyrazolo[3,4-d] as a coronary vasodilator pyrimidine.

其中：US Patent No. 4,666,908 issued May 19, 1987 to Hamilton (Hamilton) discloses pyrazolo[4,3-d]pyrimidin-7-ones of the general formula:

in:

^R includes lower alkyl of one to six carbon atoms, including lower alkylene of one to six carbons, including lower hydroxyalkyl of one to six carbons, including lower hydroxyalkylene of two to six carbons , a lower aminoalkyl group comprising one to six carbon atoms, or a lower aminoalkylene group comprising two to six carbon atoms;

n is 0-4; and

Ar is _R2 : Or 2,3, or 4-pyrimidinyl, wherein X, Y and Z are each (1) hydrogen; (2) lower alkyl comprising one to six carbons; (3) halogen; (4) hydroxyl; (5 ) lower alkoxy comprising one to six carbons; (6) nitro; (7) amino; (8) NR'R" wherein R' and R" are each (a) hydrogen or (b) comprising one to Lower alkyl of six carbons optionally substituted by (i) amino, (ii) morpholino, or (iii) cycloalkyl including five to seven carbons, (9) sulfonyl or (10) _-SO2NR'R ", wherein R' and R" are as defined above.

This patent more specifically discloses preferred compounds wherein Ar is _R2 . These compounds are useful in the treatment of cardiovascular diseases.

其中：Miyashita et al. Heterocycles (Heterocycles) 1990., 31, 1309-1341 disclose the preparation of a series of pyrazolo[3,4-d]pyrimidines of the general formula:

in:

R is phenyl or methyl; and ^R1 is hydrogen, methyl, ethyl, propyl, isopropyl, benzyl, ethyl carboxylate or phenyl. Its use is not disclosed.

Hamilton's PCT application WO88/00192, published January 14, 1988, discloses a series of 5-substituted pyrazolo[4,3-d]pyrimidin-7-one derivatives, these compounds Can be used as cardiotonic, CNS stimulant, antiallergic, antiasthmatic or recognition activator.

European patent application 0463756 published on January 2, 1992 by Bell et al. discloses a series of 5-(2,5-disubstituted phenyl)pyrazolo[4,3-d]pyrimidine-7- Ketones, these compounds can be used in the treatment of cardiovascular diseases.

US Patent 3,847,908, Breuer and Treuner, issued November 12, 1974, discloses compounds of the formula: in:

R ¹ is lower alkyl, cyclolower alkyl, or phenyl-lower alkyl;

^R is hydrogen or lower alkyl; and

R ³ is hydrogen, lower alkyl, halogen, or trifluoromethyl. Specifically disclosed are 1,3-dimethyl-6-styryl-pyrazolo[3,4-d]pyrimidine, 1-cyclopentyl-6-(2-chlorostyryl)pyrazolo[3 , 4-d]pyrimidine, 1-cyclohexyl-3-methyl-6-(styryl)pyrazolo[3,4-d]pyrimidine and 1,3-diethyl-6-(4-chloro styryl)pyrazolo[3,4-d]pyrimidine. The compound is said to be useful as an antimicrobial agent and has anti-inflammatory and membrane stabilizing properties.

US Patent 4,260,758 issued April 7, 1981 by Morrison et al. discloses the preparation of compounds of the formula: Wherein ^R is selected from lower alkyl, phenyl, phenyl substituted by one or more hydroxyl or lower alkoxy, or pyridyl. Use of this compound is not disclosed.

Canadian Patent 754,565, Burch, issued March 14, 1967, discloses a series of 4-substituted-1-alkyl-6-(2-furyl)-1H-pyrazolo[3,4-d]pyrimidines, The compound is said to be useful in the preparation of 4-substituted-1-alkyl-6-(5-nitro-2-furyl)-1H-pyrazolo[3,4-d]pyrimidines capable of inhibiting bacterial growth intermediates.

其中：US Patent No. 3,772,294 issued November 13, 1973 to Podesva et al. discloses the preparation of compounds of formula I:

in:

X represents a halogen atom, a free or substituted hydroxyl group, an amino group or a mercapto group and R represents a hydrogen atom, or a lower alkyl group or a substituted or unsubstituted aryl group. This document discloses that these compounds can be effectively used to treat hyperuricemia associated with gout, and in addition, the compounds wherein X represents a halogen atom can be used as intermediates for the synthesis of other compounds of formula I. This document also specifically discloses 4-hydroxy-6-phenyl-1-pyrazolo[3,4-d]pyrimidine.

US Patent 5,075,310 published on December 24, 1991 by Coates and Rawlings (this patent is from serial application No. 370,494 published on June 23, 1989) discloses and claims the following compound and its pharmaceutically acceptable salt; in: For the following formula (a), (b) or (c) ring: X is oxygen or sulfur; and R is _{C 1} ^-C ₆ alkyl, C _2-6 alkenyl, C _3-5 cycloalkyl-C _1-4 alkyl, or substituted by 1 to 6 fluorine C _1-4 alkyl. This document specifically discloses 6-(2-propoxyphenyl)pyrazolo[3,4-d]pyrimidin-4(5H)-one. The compound is said to act as a bronchodilator and vasodilator.

SUMMARY OF THE INVENTION The present invention relates to compounds of formula I or pharmaceutically acceptable acid addition salts and/or hydrates thereof: in:

R ¹ is tert-butyl, or cyclopentyl;

R ³ is methyl, ethyl, or benzyl;

X is _-CH2- , -O-, or -NH-; and

R ⁶ is phenyl (or from one to three, the same or different, selected from lower alkoxy, hydroxy, halogen, carboxy lower alkoxy, 4-morpholinyl lower alkoxy, 5-tetrazolyl lower Substituents of alkoxy, di-lower alkylamino, trifluoromethyl, nitro, amino, lower alkanesulfonylamino, di-lower alkylamino-lower alkylphenylcarbonyloxy, and 1-imidazolyl phenyl); or when X is -CH ₂ -, R ⁶ is additionally 2-, 3-, or 4-pyridyl, 1-pyrrolyl, 1-benzimidazolyl, 1, 2, 3, 4 -tetrahydro-2-isoquinolinyl, 1,2,3,4-tetrahydro-1-quinolinyl, hydroxyl, 1-imidazolyl, 1-lower alkyl-2-,3-,4-, or 5-pyrrolyl, 1-pyrazolyl, 3-, 4-, or 5-isoxazolyl (or 3-, 4-, or 5- isoxazolyl), 2-thienyl, or 3-thienyl.

Compounds of formula I have been found to have c-GMP-PDEV inhibitory activity and are therefore useful in the treatment of heart failure and/or hypertension. It has also been found that the compounds of formula I in combination with nitrates can be used to reverse or reduce nitrate-induced drug resistance and thus can further be used in the treatment of angina pectoris, congestive heart disease and myocardial infarction.

Preferred compounds of formula I are among them

R ¹ , R ³ and X are as defined above, while

R ⁶ is phenyl (or from one to three, the same or different, selected from lower alkoxy, hydroxy, carboxy lower alkoxy, 4-morpholino lower alkoxy, 5-tetrazolyl-lower alkane Substituents of oxy, di-lower alkylamino, trifluoromethyl, nitro, amino, lower alkylsulfonylamino, di-lower alkylamino-lower alkylphenylcarbonyloxy, and 1-imidazolyl phenyl); or when X is -CH ₂ -, R ⁶ is additionally 2-, 3-, or 4-pyridyl, 1-pyrrolyl, 1-benzimidazolyl, 1,2,3,4- Tetrahydro-2-isoquinolyl, 1,2,3,4-tetrahydro-1-quinolyl, hydroxyl, 1-imidazolyl, 1-lower alkyl-2-pyrrolyl, 1-pyrazolyl , 4-isoxazolyl), 2-thienyl, or 3-thienyl compounds substituted by lower alkyl on any possible carbon atom.

Particularly preferred compounds of formula I are those in which:

R ¹ , R ³ and X are as defined above, and

R ⁶ is phenyl (or from one to three, the same or different, selected from methoxy, hydroxyl, carboxymethoxy, 2-(4-morpholinyl) ethoxy, 1-(5-tetrazole substituted phenyl) ; or when X is -CH ₂ -, R ⁶ is additionally 2-, 3-, or 4-pyridyl, 1-pyrrolyl, 1-benzimidazolyl, 1,2,3,4-tetrahydro- 2-isoquinolinyl, 1,2,3,4-tetrahydro-1-quinolinyl, hydroxyl, 1-imidazolyl, 1-methyl-2-pyrrolyl, 1-pyrazolyl, 3,5 -Dimethyl-4-isoxazolyl), 2-thienyl, or 3-thienyl compounds.

More particularly preferred compounds of formula I above are those wherein R ¹ is cyclopentyl; R ³ is ethyl; and X and R ⁶ are as defined above.

Preferred specific compounds of the present invention are:

1-cyclopentyl-3-ethyl-6-(4-methoxybenzyl)pyrazolo[3,4-d]pyrimidin-4-one,

1-cyclopentyl-3-ethyl-6-(4-hydroxybenzyl)pyrazolo[3,4-d]pyrimidin-4-one,

1-cyclopentyl-3-ethyl-6-(benzyl)pyrazolo[3,4-d]pyrimidin-4-one, and

1-cyclopentyl-3-ethyl-6-(4-aminobenzyl)pyrazolo[3,4-d]pyrimidin-4-one.

The present invention also relates to a pharmaceutical composition comprising a compound of formula I and a pharmaceutically acceptable carrier, adjuvant, diluent, or excipient.

The present invention also relates to a method of inhibiting cGMP-phosphodiesterase in a mammal comprising administering to the animal an effective amount of a compound of formula I.

The present invention also relates to a method of treating heart failure and/or hypertension in a mammal, which comprises administering to the animal an effective amount of a compound of formula I.

The present invention also relates to a method for reversing or alleviating nitrate-induced drug resistance in a mammal receiving nitrate treatment, which comprises administering to the animal an effective amount of a compound of formula I.

The present invention also relates to a method of treating angina, congestive heart disease and myocardial infarction in a mammal comprising administering to the animal an effective amount of a compound of formula I in combination with a nitrate.

A detailed description of the preferred preparation scheme

Compounds of formula I can be combined with the corresponding enol forms: Exist in tautomeric equilibrium, except that the ketone form is considered to be dominant and all are expressed as this form, the present invention obviously includes both forms and mixtures thereof.

The term lower alkyl as used herein means a straight or branched chain hydrocarbon group having from one to about four carbon atoms, including methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, and the like.

The term lower alkoxy as used herein denotes straight or branched chain alkoxy substituents having one to about four carbon atoms, including methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxyl, et al.

The term halogen, halide or halo as used herein means bromine, chlorine, iodine or fluorine.

