IL100241A - 6-Cyclohexyl-2'-O-methyl-adenosine hydrate its preparation and pharmaceutical compositions containing it - Google Patents

Description

100241/2 6-Cyclohexyl-2'-0-methyl-adenosine hydrate, its preparation and pharmaceutical compositions containing it SANDOZ A.G.

C. 84948 100241/2 The invention relates to a new crystal form of 6-cyclohexyl-2'-0-methyl-adenosine. 6-cyclohexyl-2'-0-methyl-adenosine and its pharmacological properties are known from UK-Pat. Appl. 2226027 A. Preparation of the compound is described in this UK-Pat. Appl. and the 6-cyclohexyl-2'-0-methyl-adenosine thereby obtained contains 1 mol of ether per ol of compound and melts at 90-94°C.

In accordance with the invention, it has been found that 6-cyclo-hexyl-2'-0-methyl-adenosine is purer when obtained in the form of the hydrate than when in the form of the known etherate, and in addition is substantially better for use in galenic administration.

The hydrate of 6-cyclohexyl-2'-0-methyl-adenosine is prepared by crystallisation of the known 6-cyclohexyl-2'-0-methyl-adenosine monotherat from an aqueous solvent.

The preferred hydrate of 6-cyclohexyl-2'-0-methyl-adenosine is the 1.5 hydrate.

Preparation of the 6-cyclohexyl-2'-0-methyl-adenosine 1.5 hydrate takes place by recrystallistion of the known 6-cyclohexyl-2 ' -0-methyl-adenosine monoetherate from a mixture of ethyl alcohol and water. - 2 - 100-7708/A In the following example, in which the preparation of the 1.5 hydrate of 6-cyclohexyl-2'-0-raethyl-adenosine is described, all temperatures are given in degrees celsius and are uncorrected. 0.7 g of 6-chloro-9-purinyl-2'-0-methyl-D-ribose are heated to 80° for 2 hours in 30 ml of cyclohexylamine. The mixture is then placed under reduced pressure until dry and the residue is eluted on silica gel with a mixture of methylene chloride/ethyl alcohol 95:5. The purified product is then crystallized from methylene chloride/diethyl ether. Since the product contains diethyl ether, which cannot be removed even under high vacuum, 6-cyclohexyl--2'-0-methyl-adenosine being in form of the monoetherate is crystallized again from ethyl alcohol/water. 6-cyclohexyl-2'-0-methyl-adenosine thereby crystallizes out as the 1.5 hydrate. It has a melting point of 88-91°, [a]D20 = -57.2° (c = 1 in dimethyl formamide) .

Elementary Analysis Calculated: C : 52,3% H : 7,2% N : 17,9% 0 : 22,5% Found: C : 52,5% H : 7,2% N : 18,0% 0 : 22,4% The IR spectrum of this 1.5 hydrate, the result of DSC thermal analysis and the result of NMR analysis are attached as figures 1, 2 and 3. The results of these measurements together with the results of elementary analysis show that according to the above process 6-cyclohexyl-2'-0-methyl-adenosine .1.5 hydrate has been prepared.

The 6-cyclohexyl-2'-0-methyl-adenosine 1.5 hydrate obtained according to the invention has the same pharmacological properties as the corresponding monoetherate, but - as illustrated above - the advantage of the 1.5 hydrate is its improved purity and the better usage for galenic processing. 6-cyclohexyl-2' -O-methyl-adenosine hydrate, especially the 1.5 hydrate, can be used for the activities disclosed in UK-Pat. Appl. 2226027 A (corresponding to Israel Patent 92634) and additionally for the treatment of neurogenerative disorders, certain peripheral neuropathies such as diabetic neuropathy and of disorders associated with peripheral vascular disease and/or disorders associated with neuronal degeneration, hypertriglyceridemia, elevated free fatty acids and/or type I and type II diabetes, arrhythmias and for protection against myocardial infarction, further the compound being active against diseases caused by hypertension such as congestive heart failure, myocardial infarction or sudden cardiac death and renal insufficiency as disclosed in Europ. Pat. Appl. 378 518.

The dose of the compound used will vary in the usual way with the seriousness of the disorders, the weight of the sufferer, and the i relative efficacy of the compound. However, as a general guide suitable unit doses may be 0.1 to 1000 mg, such as 0.5 to 200, 0.5 to 100 or 0.5 to 10 mg, for example 0.5, 1, 2, 3, 4 or 5 mg; and such unit doses may be administered more than once a day, for j example 2, 3, 4, 5 or 6 times a day, but preferably 1 or 2 times per day, so that the total daily dosage for a 70 kg adult is in the range of about 0.1 to 1000 mg, that is in the range of about 0.001 to 20 mg/kg/day, such as 0.007 to 3, 0.007 to 1.4, 0.007 to 0.14 or 0.01 to 0.5 mg/kg/day, for example 0.01, 0.02, 0.04, j 0.05, 0.06, 0.08, 0.1 or 0.2 mg/kg/day; and such therapy may j extend for a number of weeks or months. | i When used herein the term "pharmaceutically acceptable" encompasses materials suitable for both human and veterinary use. 100-7708/A The following pharmacological tests have been effected to establish the activity of the compound: a) Affinity for adenosine receptors Pig striatal membranes were prepared as previously described by H. Bruns et al. in Molecular Pharmacology 29 (1986) pages 331-344. 3H-NECA, a non-selective adenosine receptor agonist was used to label both Ai and A2 receptors. IC50 values were derived from the displacement curves by weighted non-linear least-square curve fitting to the Langmuir equation and pKD values calculated.

