WO2001007043A1

WO2001007043A1 - Azetidine carboxamide derivatives for the treatment of cns disorders

Info

Publication number: WO2001007043A1
Application number: PCT/GB2000/002840
Authority: WO
Inventors: Mike Frederick Snape; Allan Fletcher; Kelly Jean Stanhope; Nathaniel Julius Monck
Original assignee: Vernalis Research Ltd
Current assignee: Ligand UK Research Ltd
Priority date: 1999-07-23
Filing date: 2000-07-21
Publication date: 2001-02-01
Anticipated expiration: 2002-01-23
Also published as: EP1196166A1; JP2003505418A; GB9917385D0; AU6007900A

Abstract

Use of a compound of formula (I) wherein R1 is aryl; and R?2, R3, R4, R5 and R6¿ which may be the same or different are selected from H, alkyl and aryl, or a pharmaceutically acceptable salt or prodrug thereof, in the manufacture of a medicament for neuroprotection in a subject or for the treatment of cerebral ischaemia, central nervous system injury or eye diseases.

Description

AZETIDINE CARBOXAMIDE DERIVATIVES FOR THE TREATMENT OF CNS DISORDERS

The present invention relates primarily to neuroprotection and to the treatment of stroke and other cerebrovascular disorders.

Stroke and other acute brain injuries are major causes of mortality and morbidity in the adult population. Stroke is the third highest cause of death in major industrialised countries and the commonest cause of permanent disability. Each year, in the US and Europe, approximately 1 million people suffer an acute stroke. Between 25% and 35% of these patients die within the first three weeks, and of the survivors 25% to 50% will be totally dependant on family or institutional care for the rest of their lives. The incidence of stroke increases with age, roughly doubling with each passing decade, with 30% of persons aged over 65 years being affected.

The statistics for stroke translate into an annual incidence of 0.1 to 0.2% in the US and Europe, with the world- wide market for stroke estimated to be worth $3 billion in 1995 and projected to rise to $10 billion in 2005. There is an unmet medical need for a cytoprotective therapy for stroke.

No effective neuroprotectant therapy is presently available for cerebrovascular disorders. The only therapy currently licensed for the treatment of ischaemic stroke is Genetech's thrombolytic recombinant tissue plasminogen activator (Activase^®, rtPA; Alteplase). Activase is indicated for the management of acute ischaemic stroke in adults for improving neurological recovery and reducing the incidence of disability. Treatment with Activase should only be initiated within 3 hours after the onset of stroke symptoms, and after exclusion of intracranial haemorrhage by a cranial computerised tomography (CT) scan or other diagnostic imaging method sensitive for the presence of haemorrhage.

The mechanisms underlying the irreversible brain damage which occurs following ischaemia are complex. Many classes of compounds are currently under investigation as treatments for cerebrovascular disorders. Acute intervention with both cytoprotective (neuroprotective) and other thrombolytic agents is undergoing active investigation. Cytoprotective neuroprotective therapy includes drugs that act to prevent cell death during ischaemia and reperfusion. These agents include calpain inhibitors, voltage-sensitive calcium- and sodium-channel antagonists, receptor-mediated calcium-channel antagonists [including N-methyl-D-aspartate (ΝMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) antagonists], glutamate-synthesis inhibitors, glutamate-release antagonists, γ-aminobenzoic acid (GABA) antagonists, 5-HT (serotonin) receptor agonists, gangliosides, antioxidants, growth factors, antiapoptotic agents, and antiadhesion molecules (Silver, B., Weber, J., Fisher, M., Clin. Neuropharmacol. 1996, 19, 101-128).

Excitotoxicity is a major determinant of neuronal death following the induction of cerebral ischaemia. Repetitive cell firing, persistent depolarisation and induction of supra-normal ionic flux across excitable membranes can initiate fatal cellular compromise via a variety of synergistic mechanisms during hypoxic excitotoxicity. Control of the state of excitability of neurons depends upon the net balance of excitatory and inhibitory influences acting on that neurone.

In general, the primary excitatory influence impinging on neurones is mediated by the glutamatergic system, whilst primary inhibition is frequently determined by GABAergic innervation, since the main endogenous inhibitory amino acid in mammalian brain is GABA. Thus increasing the inhibitory effect of GABAergic innervation, and decreasing the excitatory influence of glutamate, will reduce the net excitation of a neurone. Reducing excitation will reduce the consequences of energy depletion due to hypoxia and promote the ability of the neurone to survive hypoxic cerebral ischaemia.

Relatively few of the drugs currently under investigation as neuroprotectants for the treatment of stroke and other cerebrovascular disorders are modulators of the endogenous inhibitory amino acid, GABA.

One class of molecules which apparently possess neuroprotective properties is the GABA uptake inhibitors such as CI-966, which was shown to be effective in a gerbil ischaemia model utilising global cerebral ischaemia of 5 min. duration (Phillis, J.W., Gen. Pharmacol. 1995, 26, 1061-1064). The benzodiazepine receptor agonist diazepam has been shown to possess some neuroprotective properties (Karle, J., Witt, M. R., Nielsen, M., Brain Res. 1997, 765, 21- 29).

In rabbits with reversible spinal cord ischaemia, treatment with muscimol, a reference GABA_A agonist, at 5 mg/kg significantly prolonged P₅o time, where P₅₀ represents the duration associated with 50% probability of resultant permanent paraplegia (Madden, K.P., Stroke, 1994, 25, 2271-2275).

