NO137012B - ANALOGICAL PROCEDURE FOR THE PREPARATION OF THERAPEUTIC ACTIVE PYRAZOLO (1.5A) PYRIMIDINES - Google Patents

Description

The present invention relates to an analogous method for the preparation of therapeutically active pyrazolo-[1,5-a]pyridines with the general formula

in which R^ is alkyl with 1-8 carbon atoms or phenyl, R 2 is alkyl with 1-8 carbon atoms and R^ is fluorine, chlorine, bromine or iodine, and the distinctive feature of the analogous method according to the invention is that aminopyrazole of formula is reacted with a diketone with the general formula

wherein R 1 and have the above meaning, and the resulting 5,7-disubstituted pyrazolo/ , 5-α 7 pyrimidine is treated with a halogenating agent to obtain the desired product.

As stated in Sutherland et al, in "Cyclic AMP", Am. Fox. Biochem. 37, 1^9 (1968), cyclic adenosine monophosphate (C-AMP) has been found to be an intracellular "second messenger" that exerts many of the actions of a variety of different hormones. According to this theory, the fixed messenger hormones epinephrine and norepinephrine influence the adenyl cyclase contained at or in the cell walls to intracellularly form cyclic AMP from adenosine triphosphate upon receipt of the extracellular hormone signal. The cyclic AMP formed in turn acts as a secondary messenger and stimulates intracellular functions that are specific to the target cells for the hormone. Cyclic AMP has thus been shown to "activate" protein kinase which in turn produces physiological effects such as e.g. muscle contraction, glycogenolysis, steroidogenesis and lipolysis.

However, cyclic AMP is broken down in vivo by phosphate esterase enzymes which catalyze the hydrolysis of cyclic purine nucleotide to 5'-adenosine monophosphate with a subsequent loss of activity. It has often been suggested that sub-substituted cyclic AMP analogues which are more resistant to phosphodiesterase degradation than the naturally occurring cyclic nucleotide should be supplied to help promote the cellular processes. However, synthetic production.. of such compounds is quite expensive and it would therefore be advantageous to enhance the beneficial effects of naturally produced cyclic AMP by adding compounds capable of inhibiting

the undesired effects of phosphorus odiesterase enzymes.

Sutherland et al suggest in Circulation ^ 7. 1279 (1968), that the pharmacological effect of theophylline having the structural formula is a consequence of its ability to inhibit the action of phosphorus odiesterase enzymes. Theophylline has thus been used instead of the adenyl cyclase-stimulating hormones, epinephrine and norepinephrine, as a cardiac stimulant after cardiac arrest and in refractory asthma cases as a bronchial dilator. However, theophylline does not selectively inhibit phosphodiesterase, but rather provides a general stimulation to the central nervous system.

Consequently, the use of theophylline can make the recipient nervous and nervous

irritable and can also cause cardiovascular effects, i.e. fast heartbeats. In the same connection, theophylline is not as powerful a phosphodiesterase inhibitor as desired and must therefore be used in large quantities, and this can of course enhance the unwanted effects mentioned above.

F. L. Rose et al, in articles in J. Chem. Soc. 56^-2 (1963), 3357

(1965) and 1593 (1969) mention a number of triazoloZ2,3-c7pyrimi-

diner and triazolo/J+,3-Q7pyrazines (eg compounds I and II shown below) which are structurally related to theophylline and which are capable of protecting animals against histamine-induced bronchospasm.

Based on the possibility that the pharmacological actions of compounds I and II may be the result of the same biochemical mechanism proposed for theophylline, various substituted pyrazolo/"! , 5-α7pyrimidines were prepared and these were found to possess the ability to inhibit the enzyme 3'?5'-cyclic AMP phosphodiesterase. Further evaluation of these compounds has also shown that many of these phosphodiesterase inhibitors have significant pharmacological properties, particularly in the cardiovascular area. For example, 3-bromo-5,7- dimethylpyrazolo^Tl ,5-α7pyrimidine and 3-bromo-5-methyl-7-n-propylpyrazolo/1,5~α7pyrimidine are not only significantly more active than theophylline against various phosphodiesterase enzymes, but they also have the ability to produce a positive inotropic effect in an anesthetized dog without any particular change in heart rate or blood pressure.

