WO1995009619A2 - Substituted urea and isothiourea derivatives as no synthase inhibitors - Google Patents

Substituted urea and isothiourea derivatives as no synthase inhibitors Download PDF

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Publication number
WO1995009619A2
WO1995009619A2 PCT/GB1994/002138 GB9402138W WO9509619A2 WO 1995009619 A2 WO1995009619 A2 WO 1995009619A2 GB 9402138 W GB9402138 W GB 9402138W WO 9509619 A2 WO9509619 A2 WO 9509619A2
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ethyl
isothiourea
phenyl
group
lysine
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PCT/GB1994/002138
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French (fr)
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WO1995009619A3 (en
Inventor
Jeffrey Alan Oplinger
Barry George Shearer
Eric Cleveland Bigham
Eric Steven Furfine
Edward Patrick Garvey
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The Wellcome Foundation Limited
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Priority to AU77056/94A priority Critical patent/AU7705694A/en
Priority to JP7510678A priority patent/JPH09504278A/en
Priority to EP94927761A priority patent/EP0723438A1/en
Priority to US08/615,291 priority patent/US6090846A/en
Publication of WO1995009619A2 publication Critical patent/WO1995009619A2/en
Publication of WO1995009619A3 publication Critical patent/WO1995009619A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/75—Amino or imino radicals, acylated by carboxylic or carbonic acids, or by sulfur or nitrogen analogues thereof, e.g. carbamates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/70—Compounds containing any of the groups, e.g. isoureas
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/30—Isothioureas
    • C07C335/32—Isothioureas having sulfur atoms of isothiourea groups bound to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/08—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D277/12—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/18—Nitrogen atoms

Definitions

  • the present invention relates to N-substituted urea derivatives, to methods for their manufacture, to pharmaceutical compositions containing them and to their use in therapy, in particular their use as inhibitors of nitric oxide synthase, and in particular neuronal nitric oxide synthase.
  • endothelium-derived relaxing factor a labile humoral factor termed endothelium-derived relaxing factor (EDRF).
  • NO nitric oxide
  • NO is the endogenous stimulator of the soluble guanylate cyclase and is involved in a number of biological actions in addition to endothelium-dependent relaxation including cytotoxicity of phagocytic cells and cell-to-cell communication in the central nervous system (see Moncada et al, Biochemical Pharmacology, 23, 1709- 1715 (1989) and Moncada et al, Pharmacological reviews, 42, 109-142 (1991)). It is now thought that excess NO production may be involved in a number of conditions, particularly conditions which involve systemic hypotension such as toxic shock and therapy with certain cytokines.
  • the NO released by the constitutive enzyme acts as a transduction mechanism underlying several physiological responses.
  • the function of the NO produced by the inducible enzyme is as a cytotoxic molecule for tumour cells and invading microorganisms. It also appears that the adverse effects of excess NO production, in particular pathological vasodilation and tissue damage, may result largely from the effects of NO synthesised by the inducible NO synthase.
  • NO synthesis plays an important part in the pathology of a range of diseases of the nervous system, eg. ischemia.
  • non-selective inhibitors of NO synthases cause profound changes in blood pressure and blood flow, including cerebral blood flow.
  • ischemic injury inherently reduces the blood supply to the brain and any further decrease in blood flow caused by a non-selective NO synthase inhibitor would have a deleterious effect, potentially opposing any beneficial effect of decreased NO production within the brain.
  • studies of middle cerebral artery occlusion in both rats and mice have demonstrated a substantial protection effect of low doses of NO synthase inhibitors (see for example Nowicki et al, Eur. J Pharmacol., 1991. 204.
  • NO synthase inhibitors which are selective in the sense that they inhibit one NO synthase enzyme to a considerably greater extent compared to one or more of the other enzymes would be of even greater therapeutic benefit and much easier to use.
  • urea derivatives when used herein means "isothiourea derivatives" and “isourea derivatives”.
  • the present invention provides a method of treatment of a condition where there is an advantage in inhibiting the neuronal NO synthase enzyme with less inhibition of the endothelial or inducible NO synthase enzyme comprising administering to a mammal in need thereof a therapeutically effective amount of an N- substituted urea derivative or a salt, ester or amide thereof, other than N-(2,6- dimethylphenyl)-5,6-dihydro-4H- 1 ,3-thiazin-2-amine.
  • the present invention provides the use of a N-substituted urea derivative or salt, ester or amide thereof, other than N-(2,6-dimethylphenyl)-5,6- dihydro-4H-l,3-t_ ⁇ iazin-2-amine for the manufacture of a medicament for the treatment of a disease of the nervous system due to over production of the neuronal nitric oxide synthase enzyme.
  • diseases include cerebral ischemia, CNS trauma, epilepsy, AIDS dementia, chronic neurodegenerative disease and chronic pain, and conditions in which non-adrenergic non-cholinergic nerve may be implicated such as priapism, obesity and hyperphagia, particularly cerebral ischemia.
  • the N-substituted urea derivative is an N- substituted isothiourea derivative, other than N-(2,6-dimethylphenyl)-5,6-dihydro-4H- l,3-tl ⁇ az_n-2-amine, preferably an N,S-disubstituted isothiourea derivative.
  • the N-substituted urea derivative is an N- substituted isourea, preferably an N,O-disubstituted isourea derivative.
  • Preferred urea derivatives include those of the formula (I)
  • Q is oxygen or sulphur
  • R! is hydrogen or Ci _g hydrocarbyl
  • R2 is a mono- or bicyclic heterocyclic ring system, a C ⁇ . ⁇ Q hydrocarbyl group which may optionally contain an oxygen atom, a group S(O) n wherein n is 0, 1 or 2, or a group NR ⁇ wherein R- is hydrogen or a C ⁇ . ⁇ aliphatic group,each group R2 optionally being substituted by one to five groups independently selected from
  • Rl may be linked to the imino nitrogen to form a monocyclic heterocyclic ring; with the exception of N-(2,6-dimethylphenyl)-5,6-dihydro-4H-l,3-thiazin-2-amine.
  • One embodiment of the present invention provides compounds of formula (I) as hereinbefore defined with the proviso that R s not linked to the imino nitrogen to form a monocyclic heterocyclic ring.
  • Q is oxygen
  • Q is sulphur
  • Rl is hydrogen, C1.4 alkyl, C2-.5 alkenyl or benzyl; preferably R ⁇ is C ⁇ _4 alkyl for example ethyl;
  • R ⁇ is a 5- or 6- membered heterocyclic ring or a 9- or 10-membered bicyclic heterocyclic ring, a phenyl ring, or a C2-8 alkyl chain which optionally contains a group S(O) n as hereinbefore defined, or a C2.4 alkyl chain which contains a phenylene ring, each group R ⁇ optionally being substituted by one to five groups independently selected from (i) a C ⁇ _4 alkyl group optionally substituted by one to three fluoro atoms:
  • R1 may be linked to the imino nitrogen in the compound of formula (I) to form a thiazole or thiazoline ring.
  • Formula (I) includes compounds of formulae (I A) to (IF)
  • Z is oxygen or sulphur; Rl is as hereinbefore defined; X is a C2-9 hydrocarbyl group which may optionally contain an oxygen atom, a group S(O) n as hereinbefore defined, or a group NR ⁇ as hereinbefore defined; T is a C g hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring, or T is a C2-.4 hydrocarbyl group containing a phenylene ring; and Ar is a mono- or bicyclic aromatic ring system optionally substituted by one to five groups selected from
  • R* is a C ⁇ _ hydrocarbyl group, preferably a Cj_4 alkyl group, e.g. ethyl.
  • X is a C2-6 hydrocarbyl group and preferably a C3.5 alkylene or alkenylene group.
  • T is Cj.g hydrocarbyl containing a 5- or 6-membered heterocyclic ring; or a C2.4 hydrocarbyl group containing a phenylene ring.
  • Ar is phenyl optionally substituted by one to three substituents which may be the same or different and are selected from C 4 alkyl or C3.6 cycloalkyl groups each optionally substituted by one to three halo atoms; C1.4 alkoxy groups; hydroxy groups, benzyloxy groups; halo atoms; CO2R ⁇ groups wherein R ⁇ is hydrogen or C j _4 alkyl; groups NR ⁇ R 7 wherein R ⁇ and R 7 are independently selected from hydrogen or Cj_4 alkyl.
  • Ar is phenyl substituted by one or two substituents, preferably one substituent.
  • Ar is phenyl substituted by C 1.3 alkoxy, hydroxy, benzyloxy, halo atoms or C ⁇ _4 alkyl optionally substituted by one to three fluoro atoms, C1.3 alkoxy, hydroxy, benzyloxy, or halo atoms.
  • Suitable compounds of the formula (I) include:
  • hydrocarbyl group is meant a group that contains only carbon and hydrogen atoms and may contain double and/or triple bonds and which may be cyclic or aromatic in nature.
  • An oxygen atom, or a group S(O) n or NR ⁇ as hereinbefore defined, may optionally intersperse the carbon atoms in the hydrocarbyl chain.
  • aliphatic is meant an alkyl, alkenyl, alkynyl or cycloalkyl group.
  • alkyl, alkenyl and alkynyl are intended to include both straight and branched chain variants.
  • heterocyclic ring a cyclic compound containing one to three heteroatoms selected from oxygen, sulphur and nitrogen, and preferably nitrogen and sulphur.
  • the compounds of formula (I) may include a number of asymmetric centres in the molecule depending on the precise meaning of the various groups and the present invention is intended to include all possible isomers.
  • Certain compounds of formula (I) have also been found to have activity against the inducible NO synthase enzyme and may be of use in the treatment of systemic hypotension associated with septic and/or toxic shock induced by a wide variety of agents, therapy with cytokines such as TNF, IL-1 and IL-2 and therapy with cytokine- inducing agents such as 5,6-dimethylxanthenone acetic acid, as an adjuvant to short term immunosuppression in transplant therapy, and in the treatment of autoimmune and or inflammatory conditions affecting the joints, for example arthritis.
  • cytokines such as TNF, IL-1 and IL-2
  • cytokine- inducing agents such as 5,6-dimethylxanthenone acetic acid
  • the present invention further provides the use of a compound of formula (I) other than 5-methyl-2-(2-thiazolylamino)phenol and S-ethyl-N-phenylisothiourea for the manufacture of a medicament for the treatment of a condition requiring inhibition of the inducible NO synthase enzyme.
  • the present invention provides a N-substituted urea derivative of formula (I) other than S-ethyl-N-phenylisothiourea, S-ethyl-N-(2-chlorophenyl) isothiourea, S-ethyl-N-(2-trifluoromethylphenyl)isothiourea, 2-propenylthiourea, N- (2,6-dimethylphenyl)-5,6-dihydro-4H-l,3-thiazin-2-amine and 5-methyl-2-(2- thiazolyl amino)phenol, or a pharmaceutically acceptable salt, ester or amide thereof for use in medicine.