Felbamate, an antiepileptic drug with inter alia GABA agonist properties, provided significant neuronal protection when administered both before and after ischaemia in all regions of the brain in the gerbil model of transient forebrain ischaemia. Protection was moderate when felbamate was used before ischaemia, but was highly significant when felbamate was given 30 min. after the insult. The structural protection with felbamate was very significant when used in the post-ischaemic period (Shuaib, A., Waqaar, T., Ijaz, M.S., Kanthan, R., Wishart, T., Howlett, W., Brain Res. 1996, 727, 65-70).

Piracetam is a derivative of GABA, and was the first commercially available nootropic drug. Although widely evaluated in the treatment of senile cognitive disorders and dyslexia, piracetam has also been assessed as a treatment for deficits associated with acute stroke. Data from a number of small, short term studies in patients treated within a few days of stroke suggest that piracetam is more effective than placebo for the treatment of functional deficits (Noble, S., Benfield, P., CNS Drugs 1998, 9, 497-511).

Some combination neuroprotectant therapies have been investigated in rodent ischaemia since the excitotoxic effects of glutamate can be blocked almost completely with GABA in cell culture, tissue slices, and in some animal models. On this basis a combination of muscimol and MK 801, an NMDA receptor antagonist, was investigated and shown to be effective (Lyden, P.D., Lonzo, L., Stroke 1994, 25, 189-196).

WO-A-99/25353 discloses the use of triazolo[4,3-b]pyridazine derivatives as benzodiazepine/GABA_A modulators for the treatment of psychotic disorders and neurodegeneration. WO-A-90/09174 discloses the use of the GABAergic agent Clomethiazole (chlormethiazole) in the prevention and/or treatment of neurodegeneration. Clomethiazole is thought to act through a GABAergic pathway, whereby it augments GABA's inhibitory effect on the CNS, giving the drug both hypnotic and neuroprotectant properties.

The clinical neuroprotectant profile of clomethiazole has been reviewed (Muckle, H., IDrugs 1999, 2, 184-193). A large-scale phase III trial has been completed in which clomethiazole was evaluated for its ability to reduce nerve damage in acute cerebrovascular ischaemia. A subgroup of patients who presented with large stroke, experienced a significant benefit. Of these (n = 545), 41% of treated patients were functionally independent after 90 days, compared to 30% of patients on placebo.

The effectiveness of this GABA modulator in rat (Snape, M.F., Baldwin, H.A., Cross, A.J., Green, A.R., Neuroscience 1993, 53, 837-844) and gerbil ischaemia (Cross, A.J., Jones, J.A., Baldwin, H.A., Green, A.R., Br. J. Pharmacol. 1991, 104, 406-411) has been demonstrated. The dose in the latter paradigm was 100 mg/kg, i.p.

Azetidine-1-carboxamides and the use of these compounds in the treatment of anxiety and all forms of epilepsy is described in International Patent Applications Nos. PCT/GB99/00224, PCT/GB99/00219 and PCT/GB99/00223.

There remains a medical need for new treatments for stroke and cerebrovascular disorders. The object of the present invention is to provide such treatments.

It has now been found that certain azetidine-1-carboxamides show unexpected neuroprotectant efficacy when compared to reference GABAergic agents. In particular, certain azetidine-1-carboxamides have been shown to potentiate the action of GABA in inhibiting neurones, and have also been shown to prevent the repetitive firing induced as a consequence of activation of glutamatergic mechanisms. Such compounds are found to be neuroprotective following acute cerebral ischaemia in rats and mice, and reduced ischaemia-induced CNS damage in in vivo models of focal ischaemia in both species. According to the present invention, there is provided use of a compound of formula (I)

(I) wherein:

R¹ is aryl; and R², R³, R⁴, R⁵ and R⁶ which may be the same or different are selected from H, alkyl and aryl; or a pharmaceutically acceptable salt or prodrug thereof, in the manufacture of a medicament for neuroprotection in a subject or for the treatment of cerebral ischaemia, central nervous system injury or eye diseases.

Reference in the present specification to an "alkyl" group means a branched or unbranched, cyclic or acyclic, saturated or unsaturated (e.g. alkenyl (including allyl) or alkynyl (including propargyl)) hydrocarbyl radical. Where cyclic or acyclic the alkyl group is preferably Ci to C₁₂, more preferably C_\ to C₈ (such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert- butyl, pentyl, isopentyl, hexyl, heptyl, octyl). It will be appreciated therefore that the term "alkyl" as used herein includes alkyl (branched or unbranched), alkenyl (branched or unbranched), alkynyl (branched or unbranched), cycloalkyl, cycloalkenyl and cycloalkynyl. In a preferred embodiment, a cyclic alkyl group is preferably C₃ to Cι₂, more preferably C₅ to C₈ and an acyclic alkyl group is preferably d to C , more preferably to C₆, more preferably methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, isobutyl or tertiary- butyl) or pentyl (including n-pentyl and iso-pentyl), more preferably methyl.

Reference in the present specification to an "aryl" group means a mono or bicyclic aromatic group, such as phenyl or naphthyl.