It will be seen that the illustrative embodiments and the. description which follows that the alkyl substituents generally contain from 1 to 8 carbon atoms, preferably from 1 to 6 carbon atoms and may be branched or linear.

The compounds that can be produced according to the method and the general method are illustrated in connection with the schematic drawings that follow. The starting material used is 5-aminopyrazole (1).

The usual method for the preparation of 5?7-dialkylpyrazolo[?,5-?]pyrimidines is carried out in accordance with Y. Makisumi, Chem. Pharm. Bull. (Tokyo), IQ, 612 (1962) and is reproduced in reaction scheme I. 5-Aminopyrazole (1) is condensed with pentane-2,h-dione and gives 5,7-dimethylpyrazolo/T,5-β-pyrimidine (2). It has further been found that condensation of 5-aminopyrazole (1) with heptane-3,5-dione and. nonane->+,6-dione gives excellent yields of 5,7-diethylpyrazolo/T,5-α7pyrimidine (3) resp. 5,7-di-n-propyl-pyrazolo/T , 5-α7pyrimidine (1+).

The 3-unsubstituted-5,7-ylalkylpyrazolo[beta],5-[beta]pyrimicins

(2, 3 and h) are easily subjected to electrophilic attack in the 3-position as shown in reaction scheme II. The treatment of 5,7-dialkylpyrazolo/1,5-α/pyrimidines (2, 3 or h) with N-bromosuccinimide results in the formation of 3-bromo-5,7-dialkylpyrazolo/1,5-α7-pyrimidines (5, 9 and 10). It was further found that the treatment of (2) with N-chlorosuccinimide or with iodine mono-chloride results in the formation of the 3-chloro (6) and 3-iodo (2) derivatives of 5,7-dimethylpyrazolo/J, 5-α7pyrimidine. The reaction between 5,7-dimethylpyrazolo/1,5-α7pyrimidine (2) with trifluoroacetic acid and boron trifluoride etherate has been found to give 5,7-dimethyl-3-fluoropyrazolo/1,5-α/pyrimidine (8). That the electrophilic attack takes place in the 3-position in these 5,7-dialkylpyrazolo/1,5-α7pyrimidines has been shown by proton magnetic resonance spectra, as the proton (the up-field proton) at 6.60 in (which is coupled to the proton at 2;

J = 0.0066 i) found in the 5,7-dimethylpyrazolo/J,5-α7pyrimidine (2) is not present in these 3-substituted derivatives. The condensation of 5-aminopyrazole (1) with unsymmetrical B-diketones is shown in reaction scheme III. The reaction between 5-aminopyrazole (1) with hexane-2,^-dione gives a mixture of isomers 11 and 13. Due to the similarity in the physical properties of these two isomers, separation is difficult and for this reason the crude isomer mixture of 11 and 13 converted to high-melting derivatives 15, which can be isolated by column chromatography and fractional recrystallization. Similarly, condensation of 5-aminopyrazole (1) with heptane-2,^-dione gives a mixture of isomers 12 and 1^-, which when treated with N-bromosuccinimide gives 3-bromo-5-methyl-7-n-pro <p>yl<p>yrazolo/J , 5-a/- pyrimidine (16) which is purified by chromatography. Similarly, condensation of 1-phenyl-1,3-butane-dione with 3-aminopyrazole (1) gives an isomeric product which can be recrystallized without chromatography and gives 7-Kiethyl-5-phenylpyrazolo/J,5-a/pyrimidine (17 ) • That this isomer was obtained instead of 5-methyl-7-phenylpyrazolo/J,5-α7-pyrimidine was demonstrated by comparison of physical data

(proton magnetic resonance spectra, ultraviolet spectra and m.p.)

with the 5-methyl isomer previously discussed by H. Dorn et al, J. Prak. Chem.. 313. 969 0970-

