AZOLE DERIVATIVES AS ADENOSINE ANTAGONISTS

This invention concerns novel azole derivative-, more particularly a novel 2-heteroaryl-triazolo[l,5-a] [1,3,5]triazine and the pharmaceutically acceptable salts thereof, which have useful pharmacological properties (and in particular antagonise the actions of adenosine, such as vasodilation) . The invention also includes pharmaceutical compositions containing the novel azole derivatives for use in treating certain diseases and disorders affecting mammalian cardiac, peripheral and/or cerebral vascular systems. Also included are processes for the manufacture and formulation of the novel azole derivatives.

The compound theophylline (1,3-dimethylxanthine) has been used clinically (usually as its ethylene diamine salt, which is also known as aminophylline) as a respiratory stimulant, a centrally acting stimulant, a bronchodilator, a cardiac stimulant and as a diuretic. This diversity of clinical uses is an indication of the range of pharmacological actions which have been attributed to theophylline. These include phosphodiesterase inhibition, adenosine receptor antagonism, mobilisation of intracellular calcium and the release of catecholamines. Recently theophylline has also been reported to be useful in treating myocardial ischaemia (Maseri et al. , The Lancet, 1989, 683-686), skeletal muscle ischaemia ( Picano et al. , Angiology, 1989, 40, 1035-1039) and cerebral ischaemia (Skinhoj et al. , Acta. Neurol. Scand. , 1970, 46, 129-140). The beneficial effects of theophylline in these ischaemic disorders are believed to be due to a reduction or prevention of the phenomenon known as "vascular steal" by virtue of the compound's ability to antagonise the actions of adenosine by blocking the adenosine receptors which mediate metabolism-linked vasodilatation.

The "vascular steal" phenomenon can occur when the major artery supplying a particular vascular bed is partially or totally occluded resulting in ischaemia. In this situation, the compromised vascular bed dilates and blood flow is maintained by either an increase in flow across the narrowed vessel or by an increase in flow through the collateral vessels. However, increased metabolic activity

in adjacent vascular beds results in release of mediators such as adenosine, causing them to dilate, resulting in the limited blood flow to the compromised vascular bed being "stolen" by these adjacent areas. The loss of blood from compromised to normally perfused vascular beds by the phenomenon of "vascular steal" further diminishes the blood flow in the compromised vascular bed.

The diversity of pharmacological properties possessed by theophylline make it difficult to use in the regular treatment or prevention of occlusive diseases and conditions of the vasculature. Thus, -its associated action as a phosphodiesterase inhibitor results in cardiac stimulation which is deleterious for patients with myocardial ischaemia. Furthermore, the relatively low potency of theophylline means that dose-levels which are therapeutically useful are close to those which can cause serious central side-effects.

European patent application, publication number EP-A1-459702 discloses certain 2-heteroaryl-triazolo[l,2,4]triazolo[l,5-a] [1,3,5]- triazines and pyrazolo[2,3-a] [l,3,5]triazines that are effective antagonists of the actions of adenosine, particularly of its vasodilatory actions.

A 2-heteroaryl-triazolo[l,2,4]triazolo[l,5-a] [l,3,5]triazine compound having particularly desirable pharmacological properties has now been found.

Accordingly, the invention provides the compound 2-(2-furyl)-5-[2-(morpholino)ethylamino] [1,2,4]triazolo[l,5-a]- [l,3,5]triazin-7-amine, and the pharmaceutically acceptable salts thereof.

The compound according to the invention may be represented by the formula I, which is set out hereinafter together with the other formulae referred to herein by Roman numerals. It will be referred to hereinafter as the compound of formula I.

The compound of formula I has been found to be a selective antagonist of adenosine at the adenosine A2a receptor, the receptor which mediates the vasodilatory action of adenosine. It has also been found to possess particularly good aqueous solubility and to be surprisingly effective in vivo on oral and parenteral administration.

This combination of properties is unexpected and particularly desirable .

Particular pharmaceutically acceptable salts of the compound of formula I include, for example, salts with acids affording physiologically acceptable anions, for example, salts with acids, such as hydrochloric, hydrobromic, sulphuric, phosphoric, methanesulphonic, trifluoracetic, oxalic, citric and maleic acid.

According to another aspect, the invention provides a process for the preparation of the compound of formula I, or a pharmaceutically acceptable salt thereof, which comprises (a) reacting a compound of the formula II in which Z is a suitable leaving group, for example hydrocarbylsulphonyl such as (l-6C)alkylsulphonyl (such as raethylsulphonyl or ethylsulphonyl), aryloxy such as phenoxy, or halogeno (such as chloro or bromo), with N-(2-aminoethyl)morpholine or a salt thereof.