The alkyl and aryl groups may be substituted or unsubstituted. In one embodiment, only the alkyl and aryl groups defined above as R_\ to R may be substituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1 or 2 substituents. Substituents may include:

carbon containing groups such as alkyl aryl, arylalkyl (e.g. substituted and unsubstituted phenyl, substituted and unsubstituted benzyl); halogen atoms and halogen containing groups such as haloalkyl (e.g. trifluoromethyl); oxygen containing groups such as alcohols (e.g. hydroxy, hydroxyalkyl, (aryl)(hydroxy)alkyl), ethers (e.g. alkoxy, alkoxyalkyl, aryloxyalkyl), oxo aldehydes (e.g. carboxaldehyde), ketones (e.g. alkylcarbonyl, alkylcarbonylalkyl, arylcarbonyl, arylalkylcarbonyl, arylcarbonylalkyl), acids (e.g. carboxy, carboxyalkyl), acid derivatives such as esters

(e.g. alkoxycarbonyl, alkoxycarbonylalkyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl) and amides

(e.g. aminocarbonyl, mono- or di-alkylaminocarbonyl, aminocarbonylalkyl, mono- or di- alkylaminocarbonylalkyl, arylaminocarbonyl); nitrogen containing groups such as amines (e.g. amino, mono- or di-alkylamino, aminoalkyl, mono- or di-alkylaminoalkyl), azides, nitriles (e.g. cyano, cyanoalkyl), nitro; sulphur containing groups such as thiols, thioethers, sulphoxides and sulphones

(e.g. alkylthio, alkylsulfinyl, alkylsufonyl, alkylthioalkyl, alkylsulfinylalkyl, alkylsulfonylalkyl, arylthio, arylsulfinyl, arylsulfonyl, arylthioalkyl, arylsulfinylalkyl, arylsulfonylalkyl); and heterocyclic groups containing one or more, preferably one, heteroatom, (e.g. thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl, piperidyl, piperazinyl, mo holinyl, thionaphthyl, benzofuranyl, isobenzofuryl, indolyl, oxyindolyl, isoindolyl, indazolyl, indolinyl, 7-azaindolyl, isoindazolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolyl, isoquinolyl, naphthridinyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxadinyl, chromenyl, chromanyl, isochromanyl and carbolinyl).

Preferred substituents include alkyl, aryl, halo, or an halogen-containing group such as trifluoromethyl.

As used herein, the term "alkoxy" means alkyl-O- and "alkoyl" means alkyl-CO-.

As used herein, the term "halogen" means a fluorine, chlorine, bromine or iodine radical, preferably a fluorine or chlorine radical.

The compounds of formula (I) may exist in a number of diastereomeric and/or enantiomeric forms. Unless otherwise stated, reference in the present specification to "a compound of formula (I)" is a reference to all stereoisomeric forms of the compound and includes a reference to the unseparated stereoisomers in a mixture, racemic or non-racemic, and to each stereoisomer in its pure form.

In a preferred embodiment of the present invention, a compound of formula (I) is the (R)- enantiomer of the compound of formula (I), substantially free of its (ιS)-enantiomer.

In the compounds of formula (I), preferably R¹ is a substituted or unsubstituted aryl group selected from phenyl, naphthyl and indanyl, more preferably R¹ is a substituted phenyl, naphthyl or indanyl, more preferably R¹ is a phenyl, naphthyl or indanyl having 1 to 3 substituents and, more preferably, R¹ is a phenyl, naphthyl or indanyl having 1 or 2 substituents. It is preferred that R¹ is a mono- or di-substituted phenyl or naphthyl, preferably a mono- or di-substituted phenyl, and most preferably a mono-substituted phenyl.

Where R¹ is a phenyl having 1 substituent, the phenyl group is preferably meta- or para- substituted. Where R¹ is a phenyl having 2 substituents, the phenyl group is preferably a 3,4- disubstituted phenyl or a 3,5-disubstiruted phenyl, more preferably a 3,4-disubstituted phenyl.

Where R¹ is a naphthyl group it is preferred that R is a 2-naphthyl group. Where R¹ is an indanyl group, it is preferred that R is a 5-indanyl group.

Where R¹ is substituted, the preferred substituents are selected from chloro, fluoro, bromo, iodo, trifluoromethyl, tertiary-butyl, phenyl, CO₂Me and CN, preferably from chloro, fluoro, trifluoromethyl and tertiary-butyl, and more preferably from chloro, trifluoromethyl and tertiary-butyl.

Where R¹ is di-substituted, it is preferred that each substituent is independently selected from halo, preferably chloro and fluoro. Where R¹ is di-substituted, it is preferred that R¹ is substituted by two chloro groups or by one chloro and one fluoro group, and more preferably by two chloro groups.

The most preferred R¹ groups are selected from 3-chlorophenyl, 4-chlorophenyl, 4- fluorophenyl, 4-tert-butylphenyl, 4-(trifluoromethyl)phenyl, 3-(trifluoromethyl)phenyl, 3,4- dichlorophenyl and 3,5-dichlorophenyl.

In the compounds of formula (I), R² is selected from H, alkyl (including hydroxyalkyl such as CH₂OH) and aryl. Preferably R² is selected from H and alkyl. Where R² is alkyl, R² is preferably methyl. Where R² is aryl, R² is preferably phenyl. Preferably R² is H or methyl. More preferably R² is methyl.

