Background of the Invention Nitric oxide is produced in mammalian cells from L-arginine by the action of specific nitric oxide synthases (NOSs). These enzymes fall into two distinct classes-constitutive NOS (cNOS) and inducible NOS (iNOS). At the present time, two constitutive NOSs and one inducible NOS have been identified. Of the constitutive NOSs, an endothelial enzyme (ecNOS) is involved with smooth muscle relaxation and the regulation of blood pressure and blood flow, whereas the neuronal enzyme (ncNOS) serves as a neurotransmitter and appears to be involved in the regulation of various biological functions such as cerebral ischaemia. Inducible NOS has been implicated in the pathogenesis of inflammatory diseases. Specific regulation of these enzymes should therefore offer considerable potential in the treatment of a wide variety of disease states.

Considerable effort has been expended in efforts to identify compounds that act as specific inhibitors of one or more isoforms of the enzyme nitric oxide synthase. The use of such compounds in therapy has also been widely claimed.

WO 95/05363 discloses compounds of generic structure

wherein D represents an aromatic ring ; R represents hydrogen, alkyl C1 to 6 or halogen ; and R2 represents a variety of nitrogen containing side-chains. The compounds have nitric oxide synthase inhibitory activity.

It has now surprisingly been found that particular compounds wherein R represents certain thio derivatives, which therefore are not within the generic scope of WO 95/05363, possess unexpectedly advantageous properties. Such compounds are the subject of the present application.

Disclosure of the Invention According to the invention we provide a compound of formula (I) wherein Z represents a furan or thiophene ring, optionally substituted by one or more substituents selected from halogen, trifluoromethyl, C1 to 6 alkyl, Cl to 6 alkoxy, hydroxy, amino and S (O) aR ; X represents Cl to 6 alkyl or-CO- ; Y represents 0, S (O) b or NIO ; W represents S (O) c ; a, b and c independently represent an integer 0,1 or 2;

R1 represents hydrogen, c1 to 6 alkyl, C1 to 6 alkyl -O-R5 or C1 to 6 alkyl-NR6R7 or phenyl; said phenyl being optionally substituted by one or more substituents selected from halogen, trifluoromethyl, C1 to 6 alkyl, C1 to 6 alkoxy, hydroxy and amino; R2 and R3 independently represent hydrogen, C1 to 6 alkyl, C2 to 7 alkanoyl or -(CH2)n-phenyl ; said alkyl being optionally substituted by OR, NR R or one or more fluoro atoms; and said phenyl being optionally substituted by one or more substituents selected from halogen, trifluoromethyl, C1 to 6 alkyl, C1 to 6 alkoxy, hydroxy and amino; or the group NR2R3 represents azeitidinyl, pyrrlidinyl, piperidinyl, morpholinyl; or piperazinyl optionally 4-substituted by Cl to 6 alkyl ; each of said azacyclic rings being optionally substituted by O-R8,K NR9R10, Cl to 6 alkyl-O-R8 or Cl to 6 alkyl-NR ; or, when X represents C1 to 6 akyl and Y represents NR3, the groups X and R3 are joined together such that the groupo X-N-R3 represents a saturated 4 to 7 membered azacyclic ring; R4, R, R, R, R, R and R independently represent hydrogen or C1 to 6 alkyl ; or the groups NR R and NR R independently represent azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl; or piperazinyl optionally 4-substituted by Cl to 6 alkyl ; n represents an integer 1 to 6 ; and optical isomers, racemates and tautomers thereof and pharmaceutically acceptable salts thereof.

Preferably the substituent-W-R1 in formula (I) is in the ortho or para position relative to the amidine group. More preferably the substituent-W-R in formula (I) is in the para position relative to the amidine group.

Preferably the substituent-X-Y-R2 in formula (I) is in the meta position relative to the amidine group.

In another preferred embodiment, X represents CH2.

In another preferred embodiment, Y represents NR.

Preferably c represents 0.

Preferably R represents Cl to 6 alkyl. More preferably, c represents 0 and R represents Cl to 6 alkyl.

Preferably-W-R represents methylthio.

In one particular embodiment, Z represents unsubstituted thiophene, R represents methyl, X represents CH2 and Y represents NR.

Particular compounds of the invention include: N- (3- { [isopropylamino] methyl}-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide ; N- [3- [ (methylamino) methyl]-4- (methylsulfanyl)phenyl]-2-thiophenecarboximidamide; N- [3- { [ (2, 2-difluoroethyl) amino] methyl}-4-(methylsufanyl) phenyl]-2- thiophenecarboximidamide; N- [3- [ (3, 3,3-trifluoropropyl) amino]-4- (methylsulfanyl) phenyl]-2- thiophenecarboximidamide; N- [3- [ (methylamino) methyl]-4- (methylsulfinyl)phenyl]-2-thiophenecarboximidamide; N- [3- [(methylamino) methyl]-4-(methylsulfonyl) phenyl]-2-thiophenecarboximidamide; N- [3-{[(2-hydroxyethyl) amino] methyl}-4- (methylsulfanyl) phenyl]-2- thiophenecarboximidamide; N- [3- { [ (2-hydroxyethyl) (methyl) amino] methyl}-4- (methylsulfanyl) phenyl-2- thiophenecarboximidamide; N-[3-{[(2-hydroxyethyl) (methyl) amino] methyl}-4-(methylsulfinyl) phenyl]-2- thiophenecarboximidamide ;

N- [3- { [(2-hydroxyethyl) (methyl) amino] methyl}-4- (methylsulfonyl) phenyl]-2- thiophenecarboximidamide ; N- [3- [ (dimethylamino) methyl]-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide ; N- [3- [ (ethylamino) methyl]-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide ; N- [3- f [ (2-hydroxyethyl) (ethyl) amino] methyl}-4-(methylsulfanyl) phenyl]-2- thiophenecarboximidamide ; N-[3-{[(2-hydroxyethyl)(1-propyl)amino]methyl}-4-(methylsulf anyl)phenyl]-2- thiophenecarboximidamide ; N- 3- (aminomethyl)-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide ; N- [3- [ (benzylamino) methyl]-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide ; N- [3- (hydroxymethyl)-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide ; N- [3- { [1-propylaminolmethyl}-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide ; N- [3- { [1-butylamino] methyl}-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide ; N-[3-{[(S)-1-hydroxy-2-propylamino]methyl}-4-(methylsulfanyl )phenyl]-2- thiophenecarboximidamide; N- [3- {[ ()-2-hydroxy-1-propylamino] methyl}-4- (methylsulfanyl) phenyl]-2- thiophenecarboximidamide; N- [3-{[(2R)-2-(hydroxymethyl)pyrrolidinyl]methyl}-4-(methylsul fanyl) phenyl]-2- thiophenecarboximidamide; N- [3- { [ (3-hydroxypropyl) amino] ;-4- (methylsulfanyl) phenyl]-2- thiophenecarboximidamide; N-[3-{[(2-(dimethyl)aminoethyl)amino]methyl}-4-(methylsulfan yl)pheny]-2- thiophenecarboximidamide ; N- [3- { [bis-(3-phenylpropyl)amino]methyl}-4-(methylsulfanyl)phenyl] -2- thiophenecarboximidamide ; N- 3- [2- (dimethylamino) ethoxymethyl]-4- (methylsulfanyl) phenyl}-2- thiophenecarboximidamide; and pharmaceutically acceptable salts thereof.

