SE466104B - NEW TRIAZOLPYRIMIDINES AND PROCEDURES FOR THEIR PREPARATION - Google Patents

Description

466 104 2 R4 represents a straight or branched chain alkyl group having a chain length C4 to C9, 2,5-dioxaheptyl or 3-oxahexyl radicals; - R5 represents an H atom, an alkyl group of chain length C1 to C3, a hydroxyethyl resp. hydroxypropyl group; - R6 and R7 represent H atoms, a straight or branched chain to C5, wherein R and R can l 6 7 constitute the exclusive constituents of one of R6, R7 and N cooling group with chain length C formed heterocyclic ring, The substituents in the 5- and 7-position can also be exchanged for each other.

Among According to the formula (I), the following compounds have been found be particularly active with respect to cardiovascular effects: 5-Piperidino-7- (N- (n-pentyl) -N- (β-hydroxyethyl) -amino) -s- triazolo (1,5-a) pyrimidine 5-Diethylamino-7- (N- (n-pentyl) -N- (B-hydroxyethyl) -amino) -s- triazolo (1,5-a) pyrimidine 5-Diethylamino-7- (N- (n-hexyl) -N- (β-hydroxyethyl) -amino) -s- triazolo (1,5-a) pyrimidine 5-Piperidino-7- (N- (n-hexyl) -N- (β-hydroxyethyl) -amino) -s- triazolo (1,5-a) pyrimidine, The compounds corresponding to formula (I) are particularly suitable for use in the form of their physiologically acceptable salts. For salt formation is particularly suitable for chlorine, bromine or hydrogen iodine acid, sulfuric and nitric acid, oxalic, malonic and tartaric acid. 3 466 104 The compounds of formula (I) according to the present invention can be prepared analogously to the compounds of DD 61 269 by reacting triazolopyrimidines with the general formula (II) HoL L, l mL \ N) \ 23 wherein R 2 and R 3 have the indicated meaning and Hal represents one chlorine or bromine atom, with amines of the general formula (III) m J R (.4 or 6) (H1) i “(5 or 7) wherein R4 5 6 Oñh 7 has the specified meaning. The reactions I I V carried out in solvents such as water, water / alcohol mixtures, alcohols, toluene or petrol. The reactions proceeds in two steps with the substituted amines in question.

At temperatures below 293 K, the halogen atom is first substituted in 7-position with amines and at temperatures up to that use the boiling point of the solvent halogen atom in the 5-position.

To capture the halogenated hydrogen formed in the reaction the acids used the excess amines with it general formula (III) or triethylamine, alkali carbonates resp. alkali hydroxides. The raw products are processed in the usual way. The end products are separated and purified from by-products by extraction, distillation or recirculation tallization. Disturbing discolorations can be removed by 466 104 4 addition of adsorbents, such as activated carbon, alumina ' or bleaching earth.

The resulting compounds can be transferred with acids into their salts. ter. If stereoisomeric compounds are obtained, these can be according to known procedures are divided into their constituents.

The compounds according to the invention, in particular those mentioned individually compounds, show in animal experiments highly effective and therapeutic technically interesting properties. At the center is one inhibition of platelet aggregation in vitro and in vivo, as several times exceeds the corresponding effect of known agents such as acetylsalicylic acid or trapidil. On isolated cardiac and atrial preparations, they develop a coronary dilatation and a positive inotropic activity. Blood pressure preferably lowered at the hypertonic starting position. They prevent a deformation induced by hyperosmolar conditions loss of erythrocytes as well as their tendency to aggregate and thereby improves the flow distortions in the microcirculatory area. By the influence of arachidonic acid metabolism they reduce the formation of thromboxane A2 resp. promotes synthesis of aggregation-inhibiting prostaglandins, in particular of prostate cyclin.

The associations have been shown to be antiarrhythmic.