The results are presented in the following Table in agreement with the literature, CPA proved a potent and highly selective displacer of binding to the Aj. receptor, CV 1808 was a relatively weak but selective A2 receptor ligand and CGS 21680 showed high potency and selectivity for the A2 receptor. 6-cyclohexyl-2'-0-methyl-adenosine in the form of its 1.5 hydrate shows good affinity and high selectivity for the Aj. receptor. - 5 - 100-7708/A Table: Affinity of adenosine receptor ligands for Ax and A2 receptors Αχ A2 Ai :Aa (KD;nM) (KD;nM) selectivity n CPA* 0.74 + 0.01 33 ± 110 1260 6 CV 1808» 2460 ± 757 269 ± 70 0.1 5 CGS 21680a 4360 ± 1080 10 ± 4 0.004 4 6-cyclohexyl- 23 ± 2 24500 + 5160 1090 5 2'-0-methyl- adenosine 1.5 H20 CPA* = Cyclopentyladenosine CV 1808a = 2-Phenylaminoadenosine (Carbohydrates vol. 81 (1974) ref . 91898 K) CGS 21680» = 2-[p-(2-Carboxyethyl)phenethylamino]-5'-N-ethyl carboxamido-adenosine (FASEB J, 1989, 3 (4) Ref. 4770 and 4773) b) Arrhythmias Adenosine receptor activation reduces the incidence of supraventricular tachycardia and other arrhythmias. The test method is disclosed by C. Clarke et al. in The Pharmaceutical Journal Vol. 244, 595 to 597 (1990). In this test 6-cyclo- hexyl-2'-0-methyl-adenosine is used in form of its 1.5 hydrate. c) Mean arterial blood pressure, bradycardia and peripheral vasodilation on anesthetized rats.

The experiments were carried out on male Vistar rats, body weight 300-350 g (323 ± 3 g), under Inactin anaesthesia (150 mg/kg i.p.), according to the method of Schroeder et al.

(Measurement of the cardiodynamics, haemodynamics and the ECG in the anesthetized rat, effects of catecholamines (dopamine - 6 - 100-7708/A and isoproterenol) in Budden et al. "The Rat Electrocardiogram in Pharmacology and Toxicology" Oxford Pergamon Press 1981, 155-9) Catheters were placed in the right jugular and right femoral veins, in the left ventricle (inserted via the right carotid artery), left femoral artery, and aorta (inserted through the right femoral artery) . The following variables were measured or calculated: systolic, diastolic, and mean arterial blood pressure (mm Hg; left femoral artery, Statham pressure transducer P 23 Gb), pulse pressure (mm Hg), heart rate (beats/min; triggered from the blood pressure curve), rate of rise of left ventricular pressure (dP/dtnax, mm Hg/s; Statham pressure transducer P 23 Gb), cardiac output (ml/min/100 g body weight, thermodilution method, right jugular vein and aorta), total peripheral resistance (dynes · s cm-5/100 g body weight), ECG lead III. Arterial blood pressure, left ventricular pressure, dP/dt max, heart rate, and electrocardiogram were continuously recorded with a Schwarzer polygraph. The parameters were measured 30, 20, 10 and 2 min before administration of the substance and 1, 10, 30 and 60 min after injection of the drug into the right femoral vein. 6-cyclohexyl-2'-0-methyl-adenosine in form of its 1.5 hydrate was tested in doses of 0.003, 0.01 and 0.03 mg/kg whereby five animals per dose were used.

