The present invention relates to a novel compound and salts thereof, processes for their preparation, pharmaceutical compositions containing them and to their use in

medicine.

WO 99/62875 teaches novel cysteine derivatives which are nitric oxide synthase inhibitors. A preferred compound of that invention is (2R, 5R)-6N- (1-iminoethyl)-2, 6- diamino-5-methyl-4-thiohexanoic acid (Compound 1) and salts thereof.

A four step synthesis for the preparation of Compound 1 is described in Example 1 of WO 99/62875. The first step in the synthesis (part a) is the preparation of (2R, 5R) 1-t- butyl-2N-t-butoxycarbonyl-6-benzyloxycarbonyl-2, 6-diamino-2-methyl hexanoate (Compound 2) from the reaction of the N-t-butoxycarbonyl cysteine t-butyl ester described by Olesen et al, Med Chem. 1985, 50 (22), 4332-4336 (Compound A) NHCOOC (CH HS Co2C (Hs) 3 (Compound A) with toluene-4-sulfonic acid (S)-2-benzyloxycarbonylamino-1-methyl-ethyl ester (Compound B) o O O Mye / (Compound B) in toluène and in the presence of base 1,8-diazabicyclo[5.4.0]undec-7-ene.

We have found that that under these conditions the reaction of Compound A with Compound B yields the required compound 2 in an approximately 50:50 mixture with the corresponding (2S, 5R) diastereoisomer.

The subsequent use of this mixture ofdiastereoisomers in steps (b), (c) and (d) of Example 1 yields the required Compound (1) in an approximately 50: 50 mixture with the corresponding (2S, 5R) diastereoisomer.

We have now found that if the reaction of Compound A with Compound B is carried out in the presence of a base such as cesium carbonate or potassium t-butoxide in an appropriate solvent then the product of the reaction is the Compound (2)

substantially free of the corresponding (2S, 5R) diastereoisomer. Further the use of this product as the starting material for the preparation of Compound (1) using the specific reaction conditions described in parts (b), (c) and (d) of Example (1) of WO 99/6875 provides the required Compound (1) substantially free of the corresponding (2S, 5R) diastereoisomer.

The present invention therefore provides (2R, 5R)-6N- iminoethyl)-2, 6-diamino-5- methyl-4-thiohexanoic acid (Compound 1) and salts thereof substantially free of the corresponding (2S, 5R) diastereoisomer.

The term substantially free as used herein means that the required Compound (1) or a salt thereof contains less than 10% of the corresponding (2S, 5R) diastereoisomer and preferably 5% or less. More particularly the Compound (1) contains 2% or less of the corresponding 2S, 5R diastereoisomer and more preferably no more than 0.5%.

Suitable salts of Compound (1) include addition salts and with organic or inorganic acids and salts with inorganic and organic bases. For use in medicine such salts will be prepared using physiologically acceptable organic or inorganic acids and bases. Examples of suitable pharmaceutical acceptable acid addition salts include those formed from hydrochloric, hydrobromic, sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, oxalic, fumaric, maleic, oxaloacetic, methanesufphonic, ethanesuiphonic, p-tofuenesutphonic, benzenesuiphonic, and isethionic acids. Examples of suitable pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium and salts with organic bases such as dicyclohexyl amine and N-methyl-D-glucamine. Convenient acid addition salts salt of Compound (1) include the dihydrochloride or more particularly the monophosphate.

In a further aspect the invention provides (2R, 5R3-t-butyl-2N-t-butoxyearbonyl-6N- benzylaxycarbonyl-2, 6-diamine-2-methyl-4-thiohexanoate Compound (2) substantially free e of the corresponding (2S,5R) diastereoisomer.

The Compound (2) substantially free of the corresponding (2S,5R) diastereoisomer may be prepared by reacting the L-cysteine derivative (A) with Compound (B) in the presence of a suitable base such as cesium carbonate or alkali metal tertiary butoxides, alkali metal hexamethylsilazanes, alkali metal trimethylsilanolates or alkali metal hydrides and in a suitable aprotic solvent or solvents. The term alkali metal as used above includes lithum, sodium or potassium.