在约0℃到约室温的温度下，在合适的碱，如吡啶的存在下，非强制性地在合适的溶剂，如氯仿的存在下，用过量的合适地取代的式III酰氯，其中X′为卤素，优选氯，处理合适地取代的式II 5-氨基-1H-吡唑-4-甲腈，得到式IV羧酰胺。选择地，也可以通过在合适的溶剂，如二甲基甲酰胺(DMF)中。用式II 5-氨基-1H-吡唑-4-甲腈处理过量的式R⁶CH₂COOH酸或其酸加成盐与过量碱，如氢化钠，以及过量的合适的偶合剂，如N，N′-碳酰二咪唑的混合物而制得式IV羧酰胺。然后可在过量碱，优选氢氧化钠，或甲醇钠的存在下，在溶剂如水，低级链烷醇，或水/低级链烷醇混合物中，当甲醇钠用作碱时，优选乙醇，当氢氧化钠用作碱时，优选水/乙醇混合物，在以约0℃到所用溶剂或溶剂混合物的沸点的温度下，用过量的过氧化氢处理式IV羧酰胺，得到其中X为-CH₂-的式I化合物。The synthesis of compounds of the present invention wherein X is -CH ₂ - can be carried out as shown in Scheme A: Scheme A

In the presence of a suitable base, such as pyridine, optionally in the presence of a suitable solvent, such as chloroform, an excess of a suitably substituted acid chloride of formula III, wherein X ' is a halogen, preferably chlorine, and treatment of a suitably substituted 5-amino-1H-pyrazole-4-carbonitrile of formula II affords a carboxamide of formula IV. Alternatively, passage in a suitable solvent such as dimethylformamide (DMF) is also possible. Treatment of an excess of an acid of formula R ⁶ CH ₂ COOH or an acid addition salt thereof with 5-amino-1H-pyrazole-4-carbonitrile of formula II with excess base, such as sodium hydride, and an excess of a suitable coupling agent, such as N , the mixture of N'-carbonyl diimidazole to prepare formula IV carboxamide. Then, in the presence of excess base, preferably sodium hydroxide, or sodium methoxide, in a solvent such as water, lower alkanol, or water/lower alkanol mixture, when sodium methoxide is used as base, preferably ethanol, when hydrogen When sodium oxide is used as the base, preferably a water/ethanol mixture, treatment of the carboxamide of formula IV with an excess of hydrogen peroxide at a temperature from about 0°C to the boiling point of the solvent or solvent mixture used gives the carboxamide in which X is _-CH2- The compound of formula I.

Alternatively, compounds of formula I wherein X is -CH ₂ - can be prepared as shown in Scheme B: Scheme B In a lower alkanol solvent, preferably ethanol, at a temperature from about room temperature to the boiling point of the solvent used, with an excess of a base, such as an alkali metal lower alkanolate, preferably sodium ethoxide, and an excess of a suitably substituted formula VI Treatment of suitably substituted 5-amino-1H-pyrazole-4-carboxamides of formula V, wherein R is lower alkyl, preferably methyl or ethyl, with esters, affords compounds of formula I, wherein X is _-CH2- .

在从约80℃到所有溶剂的沸点，优选约150℃到约160℃的温度下，在合适的有机溶剂，如N-甲基-2-吡咯烷酮中，用过量的o-乙基黄原酸盐，如钾盐处理合适地取代的式V 5-氨基-1H-吡唑-4-甲酰胺，得到式V II6-硫代吡唑并[3，4-d]嘧啶-4-酮。然后在过量碱，如K₂CO₃或氢化钠的存在下。在合适的有机溶剂，如二甲基甲酰胺中，在从约0℃到约室温的温度下，用过量合适的甲基化试剂，如甲基碘或硫酸二甲酯处理式VII化合物，得到式VII 6-(甲硫基)吡唑并[3，4-d]嘧啶-4-酮。然后可在合适的溶剂，如氯仿中，在约室温的条件下，用过量合适的氧化剂，如m-氯过氧苯甲酸处理式VIII化合物，得到式IX 6-(甲基磺酰基)吡唑并[3，4-d]嘧啶-4-酮。在从约室温到约190℃，优选从约170℃到190℃的温度下，在过量碱，如氢化钠，的非强制性地存在下，用过量式X的R⁶NH₂衍生物，或过量的式XI的R⁶OH衍生物处理式IX衍生物，得到其中X为-O-或-NH-的式I化合物。Wherein X is -O-, or the compound of formula I of -NH- can be prepared as shown in Scheme C: Scheme C

O-Ethylxanthic Acid in excess of Treatment of suitably substituted 5-amino-1H-pyrazole-4-carboxamides of formula V with a salt, such as the potassium salt, affords 6-thiopyrazolo[3,4-d]pyrimidin-4-ones of formula VII. Then in the presence _of excess base, such as _K2CO3 or sodium hydride. Treatment of a compound of formula VII with an excess of a suitable methylating agent, such as methyl iodide or dimethyl sulfate, in a suitable organic solvent, such as dimethylformamide, at a temperature from about 0°C to about room temperature gives Formula VII 6-(methylthio)pyrazolo[3,4-d]pyrimidin-4-one. Compounds of formula VIII can then be treated with an excess of a suitable oxidizing agent, such as m-chloroperoxybenzoic acid, in a suitable solvent, such as chloroform, at about room temperature to afford 6-(methylsulfonyl)pyrazoles of formula IX And[3,4-d]pyrimidin-4-one. at a temperature from about room temperature to about 190°C, preferably from about 170°C to 190°C, in the optional presence of an excess of a base, such as sodium hydride, with an excess of the ^R6NH2 derivative of formula X, _or Treatment of derivatives of formula IX with excess ^R6OH derivatives of formula XI affords compounds of formula I wherein X is -O- or -NH-.

Changes in the functional groups of the compounds of formula I can be achieved using simple chemical transformations known or routine to those skilled in the art of chemistry. For example, dealkylation of aryl esters gives the corresponding phenol derivatives, treatment of aryl derivatives with formaldehyde and di-lower alkylamines gives the corresponding di-lower alkylaminomethyl derivatives, and treatment with methanesulfonate in the presence of base Treatment of a compound of formula I wherein X is _-CH2- and ^R6 is OH with an acid chloride affords the corresponding mesylate, which is treated with 1-acetylimidazole or pyrazole to yield wherein X is _-CH2- and ^R6 is The formula I compound of 1-imidazolyl or 1-pyrazolyl, catalytic reduction of nitro derivatives to obtain corresponding amines, sulfonated amines with lower alkylsulfonyl halides to obtain corresponding lower alkylsulfonamides, and in coupling agent, For example, N,N'-carbonyldiimidazole, treatment of phenol with acid in the presence gives the corresponding ester.

The compounds of formula I can be used in either free base form or in the form of acid addition salts, both of which are within the scope of the present invention. The acid addition salt form is the more convenient form to work with; indeed, the use of the salt form is itself equivalent to that of the base form. Acids useful in the preparation of acid addition salts preferably include those which, in combination with the free base, yield pharmaceutically acceptable salts, i.e., salts whose anions are relatively harmless to the animal body at pharmaceutical dosages of the salt such that The beneficial properties of the free base itself are not affected by the side effects of the anion. Forms convenient to use in the practice of this invention are the free base form or the hydrochloride, fumarate, tosylate salts. Methanesulfonate or maleate. However, other suitable pharmaceutically acceptable salts within the scope of this invention may be formed from other inorganic and organic acids. Acid addition salts of basic compounds can be prepared by standard methods known in the art including, but not limited to, dissolving the free base in an aqueous alcoholic solution containing the appropriate acid and evaporating the solution to isolate the salt, or in an organic solvent. The acid is reacted with the free base, wherein the salt is isolated directly, or precipitated with a second organic solvent, or can be obtained by concentrating the solution. The present invention includes all acid addition salts, although the pharmaceutically acceptable salts of basic compounds are preferred. All acid addition salts can be used as a source of free base forms, and even some specific salts are themselves required only as intermediates, eg, for purification or identification purposes. Alternatively, the salts are used as intermediates in the preparation of pharmaceutically acceptable salts by, for example, ion exchange.

Suitably substituted 5-amino-1H-pyrazole-4-carbonitriles of formula II and suitably substituted 5-amino-1H-pyrazole-4-carboxamides of formula V are known and can therefore be obtained as Preparation is known in the art (see, eg, US Patent 5,294,612, issued March 15, 1994, the entire contents of which are hereby adapted from reference), or may be prepared as described in the Examples below. Acid chlorides of formula III, esters of formula VI, R ⁶ NH ₂ derivatives of formula X and R ⁶ OH derivatives of formula XI can be obtained on the market, or can be prepared by methods known in the art, or can be prepared according to the following examples method of preparation.

The structures of the compounds of the present invention can be confirmed by synthetic means and by one or more methods selected from elemental analysis, infrared, nuclear magnetic resonance and mass spectrometry. The course of the reaction and the identity and homogeneity of the product can be identified by one or more methods selected from thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), or gas liquid chromatography (GLC).

The following examples will further illustrate the invention, but it does not limit the invention. All melting points (m.p.) are given in degrees Celsius (°C), uncorrected.

Example 1

(a)

Phenylacetyl chloride (10.6ml, 0.08mol) was added to stirring 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-methanol over a period of 20 minutes in an ice bath Nitrile (8.2g, 0.04mol) in solution in pyridine (100ml). The reaction mixture was stirred at this temperature for 2 hours, then at room temperature overnight. The solvent was removed in vacuo and the residue was partitioned between chloroform (200ml) and water (200ml). The organic layer was separated and concentrated in vacuo to give an oil, which was chromatographed on a silica gel column, eluting with hexane/ether (1/1), and recrystallized from ether/hexane to give 8.3 g (64%) of white flakes 1-cyclopentyl-3-ethyl-4-cyano-5-(benzylcarbonylamino)-1H-pyrazole.

(b) 1-cyclopentyl-3-ethyl-4-cyano-5-(benzylcarbonylamino)-1H-pyrazole (2.4g, 7.4mmol), ethanol (100ml), 30%H A mixture of ₂ O ₂ (4.5ml, 40mmol), NaOH (0.3g, 7.5mmol) and water (10ml) was stirred at room temperature for 1 hour, then refluxed for 1 hour. A further portion of 30% _H2O2 ( _2.5ml ) was added and the mixture was refluxed for a further 1 hour. The solvent was concentrated in vacuo, the residue was treated with water (25 mL) and acetic acid (3 mL), the yellow precipitate formed was collected by filtration, washed with water and recrystallized from isopropanol to give 0.79 g of 1-cyclopentyl-3 as orange needles -Ethyl-6-(benzyl)-pyrazolo[3,4-d]pyrimidin-4-one. mp175-176°C.

Alternatively, this product can also be prepared by dissolving sodium (2.12g) in ethanol (145ml) and adding 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4 - Formamide (10 g, 45 mmol). Ethyl phenylacetate (2.8 g) was then added and the reaction mixture was refluxed overnight. The reaction mixture was cooled to room temperature, stripped, then water was added to the residue followed by 2N HCl. The product was collected by filtration and recrystallized from ethyl acetate to give 10.52 g (73%) of 1-cyclopentyl-3-ethyl-6-(benzyl)-pyrazolo[3,4-d]pyrimidine-4 -ketone.

Example 2

(a)

A solution of phenylacetyl chloride (2.3ml, 17.2mol) in _CHCl3 (10ml) was added to stirred 1-cyclopentyl-3-benzyl-5-amino over 20 minutes under ice-bath cooling - In a solution of 1H-pyrazole-4-carbonitrile (2.3g, 8.6mmol), CHCl ₃ (50ml) and pyridine (20ml). The reaction mixture was stirred in an ice bath for 3 hours, then at room temperature for 2 hours. The solvent was removed in vacuo, the residue was partitioned between _CHCl3 (100ml) and water (50ml), and the organic layer was separated and concentrated to give a gummy solid which was recrystallized from isopropanol to give 1.1g (88 %) White needle crystal 1-cyclopentyl-3-benzyl-4-cyano-5-(benzylcarbonylamino)-1H-pyrazole, mp166-168°C.