The reaction may conveniently be carried out at a temperature in the range of from 0 to 120°C, for example from 10 to 80°. Suitable solvents for the reaction include nitriles, such as acetonitrile; alcohols, such as ethanol or propanol; ethers, such as tetrahydrofuran, 1,2-dimethoxyethane or t-butyl methyl ether; and amides such as N,N-dimethylformamide.

Examples of salts of N-(2-aminoethyl)morpholine include alkali metal salts, such as the lithium, sodium and potassium salts.

The starting materials of formula II may be obtained by procedures described in EP-A1-459702. Thus, for example, those compounds of formula II in which Z is alkylsulphonyl may be made by oxidation of the corresponding alkylthio derivative of formula III in which R is (1-6C)alkylthio, using a conventional oxidant such as a peracid, for example, peracetic, perbenzoic or chloroperbenzoic acid, conveniently at a temperature in the range, for example, 0 to 40 °C, and in a suitable solvent or diluent such as dichloromethane or chloroform. Similarly, those compounds of the formula II in which Z is chloro or bromo may be obtained, for example, by reacting an alkylthio derivative of formula III (especially in which R is methylthio or ethylthio) with chlorine or bromine in the presence of hydrogen chloride or hydrogen bromide, respectively, at a temperature

in the general range, for example, -20 to 15 °C and in a generally inert polar solvent such as ethanol or 2-propanol.

The starting alkylthio starting materials of formula III may themselves be obtained, for example, by reaction of a compound of the formula IV with the appropriate dialkyl N-cyanodithioiminocarbonate of formula V, at elevated temperature in the range, for example, 60 to 200 °C, conveniently as a melt in the absence of solvent or diluent.

The starting compounds of formula IV may themselves be obtained, for example by reacting the appropriate iminoether of the formula Q.C(0R)=NH in which Q is 2-furyl and R is (l-4C)alkyl such as methyl or ethyl (formed from the corresponding nitrile of the formula Q.CN and alcohol of the formula R.OH in the presence of an anhydrous acid such as hydrogen chloride) with an aminoguanidine salt (especially the nitrate) in the presence of a suitable base, such as pyridine or 2,6-lutidine, which may also be used as the reaction solvent, at a temperature in the range, for example, 60-120 °C.

The compounds of formula II in which Z is a phenoxy group may conveniently be prepared by reacting a 5,7-diphenoxy-[l,2,4]- triazolo[l,5-a] [1,3,5]triazine with ammonia. The process is conveniently effected at a temperature in the range of, for example, from 0 to 100°C. Suitable solvents for the process include alcohols such as ethanol and ethers such as tetrahydrofuran. It is particularly convenient to employ a solution of ammonia in an alcohol, such as ethanol, at ambient temperature.

The 5,7-diphenoxy-[l,2,4]triazolo[l,5-a] [l,3,5]triazine starting materials may be obtained by dehydrating a compound of formula VI in which each Z is a phenoxy group. Suitable dehydration agents include, for example, polyphosphoric acid silyl esters, such as polyphosphoric acid trimethylsilyl ester; phosphorus pentoxide and sulphonyl chlorides such as p-toluenesulphonylchloride. The dehydration is conveniently effected at a temperature in the range of from 60-180°C. When a polyphosphoric acid silyl ester or phosphorus pentoxide is used, convenient solvents include the aromatic hydrocarbons such as xylene or toluene. When a sulphonyl chloride is used, convenient solvents include tertiary amines such as pyridine.

The compounds of formula VI may be obtained by reacting a compound of formula VII in which each Z is a phenoxy group with a compound of formula QCOHal in which Hal is a halogen atom such as a chlorine atom. The reaction is conveniently effected at a temperature in the range of from -10 to 40°C. Suitable solvents for the reaction include halogenated hydrocarbons such as dichloromethane.

The compounds of formula VII may be obtained by reacting a compound of formula VIII in which each Z is a phenoxy group with hydrazine.

Alternatively, the compounds of formula VI may be obtained by reacting a compound of formula VIII in which each Z is a phenoxy group with a compound of formula QC0NHNH-.

According to another aspect, the invention provides another process for the preparation of the compound of formula I, or a pharmaceutically acceptable salt thereof, which comprises (b) reacting a compound of the formula IX in which Z is a suitable leaving group, for example hydrocarbylsulphonyloxy (such as p-toluenesulphonyloxy) or halogeno (such as chloro or bromo), with morpholine.