A particularly convenient base for use in this reaction is potassium tertiary butoxid.

Examples of suitable aprotic solvents include acetonitrile, acetone, tetrahydrofuran and tertiary butyl methyl ether or mixtures thereof.

The Compound (2) thus prepared may be conveniently converted into the required Compound (1) substantially free of the corresponding (2S, 5R) diasteroisomer by the route outlined below including the reaction condition described in Example 1 parts (b), (c) and (d) of WO 99/62875 which are incorporated herein by reference.

Reaction of ompound (2) with ammoniu formate in the presence of palladium hydroxide on charcoal in a solvent such as ethanol yields Compound (3) substantially free of the corresponding (2S,5R) diastereoisomer. Conveniently Compound (3) is isolated from the reaction as the formate salt thereof. Reaction of Compound (3) with 5- (1- naphthylmethyl-thioacetimidate hydrochloride or ethyl acetimidate hydrochloride in a solvent such as ethyl acetate or acetone yields gives the 1-iminoethyl derivative (4)

which may be isolated as the free base or hydrochloride salt thereof substantially free of corresponding 2S, 5R diastereoisomer.

Treatment of (4) with a suitable acid e. g. hydrogen chloride in 1, 4- dioxan or aqueous phosphoric acid in toluene yields the required Compound (1) substantially free from the corresponding 2S, 5R (diastereoisomer) as the acid addition salt thereof e. g. its dihydrochloride or monophosphate salt.

A preferred compound of the invention is the monophosphate salt of Compound (1) including the mono hydrate thereof.

The compound of formula (1) and salts thereof, substantially free of the corresponding 2S, 5R diastereoisomer has advantageous properties over the product of Example 1 of WO 99/62875 and in particular it has an improved pharmacodynamic profile.

The monophosphate salt of Compound (1) is not hygroscopic under normal atmospheric conditions and thus is particularly desirable for pharmaceutical use.

The compound of formula (1) and/or pharmaceutically acceptable salts thereof substantially free of the corresponding 2S, 5R diastereoisomer are herein after referred to as a compound of the invention or compounds of the invention.

The compounds of the invention are inhibitors of NO synthase and in particular NOS and therefore have use in the prophylaxis and treatment of clinical conditions for which an inhibitor of NO synthase is indicated, in particular, an inhibitor of iNOS. Such conditions include inflammatory conditions, shock states, immune disorders, and disorders of gastrointestinal motility. The compounds of the invention may also be of use in the prophylaxis and treatment of diseases of the central nervous system including migraine.

By shock states is meant those resulting from overproduction of NO, such as septic shock, haemorrhagic shock, traumatic shock, or shock caused by fulminant hepatic failure or by therapy with cytokines such as TNF, IL-1 and IL-2 or therapy with cytokine- inducing agents, for eampte 5, 6-dimethy) xanthenone acetic acid.

Examples of inflammatory conditions and immune disorders include those of the joint, particularly arthritis (e.g. rheumatoid arthritis, osteoarthritis, prosthetic joint failure), or the he gastrointestinal tract (e.g. ulcerative colitis, Crohn's disease, and other inflammatory bowel diseases, gastritis and mucosal inflammation resulting from infection, the enteropathy provoked by non-steroidal antiinflammatory drugs), of the lung (e. g. adult respiratory distress syndrome, asthma, cystic fibrosis, or chronic obstructive pulmonary disease), of the heart (e. g. myocarditis), of nervous tissue (e. g. multiple sclerosis), of the pancreas (e.g. diabetes melitus and complications thereof), of the kidney (e.g.

glomerulonephritis), of the skin (e. g. dermatitis, psoriasis, eczema, urticaria), of the eye (e. g. glaucoma) as well as of transplanted organs (e. g. rejection) and multi-organ diseases (e. g. systemic lupus erythematosis) and inflammatory sequelae of viral or bacterial infections. There is also evidence that iNOS inhibitors may be useful in the prophylaxis or treatment of bacterial infections (e. g. pneumonia), in particular, by reducing bacterial load in an infected mammal.