An essential feature of the new associations consists in the effect of calcium metabolism, in particular on the transmembrane Ca2 + influx in biological cells. They have been found to be calcium antagonists high specificity according to Fleckenstein (Fleckenstein A .: Calcium antagonism in heart and smooth muscle., J. Wiley & Sons, New York, 1983). Their particular advantages over known Ca2 + antagonists are of high specificity and efficacy. strength, the easily reversible effect as well as the development of an effect especially with high frequency stimulation of sensitive cells. 5,466,104 Their versatile pharmacological spectrum of action and small acute toxicity enables the use of the compounds according to invention as a therapeutic for ischemic cardiac and circulatory diseases and characterize them in comparison with the past known compounds. The use is as an injection solution in in the form of a 5% aqueous solution or in the form of according to known methods coated granules with the composition 58% active substance, 20% lactose, 19% water and 3% magnesium stearate.

The invention is illustrated by the following examples: 466 104 Example 1 18.9 g of 5,7-dichloro-s-triazolo (1,5-a) pyrimidine are dissolved resp. swish- pend in 50 ml of ethanol and add slowly at 278 to 283 K with a solution of 26.1 g of n-amylethanolamine in 25 ml ethanol. Then stir and keep the mixture after completion add another 1 hour at 283 to 288 K. Thereafter to the resulting suspension is added a solution of 17.0 g piperidine in 20 ml of ethanol at 293 to 298 K and kept in 3 hours at 313 to 323 K and the batch is evaporated. The mixture then taken up in 100 ml of methylene chloride, washed three times with each 75 ml of water and the water is separated. The methylene chloride evaporated and the residue is crystallized. After recrystallization 26.5 g of piperidino-7- (N- (n- pentyl) -N- (β-hydroxyethyl) -amino) -s-triazolo- (1,5-a) pyrimidine (80% of theory), m.p. 390 to 391 K.

Example 2 18.9 g of 5,7-dichloro-s-triazolo (1,5-s) pyrimidine are dissolved resp. swish- pend in 50 ml of ethanol and add slowly at 278 to 283 K with a solution of 26.1 g of n-amylethanolamine in 25 ml ethanol. The batch is then stirred and obtained after completion of the addition stand for another hour at 283 to 288 K. The formed sus- the pension is filtered off with suction, suspended in 50 ml of ethanol and added at 293 to 298 K with a solution of 14.6 g of diethylamine.

The batch is then kept for 3 hours under reflux and then evaporated in vacuo. The residue is taken up in 100 ml methylene chloride, the methylene chloride phase is washed with 50 ml of water and separated. The methylene chloride is evaporated, the residue crystallized and recrystallized from ether. The 5-diethylamino- formed 7- (N- (n-pentyl) -N- (β-hydroxyethyl) -amino) -s-triazolo (1,5-a) - the pyrimidine with melting point 352 to 353 K is obtained in one yield of 225 g (70% of theory). 466 104 Example 3 In 50 ml of ethanol is suspended 18.9 g of 5,7-dichloro-s-triazolo- (1,5-a) pyrimidine. At 5 to 10 ° C, it is added dropwise while stirring 29.4 g of n-hexylaminoethanol and allowing the batch temperature reach 293 K and stir for 1 hour at this temperature.

The compound formed is aspirated and resuspended in 150 ml of ethanol. At room temperature, add 15 g of diethyl amine and then heat the mixture for 3 hours to boiling.

The batch is evaporated in vacuo, the residue being taken up in 75 ml chloroform and washed with water. After washing, evaporate the organic phase, the resulting 5-diethylamino-7- (n-hexyl) - The N- (β-hydroxyethyl) -amino) -s-triazolo (1,5-a) pyrimidines are crystalline. tallied from petroleum ether. The melting point is 335 to 336 K and the yield 162 g (50% of theory).

Example 4 The reaction is carried out as described in Example 3. Instead for diethylamine, 1.7 g of piperidine were used. The 5-piper formed idino-7- (N- (n-hexyl) -N- (β-hydroxyethyl) -amino) -s-triazolo (1,5-a) the pyrimidine is recrystallized from benzene. The exchange amounts to 28 g and the melting point is 354 K.

