Compounds for Use in Therapy
The present invention relates to 1 ,2,4-triazolo[1 ,5-a]pyrimidines which are useful in the treatment and/or prophylaxis of migraine.
Migraine is a common complaint which counts 10% of the general population as sufferers (Worthington, I. (1996) Current migraine theory. Can. J. Clin. Pharm., 3, 39-51). This is an episodic disorder characterised by pulsating, severe pain (often unilateral), and may be accompanied by nausea, vomiting, photophobia and phonophobia (Goadsby, P.J. (1997) Current concepts of the pathophysiology of migraine. Neurol. Clin., 15, 27-42). Attacks last from hours to several days and this leads to significant loss of working days and revenue. Although advances have been made in the treatment of migraine in recent years (e.g. with the 5-HT antagonist, sumatriptan), this condition is still frequently undiagnosed and/or inappropriately treated (Worthington, 1996, see above). Migraine can be classified into common or "classical" subtypes. Classical migraine is associated with a visual aura before or during the onset of pain (Leone, M.L. et al, (1995) A review of the treatment of primary headaches. Ital. J. Neurol. Sci, 16, 577-586).
For many years, it was believed that migraine was caused by vasodilation of the cerebral blood vessels. However, vasodilation is not a consistent feature of migraine. The headache phase may be associated with decreased, normal or increased blood flow and slightly dilated arteries which may constrict in response to anti-migraine therapy (Olesen, J. et al. (1990) Timing and topography of cerebral blood flow, aura, and headache during migraine attacks. Ann. Neurol., 28, 791-798; Friberg et a/, 1991).
Increasing evidence now links the trigeminovascuiar system of the brainstem with the onset of migraine (Goadsby, 1997, see above). The mechanisms which activate the trigeminovascuiar system are unknown. However, cortical spreading depression first described by Leao, A#\.P. ((1944) Spreading depression of activity in the cerebral cortex. J. Neurophysiol., 7, 359-390) has been proposed as an initiator (Moskowitz, M. et al. (1993) Neocortical spreading depression provokes the expression of c-fos protein-like immunoreactivity within trigeminal nucleus caudalis via trigeminovascuiar mechanisms. J. Neurosci., 13, 1167-1 177) and is thought to underlie the visual aura in classical migraine (Lauritzen, M. (1994) Pathophysiology of the migraine aura; the spreading depression theory. Brain, 117, 199-210).
Spreading depression refers to waves of depolarisation which migrate slowly (2-3 mm/min) across brain grey matter regions (Nedergaard and Hansen, 1988). In the neurogenic theory of migraine, repeated cortical spreading depression activates the trigeminovascuiar fibres which in turn cause pain (Goadsby, 1997, see above). The characteristic sensory disturbances during migraine auras suggest that the underlying mechanism is a disturbance of the cerebral cortex (Worthington, 1996, see above).
Migraine with aura occurs in approximately 20% of migraine sufferers (Kubacka, R.T. (1994) Practical approaches to the management of migraine. Am. Pharm., N534, 34-44). Some patients may experience attacks with or without aura during different episodes (Stewart, W.F. et al, (1992) Prevalence of migraine headache in the United States. J.A.M.A., 267, 64-69, Olesen, J. et al. (1994) Migraine classification and diagnosis. Neurol., 44, 56-510). These aura symptoms develop over several minutes and last for about 1 hour (Worthington, 1996, see above).
The recurrence of this unprovoked, transient disturbance of brain function shows more similarities to epilepsy than to a vascular condition (Gowers, W. R. (1907) The border land of epilepsy, faints, vagal attacks, vertigo, and their treatment. Churchill, London; Milner, P.M. (1958) Note on a possible correspondence between the scotomas of migraine and spreading depression of Leao. Electroenceph. Clin. Neurophys., 10, 705). The spreading nature of the visual aura in classical migraine has been used by clinicians to differentiate this condition from epilepsy (Lauritzen, 1994, see above). However, the association between migraine and epilepsy has been demonstrated in an epidemiological study. This showed that migraine risk is twice as high in epileptic individuals than in the general population (Ottman, R.et al. (1994) Comorbidity of migraine and epilepsy. Neurol., 44, 2105-2110). In a separate study of 400 seizure patients, investigators found that 20% of individuals had both epilepsy and migraine (Marks, D.A. et al. (1993) Migraine-related seizures in adults with epilepsy, with EEC correlation. Neurol., 43, 2476-2483). A causal relationship was also found in 3% of these patients, all of whom had migraine with aura. GABA neurotransmission has been implicated in the pathogenesis of migraine for many years (Welch, K.M.A, et al, (1975) Cerebrospinal fluid gamma-aminobutyric acid levels in migraine. Br. I. Med., 3, 516-517).
Anticonvulsants such as sodium valproate have been employed as prophylactic treatments for migraine prevention. However, a period of 2-4 weeks is usually necessary before these drugs became effective (Rothrock, 1997). Problems were encountered due to rebound headaches caused by prolonged use of these treatments (Baumel, B. (1994) Migraine; A pharmacologic review with newer options and delivery modalities. Neurol., 44, S13-S17). Sodium valproate is potentially teratogenic and is contraindicated in pregnant females. Other side-effects include nausea, tremor, weight gain and alopecia (Rothrock, 1997).