Furthermore, there is evidence for overproduction of NO by iNOS in atherosclerosis and following hypoxic or ischaemic insults (with or without reperfusion), for example in the brain or in ischaemic heart disease.

Disorders of gastrointestinal motility include ileus, for example post-operative ileus and ileus during sepsis.

By diseases of the central nervous system is meant those for which overproduction of NO is implicated, for example migraine, psychosis, anxiety, schizophrenia, sleep disorders, cerebral ischaemia, CNS trauma, epilepsy, multiple sclerosis, AIDS dementia, chronic neurodegenerative disease (e. g. Lewy Body Dementia, Huntington's disease, Parkinson's disease, or Alzheimer's disease) and acute and chronic pain, and conditions in which non-adrenergic non-cholinergic nerve may be implicated such as priapism, obesity and hyperphagia.

Examples of acute pain include musculoskeletal pain, post operative pain and surgical pain. Examples of chronic pain include chronic inflammatory pain (e. g. rheumatoid arthritis and osteoarthritis), neuropathic pain (e. g. post heretic neuralgia, diabetic neuropathies associated with diabetes, trigeminal neuralgia, peripheral inflammatory neuropathies, pain associated with functional bowel disorders, e. g. irritable bowel syndrome, non cardiac chest pain and sympathetically maintained pain) and pain associated with cancer and fibromyatgia.

Furthermore, inhibition of NO synthase may be of advantage in preventing the lymphocyte loss associated with HIV infection, in increasing the radiosensitivity of tumours during radiotherapy and in reducing tumour growth, tumour progression, angiogenesis, and metastasis.

Accordingly, the present invention provides a method for the prophylaxis or treatment of a clinical condition in a mammal, such as a human, for which an inhibitor of nitric oxide synthase, for example, an NOS inhibitor is indicated, which comprises administration of a therapeutically effective amount of a compound of the invention In particular, the present invention provides a method for the prophylaxis or treatment of an inflammatory and/or immune disorder, such as arthritis or asthma or acute or chronic pain.) n a preferred aspect the present invention provides a method for the prophylaxis or

treatment of a clinical condition selected from arthritis, asthma, ileus, acute and chronic pain and migraine. In a further aspect, the present invention provides a method for the prophylaxis or treatment of a bacterial infection.

In the alternative, there is also provided a compound of the invention for use in medical therapy, particularly, for use in the prophylaxis or treatment of a clinical condition in a mammal, such as a human, for which an inhibitor of nitric oxide synthase, for example an iNOS inhibitor, is indicated. In particular, there is provided a compound of the invention for the prophylaxis or treatment of an inflammatory and/or immune disorder, such as arthritis or asthma. In a preferred aspect, there is provided a compound of the invention for the prophylaxis or treatment of arthritis, asthma, ileus, acute and chronic pain and migraine. In a further aspect, there is provided a compound of the invention for the prophylaxis or treatment of a bacterial infection.

It will be appreciated that the compounds of the invention may advantageously be used in conjunction with one or more other therapeutic agents. Thus for the treatment of pain they may be advantageously used in conjunction with other therapeutic agents such as COX-2 inhibitors e. g Vioxx or 2-(4-ethoxy-phenyl)-3-(4-methanesulfonyl-phenyl)- pyrazol [1, 5-b] pyridazine, gabapentin, TNF a antagonists e. g. enbrel, IL-2 antagonists and opiate analgesics such as morphine.

