WO1998055494A1

WO1998055494A1 - Treatmemt and prophylaxis of infarction by dinucleotides

Info

Publication number: WO1998055494A1
Application number: PCT/GB1998/001655
Authority: WO
Inventors: Christoph Thiemermann; George Michael Blackburn
Original assignee: William Harvey Research Limited
Priority date: 1997-06-06
Filing date: 1998-06-05
Publication date: 1998-12-10
Also published as: GB9711848D0; AU7781998A

Abstract

The present invention relates to compounds for use in the treatment or prophylaxis of infarction associated with reperfusion injury, particularly skeletal muscle infarction associated with reperfusion injury.

Description

TREATMEMT AND PROPHYLAXIS OF INFARCTION BY DINUCLEOTIDES

The present invention relates to compounds for use in the treatment or prophylaxis of infarction associated with reperfusion injury, particularly skeletal muscle infarction associated with reperfusion injury .

Infarction is most commonly due to the blockage of a nutritive blood vessel to the tissue or organ by a blood clot or thrombosis. The subsequent cessation of blood flow (ischae ia) to the tissue or organ results in the death of some of the tissue or organ.

Reperfusion by thrombolytic therapy, percutaneous transluminal angioplasty or bypass surgery, has emerged as the fundamental strategy in the management of acute ischaemic syndromes of the heart and skeletal muscle. Without question, early reperfusion is an absolute prerequisite for the survival of, for example, the ischaemic myocardium. However, there is now a substantial amount of evidence that reperfusion leads to an additional injury (Forman et al . , Circulation, 81, 69-78, (1990); Hearse & Bolli, Trends Cardiovasc. Med. , 1, 233-240, (1991); Jeroudi et al . , Am. J. Cardiol., 73, 2B-7B, (1994); Hansen, Eur . Heart. J., 16, 734-740 (1995) so that reperfusion itself can lethally damage cells. The consequences of reperfusion (leading to reperfusion injury) have been primarily investigated in the heart. It is now generally accepted that reperfusion itself triggers sudden metabolic, electrophysiologic, morphologic and functional changes which are detrimental to the myocardium. To convincingly demonstrate that a drug interferes with reperfusion-injury , injection of this drug prior to the onset of reperfusion (rather than before the onset of ischaemia) should result in a significant reduction in infarct size (Hearse & Bolli, Trends Cardiocasc. Med., l, 233-240, (1991)). The detrimental consequences of reperfusion, for example in the heart, include (i) reperfusion-induced arrhythmias, (ii) myocardial stunning (iii) lethal reperfusion injury and (iv) accelerated necrosis. Although the mechanisms leading to reperfusion injury are not entirely clear, there is now a substantial amount of evidence indicating that the generation upon reperfusion of oxygen-derived free radicals and abnormalities of calcium-homeostasis (calcium overload of cells) importantly contribute to the above manifestations of reperfusion injury. Although there is some formation of radicals during ischaemia, there is a dramatic increase in the formation of oxygen-derived free radicals in the early reperfusion period. Similarly, alterations in calcium- homeostasis occur much more frequently during the reperfusion of the ischaemic myocardium. Oxygen-derived free radicals (superoxide anions, hydroxyl-radical , hydrogen peroxide) are generated upon reperfusion and cause increased membrane permeability. The increased membrane permeability allows easier access of calcium into the myocytes leading to mitochondrial calcium overload with subsequent damage to the mitochondrial structure and loss of the ability to produce adenosine triphosphate (ATP) , which ultimately results in cell death. Thus, reperfusion injury is currently believed to be caused by a complex interaction between the generation of free radicals and the alterations in calcium homeostasis and therefore potentially amenable to a specific therapy aimed at reducing reperfusion injury.

The prior art is mainly directed to myocardial ischaemia which as a condition is distinct from reperfusion injury and especially reperfusion injury of skeletal muscle. The term myocardial ischaemia describes a condition that exists when the uptake of oxygen in the heart is insufficient to maintain the rate of cellular oxidation and metabolism. This leads to extremely complex situations, which have been extensively studied in recent years. Although there is no definite answer as to the factors determining cell death during ischaemia (without reperfusion) , it is well accepted that a fall in ATP below critical levels is of major importance. In the absence of mitochondrial injury (see above) cellular ATP levels are critically dependent on oxygen supply and oxygen demand and, hence, therapies which either reduce oxygen demand or increase oxygen supply have been shown to reduce ischaemic tissue injury. It has, however, previously been extremely difficult to delineate the mechanisms leading to ischaemic injury from the ones leading to "reperfusion-injury" as the assessment as to whether an ischaemic tissue will inevitably die can only be assessed by reperfusion of the ischaemic tissue.