  • formula (I) other than S-ethyl-N-phenylisothiourea, S-ethyl-N-(2-chlorophenyl) isothiourea, S-ethyl-N-(2-trifluoromethylphenyl)isothiourea, 2-
  • the present invention also provides a N-substituted urea derivative of formula (I) or a salt, ester or amide thereof, with the proviso that:
  • R! is methyl, R ⁇ is not a phenyl ring substituted by 3-chloro, 2-ethyl, 2-chloro-5-trifluoromethyl, 3-trifiuoromethyl, 3-methyl, 3-bromo, 4- nitro, 4-chloro, 3,4-dichloro or CO2H; or R 2 is not a group 5-chloro-2- pyridyl;
  • R! is ethyl, R 2 is not a phenyl ring or a phenyl ring substituted by 4- methoxy, 2-chloro, 4-hydroxy, 2-methoxy, 4-methyl. 2-trifluoromethyl or 3-trifluoromethyl; or
  • the present invention includes N-substituted urea derivatives in the form of salts, esters or amides, 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-pharmaceutically acceptable salts may be of utility in the preparation and purification of the compound in question.
  • preferred salts include those formed from hydrochloric, hydrobromic, sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, oxalic, fumaric, maleic, oxaloacetic, methanesulphonic, ethanesulphonic, p-toluenesulphonic, benzenesulphonic and isethionic acids.
  • Salts of N-substituted urea derivatives can be made by reacting the appropriate compound in the form of the free base with the appropriate acid.
  • Esters are pharmaceutically acceptable esters, for example Cj_4 alkyl esters.
  • treatment of a patient is intended to include prophylaxis.
  • the formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intravenous and intraarticular), 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 into association a compound of formula (I) or a pharmaceutically acceptable salt, ester or amide thereof ("active ingredient”) with the carrier which constitutes one or more accessory ingredients.
  • active ingredient a pharmaceutically acceptable salt, ester or amide thereof
  • 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 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.
  • the compounds of the invention may be administered orally or via injection at a dose of from 0.1 to 500mg/kg per day.
  • the dose range for adult humans is generally from 5mg to 35g/day, preferably 5mg to 2g/day and most preferably lOmg to lg/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.
  • the compounds of formula (I) are preferably administered orally or by injection (intravenous or subcutaneous).
  • the precise amount of compound administered to a patient will be the responsibility of the attendant physician. However the dose employed will depend on a number of factors, including the age and sex of the patient, the precise disorder being treated, and its severity. Also the route of administration may vary depending on the condition and its severity.
  • R 2 is as hereinbefore defined, with a compound having a thiocarbonyl group, followed if necessary by hydrolysis to give a compound of formula (I) wherein R* is hydrogen or a tautomer thereof, and thereafter the optional conversion to a compound of formula (I) wherein R is other than hydrogen by alkylation of the sulphur atom of the isothiourea.
  • the coupling reaction may be carried out between a compound of formula (II) and a compound having a thiocarbonyl group, for example thiophosgene followed by ammonia as described in Tet. Lett. 1991, 21 (7) 875-878 or a compound of formula (II) and an isothiocyanate, such as benzoyl isothiocyanate.
  • a polar solvent such as dichloromethane, chloroform, ethanol or acetone at a non-extreme temperature of from -78°C to 200°C, for example -5°C to 100°C and preferably room temperature.
  • the intermediate thiourea e.g.
  • R 2 is as hereinbefore defined, followed by deprotection where necessary.
  • the acid catalysed addition is suitably carried out using the alcohol (Rl-OH or water) as the solvent, at a non-extreme temperature of 0°C to 100°C, and preferably room temperature, in the presence of an acid, e.g. hydrochloric acid, conveniently in solution in ether.
  • alcohol Rl-OH or water
  • an acid e.g. hydrochloric acid
  • Compounds of formula (III) may be prepared by the reaction of a compound of formula (II) as hereinbefore defined with a compound of formula L'-CN wherein L' is a leaving group, for example a halo atom such as bromo.
  • the reaction may be carried out in ether as a solvent at a non-extreme temperature of from -20°C to 100°C, suitably 0°C.
  • the thiazoline foam was taken up into a mixture of trifluoroacetic acid (9.5ml), water (0.5ml), thioanisole (0.5ml), phenol (0.75g) and 1 ,2-ethanediol (0.25ml) at 0°C.
  • the solution was stirred for two hours at 20°C and concentrated to a volume of 3ml.
  • the solution was rapidly stired and diethyl ether (50ml) was added.
  • N-S-fiminomethoxymethyD-L-ornithine dihvdrochloride A solution of 0.75 g (1.96 mmol) N 2 -(tert-butoxycarbonyl)-N5- (iminomethoxymethyl)-L-ornithine tert-butyl ester hydrochloride in 2 mL dioxane at 0°C was treated with 15 mL of 4N hydrochloric acid in dioxane solution. The solution was stirred overnight at 22 °C, concentrated to a crude paste, and freeze-dried from 8 mL of water.
  • the product was freeze- dried a second time to yield 0.53 g N ⁇ -(iminomethoxymethyl)-L-ornithine dihydrochloride.
  • the product analyzed solvated with an additional 0.2 molar hydrochloric acid. 0.1 molar water, and 0.3 molar dioxane.
  • N6-((ber_zyloxy)carbonyl)-N 2 -(tert-butoxycarbonyl)-L-lysine tert-butyl ester was hydrogenated at 20°C under 50 psi hydrogen in 100 mL ethyl acetate for 1 h.
  • the catalyst was removed by filtration through celite and the amine intermediate isolated without further purification as an oil (3.05g).
  • the amine intermediate was taken into 40mL ether and the solution added over 10 min to a solution of l.lg (10.1 mmol) of cyanogen bromide in 50 mL ether at 0°C.
  • N 2 -(tert-butoxycarbonyl)-N ⁇ -(cyano)-L-lysine tert-butyl ester was prepared 0.95 g (91%) N 2 -(tert-butoxycarbonyl)-N6- (iminomethoxymethyl)-L-lysine tert-butyl ester hydrochloride as a foamy- solid by the method described for the preparation of N -(tert-butoxycarbonyl)- N :> -(iminomethoxymethyl)-L-ornithine tert-butyl ester hydrochloride.
  • N 2 -(tert-butoxycarbonyl)-N ⁇ -(cyano)-L-lysine tert- butyl ester was prepared 1.5 g (75%) of foamy solid N -(tert-butoxycarbonyl)- N"-(ethoxyiminomethyl)-L-lysine tert-butyl ester hydrochloride by the method described for N 2 -(tert-butoxycarbonyl)-N ⁇ -(iminomethoxymethyl)-L- ornithine tert-butyl ester hydrochloride.
  • the crude material was purified by silica gel chromatography eluting with methanol/methylene chloride/ammonium hydroxide (5/95/0.5 to 15/85/0.5) to yield 5.28g (30%) of a thick, viscous yellow oil.
  • Amion and chorion were removed from fresh placenta, which was then rinsed with 0.9% NaCl.
  • the tissue was homogenized in a Waring blender in 3 volumes of HEDS buffer (20mM Hepes pH 7.8, 0.1 mM EDTA, 5 mM DTT, 0.2 M sucrose) plus 0.1 mM PMSF.
  • the homogenate was filtered through cheesecloth and then centrifuged at lOOOg for 20 min. The supernatant was recentrifuged at 27500g for 30 min. Solid ammonium sulfate was added to the supernatant to give 32% saturation.
  • Precipitated protein was pelleted at 25,000g and then redissolved in a minimal volume of HEDS buffer plus 0.1 mM PMSF, 1 O ⁇ g/ml leupeptin and soybean trypsin inhibitor, and 1 ⁇ g/ml pepstatin. The redissolved pellet was centrigued at 15000g for 10 min. To the supernatant was added 1/20 volume of 2',5' ADP agarose resin (Sigma), and the slurry was mixed slowly overnight. In morning, slurry was packed into a column. The resin was sequentially washed with HEDS, 0.5 M NaCl in HEDS, HEDS, and then NOS was eluted with lOmM NADPH in HEDS. The enzyme could be concentrated by ultrafiltration and quick frozen and stored at -70°C without loss in activity for at least 6 months.
  • NOS was assayed for the formation of citrulline following the procedure of Schmidt et al (PNAS 88 365-369 , 1991) with these modifications: 20 mM Hepes, pH 7.4, lO ⁇ g/ml calmodulin, 2.5 mM CaCl2 2.5 mM DTT, 125 ⁇ M NADPH lO ⁇ M H4 Biopterin, 0.5 mg/ml BSA, and 1 ⁇ M L-[14C] arginine (New England Nuclear). Linearity of NOS-catalyzed rate was confirmed prior to kinetic studies that used single time point determination of rate.
  • DLD-1 (ATCC No. CCL 221) were grown at 37°C, 5% C0 2 in RPMI 1640 medium supplemented with L-glutamin, penicillin, streptomycin, and 10% heat-inactivated fetal bovine serum. Cells were grown to confluency and then the following cocktail of cytokines were added: 100 units/ml interferon-gamma, 200 units/ml interleukin-6, 10 ng/ml tumor necrosis factor, and 0.5 ng/ml interleukin-l ⁇ .
  • citrulline was assayed as described above except that 10 ⁇ M FAD was included and calmodulin and CaC12 were excluded from the assay mix.
  • Human brain NOS Human brain NOS was prepared using variations of the procedures of Schmidt et al. (TNAS 88 365-369, 1991), Mayer et al. (Ted. Eur. Biochem. Soc. 288 187-191, 1991), and Bredt and Snyder, (PNAS 87 682-685, 1990). Briefly, frozen human brain (1050 gm) was homogenized in cold buffer A (50 mM HEPES, pH 7.5 (pH at RT) and 0.5 mM EDTA, 10 mM DTT, 3.6 L total volume) with a polytron. The mixture was centrifuged at 13,000g for 1 hour and the supernatant was removed (about 2050 ml).
  • cold buffer A 50 mM HEPES, pH 7.5 (pH at RT) and 0.5 mM EDTA, 10 mM DTT, 3.6 L total volume
  • the mixture was thawed and passed through a 2',5' ADP-agarose column (0.4 g swelled in buffer A) at 4 ml/min.
  • the column was washed with 100ml buffer A. 200ml buffer A with 500 mM NaCl, 100ml Buffer A, then 30 ml buffer A with 5 mM NADPH.
  • To the enzyme eluted from the column was added glycerol to 15%, CaCl2 to 1 mM, tetrahydrobiopterin to lO ⁇ M, tween to 0.1% and FAD, FMN to 1 ⁇ M each.
  • the enzyme was then passed through a 1 ml calmodulin-agarose column which had been equilibrated in Buffer A, 15% glycerol and 1 mM CaCl2.