The amount of a compound of the invention which is required to achieve a therapeutic effect will, of course, vary with the route of administration, the subject under treatment, and the particular disorder or disease being treated. The compounds of the invention may be administered orally or via injection at a dose of from 0.1 to 1500mg/kg per day, preferably 0. 1 to 500mg/kg per day. The dose range for adult humans is generally from 5mg to 35g/day and preferably 5mg to 2g/day. Tablets or other forms of presentation provided in discrete units may conveniently contain an amount of compound of the invention which is effective at such dosage or as a multiple of the same, for instance, units containing 5mg to 500mg, usually around 10mg to 200mg.

While it is possible for the compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formutation.

Accordingly, the present invention further provides a pharmaceutical formulation comprising a compound of the invention and a pharmaceutically acceptable carrier or excipient, and optionally one or more other therapeutic ingredients.

The present invention also provides the use of a compound of the invention in the manufacture of a medicament for the prophylaxis or treatment of a clinical condition for which an inhibitor of nitric oxide synthase, for example an NOS inhibitor, is indicated, for example an inflammatory and/or immune disorder, such as arthritis or asthma, in a

preferred aspect, there is provided a compound of the invention in the manufacture of a medicament for the prophylaxis or treatment of a clinical condition selected from arthritis, asthma, ileus, and migraine. In a further aspect, there is provided a compound of the invention in the manufacture of a medicament for the prophylaxis or treatment of a bacterial infection.

Hereinafter, the term"active ingredient"means a compound of the invention.

The formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), inhalation (including fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulisers or insufflators), rectal and topical (including dermal, buccal, sublingual and intraocular) administration although the most suitable route may depend upon for example the condition and disorder of the recipient.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient ; as a powder or granules ; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid ; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or mou) ding, optionatfy with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-ftowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.

Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic ,with the blood of the intended recipient ; and

aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter or polyethylene glycol.

Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose and acacia.

Preferred unit dosage formulations are those containing an effective dose, as hereinbefore recited, or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

As stated above, the compounds of the invention may also be used in combination with other therapeutic agents. The invention thus provides, in a further aspect, a combination comprising a compound of formula (1) or a pharmaceutically acceptable derivative thereof together with a further therapeutic agent.

The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutical acceptable carrier or excipient comprise a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simuitaneousty in separate or combined pharmaceutical formuiations.

When a compound of formula (1) or a pharmaceutically acceptable derivative thereof is used in combination with a second therapeutic agent active against the same disease state the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.

The following examples are illustrative of the present invention and are not to be construed as a limitation of the scope of the invention.

Example 1 (2R, 5R)-6N- (1-iminoethv !)-2, 6-diamino-5-methvl-4-thiohexanoate dihvdrochloride (a) (2R, 5R)-t-butvl-2N-t-butoxvcarbonvl-6N-benzvloxvcarbonvl-2. 6-diamino-5-methvl- 4-thiohexanoate Method 1 Cesium carbonate (21.9g) was added portionwise over 5 mins to a stirred solution of (R)- 2-tert-butoxycarbonylamino-3-mercapto-propionic acid tert-butyl ester (18. 6g) (Olsen et al ; J. Org Chem ; 1985 ; 50 (22), 4332-4336) and toluene-4-sulfonic acid (S)-2- benzyloxycarbonylamino-1-methyl-ethyl ester (24.39g) in dry acetonitrile (800ml) at 10°C under argon. The mixture was stirred vigorously for 16h, then washed with brine (3x500ml). The combined aqueous washings were extracted with ethyl acetate (3x500ml) and the combined organic extracts dried (Na2SO4), filtered and evaporated. The residual oil was purified by chromatography on silica (Biotage). Elution with hexane/ethyl acetate 7: 1 gave the title compound as colourless solid (25.5g) 'HNMR (CDCI3 ; 600MHz) 6H 1.26 (3H, d, Me) ; 1.43 and 1.47 (ea 9H, s, CMe3), 2.97 and 2. 90 (3H, m), 3. 24 and 3. 36 (ea 1H, m), 4. 39 (1H, brm, 2H), 5. 11 (2H, s, CH2Ph), 5.31 and 5. 42 (ea I H, br, NH), 7.36 (5H, m, Ar-H) Analytical LC using Chiralpak AD, 10 micron particle size; 250 mm x 4. 6 mm i. d.; n-Hexane : Ethanol, (80: 20 v/v; pump-mixed) ; isocratic for 20 mins at 1 ml/min ; UV detection at 215 nm yielding the R, R diastereomer ; HPLC Rt 11. 57 min.