The ordered development of the migraine aura renders the vascular hypothesis of migraine origin a remote possibility (Goadsby , 1997; see above, Lauritzen, 1994, see above). Spreading depression has been observed in human cortex in vitro (Avoli, M. et al, (1991) Epileptiform activity induced by low extracellular magnesium in the human cortex maintained in vitro. Ann. Neurol, 30, 589-596) and in human hippocampus and striatum in vivo (Sramka, M. et al, (1977) Functional ablation by spreading depression: possible use in human stereotactic neurosurgery. Appl. Neurophysiol., 40, 48-61).
Spreading depression is associated with transient, elevated extracellular potassium and release of neurotransmitters (Hansen et al, 1980). It has been the belief for many years that amino acid neurotransmission is involved in this phenomenon (Van Harreveld, 1959). Substances released from cells during spreading depression may depolarise trigeminovascuiar nerve fibres and cause vascular headache (Moskowitz, M. A. (1984) The neurobiology of vascular head pain. Ann. Neurol., 16, 157-168; Moskowitz et al, 1993, see above). During migraine attacks, cortical spreading depression is thought to trigger the visual aura at the wave front where cells are depolarising and produce reduced blood flow in its wake (Lauritzen, M. (1987bJ Cortical spreading depression as migraine mechanism. Trends Neurosci., 10, 8-13).
In experimental animals, spreading depression is associated with transient elevated blood flow for 1-2 minutes and then reduced flow (oligaemia) lasting for several minutes (e.g. Mies, and Paschen (1984) Regional changes of blood-flow, glucose, and ATP content determined on brain sections during a single passage of spreading depression in rat brain cortex. Exp. Neurol, 84, 249-258). Cortical spreading depression in animals (including monkeys) has been shown to produce
changes of behaviour which resemble features of the migraine aura (Bures, J. et al, (1974) The mechanism and applications of Leao's spreading depression of electroencephalographic activity. Prague: Academia, Bures, J. et al, (1984) The meaning and significance of Leao's spreading depression. An. Acad. Bras. Ciena, 56, 385-400). In rats, this phenomenon induces contralateral sensory neglect and motor impairment of the contralateral forepaw lasting for 15-30 minutes (Bures et al, 1984, as above; Lauritzen, M. (1987a) Cerebral blood flow in migraine and cortical spreading depression. Acta Neurol. Scand. Suppi., 76, 1-40).
This is consistent with transient neurological deficits found in patients during the migraine aura (Lauritzen, 1994, as above). At the beginning of migraine attacks, reduced blood flow is found in the posterior part of the brain. This low flow region then spreads into the parietal and temporal lobes at a rate of 2-3 mm/min over the next 30-60 minutes. This has been termed, "spreading oligaemia" (Olesen, J. et al. (1981) Focal hyperaemia followed by spreading oligaemia and impaired activation of rCBF in classic migraine. Ann. Neurol., 9, 344-352). This is preceded by a phase of focal hyperaemia (Olesen et al, 1990, see above), which is analogous to the mechanism of spreading depression (Goadsby, 1997, see above). This spread of reduced blood flow does not correspond with the territories of main arteries, but follows the cortical surface. This flow change corresponds both to the rate which Lashley, K.S. (1941) Patterns of cerebral integration indicated by the scotomas of migraine. Arch. Neurol. Psychiat, 46, 331-339, estimated from plotting the progression of his own visual aura and with the rate of cortical spreading depression (Lauritzen, 1994, see above).
During migraine attacks provoked by angiography, the aura symptoms usually arose during the early spread of the oligaemia, persisted for 15-30 minutes and stopped, although the oligaemia continued to spread while the patients developed migraine headache (Olesen et al, 1981 , see above; Lauritzen, 1987a, see above). Similar changes have been observed in spontaneous migraine attacks when observed by single-photon emission computed tomography (Andersen, A.R, et al, (1988) Delayed hyperemia following hypoperfusion in classic migraine: Single photon emission computed tomographic demonstration. Arch. Neurol. 45, 154-159). In addition, aura may be experienced in patients in isolation from pain. This supports the theory that the aura arises in the central nervous system, with the vascular changes being a secondary event in classical migraine (Goadsby, 1997, see above).
Taking the above findings into consideration, there is scope in the pharmaceutical market for inhibitors of cortical spreading depression as acute migraine treatments (Lauritzen, 1994, see above; Willette, R.N. et al, (1994) A comparison of (+)SK&F 10047 and MK80I on cortical spreading depression. Brain Res., 648, 347-351 ; Schoenen, J. (1997) Acute migraine therapy; the newer drugs. Current Opinion Neurol., 10, 237-243; Goadsby, 1997, see above).
Compounds of formula A
in which R-i represents H or optionally substituted alkyl, alkoxy or alkanoyl; R2 and R3 independently represent H or optionally substituted alkyl, alkoxy, alkanoyl, alkylthio, alkylsulphinyl or sulphonyl; R and R5 independently represent H, alkyl or together with the carbon atom to which they are attached represent optionally substituted cycloalkylidene; and R6, R and R8 independently represent H, halo hydroxy, mercapto, cyano or optionally substituted alkyl, alkanoyl, alkoxy, alkoxycarbonyl, carboxy, alkanoyloxy, alkylthio, alkylsulphinyl, alkylsulphonyl, alkylsulphonylamino, sulphamoyl, carbamoyl, alkylcarbamoyl or alkanoylamino; processes for their preparation, and their use in the treatment and/or prophylaxis of seizures, neurological disorders such as epilepsy and/or conditions in which there is neurological damage such as stroke, brain trauma, head injuries and haemorrhage are described in WO95/10521 (Knoll AG). A process for preparing these compounds is disclosed in WO98/07724 (Knoll AG).