The important question as to whether a specific drug or intervention reduces infarction by interfering with the mechanisms leading to ischaemic or reperfusion injury can be assessed by comparing the reduction in infarct size afforded by this drug when given either before ischaemia (with or without reperfusion) or before reperfusion. Drugs which reduce infarct size when given just prior to the onset of reperfusion clearly reduce infarct size by interfering with the events leading to reperfusion-injury.

In contrast, drugs which reduce infarct size when given during the ischaemic period (or even before, but not prior to reperfusion) are likely to give protection by causing a reduction in ischaemic tissue injury. This applies particularly to drugs which are rapidly metabolised, as they are unlikely to interfere with the consequences of reperfusion.

Diadenosine 5 • , 5 ' ' ' -P¹ , P^-tetraphosphate (AP₄A) has been reported (European Patent Application EP-A2-0437929) as being useful in the treatment of heart disease, specifically in the treatment of arrythmia or for use as a vasodilator. Use of diadenosine 5 • , 5 • • ' -P¹ , P⁴-tetraphosphate as an anti- thrombotic agent is also discussed in International Patent Application WO89/04321 and United States Patent 5,049,550. Diadenosine 5 ' ,5 • ' '-P¹,P⁵ pentaphosphate (AP₅A) has been reported to be an inhibitor of adenylate kinase (G.E. Lienhard et al . , J. Biol. Che . , 248, 1121 (1973); S.M. Humphrey et al . , Journal of Surgical Research, 43, 1987))

Use of diadenosine 5 ' , 5 ' ' ' -P¹ , P⁴-tetraphosphate for curing ischaemic myocardial disease is disclosed in European Patent Application EP-A1-0689838. No reference to reperfusion injury or the reduction of skeletal muscle infarction is made in the application.

Some analogues of 5 ' , 5 ' ' ' -P¹ , P⁴ tetraphosphate have been disclosed in the prior art (Blackburn et al . , NAR 15, 6994- 7025, (1987)) as well as a number of analogues of S^S¹¹'- P¹ , P³-triphosphate (AP₃A) (Blackburn et al . , Tetrahedron letters, 31, 5637-5640, (1990) and Guranowski et al . , Nucleosides and Nucleotides, 14, 731-734, (1995)). However, there is no indication that the analogues may be useful in the treatment or prophylaxis of skeletal muscle infarction associated with reperfusion injury.

There remains a need for improved therapeutic compounds for use in the treatment or prophylaxis of infarction especially skeletal muscle infarction associated with reperfusion injury.

According to the present invention, there is provided use of a compound of formula (I):-

wherein X¹ and X² may be the same or different and each is a substituted, unsubstituted or modified purine base, each group represented by Y may be the same or different and each is selected from the group comprising -O- and -CZZ²- wherein Z¹ and Z² may be the same or different and each is selected from the group comprising hydrogen, halogen and alkyl groups, each atom represented by W may be the same or different and each is selected from the group comprising oxygen and sulfur, and n is 2 or 3 , or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prophylaxis of skeletal muscle infarction associated with reperfusion injury.

Infarction associated with reperfusion injury is defined as the tissue necrosis or damage caused on reperfusion of an ischaemic tissue and does not include ischaemic tissue damage, namely that caused by the cessation of blood flow to the tissue.

X¹ and X² may be the same or different. Preferably X¹ and X² are the same.

X¹ and X² may comprise a substituted, unsubstituted or modified purine base or derivative thereof. Preferably, X¹ and X² comprise adenine or a derivative thereof, or guanine or a derivative thereof. Preferably, X¹ and X² comprise adenine or guanine, more preferably adenine.

Adenine and derivatives thereof may comprise radicals of formula (II) :-

wherein R¹ and R² may be the same or different and are selected from the group comprising hydrogen, halogen, and alkyl, aryl, alkoxy, aryloxy, alkythio and arylthio groups, and R³ and R⁴ are the same or different and are selected from the group comprising hydrogen and alkyl, aryl, alkanoyl and aroyl groups.