(R, S diastereomer ; HPLC Rt7. 77 min) Method 2 (2R,5R)-t-butyl-2N-t-butoxycarbonyl-6N-benzyloxycarbonyl-2,6 -diamino-5-methyl-4- thiohexanoate (R)-2-tert-butoxycarbonylamino-3-mercapto-propionic acid tert-butyl ester (25g) was dissolved in degassed tertiary butyl methyl ether (450ml) under nitrogen and the solution is cooled to 10+3 °C. Potassium terSbutoxide in tetrahydrofuran (40 g, 20.5% w/w) is added over 5-10 minutes. When the exotherm starts to subside, toluene-4-sulfonic acid (S)-2-benzyloxycarbonylamino-1-methyl-ethyl ester (25g) was added as a solution in tertrahydrofuran (63mol), as fast as possible, followed by a tetrahydrofuran (13ml) line wash. After er the addition is complete, the reaction mixture is warmed to 22~3°C and stirred

overnight. Once the reaction was judged complete by HPLC, 15% w/w aqueous ammonium chloride solution (200ml) was added and the mixture stirred for ca 10min until a clear biphasic solution was formed. The layers were separated and the upper organic phase washed successively with 8% w/w aqueous sodium bicarbonate solution (150m1) and 30% w/w aqueous sodium chloride solution (150ml). The organic phase was dried over sodium sulfate (25 g), filtered and concentrated to ca 75 ml. tetrahydrofuran was azeotropically removed through successive distillations of tertiary butyl methyl ether so that a concentrate of 150 ml was obtained. To this, isooctane (150ml) was added at 20~3°C and after an age period of 15-30 minutes, additional isooctane (150 ml) added over 15 min. The resulting slurry is aged for 1.5 hours and the product isolated by filtration at suction. The filter cake was washed with tertiary butyl methyl ether : isooctane 1: 5 (100ml) and dried in vacuo at 353 °C overnight to give title compound (25. 8g, 80%) as a colourless solid.

(b) (R)-3-((R)-2-Amino-1-methyl-ethylsulfanyl)-2-tert-butoxycarb onylamino-propionic acid -buty) ester Method 1 Ammonium formate (1. 5g) was added to a mixture of (2R, 5R)-5-butyl-2N-t-butoxycarbonyl- 6N-benzyloxycarbonyl-2, 6-diamino-5-methyl-4-thiohexanoate (0. 55g) and palladium hydroxide (20% wt on carbon ; 0. 82g) in ethanol (20ml) and the mixture heated at 70'C for 1 h. The cooled mixture was filtered through Celite, evaporated in vacuo. and the residue purified on silica (Biotage). Elution with dichloromethane/ethanol/ammonia 200: 8 : 1 gave the title compound as colourless oil (0. 39g) sHMiViR (CDCl3; 400MHz) #H 1.26 (3H, d,Me) ; 1. 45 and 1. 48 (ea 9H, s, CMe3), 2. 68 to 2.78 (3H, m), 2.96 (2H, d), 4.43 (1H, brm), 5.60 (1H, br d, NH), Method 2 (R)-3-((R)-2-Amino-1-methyl-ethylsulfanyl)-2-tert-butoxycarb onylamino-propionic acid tert-butyl ester mono formate A mixture of (2R,5R)-5-butyl-2N-t-butoxycarbonyl-6N-benzyloxycarbonyl-2,6 -diamino-5- methyl-4-thiohexanoate (20. 0g), 20% palladium hydroxide on carbon paste (20. 0g) and ethanol (200mi) was stirred and heated to 40-45°C. A solution of ammonium formate (8. 0g) in water (20ml) and ethanol (20ml) was added dropwise over ca. 30 minutes, then the mixture was heated at 40-45°C for a further 30 minutes. The reaction was analysed