The present invention provides a method of treating migraine comprising the administration of a therapeutically effective amount of a compound of formula I
including pharmaceutically acceptable salts, solvates, racemates, enantiomers, diastereoisomers and mixtures thereof in which:
R1 represents H or one of the following groups (optionally substituted with one or more of halo, cyano, hydroxy or amino): Chalky!, C-|.6alkoxy or C^alkanoyl;
R2 and R3 independently represent H or one of the following groups (optionally substituted with one or more of halo, cyano, hydroxy or amino): Chalky!, C-|_6alkoxy,
C βalkylsulphonyl or hydroxy;
R and R5 independently represent H, Chalky! or R4 and R5 combined together with the carbon atom to which they are attached represent C3.6cycloalkylidene (each alkyl or cycloalkylidene being optionally substituted with one or more of halo, cyano, hydroxy, amino or Chalky!); and
R6, R7 and R8 independently represent H, halo, hydroxy, mercapto, nitro, cyano or one of the following groups (optionally substituted with one or more of halo, cyano, hydroxy or amino; and any nitrogen atom being optionally substituted with one or more C^ alkyl): Chalky!, C^alkanoyl, C^aikoxy, C2_6alkoxycarbonyl, carboxy, Cι_6alkanoyloxy, Ci-βalkylthio, Ci-βalkylsulphinyl, Ct.6alkylsulphonyl, C^alkyl- sulphonylamino, sulphamoyl, carbamoyl, C2.6alkylcarbamoyl or C^ealkanoylamino; to a mammal in need thereof.
It will be understood that any group mentioned herein which contains a chain of three or more carbon atoms signifies a group in which the chain may be straight or branched. For example, an alkyl group may comprise propyl which includes n-propyl and isopropyl and butyl which includes n-butyl, sec-butyl, isobutyl and tert-butyl. The total number of carbon atoms is specified herein for certain substituents, for example
C1-6 signifies an alkyl group having from 1 to 6 carbon atoms. The term 'halo' as used herein signifies fluoro, chloro, bromo and iodo. The term Optionally substituted' as used herein, unless immediately followed by a list of substituent groups, means optionally substituted with one or more group or groups selected from halo, cyano, hydroxy and amino. When the phenyl ring substituents R6, R7 and R8 are other than H, the substituent may replace any H attached to a carbon atom in the ring and may be located at any such position of the ring, ie up to three of positions 2, 3, 4 and/or 5.
Specific compounds of formula i are:-
7- 1-(4-fluorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a] pyrimidine; 7- 1-(4-chlorophenoxy)ethyl]-1,2,4-triazolo[1 ,5-a] pyrimidine; 7- 1 -(4-bromophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]-pyrimidine; 7- 1-(4-cyanophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]-pyrimidine; 7- 1 -(4-trifluoromethylphenoxy)ethyl]-1 ,2,4-triazolo [1 ,5-a]pyrimidine; 7- 1-(4-methoxyphenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a] pyrimidine; 7- 1 -(4-trifluoromethoxyphenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]pyrimidine;
7-; 1 -(4-acetylphenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]-pyrimidine;
7- 1 -[4-(methylthio)phenoxy]ethyl}-1 ,2,4-triazolo[1 ,5-a]-pyrimidine;
7- 1-(4-methylsulphinylphenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]pyrimidine;
7- 1 -(4-methylsulphonylphenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]-pyrimidine;
7 1 -[4-(ethylthio)phenoxy]ethyl}-1 ,2,4-triazolo[1 ,5-a]-pyrimidine;
7- 1 -(3-chlorophenoxy)ethyl]-1 ,2,4-triazolo [1 ,5-a]pyrimidine;
7- 1 -(2,4-difluorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]-pyrimidine;
7- 1-(2,4-dichlorophenoxy)ethyl]-1 ,2,4-triazolo [1 ,5-a]pyrimidine;
7- 1 -(3,4-dichlorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]-pyrimidine;
7- 1 -(2-chloro-4-fluorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]pyrimidine;
7- 1-(4-chlorophenoxy)ethyl]-2-methyl-1 ,2,4-triazolo [1 ,5-a]pyrimidine; l-< 4-chlorophenoxymethyl)-1 ,2,4-triazolo[1 ,5-a] pyrimidine;
7- 1-(4-chlorophenoxy)-1-methylethyl]-1 ,2,4-triazolo [1 ,5-a]pyrimidine;
7- 1 -(4-chlorophenoxy)propyl]-1 ,2,4-triazolo[1 ,5-a]-pyrimidine; and
7- 1-(4-chlorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]pyrimidin-5-ol
their stereoisomers; and pharmaceutically acceptable salts thereof.