  • the column was washed with 15ml Buffer A, 15% glycerol and 1 mM CaC_2, 15 ml of Buffer A, 15% glycerol and 5 mM EDTA, and then enzyme activity was eluted with 3 ml of Buffer A, 15% glycerol and 5 mM EDTA, 1 M NaCl.
  • To the enzyme was added tetrahydrobiopterin to 10 ⁇ M, FAD and FMN to 1 ⁇ M, and tween to 0.1%.
  • the progress curve was an exponential decay followed by a linear steady state rate.
  • Inhibition constant was calculated by dividing the steady state inhibited rate by the control uninhibited rate; percent inhibition was then used to calculate the inhibition constant assuming competitive inhibition with respect to arginine.
  • alue obtained from measuring percent inhibition at three or more concentrations of inhibitor at a single time point and assuming competitive inhibition with respect to arginine.
  • the progress curve was an exponential decay of the rate. Value is a K ⁇ determined by measuring association and dissociation rate constants for the slow onset of inhibition, as previously described (Furfine, E.S., Harmon, M.F., Paith, J.E., and Garvey, E.P. (1993) Biochemistry 32, 8512-8517).

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Abstract

The use of an N-substituted urea derivative for the manufacture of a medicament for the treatment of a condition where there is an advantage in inhibiting the NO synthase enzyme, in particular cerebral ischemia, and pharmaceutical formulations therefor are disclosed. Novel N-substituted urea derivatives and processes for the preparation thereof are also described.

Description

SUBSTITUTED UREA AND ISOTHIOUREA DERIVATIVES AS NO SYNTHASE INHIBITORS
The present invention relates to N-substituted urea derivatives, to methods for their manufacture, to pharmaceutical compositions containing them and to their use in therapy, in particular their use as inhibitors of nitric oxide synthase, and in particular neuronal nitric oxide synthase.
It has been known since the early 1980's that the vascular relaxation brought about by acetylcholine is dependent on the presence of the endothelium and this activity was ascribed to a labile humoral factor termed endothelium-derived relaxing factor (EDRF). The activity of nitric oxide (NO) as a vasodilator has been known for well over 100 years and NO is the active component of amyl nitrite, glyceryltrinitrite and other nitrovasodilators. The recent identification of EDRF as NO has coincided with the discovery of a biochemical pathway by which NO is synthesised from the amino acid L-arginine by the enzyme NO synthase.
NO is the endogenous stimulator of the soluble guanylate cyclase and is involved in a number of biological actions in addition to endothelium-dependent relaxation including cytotoxicity of phagocytic cells and cell-to-cell communication in the central nervous system (see Moncada et al, Biochemical Pharmacology, 23, 1709- 1715 (1989) and Moncada et al, Pharmacological reviews, 42, 109-142 (1991)). It is now thought that excess NO production may be involved in a number of conditions, particularly conditions which involve systemic hypotension such as toxic shock and therapy with certain cytokines.
The synthesis of NO from L-arginine can be inhibited by the L-arginine analogue NG-monomethyl-L-arginine (L-NMMA) and the therapeutic use of L-NMMA for the treatment of toxic shock and other types of systemic hypotension has been proposed (WO 91/04024 and GB-A-2240041). The therapeutic use of certain other NO synthase inhibitors apart from L-NMMA for the same purpose has also been proposed in WO 91/04024 and in EP-A-0446699. Other potent NO synthase inhibitors are described in Narayanan et al., J. Med. Chem. 21, 885-887 (1994). It has recently become apparent that there are at least three types of NO synthase enzymes as follows:-
(i) a constitutive, Ca ~H7calmodulin dependent enzyme, located in the endothelium, that releases NO in response to receptor or physical stimulation.
(ii) a constitutive, Ca++/calmodulin dependent enzyme, located in the brain, that releases NO in response to receptor or physical stimulation.
(iii) a Ca"1-*" independent enzyme which is induced after activation of vascular smooth muscle, macrophages, endothelial cells, and a number of other cells by endotoxin and cytokines. Once expressed this inducible NO synthase synthesises NO for long periods.
The NO released by the constitutive enzyme acts as a transduction mechanism underlying several physiological responses. The function of the NO produced by the inducible enzyme is as a cytotoxic molecule for tumour cells and invading microorganisms. It also appears that the adverse effects of excess NO production, in particular pathological vasodilation and tissue damage, may result largely from the effects of NO synthesised by the inducible NO synthase.
It is believed that NO synthesis plays an important part in the pathology of a range of diseases of the nervous system, eg. ischemia. However, non-selective inhibitors of NO synthases cause profound changes in blood pressure and blood flow, including cerebral blood flow. Unfortunately, ischemic injury inherently reduces the blood supply to the brain and any further decrease in blood flow caused by a non-selective NO synthase inhibitor would have a deleterious effect, potentially opposing any beneficial effect of decreased NO production within the brain. Nevertheless, studies of middle cerebral artery occlusion in both rats and mice have demonstrated a substantial protection effect of low doses of NO synthase inhibitors (see for example Nowicki et al, Eur. J Pharmacol., 1991. 204. 339-340). At high doses, or in models of global ischemia, these inhibitors fail to provide protection. Thus, there is a need for a potent inhibitor of neuronal NO synthase with preferably little or no activity against the vascular endothelial NO synthase. The NO synthase inhibitors proposed for therapeutic use so far, such as L-NMMA and L-NAME (L-nitroarginine methyl ester), are non-selective in that they inhibit all NO synthase enzymes identified to date. Use of such a non-selective NO synthase inhibitor would require great care to be taken in order to avoid the potentially serious consequences of over-inhibition of the other enzymes. Thus, whilst non-selective NO synthase inhibitors have therapeutic utility provided that appropriate precautions are taken, NO synthase inhibitors which are selective in the sense that they inhibit one NO synthase enzyme to a considerably greater extent compared to one or more of the other enzymes would be of even greater therapeutic benefit and much easier to use.
Unpublished PCT patent application PCT/GB93/02437 discloses a class of S-substituted isothiourea derivatives which inhibit the NO synthase enzymes, showing a slight selectivity of the inducible enzyme over the constitutive enzymes.
It has been found that a class of N-substituted urea derivatives or salts, esters or amides thereof are NO synthase inhibitors, showing selectivity of the neuronal NO synthase enzyme over the endothelial and inducible NO synthase enzymes. The term "urea derivatives" when used herein means "isothiourea derivatives" and "isourea derivatives".
In one aspect the present invention provides the use of an N-substituted urea derivative or a salt, ester or amide thereof, other than N-(2,6-dimethylphenyl)-5,6- dihydro-4H-l,3-thiazin-2-amine, for the manufacture of a medicament for the treatment of a condition where there is an advantage in inhibiting the neuronal NO synthase enzyme with less inhibition of the endothelial or inducible NO synthase enzymes.
In another aspect, the present invention provides a method of treatment of a condition where there is an advantage in inhibiting the neuronal NO synthase enzyme with less inhibition of the endothelial or inducible NO synthase enzyme comprising administering to a mammal in need thereof a therapeutically effective amount of an N- substituted urea derivative or a salt, ester or amide thereof, other than N-(2,6- dimethylphenyl)-5,6-dihydro-4H- 1 ,3-thiazin-2-amine. More specifically, the present invention provides the use of a N-substituted urea derivative or salt, ester or amide thereof, other than N-(2,6-dimethylphenyl)-5,6- dihydro-4H-l,3-t_ιiazin-2-amine for the manufacture of a medicament for the treatment of a disease of the nervous system due to over production of the neuronal nitric oxide synthase enzyme. Such diseases include cerebral ischemia, CNS trauma, epilepsy, AIDS dementia, chronic neurodegenerative disease and chronic pain, and conditions in which non-adrenergic non-cholinergic nerve may be implicated such as priapism, obesity and hyperphagia, particularly cerebral ischemia.
In one embodiment of the present invention the N-substituted urea derivative is an N- substituted isothiourea derivative, other than N-(2,6-dimethylphenyl)-5,6-dihydro-4H- l,3-tlύaz_n-2-amine, preferably an N,S-disubstituted isothiourea derivative. In a second embodiment of the present invention the N-substituted urea derivative is an N- substituted isourea, preferably an N,O-disubstituted isourea derivative.
Preferred urea derivatives include those of the formula (I)
Figure imgf000006_0001
wherein
Q is oxygen or sulphur
R! is hydrogen or Ci _g hydrocarbyl;
R2 is a mono- or bicyclic heterocyclic ring system, a C\.\Q hydrocarbyl group which may optionally contain an oxygen atom, a group S(O)n wherein n is 0, 1 or 2, or a group NR^ wherein R- is hydrogen or a C\.β aliphatic group,each group R2 optionally being substituted by one to five groups independently selected from
(i) C\. alkyl or C3.6 cycloalkyl each optionally substituted by one to three halo atoms; (ii) a group OR^ wherein R^ is hydrogen, C \. alkyl, phenyl or benzyl; (iii) a halo atom; (iv) a group CO2R^ wherein R^ is hydrogen or C \ .4 alkyl; (v) a group NR6R wherein R^ and R? are independently selected from hydrogen, Cj.4 alkyl or a group
Figure imgf000007_0001
wherein Q and Rl are as hereinbefore defined;
(vi) nitro; or
(vii) cyano;
or Rl may be linked to the imino nitrogen to form a monocyclic heterocyclic ring; with the exception of N-(2,6-dimethylphenyl)-5,6-dihydro-4H-l,3-thiazin-2-amine.
A preferred group of compounds are those of formula (I) with the proviso that when Q is sulphur and R^ is hydrogen or C\_5 alkyl, R^ is not an ornithine or lysine derivative optionally substituted by a Cj.g alkyl group on either the α-, β- or γ-carbon atoms, or a tautomer thereof.
One embodiment of the present invention provides compounds of formula (I) as hereinbefore defined with the proviso that R s not linked to the imino nitrogen to form a monocyclic heterocyclic ring.
In one preferred embodiment, Q is oxygen.
In a second preferred embodiment, Q is sulphur.
When Q is either oxygen or sulphur:
suitably Rl is hydrogen, C1.4 alkyl, C2-.5 alkenyl or benzyl; preferably R^ is Cι_4 alkyl for example ethyl;
suitably, R^ is a 5- or 6- membered heterocyclic ring or a 9- or 10-membered bicyclic heterocyclic ring, a phenyl ring, or a C2-8 alkyl chain which optionally contains a group S(O)n as hereinbefore defined, or a C2.4 alkyl chain which contains a phenylene ring, each group R^ optionally being substituted by one to five groups independently selected from (i) a Cι_4 alkyl group optionally substituted by one to three fluoro atoms:
(ii) a cyclohexyl ring;
(iii) a group OR^a wherein R^a is hydrogen, methyl, ethyl, phenyl or benzyl; (iv) fluoro, chloro or bromo;
(v) a group CO2R^a wherein R^a is hydrogen, methyl or ethyl; (vi) a group NR^aR^a wherein R^a and R?a are independently selected from hydrogen, methyl or ethyl or one of R^a and R?a is a group
Figure imgf000008_0001
as hereinbefore defined and the other is hydrogen; (vii) nitro; or (viii) cyano;
or R1 may be linked to the imino nitrogen in the compound of formula (I) to form a thiazole or thiazoline ring.