In the compounds of formula (I), R³ is selected from H, alkyl (including hydroxyalkyl such as CH₂OH) and aryl. Preferably R³ is selected from H and alkyl. Where R³ is alkyl, R³ is preferably methyl. Where R³ is aryl, R³ is preferably phenyl. Preferably R³ is H or methyl. In the compounds of formula (I), preferably R is selected from H and alkyl (including hydroxyalkyl). Preferably R⁴ is H or methyl. More preferably R⁴ is H.

In the compounds of formula (I), preferably R⁵ is selected from H and alkyl (including carboxy, alkoxycarbonyl and aminocarbonyl). R⁵ is preferably H or methyl. More preferably R⁵ is H.

In the compounds of formula (I), preferably R⁶ is selected from H and alkyl (including carboxy, alkoxycarbonyl and aminocarbonyl). R⁶ is preferably H or methyl. More preferably R⁶ is H.

In one embodiment of the invention R², R³, R⁴, R⁵ and R⁶ are independently selected from H and alkyl.

In a further embodiment of the invention, R² is H and R³ is methyl or R² is methyl and R³ is H.

In a further embodiment of the invention R⁴, R⁵ and R⁶ are H.

In the compounds of formula (I), R² and R⁴ may optionally be linked by a saturated divalent radical chain of carbon atoms to form a 5, 6 or 7 membered ring, preferably a 5 membered ring, such as tetrahydrofuran.

In the compounds of formula (I), R² and R³ may optionally be linked by a saturated divalent radical chain of carbon atoms to form a 5, 6 or 7 membered ring, preferably a 6 membered ring, such as cyclohexane.

In the compounds of formula (I), R² and R⁵ may optionally be linked by a saturated divalent radical chain of carbon atoms to form a 5, 6 or 7 membered ring, preferably a 6 membered ring, such as cyclohexane.

In the compounds of formula (I) wherein any of Ri to R₆, and particularly Rj, is selected from aryl substituted by two alkyl groups, particularly two alkyl groups which are in adjacent ring positions on said aryl ring, then said alkyl groups may be cychsed to form a 5, 6 or 7 membered ring, preferably a 5 or 6 membered ring, more preferably a 5 membered ring.

Particularly preferred compounds are as follows:

Chirality R¹ R² R³ R⁴ R⁵ R<

R 4-(Me₃C)-C₆H₄ H Me H H H

R 4-C1-C₆F ; H Me H H H

R 4-CF₃- H₄ H Me H H H

R 3,5-Cl₂-C -H.₃ H Me H H H

Of these, the preferred compounds are (#)-3-(4-tert-butylphenoxy)-N-(2- hydroxypropyl)azetidine- 1 -carboxamide, Cr?)-3-(4-chlorophenoxy)-N-(2- hydroxypropyl)azetidine- 1 -carboxamide, GS)-3-(4-chlorophenoxy)-N-(2- hydroxypropyl)azetidine- 1 -carboxamide, (Λ)-3-(3,4-dichlorophenoxy)-N-(2- hydroxypropyl)azetidine- 1 -carboxamide, (_JR)-3-(4-trifluoromethyl)phenoxy-N-(2- hydroxypropyl)azetidine- 1 -carboxamide and Cr?)-3-(3-trifluoromemyl)phenoxy-N-(2- hydroxypropyl)azetidine- 1 -carboxamide.

According to a further aspect of the present invention there is provided a method of neuroprotection comprising administration to a subject in need of such treatment an effective dose of the compound of formula (I), or a pharmaceutically acceptable salt or prodrug thereof.

According to a further aspect of the present invention there is provided a method of treatment of cerebral ischaemia, central nervous system injury or eye diseases comprising administration to a subject in need of such treatment an effective dose of the compound of formula (I), or a pharmaceutically acceptable salt or prodrug thereof.

The present invention may be employed in respect of a human or animal subject, more preferably a mammal, more preferably a human subject.

As used herein, the term "treatment" as used herein includes prophylactic treatment.

As used herein, the term "prodrug" means any pharmaceutically acceptable prodrug of the compound of formula (I). For example, the compound of formula (I) may be prepared in a prodrug form wherein a free -OH group is derivatised (for example, via an ester, amide or phosphate bond) with a suitable group (the group may contain, for example, an alkyl, aryl, phosphate, sugar, amine, glycol, sulfonate or acid function) which is suitably labile so as it will be removed / cleaved (eg. by hydrolysis) to reveal the compound of formula (I) sometime after administration or when exposed to the desired biological environment.

As used herein, the term "pharmaceutically acceptable salt" means any pharmaceutically acceptable salt of the compound of formula (I). Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, dichloroacetic, furnaric, gluconic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, oxalic, p-toluenesulfonic and the like. Particularly preferred are hydrochloric, hydrobromic, phosphoric, sulfuric and methanesulfonic acids, and most particularly preferred is the methanesulfonate salt. Acceptable base salts include alkali metal (e.g. sodium, potassium), alkaline earth metal (e.g. calcium, magnesium) and aluminium salts.

As used herein, the term "substantially free of its (5)-enantiomer" means that the medicament or therapeutic composition comprising the compound of formula (I) used according to the present invention contains a greater proportion of the (J?)-enantiomer of the compound of formula (I) in relation to the (5)-enantiomer of the compound of formula (I). In a preferred embodiment of the present invention the term "substantially free of its (iS)-enantiomer", as used herein, means that the composition contains at least 90 % by weight of the (ft)-enantiomer and 10 % by weight or less of the (iS)-enantiomer. In a further preferred embodiment, the term "substantially free of its (5)-enantiomer" means that the composition contains at least 99 % by weight of the (i?)-enantiomer and 1 % or less of the (6)-enantiomer. In another preferred embodiment, the term "substantially free of its (5)-enantiomer" means that the composition contains 100 % by weight of the (R)- enantiomer. The above percentages are based on the total amount of compound of formula

(I) present in the medicament or therapeutic composition used according to the present invention.