(b)

A solution of 1-cyclopentyl-3-benzyl-4-cyano-5-(benzylcarbonylamino)-1H-pyrazole (1.1g, 2.8mmol) in ice-cooled ethanol (30ml) 30% H ₂ O ₂ (2.5 ml) was added to , followed by NaOH (100 mg) dissolved in water (5 ml), the reaction mixture was stirred at this temperature for 1 hour, then at room temperature for 1 hour, then in a steam bath After heating for 3.5 hours, it was finally stirred overnight at room temperature. The reaction mixture was concentrated to dryness and partitioned between water (25ml) and _CHCl3 (50ml). The organic layer was separated, concentrated in vacuo, and the oily residue was crystallized from isopropanol to afford 234 mg (22%) of 1-cyclopentyl-3-benzyl-6-(benzyl)-pyrazolo[ 3,4-d]pyrimidin-4-one, mp 204-206°C.

Example 3

(a)

A mixture of 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carbonitrile (4.50g, 0.022mol), pyridine (5.21g, 0.066mol), and CHCl ₃ (75ml) Stir in the ice bath for 0.5 hours, then add 3,4-dimethoxyphenylacetyl chloride (9.44 g, 0.044 mol) in _CHCl3 (25 ml) over 3 hours. The reaction mixture was stirred at room temperature overnight, the solvent was removed in vacuo and the residue was partitioned between _CHCl3 (250ml) and water. After the layers were separated, the aqueous layer was extracted with _CH2Cl2 (2 x 150ml) and the combined organic layers _were concentrated in vacuo. The residue was dissolved in CHCl ₃ (20 ml) and purified by column chromatography on silica gel, eluting with ether, to give 1-cyclopentyl-3-ethane as white needles after recrystallization from hexane/CHCl ₃ (10/1). yl-4-cyano-5-[(3,4-dimethoxybenzyl)carbonylamino]-1H-pyrazole.

(b)

In 1-cyclopentyl-3-ethyl-4-cyano-5-[(3,4-dimethoxybenzyl)carbonylamino]-1H-pyrazole (1.0g, 2.7mmol), ethanol (500ml) and _NaOCH3 (0.3g) was added 30% _H2O2 ( _4ml ). The reaction mixture was stirred overnight at room temperature, an additional 30% _H2O2 (3 equiv) was added and the reaction mixture was refluxed on a steam _bath for 1 hour. Starting material _was still present, so an additional 3 equivalents of 30% _H2O2 were added and the reaction mixture was refluxed for 4 hours. The reaction mixture was stripped to dryness, treated with acetic acid and ethanol, and stripped to dryness again. The oily residue was dissolved in ethanol, water was added, and the solution was cooled with an ice bath. The resulting solid was collected by filtration and dried at 90 °C to afford 0.3 g (30%) of 1-cyclopentyl-3-ethyl-6-(3,4-dimethoxybenzyl)-pyridine as a pale yellow solid Azolo[3,4-d]pyrimidin-4-one, 1/10 hydrate, mp 148-149°C.

Example 4

(a)

NaH (1.0 g, 25 mmol, 60% dispersion in mineral oil) was added to a mixture of 4-pyridineacetic acid hydrochloride (4.3 g, 25 mmol) and DMF (50 mL) under cooling in an ice bath. The resulting mixture was stirred for 30 minutes, then N,N'-carbonyldiimidazole (4.0 g, 24.6 mmol) was added and after 30 minutes 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole was added - 4-carbonitrile (4.1 g, 20 mmol). The reaction mixture was stirred at the same temperature for 4 hours, then at room temperature for 2 days and finally heated on a steam bath for 2 hours. The reaction mixture was concentrated to dryness and water (50ml) and acetic acid (5ml) were added. The mixture was extracted with _CH2Cl2 ( _300ml ), the organic layer was concentrated in vacuo and the resulting oil was crystallized from ether and purified by column chromatography on silica gel eluting with ether to afford 2.8g (35%) of 1-cyclo as a viscous oil pentyl-3-ethyl-4-cyano-5-[(4-pyridylmethyl)carbonylamino]-1H-pyrazole.

(b)

1-cyclopentyl-3-ethyl-4-cyano-5-[(4-pyridylmethyl)carbonylamino]-1H-pyrazole (2.8g, 8.6mmol), ethanol (50ml), NaOCH A mixture of ₃ (1.0 g, 18 mmol) and 30% H ₂ O ₂ (4.5 ml) was stirred at room temperature for 20 minutes, then heated to reflux for 3.5 hours. Additional 30% _H2O2 ( _3ml ) was added and the reaction mixture was heated at reflux for 2 hours. The reaction mixture was concentrated in vacuo, the residue was partitioned between _CHCl3 (100ml) and 10% aqueous _NaHCO3 (50ml), and the organic layer was separated. The organic layer was concentrated in vacuo, the residue was crystallized from cyclohexane, and the solid was collected by filtration. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography, eluting with a solvent system of diethyl ether to 10% methanol/diethyl ether, and the resulting product was recrystallized with diethyl ether to obtain 420 mg of 1-cyclopentyl-3-ethyl-6- (4-pyridylmethyl)pyrazolo[3,4-d]pyrimidin-4-one, mp 162-164°C.

Example 5

Under ice cooling, add trimethylaluminum to a solution formed by dissolving 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (1.0g, 4.5mmol) in benzene (10ml) (4.9ml, 2M in methyl). The reaction mixture was stirred at room temperature for 1 hour, then 1-(ethoxycarbonylmethyl)pyrrole (0.7 g) dissolved in benzene (25 ml) was added and the mixture was refluxed overnight. The reaction mixture was cooled, 2N HCl was added and the mixture was extracted with _CHCl3 (3x). The organic layer was separated, dried over _MgSO4 , and concentrated in vacuo. The residue was digested with ether and recrystallized from ether to give 1-cyclopentyl-3-ethyl-6-(1-pyrrolylmethyl)-pyrazolo[3,4-d]pyrimidine-4 - Ketones, mp 170-172°C.

Example 6

Sodium spheres (207mg) were dissolved in ethanol (15ml) and 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (1.0g) was added followed by 4-methoxy Phenyl acetate (1.62 g, 9 mmol). The reaction mixture was refluxed for 16 hours, the solvent was stripped, water was added to the residue, and the product was collected by filtration and recrystallized from ether to obtain 0.89 g of 1-cyclopentyl-3-ethyl-6-(4-methoxy Phenylmethyl)pyrazolo[3,4-d]pyrimidin-4-one, m.p.172-173°C.

Example 7

1-cyclopentyl-3-ethyl-6-(4-methoxyphenylmethyl)pyrazolo[3,4-d]pyrimidin-4-one (1.0g, 2.8mmol) was dissolved in DMF (25ml) to the resulting solution was added 97% NaH (264mg) and after 20 minutes propanethiol (0.65g) was added. The reaction mixture was stirred at room temperature for 0.5 hours, then heated on a steam bath for 20 hours and finally in an oil bath at 120° C. for 8 hours. Additional 97% NaH (264mg) and propanethiol (0.77ml) were added and the reaction mixture was heated at 130°C for 32 hours. The reaction mixture was added to ice water, acetic acid was added and the formed precipitate was collected by filtration and dried, and after recrystallization from ethyl acetate, 0.69 g of 1-cyclopentyl-3-ethyl-6-(4-hydroxyphenylmethyl base) pyrazolo[3,4-d]pyrimidin-4-one, m.p.264-267°C (dec.).

Example 8

(a)

A solution of methyl 4-hydroxyphenylacetate (10 g, 0.06) in DMF (100 ml) was added to 97% NaH (1.78 g, 0.072 mol) in DMF (60 ml) in an ice bath. The reaction mixture was stirred for 0.5 h, then chloroacetonitrile (5.44 g, 0.072 mol) in DMF (50 mL) was added. The reaction mixture was stirred at room temperature for about 2 days, the solvent was removed in vacuo and the residue was partitioned between water and ether. The organic layer was separated, washed with saturated _Na2CO3 , then brine, dried over _MgSO4 , filtered, and concentrated in vacuo to yield 12.2 g of _methyl 4-(cyanomethoxy)phenylacetate.

(b)

Dissolve sodium spheres (600mg) in ethanol (15ml), add 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (1.0g), then add 4-(cyano (methoxy)phenyl acetate (1.85g). The reaction mixture was refluxed overnight, the solvent was stripped and water was added. The reaction mixture was filtered, the filtrate was acidified with 2N HCl, and the product was collected by filtration and recrystallized from ethyl acetate to give 1-cyclopentyl-3-ethyl-6-[4-(carboxymethoxy)phenylmethyl] Pyrazolo[3,4-d]pyrimidin-4-one, m.p. 218-220°C.

Example 9

Sodium metal (310mg) was dissolved in ethanol (75ml), then 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (1.1g, 5mmol) and 2-pyridine were added Methyl glycolate (1.52 g, 10 mmol). The reaction mixture was heated to reflux for 28 hours, the solvent was removed in vacuo, the residue was dissolved in water (50ml) and acidified with acetic acid. The mixture was extracted with _CHCl3 (100ml), the solvent was removed and the oily residue was crystallized from cyclohexane to give 0.85g of crude product. The product was purified by column chromatography on silica gel, eluting with diethyl ether, followed by recrystallization from cyclohexane to give 1-cyclopentyl-3-ethyl-6-(2-pyridylmethyl)pyrazolo as yellow crystals [3,4-d]pyrimidin-4-one, mp 120-122°C.

Example 10

(a)

97% NaH (3.56 g, 0.14 mol) was suspended in DMF (100 ml) and methyl 4-hydroxyphenylacetate (10 g, 0.06 mol) was added. The reaction mixture was stirred for 0.5 hours, then N-(2-chloroethyl)morpholine hydrochloride (11.2 g) was added and the reaction mixture was stirred at room temperature for about 2 days, then heated on a steam bath for 2 hours. The reaction mixture was cooled, filtered and the filtrate was stripped. The residue was partitioned between water and ether, the layers were separated and the aqueous layer was extracted with ether. The organic layers were combined, washed with brine, dried over MgSO ₄ , filtered and concentrated to afford 11.59 g (69%) of methyl 4-[2-(4-morpholinyl)ethoxy]phenylacetate.

(b)

Sodium (414mg) was dissolved in ethanol (30ml) and 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (2.0g) and 4-[2-(4 -Methyl-morpholinyl)ethoxy]phenylacetate (5.03 g, 18 mmol). The reaction mixture was refluxed overnight, the solvent was stripped and water was added to the residue followed by sufficient acetic acid to adjust the pH to 8-9. The oil was collected by decantation, treated with water and saturated NaHCO ₃ . The mixture was extracted with ethyl acetate, washed with water, then saturated _NaHCO3 (2x). The aqueous layer was extracted with another portion of ethyl acetate, and the combined organic layers were washed with 2N NaOH and brine. The organic layer was dried over _MgSO4 , filtered, stripped, digested with ether and filtered to give crude product. The crude product was subjected to several acid/base treatments followed by recrystallization from diethyl ether to afford 1.67 g of 1-cyclopentyl-3-ethyl-6-[4-[2-(4-morpholinyl)ethoxy ]phenylmethyl]pyrazolo[3,4-d]pyrimidin-4-one, mp 136-137°C.

Example 11

(a)

A mixture of methyl 4-(cyanomethoxy)phenylacetate (2.5 g, 12 mmol), NaN ₃ (0.87 g), NH ₄ Cl (0.72 g, 13 mmol) and DMF (20 ml) was heated at 125° C. for 24 Hour. The reaction mixture was cooled, water was added, and the mixture was vacuum stripped, but the solution was not stripped to dryness. This was repeated (2x) and then water was added followed by 2N HCl. The product which crystallized out of solution was collected by filtration, washed with water and air dried to give 1.7 g (60%) of methyl 4-(5-tetrazolylmethoxy)phenylacetate.