Adenine and derivatives thereof may also comprise isomers of the radicals of formula (II) , for example radicals of the formula (Ha) :-

Guanine and derivatives thereof comprise compounds of the formula (III):-

wherein R , R³ and R⁴ are as defined above, and R⁵ is selected from the groups comprising hydrogen and alkyl, aryl, alkanoyl and aroyl groups.

Guanine and derivatives thereof may also comprise isomers of the radicals of formula (III) , for example radicals of the formula (Ilia) :-

Modified purines include deazapurines. In particular, modified purines include deazaadenine and derivatives thereof which comprise radicals of formula (IV):-

wherein R², R³ and R⁴ are as defined above, and the R¹ groups are independently selected from the definition of R¹ above.

Modified purines also include deazaguanine and derivatives thereof which comprise radicals of formula (V):-

Other modified purines which will be useful in the present invention will be apparent to those skilled in the art.

Reference in the present specification to alkoxy and aryloxy groups means alky1-0- and aryl-O- groups, and their haloalkyl-O- and haloaryl-O- groups, respectively. Reference to alkanoyl and aroyl groups means alkyl-CO- and aryl-CO-, respectively. Reference in the present specification to an alkyl group means a branched or unbranched, cyclic or acyclic, saturated or unsaturated (e.g. alkenyl or alkynyl) hydrocarbyl radical. Where cyclic, the alkyl group is preferably C₃ to C₁₂, more preferably C₅ to C₁₀, more preferably C₅ to C₇. Where acyclic, the alkyl group is preferably C, to C₁₀, more preferably C, to C₆, more preferably methyl, ethyl, propyl or a halo-derivative thereof.

Reference in the present specification to an aryl group means an aromatic group, such as phenyl or naphthyl, or a heteroaromatic group containing one or more, preferably one, heteratom, such as pyridyl, pyrrolyl, furanyl and thiophenyl. Preferably, the aryl group comprises phenyl.

The alkyl and aryl groups may be substituted or unsubstituted, preferably unsubstituted . Where substituted, there will generally be 1 to 3 substituents present, preferably 1 substituent. Substituents may include halogen atoms; oxygen containing groups such as oxo, hydroxy, carboxy, carboxyalkyl, alkoxy, alkoyloxy; nitrogen containing groups such as amino, alkylamino, dialkylamino, cyano, azide and nitro; sulfur containing groups such as thiol, alkythiol, sulphonyl and sulphoxide; heterocyclic groups containing one or more, preferably one, heteratom, such as thiophenyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, i idazolinyl, pyrazolidinyl, tetrahydrofuranyl , tetrahydrothiophenyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl, piperidyl, piperazinyl, morpholinyl, thionaphthyl, benzofuranyl, isobenzofuryl, indolyl, oxyindolyl, isoindolyl, indazolyl, indolinyl, 7- azaindolyl, isoindazolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolyl, isoquinolyl, naphthyridinyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxadinyl, chromenyl, chromanyl, isochromanyl and carbolinyl; and aryl groups such as phenyl and substituted phenyl. Alkyl includes substituted and unsubstituted benzyl.

Reference in the present specification to halogen means a fluorine, chlorine, bromine or iodine radical, preferably fluorine or chlorine radical.

Each group represented by Y may be the same or different and each is selected from the group comprising -O- (oxygen) and -CZ¹Z²- (substituted or unsubstituted methylene radicals) . Preferably Y are not the same and preferably at least one is -0- and the other or others is/are CZ¹Z².

Each Y may comprise -CZ¹Z²- wherein Z¹ and Z² may be the same or different and each is selected from the group comprising hydrogen, halogen and alkyl groups. Preferably -CZ¹Z²- is CC1₂, CHC1, CF₂, CHF or CH₂.

Each atom represented by W may be the same or different and each is selected from the group comprising oxygen and sulfur. Preferably, W are the same and each are oxygen.

Preferably, the compound for the use of the present invention is diadenosine 5 ' 5 ' ' * -P¹ , P³-substituted triphosphate or diadenosine 5 ' 5 ' ' ' -P¹ , P⁴-substituted tetraphosphate, and more preferably APCC1₂PCC1₂PA, APCF₂PCF₂PA, APCHFPCHFPA, APCHC1PCHC1PA, APCH₂PCH₂PA, APCC1₂PPCC1₂PA, APCF₂PPCF₂PA, APCHFPPCHFPA, APCHC1PPCHC1PA or APCH₂PPCH₂PA.