In Examples 5 to 14, instead of the chemical terms, the following letters: A = 5-diethylamino-7- (N- (n-hexyl) -N- (β-hydroxyethyl) -amino) - s-triazolo (1,5-a) pyrimidine B = 5-Piperidino-7- (N- (n-pentyl) -N- (β-hydroxyethyl) -amino) - s-triazolo (1,5-a) pyrimidine C = 5-Piperidino-7- (N- (n-hexyl) -N- (β-hydroxyethyl) -amino) - s-triazolo (1,5-a) pyrimidine. 466 104 8 Example 5 Inhibition of transmembrane calcium influx On neuroblastoma and glioma hybrid cells of clones 108 CC 5 and 108 CC was examined by cell perfusion voltage-clamp technology. the voltage-dependent Ca2 + influx and its effect on by compound A. The electrophysiological measurement technique used niken enabled the effect of the substances on cells sensitive to the slow Ca2 + influx could directly demonstrated. The registration and evaluation of the received the information was provided by EMG-Laborcomputer and signal analyzer. Compound A was dissolved in distilled water and diluted at concentrations of 1 to 100 μm in the extracellular learn the solution (glucose 5 mM, tris HCl 130 mM, CaCl2 10 mM, MgCl 2 1 mM, KCl 4 mM, pH 7.4; intracellular solution: Diluted tris PO 4 (140 mM, pH 7.2), was extracellularly perfused by 5 to 10 ml continuous perfusion (Dauerperfusion) in graduated doses at 22 ° C. 1 to 2 minutes after extracellular application of compound A Ca 2+ influx (Ica) could be inhibited depending on the dose.

This effect was detectable over the total potential range, whereby no displacement of the voltampere characteristic could determined. The inhibition of Ica was reversible in all cases.

The time elapsed for the washout amounted to a few seconds. there. An increase in the depolarization frequency (use-dependence power) of 0.1 to 1 Hz resulted in a stronger inhibition effect of the substance on Ica.

Example 6 Arachidonic acid resp. U-46619-induced aggregation on human platelets 9 volumes of blood from donors, which for 10 previous days not taken any medication, was added with 1 volume of 3.8% tri- sodium citrate solution and centrifuged at 200 g and room temperature temperature for 10 minutes. The platelet-rich plasma (PRP) s 466 104 sucked with a silicone pipette and stored during the experimental the period of a maximum of 3 hours at room temperature.

The test compounds were dissolved in a solvent mixture of ethyl acetate. nol / chloroform (lzl), aliquots were filled into the measuring cuvette and the organic solvent mixture was blown off a stream of nitrogen gas. After addition of 0.3 ml PRP and 0.1 ml physiological NaCl solution and a pre-incubation of 2 minutes at At 37 ° C, the aggregation was effected with sodium arachidonate (0.4) to 0.8 mmol / l) resp. by the TXA2 agonist U 46619 (0.2 to 0.8 mmol / l). The measurement of the aggregation process took place according to the method of BORN (Born, 9.V.R. and Cross, U.J .: J. Physiol. lâå, 178 (1963)).

They were obtained for a 50% inhibition of aggregation necessary. the concentrations of the test compounds by immediate comparison with those for trapidil. As the experimental results in Table 1 shows, possesses the derivatives in comparison with trapidil a significantly stronger anti-aggregation effect.

Table l Effects on thrombocyte aggregation in human PRP by some derivatives compared to trapidil (pIC50 = negative logarithm of average molar inhibitory concentration) Preparation Inhibition of platelet aggregation (pIC50) AA U-46619 A 4.68 4.40 B 4.66 5.09 C 4.33 4.65 Trapidil 3.75 3.74 Example 7 Inhibition of platelet aggregation in rabbits and rats Platelet-rich plasma was extracted from citrate blood from rabbits resp. 10 466 104 rats by centrifugation at 200 g at room temperature and were stored during the experimental period in plastic syringes at room temperature. ratur. During the examination on rabbits, the test substance was added 0.9% NaCl solution (50 μl) to 0.4 ml PRP in homologous plasma and after 3 minutes of pre-incubation at 37 ° C, the aggregate was released. with arachidonic acid-Na (75-210 mmol / l).