Specific examples of the stereoisomers of compounds of formula I are:-
(+)-7-[1 -(4-fluorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]- pyrimidine; and (-)-7-[1 -(4-fluorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]- pyrimidine
(+)-7-[1 -(4-chlorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a] pyrimidine;
(-)-7-[1-(4-chlorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a] pyrimidine;
(+)-7-[1 -(4-chlorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]pyrimidin-5-ol; and (-)-7-[1-(4-chlorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]pyrimidin-5-ol
and pharmaceutically acceptable salts thereof.
Preferred compound of formula I are 7-[1-(4-chlorophenoxy)ethyl]-1 ,2,4- triazolo[1 ,5-a]pyrimidine and 7-[1-(4-chlorophenoxy)ethyl]-1 ,2,4-triazoIo[1 ,5-a]- pyrimidin-5-ol including the racemates, enantiomers and mixtures thereof, and pharmaceutically acceptable salts thereof. More preferred compounds of formula I are (R)-7-[1-(4-chlorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]pyrimidine and (S)-7-[1-(4- chlorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]pyrimidine and pharmaceutically acceptable salts thereof. A most preferred compound of formula I is (R)-7-[1-(4- chlorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]pyrimidine and pharmaceutically acceptable salts thereof.
The compounds of formula I may be prepared as described in WO95/10521 (Knoll AG) and WO98/07724 (Knoll AG).
The compound of formula I may be administered in any of the known pharmaceutical dosage forms. The amount of the compound to be administered will depend on a number of factors including the age of the patient, the severity of the condition and the past medical history of the patient and always lies within the sound discretion of the administering physician but it is generally envisaged that the dosage of the compound to be administered will be in the range 0.1 to 1000 mg preferably 1 to 500 mg per day given in one or more doses.
Oral dosage forms are the preferred compositions for use in the present invention and these are the known pharmaceutical forms for such administration, for example tablets, capsules, granules, syrups and aqueous or oil suspensions. The
excipients used in the preparation of these compositions are the excipients known in the pharmacist's art. Tablets may be prepared from a mixture of the active compound with fillers, for example calcium phosphate; disintegrating agents, for example maize starch; lubricating agents, for example magnesium stearate; binders, for example microcrystalline cellulose or polyvinylpyrrolidone and other optional ingredients known in the art to permit tableting the mixture by known methods. The tablets may, if desired, be coated using known methods and excipients which may include enteric coating using for example hydroxypropylmethylceliulose phthalate. The tablets may be formulated in a manner known to those skilled in the art so as to give a sustained release of the compounds of the present invention. Such tablets may, if desired, be provided with enteric coatings by known methods, for example by the use of cellulose acetate phthalate. Similarly, capsules, for example hard or soft gelatin capsules, containing the active compound with or without added excipients, may be prepared by known methods and, if desired, provided with enteric coatings in a known manner. The contents of the capsule may be formulated using known methods so as to give sustained release of the active compound. The tablets and capsules may conveniently each contain 1 to 500 mg of the active compound. Preferably the tablets and capsules each contain 5, 10, 15, 20, 25, 30, 50, 100 ,250 or 500mg.
Other dosage forms for oral administration include, for example, aqueous suspensions containing the active compound in an aqueous medium in the presence of a non-toxic suspending agent such as sodium carboxy-methylcellulose, and oily suspensions containing a compound of the present invention in a suitable vegetable oil, for example arachis oil. The active compound may be formulated into granules with or without additional excipients. The granules may be ingested directly by the patient or they may be added to a suitable liquid carrier (for example, water) before ingestion. The granules may contain disintegrants, eg an effervescent couple formed from an acid and a carbonate or bicarbonate salt to facilitate dispersion in the liquid medium.
The therapeutically active compounds of formula I may be formulated into a composition which the patient retains in his mouth so that the active compound is administered through the mucosa of the mouth.
Dosage forms suitable for rectal administration are the known pharmaceutical forms for such administration, for example, suppositories with cocoa butter or polyethylene glycol bases.
Dosage forms suitable for parenteral administration are the known pharmaceutical forms for such administration, for example sterile suspensions or sterile solutions in a suitable solvent.
Dosage forms for topical administration may comprise a matrix in which the pharmacologically active compounds of the present invention are dispersed so that the compounds are held in contact with the skin in order to administer the compounds transdermally. A suitable transdermal composition may be prepared by mixing the pharmaceutically active compound with a topical vehicle, such as a mineral oil, petrolatum and/or a wax, e.g. paraffin wax or beeswax, together with a potential transdermal accelerant such as dimethyl sulphoxide or propylene glycol.
Alternatively the active compounds may be dispersed in a pharmaceutically acceptable cream, gel or ointment base. The amount of active compound contained in a topical formulation should be such that a therapeutically effective amount of the compound is delivered during the period of time for which the topical formulation is intended to be on the skin.
The therapeutically active compound of formula I may be formulated into a composition which is dispersed as an aerosol into the patients oral or nasal cavity. Such aerosols may be administered from a pump pack or from a pressurised pack containing a volatile propellant.
The therapeutically active compounds of formula I used in the method of the present invention may also be administered by continuous infusion either from an external source, for example by intravenous infusion or from a source of the compound placed within the body. Internal sources include implanted reservoirs containing the compound to be infused which is continuously released for example by osmosis and implants which may be (a) liquid such as an oily suspension of the compound to be infused for example in the form of a very sparingly water-soluble derivative such as a dodecanoate salt or a lipophilic ester or (b) solid in the form of an implanted support, for example of a synthetic resin or waxy material, for the compound to be infused. The support may be a single body containing all of the
compound or a series of several bodies each containing part of the compound to be delivered. The amount of active compound present in an internal source should be such that a therapeutically effective amount of the compound is delivered over a long period of time.