The structural formula given for compounds 15-17 is based on the poroton magnetic resonance spectra for the well-separated C^ and C^ methyl signals. It has been demonstrated (H. Reimlinger, Chem. Ber.. 103, 1900 and 3252 (1970), IQjf, 2232 and 2237 (1971))

that in 5,7-dimethylpyrazolo/JT ,5-α7pyrimidine (2), the C^-methyl signal, as this lies next to the bridgehead nitrogen, is uncovered to a greater extent than the C^-methyl signal (up to the N^-nitrogen) and appears thus in a lower field. The values are 2.56 & for C^-methyl and 2.73 for C^-methyl in deuterochloroform. Since the replacement by a longer alkyl chain (ethyl, propyl, etc.) of a methyl group at either CV or C^ was not expected to change the chemical shift of the methyl group remaining therein, 5-methyl-7-alkyl-is orne can be distinguished from 7-Kiethyl-5-alkyl isomer one by means of pmr (in deuterochloroform) and the percentage content of each isomer in the isomeric mixture can be judged from integration of the signals. In a similar way, the C^-methyl signal appears at 2.6o£ and the C^-methyl signal at 2.72 & (in deuterochloroform) for the pmr spectrum of 3-bromo-5,7-dimethylpyrazolo/Jl,5~a7pyrimidine (5) , which allows the identification of the separated products 15 and 16 from the bromination (and subsequent chromatography) of the isomeric mixtures 11 and 13 or 12 and 1^f.

The invention shall be further described and illustrated in the following examples, in which all parts and proportions are by weight and all temperatures are given in °C unless otherwise stated. Melting points were made on a Thomas-Hoover type melting point apparatus and are uncorrected. Infrared and nuclear magnetic resonance spectra were determined on a Perkin-Elmer-257 infrared spectrophotometer respectively. on a Hitachi Perkin-Elmer R-20A nuclear magnetic resonance spectrophotometer with high resolution. Hydrogenations were carried out on a Parr hydrogenator at room temperature and an output pressure of 2.9 kg/cm hydrogen. All samples gave a single spot by thin-layer chromatography and were analyzed by Heterocyclic Chemical Corporation, Harrisonville, Missouri, USA. Where analyzes are indicated only by the signs for the elements or functions, the analytical results obtained for these elements or functions were within + 0.h% of the theoretical values.

EXAMPLE I

5,7-Diethylpyrazolo/J,5-α7pyrimidine (3)

This compound was prepared from 6.5 g (0.05 mol) of heptane-3.,5-diol and the yield of chromatographed material (white needles with m.p. i+3 - V+°C from petroleum ether) was 6.3 g (72%).

Analysis calculated for C^H^N^ (MW 175): C, 68.5>+; H, 7.^8;

W, 23, 98

Found: C, 68.52; H, 7.58; N, 2^,25

NMR (CDCl₂) m, 1,<l>f£ (both C₁-ethyl and C₁-ethyl triplets);

m, 3.1$ (both C₁-ethyl and C₁-ethyl quartets); p, 6 .58&(Cg-H);

s, 6.6£ (C3~H^ °S s, 8.1(C2-H), the last two protons being paired with J0 = 1.9 eps.

EXAMPLE II

5,7-di-n-propylpyrazolo/JI,5-α7pyrimidine (h-)

This compound was synthesized from nonane-^-,6-dione (15.6 g,

0.10 mol) and after chromatography gave 17.0 g (8w%) of a pale yellow oil.

Analysis calculated for C12<H>17N3 (MW 203): C, 70.93; H,..,N,20,68

Found: C, 71.16; H, 8.25; N,20,85

Kp. 155-158°C (0j1 mm)

EXAMPLE III

3-Bromo-5,7-dimethyl-pyrazolo[in],5-[alpha]-pyrimidine (5)

To a solution of 2.0 g (13.6 mmol) of 5,7-dimethylpyrazolo-Zl,5-a/pyrimidine (Y. Makisumi, Chem. Pharm. Bull. (Tokyo) 10, 612 (1962)) in CHCl ^ (25 ml) was added N-bromosuccinimide (NBS) (2.2 g (13.6 mmol)). This mixture was heated on a steam bath for 10 min. and then allowed to cool down to room temperature. The clear yellow solution was then added to an ice-cold solution of potassium hydroxide (50 mL 2N) with good stirring. The CHCl 4 layer was dried over Na 2 SO 4 and then chromatographed on basic alumina. Evaporation of the CHCl 4 eluent gave a white solid which was further purified by recrystallization from petroleum ether (30-60°) to give 1.7 g (56%) of analytically pure product with m.p. 115 - 116°C.