Platelets from rats were separated and suspended in Michaelis buffer (pH 7.4) and defibrinated homologous plasma (l: l). To 1.2 ml of this platelet suspension was added 10 ul of preparation solution in ethanol and after 2 minutes pre-incubation at 37 ° C, aggregation was started by adding arachis donic acid (2 mmol / l final concentration in the cuvette). The measurement of the aggregation was nephelometric according to the method of BORN (Born, 9.V.R. and Cross, U.J .: J. Physiol. Lâå, 178 (1963)).

A comparison with the mean molar inhibitory concentration (Table 2) also shows in these experiments a clearly stronger pronounced anti-aggregatory effect of the new test compounds in comparison with trapidil.

Table 2 Inhibition of platelet aggregation induced by arachidonic acid in rabbits and rats in vitro Preparation pIC50 Rabbits Rats Trapidil 4.05 2.49 A 5.47 3.72 B 4.97 3.75 C 5.05 3.78 Example 3 Impact on platelet aggregation in vivo Intravenous application of arachidonic acid caused in rabbits lvl IN) 11 466 104 via platelet aggregation, especially in the pulmonary vessels symptoms and lethal effect. Already in low, asymptomatic tolerated concentrations, the aggregatory effect of arachidonic acid due to a transient, immediately after the injection detectable reduction in the number of free circulating platelets in the peripheral blood is obtained quantitatively and the effect of previously administered drugs are detected. On non-anesthetized Rabbits of both sexes were determined after blood tests from the ear vein the number of platelets by phase contrast microscopy. Then arachidonic acid-Na was injected at a dose of 0.1 mg / kg i.v. and the platelet count was then re-determined after 1/2, 1, 2, 3, 5 and 10 minutes. As a quantitative criterion for arachidonic acid-induced thrombocytopenia, they were used by the percentage the decrease in the number of platelet enclosed surfaces and their value for the animals in the control group was set at 100%. For testing of the in vivo effect of the test preparation was given orally after obtaining the individual unaffected The AA reaction as suspension in ultra-swelling cellulose by flexible pharyngeal probe and in the manner indicated above, the the thrombocytopenin after arachidonic acid injection 1, 2, 4, 6 and 24 hours after drug administration.

Table 3 Impact of arachidonic acid-induced aggregation on rabbits in live Preparation Dose Inhibition of AA-induced thrombocytopenia (%) mg / kg hours after administration p.o. l 2 4 6 24 Trapidil 20 14 24 6 0 0 50 90 96 74 72 17 A 10 97 89 40 51 22 B 10 45 67 71 48 41 C 10 27 53 8 0 0 The results (Table 3) confirm it in comparison with trapidil 12 466 104 superior anti-aggregation effect of the new derivatives and at the same time determines their effectiveness even during in vivo conditions.