Formula (I) includes compounds of formulae (I A) to (IF)
Figure imgf000008_0002
Figure imgf000009_0001
wherein Z is oxygen or sulphur; Rl is as hereinbefore defined; X is a C2-9 hydrocarbyl group which may optionally contain an oxygen atom, a group S(O)n as hereinbefore defined, or a group NR^ as hereinbefore defined; T is a C g hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring, or T is a C2-.4 hydrocarbyl group containing a phenylene ring; and Ar is a mono- or bicyclic aromatic ring system optionally substituted by one to five groups selected from
(i) C\. alkyl or C3.6 cycloalkyl each optionally substituted by one to three halo atoms; (ii) a group OR^ wherein R^ is hydrogen, C .β alkyl, phenyl or benzyl; (iii) a halo atom;
(iv) a group CO2R^ wherein R^ is hydrogen or C 1.4 alkyl; (v) a group NR^R7 wherein R^ and R~ are independently selected from hydrogen, C-[_4 alkyl or a group
Figure imgf000009_0002
wherein Q and R* are as hereinbefore defined; (vi) nitro; or (vii) cyano;
In the formulae (IA) to (IF);
Suitably, R* is a C\_ hydrocarbyl group, preferably a Cj_4 alkyl group, e.g. ethyl.
Suitably, X is a C2-6 hydrocarbyl group and preferably a C3.5 alkylene or alkenylene group. Suitably, T is Cj.g hydrocarbyl containing a 5- or 6-membered heterocyclic ring; or a C2.4 hydrocarbyl group containing a phenylene ring.
Suitably Ar is phenyl optionally substituted by one to three substituents which may be the same or different and are selected from C 4 alkyl or C3.6 cycloalkyl groups each optionally substituted by one to three halo atoms; C1.4 alkoxy groups; hydroxy groups, benzyloxy groups; halo atoms; CO2R^ groups wherein R^ is hydrogen or Cj_4 alkyl; groups NR^R7 wherein R^ and R7 are independently selected from hydrogen or Cj_4 alkyl. Most suitably Ar is phenyl substituted by one or two substituents, preferably one substituent.
Preferably Ar is phenyl substituted by C 1.3 alkoxy, hydroxy, benzyloxy, halo atoms or Cι_4 alkyl optionally substituted by one to three fluoro atoms, C1.3 alkoxy, hydroxy, benzyloxy, or halo atoms.
One preferred embodiment of the present invention includes compounds of formula (IA) wherein X is a C2.9 hydrocarbyl group which contains an oxygen atom, a group S(O)n or NR3 wherein n and R^ are as hereinbefore defined, and compounds of formulae (IB) to (IF) as hereinbefore defined.
Suitable compounds of the formula (I) include:
S-Ethyl-N-(4-phenoxyphenyl)isothiourea
S-ethyl-N-(3-methoxyphenyl)isothiourea
S-ethyl-N-[4-(benzyloxy)phenyl]isothiourea
S-ethyl-N-[4-(ethoxycarbonyl)phenyl]isothiourea
S-ethyl-N-(4-carboxyphenyl)isothiourea
S-ethyl-N-(3-carboxyphenyl)isothiourea
S-ethyl-N-(2-bromophenyl)isothiourea
S-ethyl-N-(4-dimethylaminophenyl)isothiourea
S-ethyl-N-(4-cyclohexylphenyl)isothiourea
S-ethyl-N-(4-hydroxyphenyl)isothiourea
S-ethyl-N-(4-methoxyphenyl)isothiourea
S-ethyl-N-(2-pyridyl)isothiourea
S-Ethyl-N-[4-trifluoromethyl)phenyl]isothiourea S-Benzyl-N-[4-(trifluoromethyl)phenyl]isothiourea
S-Ethyl-N-(3-chlorophenyl)isothiourea
S-Ethyl-N-(2-isopropylphenyl)isothiourea
S-Ethyl-N-(4-isopropylphenyl)isothiourea
S-Ethyl-N-[3-(trifluoromethyl)phenyl]isothiourea
S-Ethyl-N-[2-(trifluoromethyl)phenyl]isothiourea
S-Ethyl-N-[2-(chlorophenyl)isothiourea
S-Ethyl-N-(2-methoxyphenyl)isothiourea
S-Ethyl-N-(4-methylphenyl)isothiourea
S-Ethyl-N-(3-pyridyl)isothiourea
S-Ethyl-N-(4-chloro-3-(trifluoromethyl)phenyl) isothiourea
S-Ethyl-N-(2-chloro-5-(trifluoromethyl)phenyl) isothiourea
S-Ethyl-N-(3-pyridyl)isothiourea
S-Ethyl-N-(4-pyridyl)isothiourea
O-Methyl-N-(4-(trifluoromethyl)phenyl)isourea
O-Ethyl-N-(4-(trifluoromethyl)phenyl)isourea
S-Ethyl-N-[4-(trifluoromethoxy)phenyl]isothiourea
N5-(2-thiazolin-2-yl)-L-ornithine
N6-(2-thiazolin-2-yl)-L-lysine
N, N'-((methylthio)iminomethyl)-m-xylylenediamine
N, N-((methylthio)iminomethyl)-p-xylylenediamine
N5-(imino(methylthio)methyl-L-ornithine
N5-((ethylthio)iminomethyl)-L-ornithine
N6-(imino(methylthio)methyl)-L-lysine
N6-((ethylthio)iminomethyl)-L-lysine
N5 -(imino( 1 -methy lethy lthio)methyl)-L-ornithine
N6-(imino( 1 -methylethylthio)methyl)-L-lysine
N5-(imino(2-methylpropylthio)methyl)-L-ornithine
N6-(imino(2-methylpropylthio)methyl)-L-lysine
N5-((methylthio)iminomethyl)-D-ornithine
N6-((methylthio)iminomethyl)-D-lysine
N5-((ethylthio)iminomethyl)-D-ornithine
N6-((ethylthio)iminomethyl)-D-lysine
N5-(imino( 1 -methylethylthio)methyl)-D-ornithine
N6-(( 1 -methylethylthio)iminomethyl)-D-lysine N5-((2-methylpropylthio)iminomethyl)-D-orni thine
N6-((methylpropylthio)iminomethyl)-D-lysine
N5-(iminomethoxymethyl)-L-orni thine
N5-(ethoxyiminomethyl)-L-orni thine
N5-(iminoisopropoxymethyl)-L-ornithine
N6-iminomethoxymethyl)-L-lysine
N6-(ethoxyiminomethyl)-L-lysine
1 -(3 -(Aminomethyl)benzyl)-0-ethylisourea l-(3-(Aminomethyl)ber_-_yl)-S-methylisothiourea
1 -(3 -(Aminomethyl)benzyl)-S-ethylisothiourea l-(4-(Aminomethyl)benzyl)-S-methylisothiourea l-(4-(Aminomethyl)benzyl)-S-ethylisothiourea
S-ethyl-N-(4-diethylamino)phenyl) isothiourea
S-ethyl-N-(5-chloro-2-pyridyl)isothiourea
S-ethyl-N-(4-nitrophenyl) isothiourea
S-ethyl-N-(4-chlorophenyl) isothiourea
S-ethyl-N-(3, 4-dichlorophenyl) isothiourea
S-benzyl-N-phenyl isothiourea
S-ethyl-N-phenyl isothiourea
and salts, esters or amides thereof.
By the term "hydrocarbyl" group is meant a group that contains only carbon and hydrogen atoms and may contain double and/or triple bonds and which may be cyclic or aromatic in nature. An oxygen atom, or a group S(O)n or NR^ as hereinbefore defined, may optionally intersperse the carbon atoms in the hydrocarbyl chain.
By the term "aliphatic" is meant an alkyl, alkenyl, alkynyl or cycloalkyl group. The terms alkyl, alkenyl and alkynyl are intended to include both straight and branched chain variants.
By the term "heterocyclic ring" is meant a cyclic compound containing one to three heteroatoms selected from oxygen, sulphur and nitrogen, and preferably nitrogen and sulphur. The compounds of formula (I) may include a number of asymmetric centres in the molecule depending on the precise meaning of the various groups and the present invention is intended to include all possible isomers.
When R! is hydrogen, compounds of formula (I) may exist in tautomeric form and the present invention includes all such forms.
Certain compounds of formula (I) have also been found to have activity against the inducible NO synthase enzyme and may be of use in the treatment of systemic hypotension associated with septic and/or toxic shock induced by a wide variety of agents, therapy with cytokines such as TNF, IL-1 and IL-2 and therapy with cytokine- inducing agents such as 5,6-dimethylxanthenone acetic acid, as an adjuvant to short term immunosuppression in transplant therapy, and in the treatment of autoimmune and or inflammatory conditions affecting the joints, for example arthritis. Accordingly, the present invention further provides the use of a compound of formula (I) other than 5-methyl-2-(2-thiazolylamino)phenol and S-ethyl-N-phenylisothiourea for the manufacture of a medicament for the treatment of a condition requiring inhibition of the inducible NO synthase enzyme.
In a further aspect the present invention provides a N-substituted urea derivative of formula (I) other than S-ethyl-N-phenylisothiourea, S-ethyl-N-(2-chlorophenyl) isothiourea, S-ethyl-N-(2-trifluoromethylphenyl)isothiourea, 2-propenylthiourea, N- (2,6-dimethylphenyl)-5,6-dihydro-4H-l,3-thiazin-2-amine and 5-methyl-2-(2- thiazolyl amino)phenol, or a pharmaceutically acceptable salt, ester or amide thereof for use in medicine.
The present invention also provides a N-substituted urea derivative of formula (I) or a salt, ester or amide thereof, with the proviso that:
(a) when Q is sulphur and
(i) R! is methyl, R^ is not a phenyl ring substituted by 3-chloro, 2-ethyl, 2-chloro-5-trifluoromethyl, 3-trifiuoromethyl, 3-methyl, 3-bromo, 4- nitro, 4-chloro, 3,4-dichloro or CO2H; or R2 is not a group 5-chloro-2- pyridyl; (ii) R! is ethyl, R2 is not a phenyl ring or a phenyl ring substituted by 4- methoxy, 2-chloro, 4-hydroxy, 2-methoxy, 4-methyl. 2-trifluoromethyl or 3-trifluoromethyl; or
(b) the compound of formula (I) is not 2-propenylthiourea
N-(2,6-dimethylphenyl)-5,6-dihydro-4H- 1 ,3-thiazin-2-amine 5-methyl-2-(2-thiazolylamino)phenol.