In some formulations it may be beneficial to use the compounds of the present invention in the form of particles of very small size, for example as obtained by fluid energy milling.
In the compositions of the present invention the compound of formula I may, if desired, be associated with other compatible pharmacologically active ingredients, for example an analgesic e.g. aspirin, ibuprofen, paracetamol or codeine phosphate; or an anti-emetic for example metoclopramide.
The following in vivo test supports the finding that compounds of formula I have efficacy in the treatment of migraine.
An investigation of the roles of excitatory and inhibitory amino acid neurotransmitters in an experimental model of cerebral ischaemia, namely middle cerebral artery occlusion, in freely-moving rats using in vivo microdialysis was carried out. Bipolar diathermy was used to cauterise the artery as a model of permanent focal ischaemia. Sham-operated control rats were subjected to cauterisation of the cortical surface adjacent to this artery, avoiding non-specific damage to the cortex. Relatively short sampling times of 10 minutes over a four hour period post-insult were used. An interesting finding in the sham-operated animals was apparently random, transient and repeated, increased extracellular levels of glutamate, glycine and D/L-serine. Damage after this procedure was limited to a small area around the surgical site which did not overlap the microdialysis probe sampling region. These changes resembled spreading depression-like depolarisations migrating through the cortex in response to the remote noxious stimuli (Smith, S.L. et al, (1997a) Cortical amino acid neurotransmitter levels in freely-moving rats after focal ischaemia. Proceedings of the 27th Annual Meeting Society for Neuroscience, New Orleans, 1997; Smith, S.L. et al, (1998) Characterisation of aspartate and glutamate involvement in focal ischaemia and spreading depression: an in vivo microdialysis study in freely-moving rats. Brit. J: Pharmacol., 123, P205. Amino acid neurotransmission has long been believed to be involved in spreading depression
propagation (Van Harreveld, 1959). However, this is the first clear demonstration of spreading depression in conscious rat cortex using in vivo microdialysis. This phenomenon has previously been demonstrated by electrophysiological means (e.g. Nedergaard, M. et al. (1988) Spreading depression is not associated with neuronal injury in the normal brain. Brain Res., 449, 395-398). However, unlike electrophysiological studies, microdialysis provides information about which amino acid neurotransmitters are involved in this mechanism. Fabricius, M. et al, (1993) Microdialysis of interstitial amino acids during spreading depression and anoxic depolarisation in rat neocortex. Brain Res., 612, 61-69, reported transient increased levels of various amino acid neurotransmitters after high K+-induced spreading depression in anaesthetised rat cortex. However, these were combined results taken from groups of 6-8 rats. The random nature of spreading depression renders combined data from animals difficult to interpret. In addition, the reported increased levels were very small (116-170% compared with basal values).
Following on from these observations, it was decided to investigate whether the compounds of the present invention would inhibit the transient elevated activity of glutamate, glycine and D/L-serine. The compounds of the present invention interact with the GABAA receptor to enhance the function of this neurotransmitter. Therefore, concentrations of GABA in microdialysis samples were also measured.
METHODS
( )-7-[1-(4-Chlorophenoxy)ethyi]-1 ,2,4-triazolo[1 ,5-a]pyrimidine was used as the test compound and is referred to hereinafter as the test compound
i) Under halothane anaesthesia (5% to induce, 2-2.25% to maintain), microdialysis probes (2 mm length sealed into 24 g cannulae; Hospal membrane, AN69) were implanted in the right cortex (mm from bregma; AP +1.2, L-5.5, V-6.0, jawbar set at -3.3 mm). Animals were allowed to recover for at least 20 hours and had free access to food and water. Three groups of rats were used, Group 1 ; anaesthetic plus vehicle (n=7), Group 2; cortex cauterisation plus vehicle (n=8), Group 3; cortex cauterisation plus the test compound (n=8).
ii) The perfusion rate was increased from an overnight rate of 0.8 μl/minute to
2 μl/minute at least 2 hours before starting the experiment. Artificial CSF comprised
l40mM NaCI, 3mM KCI, 0.27mM NaH2PO4, 1.2mM Na2HP04, 1 mM MgCI2, 1.2mM CaCl2 (pH 7.4). Three initial basal samples of 10 minute duration were collected from each group.
iii) The animals were anaesthestised with halothane and wrapped in a thermostatically-controlled heated operating blanket to maintain body temperature at 37°C±0.5°C throughout the surgical procedure. A scalp incision was made between the right ear and right eye. The temporalis muscle was retracted and a burr hole drilled (size 5 round drill bit) rostral to the fusion of the squamosal and zygomatic skull bones. The dura and pia-arachnoid membranes were incised and peeled from the surface of the cortex. Care was taken to avoid causing non-specific damage to the cortex or bleeding from minor blood vessels. The surface of the cortex was lightly cauterised using bipolar diathermy. The anaesthetic was immediately reduced to 0.5% to maintain the rats under light anaesthesia until dosed with the drug.