Example 9 Inhibition of thromboxane A2 synthesis The formation of TXA2 and its effect were studied on rabbit blood platelets after aggregation elimination with arachidonic acid. Forward the position of the PRP and the aggregation rate corresponded to those in the conditions set out in Example 9. 90 seconds after AA batch used aliquot portions of the cuvette contents for biological determination of the TXA2 content. The content determination on the thrombox A2 occurred on helically cut vascular strips from A. mesenterica of rabbits in superfusion technology. As a superfusion medium served Tyrode solution, which to increase selectivity contained propranolol (5 pg / ml), pentolamine (1 ug / ml), atropine (μg / ml), antazoline (0.25 pg / ml), methysegide (20 μg / ml) and indomethacin (1 pg / ml) as blocking substances. Vessel strap sensitivity of the cells was tested with EMA (9,11-epoxymethano-15 (S) - -hydroxy-prosta-5,13-dienoic acid = U-44069) in a dose range of 1 to 25 pg and the contraction was recorded over an inductive one converter on a printer. In comparison with trapidil distinguishes the new derivatives of a more intense inhibition of thromboxane A2 biosynthesis (Table 4). 13 466 104 Table 4 TXA2 formation in arachidonic acid stimulated platelets from rabbits under the influence of the test preparations Preparation Dose Inhibition (%) of pIC5O umol / l TXA2 formation Trapidil 50 23.8 l00 52.8 150 75.2 4.03 A 5 22.6 46.3 88.7 5.01 B 5 22.6 l0 62.4 94.9 5.09 C 19.0 5 50.9 83.5.5.42 Example 10 Inotropic effect The influence on the contractile force of the heart was examined in isolation. atrophy from guinea pigs. Right, spontaneously striking atrial- preparations were suspended in 25 ml organ baths in O 2 throughput Tyrode solution and their contractions were measured semi-isometrically by mechanical-electrical converter.

All surveyed associations were marked by a positive inotropic effect on isolated atrial preparations from guinea pigs. The for a 50% increase in the contractile force required the doses, described as negative logarithms of the corresponding molar concentrations, are given in Table 5 and determine one in comparison with trapidil superior effect. 466 104 14 Table 5 Increase in the contractile force in isolated atria (guinea pigs) Preparation Contraction reinforcement when isolated atrial drug (pED5C) * Trapidil 3.84 A 4.46 B 4.20 C 4.27 Example ll Blood pressure effect From spontaneous hypertensive rats (okamoto-Aoki) were pre-prepared under pentobarbital anesthesia (60 mg / kg i.p.) A. carotis communis dextra and a polyethylene catheter were inserted. The arterial blood the pressure was recorded across a mechanoelectric converter on a polygraph (EMT 34; Minograph 81, Elema-Schönander, Stockholm).

The drug application took place in V. subclavia sinistra in a volume of 0.6 ml in physiological NaCl solution.

In spontaneously hypertensive rats, in which trapidil only resulted a short-term decrease in mean arterial pressure, they provide new test compounds a long-lasting anti-hyper- tensile effect. As in this respect most effective derivatives shows the compounds A and C, which lower the arterial the average pressure for 2 to 4 hours by 15 to 20%.

Example 12 Inhibition of phosphodiesterase f O) The activity of PDE was determined according to BUTCHER and SUTHERLAND (Butcher, R. W. and E. W. Sutherland: J. Biol. Chem. 221, 1244 (1962)). The batch (0.9 ml) had the following composition 1.8 mM MgSO 4, 36 mM Tris, 0.5 mM cAMP (Boehringer, 15 466 104 Mannheim) and 15 pg phosphodiesterase from bovine heart muscle (Fa. Boehringer, Mannheim). The reaction took place at pH 8.0 and 37 ° C and stopped after 10 minutes by heating in 1 minute in boiling water bath. Then 0.1 ml was added Crotalus atrox (Sigma, USA) and further incubated 10 minutes at 37 ° C. To determine the orthophosphate was stopped the incubation by adding 0.1 ml of 5 N HClO4 and after centrifugation, the resulting phosphate was taken up in a semi-micro- procedure according to ALLEN (Allen, R.I.L .: Biochem J. âå, 858 (1940)).

For example, in derivatives A and C, it appears that the new are significantly more potent inhibitors of phosphodiester late than trapidil.

As inhibitory constants were obtained: 3 Trapidil Ki = 1.3 x 10 'M A 5.4 x 1o'5 M c 2.0 x 10.5 M Example 13 Acute toxicity The acute toxicity of the test compounds was obtained on NMRI male mice after intraperitoneal resp. intravenous appli- cation. The compounds were given suspended in ultra-swelling cellulose (i.p.) or dissolved tartaric acid (i.v.). The determination of LD took place according to the procedure of LITCHFIELD and WILCOXON. 50 466 104 16 Table 6 Acute toxicity of test compounds to mice Preparation 305% i.p. i.v.