Unless otherwise indicated, the term"C 1 to 6 alkyl"referred to herein denotes a straight or branched chain alkyl group having from 1 to 6 carbon atoms and/or a cyclic alkyl group having from 3 to 6 carbon atoms. Examples of such groups include methyl, ethyl, n-propyl,

i-propyl, n-butyl, i-butyl, t-butyl, cyclopropyl, cyclopropylmethyl, cyclopentyl, methylcyclopentyl, cyclopentylmethyl and cyclohexyl.

Unless otherwise indicated, the term"C2 to 7 alkanoyl"referred to herein denotes a straight or branched chain alkyl group having from 1 to 6 carbon atoms or a cyclic alkyl group having from 3 to 6 carbon atoms bonded to a carbonyl (CO) group. Examples of such groups include acetyl, propionyl, iso-butyryl, valeryl, pivaloyl, cyclopentanoyl and cyclohexanoyl.

Unless otherwise indicated, the tenn"C 1 to 6 alkoxy"referred to herein denotes an oxygen substituent bonded to a straight or branched chain alkyl group having from 1 to 6 carbon atoms and/or a cyclic alkyl group having from 3 to 6 carbon atoms. Examples of such groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, cyclopropyloxy, cyclopropylmethoxy, cyclopentyloxy, methylcyclopentyloxy, cyclopentylmethoxy and cyclohexyloxy.

Unless otherwise indicated, the tenn"halogen"referred to herein denotes fluorine, chlorine, bromine and iodine.

The present invention includes compounds of formula (I) in the form of salts, in particular acid addition salts. Suitable salts include those formed with both organic and inorganic acids. Such acid addition salts will normally be pharmaceutically acceptable although salts of non-phannaceutically acceptable acids may be of utility in the preparation and purification of the compound in question. Thus, preferred salts include those formed from hydrochloric, hydrobromic, sulphuric, phosphoric, citric, tartaric, lactic, pyruvic, acetic, succinic, fumaric, maleic, methanesulphonic and benzenesulphonic acids.

According to the invention, we further provide a process for the preparation of compounds of formula (I), and optical isomers and racemates thereof and pharmaceutically acceptable salts thereof, which comprises preparing a compound of formula (I) by: (a) reacting a corresponding compound of formula (II) or a salt thereof wherein R, R, X, Y and W are as defined above, with a compound of formula (III) or a salt thereof wherein Z is as defined above and L represents a leaving group; or (b) reacting a corresponding compound of formula (IV) or a salt thereof wherein R, X, W and Z are as defined above and L is a leaving group, with a compound of formula (V) or a salt thereof H-Y-R2 (V) whereinR2 and Y are as defined above; or

(c) preparing a compound of formula (I) wherein X represents-CH2-by reduction of a corresponding compound wherein X represents-CO- (formula VI)

and wherein R1,R2,W,Y and Z are as defined above; and where desired or necessary converting the resultant compound of formula (I), or another salt thereof, into a pharmaceutically acceptable salt thereof ; or converting one compound of formula (I) into another compound of formula (I) ; and where desired converting the resultant compound of formula (I) into an optical isomer thereof.

In process (a), the reaction will take place on stirring a mixture of the reactants in a suitable solvent, for example a lower alkanol such as ethanol, 2-propanol or tert-butanol, at a temperature between room temperature and the reflux temperature of the solvent. The reaction may optionally be carried out under an atmosphere of an inert gas such as nitrogen or argon. The reaction time will depend inter alia on the solvent and the nature of the leaving group, and may be up to 48 hours; however it will typically be from 1 to 5 hours.

Suitable leaving groups L include thioalkyl, sulfonate, trifluoromethylsulfonate, halide, alkoxide, aryloxide and tosylate groups; others are recited in"Advanced Organic Chemistry", J. March (1985) 3rd Edition on page 315 and are well known in the art. We find thioalkyl, especially thiomethyl or thioethyl, to be particularly useful.

In process (b), the displacement reaction is performed by reacting a compound of formula (IV) with a compound of formula (V) in an inert solvent. Suitable leaving groups include sulfonate, trifluorosulfonate, tosylate, and halides selected from the group chloride, bromide or iodide. The reaction is generally carried out in the presence of a base. This base can be an additive to the reaction mixture, or in the case wherein Y represents NR,

can alternatively be an excess of the amine nucleophile. Potential basic additives are metal carbonate, especially alkali metal carbonates, metal oxides and hydroxides, and tertiary amine bases such as diisopropylethylamine. Suitable organic solvents are those such as acetonitrile, dioxane, N, N-dimethylformamide, N-methyl-2-pyrrolidinone, tetrahydrofuran, dimethylsulfoxide, sulfolan and Cl to 4 alcohols. In a preferred embodiment, the leaving group is chloride.

In process (c), the reduction is generally performed using a suitable reducing agent in a inert solvent. Such methods are generally well known in the art. For example, the reducing agent may be diborane or lithium aluminium hydride, and the solvent may be an ether such as diethyl ether or tetrahydrofuran.

Compounds of formula (I) wherein W represents-S (O) c-and c represents 1 or 2 may be prepared by oxidation of the corresponding compound of formula (I) wherein c represents 0 using a suitable selective oxidising agent. Such methods are generally well known in the art. For example, compounds wherein c represents 1 may be prepared using one equivalent of sodium periodate as oxidising agent. And compounds wherein c represents 2 may be prepared using two or more equivalents of oxone or of sodium periodate as the oxidising agent.

Salts of compounds of formula (I) may be formed by reacting the free base or a salt, enantiomer, tautomer or protected derivative thereof, with one or more equivalents of the appropriate acid. The reaction may be carried out in a solvent or medium in which the salt is insoluble, or in a solvent in which the salt is soluble followed by subsequent removal of the solvent in vacuo or by freeze drying. Suitable solvents include, for example, water, dioxan, ethanol, 2-propanol, tetrahydrofuran or diethyl ether, or mixtures thereof. The reaction may be a metathetical process or it may be carried out on an ion exchange resin.

Certain novel intermediates of formulae (II), (IV) and (VI) form another aspect of the invention. Compounds of formula (II) may be prepared by methods that will be generally apparent to the man skilled in the art. In particular, these methods include the reduction of a corresponding compound of formula (VII)

wherein R, R, X, Y and W are as defined above.

Such reductions may be achieved using various methods that are well known in the art.

Compounds of formula (III) are either known or may be prepared by known methods. For example, compounds of formula (III) in which L represents thioalkyl may be prepared by treatment of the corresponding thioamide of formula (VIII) wherein Z is as defined above; with an alkyliodide.