by HPLC to check for complete consumption of starting material. The mixture was filtered through a bed of celite and the celite bed washed with ethanol (100moi). The ethanol was removed by evaporation under reduced pressure, and the residue dissolved in ethyl acetate (150ml). The solution was washed with 10% aqueous sodium carbonate (200ml), then the aqueous phase extracted with ethyl acetate (100ml). The organic solutions were combined, washed with water (100moi) then evaporated under reduced pressure. The residue was redissolved in ethyl acetate (150mL) and the solution was cooled to 5°C. <BR> <BR> <BR> <BR> <P>Formic acid (2. 0g) was added and the mixture stirred at 5°C for 1 hour to crystallise the product. The product was isolated by filtration, washed with ethyl acetate (50mL) and dried in vacuo at 40°C to give the title comPound (11. 99) as a white solid.

(c) (2R, 5R)-t-butyl-2N-t-butoxvcarbonvl-6N- (1-iminoethvl)-2, 6-diamino-5-methvl- 4-thiohexanoate hvdrochloride S- (l-naphthylmethyl) thioacetimidate hydrochloride (18. 15g) was added in one portion to a solution of (R)-3-((R)-2-Amino-1-methyl-ethylsulfanyl)-2-tert-butoxycarb onylamino- propionic acid tert-butyl ester (11.47g) in dry THF (200mi) and the mixture stirred at room temperature for 18h. The mixture was fitered and the filtrate evaporated in vacuo. The residue was purified on silica (Biotage). Elution with ethyl acetate/methanol 4: 1, followed by filtration of the appropriate combined, evaporated fractions through Celite gave the title compound as a colourless foam (11. 6g) 1HNMR (DMSO ; 400 MHz) 6H 1. 24 (3H, d, Me) ; 1. 39 and 1. 41 (ea 9H, s, CMe3), 2. 17 (3H, s, Me), 2. 77-2. 83 (2H, m, CH2), 3. 02 (1 H, m, CH), 3. 34 (2H, m, CH2), 3. 95 (1 H, m, CH), 7. 24 (1H, d, NH), 8.75 (1H, s, NH), 27 (1 H, s, NH), 9.55 (1H, s, NH) Method 2 To a suspension of (R)-3-((R)-2-Amino-1-methyl-ethylsulfanyl)-2-tert- butoxycarbonylamino-propionic acid tert-butyl este rmono formate (45.5g) in ethyl acetate (432ml) was added added S-(1-naphthylmethyl)thioacetimidate hydrochloride (30.8g) followed by an ethyl acetate (23ml) line wash. The mixture is stirred for at least 18 hour at room temperature and then analysed by HPLC. The reaction mixture was heated to 45°C for 1 h, cooled to 20°G and stirred for 30min and filtered under vacuum, and the filter cake is washed successively with ethyl acetate (137mol) then methyl isobutyl ketone (137ml). The wet cake was then dried at 45°C in vacuo overnight to afford the title compound (44.5g) as a white crystalline solid.