The diseases, disorders and medical treatments/procedures to which the present invention is directed are:

Cerebral Ischaemia, including transient ischaemic attack, stroke (thrombotic stroke, ischaemic stroke, embolic stroke, haemorrhagic stroke, lacunar stroke), subarachnoid haemorrhage, cerebral vasospasm, neuroprotection for stroke, peri-natal asphyxia, drowning, carbon monoxide poisoning, cardiac arrest and subdural haematoma;

Central Nervous System Injury, including traumatic brain injury, neurosurgery (surgical trauma), neuroprotection for head injury, raised intracranial pressure, cerebral oedema, hydrocephalus and spinal cord injury; and

Eye Diseases, including drug-induced optic neuritis, cataract, diabetic neuropathy, ischaemic retinopathy, retinal haemorrhage, retinitis pigmentosa, acute glaucoma, chronic glaucoma, macular degeneration, retinal artery occlusion and retinitis.

Additionally, the compound of formula (I) may also be used to potentiate the effects of other treatments, for example to potentiate the neuroprotective effects of brain derived nerve growth factor.

The invention is particularly directed to the treatment of cerebral ischaemia and central nervous system injury. The invention is also particularly directed to the treatment of post- asphyxial brain damage in new-born subjects. The compound of formula (I) may be used in combination with one or more additional drugs useful in the treatment of the disorders mentioned above, the components being in the same formulation or in separate formulations for administration simultaneously cr sequentially.

Compounds of formula (I) may be prepared according to the reaction scheme (where P is a nitrogen protecting group). R¹, R², R³, R⁴, R⁵, R⁶ are as previously defined. The 3- (aryloxy)azetidine (m) may be formed by reaction of the azetidinol (II) either with an arylalkanol (R'OH) and diethylazo dicarboxylate and triphenyl phosphine or with a substituted aryl fluoride (R'F) and a strong base such as sodium hydride. Formation of the azetidine (IV) is achieved by reaction of (LTf) with a suitable nitrogen deprotection agent. For example, if P is a diphenylmethyl group, then deprotection may be carried out by treatment with 1 -chloroethyl chloroformate followed by methanol. The urea (I) is formed by reaction of azetidine (TV) with an N-alkylisocyanate or an N-alkylcarbamoyl chloride and a base such as triethylarnine or potassium carbonate. Alternatively, the urea may be prepared directly from the azetidine (HI) without isolation of an intermediate such as the secondary amine (TV). For example, when P is a diphenylmethyl group, azetidine (Lπ) may be treated with phosgene followed by alkylamine R⁴O.CR²R³.CR⁵R⁶.NH₂ to give urea (I) directly.

Reaction Scheme

The invention further provides a pharmaceutical composition comprising an effective amount of the compound of formula (I) in combination with a pharmaceutically acceptable carrier or excipient and a method of making such a composition comprising combining an effective amount of the compound of formula (I) with a pharmaceutically acceptable carrier or excipient. To further increase efficacy, the composition may contain components such as dextrans or cyclodextrins or ether derivatives thereof, which aid stability and dispersion, and decrease metabolism of the active ingredient.

For compositions in which the pharmaceutically acceptable carrier comprises a cyclodextrin or an ether derivative thereof, the active ingredient is intimately mixed with an aqueous solution of the cyclodextrin or ether derivative thereof, with optional addition of further pharmaceutically acceptable ingredients before, during or after said mixing. The thus obtained solution is optionally lyophilized, and the lyophilized residue is optionally reconstituted with water.

In an embodiment of the present invention, the composition further comprises a buffer system, an isotonizing agent and water.

Compounds of formula (I) may be administered in a form suitable for oral use, for example a tablet, capsule, aqueous or oily solution, suspension or emulsion; for topical use including transmucosal and transdermal use, for example a cream, ointment, gel, aqueous or oil solution or suspension, salve, patch or plaster; for nasal use, for a example a snuff, nasal spray or nasal drops; for vaginal or rectal use, for example a suppository; for administration by inhalation, for example a finely divided powder or a liquid aerosol; for sub-lingual or buccal use, for example a tablet or capsule; or for parenteral use (including intravenous, subcutaneous, intramuscular, intravascular or infusion), for example a sterile aqueous or oil solution or suspension. In general the above compositions may be prepared in a conventional manner using conventional excipients, using standard techniques well known to those skilled in the art of pharmacy. Preferably, the compound is administered orally.

For oral administration, the compounds of formula (I) 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 ingredient 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 co 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 formula (I) 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 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.

It will be appreciated that the dosage levels used may vary over quite a wide range depending upon the compound used, the severity of the symptoms exhibited by the patient and the patient's body weight.

The invention will now be described in detail with reference to the following pharmacological examples. It will be appreciated that the examples are intended to illustrate and not to limit the scope of the present invention.