The process is conveniently performed at a temperature in the range of, for example, 10 to 120°C, more conveniently in the range 30 to 80°C. Suitable solvents for the reaction include, for example alcohols such as ethanol, nitriles such as acetonitrile, amides such as N,N-dimethylformamide and ethers such as tetrahydrofuran, 1,2-dimethoxyethane and t-butyl methyl ether.

The starting materials of formula IX may be obtained by standard procedures well known in the art from the corresponding compound of formula IX in which Z is a hydroxy group. This compound may itself be prepared by reacting a compound of formula II with 2-aminoethanol according to the method of process (a) described hereinabove.

According to another aspect, the invention provides another process for the preparation of the compound of formula I, or a pharmaceutically acceptable salt thereof, which comprises

2

(c) reacting a compound of formula X in which Z is a suitable leaving group, for example aryloxy (such as phenoxy), alkylthio (such as methylthio) or halogeno (such as chloro or bromo) with ammonia.

The reaction is conveniently effected at a temperature in the range of, for example, from 0 to 100°C. Suitable solvents for the process include water, alcohols such as ethanol and ethers such as tetrahydrofuran.

The starting materials of formula X may be obtained by dehydrating a compound of formula XI. Suitable dehydration agents include, for example, phosphorus pentoxide, a polyphosphoric acid silyl ester such as polyphosphoric acid trimethylsilyl ester or a sulphonyl chloride such as p-toluenesulphonylchloride. The dehydration is conveniently effected at a temperature in the range of from 60-180°C. When phosphorus pentoxide is used, convenient solvents include the aromatic hydrocarbons such as xylene or toluene. When a sulphonyl chloride is used, convenient solvents include tertiary amines such as pyridine.

The compounds of formula XI may be obtained by reacting a compound of formula VI with N-(2-aminoethyl)morpholine.

According to another aspect, the invention provides another process for the preparation of the compound of formula I, or a pharmaceutically acceptable salt thereof, which comprises

(d) dehydrating a compound of formula XII Suitable dehydration agents include, for example, polyphosphoric acid silyl esters*, such as polyphosphoric acid trimethylsilyl ester; phosphorus pentoxide and sulphonyl chlorides such as p-toluenesulphonylchloride. The dehydration is conveniently effected at a temperature in the range of from 60-180°C. When a polyphosphoric acid silyl ester or phosphorus pentoxide is used, convenient solvents include the aromatic hydrocarbons such as xylene or toluene. When a sulphonyl chloride is used, convenient solvents include tertiary amines such as pyridine.

The starting materials of formula XII may be prepared from a compound of formula VIII by reaction, in any convenient order, with ammonia, N-(2-aminoethyl)morpholine and 2-furoic acid hydrazide.

Whereafter, when a pharmaceutically acceptable salt is required, it may be obtained, for example, by reacting a compound of formula I with the appropriate acid or base affording a physiologically acceptable ion or another conventional procedure.

As stated above, the compounds of the invention possess the property of antagonising one or more of the physiological actions of adenosine and are valuable in the treatment of diseases and medical conditions affecting the mammalian cardiac, peripheral and/or cerebral vascular systems, such as ischaemic heart disease (angina), peripheral vascular disease (claudication) and cerebral ischaemia. The compounds may also be useful in the treatment of migraine.

The effects of compounds of formula I as adenosine receptor antagonists may be demonstrated in one or more of the following standard in vitro and/or in vivo tests.

(a) A„ Adenosine receptor affinity test

This test involves the ability of a test adenosine antagonist to displace the known adenosine mimetic agent 3 [ H]-N-ethylcarboxamidoadenosine (NECA) from binding sites on membrane preparations derived from the rat phaeochromocytoma cell line PC 12

(available from the Beatson Institute, Glasgow). The basic procedure has been described by Williams e_t al. (J. Neurochemistry, 1987, 48(2),

498-502).

The membrane preparation is obtained as follows:

Frozen pellets of PC12 cells are washed twice with ice cold, buffered, physiological saline and the cells recovered by centrifugation (1500G) at 3°C. The separated cells are then suspended in hypotonic solution

(distilled water), allowed to stand on ice for 30 minutes and are then carefully homogenized using a standard high-speed homogeniser with periodic ice-cooling to obtain a fine suspension. The homogenate is centrifuged (48000G) and the pellet is resuspended in 50 mM tris-HCl buffer, pH 7.4 containing adenosine deaminase (5 units/ml, Type VII from calf intestinal mucosa, available from Sigma Chemical

Corporation, under reference no. A1280). The mixture is then incubated at 37°C. After 20 minutes, the reaction is terminated by dilution with ice-cold buffer and transfer onto ice. The material

obtained containing the cell membranes is recovered by centrifugation and washed by resuspension in buffer and recentrifugation. The pellet produced is then resuspended in ice-cold buffer using a hand-driven homogenizer. The resultant membrane suspension is frozen and stored under liquid nitrogen until required.