iv) The test compound was sonicated in 0.5% carboxymethylcellulose to form a suspension. Animals were dosed (50 mg/kg or equivalent volume of vehicle by oral gavage) 15 mm after cauterisation of the cortex and the anaesthetic was terminated. Animals subjected to halothane alone were anaesthetised for the same duration as the other surgical interventions (15 minutes). The rats were placed back into the home cage on a warmed pad until they regained consciousness.
v) Dialysate samples of 10 mm duration were collected from freely-moving rats for 3 hours from the point of cauterisation of the cortex.
vi) On termination of the experiment, a random sample of 6 animals were selected from Group 2 (cortex cauterisation/ vehicle). The rats were perfused trans- cardially with cold (4°C) phosphate-buffered saline (Oxoid tablets), then with cold 4% formaldehyde in phosphate-buffered saline, pH 7.4. Brains were fixed for at least 24 hours at 4°C before being sectioned at 100 μm intervals using a vibratome (General Scientific, Series 1000). Sections were stained with cresyl fast violet to determine extent of brain damage from the cortex cauterisation and distance of this damage from the microdialysis probe.
vii) Microdialysis samples were analysed for D/L-serine, glutamate, glycine and GABA content by HPLC with electrochemical detection as previously described
(Smith, S.L. et al, (1997b) Determination of multiple neurotransmitter amino acids: an in vivo microdialysis study in conscious rats. Brit. J. Pharmacol., 120, P368).
viii) Due to the random nature of the changes in amino acid neurotransmitter levels, data from each animal were analysed individually. This was by calculating area under the curve (AUC) of activity over 150% or under 50% of basal levels and also the number of fractions greater than these thresholds, using Wiicoxon rank-sum test. The analysis for AUC is depicted in Fig. 1. Units for AUC are expressed as percentage change from basal values above or below the thresholds, multiplied by time in minutes. Significance was determined using exact critical values. Differences were analysed between the groups after the drug reached peak brain levels (30 minutes after drug administration). All data are quoted as mean ± standard error of the mean. The statistical analysis performed on this data, which considers occasional peaks over certain thresholds necessarily results in large standard errors of the means.
RESULTS
i) After surgery, rats rapidly regained consciousness from anaesthesia (<10 minutes) and did not show untoward behavioural effects, such as seizures, after cortex cauterisation. The animals did not show obvious sedation after treatment with the test compound.
ii) Damage due to cortex cauterisation was localised to the surgical site (Fig. 2).
Histological examination of brain sections showed the microdialysis probe to be located 3.2±0.3 mm away from the site of cortex cauterisation (Fig. 2). No other cortical damage was found (40 x magnification) from +5.2 mm to +0.2 mm relative to bregma.
iii) Initial basal values of excitatory and inhibitory amino acids, D/L-serine, glutamate, glycine and GABA are listed in Table 1. Basal efflux of these neurotransmitters was the same in all groups; Group 1 , anaesthetic/vehicle, Group 2, cortex cauterisation/vehicle, Group 3, cortex cauterisation/the test compound .
iv) In the anaesthetic/vehicle control group, there were no changes from basal values for any of the neurotransmitters. Combined results forthis group of 7 rats are shown in Tables 2 to 5.
v) In rats subjected to cortical cauterisation/vehicle, this procedure gave rise to sporadic and sharply-defined, simultaneous increases in D/L-serine, glutamate and glycine when compared with the anaesthetic/vehicle group. However, no change occurred in GABA efflux. Combined results for this group of 8 rats are shown in Tables 2 and 3.
vi) When the test compound (50 mg/kg, given orally) was administered 15 minutes after cortex cauterisation, this drug abolished the increases in D/L-serine, glutamate and glycine resulting from the cortical insult. This was the case after the drug reached peak brain levels, 30 mm after administration. This effect of the test compound was highly significant (P<0.05 to P<0.001) irrespective of whether the results were analysed as either AUC (Table 2) or by the number of microdialysis samples which exceeded the 150% of basal values threshold (Table 3). The test compound did not influence the lack of effect of cortical cauterisation on GABA efflux. Combined results for this group of 8 rats are shown in Tables 2 and 3.
vii) In addition to preventing the increase of glutamate evoked by the cortical insult, the test compound was also found to decrease the level of glutamate efflux in comparison to the basal values in the anaesthetic/vehicle group. Combined results for this group of 8 rats are shown in Tables 4 and 5.
These results demonstrate that the test compound abolished the propagation of cortical spreading depression in rats (from the time of peak brain levels), when given orally, 15 minutes after the initiation of this mechanism. This was shown by inhibition of the sharply delineated increases of amino acid neurotransmitters, after the remote cortical insult. No alterations were found in extracellular levels of GABA after treatment with the test compound, although this compound enhances GABA function at the GABAA receptor. The test compound also decreased extracellular levels of glutamate compared with basal levels in the group given anaesthetic/vehicle treatment alone. Tissue damage from cortex cauterisation was localised to the surgical site and did not overlap with the microdialysis probe sampling region when examined histologically. No other damage was found in the cortex when cresyl violet-stained brain sections were examined. This is in agreement with previous electrophysiological studies (e.g. Nedergaard and Hansen, 1988, see above) that spreading depression in the healthy brain does not cause cell damage.