Reference to skeletal muscle infarction associated with reperfusion injury means tissue necrosis or damage to skeletal muscle arising from the reperfusion of an ischaemic skeletal muscle with blood. "Reperfusion injury", as indicated previously, is thought to be due to the invasion of injured tissue with neutrophils (white blood cells) , which then become activated and cause the release of oxygen radicals and enzymes. A particular feature of the present invention is the prophylactic protection of skeletal muscle afforded by the compounds of the invention against infarction associated with reperfusion injury. This feature makes the compounds of the invention particularly useful in the prophylaxis of infarction associated with reperfusion injury during elective surgery requiring interruption to the blood supply to skeletal muscle and subsequent reperfusion.

The medicaments employed in the present invention can be administered by oral or parenteral route, including intravenous, intramuscular, intraperitoneal , subcutaneous, transdermal, airway (aerosol) , rectal and topical administration .

For oral administration, the compounds of the invention will generally be provided in the form of tablets or capsules or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredients mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

For intramuscular, intraperitoneal, subcutaneous and intravenous use, the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl- pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

The compounds of the invention may also be presented as liposome formulations.

The compounds of the present invention may be presented alone or in combination with thro bolytic agents such as t- PA or streptokinase, or with agents such as prostacyclin, nitric oxide donors, organic nitrates, calcium antagonists, inhibitors of the activity of poly (ADP-ribose) synthetase (PARS) or nitric oxide synthase inhibitors.

The invention further provides use of a compound of formula (I) or an analogue thereof or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prophylaxis of inflammation associated with reperfusion injury of skeletal muscle.

According to a further aspect of the present invention there is a method of treatment or prophylaxis of skeletal muscle infarction or inflammation associated with reperfusion injury comprising adminstration to a patient, an effective dose of a compound of formula (I) or a pharmaceutically acceptable salt thereof. According to a further aspect of the present invention there is provided a compound of formula (I) wherein n is 2 and at least one of the atoms represented by W is S, or a pharmaceutically acceptable salt thereof. Preferably, the compound of the present invention is APSPCCl₂PA, APSPCF₂PA, APSPCHC1PA or APSPCH₂PA.

The compounds of the present invention and the compounds used in the present invention may exist as a number of stereoisomers. For example, APSPCHC1PA has stereoisomeric centres at P and C in the phosphate moiety, which are marked with an asterisk in the formula below, and therefore has 4 stereoisomers for this phosphate moiety.

The invention will now be described with reference to the following examples and to the figures in which :-

Figure 1 illustrates the effect of injection of vehicle (control) compounds and AP₃A compounds of the present invention on infarct size in an anaesthetized rabbit model of skeletal muscle infarction associated with reperfusion (4 hour occlusion, 3 hour reperfusion) (*:P<0.05).

Figure 2 illustrates the effect of control compounds and AP₄A compounds of the present invention on infarct size in an anaesthetized rabbit model of skeletal muscle infarction associated with reperfusion (4 hour occlusion, 3 hour reperfusion) (*:P<0.05).

Figure 3 illustrates the effect of control compounds and AP₃A and AP₄A compounds of the present invention on infarct size in an anaesthetized rabbit model of skeletal muscle infarction associated with reperfusion (4 hour occlusion, 3 hour reperfusion) (*:P<0.05).

Figure 4 illustrates the effect of control compounds and AP₃A and AP₄A compounds of the present invention of infarct size in an anaesthetized rabbit model of skeletal muscle infarction associated with reperfusion (4 hour occlusion, 3 hour reperfusion) (*:P<0.05).

Figure 5 illustrates the effect of control compounds and diadenosine polyphosphates in an anaesthetized rabbit model of skeletal infarction associated with reperfusion (4 hour occlusion, 3 hour reperfusion) (*:P<0.05).

Figure 6 illustrates the effect of APA analogues on infarct size in a rabbit model of acute myocardial ischaemia and reperfusion. (45 minute occlusion, 2 hour reperfusion) (*:P<0.05) .

It will be appreciated that what follows is by way of example only and that modifications to detail may be made whilst still falling within the scope of the invention.