Binding studies are carried out in microtitre plates, the assay mixtures being buffered in 50 mH tris-HCl, pH 7.4 at room temperature. The test compound is dissolved in dimethyl sulphoxide (DMSO) and then diluted with assay buffer to give the test solutions. [The final concentration of DMSO is not allowed to exceed 1% by volume, at which level it does not affect radioligand binding to the membrane receptor.] Incubations are performed at 30°C for 90 minutes in a total volume of 150 μl comprising the test solution or buffer (50 μl), tritiated NECA (50 μl) and membrane suspension (50 μl). After incubation, the samples are rapidly filtered over glass-fibre mats and the filter mats are washed to remove non-receptor-bound radioligand. Receptor-bound radioligand entrapped on the filter mats is then determined by liquid scintillation counting. Filtration and washing are carried out using a conventional vacuum filtration cell harvester.

The specific binding (defined as the difference between the total binding and the non-specific binding) in the presence of the particular test compound is determined and compared with the control value. Results are conveniently expressed as the negative logarithm of the concentration required to cause a 502 displacement of control specific binding (pIC- ₀ ).

The compound of Example 1 herein shows a pIC _c0 of 7.6. Using the same test procedure, the known compound 1,3-dimethylxanthine typically shows a pICc _n °- about 5.

(b) Guinea-pig Atrial Bradycardic Test

This test has also been described by Collis et al. (British J^ Pharmacology, 1989, 9]_, 1274-1278) and involves the ability of a test compound to antagonise the bradycardic effect of the adenosine mimetic, 2-chloroadenosine, in a beating guinea-pig atrial preparation, an effect mediated via the adenosine receptor known as A,.

The atrial pair preparation may be obtained as follows:- Atrial pairs are obtained from guinea-pigs (Dunkin Hartley strain, 250-400g males) and mounted in organ baths containing oxygenated Krebs buffer solution (952 0-; 52 C0 ₂ ) at 37°C. The spontaneously beating atria are then placed under a resting tension of 1 g and allowed to equilibrate for 50 minutes with continuous overflow. Overflow is then stopped and adenosine (1 Unit\ml) added to prevent the accumulation of endogenously produced adenosine. After equilibration for 15 minutes, a cumulative dose response curve to the adenosine

-8 -4 mimeςic, 2-chloroadenosine (10 H to 10 M) is administered to produce a maximal slowing of atrial rate. After washout during 30 minutes, adenosine deaminase is readministered to the bath which is allowed to equilibrate for 15 minutes. A 10 ^" M solution of the test compound in

DMSO is then added to the bath which is left to incubate for 30 minutes. Any effect on the beating rate due to the test compound is noted before the dose response curve to 2-chloroadenosine is repeated.

Compounds which are adenosine antagonists attenuate the

2-chloroadenosine response.

Test compounds are assessed by comparing dose response curves to 2-chloroadenosine alone with those obtained in the presence of the compound. Competitive adenosine antagonists produce a parallel shift in the 2-chloroadenosine dose response curve. The dose ratio

(DR) is calculated from the ratio of the concentration of

2-chloroadenosine to produce a 50% reduction in atrial rate (ED,- _n ) in the presence of the test compound divided by the ED concentration of

2-chloroadenosine in the absence of the test compound for each atrial pair. The pA2 is then calculated using a standard computational technique. The compound of Example 1 herein has a pA2 of 5.5. The known compound, 1,3-dimethylxanthine, typically shows a pA2 of about

5.

If the pA2 value obtained for the compound of Example 1 in this test is compared with the pIC,- ₀ obtained for the same compound in test (a), it can be seen that the compound is over one hundred fold selective for the A„ receptor over the A ₁ receptor. This is particularly desirable, because antagonism of adenosine at the A..

receptor is associated with effects on the kidney, adipocytes, heart and central nervous system.

(c) Anaesthetised dog Test

This test involves the assessment of the effects of a test compound on antagonising the actions of adenosine in lowering heart rate and increasing vasodilation (as measured by a fall in hind-limb perfusion pressure).