The present invention includes N-substituted urea derivatives in the form of salts, esters or amides, 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-pharmaceutically acceptable salts may be of utility in the preparation and purification of the compound in question. Thus, preferred salts include those formed from hydrochloric, hydrobromic, sulphuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, oxalic, fumaric, maleic, oxaloacetic, methanesulphonic, ethanesulphonic, p-toluenesulphonic, benzenesulphonic and isethionic acids. Salts of N-substituted urea derivatives can be made by reacting the appropriate compound in the form of the free base with the appropriate acid. Esters are pharmaceutically acceptable esters, for example Cj_4 alkyl esters.
As used herein, reference to "treatment" of a patient is intended to include prophylaxis.
The activity of compounds of formula (I) as inhibitors of isolated NO synthase enzymes has been demonstrated against NO synthase enzymes isolated from the human placenta, the human brain and carcinoma cells.
Whilst it may be possible for the compounds of formula (I) to be administered as the raw chemical, it is preferable to present them as a pharmaceutical formulation. According to a further aspect, the present invention provides a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt, ester or amide thereof, other than S-ethyl-N-phenylisothiourea, S-ethyl-N-(2- chlorophenyl)isothiourea, S-ethyl-N-(2-trifluoro methylphenyl)isothiourea, 2- propenyl thiourea, N-(2,6-clrαethylphenyl)-5,6-dihydro-4H-l,3-thiazin-2-amine and 5-methyl-2-(2-thiazolylamino)phenol, together with one or more pharmaceutically acceptable carriers therefor and optionally one or more other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intravenous and intraarticular), 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 into association a compound of formula (I) or a pharmaceutically acceptable salt, ester or amide thereof ("active ingredient") 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 moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing 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 hereinbelow 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.
The compounds of the invention may be administered orally or via injection at a dose of from 0.1 to 500mg/kg per day. The dose range for adult humans is generally from 5mg to 35g/day, preferably 5mg to 2g/day and most preferably lOmg to lg/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. The compounds of formula (I) are preferably administered orally or by injection (intravenous or subcutaneous). The precise amount of compound administered to a patient will be the responsibility of the attendant physician. However the dose employed will depend on a number of factors, including the age and sex of the patient, the precise disorder being treated, and its severity. Also the route of administration may vary depending on the condition and its severity.
The invention further includes a process for the preparation of the novel compounds of formula (I), analagous to those known in the art for preparing N-substituted ureas.
Thus:
(a) Compounds of formula (I) wherein Q is S may be prepared by the reaction of a compound of formula (II)
H2N-R2 (II)
wherein R2 is as hereinbefore defined, with a compound having a thiocarbonyl group, followed if necessary by hydrolysis to give a compound of formula (I) wherein R* is hydrogen or a tautomer thereof, and thereafter the optional conversion to a compound of formula (I) wherein R is other than hydrogen by alkylation of the sulphur atom of the isothiourea.
The coupling reaction may be carried out between a compound of formula (II) and a compound having a thiocarbonyl group, for example thiophosgene followed by ammonia as described in Tet. Lett. 1991, 21 (7) 875-878 or a compound of formula (II) and an isothiocyanate, such as benzoyl isothiocyanate. Suitably the reaction is carried out in a polar solvent, such as dichloromethane, chloroform, ethanol or acetone at a non-extreme temperature of from -78°C to 200°C, for example -5°C to 100°C and preferably room temperature. The intermediate thiourea, e.g. benzoylthiourea, may be hydro lysed in a polar solvent such as 10% sodium hydroxide solution at a non- extreme temperature of from -20°C to 200°C, such as the refluxing solvent temperature. The alkylation is generally carried out using a compound of formula R -L, wherein Rl is as hereinbefore defined other than hydrogen and L is a suitable leaving group. Suitable leaving groups include a halo atom, for example iodo.
Compounds of formula (II) are commercially available or may be prepared by methods known in the art.
(b) Compounds of formula (I) wherein Q is O may be prepared by the acid catalysed addition of (i) an alcohol of formula R -OH to prepare a compound wherein R* is as hereinbefore defined other than hydrogen; or (ii) water to prepare a compound wherein R s hydrogen
to a compound of formula (III)
Figure imgf000018_0001
or a protected derivative thereof, wherein R2 is as hereinbefore defined, followed by deprotection where necessary.
The acid catalysed addition is suitably carried out using the alcohol (Rl-OH or water) as the solvent, at a non-extreme temperature of 0°C to 100°C, and preferably room temperature, in the presence of an acid, e.g. hydrochloric acid, conveniently in solution in ether.
Compounds of formula (III) may be prepared by the reaction of a compound of formula (II) as hereinbefore defined with a compound of formula L'-CN wherein L' is a leaving group, for example a halo atom such as bromo. The reaction may be carried out in ether as a solvent at a non-extreme temperature of from -20°C to 100°C, suitably 0°C. The present invention will now be illustrated by way of example only.
Proton (*H) NMR analysis was consistent with the proposed structure in all cases.
Example 1
Preparation of S-Ethyl-N-f4-phenoxyphenyDisothiourea
To a stirred solution of l-(4-phenoxyphenyl)-2 -thiourea (2.00g, 8.19 mmol) in acetone (20ml) was added iodoethane (7.6 lg, 48.7 mmol). The mixture was heated to reflux for three hours, cooled to room temperature and concentrated at reduced pressure. The resulting viscous oil was crystallized from acetone-pentane to afford S-ethyl-N- (4-phenoxyphenyl)isothiourea hydroiodide as a beige solid. Mp = 140-143°C.
The following compounds were made by an analagous method:
Ex. No. Compound MP°C
1A S-ethyl-N-(3-methoxyphenyl)isothiourea 75-77a
IB S-ethyl-N-[4-(benzyloxy)phenyl]isothiourea 172-174
1C S-ethyl-N-[4-(ethoxycarbonyl)phenyl]isothiourea 80-85a
ID S-ethyl-N-(4-carboxyphenyl)isothiourea 214-215a
IE S-ethyl-N-(3-carboxyphenyl)isothiourea 149-215c
IF S-ethyl-N-(2-bromophenyl)isothiourea 89-100d
IG S-ethyl-N-(4-dimethylaminophenyl)isothiourea 138-141e
1H S-ethyl-N-(4-cyclohexylphenyl)isothiourea 174-175a
II S-ethyl-N-(4-hydroxyphenyl)isothiourea 135e
1J S-ethyl-N-(4-methoxyphenyl)isothiourea 127-128*
IK S-ethyl-N-(2-pyridyl)isothiourea 161-1638
a = Recrystallized from acetone-pentane b = Triturate with pentane c = Triturated with pentane, followed by hot ethyl acetate d = Triturated with hot ethyl acetate e = Recrystallized from acetone-pentane, and then from ethanol-ethyl acetate f = Recrystallized from ethyl acetate-hexane g= Recrystallized from methanol-ether
Example 2
Preparation of S-Ethyl-N-[4-trifluoromethyl .phenyljisothiourea
To a stirred suspension of l-[4-trifluoromethyl)phenyl]-2-thiourea (3.00 g, 13.6 mmol) in acetone (50 mL) was added iodoethane (6.63 g, 42.5 mmol). The mixture was heated to reflux and stirred overnight. After cooling to room temperature, the mixture was concentrated at reduced pressure. The resulting red viscous oil was poured into saturated NaHCO, and extracted with ether. The organic layer was dried over magnesium sulphate and filtered. The filtrate was acidified with IN hydrochloric acid in ether, diluted with pentane and stirred for 20 minutes. The yellow solid was collected and recrystallized from ethanol-ether to afford S-ethyl-N- [4-(trifluoromethyl)phenyl]isothiourea hydrochloride (2.71 g, 70%) as a white solid. Mp=127°C. Anal. Calcd for C^Hj ^SF^HCl: C, 42.18; H,4.25; N, 9.84; S, 11.26; Cl, 12.45. Found: C, 42.22; H, 4.20; N, 9.85; S, 11.20; Cl, 12.43.
The following compounds were made by an analagous method:
Ex.No. Compound MP°C
2A S-Benzyl-N-[4-(trifluoromethyl)phenyl]isothiourea 71-72a
2B S-Ethyl-N-(3-chlorophenyl)isothiourea 95-96b
2C S-Ethyl-N-(2-isopropylphenyl)isothiourea 66-68a
2D S-Ethyl-N-(4-isopropylphenyl)isothiourea 113- 116a
2E S-Ethyl-N-[3-(trifluoromethyl)phenyl]isothiourea 102a
2F S-Ethyl-N-[2-(trifluoromethyl)phenyl]isothiourea 75-77a
2G S-Ethyl-N-[2-(chlorophenyl)isothiourea 87-90a
2H S-Ethyl-N-(2-methoxyphenyl)isothiourea 75-78b
21 S-Ethyl-N-(4-methylphenyl)isothiourea 138-141 a
2J S-Ethyl-N-(3-pyridyl)isothiourea 180-182c
2K S-Ethyl-N-(4-chloro-3-(trifluoromethyl)phenyl) 143a isothiourea
2L S-Ethyl-N-(2-chloro-5-(trifluoromethyl)phenyl) 155-156d isothiourea 2M S-Ethyl-N-(4-pyridyl)isothiourea 151-152e
a = Triturated with pentane b = Recrystallised from acetone-pentane c = Recrystallised from ethanol-ether d = Recrystallisation not required e = Free base purified by silica gel chromatography
Example 3
Preparation of Q-Methyl-N-f4-ftrifluoromethyDphenyl sourea
To a stirred, cooled (0°C) solution of 4-aminobenzotrifluoride (1.61g, lO.Ommol) in methanol (10ml) was added a solution of cyanogen bromide (1,17g, l l.Ommol) in methanol (10ml) dropwise. The mixture was allowed to warm to room temperature and stirred for 5 days. The solvent was removed at reduced pressure and the residue was partitioned between diethyl ether and water. The organic layer was dried over sodium sulphate and filtered. Solvent was removed at reduced pressure. The residue was chromatographed on silica gel eluting with 20% ethyl acetate in hexane to give 660mg of a solid (mp = 117-126°C).
To a stirred, cooled (0°C) solution of the above solid (172mg, 0.93mmol) in methanol (10ml) was added IN hydrochloric acid in diethyl ether (0.93ml, 0.93mmol). After 20 minutes the mixture was warmed to room temperature, stirred for 10 hours, and concentrated at reduced pressure. The residue was triturated with diethyl ether and filtered to afford 149mg (63%) of O-Methyl-N-(4-(trifluoromethyl)phenyl)isourea as a light beige solid. Mp = 121-123 °C.
The following compound was prepared by an analagous method:
3 A O-Ethyl-N-(4-(trifluoromethyl)phenyl)isourea 117-119°C
Example 4
Preparation of S-Ethyl-N-[4-ftrifluoromethoxy)phenyl] isothiourea To a stirred solution of 4-(trifluoromethoxy)aniline (5.24g, 29.6 mmol) in acetone (100ml) was added benzoyl isothiocyanate (5.46g, 33.5 mmol). After strirring overnight at room temperature, the mixture was concentrated at reduced pressure giving a yellow solid. Recrystallization from ethyl acetate-hexane afforded 9.29g (92%) of a pale yellow solid. Mp=124-125°C.