Trapidil l55 (l24-l94) ll5 (104-l27) A 285 (238-342) 56 (48-66) B 230 (193-274) 69 (62-75) C 720 (654-792) 59 (54-65) The new derivatives show up in terms of their acute toxicity not significantly more toxic compared to trapezoid dil, so that in view of their intensive pharmacodynamic effects is a greater therapeutic breadth to expect.

Example 14 For clinical use of the active substances according to the finning was mixed in the usual way 50 g of active substance 28 g of sucrose 3 g of lactose 35.4 g potato starch 3 g of talc 0.6 g magnesium stearate with each other and were granulated after the addition of liquid. After drying and intentioning, talc and magnesium stearate were added and the mixture was compressed into tablets, which per tablet contained 50 mg of active substance.

Claims (4)

f; 466 104 PATENT REQUIREMENTS 1. New s-triazolo (1,5-a) pyrimidines of the general formula 94 \ N / 25 Rc ___? Wherein the substituents have the following meanings: - R 2 and R 3 represent an H atom, an alkyl group of chain length C1 to C3 or a halogen atom; - R 4 represents a straight or branched chain alkyl group of chain length C 4 to C 9, 2,5-dioxaheptyl or 3-oxahexyl radicals; - R5 represents an H atom, an alkyl group of chain length C1 to C3, a hydroxyethyl or hydroxypropyl group; - R 6 and R 7 represent H atoms, a straight or branched chain alkyl group of chain length C1 to C5, wherein R6 and R7 may be the exclusive constituents of a heterocyclic ring formed by R6, R7 and N; the substituents in the 5- and 7-position can also be exchanged for each other. Novel s-triazolo (1,5-a) pyrimidines according to claim 1, in particular. 5-Piperidino-7- (N- (n-pentyl) -N- (β-hydroxyethyl) -amino) -s-triazolo (1,5-a) pyrimidine-5-diethylamino-7- (N- (n -pentyl) -N- (β-hydroxyethyl) -amino) -s-triazolo (1,5-a) pyrimidine-5-diethylamino-7- (N- (n-hexyl) -N- (β-hydroxyethyl) - amino) -s-triazolo (1,5-a) pyrimidine-5-piperidino-7- (n-hexyl) -N- (β-hydroxyethyl) -aminoH0) 'Stria: m zol0 (1,5-a ) pyrimidine. 466 104 ”Y Process for the preparation of the compounds of formula (I), in particular the compounds according to claim 2, characterized in that compounds of the general formula (II) Hcl-Rlfx "Hbb \ fl; / l§¶v" J Wherein R 2 and R 3 have the indicated meaning and Hal represents a chlorine or bromine atom, are reacted with amines of the general formula (III) HN ..- t '"(4 or 6) (111)" (5 or 7) wherein R 4 to R 7 have the indicated meaning, in two steps in the presence of solvent, the first of which is carried out at temperatures up to 20 ° C and the second at temperatures up to the boiling point of the solvent. IN Therapeutic composition, characterized in that it contains one of the compounds of formula (I), especially one of the compounds: - 5-piperidino-7- (N- (n-pentyl) -N- (β-) hydroxyethyl) -amino) -s-triazolo (1,5-a) pyrimidine-5-diethylamino-7- (N- (n-pentyl) -N- (β-hydroxyethyl) -amino) -s-triazolo (1,5-a) 5-a) Pyrimidine-5-diethylamino-7- (N- (n-hexyl) -N- (β-hydroxyethyl) -amino) -s-triazolo (1,5-a) pyrimidine-5-piperidino-7- (n-hexyl) -N- (β-hydroxyethyl) -amino) -s-triazolo (1,5-a) pyrimidine-