Beagles (12 - 18 kg) are anaesthetised with sodium pentobarbitone (50 mg/kg, iv). The following blood vessels are catheterised: right jugular vein (for infusion of the anaesthetic at approximately 112 mg per hour as a 3 mg/ml solution in isotonic saline), right brachial vein (for administration of drugs and test agents), right brachial artery (for measurement of systemic blood pressure and pulse rate) and the left carotid artery (for administration of adenosine into the left ventricle). Both vagi, the right femoral and sciatic nerves are ligated and severed. A bolus injection of 1250 U heparin is administered before perfusing the right hindlimb at constant blood flow with blood from the iliac artery. The right leg is tied just below the ankle. Xamoterol (1 mg/kg) is then administered to the animal to stabilise heart rate at a high level and nitrobenzylthioinosine (NBTI, 0.5 mg/kg) to inhibit the uptake of adenosine. The animal is sensitised to adenosine during the equilibration time following NBTI by carrying out a dose response curve (DRC). During this time any blood gas or pH imbalance is corrected. A control DRC to adenosine is performed followed by up to three DRC's after cumulative administration of the test compound administered in a mixture of 50% v/v polyethylene glycol (PGE) 400 and 0.1M sodium hydroxide. Each DRC is carried out 15 minutes after administration of test compound and after the measured parameters of heart rate and hindlimb perfusion pressure have returned to a stable state. Similarly, blood gases and pH are maintained within physiological limits throughout the evaluation.

The amount of adenosine required to cause a 50% fall in measured parameter (ED ) i.e. heart rate and hindlimb perfusion pressure is calculated for each does of test compound and a Schild plot constructed. From this plot a K _D value is determined for

D

antagonism of heart rate response and vasodilator response to adenosine. The compound of Example 1 has been found to give a K_ in the range of from 0.01 - 0.24 mg/kg for antagonism of vasodilator response to adenosine with no indication of toxic or other untoward properties at doses several times greater than the minimum effective dose.

(d) Conscious cat blood pressure test.

This test assesses the ability of a test compound to antagonise the fall in diastolic blood pressure produced by administration of adenosine.

Male cats (2 - 4.5kg) are selected and trained to sit quietly and calmly in an open fronted box. Suitable animals are then prepared for chronic catheterisation. Anaesthesia is induced with alphalaxone/alphadolone (18mg, iv) and maintained with halothane (1.5-3%) supplemented oxygen. The dorsal and ventral regions of the neck are clipped then swabbed with alcohol/chlorhexidine/water (70/20/10% v/v) solution. The right carotid artery (for measurement of systemic arterial blood pressure) and the right jugular vein (for administration of adenosine or test substance) are catheterised and exteriorised. Both catheters are sutured in and the incision closed using suture. The cats are allowed to regain consciousness before being transported to the animals rooms where they are housed individually. A minimum post surgical recovery time of one week is allowed before any of the animals are used for test.

All the surgery is performed using aseptic techniques, all instruments are autoclaved before use. Catheters are soaked in alcohol/chlorhexidine solution for at least one hour prior to implantation, the catheters are then washed with sterile saline to avoid tissue necrosis caused by the alcohol/chlorhexidine solution.

The cats are placed individually, unrestrained into open fronted boxes. After a short period of acclimatisation a siting dose of 0.6mg/kg adenosine is administered to assess the sensitivity of the cat towards adenosine. Three consecutive bolus injections of either 0.6, 1.0 or lmg/kg adenosine are administered and the fall in diastolic blood pressure is measured. Cats are then dosed with the

test compound either as a solid or as a solution in polyethylene glycol (PEG) 400, p.o. or in solution for intravenous administration.

The adenosine challenges are repeated at between 15 min and 24 hours post administration of the test compound. Activity of the test compound is expressed as a percentage inhibition of the diastolic blood pressure response. The compound of Example 1 has been found to have significant adenosine antagonist acitivity at a dose of 0.3 - lmg/kg without any sign of overt toxicity at several times the minimum effective dose.

The compounds of the invention are generally best administered to warm-blooded animals for therapeutic or prophylactic purposes in the treatment or prevention of cardiovascular diseases and adverse conditions in the form of a pharmaceutical composition comprising said compound of formula I or a pharmaceutically acceptable salt thereof, in admixture or together with a pharmaceutically acceptable diluent or carrier. Such compositions are provided as a further feature of the invention.

In general, it is envisaged that a compound of formula I will be administered orally, intravenously or by some other medically acceptable route (such as by inhalation, insufflation, sub-lingual or transdermal means) so that a dose in the general range, for example, 0.001 mg to 10 (and more particularly in the range, for example, 0.05 to 5 mg/kg) mg/kg body weight is received. However, it will be understood that the precise dose administered will necessarily vary according to the nature and severity of the disease or condition being treated and on the age and sex of the patient.