To a stirred solution of the above solid (7.80g, 22.9 mmol) in tetrahydrofuran (100ml) was added 2.0N aqueous sodium hydroxide (25ml, 50.0 mmol). The mixture was heated to reflux for 3h, cooled to room temperature and then concentrated at reduced pressure. The residue was suspended in water and extracted repeatedly with dichloromethane. The combined organic layers were washed with brine and dried over magnesium sulphate. Solvent was removed at reduced pressure giving a solid. Recrystallization from ethyl acetate-hexane afforded 3.77 g (70%) of a white solid. Mp=136-137°C.
To a stirred solution of the above white solid (3.00g, 12.7 mmol) in acetone (100ml) was added iodoethane (5.85g, 37.5 mmol). The mixture was heated to reflux and stirred overnight. After cooling to ambient temperature, the mixture was concentrated at reduced pressure giving an oil. The oil was dissolved in water and washed with pentane. The aqueous layer was poured into saturated aqueous sodium hydrogen carbonate (75ml) and extracted with ether. The organic layer was dried over magnesium sulphate, concentrated at reduced pressure to a volume of 200mL and treated with IN hydrochloric acid (15ml, 15.0 mmol) in ether. This mixture was stirred for 20 minutes, concentrated at reduced pressure and placed in vacuo overnight to give a gummy foam. Trituration of the foam with pentane gave a solid which was collected and dried in vacuo to afford S-ethyl-N-[4-(trifluoromethoxy) phenyljisothiourea hydrochloride (3.35g, 88%) as a white solid. Mp= 96-97°C.
Example 5
Preparation ofN-S-^-thiazolin^-yiyL-ornithine
To a solution of N5-carbonylbenzyloxy-N2-tert-butyloxycarbonyl-L-ornithine tert- butyl ester (2.2g, 5.2 mmol) (Feldman, Tet. Lett. 1991, 21 (7), 875-878) in ethyl acetate (50ml) was added 10% palladium on carbon (l.Og). The suspension was shaken at 22°C under 50 psi H2 for 30 minutes in a 500ml Parr bottle. The catalyst was removed by filtration through celite. The resulting solution was concentrated to yield the amine intermediate (1.5g) as a crude oil. To a solution of the amine intermediate in tetrahydrofuran (20ml) and triethylamine (1ml) was added chloroethylisothiocyanate (650mg, 5.35 mmol) as a solution in tetrahydrofuran (10ml) at 20°C. The mixture was stirred for 16 hours, filtered, concentrated, and purified by silica gel chromatography eluting with ethyl acetate to yield the thiazoline intermediate (1.2 g, 62%) as a white foamy solid. The thiazoline foam was taken up into a mixture of trifluoroacetic acid (9.5ml), water (0.5ml), thioanisole (0.5ml), phenol (0.75g) and 1 ,2-ethanediol (0.25ml) at 0°C. The solution was stirred for two hours at 20°C and concentrated to a volume of 3ml. The solution was rapidly stired and diethyl ether (50ml) was added. After decanting and washing with ether, the residue was purified by silica gel chromatography (ammonium hydroxide: methanol, gradient, 0:100 to 3:97) to yield N5-(2-thiazolin-2-yl)-L-omithine (480mg, 69%) as a white solid, 1H NMR (300MHZ, D2O) δ 3.36 (t, J=7.4 Hz, 2H), 3.54 (m,lH), 3.38 (t,J= 7.4 Hz, 2H), 3.27 (t, J=6.7Hz, 2H), 1.8-1.55 (m,4H).
The following compound was made by an analagous method:
5A N6-(2-thiazolin-2-yl)-L-lysine
1H NMR (200 MHz, D2O) δ 3.85(t, J=7.3 Hz, 2H) 3.53 (m, 1H), 3,37 (t, J=7.4 Hz, 2H), 3.24 (t,J=6.8Hz, 2H), 1.8-1.65(m,2H), 1.65-1.5 (m, 2H), 1.45-1.3 (m,2H)
Example 6
Preparation of N. N frnethylthio minomethylVm-xylylenediamine
To a stirred solution of p-xylylene diamine (3.3ml, 25mmol) in dichloromethane (100ml) at 0°C was added benzoylisothiocyanate (7.0ml, 52.0mmol). The reaction mixture was stirred for 16 hours with gradual warming to room temperature. The solvent was removed under reduced pressure and the yellow-coloured solid slurried in hot ethanol to yield a pale yellow solid. This solid was suspended in lOOmL of 10% sodium hydroxide solution and heated to reflux for exactly five minutes. The solution was acidfied with concentrated hydrochloric acid, then made basic with concentrated ammonium hydroxide. As the solution cools, an off-white, granular solid forms that is washed with hot 95% ethanol and dried to give a bis-thiourea intermediate (4.82g). The bis-thiourea intermediate (2.54g, lOmmol) was stirred at 22°C in dimethyl formamide (25 ml) with iodomethane (5.0ml, 80.0mmol) for 72 hours. The solution was concentrated to a thick oil and purified by preparative C18 reverse phase chromatography. Elution with 95:5:0.1 water: methanol: trifluoroacetic acid gave an oil. The oil was taken up into hot absolute ethanol and ethyl acetate. Upon cooling, N,N'-((methylthio)iminomethyl)-m-xylyenediamine (4.28g) was isolated as a white solid (m.p. = 164-167 °C).
Example 7
Preparation of N. N-.fmethylthio minomethyD-p-xylylenediamine
The intermediate bis-thiourea (5.92g) was prepared from p-xylylenediamine (3.4G, 25mmol) as described in Example 5. To a solution of the bis-thiourea intermediate (1.27g, 5mmol) in dimethylformamide (25ml) was added iodomethane (2.5ml, 40mmol). The solution was stirred at 22°C for 72 hours, and the solvent was removed under reduced pressure to yield a crude, amber-coloured oil. The crude product was taken up into hot ethanol (40 mL) and treated with 160mL of ethyl acetate with scratching to induce crystallization. The solution was cooled overnight, filtered, and dried in vacuo at 60°C for 48 hours to yield N,N-((methylthio)iminomethyl)-p- xylylenediamine (2.29g, 87%) as a light yellow solid (m.p.=209-212°C).
Example 8
Preparation of N^-fiminofmethylthio^methyl-L-omithine
N -Tertbutoxycarbonyl-L-thiocitrulline tert-butyl ester (Tett. Lett. (1991) 21(7), 875- 878) (4g, 12.0 mmol) was treated with 7.5 mL (120 mmol) iodomethane in 30 mL anhydrous acetonitrile at 20°C with stirring for 4 hours. The solution was concentrated to a yellow foam and stirred with a 0°C mixture of trifluoroacetic acid (30mL), phenol (2.25g), water (l.OmL), thioanisole (1.0 mL), and 1 ,2-ethanedithiol (0.5 mL) for 1.5 hours. The mixture was concentrated under reduced pressure to a volume of 5 mL and 80mL ether was added with rapid stirring. The resulting gummy residue was washed with ether several times and then dissolved into a minimum amount of methanol (lOmL). A 50/50 solution of ammonium hydroxide and methanol was added until initial spotting on litmus paper indicated a pH of 8. Upon standing at 20°C for 16 hours, a white precipitate was collected by filtration and dried under reduced pressure at 100°C to yield N5-(imino(methylthio)methyl-L-ornithine (2.15g, 56%) as a white solid as the mono-trifluoroacetic acid salt (m.p.=207-208, dec).
Example 9
Preparation ofN-^(Yethylthio minomethylVL-ornithine
N2-tert-butoxycarbonyl-L-thiocitrulline tert-butyl ester (1.3g, 3.89 mmol) in 20mL anhydrous acetonitrile was stirred with 1.2 lg (7.75 mmol) iodoethane at 20°C for 60 hours. The solution was concentrated to a tan-coloured foam (1.75g). This crude solid was treated at 0°C with stirring for 2 hours with a solution of 14 mL trifluoroacetic acid, 2.25g phenol, 1.5mL thioanisole, 1.5mL water, and 0.75 mL 1,2-ethanedithiol. The mixture was partially concentrated and lOOmL diethylether was added. The resulting residue was washed with ether several times and purified by silica gel chromatography eluting with methanol followed by 1% ammonium hydroxide in methanol solution. The pooled product fractions were concentrated and passed through a reverse phase C18 column eluting with methanol/water mixtures containing 0.1% heptafluorobutyric acid. After freeze-drying, the material was passed through a second C18 column eluting with methanol/water mixtures containing 0.1% trifluoroacetic acid. Concentration of the pooled product fraction gave 336mg of the bis-TFA salt (19% overall yield) as a hygroscopic glass. !H NMR (300 MHz, D2O) δ 3.92 (t, J=6.1Hz,lH), 3.41 (t, J=6.6Hz, 2H), 3.09 (q, J=7.3Hz, 2H), 1.95 (m, 2H), 1.75 (m, 2H), 1.32 (t, J=7.3Hz, 3H).
The following compounds were prepared by an analogous method:
9A N^-(imino(methylthio)methyl)-L-lysine prepared from N2-tert-butoxy- carbonyl-L-homothiocitrulline tert-butyl ester
Mass spectrum (Cl) 220 (MH+, 100%) 9B N^-((ethylthio)iminomethyl)-L-lysine
Mass spectrum (Cl) 234 (MH+ 70%) 9C N^-(imino(l-methylethylthio)methyl)-L-omithine
Mass spectrum (FAB) 234.2 (MH+) 9D N"-(imino( 1 -methylethylthio)methyl)-L-ly sine
Mass spectrum (FAB) 248.2 (MH+) 9E N:)-(imino(2-methylpropylthio)methyl)-L-omithine
Mass spectrum (FAB) 248.2 (MH+) 9F N°-(imino(2-methylpropylthio)methyl)-L-lysine
Mass spectrum (FAB) 262.3 (MH+) 9G N^ -((methylthio)iminomethyl)-D-ornithine
Mass spectrum (Cl) 205.9 (MH+, 79%) 9H N^-((methylthio)iminomethyl)-D-lysine
Mass spectrum (Cl) 220.0 (MH+, 97%) 91 N^ -((ethy lthio)iminomethyl)-D-ornithine
Mass spectrum (FAB) 220.2 (MH+, 100%) 9J N°-((ethylthio)iminomethyl)-D-lysine
Mass spectrum (FAB) 234.0 (MH+ 100%) 9K N^-(imino( 1 -methylethylthio)methyl)-D-ornithine
TLC: 2% NH4OH/MeOH on silica gel, Rf = 0.34 9L N^-((l -methylethy lthio)iminomethy l)-D-ly sine
TLC: 2% NH OH/MeOH on silica gel, Rf = 0.34 9M N5-((2-methylpropylthio)iminomethyl)-D-ornithine
Mass spectrum (FAB) 248.1 (MH+, 100%) 9N N"-((me ylpropylmio)iminomethyl)-D-lysine
TLC: 2% NH4OH/MeOH on silica gel, Rf = 0.38
Example 10
Preparation of N^fiminomethoxymethylVL-ornithine dihydrochloride
a. N^-ftert-butoxycarbonylVN-^cyano-L-ornithine tert-butyl ester
To a solution of 6.1 g (14.44 mmol) N^-((Benzyloxy)carbonyl)-N2-(tert- butoxycarbonyl)-L-ornithine tert-butyl ester in 100 mL ethyl acetate was added 1.0 g 10% palladium on carbon. The suspension was shaken at 22 °C under 50 psi H2 for 45 minutes in a 500 mL Parr bottle. The catalyst was removed by filtration through celite. The resulting solution was concentrated to yield a crude oil. The oil was dissolved into 50 mL of diethyl ether and added dropwise to a 0°C stirred solution of 1.52 g (14.3 mmol) of cyanogen bromide in 30 mL ether. The solution was stirred for 2 h, poured into aqueous sodium bicarbonate, and extracted with ether. The ether solution was dried (magnesium sulfate), concentrated, and purified by silica gel chromatography (100 g, 230-400 mesh silica gel). Elution with a gradient of ethyl acetate in hexanes (30% - 60%) gave 3.0 g (67% yield) N2-(tert-butoxycarbonyl)-N5- cyano-L-ornithine tert-butyl ester as an oil.