EXPERIMENTAL

Animal selection and exclusion criteria

The study was carried out on male rabbits (New Zealand White SPF rabbits, Regal, Great Bookham, U.K.) weighing 2.0 to 3.5kg receiving a standard diet and water ad libitum . All data obtained from rabbits which died during the course of the experiment due to for example, ventricular fibrillation (n = 0) or cardiac failure (n = 0) were excluded from the study.

Surgical procedures Ten minutes before surgery, all animals were premedicated with 0.1 ml kg^'1 Hypnorm s.c. (containing 0.315 mg ml"¹ fentanyl citrate and 10 mg ml^"1 fluanisone; Janssen Pharmaceutical Ltd.). General anaesthesia was then induced with sodium pentobarbitone (20 mg kg^"1), i.v.; Sagatal, May & Baker) injected into the left marginal ear vein and maintained with supplementary doses of sodium pentobarbitone as required. Lignocaine (Xylocaine 2%, Astra Pharmaceuticals) was also used for local anaesthesia. The rabbits were intubated and ventilated with room air from a Harvard ventilator at a rate of 36-40 strokes per minute and a tidal volume of 18-20 ml. Body temperature was maintained at 38 ± It by means of a rectal probe thermometer attached to a homeothermic blanket control unit (Harvard Apparatus Ltd.) .

The left femoral artery was cannulated and connected to a pressure transducer (Spectramed P23XL) to monitor mean arterial blood pressure and for the withdrawal of blood samples. Whilst monitoring pressure, another catheter was placed in the left ventricle, via the right common carotid artery, for measurement of left ventricular systolic pressure (LVSP) and for drug administration.

Experimental protocol and drug regimen

After surgery, all animals were allowed to stabilise for 30 minutes. In experiments designed to evaluate whether a compound causes a reduction in myocardial infarction size, the compounds (1 mg/kg, bolus, i.a.) were administered slowly via the right common carotid artery into the left ventricle. In experiments designed to evaluate whether a compound causes a reduction in skeletal muscle necrosis, compounds were administered (lmg/kg i.v.) into the marginal ear vein.

Inducing skeketal muscle ischaemia and reperfusion, and determination of myocardial infarct size (in vivo) The method of distal aorta occlusion/reperfusion in the anaesthetized rabbit was performed according to Thiemermann et al . PNAS USA. 94, 679-683, (1997).

For inducing skeletal muscle ischaemia and reperfusion, rabbits were surgically prepared as described above for haemodynamic recordings. Subsequently, after a midline laparotomy, the bifurcation of the aorta was isolated. Thirty minutes later, the distal aorta was occluded (4 h) and reperfused (3 h) . The left and right gracilis muscles were excised in toto and % necrosis was determined by staining of muscle slices with p-nitro-blue tetrazoliu (NBT, 0.5 mg-ml^"1, 20 min at 37C) and weighing stained (alive) and unstained (infarcted) tissue.

Inducing myocardial ischaemia and reperfusion and determination of myocardial infarct size (in vivo)

The method of coronary artery occlusion/reperfusion in the anaesthetized rabbit was performed according to Thiemermann et al . Br. J. Pharmacol. 97, 401-408 (1989).

Rabbits were surgically prepared as described above for haemodynamic recordings and sampling of blood. Subsequently, a 2-3 cm left intercostal thoracotomy (4th intercostal space) was performed and the heart was suspended in a temporary pericardial cradle. A snare occluder was placed around the first antero-lateral branch of the left coronary artery (LAL) (Maxwell et al . , Cardiovasc. Res., 21, 737-746, 1987) 1 cm distal from its origin. In contrast to other species, the rabbit LAL supplies most of the left ventricle and apex of the left ventricular myocardium (Flores et al . , Int. J. Cardiol., 6, 459-471, 1984). Care was taken not to include any veins draining blood from this area. Upon completion of the surgical procedure the animals were allowed to stabilize for 30 min before LAL ligation. Haemodynamic parameters (left ventricular systolic pressure, LVSP; mean arterial blood pressure, MAP; heart rate; pressure rate index, PRI (an indicator of myocardial oxygen consumption)) were measured.

The coronary artery was occluded at time 0 by tightening of the occluder. This was associated with the typical electrocardiographic (ST-segment elevation and increase in R-wave amplitude) and haemodynamic (transient fall in LVSP) changes of myocardial ischaemia. After 45 min of acute myocardial ischaemia, the occluder was re-opened to allow a 2h reperfusion, which was confirmed by the appearance of an "epicardial blush".