A composition according to the invention may be in a variety of dosage forms. For example, it may be in the form of tablets, capsules, solutions or suspensions for oral administration; in the form of a suppository for rectal administration; in the form of a sterile solution or suspension for administration by intravenous or intramuscular injection; in the form of an aerosol or a nebuliser solution or suspension, for administration by inhalation; in the form of a powder, together with pharmaceutically acceptable inert solid diluents such as lactose, for administration by insufflation; or in the form of a skin patch for transdermal administration. The

compositions may conveniently be in unit dose from containing, for example, 5 - 200 mg of the compound of formula I or an equivalent amount of a pharmaceutically acceptable salt thereof.

The percentage by weight of the compound of formula I present in the composition according to the invention will vary according to' such consideration on the route of adminstration, For example, the composition may comprise from 0.1 to 99.5% by weight of the compound of formula I, or a pharmaceutically acceptable salt thereof.

The compositions may be obtained by conventional procedures using pharmaceutically acceptable diluents and carriers well known in the art. Tablets and capsules for oral administration may conveniently be formed with an enteric coating (such as one based on cellulose acetate phthalate) to minimise the contact of the active ingredient of formula I with stomach acids.

The compositions of the invention may also contain one or more agents known to be of value in the diseases or conditions of the cardiovasculature intended to be treated. Thus, they may contain, in addition to the compound of formula I, for example: a known platelet aggregation inhibitor, prostanoid constrictor antagonist or synthase inhibitor (thromboxane A ₂ antagonist or synthase inhibitor), cyclooxygenase inhibitor, hypolipidemic agent, inotropic agent or thrombolytic agent.

In addition to their use in therapeutic medicine, the compounds of formula I are also* useful as pharmacological tools in the development and standardisation of test systems for the evaluation of new cardiovascular agents in laboratory animals such as cats, dogs, rabbits, monkeys, rats and mice.

The invention will now be illustrated by the following non-limiting Examples in which, unless otherwise stated:- (i) evaporations were carried out by rotary evaporation in vacuo;

(ii) operations were carried out at room temperature, that is in the range 18-26°C;

(iii) flash column chromatography or medium pressure liquid chromatography (MPLC) was performed on silica gel [either Fluka ieselgel 60 (catalogue no. 60738) obtained from Fluka AG, Buchs, Switzerland, or Merck Kieselgel Art. 9385, obtained from E Merck, Darmstadt, Germany] ;

(iv) yields are given for illustration only and are not necessarily the maximum attainable by diligent process development; (v) proton NMR spectra were normally determined at 200 MHz in deuterated dimethyl sulphoxide as solvent, using tetramethylsilane (TMS) as an internal standard, and are expressed as chemical shifts (delta values) in parts per million relative to TMS using conventional abbreviations for designation of major peaks: s, singlet; m, multiplet; t, triplet; br, broad; d,doublet; q,quartet; and (vi) all end-products were characterised by microanalysis, NMR and/or mass spectroscopy.

Example 1

N-(2-Aminoethyl)morpholine (2.60g) was added to a stirred suspension of 7-amino-2-(2-furyl)-5-methylsulphonyl- -[l,2,4]triazolo[l,5-a] [l,3,5]triazine (3.36g) in acetonitrile 200ml and stirring was continued for 2 hours. The solvent was evaporated and the residue was purified by chromatography on silica (200g) eluting with dichloromethane containing methanol (10% v/v). The solid (4.0g) obtained was crystallised from ethanol to give 2-(2-furyl)-5-[2-(morpholino)ethylamino] [1,2,4]triazoloIl,5-a] [1,3,5]- triazin-7-amine (3.03g), m.p. 242-4°C; microanalysis, found: C,50.7; H,5.40; N,33.7%; C^H^N^ requires C 50.9 H 5.5 N 33.9% NMR: 2.47 (broad, 6H, CH_ ₂ N(CH ₂ ) ₂ ), 3.43 (q,2H, CH ₂ NH) , 3.61(m,4H,CH ₂ 0CH ₂ ) , 6.68 (d of d, 1H, furyl-4H , 7.06(d, 1H, furyl-3H) , 7.27(br, 1H, NH) , 7.86(d,lH, furyl-5H) and 8.15 (br, 2H, NH ₂ ); m/e 331(M+H) ⁺ .