Analysis (CgHgN^Br) C, H, N.

NMR (CDCl 3 ); four singlets in a 3:3:1:1 ratio at $2.60

(CH^), 2.72£> (CB^), 6.62l (H in the 6-position) and 8.10£ (H in the 2-position). The spectrum of the starting material exhibits peaks at 2.56i> (CH 3 at C?) , 2.73 S (CH 3 at. C^ ) , 6.58fc (Cg-H) , . 6.60 £ (C^-H), 8.11 £ (C2-H) (protons at C2 and C^ were paired,

J = 2.1 eps.)

EXAMPLE IV

3-Chloro-5,7-dimethyl-pyrazolo/J*1,5-α7pyrimidine (6)

In a similar manner to the preparation in Example III, the treatment of 5,7-dimethyl-pyrazolo,£1,5-a^pyrimidine (1.20 g (8.15 mmol)) with N-chlorosuccinimide (NOS) (1.33 g (10.0 mmol)) 963 mg (65%) of analytically pure product with m.p. 89 - 90 C.

Analysis (CgHg^Cl) C, H, N.

EXAMPLE V

5,7-Dimethyl-3-iodo-pyrazolo[1,5-a]pyrimidine (7)

A release of. ICI (5.0 g (27 mmol)) in CHCl 2 (50 mL) was added to a stirred solution of 5,7-dimethylpyrazolo/JI,5-a/pyrimidine (2.96 g (20 mmol)) in CHCl ^ (50 ml). In the course of a few minutes the mixture warmed and crystals of the hydrochloride salt of the desired compound began to separate. The mixture was heated on a steam bath for 2-3 min. for complete circulation and was put away in a refrigerator overnight. The yellow hydrochloride salt was separated by filtration, washed with E2O and then air dried. The yellow solid, weighing h.k g, was dissolved in water (100 ml) and this solution was made alkaline by the addition of NaOH solution (2.5 N). The alkaline solution was extracted with CHCl 2 (3 x 25 mL) and the CHCl 2 extracts were dried over Na 2 SO 4 . The CHCl₂ extract was chromatographed on basic alumina and the CHCl₂ eluent evaporated to dryness. The residue was recrystallized from petroleum ether (30-60 ) and gave 2.02 g (37%) analytically pure product with m.p. (120-122°C).

Analysis (CgHgN^I) C, H, N.

EXAMPLE VI

5,7-dimethyl-3-fluoro-pyrazolo/1,5-a/pyrimidine (8)

A mixture of 5,7-dimethyl-pyrazolo/J,5-a/pyrimidine (1.7 g

(10 mmol)) trifluoroacetic anhydride (2.0 mL) and boron trifluoride etherate (2.0 mL) in CB₂Cl₂ (30 mL) were heated under reflux for 2 h. At the end of this time, the red solution was cooled and added to an ice-cold solution of NaOH (30 mL),

2N). The organic layer was separated and the alkaline layer was extracted with CH 2 Cl 2 (3 x 20 mL). The combined CB₂C₂ extracts were washed with water (2 x 20 mL) and dried over Na₂SO₂₂. The CH 2 Cl 2 extract was evaporated and the residue covered with

n-pentane and brackish water. The yellowish-white crystalline plates were separated by filtration and recrystallized from n-heptane to give an analytically pure product with m.p. 129 - 130°C.

Analysis (CgHgN^F) C, H, N, F.

NMR (CDCl^) four singlets in a ratio of 3:3:1:1 at

2.55 b (CH^), 2.60 b (CH3), 6.60^ (H in 6-position) and 8.60 i>

(H at 2 position).

EXAMPLE VII

3-bromo-5,7-diethylpyrazolo[1,5-a]pyrimidine (9)

This compound was prepared by bromination of 1.75 g

(0.01 mole) of the dialkyl compound. Chromatography on basic alumina with chloroform gave 2.0 g (79%) of white needles, m.p. 6!+ - 65°C

Analysis calculated for C^R^N^r (MW 2$ k): C, ^7.2^; H, ^.72;

N, 16.53-

Found: C, ^7.10; H, h, 63; N, 16.71.