(b)

Sodium (0.253g) was dissolved in ethanol (15ml), then 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (0.813g, 3.7mmol) and 4-( Methyl 5-tetrazolylmethoxy)phenylacetate (17 g, 7.3 mmol). The reaction mixture was refluxed for about 2 days, cooled, and the solvent was removed in vacuo. The residue was treated with water and acetic acid and the precipitate formed was collected by filtration and dried under vacuum at 60°C. The product was dissolved in ethyl acetate, extracted with _Na2CO3 (4 x 200ml), the aqueous layer was acidified to pH _2.5 with concentrated HCl, then the aqueous layer was extracted with ethyl acetate (2 x 300ml), and the organic The layers were dried over _MgSO4 , filtered and concentrated in vacuo. The solid residue was recrystallized from ethyl acetate to obtain 0.21 g of 1-cyclopentyl-3-ethyl-6-[4-(5-tetrazolylmethoxy)phenylmethyl]pyrazolo[3 , 4-d] pyrimidin-4-one, 2/5 hydrate, mp 240-242°C.

Example 12

(a)

A suspension of 97% NaH (3.0 g, 0.12 mol) in DMF (100 mL) was stirred for 0.5 h, then benzimidazole (11.8 g, 0.1 mol) in DMF (50 mL) was added over 30 min, Then ethyl bromoacetate (20 g, 0.12 mol) in DMF (50 mL) was added. The reaction mixture was stirred at room temperature overnight, _NH4Cl was added and the reaction mixture was stripped, water was added to the residue and it was extracted with _CH2Cl2 (3 x 200 mL) _. The organic layer was dried over _MgSO4 , filtered and concentrated in vacuo. The residue was treated with ethyl acetate, the white solid was collected by filtration, the filtrate was concentrated in vacuo, and the residue was recrystallized from water to give 8.0 g of 1-(ethoxycarbonylmethyl)benzimidazole, mp.63-64°C.

(b)

Dissolve sodium (338 mg) in ethanol (30 mL), add 1-(ethoxycarbonylmethyl)benzimidazole (3.0 g, 14.7 mmol) and -1-cyclopentyl-3-ethyl-5-amino- 1H-Pyrazole-4-carboxamide (1.6 g). The reaction mixture was refluxed overnight, the solvent was stripped and the residue was treated with water and acetic acid. The formed precipitate was collected by filtration and washed with water to give 1.96 g (75%) of 1-cyclopentyl-3-ethyl-6-[1-benzimidazolylmethyl]pyrazolo[3,4-d] Pyrimidin-4-one, m.p. 256-259°C.

Example 13

Dissolve sodium (0.11g) in ethanol, then add 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (0.49g, 2.21mmol), and then add the Ethyl 3-pyridylacetate (0.73 g) in ethanol (2 mL). The reaction mixture was refluxed overnight, further 3-pyridyl acetate (0.35 g) was added, and the reaction mixture was refluxed again overnight. The reaction mixture was cooled to room temperature, the solvent was stripped, and the residue was dissolved in water and treated with acetic acid (to pH 7). The mixture was extracted with _CHCl3 (3 x 50 mL), the organic layers were combined, dried over _MgSO4 and stripped to give a light yellow oil. The oil was crystallized by adding diethyl ether, and the product was collected by filtration and washed with diethyl ether to obtain 0.4 g of 1-cyclopentyl-3-ethyl-6-[3-pyridylmethyl]pyrazolo[3,4-d ] pyrimidin-4-one.

Example 14

37% formaldehyde (5 mL) was added to 1-cyclopentyl-3-ethyl-6-[4-hydroxybenzyl]pyrazolo[3,4-d]pyrimidin-4-one (2.68 g, 7.9 mmol) and 40% diethylamine (5 mL) in suspension in acetic acid (50 mL). The reaction mixture was stirred at room temperature for 2 hours, then it was heated on a steam bath at 100 °C for 4 hours. Strip the solvent, add ethanol to the residue, then strip the ethanol, purify the residue by preparative thin-layer chromatography, develop with 5% isopropylamine/ethyl acetate, and recrystallize the product with ether to obtain 0.79g 1 -Cyclopentyl-3-ethyl-6-[3,5-dimethylamino-4-hydroxybenzyl]pyrazolo[3,4-d]pyrimidin-4-one, m.p.164-166°C .

Example 15

(a)

A solution of ethyl bromoacetate (20.9 g) in acetonitrile (30 mL) was added to K ₂ CO ₃ (27.6 g, 0.2 mol) and 1,2,3,4-tetrahydro-2-isoquinoline (13.3 g , 0.1 mol) in a mixture of acetonitrile (220 mL). The reaction mixture was stirred at room temperature for about 2 days, then refluxed for 4 hours. The reaction mixture was filtered, the filtrate was concentrated in vacuo, and the residue was partitioned between ether and water. The ether layer was separated, dried over _MgSO4 and concentrated in vacuo to give a liquid which was dissolved in ether (200 mL), cooled and treated with _Et2O ·HCl to give 12.5 g (77%) of 1,2,3 , 4-tetrahydro-2-isoquinolyl ethyl acetate hydrochloride.

(b)

Ethyl 1,2,3,4-tetrahydro-2-isoquinolinyl acetate hydrochloride (3.63 g) was dissolved in water (100 mL), treated with NaHCO ₃ and extracted with diethyl ether (3×75 mL). The combined organic layers were dried over _MgSO4 , filtered and stripped to give 3.02 g of ethyl 1,2,3,4-tetrahydro-2-isoquinolylacetate as a pale yellow liquid.

(c)

Sodium (0.24 g) was dissolved in ethanol (25 mL) and 1-cyclopentyl-3-ethyl-5-amino-1 H-pyrazole-4-carboxamide (1.09 g, 4.91 mmol), followed by the addition of 1, Ethyl 2,3,4-tetrahydro-2-isoquinolyl acetate (2.15 g). The reaction mixture was refluxed for about 2 days, the solvent was stripped and the residue was treated with water and acetic acid. The gum formed was extracted with _CHCl3 (3 x 100ml) and the combined organic layers were dried over _MgSO4 , filtered and stripped to give a yellow solid which was washed with diethyl ether to give the crude product (0.6g). The crude product was combined with that from a similar experimental route, and the mixture was purified by column chromatography on silica gel, eluting with ethyl acetate/hexane (1/1). Another portion of the product was also obtained from the concentration of the above diethyl ether filtrate and purified by column chromatography as above. The product fractions were precipitated and recrystallized from ether to afford 1-cyclopentyl-3-ethyl-6-(1,2,3,4-tetrahydro-2-isoquinolylmethyl)pyrazole as a white solid And[3,4-d]pyrimidin-4-one, mp 149-151°C.

Example 16

(a)

With stirring, ethyl bromoacetate (8.4 mL) was added dropwise into K ₂ CO ₃ (20.7 g, 150 mmol) and 1,2,3,4-tetrahydro-1-quinoline (10.0 g, 75.08 mmol) in acetonitrile (150 mL), and the mixture was stirred at room temperature for 48 hours. The reaction mixture was filtered, the filtrate was concentrated in vacuo, and the residue was partitioned between ether and water. The organic layer was separated, washed with brine, dried over _MgSO4 and evaporated to give 15.0 g (91%) of ethyl 1,2,3,4-tetrahydro-1-quinolylacetate as an amber liquid.

(b)

Sodium (0.33 g) was dissolved in ethanol (30 mL) and 1-cyclopentyl-3-ethyl-5-amino-1 H-pyrazole-4-carboxamide (1.54 g, 6.94 mmol), followed by the addition of ethanol ( 5-10 mL) of ethyl 1,2,3,4-tetrahydro-1-quinolyl acetate (3.04 g). The reaction mixture was heated to reflux for about two days, the solvent was stripped and the resulting oily residue was treated with water and neutralized with acetic acid. The formed precipitate was extracted with CHCl ₃ , dried over MgSO ₄ and stripped to give an amber oil. The oil was purified by column chromatography on silica gel, eluting with ethyl acetate/hexane (1/1), followed by recrystallization from acetonitrile to give 1-cyclopentyl-3-ethyl-6-(1 , 2,3,4-tetrahydro-1-quinolylmethyl)pyrazolo[3,4-d]pyrimidin-4-one.

Example 17

Dissolve sodium (0.68g) in ethanol, add 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (3.0g, 13.5mmol), ethyl glycolate successively (2.8 g, 27 mmol). The reaction mixture was refluxed overnight, the ethanol was stripped, water and then 2N HCl were added to the residue. The formed precipitate was collected by filtration, washed with saturated NaHCO ₃ and then water. The product was successively recrystallized from ethyl acetate and diethyl ether to obtain 0.83 g of 1-cyclopentyl-3-ethyl-6-(hydroxymethyl)pyrazolo[3,4-d]pyrimidin-4-one mp183- 184°C.

Example 18

(a)

At -50°C, in 1-cyclopentyl-3-ethyl-6-(hydroxymethyl)pyrazolo[3,4-d]pyrimidin-4-one (1.0g, 3.8mmol), CH ₂ To a mixture of _Cl2 (25 mL) and triethylamine (0.77 g, 7.6 mmol) was added methanesulfonyl chloride (0.44 g, 3.8 _mmol ) in _CH2Cl2 (5 mL). The reaction mixture was stirred for 1 hour, _CH2Cl2 and water were added, the organic layer was separated, dried over _MgSO4 , _filtered and concentrated in vacuo to give 1.28 g of crude product. The crude product was combined with the product from a similar experimental route, and the mixture was recrystallized from diethyl ether (2x) to give 1-cyclopentyl-3-ethyl-6-(methylsulfonyloxymethyl)pyrazolo [3,4-d]pyrimidin-4-one, mp 138-140°C.

(b)

1-cyclopentyl-3-ethyl-6-(methylsulfonyloxymethyl)pyrazolo[3,4-d]pyrimidin-4-one (1.8g, 15.3mmol), 1-(methylcarbonyl ) imidazole (640 mg, 5.8 mmol) and _CH3CN (36 mL) was heated at reflux for 6 hours. The solvent was stripped, ice water and saturated _NaHCO3 were added, and the mixture was extracted with ethyl acetate, dried over _MgSO4 , filtered and stripped. This product was combined with that from a similar experimental route, and the mixture was recrystallized from ethyl acetate. The product was treated with 2N HCl, extracted with ethyl acetate, and the aqueous layer was neutralized with NaHCO ₃ , extracted with ethyl acetate, dried over MgSO ₄ , filtered and stripped. The residue was recrystallized from ethyl acetate to give 0.54 g of 1-cyclopentyl-3-ethyl-6-(1-imidazolylmethyl)pyrazolo[3,4-d]pyrimidin-4-one, mp252-253°C.

Example 19

Dissolve sodium (1.19g) in ethanol (85mL), add 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (5.7g, 26mmol) and 3-methyl Ethyl oxyphenylacetate (10 g). The reaction mixture was refluxed overnight, cooled to room temperature, and stripped of solvent. Water and 2N HCl were successively added to the residue, and the formed precipitate was collected by filtration and recrystallized from ethyl acetate to obtain 5.08 g of 1-cyclopentyl-3-ethyl-6-(3-methoxybenzene Methyl)pyrazolo[3,4-d]pyrimidin-4-one, m.p.148-150°C.