The test compound inhibited cortical spreading depression propagation in rats. This effect was rapid in onset (by 30 minutes after administration) and was prolonged (at least 2.25 hours). The animals did not show obvious sedation after a single 50mg/ kg oral dose. Therefore, this study supports the use of the compounds of the present invention as inhibitors of spreading depression, to prevent classical migraine attacks before (or very shortly after) pain commences. A compound of the present invention is suitable for oral administration at the time of the aura, before feelings of nausea and vomiting commence.
The results from this in vivo microdialysis study, demonstrate that the compounds of the present invention, and in particular the test compound, are effective inhibitors of the propagation of spreading depression at a non-sedating dose of this drug. The compounds of the present invention have important therapeutic value as acute agents for the prevention of classical migraine attacks. In addition, if the compounds of the present invention are taken repeatedly for short periods of time, they may also prevent the relapse headaches that may occur within 48 to 72 hours of the initial migraine attack.
Accordingly the present invention further provides a method for, and the use of a compound of formula I in the manufacture of the medicament for, thetreament of migraine. The present invention also provides a method for, and the use of a compound of formula I in the manufacture of a medicament for, the prophylaxis of migraine.
In an alternative embodiment the present invention provides a pharmaceutical composition for the treatment and/or the prevention of migraine comprising a therapeutically effective amount of a compound of formula I including enantiomers and pharmaceutically acceptable salts thereof in conjunction with a pharmaceutically acceptable diluent or carrier.
In an alternative embodiment the present invention provides a method for the prophylaxis of migraine comprising the administration of a compound of formula I including enantiomers and pharmaceutically acceptable salts thereof in conjunction with a pharmaceutically acceptable diluent or carrier to a mammal in need thereof.
The compounds of the present invention are also useful for the treatment of conditions which are known to those skilled in the art to be related to migraine and for treating the physical discomforts associated with migraine which are described in the first five pages of this document. The compounds of the present invention are also useful for the treatment of spreading depression and particularly for inhibiting cortical spreading depression.
The invention is illustrated by the following Example which are given by way of example only. The final product was characterised by the following procedures: gas- liquid chromatography; high performance liquid chromatography; elemental analysis; nuclear magnetic resonance spectroscopy and infrared spectroscopy.
Other compounds of the present invention were prepared as described in WO95/10521 (Knoll AG) and WO98/07724 (Knoll AG) which are incorporated herein by reference.
Example 1
1 a) A mixture of 2-(4-chlorophenoxy)propionic acid (30.0 g) in toluene (300 ml) was added to a solution of thionyl chloride (22.0 ml) and dimethylformamide (2 ml) in toluene (150 ml) at 60-70°C with stirring. The mixture was stirred for 18 hours at 70-80°C and then evaporated under reduced pressure to give the acid chloride. A solution of Meldrum's acid (23.5 g) in dichloromethane (90 ml) was cooled to 0-5°C under nitrogen and pyridine (33 ml) was added at 0-5°C. To this mixture was added a solution of the acid chloride prepared above in dichloromethane (90 ml) dropwise keeping the temperature below 5°C. The mixture was stirred for 1 hour at 0-5°C and then at ambient temperature for 18 hours. The mixture was diluted with dichloromethane (150 ml) and washed with 2M hydrochloric acid and then with saturated sodium bicarbonate solution and then water. The bicarbonate washes were acidified with 5M hydrochloric acid and extracted into dichloromethane. These dichloromethane extracts were combined with the original dichloromethane solution and dried and evaporated to give the crude intermediate Meldrum's acid derivative. This derivative was boiled under reflux with methanol (400 ml) for 6 hours and then left to stand at ambient
temperature for 66 hours with methanolic hydrogen chloride solution (10 ml). The mixture was evaporated under reduced pressure and the residue was partitioned between ethyl acetate and water. The ethyl acetate layer was separated off, washed with saturated sodium bicarbonate solution, brine and then dried and evaporated under reduced pressure to give an oil which was distilled under high vacuum. The distillate was purified by flash column chromatography on silica using petroleum ether, b.p. 60-80°C / ethyl acetate 20:1 as the mobile phase to give methyl 4-(4-chlorophenoxy)-3- oxopentanoate as an oil.
b) Guanidine hydrochloride (1.87 g) was added with stirring to a solution of sodium (0.41 g) in ethanol (15 ml). The mixture was stirred for 15 minutes and then to this mixture was added a solution of methyl 4-(4-chlorophenoxy)- 3-oxopentanoate (5.0 g) in ethanol (15 ml). The mixture was stirred and boiled under reflux for 16 hours. The mixture was cooled and then evaporated to dryness under reduced pressure to give a solid. The solid was triturated with water (10 ml) containing glacial acetic acid (2 ml) and dichloromethane (20 ml) for 1 hour and then filtered. The residue obtained was washed with water and then with dichloromethane to give 2-amino-4-[1- (4-chlorophenoxy)ethyl-6-hydroxypyrimidine, m.p. 125°C.
c) A mixture of 2-amino-4-[1-(4-chlorophenoxy)ethyl-6-hydroxypyrimidine (0.5 g), dimethylformamide dimethyl acetal (0.5 ml) and toluene (5 ml) was stirred and boiled under reflux for 8 hours. The mixture was evaporated for 8 hours. The mixture was allowed to stand at ambient temperature for 24 hours and then evaporated to dryness under reduced pressure to give a solid which was triturated with ether and filtered to give the amidine as a solid.