EXAMPLES

Synthetic Examples

Preparation of l-(Diphenylmethyl)-3-azetidinol

This compound was prepared according to the method of Anderson and Lok (J. Org. Chem., 1972, 37, 3953, the disclosure of which is incorporated herein by reference), m.p. 111-112 °C (lit. m.p. 113 °C). Preparation of 3-(4-te/?-Butylphenoxy)-l -(diphenylmethyl) azetidine (1)

Triphenylphosphine (13.11 g, 50.0 mmol) was added to a stirred solution of 1- (diphenylmethyl)-3-azetidinol (11.97 g, 50.0 mmol) and 4-tert-butylphenol (7.50 g, 50.0 mmol) in acetonitrile (200 mL). Diethyl azodicarboxylate (7.9 mL, 8.7 g, 50.0 mmol) was added dropwise to the solution with water cooling. The suspension was heated under reflux for 4 h, the solution allowed to cool and concentrated in vacuo. The gum was suspended in ether (200 mL), and the suspension was refrigerated overnight. The precipitated triphenylphosphine oxide was filtered off and washed with ether, and the combined filtrate and washings were concentrated. The solid residue was dissolved in dichloromethane (200 mL), and the solution was washed with 1-N sodium hydroxide (50 mL), dried (Na₂SO₄), and concentrated in vacuo. The crude product was purified by chromatography [SiO₂; ethyl acetate-hexane (1:4)] and triturated with cyclohexane to give the title compound (9.39 g, 51 %) as a white solid, m.p. 126-127 °C. Found: C, 83.9; H, 7.9; N, 3.75. C₂₆H₂₉NO requires C, 84.1; H, 7.9; N, 3.8%.

Example 1. (R)-3-(4-te/f-Butylphenoxy)-N-(2-hydroxypropyl) azetidine-1 -carboxamide

(2)

A solution of compound (1) (2.30 g, 6.19 mmol) in dichloromethane (25 mL) was treated with phosgene solution (ca. 20 wt. % in toluene; 3.28 mL, 6.2 mmol) with water cooling. The solution was stirred for 1 h, cooled to 0 °C, and (i?)-l-amino-2-propanol (1.08 mL, 1.03 g, 13.7 mmol) added dropwise. The solution was stirred for 18 h. Dichloromethane (25 mL) was added, and the solution was washed with 1-M HC1 (25 mL), dried (Na₂SO₄), and concentrated in vacuo. The crude product was purified by chromatography [SiO₂; MeOH- ethyl acetate-hexane (0:2:3→0:1:0→ 1:9:0)] and triturated with ether to give the title compound (1.15 g, 61 %) as a white solid, m.p. 98-99 °C. Found: C, 66.5; H, 8.8; N, 9.1. C,₇H₂₆N₂O₃ requires C, 66.6; H, 8.55; N, 9.1 %.

Example 2. (R)-3-(4-Chlorophenoxy)-N-(2-hydroxypropyl) azetidine-1-carboxamide (3) This product was prepared from 3-(4-chlorophenoxy)-l-(diphenylmethyl)azetidine (as described in US-4,956,359, the disclosure of which is incorporated herein by reference) and (i?)-l-amino-2-propanol using the procedure described for compound (2) (69 % yield), m.p. 104-105 °C. Found: C, 54.9; H, 6.0; N, 9.7. C₁₃H₁₇N₂O₃ requires C, 54.8; H, 6.0; N, 9.8 %.

Example 3. (S)-3-(4-Chlorophenoxy)-N-(2-hydroxypropyl) azetidine-1 -carboxamide (4)

This product was prepared from from 3-(4-chlorophenoxy)-l-(diphenylmethyl)azetidine and (5)-l-amino-2-propanol using the procedure described for compound (2) (57 % yield), m.p. 104-105 °C. Found: C, 54.7; H, 6.0; N, 9.7. Cι₃H,₇N₂O₃ requires C, 54.8; H, 6.0; N, 9.8%.

Example 4. 3-(4-Chlorophenoxy)-N-(2-hydroxy-2-methylpropyl) azetidine-1- carboxamide (5)

This product was prepared from 3-(4-chlorophenoxy)-l-(diphenylmethyl)azetidine and 1- amino-2-methyl-2-propanol using the procedure described for compound (2) (35 % yield), m.p. 121-122 °C. Found: C, 56.0; H, 6.6; N, 8.85. C₁₄Hι₉N₂O₃ requires C, 56.3; H, 6.4; N, 9.4%.

Example 5. (R)-3-(4-Fluorophenoxy)-7V-(2-hydroxvpropyl) azetidine-1- carboxamide (6)

This product was prepared from 3-(4-fluorophenoxy)-l-(diphenylmethyl)azetidine (as described in US-4,956,359, the disclosure of which is incorporated herein by reference) and (./?)- l-amino-2-propanol using the procedure described for compound (2) (76 % yield), m.p. 112-114 °C. Found: C, 58.3; H, 6.4; N, 10.4. C_]3Hι₇FN₂O₃ requires C, 58.2; H, 6.4; N, 10.4%.

Example 6. (R)-3-(3,4-Dichlorophenoxy)-N-(2-hydroxypropyl)azetidine-l- carboxamide (7) This product was prepared from 3 -(3 ,4-dichlorophenoxy)-l -(diphenylmethyl) azetidine (as described in US-4,956,359, the disclosure of which is incorporated herein by reference) and (i?)-l-amino-2-propanol using the procedure described for compound (2) (88 % yield), m.p. 83-84 °C. Found: C, 49.8; H, 5.05; N, 8.35. Cι₃H₁₇N₂O₃ requires C, 48.9; H, 5.05; N, 8.8%.