Example 20 Add 97% NaH (1.26g) to 1-cyclopentyl-3-ethyl-6-(3-methoxybenzyl)pyrazolo[3,4-d]pyrimidin-4-one (4.49 g, 13 mmol) in DMF (123 mL), after 20 min, propanethiol (2.96 g, 39 mmol) was added. The reaction mixture was heated at 130°C overnight, cooled to room temperature, and then ice water and acetic acid were added successively. The formed precipitate was collected by filtration, recrystallized with ether, and dried at 110° C. and 2 mmHg to obtain 2.72 g of 1-cyclopentyl-3-ethyl-6-(3-hydroxybenzyl) pyrazolo[3,4 -d] pyrimidin-4-one, m.p. 195-197°C.

Example 21

(a)

A mixture of 3 _- hydroxyphenylacetic acid (10 g, 66 mmol), HCl ethanolic solution (160 mL) and _H2SO4 (1 mL) was refluxed overnight. The solvent was stripped to yield 11.66 g (98%) of ethyl 3-hydroxyphenylacetate.

(b)

A suspension of 97% NaH (1.75 g, 0.07 mol) in DMF (50 mL) was stirred for 15 minutes, then ethyl 3-hydroxyphenylacetate (5.4 g, 0.03 mol) in DMF (25 mL) was added. The reaction mixture was stirred for 0.5 hours, then cooled in an ice bath, and N-(2-chloroethyl)morpholine hydrochloride (5.6 g, 0.03 mol) was added. The reaction mixture was left at room temperature overnight and then heated on a steam bath for four hours. The solvent was stripped, the residue was partitioned between cold water and ether, the organic layer was separated, and the aqueous layer was extracted with ether. The organic layers were combined, dried over MgSO, filtered and stripped to give the crude product, which was purified by column chromatography on silica gel, eluting with ethyl acetate, to give _4.64 g of 3-[2-(4-morpholino)ethane Oxy] ethyl phenylacetate.

(c)

Sodium (364 mg) was dissolved in ethanol (30 mL) and then 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (1.75 g) and 3 - Ethyl [2-(4-morpholinyl)ethoxy]phenylacetate (4.64 g). The reaction mixture was refluxed for 2 days, the solvent was stripped, and water and acetic acid were added successively to the residue. The mixture was treated with NaHCO ₃ , extracted with ethyl acetate (2×300 mL), the organic layers were combined and washed with saturated Na ₂ CO ₃ , then brine. The organic layer was dried over _MgSO4 , filtered and stripped to give 2.89 g of crude product. The crude product was purified by silica gel column chromatography, eluted with ethyl acetate, recrystallized with ether, and dried at 75° C. and 0.2 mmHg to obtain 1.21 g of 1-cyclopentyl-3-ethyl-6-[3-[ 2-(4-morpholinyl)ethoxy]benzyl]pyrazolo[3,4-d]pyrimidin-4-one.

Example 22

Sodium pellets (1.0 g, 43.5 mmol) were dissolved in ethanol (50 mL) under reflux, and then 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (2.2 g, 10 mmol) and methyl 1-methylpyrrole-2-acetate (3.06 g, 20 mmol) in ethanol (30 ml). The reaction mixture was refluxed overnight under argon atmosphere, cooled to room temperature and evaporated to dryness. The residue was dissolved in water and the solution was cooled to collect the resulting solid by filtration to give recovered starting material. The filtrate was frozen and the solid formed was collected by filtration, slurried with water, and acidified with 2N HCl. The mixture was cooled, and the product was collected by filtration and dried in vacuo at 110° C. to give 1.2 g of 1-cyclopentyl-3-ethyl-6-(1-methyl-2-pyrrolylmethyl)pyrazolo[3, 4-d] Pyrimidin-4-one, m.p. 214.5-216.5°C. The filtrate was acidified by acetic acid and the product was collected by filtration and recrystallized from ethyl acetate to give another crop (0.6g).

Example 23

(a)

A solution of 1-cyclopentyl-3-ethyl-6-(hydroxymethyl)pyrazolo[3,4-d]pyrimidin-4-one (5.15 g) in CH ₂ Cl ₂ (1 20 mL) Cool to -50°C and add triethylamine (4.7 mL), methanesulfonyl chloride (2.09 g, 18 mmol) in _CH2Cl2 (24 mL) _sequentially . The reaction mixture was warmed to room temperature and stirred for 3 hours. Water and _CH2Cl2 were added to the reaction mixture, the layers were separated, and the _aqueous layer _was extracted with _CH2Cl2 . The organic layers were combined, dried over _MgSO4 , filtered and stripped. The residue was taken up in ether, the oil which separated was decanted, the filtrate was treated with charcoal, filtered and concentrated in vacuo. The residue was recrystallized with ether and dried at 75°C and 0.2 mmHg to obtain 1.58 g of l-cyclopentyl-3-ethyl-4-methylsulfonylamino-6-(methylsulfonyloxymethyl)pyrazole And[3,4-d]pyrimidine, m.p114-116°C.

(c)

97% NaH (0.158g) was stirred in DMF (25mL) for 15 minutes, then pyrazole (0.372g, 58mmol), and 1-cyclopentyl-3-ethyl-4-methanesulfonyloxy- 6-(Methanesulfonyloxymethyl)pyrazolo[3,4-d]pyrimidine (1.22 g, 2.9 mmol). The reaction mixture was stirred at room temperature for 2 hours, then heated on a steam bath overnight. Water was added to the reaction mixture, followed by the addition of sufficient 2N HCl to acidify the mixture. The mixture was left for 1.5 hours and the product was collected by filtration and washed with water. The product was purified by preparative thin-layer chromatography on silica gel, developed with 50% ethyl acetate/hexane, recrystallized with ether, and dried at 75°C and 0.2mmHg to obtain 0.26g of 1-cyclopentyl-3-ethane yl-6-(1-pyrazolylmethyl)pyrazolo[3,4-d]pyrimidin-4-one, m.p.130-131°C.

Example 24

(a)

A mixture of 4 _- trifluoromethylphenylacetic acid (15 g), HCl ethanolic solution (200 mL) and _H2SO4 (1 mL) was refluxed overnight. The solvent was stripped, the residue was partitioned between ethyl acetate and saturated NaHCO ₃ , the organic layer was separated, washed with brine, dried over MgSO ₄ , filtered and concentrated to give 15.35 g (90%) of 4-trifluoromethylbenzene ethyl acetate.

(b)

Sodium (622 mg) was dissolved in ethanol (45 mL), then 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (3.0 g, 13.5 mmol) and 4- Ethyl trifluoromethylphenylacetate (6.27 g, 27 mmol). The reaction mixture was refluxed overnight, cooled to room temperature, and stripped of solvent. Water and 2N HCl were added successively to the residue, the product was collected by filtration, recrystallized with ether, and dried at 110°C and 0.2mmHg to obtain 3.03g of 1-cyclopentyl-3-ethyl-6-(4-trifluoromethyl phenylmethyl)pyrazolo[3,4-d]pyrimidin-4-one, m.p.212-213°C.

Example 25

(a) and (b)

To 90% _HNO3 (95 mL) was added 1-cyclopentyl-3-ethyl-6-(benzyl)pyrazolo[3,4-d]pyrimidine-4 at -10 to -15°C - Ketone (9.62 g, 30 mmol). The reaction mixture was stirred at the above temperature for 1.5 hours, then it was poured into ice water. The formed precipitate was collected by filtration, recrystallized with ethyl acetate, and dried at 100° C. and 0.2 mmHg to obtain 2.31 g of 1-cyclopentyl-3-ethyl-6-(4-nitrobenzyl)pyrazolo [3,4-d]pyrimidin-4-one, mp 221-223°C [labeled as Example 25(a)]. The mother liquors were combined and concentrated in vacuo to give 1-cyclopentyl-3 ethyl-6-(2-nitrobenzyl)pyrazolo[3,4-d]pyrimidin-4-one and 1-cyclopentyl - Mixture of 3-ethyl-6-(3-nitrobenzyl)pyrazolo[3,4-d]pyrimidine-4- [labeled as Example 25(b)].

Example 26

1-Cyclopentyl-3-ethyl-6-(4-nitrobenzyl)pyrazolo[3,4- d] Pyrimidin-4-one (2.12 g, 5.8 mmol). The reaction mixture was filtered through ^SUPERCELL® , the filtrate was concentrated in vacuo, the residue was recrystallized from ethyl acetate, and after drying at 100° C. and 0.2 mmHg, 1.28 g of 1-cyclopentyl-3-ethyl-6-(4- Aminobenzyl)pyrazolo[3,4-d]pyrimidin-4-one 1/4 hydrate, mp198-199°C.

Example 27

(a)

To a solution of 2-hydroxyphenylacetic acid (25 g, 0.16 mol) in DMF (300 mL) was added K ₂ CO ₃ (56.7 g, 0.41 mol) and methyl iodide (46.7 g, 0.32 mol) sequentially. The reaction mixture was stirred for about 3 days, filtered and the filtrate was stripped. The residue was taken into ethyl acetate, washed with water saturated _Na2CO3 and brine, the organic layer was dried over _MgSO4 , filtered and stripped to give 26.4 _g (92%) of methyl 2-methoxyphenylacetate.

(b)

Dissolve sodium (590mg) in ethanol (45mL), then add 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (2.65g, 12mmol), and 2-methyl Methyl oxyphenylacetate (4.7 g, 26 mmol). The reaction mixture was refluxed overnight, the solvent was stripped and the residue was treated with water followed by 2N HCl. The product was collected by filtration and recrystallized from ethyl acetate to obtain 2.23 g of 1-cyclopentyl-3-ethyl-6-(2-methoxybenzyl)pyrazolo[3,4-d]pyrimidine-4 - Ketone, m.p. 145-146°C.

Example 28

(a)

3-Acetyl-4-oxopentanoic acid ethyl ester (37.24g, 0.2mol), hydroxylamine hydrochloride (14.6g, 0.21mol), NaOAc (17.23g, 0.21mol) and ethanol (500mL) The mixture was refluxed for 4 hours. The reaction mixture was filtered, the filtrate was concentrated in vacuo, and the residue was heated in acetic acid (12.61 g, 0.21 mol) and toluene (300 mL) for 6 to 6.5 hours to remove water. The reaction mixture was filtered and the filtrate was concentrated in vacuo to give a tan oil which crystallized on cooling. The product was collected by filtration and washed with ether to give 1.26 g of ethyl 3,5-dimethyl-4-isoxazolylacetate, m.p. 180-182°C. An additional crop of product, 31.18 g (85%), was obtained by concentrating the ether filtrate and distilling the residue at 69-81.5°C and 0.05 mmHg.

(b)

Sodium pellets (0.23g) were dissolved in refluxing ethanol (50mL), then 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (1.1g, 5mmol) was added sequentially and ethyl 3,5-dimethyl-4-isoxazolylacetate (1.83 g, 10 mmol). The reaction mixture was refluxed under argon atmosphere for 72 hours, acidified with acetic acid, then concentrated in vacuo. The residue was extracted with ether, the ether layer was washed with water, dried over _MgSO4 , filtered, evaporated and recrystallized from _CH3CN /ether to give 0.65 g of 1-cyclopentyl-3-ethyl-6- (3,5-Dimethyl-4-isoxazolylmethyl)pyrazolo[3,4-d]pyrimidin-4-one, mp 179.5-180°C.