N^-ftert-butoxycarbonylVN-^fiminomethoxymethylVL-ornithine tert-butyl ester hydrochloride
To a solution of 1.05 g (3.35 mmol) N2-(tert-butoxycarbonyl)-N5-cyano-L- ornithine tert-butyl ester in 25 mL methanol at 0°C was added 3.35 mL of 1.0 M hydrochloric acid in ether solution. The solution was stirred overnight (14 h), concentrated, and purified by silica gel chromatography. Elution with a gradient of methanol in dichloromethane (0 % - 15 %) gave 0.95 g (74 % yield) N2-(tert-butoxycarbonyl)-N^-(iminomethoxymethyl)-L-ornithine tert- butyl ester hydrochloride .
By the method described above for the preparation of N -(tert- butoxycarbonyl)-N5-(iminomethoxymethyl)-L-ornithine tert-butyl ester hydrochloride , 1.05 g (3.35 mmol) of N2-(tert-butoxycarbonyl)-N5-cyano-L- ornithine tert-butyl ester produced 0.95 g (75 %) N2-(tert-butoxycarbonyl)- N5-(ethoxyin-inomemyl)-L-ornithine tert-butyl ester hydrochloride (TLC, silica gel, methanol: dichloromethane/ 1 :4, Rf = 0.61) and 1.195 g (3.2 mmol) of N -(tert-butoxycarbonyl)-N^-cyano-L-oπ_ithine tert-butyl ester produced 0.63g (63%) N -(tert-butoxycarbonyl)-N5-(iminoisopropoxymethyl)-L- ornithine tert-butyl ester hydrochloride (TLC, silica gel, methanol: dichloro methane /1 :4, R_M).53). Mass Spectrum (Cl) 360 (MH+, 100%).
i) Preparation of N-S-fiminomethoxymethyD-L-ornithine dihvdrochloride. A solution of 0.75 g (1.96 mmol) N2-(tert-butoxycarbonyl)-N5- (iminomethoxymethyl)-L-ornithine tert-butyl ester hydrochloride in 2 mL dioxane at 0°C was treated with 15 mL of 4N hydrochloric acid in dioxane solution. The solution was stirred overnight at 22 °C, concentrated to a crude paste, and freeze-dried from 8 mL of water. The product was freeze- dried a second time to yield 0.53 g N^-(iminomethoxymethyl)-L-ornithine dihydrochloride. The product analyzed solvated with an additional 0.2 molar hydrochloric acid. 0.1 molar water, and 0.3 molar dioxane.
ii) Preparation of N^-fethoxyiminomethyD-L-ornithine dihydrochloride. By the method described above for the preparation of N^- (iminomethoxymethyl)-L-ornithine dihydrochloride, 0.78 g (1.97 mmol) N2-(tert-butoxycarbonyl)-N^-(ethoxyiminomethyl)-L-ornithine tert-butyl ester hydrochloride was deprotected to yield 0.51 g (94 %) N^- (ethoxyiminomethyl)-L-omithine dihydrochloride monohydrate. TLC (silica gel, ammonium hydroxide:methanol /1 :25) Rf = 0.24. Mass spectrum (Cl) 204 (MH÷ 74%).
iii) Preparation of N^- iminoisopropoxymethylVL-ornithine dihydrochloride.
To a 0°C stirred solution of 0.54 g (1.32 mmol) N2-(tert-butoxycarbonyl)- N5-(iπύnoisopropoxymethyl)-L-omithine tert-butyl ester hydrochloride in 2ml dioxane was added 10 mL of 4N hydrochloric acid in dioxane solution. The solution was stirred 14 h leaving a pale yellow precipitate. The dioxane was removed under reduced pressure and the residue suspended in ether. After stirring for 3 h, the ether was decanted and the solids dried under vacuum to give 0.52 g N5-(iminoisopropoxymethyl)-L- ornithine dihydrochloride. TLC (silica gel, ammonium hydroxide:methanol/l:25) Rf = 0.27.
Example 11
Preparation of N^-iminomethoxymethylVL-lysine dihydrochloride
a. N^-ftert-butoxycarbonyl .-N&-fcyano)-L-lysine tert-butyl ester
N6-((ber_zyloxy)carbonyl)-N2-(tert-butoxycarbonyl)-L-lysine tert-butyl ester was hydrogenated at 20°C under 50 psi hydrogen in 100 mL ethyl acetate for 1 h. The catalyst was removed by filtration through celite and the amine intermediate isolated without further purification as an oil (3.05g). The amine intermediate was taken into 40mL ether and the solution added over 10 min to a solution of l.lg (10.1 mmol) of cyanogen bromide in 50 mL ether at 0°C. The solution was stirred for 2h, poured into aqueous sodium bicarbonate, dried over magnesium sulfate, and concentrated to give an oil. The crude product was purified by silica gel chromatography eluting with ethyl acetate in hexanes (30% - 50%). Concentration gave 2.7g (80%) of N2-(tert- butoxycarbonyl)-N"-(cyano)-L-lysine tert-butyl ester as a colorless, viscous oil. TLC (silica gel, ethyl acetate:hexanes/l:l)Rf = 0.5. IR (neat film) 2222 cm"1 (CN). Mass Spectrum (Cl 328 (MH+, 44%).
b. N --rtert-butoxycarbonyiyN6-.iminomethoxymethylVL-lysine tert-butyl ester hydrochloride
From 0.86 g (2.63 mmol) N2-(tert-butoxycarbonyl)-N^-(cyano)-L-lysine tert- butyl ester was prepared 0.95 g (91%) N2-(tert-butoxycarbonyl)-N6- (iminomethoxymethyl)-L-lysine tert-butyl ester hydrochloride as a foamy- solid by the method described for the preparation of N -(tert-butoxycarbonyl)- N:>-(iminomethoxymethyl)-L-ornithine tert-butyl ester hydrochloride. TLC (methanol: dichloromethane/ 1 :9) Rf = 0.36. Mass spectrum (Cl) 360 (MH+, 60%).
From 1.6 g (4.89 mmol) N2-(tert-butoxycarbonyl)-N^-(cyano)-L-lysine tert- butyl ester was prepared 1.5 g (75%) of foamy solid N -(tert-butoxycarbonyl)- N"-(ethoxyiminomethyl)-L-lysine tert-butyl ester hydrochloride by the method described for N2-(tert-butoxycarbonyl)-N^-(iminomethoxymethyl)-L- ornithine tert-butyl ester hydrochloride. TLC (methanol:dichloromethane/l:9) Rf = 0.36. Mass spectrum (Cl) 360 (MH+, 60%).
c(i) Ng-fiminomethoxymethylVL-lysine dihydrochloride
From 0.70 g (1.77 mmol) N2-ftert-butoxycarbonyl)-N6- (iminomethoxymethyl)-L-lysine tert-butyl ester hydrochloride was prepared 0.38g (78%) N^-(iminomethoxymethyl)-L-lysine dihydrochloride by the method described above for the preparation of N^-(iminomethoxymethyl)-L- ornithine dihydrochloride TLC (silica gel, ammonium hydroxide :methanol/ 1:25) Rf = 0.24.
c.(ii) Ng-fethoxyiminomethylVL-lysine dihydrochloride From 1.3g (3.17 mmol) N2-(tert-butoxycarbonyl)-N6-(ethoxyiminomethyl)-L- lysine tert-butyl ester hydrochloride was prepared 0.82 g (89%) N^- (ethoxyiminomethyl)-L-lysine dihydrochloride by the method described above for the preparation of N:'-(iminomethoxymethyl)-L-ornithine dihydrochloride. TLC (4% ammonium hydroxide: methanol) Rf = 0.24. Mass spectrum (Cl) 218 (MH+, 92%).
Example 12
Preparation of l-f3-fAminomethyl)benzylVO-ethylisourea
a. Tert-butyl N-(3 - . aminomethvDbenzyPcarbamate lOg (73.42mmol) of m-xylenediamine was added to 5.1ml (36.71mmol) of triethylamine (Kodak) and 200 ml of anhydrous methanol. To this solution at 0°C was added a solution of 8.0g (36.71mmol) of di-t-butyldicarbonate in 60ml of tetrahydrofuran dropwise over 60 minutes. The solution was stirred and additional two hours at 0°C, filtered, and concentrated to dryness. The crude material was purified by silica gel chromatography eluting with methanol/methylene chloride/ammonium hydroxide (5/95/0.5 to 15/85/0.5) to yield 5.28g (30%) of a thick, viscous yellow oil.
b. l-fS-fAminomethyDbenzyl .-O-ethylisourea
1.2g (5.08mmol) of tert-butyl N-(3-(aminomethyl)benzyl)carbamate in 20ml ether was cooled to 0°C and 538mg (5.08mmol) of cyanogen bromide was added. The mixture was stirred for two hours, poured into saturated sodium bicarbonate solution, and extracted with ether (2x100ml) and ethyl acetate (100ml). The organic solutions were dried (sodium sulphate), concentrated, and the resulting crude product was purified by silica gel chromatography. Elution with methylene chloride followed by 5% to 10% methanol in methylene chloride and concentration of the product fractions gave cyanamide intermediate as a white solid (91.08g, 82% yield). To a solution of l.Og (3.83mmol) of the cyanamide intermediate in 10ml of ethanol at 0°C was added 3.83ml (3.83mmol) of 1.0M hydrochloric acid solution in anhydrous ether. The solution was stirred for 16 hours, concentrated, and the resulting crude oil was purified by silica gel chromatography eluting with methylene chloride followed by 5% to 20% methanol in methylene chloride to yield 1.22g (92%) of ethyl acetimidate intermediate as a white coloured foam. To l.Og (2.9mmol) of this foam in 5ml dioxane chilled to 10°C was added 10ml (40mmol) of 4N hydrochloric acid in dioxane solution. After stirring for 6 hours, most of the solvent was removed under reduced pressure and the residue remaining was treated with 20ml of ether with rapid stirring. The resulting white solids were collected by filtration to yield 0.73g (89%) of l-(3- (Aminomethyl)benzyl)-O-ethylisourea. Mass spectrum (Cl) 208.0 (MH+, 68%).