Example 7. (R*S)-3-(4-Chlorophenoxy)-N-(2-hydroxypropyl)azetidine-l- carboximide (8)

This product was prepared from 3-(4-chlorophenoxy)-l-(diphenylmethyl)azetidine and (d,l)- l-amino-2-propanol using the procedure described for compound (2) (81% yield), m.p. 104- 105°C. Found: C, 55.1; H, 6.1; N, 9.8. Cι₃H₁₇N₂O₃ requires C, 54.8; H, 6.0; N, 9.8%.

Example 8. (R 3-(4-(TrifluorornethyI)phenoxy)-iV-(2- hydroxypropyl)azetidine-l -carboxamide (9)

This product was prepared from 3-(4-trifluoromethyl)phenoxy)-l-(diphenylmethyl)azetidine and (i^-l-amino-2-propanol using the procedure described for compound (2) (60% yield), m.p. 112.5-113 °C. Found; C, 52.9; H, 5.4; N, 8.7. C_]4H₁₇F₃N₂O₃ requires C, 52.8; H, 5.4; N, 8.8%.

Examples 9 to 58 (see Table 1)

These products were prepared using the procedure described for compound (2).

Table 1

t

CO

Footnotes to Table 1.

Footnote a: IR: 3300, 2927, 1638, 1545, 1490, 1466, 1378, 1242, 1091, 825, 665 cm^"1. Footnote b: IR: 3519, 3271, 2926, 2855, 2233, 1624, 1606, 1506, 1458, 1397, 1260, 1171, 1126, 832, 550 cm^"1.

Footnote c: IR: 3350, 2925, 2854, 1627, 1549, 1488, 1469, 1397, 1374, 1326, 1273, 1149, 1137, 1088, 803 cm^"1.

Testing Procedures

Rat transient middle cerebral artery occlusion (MCAo) ischaemia model

This model of middle cerebral artery occlusion used relies on an intraluminal filament technique in the rat (Zhao Q. et al, Ada Physiol. Scand. 1994, 152, 349-350). Male Lister Hooded rats were used in these experiments and were divided into three groups (Group 1: vehicle; Group 2: chlomethiazole (CMZ); Group 3: a compound of formula (I)). The sample size in each was 11 to 15. The animal was anaesthetised and the carotid artery exposed. A heat-rounded dermalon suture (3/0) of a specified diameter was introduced into the ligated carotid artery, past the bifurcations of the external and common carotid, the internal carotid and the pterygopalatine artery, into the intracranial circulation. The filament then lodged in the narrow proximal anterior carotid occluding the middle cerebral artery. After 90 min. of middle cerebral artery occlusion, the filament was removed, allowing re-circulation.

22.5 h following reperfiision, the animal was perfused via the transaortic route, using 200 ml of a 4 percent solution of tetrazolium chloride warmed to 37° C. Following perfusion, the brain was removed and immersion fixed in 10 percent formalin/saline for at least 48 h. Following fixation, the brain was sliced into 0.5 mm sections on a vibroslice. Using this technique, viable tissue was stained dark red and infarcted tissue remains unstained. The area of infarction on each section was measured, and the total volume of infarction in the hemisphere, cortex and striatum computed, using the Kontron image analysis system. Mouse permanent middle cerebral artery occlusion ischaemia model

Adult male C57B1 mice (20-25 g, n = 10 per group) were administered a compound of formula (I) (10 mg/kg) or vehicle (60% PEG400 in water) i.p. 30 minutes prior to middle cerebral artery (MCA) occlusion. Under halothane anaesthesia (1.5% halothane in nitrous oxide: oxygen (70:30)), a small craniectomy was made to expose the left MCA. The distal portion of the MCA was occluded by electrocoagulation. The incision site was sutured and anaesthetics withdrawn. 24 h following MCA occlusion, the mouse was euthanised, the brain removed and immersed in 4% tetrazolium chloride to visualise the area of infarction (Backhaus C. et al, J. Pharm Methods 1992, 27, 27-32). Brains were then stored in 10% formalin/saline. The area of infarction as visible on the cortical surface was then computed using a PC digital imaging system (KS300, Imaging Associates, UK). Data generated is absolute area of infraction in mm² for each animal. Mean infarct areas were compared by unpaired t-tests with significance taken at p < 0.05.

The experimental results are displayed in Figures 1 and 2 which show the effects of (i) vehicle; and (ii) 60 mg/kg i.p. of compound 10 or compound 58 on infarction after permanent middle cerebral artery occlusion.

Figure 1 shows that compound 10 exhibits significant neuroprotection at a dose of 60 mg/kg i.p. in the mouse permanent MCAo model.

Figure 2 shows that compound 58 exhibits significant neuroprotection at a dose of 60 mg/kg i.p. in the mouse permanent MCAo model.

Claims

1. Use of a compound of formula (I)

(I) wherein R¹ is aryl; and

R², R³, R⁴, R⁵ and R⁶ which may be the same or different are selected from H, alkyl and aryl; or a pharmaceutically acceptable salt or prodrug thereof, in the manufacture of a medicament for neuroprotection in a subject or for the treatment of cerebral ischaemia, central nervous system injury or eye diseases.