Example 29

Under ice-bath cooling, triethylamine (0.54 mL, 3.8 mmol) was added to 1-cyclopentyl-3-ethyl-6-(4-aminobenzyl)pyrazolo[3,4-d]pyrimidine - To a solution of 4-one (1.3 g, 3.8 mmol) in pyridine (30 mL) was added methanesulfonyl chloride (0.52 g, 4.6 mmol). The reaction mixture was stirred at the same temperature for 3 hours and then at room temperature overnight. Water was added to the reaction mixture, and the crude product was collected by filtration. The crude product was taken up in ethyl acetate, washed with 2N HCl, and the mixture was filtered. The filtrate was dried with MgSO ₄ , filtered, stripped, recrystallized with ethyl acetate, and dried at 110° C. and 0.2 mmHg to obtain 0.65 g of 1-cyclopentyl-3-ethyl-6-[4-(methylsulfonate Amino)benzyl]pyrazolo[3,4-d]pyrimidin-4-one, mp 242-243°C.

Example 30

1-cyclopentyl-3-ethyl-6-(2-methoxybenzyl)pyrazolo[3,4-d]pyrimidin-4-one (1.64g, 4.6mmol) in DMF (45ml ) was treated with 97% NaH (0.46 g, 19 mmol) followed by the addition of 3-propanethiol (1.08 g, 14 mmol). The reaction mixture was heated at 130 °C overnight and cooled to room temperature. Ice water and acetic acid were added successively, and the product was collected by filtration and washed with water. The product was recrystallized from ethyl acetate, dried at 100°C and 0.2 mmHg to obtain 1.27 g (8.2%) of 1-cyclopentyl-3-ethyl-6-(2-(2-hydroxybenzyl)pyridine Azolo[3,4-d]pyrimidin-4-one, m.p. 191-193°C.

Example 31

Dissolve sodium (414mg) in ethanol (45mL), add 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (2g, 9mmol), 3-thienylacetic acid Ethyl ester (3.1 g, 18 mmol). The reaction mixture was refluxed for about 2 days and the solvent was stripped. The residue was treated with water followed by dilute HCl and the product was collected by filtration. The product was recrystallized with ethyl acetate, dried at 100°C and 0.2 mmHg to obtain 1.25 g of 1-cyclopentyl-3-ethyl-6-(3-thienyl)pyrazolo[3,4-d ] pyrimidin-4-one, m.p.210-211

Example 32

Sodium (414 mg) was dissolved in ethanol (45 mL), 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (2 g, 9 mmol) and 2-thienylacetic acid were added sequentially Ethyl ester (3.1 g, 18 mmol). The reaction mixture was refluxed overnight, cooled to room temperature and stripped of ethanol. Water and 2N HCl were added successively to the residue, the product was collected by filtration and washed with water, the product was recrystallized with ethyl acetate, dried at 90°C and 0.2mmHg to obtain 119g of 1-cyclopentyl-3-ethyl- 6-(2-Thienylmethyl)pyrazolo[3,4-d]pyrimidin-4-one, m.p. 173-174°C.

Example 33

(a)

Under cooling in an ice bath, diethylamine (32.2 g, 0.44 mol) was added dropwise to a solution of 4-chloromethylbenzoic acid (17.1 g, 0.1 mol) in ethanol (200 ml) over 20 minutes. The reaction mixture was refluxed for 17 hours, cooled to room temperature and stripped of solvent. The residue was dissolved in 1N NaOH (50ml). Extract with diethyl ether (50-100ml), and acidify the aqueous layer to pH3 with 2N HCl. The aqueous layer was stripped, the residue was treated with ethanol, filtered and the ethanol was stripped. The residue was recrystallized from isopropanol (3x) to give 13.54 g of 4-diethylaminomethylbenzoic acid hydrochloride, m.p. 189-191°C.

(b)

A mixture of 4-diethylaminomethylbenzoic acid hydrochloride (360mg, 1.5mmol), N,N'-carbonyldiimidazole (264mg, 1.5mmol) and dioxane (20ml) was heated on an oil bath 1 hour. The reaction mixture was cooled to room temperature, 1-cyclopentyl-3-ethyl-6-(4-hydroxybenzyl)pyrazolo[3,4-d]pyrimidin-4-one (500mg, 105mmol) was added and Dioxane (10ml) and the mixture was heated at 100°C overnight. The reaction mixture was cooled, the solvent was stripped, then water and ethyl acetate were added. The ethyl acetate layer was separated and washed with 2N HCl (4 x 150ml). The aqueous layers were combined, treated with concentrated _NH4OH , then extracted with ethyl acetate (2x). The ethyl acetate layer was washed with brine, dried over _MgSO4 , filtered and stripped to give crude product. The crude product was combined with that from a similar experimental route and the mixture was recrystallized from ether to give 0.46 g of 1-cyclopentyl-3-ethyl-6-[4-[4-(diethylaminomethyl) Phenylcarbonyloxy]benzyl]pyrazolo[3,4-d]pyrimidin-4-one, mp 143-145°C.

Example 34

Hydrogenation of 1-cyclopentyl-3-ethyl-6-(2-nitrobenzyl)pyrazolo[3,4-d]pyrimidine at 55 psi in the presence of 10% palladium on carbon (700 mg) -4-one and 1-cyclopentyl-3-ethyl-6-(3-nitrobenzyl)pyrazolo[3,4-d]pyrimidin-4-one (7.1g, 19mmol) in DMF (300 mL), the reaction mixture was filtered through ^SUPERCELL® , and the filtrate was stripped to give the crude product. This crude product was combined with crude products from two other similar experimental pathways, the mixture was recrystallized from ethyl acetate, and then purified by silica gel chromatography, eluting with 50% ethyl acetate/hexane, followed by ethyl acetate Recrystallization gave 111 g of 1-cyclopentyl-3-ethyl-6-(2-aminobenzyl)pyrazolo[3,4-d]pyrimidin-4-one, mp 190-192°C.

Example 35

(a)

A mixture (1 mL) of 4-dimethylaminophenylacetic acid (10 g, 56 mmol), HCl in ethanol (160 mL) and concentrated _H2SO4 (1 _mL ) was refluxed overnight. The solvent was stripped, ethyl acetate and dilute _NH4OH were added to the residue, and the layers were separated. The organic layer was washed with brine, dried over _MgSO4 , filtered and stripped to give 8.32 g (72%) of ethyl 4-dimethylaminophenylacetate.

(b)

Dissolve sodium (920mg) in ethanol (67mL), add ethyl 4-dimethylaminophenylacetate (8.32g, 40mmol) and 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole- 4-Carboxamide (4.44 g, 20 mmol), the reaction mixture was refluxed for about 2 days. The reaction mixture was cooled to room temperature, ethanol was stripped, and water was added to the residue. The product was collected by filtration, washed with water, recrystallized with ethyl acetate (2x), and dried at 100° C. and 0.2 mmHg to obtain 3.41 g of 1-cyclopentyl-3-ethyl-6-(4-dimethylaminobenzyl base) pyrazolo[3,4-d]pyrimidin-4-one, m.p.225-226°C. Example 36

(a)

4-(1-Imidazolyl)benzaldehyde (5.08 g, 29.5 mmol) and methyl(methylthio)methyl sulfoxide (2.57 g, 21 mmol) were treated with 40% ^TRITON® B in methanol (3 mL) in THF (5 mL). The reaction mixture was refluxed for 4 hours, another portion of methyl(methylthio)methylsulfoxide (1.09 g, 8.5 mmol) was added, and the reaction mixture was refluxed for another 2 hours. The reaction mixture was cooled to room temperature, _CH2Cl2 was added, the organic layer was washed successively _with water and brine, dried over _MgSO4 , filtered and stripped. The residue was transferred into ethanolic HCl (180 mL) and refluxed for 16 hours. The reaction mixture was cooled, the ethanol was stripped, the residue was treated with water (200 mL) and extracted with ethyl acetate (250 mL). The aqueous layer was treated with dilute _NH4OH , extracted with ethyl acetate (2x). The organic layer was then washed with brine, dried over _MgSO4 , filtered and stripped to give crude product. The crude product was taken into ethyl acetate and shaken with 25% sodium bisulfite followed by brine. The aqueous layer was extracted with ethyl acetate, the combined organic layers were dried over MgSO ₄ , filtered and stripped to give 193 g of ethyl 4-(1-imidazolyl)phenylacetate.

(b)

Dissolve sodium (193mg) in ethanol (25mL), add 1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (0.93g, 4.2mmol), and ethanol (7mL ) ethyl 4-(1-imidazolyl)phenylacetate (1.93 g, 8.4 mmol). The reaction mixture was refluxed overnight, the ethanol was stripped and water was added to the residue. The mixture was cooled and the crude product was collected by filtration. The crude product was stirred with 1N HCl and the product was collected by filtration as the hydrochloride salt. The hydrochloride was treated with dilute _NH4OH and the resulting precipitate was collected by filtration, recrystallized from ethyl acetate and dried at 100°C to 0.2 mmHg to give 0.4 g of 1-cyclopentyl-3-ethyl-6 -[4-(1-Imidazolyl)benzyl)pyrazolo[3,4-d]pyrimidin-4-one, mp 232-234°C.

Example 37

(a)

Add triethylamine (70 mL, 0.5 mol) to (1-ethoxyethylene) malononitrile (68 g, 0.5 mol) and tert-butylhydrazine hydrochloride (62.3 g, 0.5 mol) in ethanol (500 mL) in the mixture. The reaction mixture was stirred at room temperature for 2 hours, then cooled in ice, and the product was collected by filtration and washed with ether to give 85.9 g of 1-tert-butyl-3-methyl-5-amino-1H-pyrazole-4-carbonitrile 2/3 _Et3N -HCl. An additional 46.2 g of the desired product were obtained by concentrating the mother liquor and recrystallizing the residue from ethanol.

(b)

A mixture of 1-tert-butyl-3-methyl-5-amino-1H-pyrazole-4-carbonitrile _2/3 Et3N-HCl (38 g, 0.12 mol) and water (250 mL) was heated to 85 °C. The product precipitated from the reaction mixture was collected by filtration to give 23.25 g of 1-tert-butyl 3-methyl-5-amino-1H-pyrazole-4-carbonitrile, mp 156-157°C.

(c)

To a mixture of water (200 mL), ethanol (120 mL) and KOH (37 g, 0.56 mol) were added successively 30% H ₂ O ₂ (89.1 g, 0.786 mol) and 1-tert-butyl-3-methyl Base-5-amino-1H-pyrazole-4-carbonitrile (20.0g, 0.112mol), the reaction mixture was stirred for 4 hours, the product was collected by filtration, washed with water, and dried to obtain 20.64g (94%) of white needle crystals 1-tert-butyl-3-methyl-5-amino-1H-pyrazole-4-carboxamide, mp 195-196°C.

(d)

1-tert-butyl-3-methyl-5-amino-1H-pyrazole-4-carboxamide (1.0 g, 5.1 mmol), ethyl phenylacetate (1.67 g, 10.2 mmol), NaOCH ₃ (1.74 g , 31 mmol) and ethanol (50 mL) was refluxed for about 3 days. The reaction mixture was stripped to dryness and the residue was treated with water and acidified with acetic acid. The product was collected by filtration, washed with water, and dried to give 0.53 g (35%) of 1-tert-butyl-3-methyl-6-(benzyl)pyrazolo[3,4-d]pyrimidin-4-one 1 /5 hydrate, mp 196-197°C.