Blood flow through ischaemic skeletal muscle Male normotensive rats (ca. 300 g) were anaesthetised with Evipan Na (160 mg/kg i.p.) and the right femoral artery was ligated. Three weeks after ligation the rats were anaesthetised with pentothal (Thiopental-Na, 40 mg/kg i.p.) and placed on a special holder for measurement of the resonances of phosphocreatine (PCr), inorganic phosphate (Pi), of the three phosphates of ATP, and of phosphomono-esters and sugar phosphates (PM) by use of a Bruker Biospec 47/15 spectrometer equipped with a a 4.7 T horizontal magnet of 15 cm clear bore. The blood supply to the right hindleg was then - 7 - 100-7708/A temporarily occluded for about 30-40 min by a tourniquet placed around the upper hindleg, until the PCr levels had fallen to about 1/3 of the original level. Thereafter the occlusion was released and the rate of recovery (τχ) of PCr was followed. Thirty minutes later the same procedure was repeated without and after intravenous administration of the drug e.g. 6-cyclohexyl-2'-0-methyl-adenosine in form of its 1.5 hydrate under investigation, the rate of recovery or the PCr level (τ2) being followed again. The ratio of both recovery times (τ2/τι) was taken as a parameter for the drug effect on the peripheral circulation. Some animals were tested with the sympathetic neurotoxin 6-hydroxydopamine, at the time of femoral artery ligation and the effects of 6-cyclohexyl- -2'-0-methyl-adenosine in the form of its 1.5 hydrate on the induced peripheral vascular insufficiency determined. e) Glucose Transport in Rat Adipocytes Adipocytes were isolated from the epididymal fat pads of normal chow-fed rats by digestion with collagenase. Cells (final concentration 2% v/v) were pre-incubated with adenosine deaminase (1 U/ml), test compound 6-cyclohexyl-2'-0- methyl-adenosine in form of its 1.5 hydrate and other additions as indicated for 30 min at 37°C. [3-3H]Glucose (final concentration, 50 uM, 0.5 uCi/ml) was then added, and incubation was continued for a further 60 min. Incorporation of radioactivity from [3-3H]glucose into cell lipids (a measure of glucose transport) was evaluated by extraction of the cell suspension (0.5 ml) with 5 ml of toluene-based scintillant, followed by liquid scintillation counting; water-soluble metabolites and residual [3-3H] glucose remain in the aqueous phase and are not detected. f) Dyslipidemias characterised by elevated serum triglycerides Several studies have shown a positive correlation between - 8 - 100-7708/A serum triglyceride levels and the risk for coronary heart disease (CHD) (Grundy, in Cholesterol and Atherosclerosis: Diagnosis and Treatment, Lippincott, Philadelphia (1990)). The value of reducing elevated triglyceride levels as an approach to reducing the risk of CHD emerged from the Helsinki Heart Study where, following treatment with gemfibrozil, the greatest reduction in serious coronary events occured in Type IIB hyperlipidemic patients in whom both LDL-cholesterol and total serum triglycerides were elevated. 6-cyclohexyl-2'-0-methyl-adenosine in the form of its 1.5 hydrate produced substantial and long-lasting falls in plasma free fatty acids and triglycerides in the Rhesus monkey.

Protection against infarction by preconditioning Preconditioning (5 minutes of ischaemia followed by 10 minutes of recovery) renders the heart very resistant to infarction from subsequent ischaemia. The test method is disclosed in the article of G.S. Liu et al. in Circulation 84, 1, 350 to 356 (1991). In this test 6-cyclohexyl-2'-0-methyl-adenosine is used in form of its 1.5 hydrate. 100241/3 - 9 -

Claims (6)

1. 6-cyclohexy1-2'-O-methyl-adenosine hydrate.

2. Process for the preparation of 6-cyclohexyl-2' -O-methyl- adenosine. hydrate according to claim 1 characterized in that 6-cyclohexyl-2'-0-methyl -adenosine- onoetherate is crystallized from an aqueous solvent.

3. 6-cyclohexyl-2'-0-methyl-adenosine hydrate according to claim 1 characterized in that the hydrate is a 1.5 hydrate.

4. Process for the production of the 1.5 hydrate of 6-cyclo- hexyl-2'-0-meth l-adenosine according to claim 3, characterized in that- the 6-cyclohexyl-2' -O-methyi-aden- osine. onoetherate is crystallized from a mixture of ethyl alcohol and vater.

5. Use of 6-cyclohexyl-2' -O-methyl-adenosine hydrate for the preparation, substantially as described in the specification, of medicaments useful against high blood pressure, as coronary vasodilators, for inhibition of both thrombocyte aggregation and activation of leucocytes, for lowering blood lipid levels, against congestive heart failure, myocardial infarction, sudden, cardiac death, renal insufficiency, for the treatment of neurodegenerative disorders, certain peripheral neuropathies such as diabetic neuropathy and of disorders associated vith peripheral vascular disease and/or disorders associated vith neuronal degeneration, hypertriglyceridemia, elevated free fatty acids and/or type I and type II diabetes, arrhythmias and for protection against myocardial infarction.

6. A pharmaceutical composition comprising a therapeutically effective amount of 6-cyclohex l-2' -O-methyl-adenosine . hydrate and a pharmaceutically acceptable carrier thereof suitable for the treatment of high blood pressure, as 100241/2 coronary vasodilator, for inhibition of both thrombocyte aggregation and activation of leucocytes, for lowering blood lipid levels, against congestive heart failure, myocardial infarction, sudden cardiac death, renal insuffic ency> for the treatment of neurodegenerative disorders, certain peripheral neuropathies such as diabetic neuropathy and of disorders associated vith peripheral vascular disease and/or disorders associated vith neuronal degeneration, hypertriglyceridemia, elevated free fatty acids and/or type I and type II diabetes, arrhythmias and for protection against myocardial infarction. For ih& Applicants DR. REINHOLD CQfW ND PARTNERS Byi