The necessary starting material was prepared as follows:- (1) Hydrogen chloride gas (20.Og) was bubbled into an ice-cooled mixture of 2-furonitrile (46.5 g) and absolute ethanol (23.0 g). After addition of the gas, solid crystallised from the mixture. The crystalline solid was collected by filtration and heated in pyridine (300 ml) with aminoguanidine nitrate (56.0 g) under reflux for 4

hours. The mixture was cooled, solid material removed by filtration and the filtrate evaporated to give crude 3-amino-5-(2-furyl)-1,2,4- triazole. This material was purified by treatment with nitric acid (400 ml of 50% v/v). The crystalline salt which formed was collected by filtration, washed sequentially with water (100 ml) and ethanol (50 ml) and air dried to give 3-amino-5-(2-furyl)-l,2,4-triazole nitrate (45.0 g), m.p. 130-133°C (decomp.). Several batches (184.Og) of this salt (184 g) were suspended in hot water (400 ml) and sodium carbonate (46.0 g) was added in portions. The basic solution obtained was allowed to cool to give 3-amino-5-(2-furyl)-l,2,4-triazole (82.0 g) as colourless prisms, m.p. 204-206°C; NMR 6.05(s, 2H, NH ₂ ), 6.6(s, 1H, furyl-4H), 6.7(s, 1H, furyl-3H) , 7.7(s,lH, furyl-5H) , 12.05(br s,lH NH).

(2) An intimate mixture of 3-amino-5-(2-furyl)-l,2,4-triazole (33.0 g) and dimethyl N-cyanodithioiminocarbonate (33.0 g) was heated at 170°C for 1 hour, under a slow stream of argon. After cooling, the resulting solid was purified by column chromatography on silica

(600 g) eluting with an increasing amount of ethyl acetate in dichloromethane (5-10% v/v) to give 7-amino-2-(2-furyl)- 5-methylthio-[l,2,4]triazolo[l,5-a] [l,3,5]triazine as a colourless solid (11.1 g), essentially pure by T C, which was used without further purification. [A small amount of the above solid was recrystallised from ethanol to give, crystals, m.p. 238-240°C; microanalysis, found: C,44.0; H,3.3; N,33.7; CgHgNgSO. 0.05C ₂ H,-OH requires C,43.6; H,3.3; N,33.6; NMR 1.05 and 3.4 (t+q, ethanol of crystallisation), 2.5 (s, 3H, CH ₃ S-), 6.7(dd, 1H, furyl-4H) , 7.2(d, 1H, furyl-3H), 7.7(d, 1H, furyl-5H) 8.7-9.0(br d, 2H, NH ₂ ); m/e 248 (M ⁺ ).

(3) A solution of 3-chloroperoxybenzoic acid (50% strength, 45.Og) in dichloromethane (300 ml) was added to a stirred,ice-cooled suspension of 7-amino-2-(2-furyl)-5-methylthio-[1,2,4]triazolo- [1,5-a] [1,3,5]triazine (8.0 g) in dichloromethane (300 ml). The residual aqueous layer was discarded. The resulting suspension was allowed to warm to ambient temperature and stirred for 16 hours. The solvent was evaporated and ethanol (150 ml) was added to the residue. The suspension obtained was left to stand for 30 minutes with

occasional swirling. The solid was then collected by fitration, washed with ethanol and dried to give 7-amino-2-(2-furyl)- 5-methylsulphonyl-[l,2,4]triazolo[l,5-a] [l,3,5]triazine (6.6 g) as colourless solid, NMR: 3.3(s, 3H) , CH ₃ -S0 ₂ ), 6.7(q, 1H, furyl-4H) , 7.3(q, 1H, furyl-3H), 7.9(q, 1H, furyl-5H) , 9.4-9.8(d, 2H, NH ₂ ), which was used without further purification.

Example 2

A mixture of 7-amino-2-(2-furyl)-5-phenoxy- -[l,2,4]triazolo[l,5-a] [l,3,5]triazine (1.47g) and N-(2-aminoethyl)- -morpholine (0.78g) in propanol (75ml) was heated under reflux for 48 hours, until thin layer chromatography indicated that the reaction was essentially complete. The solvent was removed and the residue purified by chromatography on silica eluting with methylene chloride methanol 10% v/v. The solid (l.lg) obtained was crystallised from ethanol to give 2-(2-furyl)-5-[2-(morpholino)ethylamino] [1,2,4]- triazolo[l,5-a] [l,3,5]-triazin-7-amine(0.69g) m.p. 243-6°C identical in all respects to the product obtained in Example 1. The necessary starting material may be prepared as follows:-