Area at risk (AAR) was determined by staining of the perfused myocardium (Evans blue dye) , and infarct size by staining of the AAR with p-nitro-blue tetrazolium (NBT, 0.5 mg-ml^"1, 20 min at 30C) as previously described by Maxwell et al , (Cardiovasc. Res., 21, 737746 (1984). The AAR (all animal groups) ranged from 33±4% to 40±4% (P>0.05, ANOVA followed by Bonfevoni ' s test).

Haemodynamic measurements and electrocardiogram:

Haemodynamic parameters, including mean arterial pressure (MAP) , heart rate (HR) , systolic and diastolic pressure (PA_d) and left ventricular systolic pressure (LVSP) were continuously recorded on a 4-channel Grass 7D polygraph recorder (Quincy, Mass., U.S.A.). However, detailed data analysis was only performed at -10 min (baseline) , -5 min (immediately after bolus adminstration of compound) , 15, 30, 45, 60 min (occlusion period) and every hour during the subsequent reperfusion period. Lead II electrocardiograms (ECCs) were recorded from sub-dermal platinum electrodes on a 7P4H Grass ECG-amplifier attached to Grass 4-channel recorder (Grass, Mass., U.S.A.). The heart rate was automatically calculated from left ventricular systolic pulse curves by means of a Grass 7P4H tachograph. The MAP was calculated as (LVSP-PA + PA_d.

3 The pressure rate index, a relative indicator of myocardial oxygen consumption (Bailer et al . , Basic Res. Cardiol., 76, 163-181, 1981) was calculated as the product of MAP and HR, and expressed in mmHg min^"1.

Statistical comparison: All values in the text and figures are expressed as the mean±s.e.mean of n observations. Statistical comparisons between groups were made by ANOVA followed by Bonferoni's test (myocardial ischaemia experiments) or Dunnet ^• s post hoc test (skeletal muscle experiments) for multiple comparisons. A p value of less than 0.05 was considered statistically significant.

Synthesis of Analogues AP_AA and AP₃A

Compounds of the present invention which are not commercially available may be prepared according to conventional techniques, such as described in Blackburn et al . , NAR, 15, 6991-2025, (1987); Guranowski et al . , Nucleosides and Nucleotides, 14, 731-734, (1995); and Blackburn et al . , Tetrahedron Letters, 31, 5637-5640, (1990) the teachings of which are incorporated herein by reference.

Preparation of Adenosine 5 ' -P¹ , P²-dichloromethylene bisphosphate

Adenosine 5 ' -P¹ ,P²-dichloromethylenebisphosphate was prepared from adenosine (as described in Davisson, V.J. et al . , J . Org. Chem. , 52, 1794-1801 (1987) for adenosine 5 ' -α ,3-difluoromethylene-bisphosphate) but using dichloromethylenebisphosphonate in place of difluoromethylenebisphosphonate. Yield 46 %. Analytical data: NMR δ (D₂0) : 11.1 (d, J 16.7 Hz, P¹) and 8.75 (d, J 16.7 Hz, P²) . 5_H (D₂0) : 8.47 (s, H⁸) , 7.95 (s, H²),6.0 (d, J5, H¹), 4.70 (t, J4.3, H²'), 4.55 (t, J4.4 , H³'), 4.43 (t, J4.4, H⁵',H⁵"), and 4.38 (m, H⁴'). FAB-MS (negative) : m/z 562 (10%, M-H⁺) , 560 (22%, M-H⁺),558 (22%, M-H⁺) , 540 (29%, M-Na⁺) , 538 (89%, M-Na⁺)and536 (100%, M-Na⁺) . C^H^Cl^O^Na_j has MW 563, 561, 559 for the three isotopes of chlorine.

Preparation of Diadenosine 5' ,5' ' ' - (P¹ , P²-dichloromethylene- P³-thio) -P¹. P³-trisphosphate fAPSPCCl.PA) .