NMR (CDCl 3 ) t, 1.35be and t, 1.!+3£ (from Cy and C₁ethyl);

q, 2.98b and q, 3.105 (from C₁- and Cy-ethyl); s, 6.63å (Cg-H) and s, 8.08b (C2-H) in the ratio 3:3:2:2:1:1. No link was observed for singlets at 6.63 tø and 8.08 .

EXAMPLE VIII

3-bromo-5,7-di-n-propylpyrazolo[beta],5-a7pyrimidine (10)

This compound was prepared by bromination of ^-.06 g

(0.02 mol) of the 5,7-di-n-propyl starting compound to obtain

-, C I C^ Ol\ V,,,-; 4-^ .Sl^^ ^ rv, r, & 0°r ottor. Ir

petroleum ether.

•Analysis calculated for C^H^N^Br (MW 282): C, 51 .06; H, 5.67;

N, l<*>f,89.

Found: C, 50.85; H, 5.92; N, 15,11-.

NMR (CDCl^) propyl groups appear as overlapping multiplets

at 1,2b , 1,8b and 3,Oi ; s, 6.6o£> (C8-H); s, 8.8 Å (C-H) .

In the ratio 6:^:^:1:1.

EXAMPLE IX

5-methyl(n-propyl)-7-n-propyl(methyl)pyrazolo/1,5-α7pyrimidine (12%

This compound was prepared from heptane-2,<1>+-dione (8.2 g

0.06<1>+ mol) and the predominant isomer was found to be 5-methyl-7-n-propyl. Yield: 15 g of mixed isomers (86%) colorless semi-solid with m.p. ho - Lf5°C pg kp. 165 - 169°C/

0.1 mm.

Mass spectrum M+ = 175

Analysis calculated for' C^H^N^MW 175): C, 68.5^; H, 7,W;

N, 23.98.

Found: C, 68.31; H, 7.56; N, 23.79'.

NMR (CDCl 2 ) m, 1.2 b (C 1 , C 2 -propyl); m, 1.8 (C 1 , C 2 -propyl);

m, 3,OS(C5,C7-propyl); s, 2.58 b (C^-CH^ and s, 2.72&

(C7-CH3)'; s, 6.58S(C6-H) and coupled s, 6.63& (C3~H) with coupled s, 8.10fc (C2-H) H2?3 = 1.9 eps.

EXAMPLE X

3-bromo-7-ethyl-5-methylpyrazolo[/i,5-a]pyrimidine (15)

This compound was prepared from 2.6 g (0.063 mol) of the isomeric mixture of the dialkyl compound and gave 2.5 g (6w%) of the pure 7-ethyl-5-methyl isomer with m.p. 78 - 79°C. -Analysis calculated for C^H^N^Br (MW 2^0): C, ^5.00; H, M-, 16;

N, 17.50.

Found: C, ¥f.79; H, ^.38; N, 17,<1>+0.

NMR (CDCl 3 ) t, 1,<l>f 3> (ethyl); s, 2.6 in (C^-CH^ ; q, 3.10$, (ethyl); s, 6.63S (C6-H); s, 8.10i(C2-H).

The determination of the isolated isomer is based on nmr data for 3-bromo-5,7-dimethyl-pyrazolo/"1,5-a/pyrimidine and which has methyl peaks at 2, 60b and 2.72Å. The peak at 2, 6oå is due to C^-CH^ i.e. the methyl group closest to the N^-nitrogen which is not included in the bridgehead. The peak at 2.72fc is due to 0,-,-CH^ i.e. the methyl group closest to the bridgehead nitrogen due to its greater shielding effect (English : "deshielding effect") which is consistent with studies by other authors (Y. Makisumi, et al, Chem. Pharm. Bull.. J2, 20h (196^)).

etc

EXAMPLE XI

3-bromo-5-methyl-7-n-propylpyrazolo(/"1 , 5-a/pyrimidine (16)

This compound was prepared from 1.75 g (0.01 mol) of the 5,7-dialkyl compound (mixed isomers) which was purified by chromatography on basic alumina with chloroform. A colorless oil was obtained and this rapidly solidified into white crystals which, on recrystallization from benzene-petroleum (1:25), gave white needles with m.p. 88 - 89°C in a quantity of 1.20 g (h8%).