Compounds of formula (VII) may be prepared by methods that will be generally apparent to the man skilled in the art. Such methods include: (a) reaction of a compound of formula (IX) wherein R, X and W are as defined above and Hal represents a halogen,

with a nucleophile of formula (V); and (b) when X represents alkyl and Y represents NR, by reductive amination of a compound of formula (X) wherein X1 represents an alkyl group having one less CH2 group than X, and R1,X and W are as defined above; with an amine of formula HNR2R3.

Compounds of formula (IV) may be prepared from corresponding compounds of formula (XI) wherein R, X, W and Z are as defined above, using methods that are generally well known in the art.

Compounds of formula (VI) may be prepared by reaction of a compound of formula (XII) where X,Y and R2 are as defined abouve and Hal represents a halogen,

with a metal thioalkoxide, M-SR, wherein R is as defined above and M represents a metal, particularly an alkali or alkaline earth metal such as sodium or potassium; followed by reduction of the nitro group and then amidination of the resulting amine.

Compounds of formulae (V), (VII), (VIII), (IX), (X) and (XI) are either known or may be prepared by conventional methods known per se.

Intermediate compounds may be prepared as such or in protected form. In particular amine and hydroxy groups may be protected. Suitable protecting groups are described in the standard text"Protective Groups in Organic Synthesis", 3rd Edition (1999) by Greene and Wuts. Amine protecting groups which may be mentioned include alkyloxycarbonyl such as t-butyloxycarbonyl, phenylalkyloxycarbonyl such as benzyloxycarbonyl, or trifluoroacetate. Deprotection will normally take place on treatment with aqueous base or aqueous acid.

The compounds of the invention and intermediates may be isolated from their reaction mixtures, and if necessary further purified, by using standard techniques.

The compounds of formula (I) may exist in tautomeric, enantiomeric or diastereoisomeric forms, all of which are included within the scope of the invention. The various optical isomers may be isolated by separation of a racemic mixture of the compounds using conventional techniques, for example, fractional crystallisation or HPLC. Alternatively, the individual enantiomers may be made by reaction of the appropriate optically active starting materials under reaction conditions that will not cause racemisation.

Intermediate compounds may also exist in enantiomeric forms and may be used as purified enantiomers, diastereomers, racemates or mixtures.

The compounds of formula (I), and their pharmaceutically acceptable salts, enantiomers, racemates and tautomers, are useful because they possess pharmacological activity in animals.

In particular, the compounds are active as inhibitors of the enzyme nitric oxide synthase and

as such are predicted to be useful in therapy. More particularly, they are in general selective inhibitors of the neuronal isoform of the enzyme nitric oxide synthase.

The compounds and their pharmaceutically acceptable salts, enantiomers, racemates and tautomers are indicated for use in the treatment or prophylaxis of diseases or conditions in which synthesis or oversynthesis of nitric oxide synthase forms a contributory part ; Examples of such diseases or conditions include hypoxia, such as in cases of cardiac arrest, stroke and neonatal hypoxia, neurodegenerative conditions including nerve degeneration and/or nerve necrosis in disorders such as ischaemia, hypoxia, hypoglycemia, epilepsy, and in external wounds (such as spinal cord and head injury), hyperbaric oxygen convulsions and toxicity, dementia, for example, pre-senile dementia, Alzheimer's disease and AIDS-related dementia, Sydenham's chorea, Parkinson's disease, Huntington's disease, multiple sclerosis, Amyotrophic Lateral Sclerosis, Korsakoffs disease, imbecility relating to a cerebral vessel disorder, sleeping disorders, schizophrenia, anxiety, depression, seasonal affective disorder, jet-lag, depression or other symptoms associated with Premenstrual Syndrome (PMS), anxiety and septic shock.

The compounds of formula (I) are also useful in the treatment and alleviation of acute or persistent inflammatory or neuropathic pain, or pain of central origin.

The compounds of formula (I) may also be useful in the treatment or prophylaxis of inflammation. Conditions that may be specifically mentioned include osteoarthritis, rheumatoid arthritis, rheumatoid spondylitis, gouty arthritis and other arthritic conditions, inflamed joints; eczema, psoriasis, dermatitis or other inflammatory skin conditions such as sunburn; inflammatory eye conditions including uveitis and conjunctivitis; lung disorders in which inflammation is involved, for example, asthma, bronchitis, chronic obstructive pulmonary disease, pigeon fancier's disease, farmer's lung, acute respiratory distress syndrome; bacteraemia, endotoxaemia (septic shock), aphthous ulcers, gingivitis, pyresis, pain and pancreatitis; conditions of the gastrointestinal tract including inflammatory bowel disease, Crohn's disease, atrophic gastritis, gastritis varialoforme, ulcerative colitis, coeliac disease, regional ileitis, peptic ulceration, irritable bowel syndrome, damage to the

gastrointestinal tract resulting from infections by, for example, Helicobacterpylori, or from treatments with non-steroidal anti-inflammatory drugs; and other conditions associated with inflammation.

The compounds of formula (I) and their pharmaceutically acceptable salts, enantiomers, racemates and tautomers may also be useful in the treatment or prophylaxis of diseases or conditions in addition to those mentioned above. For example, the compounds may be useful in the treatment of atherosclerosis, cystic fibrosis, hypotension associated with septic and/or toxic shock, in the treatment of dysfunction of the immune system, as an adjuvant to short- term immunosuppression in organ transplant therapy, in the treatment of vascular complications associated with diabetes and in cotherapy with cytokines, for example TNF or interleukins.

Compounds of formula (I) are also predicted to show activity in the prevention and reversal of tolerance to opiates and diazepines, treatment of drug addiction and treatment of migraine and other vascular headaches. The compounds of the present invention may also show useful immunosuppressive activity, and be useful in the treatment of gastrointestinal motility disorders, and in the induction of labour. The compounds may also be useful in the treatment of cancers that express nitric oxide synthase.

Compounds of formula (I) are predicted to be particularly useful in the treatment or prophylaxis of hypoxia or stroke or ischaemia or neurodegenerative conditions or schizophrenia or migraine or for the treatment of pain and especially in the treatment or prophylaxis of hypoxia or stroke or ischaemia or neurodegenerative disorders or schizophrenia or pain. We are particularly interested in the conditions selected from the group consisting of hypoxia, ischaemia, stroke, pain, anxiety, schizophrenia, Parkinson's disease, Huntington's disease and migraine and other vascular headaches.

For the treatment of Parkinson's disease, the compounds of formula (I) are expected to be particularly useful either alone, or in combination with other agents such as L-Dopa.

Prophylaxis is expected to be particularly relevant to the treatment of persons who have suffered a previous episode of, or are otherwise considered to be at increased risk of, the disease or condition in question. Persons at risk of developing a particular disease or condition generally include those having a family history of the disease or condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the disease or condition.