2. A use according to claim 1 wherein R¹ is a substituted or unsubstituted aryl group selected from phenyl, naphthyl and indanyl.

3. A use according to claim 1 or 2 wherein R¹ has 1, 2 or 3 substituent groups.

4. A use according to claim 1 wherein R¹ is a para-substituted phenyl group.

5. A use according to claim 1 wherein R¹ is a meta-substituted phenyl group.

6. A use according to claim 1 wherein R¹ is a 3,4-disubstituted phenyl group or a 3,5- disubstituted phenyl group.

7. A use according to any preceding claim wherein R¹ is substituted by one or more groups selected from chloro, fluoro, bromo, iodo, trifluoromethyl, tertiary-butyl, phenyl, CO₂Me and CN.

8. A use according to claim 1 wherein R¹ is selected from 3-chlorophenyl, 4- chlorophenyl, 4-fluorophenyl, 4-tert-butylphenyl, 4-(trifluoromethyl)phenyl and 3- (trifluoromethyl)phenyl.

9. A use according to claim 3 or claim 6 wherein R¹ has 2 substituent groups each of which are independently selected from halo.

10. A use according to claim 1 wherein R¹ is 3,4-dichlorophenyl, 3-chloro-4-fluorophenyl, 4-chloro-3 -fluorophenyl or 3,5-dichlorophenyl.

11. A use according to any one of claims 1 to 10 wherein R², R³, R⁴, R⁵ and R⁶ are independently selected from H and alkyl.

12. A use according to any one of claims 1 to 11 wherein R², R³, R⁴, R⁵ and R⁶ are independently selected from H and methyl.

13. A use according to any one of claims 1 to 11 wherein one or both of R² and R³ are hydroxyalkyl.

14. A use according to any one of claims 1 to 10 wherein one or both of R² and R³ are phenyl.

15. A use according to any preceding claim wherein R⁴ is hydroxyalkyl.

16. A use according to any preceding claim wherein R⁵ and R⁶ are independently selected from carboxy, alkoxycarbonyl and aminocarbonyl.

17. A use compound according to any one of claims 1 to 14 wherein R , R and R are hydrogen.

18. A use according to any one of claims 1 to 10 wherein R² is hydrogen and R³ is methyl or R is methyl and R is hydrogen.

19. A use according to claim 1 wherein the compound is selected from (R)-3-(4-tert- butylphenoxy)-N-(2-hydroxypropyl)azetidine- 1 -carboxamide, (K)-3-(4-chlorophenoxy)-N-(2- hydroxypropyl)azetidine- 1 -carboxamide, (S)-3-(4-chlorophenoxy)-N-(2- hydroxypropyl)azetidine- 1 -carboxamide, 3-(4-chlorophenoxy)-N-(2-hydroxy-2- methylpropyl)azetidine- 1 -carboxamide, (i?)-3-(4-fluorophenoxy)-N-(2- hydroxypropyl)azetidine- 1 -carboxamide, (i?)-3-(4-trifluoromethyl)phenoxy-N-(2- hydroxypropyl)azetidine- 1 -carboxamide, (i?)-3-(3,4-dichlorophenoxy)-N-(2- hydroxypropyl)azetidine- 1 -carboxamide, (i?)-3-(3-trifluoromethyl)phenoxy-N-(2- hydroxypropyl)azetidine- 1 -carboxamide, (i?)-3-(3-chlorophenoxy)-N-(2- hydroxypropyl)azetidine- 1 -carboxamide and (R)-3-(3,5-dichlorophenoxy)-N-(2- hydroxypropyl)azetidine- 1 -carboxamide.

20. A use according to any one of claims 1 to 10 wherein R² and R⁴ are linked by a saturated divalent radical chain of carbon atoms to form a 5, 6 or 7 membered ring.

21. A use according to any one of claims 1 to 10 wherein R² and R³ are linked by a saturated divalent radical chain of carbon atoms to form a 5, 6 or 7 membered ring.

22. A use according to any one of claims 1 to 10 wherein R and R are linked by a saturated divalent radical chain of carbon atoms to form a 5, 6 or 7 membered ring.

23. A use according to any preceding claim wherein said medicament comprises a pharmaceutically acceptable carrier and as active ingredient an effective amount of a compound of formula (I).

24. A use according to claim 23 wherein said carrier comprises a cyclodextrin or an ether derivative thereof.

25. A use according to any preceding claim wherein the medicament further comprises a buffer system, an isotonizing agent and water.

26. Use according to any of preceding claim wherein the compound of formula (I) is in combination with one or more additional drugs useful in neuroprotection or the treatment of cerebral ischaemia, central nervous system injury or eye diseases, the components being in the same formulation or in separate formulations for administration simultaneously or sequentially.

27. A method of neuroprotection comprising administration to a subject in need of such treatment an effective dose of a compound of formula (I) as defined in any of claims 1 to 22, or a pharmaceutically acceptable salt or prodrug thereof.

28. A method of treatment of cerebral ischaemia, central nervous system injury or eye diseases comprising administration to a subject in need of such treatment an effective dose of a compound of formula (I) as defined in any of claims 1 to 22, or a pharmaceutically acceptable salt or prodrug thereof.

29. A method according to claim 27 or 28 wherein the compound of formula (I) is administered in the form as set out in any of claims 23, 24, 25 or 26.