Analysis calculated for C^ qE^ N^ Bv (MW 2^): C, , 2h-, H, ^.72;

N, 16.53-

Found: C, M-7.30; H, k,81 ; N, 16.60.

NMR (CDCl 3 ) t, 1.05 (propyl); m, 1 .8£ (propyl); pp, 2.6^

(C5-CH3); t, 3.10i (propyl); - s, 6.59Å (Cg-H); p, 8.05i (C2-H).

EXAMPLE XII

5-phenyl-7-methyl-pyrazolo[1,5-α7pyrimidine (17)

This compound was prepared from 1-phenylbutane-1,3-dione

(8.1 g 0.05 mol) in the usual manner to give a crude product which was chromatographed on basic alumina with ether chloroform (9:2) to give white plates, 1.3 g (12.7%) at m.p. 70 - 71°C

after recrystallization from petroleum ether. This product was shown to be the pure 5-methyl-7-phenyl isomer by nmr.

Analysis calculated for C^H^N^ (MW 209.3): C, 7^.62; H, 5.30;

N, 20.08.

Found: C, 7^.77; H, 5.20; N, 20,21.

NMR (CDCX 1 ): s, 2.82 (C 2 -CH 2 ); coupled s, 6.7U (C^-H);

s, 7.10i (C6-H) m, 7.80bog 8.15& (C^-phenyl ABX monster) and coupled s, 8.03S (C2-H). J2 3 = 2>1 CPS-

EXAMPLE XIII

The compounds according to the invention were tested for inhibition of phosphodiesterase by the following method.

3',5'-cyclic-AMP phosphodiesterase (PDE) was isolated and purified from three different tissues as follows. Homogenates of rabbit kidney, rabbit lung and bovine heart were prepared in sucrose-tris-magnesium buffer and subjected to low-speed centrifugation to remove nuclei and cell debris. The liquid fractions were then centrifuged at 105,000 x g for 30 min. and the liquid phase after the 105,000 x g was then fractionated using ammonium sulfate. The precipitates formed at 0-30% saturation are collected by centrifugation at 20,000 x g, dissolved in trismagnesium buffer and

dialyzed overnight against the same buffer. Another (NH^^SO^ fraction is obtained by raising the concentration in the first liquid phase to 50%. These two (NHIf)2S01+ fractions as well as the liquid phase from 30 to 50% fraction were then tested on PDE- activity using the method of Appleman, Biochem. 10, 311 (1971). The first fraction obtained from both kidney and lung tissue contains a PDE with a low affinity for 3',5'-c-AMP (high Km). The second fraction exhibits a biphasic curve when the Lineweaver-Burk method of analysis is used, indicating either the presence of two separate enzymes, one with a high and the other with a low affinity for C-AMP, or a protein with two separate positions. Appleman, supra, shows that extracts of brain yield two separate enzymes (a high Km and a low Km) which can be separated by separose gel chromatography.

The inhibition studies reproduced in Tables I and II were carried out with the low affinity enzyme (fraction I, high Km) obtained from rabbit kidney or rabbit lung. The attempts that are

reproduced in tables III-VI was carried out with the enzyme with high affinity (fraction II, low Km) obtained from rabbit lung or kidney or bovine heart. I^Q values were calculated in some cases by plotting long I against percent I in experiments in which the inhibitor concentration is varied over a wide range, at a constant 3',5'-cyclic AMP concentration of about 5 x 10< -I>fM (Tables I and II) or 1 .6 x 10~^M

(Tables III and IV). The relative inhibitory activity of each compound compared to theophylline is expressed as an α value. This value is obtained by dividing the I^Q value obtained for the particular compound under consideration.

In most cases, α-values were calculated from inhibition experiments carried out with a single concentration for the test compound as long as the inhibition produced at this concentration was from 20 to 80%. In this case, the a value was calculated as

concentration of theophylline that gives (x%) inhibition concentration of test substance that gives the same (x%) inhibition

The validity of this method was checked, ve-.; comparison of the oc-values obtained by (1) measurements at a single concentration of inhibitor and (2) measurements at four concentrations of inhibitor (Ic~ determinations), the a-vc-rdies

yes .. compared in this'way have been found to match within 10% of each other.