Example 38

(a)

1-cyclopentyl-3-ethyl-5-amino-1H-pyrazole-4-carboxamide (4.6g, 20.7mmol), o-ethyl xanthate potassium salt (6.4g, 40mmol) and N - A mixture of methyl-2-pyrrolidone (15 mL) was heated at 150-160°C for 5 hours. The reaction mixture was cooled. Water was added, and the mixture was filtered. The filtrate was treated with charcoal and then acidified with acetic acid. The formed precipitate was collected by filtration, washed with water and dried at 90-95 °C to obtain 4.7 g of 1-cyclopentyl-3-ethyl-6-(thio)pyrazolo[3,4-d]pyrimidine-4- Ketones, m.p. 249-251°C.

(b)

1-cyclopentyl-3-ethyl-6-(thio)pyrazolo[3,4-d]pyrimidin-4-one (5.2 g, 19.6 mmol), DMF (50 mL) and K ₂ CO ₃ (2.76 g, 20 mmol) mixture was stirred at room temperature for 25 minutes, then dimethyl sulfate (3.88 mL, 40 mmol) was added. The reaction mixture was stirred for 2 hours, then poured into cold water, the product was collected by filtration, washed with water, recrystallized from cyclohexane/ether, and dried under vacuum at 70-75°C to obtain 4.6 g of 1-cyclopentyl-3-ethyl- 6-(Methylthio)pyrazolo[3,4-d]pyrimidin-4-one, mp 200-202°C.

Alternatively, this product can also be prepared by adding NaH (4.0 g, 0.1 mol, 60% dispersion in mineral oil) to 1-cyclopentyl-3-ethyl- 6-(Thio)pyrazolo[3,4-d]pyrimidin-4-one (23.4 g, 0.09 mol) in a mixture in DMF (250 mL). The resulting mixture was cooled in an ice bath, then methyl iodide (6.3 mL, 0.1 mol) was added within 20 minutes, and the resulting mixture was stirred for 3 hours. The reaction mixture was poured into ice water (400 mL), and the formed precipitate was collected by filtration, washed with water and hexane successively, and dried under vacuum at 80-85 °C to obtain 18.6 g (74%) of 1-cyclopentyl-3 ethyl-6 -(Methylthio)pyrazolo[3,4-d]pyrimidin-4-one, m.p. 203-205°C.

(c)

1-cyclopentyl-3-ethyl-6-(methylthio)pyrazolo[3,4-d]pyrimidin-4-one (4g), CHCl ₃ (100mL) and m-chloroperoxybenzene The mixture of formic acid (10.3g) was stirred overnight. The reaction mixture was extracted with saturated aqueous NaHCO ₃ (100 mL), the CHCl ₃ layer was dried over MgSO ₄ , filtered and concentrated to dryness. Allow the oily residue to crystallize, recrystallize from cyclohexane, and dry under vacuum at 70-75°C to give 1-cyclopentyl-3-ethyl-6-(methylsulfonyl)pyrazolo[3,4-d ] pyrimidin-4-one, mp>300°C.

(d)

Mixture of 1-cyclopentyl-3-ethyl-6-(methylsulfonyl)pyrazolo[3,4-d]pyrimidin-4-one (2.3g, 7.4mmol) and aniline (1.2g, 13mmol) Heat at 180-190°C for 3 hours. The reaction mixture was cooled to room temperature and left overnight at room temperature. Diethyl ether was added to the reaction mixture, and the product was collected by filtration, washed with diethyl ether, and dried under vacuum at 80-85°C to obtain a crude product. The crude product was treated with saturated _NaHCO3 (25 mL), and the insoluble material was collected by filtration, dissolved in hot isopropanol, treated with charcoal, and concentrated in vacuo. The residue was crystallized from ether and dried under vacuum at 80-85°C to give 0.58 g (25%) of 1-cyclopentyl-3-ethyl-6-(anilino)pyrazolo[3,4-d]pyrimidine -4-one.

Example 39

1-cyclopentyl-3-ethyl-6-(methylsulfonyl)pyrazolo[3,4-d]pyrimidin-4-one (3g, 9.6mmol), phenol (6g, 64mmol) and NaH( 0.5 g, 12.8 mmol, 60% dispersion in mineral oil) mixture was heated at 170-175°C for 5 hours. The reaction mixture was poured into water (50 mL), extracted with CHCl ₃ (100 mL). The CHCl ₃ layers were concentrated in vacuo and the residue was purified by silica gel column chromatography eluting with 10% ether/hexanes to afford 2.1 g (68%) of 1-cyclopentyl-3-ethyl-6-( Phenoxy)pyrazolo[3,4-d]pyrimidin-4-one, mp 175-177°C.

Biological test results

In standard biological assay procedures, the compounds of formula I have been found to have c-GMP-PDEV (formerly known as c-GMP-PDEI) inhibitory activity and are therefore useful in the treatment of heart failure and hypertension. It has now been found that compounds of formula I in combination with nitrates can be used to reverse or reduce the tolerance induced by nitrates and thus be useful in the treatment of angina pectoris, congestive heart disease and myocardial infarction.

Multiple isozyme forms of cyclic nucleotide phosphodiesterases (PDEs) in mammalian cells have been identified. These isozymes hydrolyze cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP) to 5'-nucleoside phosphates which may not be biologically active. Elevation of intracellular cGMP in vascular smooth muscle causes a series of changes leading to a reduction in muscle tension. At the same time, elevated renal tubular cell cGMP stimulates natriuresis and diuresis. Vascular smooth muscle and kidney cells contain a phosphodiesterase isozyme with low km (1 μM) for cGMP hydrolysis, which has been termed cGMP-PDE or cGMP-PDE V (formerly cGMP-PD I, Because it was in the first PDE activity peak when eluted from anion exchange agarose resin with sodium acetate concentrations between 150-200 mM). Inhibition of the cGMP-PDE isoenzyme is therefore a viable subcellular mechanism, resulting in a decrease in overall peripheral resistance and stimulation of natriuresis and diuresis due to an increase in cGMP. The development of cGMP-PDE inhibitors heralds the discovery of agents useful in the treatment of heart failure and hypertension. For example, compounds with high inhibitory effects on cGMP-PDE are believed to lower blood pressure and induce natriuresis and polyuria.

The cGMP-PDEV inhibitory activity of typical compounds of the present invention can be demonstrated by the following steps.

cGMP-PDE and other PDE isozymes can be obtained from various animal and human cardiovascular tissues (heart) by anion-exchange and affinity chromatography as described in Silver et al., Sec. and aorta), and the PDE activity in the presence or absence of test compounds was determined essentially as described by Thompson et al., Adv Cyclic Nucleotide Res. 10:69-92. To determine the potency and selectivity of compounds as PDE inhibitors, their effect on the hydrolysis of cyclic nucleosides at 10 [mu]M can be assayed. If > 50% inhibition of PDE activity was observed, _IC50 values (concentration of compound resulting in 50% reduction in PDE activity) and corresponding 95% confidence intervals were derived. _IC50 can be as described by Tallarida and Murray, Manual of Pharmacologic Calculations with Computer Programs, Procedure 8, Graded Dose-response, PP. 14-19, Springer-Verlag, New York, 1981 ("Guidelines for Pharmacologic Calculations with Computer Programs") The method is calculated from the corresponding concentration curve.

The following table summarizes the results obtained from testing representative compounds of the invention.

Examples of percent inhibition in μM or _IC50 (nM) as given cGMP-PDEV1(b) 102(b) 9% (1 μM)3(b) 224(b) 6505 3106 237 198(b) 12009 54010(b) 27011(b) 31012(b) 39% (1 μM) or 17% (0.1 μM)13 66014 77% (10 μM) or 28% (1 μM)15(c) 177016(b) 90% (10 μM) or 32% (1 μM)17 61% (10 μM) or 20% (1 μM)18(b) 33% (1 μM)19 3320 8621(c) 42022 30023(b) 33% (1 μM)24(b) 18025(a ) 7626 8.727(b) 73% (1 μM) or 25% (0.1 μM) 28(b) 48% (1 μM) 29 94% (0.1 μM) or 77% (0.01 μM) 30 73% (1 μM) or 43% (0.1μM)

Examples of Percent Inhibition in the form of the given μM or _IC50 (nM) cGMP-PDEV31 3032 89% (1 μM) or 58% (0.1 μM)

Or 24 % (0.01 μm) 33 (b) 56 % (0.1 μm) 34 78 % (1 μm) or 45 % (0.1 μm) 35 53 % (1 μm) or 34 % (0.1 μm) 36 (b) 62 % (B) 62 % ( 1μM) or 26% (0.1μM)37(d) 78% (1μM) or 36% (0.1μM)38(d) 27039 μ .1μM or 74%3(1%)

The antihypertensive activity of typical compounds of the present invention was demonstrated in the following procedure.

Spontaneously hypertensive rats (SHR) were anesthetized with sodium pentobarbital (50 mg/kg, ip), catheterized inferiorly to the vena cava and abdominal aorta to administer drugs and record arterial blood pressure and heart rate, respectively. After 2 days of postoperative recovery, three baseline blood pressure measurements were taken in awake SHR at 5-minute intervals. Test compound or vehicle was infused intravenously in a dose-related manner (0.3-10 mg main dose/kg) while arterial blood pressure was continuously recorded with multiple polygraphs. Mean blood pressure responses were measured 5 minutes after administration of each dose of test compound and the next dose in a cumulative dose fashion. Responses to each dose of test compound were calculated as the difference from the mean of the three baseline measurements.

The following table summarizes the results obtained from testing representative compounds of the invention.

SHR iv

Example Percent Change in Mean Arterial Blood Pressure Expressed in mg/kg or ED ₂₅ (mg/kg) 1(b) -15% (1 mg/kg) or -29% (30 mg/kg)6 8.4 or - 30% (10mg/kg)7 4.6 10(b) -17% (10mg/kg)11(b) -4% (10mg/kg)19 11.0 or -21% (10mg/kg)20 10.1 or -23% ( 10mg/kg)24(b) -8% (10mg/kg)25(a) -5% (10mg/kg)26 4.2 or -46% (10mg/kg) or

-56% (10mg/kg, po)33(b) 9.3 or -27% (10mg/kg)

The activity of representative compounds of the present invention to reverse or slow down the drug resistance induced by nitrates can be demonstrated in the following steps:

Spontaneously hypertensive rats (17-25 weeks old) were resistant to nitroglycerin by repeated administration of high doses of nitroglycerin (100 mg/kg, sc, 3 times/day, for 3 consecutive days). To further confirm drug resistance, nitroglycerin was administered intravenously in a dose range of 1-300 μg/kg, and the maximum change in mean arterial blood pressure (MAP) was recorded for each dose. 5-10 minutes before the administration of the challenge dose of nitroglycerin, the compounds of the present invention (drug resistance pretreatment group) or excipients (0.05N NaOH) (drug resistance excipient pretreatment group) were divided into groups Drug-resistant rats were pretreated with intravenous administration. Administration of a challenge dose of nitroglycerin to non-resistant rats (non-resistant group) resulted in a dose-related decrease in MAP between 10 and 40 mmHg. Administration of a challenge dose of nitroglycerin to the resistant vehicle-pretreated group resulted in attenuation of the marked hypertensive response. Administration of challenge doses of nitroglycerin to drug-resistant rats pretreated with the compound of the present invention (drug-resistant pretreatment group) resulted in different degrees of recovery of hypertensive responses. The area under the dose-MAP curve was calculated for the non-drug-resistant group, as well as the drug-resistant vehicle pretreatment group and the drug-resistant pretreatment group. The percent reversal rate of phosphate-induced drug resistance was calculated according to the formula: Percent reversal rate=(AUC _{tol-pretreated} -AUC _(tol-veh) /(AUC _nontol -AUC _tol-veh )×100 where: AUC _nontol = Area under the dose-MAP curve of the non-resistant group.