Thus according to a further aspect of the invention we provide a compound of formula {I}, or an optical isomer or racemate thereof or a pharmaceutically acceptable salt thereof, for use as a medicament.

According to another feature of the invention we provide the use of a compound of formula (I) or an optical isomer or racemate thereof or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prophylaxis of the aforementioned diseases or conditions ; and a method of treatment or prophylaxis of one of the aforementioned diseases or conditions which comprises administering a therapeutically effective amount of a compound of formula (1), or an optical isomer or racemate thereof or a pharmaceutically acceptable salt thereof, to a person suffering from or susceptible to such a disease or condition.

For the above mentioned therapeutic indications, the dosage administered will, of course, vary with the compound employed, the mode of administration and the treatment desired.

However, in general, satisfactory results are obtained when the compounds are administered to a human at a daily dosage of between 0.5 mg and 2000 mg (measured as the active ingredient) per day, particularly at a daily dosage of between 2 mg and 500 mg.

The compounds of formula (1), and optical isomers and racemates thereof and pharmaceutically acceptable salts thereof, may be used on their own, or in the form of appropriate medicinal formulations. Administration may be by, but is not limited to, enteral (including oral, sublingual or rectal), intranasal, or topical or other parenteral routes. Conventional procedures for the selection and preparation of suitable

pharmaceutical formulations are described in, for example,"Pharmaceuticals-The Science of Dosage Form Designs", M. E. Aulton, Churchill Livingstone, 1988.

According to the invention, there is provided a pharmaceutical formulation comprising preferably less than 95% by weight and more preferably less than 50% by weight of a compound of formula (I), or an optical isomer or racemate thereof or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable diluent or carrier.

The formulation may optionally also contain a second pharmacologically active ingredient such as L-Dopa.

The compounds of formula (I), and pharmaceutically acceptable derivatives thereof, may also be advantageously used in combination with a COX-2 inhibitor. Particularly preferred COX-2 inhibitors are Celecoxib and MK-966. The NOS inhibitor and the COX-2 inhibitor may either be formulated together within the same pharmaceutical composition for administration in a single dosage unit, or each component may be individually formulated such that separate dosages may be administered either simultaneously or sequentially.

We also provide a method of preparation of such pharmaceutical formulations which comprises mixing the ingredients.

Examples of such diluents and carriers are: for tablets and dragees: lactose, starch, talc, stearic acid; for capsules: tartaric acid or lactose; for injectable solutions: water, alcohols, glycerin, vegetable oils; for suppositories: natural or hardened oils or waxes.

Compositions in a form suitable for oral, that is oesophageal, administration include: tablets, capsules and dragees; sustained release compositions include those in which the active ingredient is bound to an ion exchange resin which is optionally coated with a diffusion barrier to modify the release properties of the resin.

The enzyme nitric oxide synthase has a number of isoforms and compounds of formula (I), and optical isomers and racemates thereof and pharmaceutically acceptable salts thereof, may be screened for nitric oxide synthase inhibiting activity by following procedures based

on those of Bredt and Snyder in Proc. Natl. Acad. Sci., 1990,87,682-685. Nitric oxide synthase converts 3H-L-arginine into 3H-L-citrulline which can be separated by cation exchange chromatography and quantified by scintillation counting.

Screen for neuronal nitric oxide synthase inhibiting activity The enzyme is isolated from rat hippocampus or cerebellum. The cerebellum or hippocampus of a male Sprague-Dawley rat (250-275g) is removed following C02 anaesthesia of the animal and decapitation. Cerebellar or hippocampal supernatant is prepared by homogenisation in 50 mM Tris-HCl with 1 mM EDTA buffer (pH 7.2 at 25 °C) and centifugation for 15 minutes at 20,000 g. Residual L-arginine is removed from the supernatant by chromatography through Dowex AG-50W-X8 sodium form and hydrogen form columns successively, and further centrifugation at 1000 g for 30 seconds.

For the assay, 25 pi of the final supernatant is added to each of 96 wells (of a 96 well filter plate) containing either 25 pi of an assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl2, pH 7.4) or 25 pi of test compound in the buffer at 22 °C and 25 pi of complete assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl2, 1 mM DTT, 100 uM NADPH, 10 pg/ml calmodulin, pH 7.4). Following a 10 minute equilibration period, 25 pi of an L-arginine solution (of concentration 18 pM'H-L-arginine, 96 nM 3H-L-arginine) is added to each well to initiate the reaction. The reaction is stopped after 10 minutes by addition of 200 pi of a slurry of termination buffer (20 mM HEPES, 2 mM EDTA, pH 5.5) and Dowex AG-50W-X8 200-400. mesh.

Labelled L-citrulline is separated from labelled L-arginine by filtering each filter plate and 75pl of each terminated reaction is added to 3 ml of scintillation cocktail. The L-citrulline is then quantified by scintillation counting.

In a typical experiment using the cerebellar supernatant, basal activity is increased by 20,000 dpm/ml of sample above a reagent blank that has an activity of 7,000 dpm/ml. A reference standard, N-nitro-L-arginine, which gives 80% inhibition of nitric oxide synthase at a concentration of 1 pM, is tested in the assay to verify the procedure.

Screen for human neuronal nitric oxide synthase inhibiting activity Enzyme was isolated from human hippocampus, cortex or cerebellum. Cerebellar, cortical or hippocampal supernatant is prepared by homogenisation of frozen human tissue

(1 to 5 g) in 50 mM Tris-HCl with 1 mM EDTA buffer (pH 7.2 at 25 °C) and centrifugation for 15 minutes at 20,000 g. Residual L-arginine is removed from the supernatant by chromatography through Dowex AG-SOW-X8 sodium form and hydrogen form columns successively and further centrifugation at 1000 g for 30 seconds.

Subsequently, the supernatant is passed through 2'-5'ADP Sepharose and the human nNOS eluted with NADPH.

For the assay, 25 pi of the final supernatant is added to each of 96 wells (of a 96 well filter plate) containing either 25 pi of an assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl2, pH 7.4) or 25 pi of test compound in the buffer at 22 °C and 25 pi of complete assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl2, 1 mM DTT, 100 pM NADPH, 10 pg/ml calmodulin, pH 7.4). Following a 30 minute equilibration period, 25 pi of an L-arginine solution (of concentration 12 pM'H-L-arginine, 96 nM 3H-L-arginine) is added to each test tube to initiate the reaction. The reaction is stopped after 30 minutes by addition of 200 pi of a slurry of termination buffer (20 mM HEPES, 2 mM EDTA, pH 5.5) and Dowex AG-50W-X8 200-400 mesh.

Labelled L-citrulline is separated from labelled L-arginine by filtering each filter plate and 75pl of each terminated reaction is added to 3 ml of scintillation cocktail. The L-citrulline is then quantified by scintillation counting.

In a typical experiment using the cerebellar supernatant, basal activity is increased by 20,000 dpm/ml of sample above a reagent blank that has an activity of 7,000 dpm/ml. A reference standard, N-nitro-L-arginine, which gives 80% inhibition of nitric oxide synthase at a concentration of 1 pM, is tested in the assay to verify the procedure.