The basic incubation mixture contained f 61gend.e.-substances (amounts in /imole). ^H-cAMP (specific activity about 2.180 cmp/pmol), 0.00016; Tris pH 7.5, <0; HgCl 2 , 0.5; enzyme (cAMP phosphodiesterase), 5-50 µg protein; and 10~^ to 10~<6 >molar concentrations of inhibitor; incubation time 10 min. at 30°C. At the end of incubation, the mixture was heated to 90°C for 2 min. and 100 µg of snake venom phosphodiesterase from Crotalus atrox was added and the tubes were incubated for 10 min.

at 30°C. The mixture was then cooled in 1 ml of "Dowex" 1-2X, 200-^fOO mesh suspension, prepared by mixing 100 g of the resin in 200 g of H 2 O, was added and the mixture centrifuged. A sample of the liquid phase was used to determine units per min using a liquid scintillation spectrometer Zero time values were obtained using incubations in which the cAMP phosphodiesterase was omitted from the first incubation.

The results shown in the preceding tables show that several of the compounds which can be prepared by the method according to the invention are many times more effective as inhibitors of phosphodiesterase enzyme than theophylline. For the same reason, the results show that the compounds are capable of selective inhibition.

In general, the compounds that act as phosphodiesterase inhibitors can find application in the treatment of disorders that can be influenced by the supply of epinephrine or norepinephrine, since in both cases the result is the maintenance of greater levels of C-AMP- in the first case by slowing down C- AMP degradation and in the second case by stimulating its production.

Several of the compounds in question have been tested in vivo and show

a series of activities indicating selective transport to specific tissues. 5?7-Dimethyl-3-bromo-pyrazolo/1,5-a/pyrimidine demonstrated anti-edema and inhibitory effects against ADP-induced platelet aggregation, and also had muscle relaxant properties at 300 mg/kg 3 hours after administration. This compound also had a positive inopropionate effect on the heart.

Preliminary pharmacological evaluation has shown that 5?7-dimethyl-3-iodopyrazolo/1,5-?7pyrimidine and 3-bromo-5,7-dimethylpyrazolo-?1,5-?7pyrimidine have potent cardiovascular properties. In the preparation of isolated Langendorf heart, these compounds cause coronary dilation and/or produce a positive inotropic effect at a concentration of 2.5 mg/ml.

When administered orally to rats at a dose of 50 mg/kg body weight, 3-Dronlo-5,7-dimethylpyrazolo/*1,5-j!?>yrimidine and 5,7-dimethyl-3-iodo-pyrazolo //1 ,5-a7pyrimidine considerably the blood pressure (10% or more). At an oral dose of 25 mg/kg, 5,7<_>dimethyl-3-iodo<py>razolo/f,5-α7pyrimidine lowered blood pressure by 10% for periods of up to 6 hours.

In anesthetized dogs, 3-t>romo-5,7-climethyl-pyrazolo[1,5-a]-pyrimidine (5 mg/kg i.v. infusion) caused a strong increase in cardiac

performance at both 30 and 60 min. after starting infusion, (p 0.05).

The increased effect was respectively 21 and 20%. The cardiac effect was maintained

above baseline values for 2 hours after the infusion was stopped. A similar increase (p=0.005) was observed in stroke volume below that

same time period. No particular changes in arterial pressure, central venous pressure, or heart rate were noted.

Claims (1)

1. Analogous process for the preparation of therapeutically active pyrazolo/1,5-β-pyrimidines of the general formula wherein R 1 is alkyl with 1-8 carbon atoms or phenyl, R 2 is alkyl with 1-8 carbon atoms and. R^ is fluorine, chlorine, bromine or iodine, characterized in that the aminopyrazole of formula is reacted with a diketone of the general formula in which IL and R2 have the above meaning, and the resulting 5,7-disubstituted pyrazoloZ/1,5-ylpyrimidine is treated with a halogenating agent to obtain the desired product.