d) Sodium hydride (62 mg of a 60% dispersion in mineral oil from which the mineral oil had been removed by washing with petrol) was dissolved in methanol (10 ml) and hydroxylamine hydrochloride (0.12 g) was added. The mixture was stirred for 5 minutes and then added to the amidinopyrimidine (0.5 g) obtained in c). The mixture was stirred at ambient temperature for 72 hours and then evaporated to dryness under reduced pressure to give a residue which was washed with water and dried to give the formamidoxine.
e) The product from d) (5.0 g) and polyphosphoric acid (100 g) were stirred and heated on a steam bath for 4 hours. The mixture was allowed to cool to ambient temperature and then ice (100 g) and ethyl acetate (100 ml) were added. A solution of potassium carbonate (110 g) in water (total volume 100 ml) was added dropwise over 15 minutes to neutralise the mixture. The mixture was separated and the aqueous layer was extracted with ethyl acetate. The combined ethyl acetate layers were dried and evaporated to give a solid which was purified by flash column chromatography on silica using dichloromethane / methanol (75:3) as the mobile phase to give 7-[1-(4- chlorophenoxy)ethyl]-1 ,2,4-triazolo[1 ,5-a]pyrimidin-5-ol, m.p. 190°C.
Example A
The use of compounds of the present invention in the manufacture of pharmaceutical compositions is illustrated by the following description. In this description the term "active compound" denotes any compound of the invention but particularly any compound which is the final product of one of the preceding
Examples.
a) Capsules
In the preparation of capsules, 10 parts by weight of active compound and 240 parts by weight of lactose are de-aggregated and blended. The mixture is filled into hard gelatin capsules, each capsule containing a unit dose or part of a unit dose of active compound.
b) Tablets
Tablets are prepared from the following ingredients. Parts by weight
Active compound 10
Lactose 190
Maize starch 22
Polyvinylpyrrolidone 10 Magnesium stearate 3
The active compound, the lactose and some of the starch are de-aggregated, blended and the resulting mixture is granulated with a solution of the polyvinyl- pyrrolidone in ethanol. The dry granulate is blended with the magnesium stearate and the rest of the starch. The mixture is then compressed in atabletting machine to give tablets each containing a unit dose or a part of a unit dose of active compound.
c) Enteric coated tablets
Tablets are prepared by the method described in (b) above. The tablets are enteric coated in a conventional manner using a solution of 20% cellulose acetate phthalate and 3% diethyl phthalate in ethano dichioromethane (1 :1).
d) Suppositories
In the preparation of suppositories, 100 parts by weight of active compound is incorporated in 1300 parts by weight of triglyceride suppository base and the mixture formed into suppositories each containing a therapeutically effective amount of active ingredient.
NEUROTRANSMITTER
TABLE 1
Table 1. Initial basal values of amino acid neurotransmitters for each of the three groups of rats. Values were picomoles / 25μl injected onto the column.
NEUROTRANSMITTER
TABLE 2
Table 2. Analysis of AUC activity above the threshold of 150% of initial basal values. Units are expressed in % change from basal values above or below the thresholds x time (minutes). Samples were analysed statistically from the time of peak brain levels of test compound, 30 minutes after administration. ***P<0.001 compared with the anaesthetic / vehicle control group (Group 1 ; n = 7). "PO.01 , •••P<001 compared with the cortical insult / vehicle group (Group 2; n = 8). This table shows elevated activity of D/L-serine, glutamate and glycine in rats subjected to a cortical insult. This activity was abolished in the cortex cauterised / test compound treated groups (Group 3; n = 8). GABA levels were unchanged in all groups.
NEUROTRANSMITTER
TABLE 3
Table 3. Analysis of the number of samples above the threshold of 15% of initial basal values. Samples were analysed statistically from the time of peak brain levels of test compound, 30 minutes after administration. **P<0.01; ***P<0.001 compared with the anaesthetic / vehicle control group (Group 1 ; n = 7). *P<0.05, "PO.01 , *"P<0.001 compared with the cortical insult / vehicle group (Group 2; n = 8). This table shows elevated activity of D/L-serine, glutamate and glycine in rats subjected to a cortical insult. This activity was abolished in the cortex cauterised test compound treated group (Group 3; n = 8). GABA levels were unchanged in all groups.
NEUROTRANSMITTER
TABLE 4
Table 4. Analysis of AUC below the threshold of 50% of initial basal values. Units are expressed in % change from basal values above or below the thresholds x time (minutes). Samples were analysed statistically from the time of peak brain levels of test compound, 30 minutes after administration. **P<0.01 compared with the anaesthetic / vehicle control group (Group 1 ; n = 7). This table shows decreased levels of gluamate after test compound administration (Group 3; n = 8).
NEUROTRANSMITTER
TABLE 5
Table 5. Analysis of the number of samples below the threshold of 50% of initial basal values. Samples were analysed statistically from the time of peak brain levels of test compound, 30 minutes after administration. ** P<0.01 compared with the anaesthetic / vehicle control group (Group 1 ; n = 7). This table shows decreased levels of glutamate after test compound administration (Group 3; n = 8).