AUC _tol-veh = surface under the dose-MAP curve in the drug-resistant excipient pretreatment group

Product

AUC _{tol-pretreated} = area under the dose-MAP curve in the resistance pretreatment group A percent reversal rate of 100% or greater indicates complete reversal of nitrate-induced resistance, while a percent reversal rate of 0% indicates The resulting nitrate-induced resistance was not reversed at all. The following table summarizes the results obtained in tests with representative compounds of the invention. Example Dose (mg/kg) Percent reversal of nitroglycerin-induced resistance6 1.0 697 3.0 140

0.3 46

1.0 45

The compound of the present invention can be prepared into a pharmaceutical composition suitable for pharmaceutical use by conventional pharmaceutical methods known in the art; Compositions of carriers, adjuvants, diluents or excipients, prepared in solid or liquid form for oral administration, for parenteral administration, topical administration or aerosol inhalation administration, etc. .

Solid compositions for oral administration include compressed tablets, pills, powders and granules. In these solid compositions the active compound is admixed with at least one inert diluent such as starch, calcium carbonate, sucrose or lactose. These compositions may also contain substances other than diluents, for example, lubricating agents such as magnesium stearate, talc and the like.

Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs, which contain inert diluents commonly used in the art, such as water and liquid paraffin. Besides inert diluents, these compositions can also contain adjuvants, such as wetting and suspending agents, and sweetening, flavoring, perfuming and preservative agents. According to the present invention, the compounds for oral administration may also be contained in capsules of ingestible material, such as gelatin, which capsules contain the active ingredient as described above with or without additional diluents or excipients.

Formulations for parenteral administration of the present invention include sterile aqueous, aqueous-organic, and organic solutions, suspensions, and emulsions. Examples of organic solvents or suspending media include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and organic esters for injection such as ethyl oleate. These compositions may also contain adjuvants such as stabilizers, preservatives, wetting agents, emulsifiers and dispersants.

The preparations for topical administration or aerosol inhalation of the present invention include preparations in which the compound of the present invention is dissolved or dispersed in pharmaceutically acceptable excipients such as water, hydroalcohol, glycol, oil solution or oil-water emulsion and the like.

If desired, the compounds of the present invention can be further incorporated into sustained-release or targeted-release systems such as polymer matrices, liposomes, and microspheres.

The percentage of active ingredient in these compositions may be varied so that a suitable dosage will be obtained. The dosage administered for a particular patient may vary according to the judgment of the clinician, using such criteria as: route of administration, duration of treatment, size and physical condition of the patient, potency of the active ingredient and response of the patient. The effective dosage of the active ingredient can thus be readily determined by the clinician using his best judgment regarding the performance of the patient taking into account all criteria.

Claims (36)

1. following formula: compound or its pharmaceutically-acceptable acid addition and/or hydrate:

Wherein:

R ¹Be the tertiary butyl, or cyclopentyl;

R ³Be methyl, ethyl, or phenmethyl;

X is-CH ₂-,-O-, or-NH-; And

R ⁶For phenyl (or by from one to three, identical or different, be selected from lower alkoxy, hydroxyl, halogen, carboxyl lower alkoxy, 4-morpholinyl lower alkoxy, 5-tetrazyl lower alkoxy, two elementary alkyl amido, trifluoromethyl, nitro, amino, low alkyl group sulfonamido, the phenyl that the substituting group of two elementary alkyl amido-low alkyl group phenyl carbonyl oxygen base and 1-imidazolyl replaces); Perhaps working as X is-CH ₂-time, R ⁶Be 2-in addition, 3-, or 4-pyridyl, 1-pyrryl, 1-benzimidazolyl-, 1,2,3,4-tetrahydrochysene-2-isoquinolyl, 1,2,3,4-tetrahydrochysene-1-quinolyl, hydroxyl, 1-imidazolyl, 1-low alkyl group-2,3,4 or the 5-pyrryl, 1-pyrazolyl, 3-, 4-, or the 5-isoxazolyl (or on its any possible carbon atom by low alkyl group replace 3,4, or the 5-isoxazolyl), 2-thienyl or 3-thienyl.

2. according to the compound of claim 1, R wherein ⁶For phenyl (or by from one to three, identical or different, be selected from lower alkoxy, hydroxyl, carboxyl lower alkoxy, 4-morpholinyl-lower alkoxy, 5-tetrazyl-lower alkoxy, two elementary alkyl amido, trifluoromethyl, nitro, amino, the low alkyl group sulfonamido, the phenyl that the substituting group of two elementary alkyl amido-low alkyl group phenyl carbonyl oxygen base and 1-imidazolyl replaces); Perhaps working as X is-CH ₂-time, R ⁶Be 2,3 in addition, or the 4-pyridyl, the 1-pyrryl, the 1-benzimidazolyl-, 1,2,3,4-tetrahydrochysene-2-isoquinolyl, 1,2,3,4-tetrahydrochysene-1-quinolyl, hydroxyl, the 1-imidazolyl, 1-low alkyl group-2-pyrryl, 1-pyrazolyl, the 4-isoxazolyl that on its any possible carbon atom, replaces), 2-thienyl, or 3-thienyl by low alkyl group.

3. according to the compound of claim 2, R wherein ⁶For phenyl (or from one to three, identical or different, be selected from methoxyl group, hydroxyl, carboxyl methoxyl group, 2-(4-morpholinyl) oxyethyl group, 1-(5-tetrazyl)-methoxyl group, dimethylamino, trifluoromethyl, nitro, amino, sulfonyloxy methyl amino, the phenyl that the substituting group of diethylin aminomethyl phenyl carbonyl oxygen base and 1-imidazolyl replaces); Perhaps working as X is-CH ₂-time, R ⁶Be 2-in addition, 3-, or 4-pyridyl, 1-pyrryl, 1-benzimidazolyl-, 1,2,3,4-tetrahydrochysene-2-isoquinolyl, 1,2,3,4-tetrahydrochysene-1-quinolyl, hydroxyl, the 1-imidazolyl, 1-methyl-2-pyrryl, 1-pyrazolyl, 3,5-dimethyl-4-isoxazolyl), 2-thienyl, or 3-thienyl.

4. according to the compound of claim 3, R wherein ³Be methyl, or ethyl.

5. according to the compound of claim 4, R wherein ⁷Be cyclopentyl, and R ³Be ethyl.

6. according to the compound of claim 5, wherein be selected from:

1-cyclopentyl-3-ethyl-6-(4-methoxyphenyl methyl) pyrazolo [3,4-d] pyrimidin-4-one,

1-cyclopentyl-3-ethyl-6-(4-hydroxy phenyl methyl) pyrazolo [3,4-d] pyrimidin-4-one,

1-cyclopentyl-3-ethyl-6-(phenmethyl) pyrazolo [3,4-d] pyrimidin-4-one and

1-cyclopentyl-3-ethyl-6-(4-amino-benzene methyl) pyrazolo [3,4-d] pyrimidin-4-one.

7. pharmaceutical composition is comprising compound and the pharmaceutically acceptable carrier according to claim 1, auxiliary, thinner, or vehicle.

8. pharmaceutical composition is comprising compound and the pharmaceutically acceptable carrier according to claim 2, auxiliary, thinner, or vehicle.

9. pharmaceutical composition is comprising compound and the pharmaceutically acceptable carrier according to claim 3, auxiliary, thinner, or vehicle.

10. pharmaceutical composition is comprising compound and the pharmaceutically acceptable carrier according to claim 4, auxiliary, thinner, or vehicle.

11. a pharmaceutical composition is comprising compound and the pharmaceutically acceptable carrier according to claim 5, auxiliary, thinner, or vehicle.

12. a pharmaceutical composition is comprising compound and the pharmaceutically acceptable carrier according to claim 6, auxiliary, thinner, or vehicle.

13. a method that suppresses Mammals cGMP-phosphodiesterase is comprising the compound that this animal is used the claim 1 of significant quantity.

14. a method that suppresses Mammals cGMP-phosphodiesterase is comprising the compound that this animal is used the claim 2 of significant quantity.

15. a method that suppresses Mammals cGMP-phosphodiesterase is comprising the compound that this animal is used the claim 3 of significant quantity.

16. a method that suppresses Mammals cGMP-phosphodiesterase is comprising the compound that this animal is used the claim 4 of significant quantity.

17. a method that suppresses Mammals cGMP-phosphodiesterase is comprising the compound that this animal is used the claim 5 of significant quantity.

18. a method that suppresses Mammals cGMP-phosphodiesterase is comprising the compound that this animal is used the claim 6 of significant quantity.

19. the in heart failure and/or hypertensive method of treatment Mammals is comprising the compound that this animal is used the claim 1 of significant quantity.

20. the in heart failure and/or hypertensive method of treatment Mammals is comprising the compound that this animal is used the claim 2 of significant quantity.

21. the in heart failure and/or hypertensive method of treatment Mammals is comprising the compound that this animal is used the claim 3 of significant quantity.

22. the in heart failure and/or hypertensive method of treatment Mammals is comprising the compound that this animal is used the claim 4 of significant quantity.

23. the in heart failure and/or hypertensive method of treatment Mammals is comprising the compound that this animal is used the claim 5 of significant quantity.

24. the in heart failure and/or hypertensive method of treatment Mammals is comprising the compound that this animal is used the claim 6 of significant quantity.

25. one kind reverse or alleviate Mammals in accepting the process of nitric ether treatment by the chemical sproof method of nitric ether inductive, comprising the compound that this animal is used the claim 1 of significant quantity.

26. one kind reverse or alleviate Mammals in accepting the process of nitric ether treatment by the chemical sproof method of nitric ether inductive, comprising the compound that this animal is used the claim 2 of significant quantity.

27. one kind reverse or alleviate Mammals in accepting the process of nitric ether treatment by the chemical sproof method of nitric ether inductive, comprising the compound that this animal is used the claim 3 of significant quantity.

28. one kind reverse or alleviate Mammals in accepting the process of nitric ether treatment by the chemical sproof method of nitric ether inductive, comprising the compound that this animal is used the claim 4 of significant quantity.

29. one kind reverse or alleviate Mammals in accepting the process of nitric ether treatment by the chemical sproof method of nitric ether inductive, comprising the compound that this animal is used the claim 5 of significant quantity.

30. one kind reverse or alleviate Mammals in accepting the process of nitric ether treatment by the chemical sproof method of nitric ether inductive, comprising the compound that this animal is used the claim 6 of significant quantity.

31. a treatment Mammals stenocardia, the method for congestive heart disease and myocardial infarction is comprising the compound that this animal is used in combination the claim 1 of significant quantity with nitric ether.

32. a treatment Mammals stenocardia, the method for congestive heart disease and myocardial infarction is comprising the compound that this animal is used in combination the claim 2 of significant quantity with nitric ether.

33. a treatment Mammals stenocardia, the method for congestive heart disease and myocardial infarction is comprising the compound that this animal is used in combination the claim 3 of significant quantity with nitric ether.

34. a treatment Mammals stenocardia, the method for congestive heart disease and myocardial infarction is comprising the compound that this animal is used in combination the claim 4 of significant quantity with nitric ether.

35. a treatment Mammals stenocardia, the method for congestive heart disease and myocardial infarction is comprising the compound that this animal is used in combination the claim 5 of significant quantity with nitric ether.

36. a treatment Mammals stenocardia, the method for congestive heart disease and myocardial infarction is comprising the compound that this animal is used in combination the claim 6 of significant quantity with nitric ether.