(d) (2R, 5R)-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoate dihydrochloride HCI (4M in 1, 4- dioxan ; 115ml) was added dropwise over 10mins to a vigorously stirred solution of (2R, 5R)-t-butyl-2N-t-butoxycarbonyl-6N- (1-iminoethyl)-2, 6-diamino-5-methyl-4- thiohexanoate hydrochloride (18. 9g) in dry 1,4-dioxan (100ml) at 50 under argon. The mixture was stirred for 16h and the resulting precipitate concentrated in vacuo, triturated under ether (2x100ml) and filtered to give the title compound as a colourless solid (13. 4g) 'HNMR (DMSO; 600 MHz) #H 1.27 (3H, d, 7Hz, Me); 2.21 (3H, s, Me), 3.14 (2H, d, 6Hz, CH2), 3. 18 (1H, m, CH), 3.43 and 3.36 (2H, m, CH2), 4. 16 (1H, m, CH), 8. 65 (3H, s, NH3), 8.87 (1H, s, NH), 9.80 and 9.30 (2H, s, NH2), 14.0 (1H, brs, OH) Analytical LC using Crownpak CR (+), 5 micron particle size ; 150 mm x 4. 0 mm i. d.; Water : Perchloric acid (70%, 16. 3g / litre ; pH1), ambient for 20 mins at 0. 5ml/min ; UV detection at 205 nm yielding the R, R diastereomer ; HPLC Rt 7. 58 min.

(R, S diastereomer ; HPLC Rt 5. 34 min).

Examte 2 (2R,5R)-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoa te monophosphate monohydrate To a solution of (2R, 5R)-t-butyl-2N-t-butoxycarbonyl-6N- (1-iminoethyl)-2, 6-diamino-5- methyl-4-thiohexanoate hydrochloride (15g) in water (52. 5moi) was added toluene (52. 5ml). The mixture was stirred vigorously and cooled to 53°C. A solution of potassium carbonate (18g) in water (18ml) was added over at least 15 min at 53°C. The <BR> <BR> <BR> <BR> <BR> <BR> <BR> biphasic mixture was stirred at 5~3°C for 30min, allowed to settle and the lower aqueous<BR> layer sampled for analysis by hplc to check no residual input material remains. The phases were separated and to the organic layer at 5~3°C was added a solution of phosphoric acid (3.6g) in water (36ml) over 10min. The biphasic mixture was stirred at 702°C for 18hr, allowed to settle and the aqueous layer sampled for analysis by hplc.

The mixture was cooled to 200C and the layers separated.

A mixture of acetone (60ml) and water (12ml) was heated to 40~2°C and seeded with the title compound (45mgs). To this mixture a portion (1.5ml) of the above aqueous layer

containing the product was added over 30min. Once the crystallization had been established the resulting suspension is stirred at 40~2°C for 15 min. The remaining aqueous layer was added to the suspension over 1 h keeping the temperature at 40~2°C during the addition. Further acetone (36ml) was added to the suspension at 40~2°C over 45min, the mixture stirred at 40~2°C for a further 15min, cooled to 52°C and held at this temperature for at least 1hr. The product was filtered at suction and the filter cake washed with acetone-water 3: 1 (75ml). The cake was sucked free of extraneous solvent under minimum vacuum and dried at 60°C at 10mbar in vacuo. This product tumbled in presence of wet nitrogen (bubbled through a saturated brine solution) for 2h in a rotary evaporator at atmospheric pressure to afford the title compound (8. 54g).

Example 3 (2R,5R)-6N-(1-iminoethyl)-2,6-diamino-5-methyl-4-thiohexanoa te monophosphate monohydrate To a solution of (2R, 5R)-5-butyl-2N-t-butoxycarbonyl-6N-(1-iminoethyl)-2,6-diamin o-5- methyl-4-thiohexanoate hydrochloride (2. 5g) in water (8. 75ml) was added toluene (8. 75mi). The mixture was stirred vigorously and cooled to 10~3°C. A solution of potassium carbonate (3g) in water (2. 5ml) was added dropwise at 103°C. The biphasic mixture was allowed to come to 20~3°C over 30min, and the lower aqueous layer <BR> <BR> <BR> sampled for analysis by hpic to check no residua) input materia) remains. The phases were separated and to the organic layer at 203°C was added dropwise a solution of phosphoric acid (0.6g) in water (6. 25ml). The biphasic mixture was stirred at 74~2°C for 23hr, allowed to settle and the aqueous layer sampled for analysis by hpic. The mixture was cooled to 20°C and the layers separated.