Screen for human inducible nitric oxide synthase inhibiting activity Partially purified iNOS was prepared from cultured and lysed human DLD 1 cells which had been activated with TNF-alpha, interferon gamma, and LPS. Centrifugation at 1000g removed cellular debris and residual L-arginine was removed from the supernatant by chromatography through Dowex AG-50W-X8 sodium form and hydrogen form columns successively.

For the assay, 25 pi of the final supernatant is added to each of 96 wells (of a. 96 well filter plate) containing either 25 p1 of an assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM

CaCl2, pH 7.4) or 25 ul of test compound in the buffer at 22 °C and 25 pi of complete assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl2, 1 mM DTT, 100 pM NADPH, 10 pg/ml calmodulin, pH 7.4). Following a 30 minute equilibration period, 25 pl of an L-arginine solution (of concentration 12 pM tH-L-arginine, 96 nM 3H-L-arginine) is added to each test tube to initiate the reaction. The reaction is stopped after 30 minutes by addition of 200 ul of a slurry of termination buffer (20 mM HEPES, 2 mM EDTA, pH 5.5) and Dowex AG-50W-X8 200-400 mesh.

Labelled L-citrulline is separated from labelled L-arginine by filtering each filter plate and 75pl of each terminated reaction is added to 3 ml of scintillation cocktail. The L-citrulline is then quantified by scintillation counting.

In a typical experiment using the DLD 1 supernatant, basal activity is increased by 10,000 dpm/ml of sample above a reagent blank that has an activity of 5,000 dpm/ml. A reference standard, N-methyl-L-arginine, which gives 80% inhibition of nitric oxide synthase at a concentration of 1 pM, is tested in the assay to verify the procedure.

Screen for endothelial nitric oxide synthase inhibiting activity The enzyme is isolated from human umbilical vein endothelial cells (HUVECs) by a procedure based on that of Pollock et al in Proc. Natl. Acad. Sci., 1991,88,10480-10484.

HUVECs were purchased from Clonetics Corp (San Diego, CA, USA) and cultured to confluency. Cells can be maintained to passage 35-40 without significant loss of yield of nitric oxide synthase. When cells reach confluency, they are resuspended in Dulbecco's phosphate buffered saline, centrifuged at 800 rpm for 10 minutes, and the cell pellet is then homogenised in ice-cold 50 mM Tris-HCl, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulphonylfluoride, 2 µM leupeptin at pH 4.2. Following centrifugation at 34,000 rpm for 60 minutes, the pellet is solubilised in the homogenisation buffer which also contains 20 mM CHAPS. After a 30 minute incubation on ice, the suspension is centrifuged at 34,000 rpm for 30 minutes. The resulting supernatant is stored at-80 °C until use.

For the assay, 25 pl of the final supernatant is added to each of 12 test tubes containing 25 jj. l L-arginine solution (of concentration 12 uM'H-L-arginine, 64 nM 3H-L-arginine) and either 25 u. l of an assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl2, pH 7.4) or

25 Ll of test compound in the buffer at 22 °C. To each test tube was added 25 Ll of complete assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl2, 1 mM DTT, 100 uM NADPH, 10 pLg/ml calmodulin, 12 ; je tetrahydrobiopterin, pH 7.4) to initiate the reaction and the reaction is stopped after 10 minutes by addition of 2 ml of a termination buffer (20 mM HEPES, 2 mM EDTA, pH 5.5).

Labelled L-citrulline is separated from labelled L-arginine by chromatography over a Dowex AG-SOW-X8 200-400 mesh column. A 1 ml portion of each terminated reaction mixture is added to an individual 1 ml column and the eluant combined with that from two 1 ml distilled water washes and 16 ml of scintillation cocktail. The L-citrulline is then quantified by scintillation counting.

In a typical experiment, basal activity is increased by 5,000 dpm/ml of sample above a reagent blank that has an activity of 1500 dpm/ml. A reference standard, N-nitro-L- arginine, which gives 70-90% inhibition of nitric oxide synthetase at a concentration of 1 uM, is tested in the assay to verify the procedure.

In the screens for nitric oxide synthase inhibition activity, compound activity is expressed as ICso (the concentration of drug substance which gives 50% enzyme inhibition in the assay). IC50 values for test compounds were initially estimated from the inhibiting activity of 1, 10 and 100 pM solutions of the compounds. Compounds that inhibited the enzyme by at least 50% at 10 pM were re-tested using more appropriate concentrations so that an IC5o could be determined.

When tested in the above screens, the compounds of Examples 1 to 26 below showed ICso values for inhibition of neuronal nitric oxide synthase of less than 10 jDvt and good selectivity compared to inhibition of the endothelial isoform of the enzyme, indicating that they are predicted to show particularly useful therapeutic activity.

The invention is illustrated but in no way limited by the following examples:

Preparation 1 2-Thiophenecarboximidothioic acid ethyl ester hydrochloride To a stirred solution of ethanethiol (28.4 g, 450 mmol) in dichloromethane (500 mL) at 10 °C under nitrogen was added 2-thiophenecarbonitrile (50.0 g, 450 mmol). The solution was treated with a slow stream of hydrogen chloride gas for 6 h. The reaction mixture was then allowed to warm to room temperature. After 18 h diethyl ether (200 mL) was added and a white solid crystallized out. The solid 2-thiophenecarboximidothioic acid ethyl ester hydrochloride was collected by filtration and air dried (65.8 g, 83%); m. p. 196-197 °C.

Preparation 2 2-Furancarboximidothioic acid ethyl ester hydrochloride Following the procedure described in Preparation 1 but substituting 2-furancarbonitrile for 2-thiophenecarbonitrile, the title compound was prepared as a white solid in 23% yield.

MS: m/z 156 [M + H] +.

Preparation 3 2-Methylsulfanyl-5-nitrobenzaldehyde 2-Chloro-5-nitrobenzaldehyde (9.99 g, 54 mmol) was stirred in methanol (200 ml) under nitrogen. Sodium methanethiolate (4.50 g, 64 mmol) in methanol (50 ml) was added. The solution was stirred at room temperature overnight under nitrogen. The solution was then evaporated using a rotary evaporator and the residue was partitioned between chloroform and dilute aqueous sodium hydroxide solution. The organic layer was collected, then dried (magnesium sulfate), filtered and evaporated. The residue was recrystallised from ethyl acetate/hexane to give the title compound as a yellow solid (8.68 g, 44 mmol, 81%).

M. p. 163 °C ; MS (ES+)/z 198 (MH+).

Preparation 4 [2-Methylsulfanyl-5-nitrophenvll methanol To 2-methylsulfanyl-5-nitrobenzaldehyde (10.45 g, 53 mmol) in methanol was added sodium borohydride (2 g, 52.9 mmol). The mixture was stirred for 1 h at room temperature. The mixture was diluted with water. The precipitate was collected by filtration and dried to give the title compound as a solid (10.05 g, 50.4 mmol, 95%).