Hexamethyldisiloxane (3.0 mole) is charged to a slurry of phosphorus pentoxide (1.5 mole, measured as P.O.,.) in xylene. The mixture is then heated to 90°C over 1.5 hours and then stirred for 1 hour at 90°C during which time all of the solid dissolves. N-2-(4,6-diphenox )-[1,3,5]triazinyl]-N'-(2-furoyl)hydrazine (1.0 mole) is then charged to the solution and the temperature is increased to reflux. The solid dissolves, but during the course of the cyclisation a second solid is precipitated. The cyclisation is normally complete in 2.5 hours at which point the mixture is cooled to 25°C, and can for convenience be held overnight. Acetonitrile is then added and the temperature reduced to 15°C. Water is then added. The mixture is then cooled back to 15°C and 0.91 ammonia solution is added, keeping the temperature below 25°C. Once the addition is complete the temperature is increased to 40°C for 1 hour. The reaction mixture is then cooled to 25°C, the solid filtered off and washed with a large volume of water. The yield is approximately 85%.

The requisite N-2-(4,6-diphenoxy)-[l,3,5]triazinyl-N'-(2- furoyl)hydrazine was prepared as follows:-

A solution of 2,4,6-triphenoxy-l,3,5-triazine (7.2 g) and 2-furoic acid hydrazide (2.5 g) in xylene (60 ml) was heated under reflux for 3 hours. The solvent was then removed by evaporation and the residue was purified by chromatography on silica gel (400 g), eluting with methylene chloride/methanol (2-3% v/v). A solid was obtained, and this was crystallised from isopropanol to give N-2- (4,6-diphenoxy)-[1,3,5]triazinyl-N'-(2-furoyl)hydrazine as colourless prisms; m.p. 182-4 °C; microanalysis; found C, 61.4; H, 3.8; N, 17.7%;

^C 20 ^H 15 ^N 5°4 ^re( J ^{uires C} ' ⁶¹ ' ⁷ '> ^H ' ³ ' ⁹ 5 ^N > 18-02; NMR 6.63 (d of d, 1H, 4-furyl H) , 7.05-7.5 (complex, 1H, 3-furyl H and phenyl H) , 7.87 (s, 1H, 5-furyl H), 9.96 (s, 1H, NH) and 10.34 (s, 1H, NH) ; m/e 390 (M+H) ⁺ .

Example 3

2-(2-Furyl)-5-[2-(morpholino)ethylamino] [1,2,4]- triazolo[l,5-a] [1,3,5]-triazin-7-amine (14.2g) was dissolved in warm methanol (1,400ml) and acidified to to pH3 with 7N hydrogen chloride in dioxane. The mixture was allowed to remain at pH3 for 30 minutes. The solvent was evaporated and replaced with fresh methanol, which was then evaporated. This process was repeated twice more.

The residue was recrystallised from methanol (80ml) to give, after drying at 80°/vacuum/16 hours, 2-(2-furyl)-5-[2-(morpholino)- ethylamino] [l,2,4]triazolo-[l,5-a] [l,3,5]triazin-7-amine, bis hydrochloride (15.6g) m.p. 313-15 dec. microanalysis; found: C, 39.6; H,5.4; N,26.6: Cl, 15.9; H20, 5.1%; C^H-^NgO _j - 2HC1 - 1.2 H.,0 - 0.05 CH ₃ 0H requires: C, 39.57; H, 5.34; N, 26.27; Cl, 16.63; H ₂ 0, 5.06%; MS 331(M+H) . NMR: at room temperature the spectrum was complicated by the presence of rotamers. At 373°K δ 3.3(m,6H, 3 x CH ₂ N) ; 3.75(t,2H,CH ₂ NH); 3.9(t,4H,CH ₂ 0CH ₂ ) ; 6.65(m,lH,furyl-4H) ; 7.09(d,lH,furyl-3H); 7.5(brs, 1H, NHCH ₂ ); 7.8(d,lH,furyl-5H); 8.0(brs, 2H,NΗ ₂ ); also at δ 3.2(s, methanol of crystallisation).

Example 4

The following illustrate representative pharmaceutical dosage forms containing the compound of formula I, or a pharmaceutically acceptable salt thereof (hereafter referred to as "compound X"), for therapeutic or prophylactic use in humans:-

(a) Tablet mg/tablet

Compound X 50

Lactose Ph.Eur 223.75

Croscarmellose sodium 6.0

Maize starch 15.0

Polyvinylpyrrolidone (5% w/v paste) 2.25

Magnesium stearate 3.0

(b) Capsule mg/capsule

Compound X 10

Lactose Ph.Eur 488.5

Magnesium stearate 1.5

The above formulations may be obtained by conventional procedures well- known in the pharmaceutical art. The tablets may be enteric coated by conventional means, for example to provide a coating of cellulose acetate phthalate.

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