A solution of acetone (25ml) was heated to 402°C, to this the above aqueous layer containing the product was added dropwise. The resulting suspension is stirred at 45~2° for 15 min and cooled to 202°C. The product was filtered at suction and the filter cake washed with acetone (2.5ml). The cake was sucked free of extraneous solvent under minimum vacuum and dried in vacuo at 60°C overnight to afford title compound (1.49g) as a white crystalline solid.

Example 4 (2R,5R)-6N-(1-iminoethyl)-2.6-diamino-5-methyl-4-thiohexanoa te monophosphate To a cloudy solution of (2R, 5R)-t-butyl-2N-t-butoxycarbonyl-6N- (1-iminoethyl)-2, 6- diamino-5-methyl-4-thiohexanoate hydrochloride (3.67g) in ethyl acetate (20mut) was added saturated NaHCO3 (10ml). The mixture was stirred vigorously and the layers allowed to settle. The aqueous layer was then removed and extracted with further ethyl acetate (20ml). The combined organic phase was concentrated i vacuo and then re-

dissolved in toluene (1 8mol). To this toluene layer at 20~3°C a solution of phosphoric acid (0.96g) in water (11ml) was added dropwise. The mixture was then stirred at 70~2°C for at least 6hr and aqueous layer sampled for analysis by hplc. The mixture was cooled to 20~3°C and the layers separated. The pH of the resulting aqueous was adjusted to pH 5. 54 using concentrated ammonia this is followed by dropwise addition of ethyl acetate (10mi). The resulting solution was then seeded with few crystals of title compound and cooled and left standing overnight at 10~3°C. The product mixture was filtered under vacuum and the damp product was dried at 602°C in vacuo for 18hrs to afford title compound (1. 64g) as a white crystalline solid.

'HNMR (D2O ; 400 MHz) 6H 1. 19# (3H) d, J=6.9Hz ; 2. 138, (2. 178, minor rotamer, s) (3H) s; 3. 008 (1H) d of d, J=14.9Hz, 4.7Hz ; 3.03-3. 13# (2H) m; 3. 288 (1H) d of d, J=14.2Hz, 6. 6Hz ; 3. 368 (1H) d of d, J=14. 2Hz, 6.6Hz ; 3. 858 (1H) m; Pharmacy Example Tablet.

Active* 382 mg (equivalent to 250 mg as free base) Microcrystalline Cellulose 200 Sodium Starch Glycolat 37.5 Mannitol 121. 13 Colloidal Silicon Dioxide 1. 875 Magnesium Stearate 7. 5 mg Total Tablet Weight 750 mg The active* ((2R, 5R)-6N-(1-iminoethyl)-2, 6-diamino-5-methyl-4-thiohexanoate monophosphate monohydrate) and excipients are mixed using a conventional dry granulation process and the resultant granules compressed to form a 750mg tablet using a coneventional tablet press. The resultant tablet may be film coated using a conventional aqueous film coat e. g. Propriety Brand, Opadry.

Aqueous Film Coat (Proprietary Brand, Opadry) 3% coat applied of above tablet weight (22.5 mg of film coat applied) The potency and selectivity of the compound of the invention for human NOS enzyme was evaluated using standard in vitro nitric oxide synthase (NOS) methodology. Thus the iNOS activity was determined using quantitative absorption changes of haemoglobin as described by R. G. Knowles and J. Dawson in"A

Microtitreplate Assay of Human NOS Isoforms"in Methods in Molecular Biology, 1998, Volume 100,237-242, Nitric Oxide Protocols, Ed M. A. Titheradge, Humana Press, Totowa NJ.

In this test the compound of Example 1 had a plC5o of 5.8. Further the compound was found to be approximately 100 fold more selective for iNOS over eNOS and approximately 40 fold more selective for NOS over nNOS.

The ability of the compound of the invention to alleviate chronic pain can readily demonstrated in rats treated with Freunds complete adjuvant for example using the procedure described in the Journal of Pharmacology and experimental therapeutic 2003, Vol 304, (1) 56-62.