MS (ES+)/z 200 (MH+).

Preparation 5 [5-Amino-2-(methylsulfanyl) phenyllmethanol To [2-methylsulfanyl-5-nitrophenyl] methanol (9.94 g, 49.9 mmol) in ethanol (150 ml) was added 10% palladium-C (1 g). The mixture was hydrogenated at 50 psi for 20 h. The catalyst was filtered off through celite. The filtrate was evaporated to give the title compound as a solid.

MS (ES+) m/z 170 (MH+).

Preparation 6 [3-(Chloromethyl)-4-(methylsulfanyl)phenyl]-2-thiophenecarbo ximidamide hydrochloride To [3- (hydroxymethyl)-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide (Example 17) in dichloromethane (200 ml) was added thionyl chloride (11 ml). The mixture was stirred at room temperature for 4 h. Ether was added and the resulting solid was filtered off and dried to give the title compound as an off white solid (15.5 g, 46.5 mmol, 93% overall yield from [2-methylsulfanyl-5-nitrophenyl] methanol).

MS (ES+) m/z 298 (MH+).

Example 1 N-(3-{[Isopropylamino]methyl}-4-(methylsulfanyl)phenyl]-2-th iophenecarboximidamide A mixture of [3- (chloromethyl)-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide hydrochloride (534 mg, 1.6 mmol), isopropylamine (0.77 ml, 9.0 mmol) and diisopropylethylamine (1.56 ml, 9.0 mmol) inN, N-dimethylformamide (10 ml) was stirred at room temperature for 16 h. The mixture was diluted with water. Potassium carbonate was added and a white precipitate was formed. The solid was collected by filtration and washed with water, then dried to give the title compound as a white solid (360 mg, 1.13 mmol, 70%).

MS (ES+) m/z 320 (MH+).

. Example 2 N-[3-[(Methylamino)methyl]-4-(methylsulfanyl)phenyl]-2-thiop henecarboximidamide Prepared by a method analogous to that described in Example 1 and using [3- (chloromethyl)-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide hydrochloride, methylamine (2M) and diisopropylethylamine in N, N-dimethylformamide. The compound was purified by reverse phase HPLC on a Waters Bondapak Cis column using a gradient of acetonitrile and 0.1 % aqueous trifluoroacetic acid as the eluent. The free base product was prepared by basification of the product-containing fractions with potassium carbonate and extraction with dichloromethane. The organic extracts were dried (magnesium sulfate) and evaporated to give the title compound as a white solid (43%).

MS (ES+)/z 292 (MH+).

Example 3 N-r3-f [(2*2-Difluoroethyl) aminolmethyl}-4-(methylsufanyl) phenyll-2 thiophenecarboximidamide Prepared by a method analogous to that described in Example 1 and using

[3- (chloromethyl)-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide hydrochloride, 2,2-difluoroethylamine and diisopropylethylamine in N, N-dimethylformamide. The compound was purified by reverse phase HPLC on a Waters Bondapak Cis column using a gradient of acetonitrile and 0.1% aqueous trifluoroacetic acid as the eluent. The product- containing fractions were basified with potassium carbonate. The resulting precipitate was filtered off, washed with water and dried to give the title compound as a white solid (48%).

MS (ES+) 342 m/z (MH+).

Example 4 N-[3-[(3,3,3-Trifluoropropyl)amino]-4-(methylsulfanyl)phenyl ]-2- thiophenecarboximidamide Prepared by a method analogous to that described in Example 1 and using <BR> <BR> <BR> <BR> [3- (chloromethyl)-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide hydrochloride, 3,3,3-trifluoropropylamine and diisopropylethylamine in N, N-dimethylformamide. The compound was purified by reverse phase HPLC on a Waters Bondapak Cis column using a gradient of acetonitrile and 0.1 % aqueous trifluoroacetic acid as the eluent. The product- containing fractions were basified with potassium carbonate. The resulting precipitate was filtered off, washed with water and dried to give the title compound as a white solid (39%).

MS (ES+)/z 374 (MH+).

Example 5 N-r3-r(Methvlamino) methyll-4- (methylsulfinvllphenvll-2-thiophenecarboximidamide<BR> <BR> <BR> dihydrochloride To a solution of N- [3- [ (methylamino) methyl]-4- (methylsulfanyl) phenyl]-2- thiophenecarboximidamide (313.8 mg, 1.08 mmol) in methanol (10 ml) and water (0.5 ml) was added sodium periodate (238. 3 mg, 1.11 mmol). The mixture was stirred for 20 h at room temperature. The reaction was diluted with water. Solid potassium carbonate was added. The aqueous layer was extracted with dichloromethane. The organic layer was

collected, then dried (magnesium sulfate), filtered, and evaporated. The compound was purified by reverse phase HPLC on a Waters Bondapak Cis column using a gradient of acetonitrile and 0.1 % aqueous trifluoroacetic acid as the eluent. The free base was prepared by basification of the product-containing fractions with potassium carbonate and extraction with dichloromethane. The organic extract was dried (magnesium sulfate), filtered and evaporated. Dissolution of the residue in methanol, addition of excess hydrogen chloride solution (1M in diethyl ether) and evaporation gave the title compound as a yellow solid (120 mg, 0.32 mmol, 29%).

MS (ES+) m/z 308 (MH+).

Example 6 <BR> <BR> <BR> <BR> <BR> N-3-f (Methylamino) methyll-4- (methylsulfonyl) phenyll-2-thiophenecarboximidamide<BR> <BR> <BR> <BR> dihydrochloride To a solution of N- [3- [ (methylamino) methyl]-4- (methylsulfanyl) phenyl]-2- thiophenecarboximidamide (460 mg, 1.58 mmol) in methanol (15 ml) was added oxone (1.89 g, 3.07 mmol). The residue was partitioned between water and dichloromethane. The organic layer was collected, then dried (magnesium sulfate), filtered, and evaporated. The compound was purified by reverse phase HPLC on a Waters Bondapak Cis column using a gradient of acetonitrile and 0.1 % aqueous trifluoroacetic acid as the eluent. The free base was prepared by basification of the product-containing fractions with potassium carbonate and extraction with dichloromethane. The organic extract was dried (magnesium sulfate), filtered and evaporated. Dissolution of the residue in methanol, addition of excess hydrogen chloride solution (1M in diethyl ether) and evaporation gave the title compound as a yellow solid (95 mg, 24%).

MS (ES+) m/z 324 (MH+).