Adenosine 5 ' -thiophosphate (180 mg, 0.308 mmol) as its bis-triethylammonium salt and tri-n-octylamine (0.141 ml, 0.323 mmol) were shaken in methanol (7 ml) until dissolution was achieved. The solution was evaporated under reduced pressure. The residue was then coevaporated with pyridine (3 x 10 ml) and further dried under vacuum over P₂0₅ for 12 h. The oily residue was then dissolved in dry dioxane (3 ml). Diphenyl phosphorochloridate (0.098 ml, 0.471 mmol) and tri-n-butylamine (0.176 ml, 0.739 mmol) were added. The mixture was stirred at rt. and the initial cloudy solution gradually became clear. After 3.5 h, the solvent was evaporated and the oily residue was washed with dry diethyl ether (3 x 10 ml) and then coevaporated with dry pyridine (2 x 10 ml) . Adenosine 5 ' - (P¹ , P²-dichloromethylene) diphosphate (165 mg, 0.237 mmol, prepared as described in Davisson et al.) as its tris-triethylammonium salt and tri-n-butylamine (0.113 ml, 0.474 mmol) were shaken in dry methanol (5 ml) until dissolution was achieved, then the solution was evaporated under reduced pressure. The residue was coevaporated with pyridine (3 x 10 ml) and further dried in vacuo over P₂0₅ overnight. The resulting oil was dissolved in dry pyridine (3.6 ml) and this solution was added to the nucleoside activated as above. The reaction mixture was stirred overnight at rt., then evaporated under reduced pressure. The oily residue was partitioned between dichloromethane (2 x 15 ml) and water (50 ml) , the aqueous layer was evaporated under reduced pressure and the residue was chromatographed on a DEAE A-25 Sephadex column with gradient elution using aqueous triethylammonium hydrogen carbonate (TEAB) solution pH 7.6 from 0.05 M to 0.5 M in 4 litres. The product, as its triethylammonium salt, was eluted at a concentration of 0.37 M TEAB. The product-containing fractions were pooled and evaporated under reduced pressure. The residue was coevaporated with methanol (3 x 15 ml) and the compound was obtained as its triethylammonium salt. To converted this into its sodium salt, the product was dissolved in 2 ml methanol and added dropwise into a stirred solution of Nal in acetone (50 ml, 1 M) . The precipitate was collected by centrifugation and washed with acetone (4 x 50 ml) . Yield 97 mg (45% as trisodium salt) . Spectroscopic and analytical data are as follows:

NMR <S_p (D₂0) : 43.8 (d, J 34.5) and 43.5 (d, J 34.5) (P³, two diastereoisomers) , 8.5 (d, J 20.0, P¹), -1.4 (dd, J 20.0 and 34.5) and -1.5 (dd, J 20.0 and 34.5) (P², two diastereoisomers). δ_H (D₂0) : 8.45 (s) , 8.42 (s) , 8.40 (s) and 8.36(s) [2H in total], 8.04 (m, 2H) , 5.97 (m, 2H) and 4.56-4.25 (m, 10H) . FAB-MS (positive) : m/z 839 (M+H⁺) , 861 (M+Na⁺) and 883 (M+2Na⁺-H⁺) .

Preparation of Diadenosine 5' ,5' ' ' - (P¹ , P²-methylene-P³-thio) -P¹.P³-trisphosphate (APSPCH-.PA) .

This compound was prepared similarly to diadenosine 5 ' , 5 • ' ' - (P , P-dichloromethylene-P³-thio) -P¹ , P³-trisphosphate (above). Adenosine 5 ' -thiophosphate (226 mg, 0.4 mmole) as its tris-triethylammonium salt and tri-n-octylamine (179 mg, 0.48 mmole) was activated with diphenyl phosphorochloridate (252 mg, 1.2 mmole) and tri-n-butylamine (0.48 ml) using the methods described for the preparation of diadenosine 5 • , 5 ' * ' - (P¹ , P²-dichloromethylene-P³-thio) -P¹ , P³-trisphosphate in the above section. This was condensed with adenosine 5 ' -methylenebisphosphonate (221 mg, 0.4 mmole) as its tri-n-butylammonium salt as described above. Chromatography of the crude product on Sephadex A-25 with a TEAB gradient from 0.05 M to 0.6 M gave the pure product as a white powder (75 mg, 20 %) with recovery of 61% of unreacted adenosine 5 ' -P¹ , P²-methylenebisphosphonate as its tris-triethylammonium salt (136 mg, 0.25 mmol). Analytical hplc showed this material to be a mixture of 2 diastereoisomers, as also evident in the ³¹P NMR signals. This was converted into the trisodiu salt as described above (58 mg, 18 % yield) . Analytical data are: δ_p (D₂0) : 43.0 ( d, J 31.4) and 43.3 (d, J 31.4) (P³, two diastereoisomers), 17.9 (d J 8.4) and 18.0 (d, J 7.8) (P¹, two diastereoisomers), 7.6 (dd, J 31.2 and 7.9, P²) . _<S„ (D₂0) : 8.50 (s) , 8.43 (s) , 8.35 (s) and 8.31 (s) [2H in total], 8.02 (m, 2H) , 5.91-6.01 (m, 2H) , 4.05-4.74 (m, 10H) , 3.28 (m, 2H, PCH₂P) . FAB-MS (positive): m/z 771 (M+H⁺) , 793 (M+Na⁺) , 815 (M+2Na⁺-H⁺) , 837 (M+3Na⁺-2H⁺) .