Example 7 N-[3-{[(2-Hydroxyethyl)amino]-4-(methylsulfanyl)phenyl]-2- thiophenecarboximidamide dihydrochloride A mixture of [3- (chloromethyl)-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide hydrochloride (500 mg, 1.5 mmol), ethanolamine (513.1 mg, 8.4 mmol) and diisopropylethylamine (1.46 ml, 8.4 mmol) in N, N-dimethylformamide (10 ml) was stirred for 6 h at room temperature. The reaction was diluted with water. Excess solid potassium carbonate was added. The aqueous layer was extracted with dichloromethane. The organic layer was dried (magnesium sulfate), filtered and evaporated. The compound was purified by reverse phase HPLC on a Waters Bondapak C18 column using a gradient of acetonitrile and 0.1 % aqueous trifluoroacetic acid as the eluent. The free base was prepared by basification of the product-containing fractions with potassium carbonate and extraction with dichloromethane. The organic extract was dried (magnesium sulfate), filtered and evaporated. Dissolution of the residue in methanol, addition of excess hydrogen chloride solution (1M in diethyl ether) and evaporation gave the title compound as a yellow solid (109 mg, 0.28 mmol, 19%).

MS (ES) m/z 322 (MH).

Example 8 N-r3-{m-Hydroxyethyl) (methvl) aminolmethyl}-4-(methylSulfanyl) phenyl-2- thiophenecarboximidamide Prepared by a method analogous to that described in Example 7 and using <BR> <BR> <BR> [3- (chloromethyl)-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide hydrochloride, 2- (methylamino) ethanol and diisopropylethylamine in N, N-dimethylformamide. The solution was diluted with water. Potassium carbonate was added and a precipitate was formed. The solid was collected by filtration and washed with water, then dried, to give the title compound as a yellow solid (1.77 g, 5. 28 mmol, 85%).

MS (ES+) m/z 336 (MH+).

Example 9 <BR> <BR> <BR> <BR> <BR> <BR> N- !'3- {r ('2-HvdroxvethvlVmethvl) amino1methvl}-4- ('methvlsulfinyl) phenvn-2-<BR> <BR> <BR> <BR> <BR> <BR> thiophenecarboximidamide dihydrochloride To the solution of N-(3-{[(2-hydroxyethyl) (methyl) amino] methyl}-4- (methylsulfanyl) phenyl-2-thiophenecarboximidamide (659.8 mg, 1.96 mmol) in a mixture of methanol, dichloromethane and water (10,5 and 1 ml respectively) was added sodium periodate (430.2 mg, 2.0 mmol). The mixture was stirred for 7 h. The solution was evaporated. The residue was partitioned between water and dichloromethane. The organic layer was collected, then dried (magnesium sulfate), filtered and evaporated. The compound was purified by reverse phase HPLC on a Waters Bondapak Cl8 column using a gradient of acetonitrile and 0.1 % aqueous trifluoroacetic acid as the eluent. The free base was prepared by basification of the product-containing fractions with potassium carbonate and extraction with dichloromethane. The organic extract was dried (magnesium sulfate), filtered and evaporated. Dissolution of the residue in methanol, addition of excess hydrogen chloride solution (1M in diethyl ether) and evaporation gave the title compound as a white solid (398. 3 mg, 1.13 mmol, 58%).

MS (ES+)/z 352 (MH+).

Example 10 N-r3-f [(2-Hydroxyethyl)methyl)amino]-4-(methylsulfanyl)phenyl]-2- thiophenecarboximidamide dihydrochloride To the solution of N-(3-{[(2-hydroxyethyl) (methyl) amino] methyl}-4- (methylsulfanyl) phenyl-2-thiophenecarboximidamide (632.6 mg, 1.88 mmol) in a mixture of methanol, dichloromethane and water (25,7 and 3 ml respectively) was added sodium periodate (2.03 g, 9.5 mmol). The mixture was refluxed for 8 h and then left at room temperature for 18 h. The solution was evaporated. The residue was partitioned between water and n-bufanol. The organic layer was collected, then dried (magnesium sulfate), filtered and evaporated. The compound was purified by reverse phase HPLC on a Waters Bondapak Cis column using a gradient of acetonitrile and 0.1 % aqueous trifluoroacetic

acid as the eluent. The hydrochloride salt was prepared by evaporation of the product- containing fractions, dissolution of the residue in methanol, addition of excess hydrogen chloride solution (1M in diethyl ether) and evaporation. The title compound was obtained as a yellow solid (138.6 mg, 0.31 mmol, 17%).

MS (ES+) m/z 368 (MH+).

Example 11 N-[3-[(Dimethylamino)methyl]-4-(methylsulfanyl)phenyl]-2-thi ophenecarboximidamide To a suspension of [3- (chloromethyl)-4- (methylsulfanyl) phenyl]-2- thiophenecarboximidamide hydrochloride (1.33 g, 4.0 mmol) in acetonitrile (6 ml) was added dimethylamine (2.0Min tetrahydrofuran, 4.4 ml, 8.8 mmol) and the reaction was stirred overnight at room temperature. Water was then added and the product was extracted with dichloromethane and the combined organics were dried over anhydrous sodium sulfate. Removal of solvents yielded a brown liquid which was purified by silica gel chromatography (20: 1 then 10: 1 dichloromethane/2M ammonia in methanol). The isolated solid was then triturated in ether/hexane (1: 1) and a pale yellow solid was obtained following drying (584 mg).

MS :/z 306 (M+1) +.

Example 12 N-F3-r (Ethylamino) methyll-4-(methelsulfanyl) phenY11-2-thiophenecarboximidamide Prepared by a method analogous to Example 11 but using ethylamine (2.0Min tetrahydrofuran, 10 equivalents) instead of dimethylamine. The title compound was obtained as a pale yellow solid (679 mg).

MS : m/z 306 (M+1) +.

Examole 13 N-r3- {F (2-Hydroxyethyl ! (ethyl) aminolmethyl}-4-(methYlsulfanyl ! phenyll-2- thiophenecarboximidamide Prepared by a method analogous to Example 11 but using 2- (ethylamino) ethanol (2 equivalents) instead of dimethylamine. Following purification by silica gel chromatography, the title compound was obtained as a yellow solid (940 mg).

MS: m/z 350 (M+1)+.

Example 14 N-[3-{[(2-Hydroxyethyl)(1-propyl)amino]methyl]-4-(methylsulf anyl)phenyl]-2- thiophenecarboximidamide dihydrochloride Prepared by a method analogous to Example 11 but using 2- (propylamino) ethanol (2 equivalents) instead of dimethylamine. Following silica gel chromatography, the isolated product was taken up in methanol and treated with an excess of hydrogen chloride (1N in diethyl ether). Solvents were removed and the residue was dried to yield the title compound (622 mg).

MS :/z 364 (M+1) +.

Example 15 N-[3-(Aminomethyl)-4-(methylsulfanyl)phenyl]-2-thiophenecarb oximidamide A solution of [3- (chloromethyl)-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide hydrochloride (1.00 g, 3.00 mmol) in methanol (16 ml) was added to 2.0M ammonia in methanol (150 ml, 300 mmol) over 2 h. After 20 h, additional 2.0M ammonia in methanol (75 ml, 150 mmol) was added to the mixture and stirring continued for 48 h. The mixture was concentrated and partitioned between aqueous potassium carbonate and dichloromethane. The aqueous portion was extracted with additional dichloromethane.