Results

AP₃A, AP^A and their analogues reduce the degree of skeletal muscle necrosis caused by ischaemia and reperfusion in the hind limb of the rabbit. Occlusion of aorta (4 h) and reperfusion (3 h) resulted in an infarct size of 52% of the gracilis muscle (Figures 1 to 5) . Intravenous injection (1 min) prior to reperfusion of APA (1 mg/kg) caused a significant reduction in the degree of skeletal muscle necrosis caused by ischemia-reperfusion of the hind limb. Similarly, several analogues of AP^A and AP₃A including APSPCH₂PA, APSPCC1₂PA, APPCF₂PA, and APCHC1PPCHC1PA, caused a substantial reduction in infarct size (Figures 1 to 4) .

Results

AP₃A and AP₄A and their analogues do not reduce the infarct size caused by myocardial ischaemia and reperfusion. Occlusion (1 h) and reperfusion (2 h) of the LAL coronary artery resulted in an infarct size of approximately 50% of the area at risk. Neither AP₃A, AP₄A nor any of their analogues (given at 1 mg/kg 5 min prior to reperfusion of the ischaemic myocardium) had any significant effect on infarct size (Figure 6.)

Claims

CLAIMS t

Use of a compound of formula (I)

wherein X and X^ may be the same or different and each is a substituted, unsubstituted or modified purine base, each group represented by Y may be the same or different and each is selected from the group comprising -0- and -CZ¹Z²- wherein Z¹ and Z² may be the same or different and each is selected from the group comprising hydrogen, halogen and alkyl groups, each atom represented by W may be the same or different and each is selected from the group comprising oxygen and sulfur, and n is 2 or 3, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prophylaxis of skeletal muscle infarction associated with reperfusion injury.

2. Use of a compound according to claim 1 where X¹ and X² may be the same or different and is each adenine or a derivative thereof or guanine or a derivative thereof.

3. Use of a compound according to claim 1 where X¹ and X² may be the same or different and is each a modified adenine or a modified guanine.

4. Use of a compound according to any preceding claim, wherein X¹ and X² are the same.

5. Use of a compound according to claim 4 wherein the compound is diadenosine 5', 5 ' ' ' -P¹ ,P³-triphosphate or diadenosine 5', 5 ΓÇó ' ΓÇó -P¹ , P⁴-tetraphosphate.

6. Use of the compound according to claim 4 wherein the compound is APCC1₂PCC1₂PA, APCF₂PCF₂PA, APCHFPCHFPA,

APCHC1PCHC1PA, APCH-,PCH₂PA, APCCl₂PPCCl₂PA, APCF₂PPCF₂PA, APCHFPPCHFPA, APCHC1PPCHC1PA or APCH₂PPCH₂PA.

7. A compound of formula (I)

wherein X¹ and X² may be the same or different and each is a substituted, unsubstituted or modified purine base, each group represented by Y may be the same or different and each is selected from the group comprising -O- and -CZ¹Z²- wherein Z¹ and Z² may be the same or different and each is selected from the group comprising hydrogen, halogen and alkyl groups, each atom represented by W may be the same or different and each is selected from the group comprising oxygen and sulfur provided at least one atom is sulfur, and is 2 or 3, or a pharmaceutically acceptable salt thereof.

8. A compound according to claim 7 for use in a method of treatment or prophylaxis of skeletal muscle infarction associated with reperfusion injury.

9. A method of treatment or prophylaxis of skeletal muscle infarction associated with reperfusion injury comprising administration to a patient or organ, an effective dose of a compound of formula (I) or pharmaceutically acceptable salt thereof.