The combined organic portions were washed (brine), dried, and evaporated to a crude

yellow oil which was purified by silica gel chromatography, eluting with 10% 2.0M ammonia in methanol: 90% dichloromethane (v/v), to give the product as a yellow foam (0.40 g, 48%).

MS:/z 278 (M+1) +.

Example 16 N-[3-[(Benzylamino)methyl]-4-(methylsulfanyl)phenyl]-2-thiop henecarboximidamide A mixture of N-[3-(aminomethyl)-4-(methylsulfanyl) phenyl]-2-thiophenecarboximidamide (0.185 g, 0.67 mmol), benzaldehyde (0.081 g, 0.76 mmol) and glacial acetic acid (38 p1, 0.67 mmol) in 1,2-dichloroethane (5 ml) was treated with sodium triacetoxyborohydride (0.222 g, 1.05 mmol). After 4 h, additional benzaldehyde (68 Ll, 0.67 mmol) was added and stirring continued for 2 h. The mixture was partitioned between aqueous potassium carbonate and dichloromethane. The aqueous portion was extracted with additional dichloromethane. The combined organic portions were washed (brine), dried, and evaporated to a crude yellow oil which was purified by silica gel chromatography, eluting with 5% 2.0M ammonia in methanol: 95% dichloromethane (v/v), to give the product as a yellow solid (0.134 g, 54%).

MS: m/z 368 (M+1) +.

Example 17 N-r3- (Hydroxvmethyl)-4- (methvlsulfanvl) phenyll-2-thiophenecarboximidamide To the [5-amino-2-(methylsulfanyl) phenyl] methanol prepared in Preparation 5 in ethanol (100 ml) was added 2-thiophenecarboximidothioic acid ethyl ester hydrochloride (15 g, 72.2 mmol). The solution was heated under reflux for 6 h and then left at room temperature for 3 days. The solution was then evaporated and gave the title compound as a solid.

MS (ES+)/z 280 (MH+).

Example 18 N-[3-{[1-Propylamino]methyl}-4-(methylsulfanyl)phenyl]-2-thi ophenecarboximidamide Prepared by a method analogous to Example 11 but using propylamine (4 equivalents) instead of dimethylamine. An off-white solid was obtained following silica gel chromatography (35%).

MS : m/z 320 (M+1) +.

Example 19 <BR> <BR> <BR> <BR> <BR> <BR> <BR> N-f3- {f 1-Butylaminojmethyl}-4- (methvlsulfanvl) phenvn-2-thiophenecarboximidamide Prepared by a method analogous to Example 11 but using butylamine (4 equivalents) instead of dimethylamine. An off-white solid was obtained following silica gel chromatography (37%).

MS : m/z 334 (M+1) +.

Example 20 N-f3- {j S)-l-Hydroxy-2-propylaminolmethyl}-4- (methylsulfanvl) phenyll-2- thiophenecarboximidamide Prepared by a method analogous to Example 11 but using (S)- (+)-2-amino-1-propanol (4 equivalents) instead of dimethylamine. A yellow solid was obtained following silica gel chromatography (55%).

MS :/z 336 (M+1) +.

Example 21 N-[3-{[(S)-2-Hydroxy-1-propylamino]methyl}-4-(methylsulfanyl )phenyl]-2- thiophenecarboximidamide Prepared by a method analogous to Example 11 but using (S)- (+)-l-amino-2-propanol (4 equivalents) instead of dimethylamine. A yellow solid was obtained following silica gel chromatography (51%).

MS : m/z 336 (M+1) +.

Example 22 N-[3-{[(2R)-2-(hydroxymethyl)pyrrolidinyl]methyl}-4-(methyls ulfanyl)phenyl]-2- thiophenecarboximidamide Prepared by a method analogous to Example 11 but using (R)- (-)-2-pyrrolidinemethanol (4 equivalents) instead of dimethylamine. A yellow solid was obtained following silica gel chromatography (72%).

MS : m/z 362 (M+1) +.

Example 23 N-[3-{[(3-Hydroxypropyl)amino]methyl}-4-(methylsulfanyl)phen yl]-2- thiophenecarboximidamide Prepared by a method analogous to Example 11 but using 3-amino-1-propanol (4 equivalents) instead of dimethylamine. A light brown solid was obtained following silica gel chromatography (49%).

MS : m/z 336 (M+1) +.

Example 24 N-F3-f (2- (Dimethvl) aminoethvl) aminolmethyl}-4- (methylsulfan) phenvll-2- thiophenecarboximidamide A suspension of [3- (chloromethyl)-4-methylsulfanyl) phenyl]-2-thiophenecarboximidamide hydrochloride (0.394 g, 1.18 mmol) in acetonitrile (10 ml) was treated with N, N-dimethylethylenediamine (2.0 ml, 18.2 mmol). After 4 h, the mixture was concentrated and partitioned between aqueous potassium carbonate and dichloromethane.

The aqueous portion was extracted with additional dichloromethane. The combined organic portions were washed (brine), dried and evaporated to a crude yellow oil which was purified by silica gel chromatography, eluting with 10% 2.0M ammonia in methanol: 90% dichloromethane (v/v), to give the product as a yellow solid (0.106 g, 25%).

MS : m/z 349 (M+1) +.

Example 25 N-[3-{[Bis-(3-phenhylpropyl)amino]methyl}-4-(methylsulfanyl) phenyl]-2- thiophenecarboximidamide Prepared by a method analogous to Example 16 but using 3-phenylpropionaldehyde (2 equivalents) instead of benzaldehyde. The crude oil was purified by silica gel chromatography, eluting with 5% 2.0M ammonia in methanol: 95% dichloromethane followed by 1: 1 hexane: ethyl acetate (v/v), to give the product as a tan oil (68 mg, 25%).

MS:/z 514 (M+1) +.

Example 26 N-3-[2-(Dimethylamino)ethoxymethyl]-4-(methylsulfanyl)phenyl }-2- thiophenecarboximidamide [3- (Hydroxymethyl)-4- (methylsulfanyl) phenyl]-2-thiophenecarboximidamide (0.295 g, 1.06 mmol) was added to a mixture of sodium hydride (60% dispersion, 0.443 g,

11.2 mmol) in N, N-dimethylformamide (8 ml) and stirred for 1.5 h. The suspension was treated with 2- (dimethylamino) ethyl chloride hydrochloride (0.462 g, 3.21 mmol) and stirred at ambient temperature for 16 h. The mixture was partitioned between saturated aqueous ammonium chloride solution and dichloromethane. The aqueous portion was extracted with additional dichloromethane. The combined organic portions were washed (brine), dried and evaporated to a crude yellow oil which was purified by silica gel chromatography, eluting with 10% 2.0M ammonia in methanol: 90% dichloromethane (v/v), to give the product as a yellow solid (0.065 g, 17%).

MS: m/z 350 (M+1) +.