Example 13
Preparation of l-(3-(Aminomethyl .benzylVS-methylisothiourea
a. Tert-butyl N- -ffthioureido^methyDbenzyl .carbamate
A solution of 0.96g (4.06mmol) of tert-butyl N-(3-(aminomethyl)benzyl) carbamate in 20ml chloroform was added to a 0°C stirred mixture of 0.975g (9.75mmol) calcium carbonate, 0.37ml (4,87mmol) thiophosgene, 10ml water, and 10ml chloroform. After three hours the mixture was filtered and the aqueous phase was washed with chloroform. The organics were dried (sodium sulphate), concentrated to an oil, and dissolved into 50ml of anhydrous methanol cooled to 0°C. Ammonia was bubbled for 5 minutes and the solution was stirred for two hours. .After concentration to an oil, the crude product was dissolved into 50ml ethyl acetate, washed with 50ml water, dried (sodium sulphate), concentrated, and purified by silica gel chromatography eluting with 50% to 80% ethyl acetate in hexanes to provide 0.96g (80%) of Tert-butyl N- (3-((thioureido)methyl)benzyl)carbamate as a white foam.
b. l-G-fAminomethyDbenzylVS-methylisothiourea
A solution of 0.34g (1.15mmol) tert-butyl N-(3-((thioureido)methyl)benzyl) carbamate in 25ml acetonitrile was treated with 0.72ml (11.51mmol) iodomethane. The solution was stirred for 22 hours and concentrated to yield 0.49g (98%) of isothiourea as a foamy solid. This isothiourea intermediate (0.49g) in 30ml dioxane was treated with 1.4ml (5.6mmol) of 4N hydrochloric acid in dioxane solution. The reaction was stirred for five hours at 20°C and concentrated. The residue was freeze-dried from water (25ml) and then purified by preparative reverse phase HPLC eluting with methanol/water/trifluoroacetic acid (5/95/0.1) to yield 260mg of clear colourless l-(3-(Aminomethyl)benzyl)-S-methylisothiourea (46% yield, trifluoroacetic acid salt). Mass Spectrum (Cl) 210.0 (MH+, 71.4%)
The following compound was made by an analogous method:
13 A 1 -(3-(Aminomethyl)benzyl)-S-ethylisothiourea
Mass Spectrum (Cl) 224.0 (MH+, 74.6%)
Example 14
Preparation of 1 -f4-f AminomethyDben_y IVS-methylisothiourea
a. Tert-butyl N-f~4-faminornethyl .benzyDcarbamate
To a 0°C stirred solution of 5.0g (36.71mmol) p-xylenediamine in 50ml tetrahydrofuran and 10ml triethylamine was added 8.01g (36.71mmol) di-t- butyldicarbonate. The solution was stirred for six hours, concentrated, and extracted from water with ethyl acetate. The organics were dried (sodium sulphate) and purified by silica gel chromatography eluting with methanol in methylene chloride (0-60%). Concentration of the product fractions gave 0.96g (11% yield) of tert-butyl N-(4-(aminomethyl)bei_zyl)carbamate as a yellow solid.
b. l-. -fAminomethyDbenzylVS-methylisothiourea
From 2.3 lg (9.77mmol) of tert-butyl N-(4-(aminomethyl)benzyl)carbamate was prepared 0.43g of l-(4-(Aminomethyl)be__zyl)-S-methylisothiourea by the method described in example 13. Mass Spectrum (Cl) 209.9 (MH+, 41%).
The following compound was prepared by an analagous method:
14 A 1 -(4-( Aminomethyl)benzyl)-S-ethylisothiourea
Mass Spectrum (Cl) 224.0 (MH+, 60%)
Example 15 Biological Activity
The activity of representative compounds of the present invention was determined in accordance with the assay herein described.
Purification of NOS from human placenta.
Amion and chorion were removed from fresh placenta, which was then rinsed with 0.9% NaCl. The tissue was homogenized in a Waring blender in 3 volumes of HEDS buffer (20mM Hepes pH 7.8, 0.1 mM EDTA, 5 mM DTT, 0.2 M sucrose) plus 0.1 mM PMSF. The homogenate was filtered through cheesecloth and then centrifuged at lOOOg for 20 min. The supernatant was recentrifuged at 27500g for 30 min. Solid ammonium sulfate was added to the supernatant to give 32% saturation. Precipitated protein was pelleted at 25,000g and then redissolved in a minimal volume of HEDS buffer plus 0.1 mM PMSF, 1 Oμg/ml leupeptin and soybean trypsin inhibitor, and 1 μ g/ml pepstatin. The redissolved pellet was centrigued at 15000g for 10 min. To the supernatant was added 1/20 volume of 2',5' ADP agarose resin (Sigma), and the slurry was mixed slowly overnight. In morning, slurry was packed into a column. The resin was sequentially washed with HEDS, 0.5 M NaCl in HEDS, HEDS, and then NOS was eluted with lOmM NADPH in HEDS. The enzyme could be concentrated by ultrafiltration and quick frozen and stored at -70°C without loss in activity for at least 6 months.
Assay for human placental NOS
NOS was assayed for the formation of citrulline following the procedure of Schmidt et al (PNAS 88 365-369 , 1991) with these modifications: 20 mM Hepes, pH 7.4, lOμ g/ml calmodulin, 2.5 mM CaCl2 2.5 mM DTT, 125 μM NADPH lOμM H4 Biopterin, 0.5 mg/ml BSA, and 1 μM L-[14C] arginine (New England Nuclear). Linearity of NOS-catalyzed rate was confirmed prior to kinetic studies that used single time point determination of rate.
Purification of NOS cytokine-induced human colorectal adenocarcinoma DLD-1 cells. DLD-1 (ATCC No. CCL 221) were grown at 37°C, 5% C02 in RPMI 1640 medium supplemented with L-glutamin, penicillin, streptomycin, and 10% heat-inactivated fetal bovine serum. Cells were grown to confluency and then the following cocktail of cytokines were added: 100 units/ml interferon-gamma, 200 units/ml interleukin-6, 10 ng/ml tumor necrosis factor, and 0.5 ng/ml interleukin-lβ. At 10-24 hr post- induction, cells were harvested by scraping and washed with phosphate-buffered saline. Pelleted cells were stored at -70°C. Purification of the induced NOS was performed at 4°C. Crude extract was prepared by three cycles of freeze/thawing cells in TDGB (20 mM tris pH 7.5, 10% glycerol, ImM DTT, 2 μM tetrahydrobiopterin). Extract was applied directly onto a column of 2',5' ADP sepharse (Pharmacia). Resin was sequentially washed with TDGB, 0.5 M NaCl in TDGB, TDGB. NOS was eluted with 2 mM NADPH in TDGB. BSA was immediately added to give a final concentration of 1 mg/ml. NOS could be quick frozen and stored at -70°C without loss in activity for at least 2 months.
Assay for inducible human NOS.
The formation of citrulline was assayed as described above except that 10 μM FAD was included and calmodulin and CaC12 were excluded from the assay mix.
Purification of NOS from human brain
Human brain NOS was prepared using variations of the procedures of Schmidt et al. (TNAS 88 365-369, 1991), Mayer et al. (Ted. Eur. Biochem. Soc. 288 187-191, 1991), and Bredt and Snyder, (PNAS 87 682-685, 1990). Briefly, frozen human brain (1050 gm) was homogenized in cold buffer A (50 mM HEPES, pH 7.5 (pH at RT) and 0.5 mM EDTA, 10 mM DTT, 3.6 L total volume) with a polytron. The mixture was centrifuged at 13,000g for 1 hour and the supernatant was removed (about 2050 ml). To the supernatant, solid ammonium sulfate (365 gm, about 30% of saturation) was added and stirred slowly for a total of 30 minutes. The precipitate was pelleted at 13,000g for 30 minutes and the pellet was resuspended in -400 mis of buffer A with 4 μM tetrahydrobiopterin, lμM FAD (Sigma), 1 μM FMN (Sigma). The solution was centrifuged at 41,000g for 60 minutes. The supernatant was removed, frozen by pouring into liquid nitrogen, and stored overnight at -70°C. The mixture was thawed and passed through a 2',5' ADP-agarose column (0.4 g swelled in buffer A) at 4 ml/min. The column was washed with 100ml buffer A. 200ml buffer A with 500 mM NaCl, 100ml Buffer A, then 30 ml buffer A with 5 mM NADPH. To the enzyme eluted from the column was added glycerol to 15%, CaCl2 to 1 mM, tetrahydrobiopterin to lOμM, tween to 0.1% and FAD, FMN to 1 μM each. The enzyme was then passed through a 1 ml calmodulin-agarose column which had been equilibrated in Buffer A, 15% glycerol and 1 mM CaCl2. The column was washed with 15ml Buffer A, 15% glycerol and 1 mM CaC_2, 15 ml of Buffer A, 15% glycerol and 5 mM EDTA, and then enzyme activity was eluted with 3 ml of Buffer A, 15% glycerol and 5 mM EDTA, 1 M NaCl. To the enzyme was added tetrahydrobiopterin to 10 μM, FAD and FMN to 1 μM, and tween to 0.1%. This solution was concentrated by centriprep-30 to a volume of approximately 500 μl. Human NOS was prepared completely analogously except the calmodulin-agarose column was not used. Enzyme activity was determined as described by Schmidt et al. 1991, except that lOμ M tetrahydrobiopterin was included in the assay. The results are as given in Table 1.
TABLE 1.
Example Human Human Human
Inducible Placental Brain
Ki/μM Ki/μM Ki/μM
1 3.1 4.0 0.19 ± 0.01
1A 3.2 3.1 0.56 ± 0.02
IB 6.6 2.8 0.21 ± 0.01
1C >25 12 1.6
ID 0 14% @ 25μm 30%@ 25μm
IE 44 14 11
IF 4.7 2.0 0.25 ± 0.02
1G 29 9.0 1.4 ± 0.01
1H 28 14 1.5 ± 0.07
11 2.5 2.7 0.34
1J 4.9 2.7 0.29 ± 0.04
IK 48%@25 μM 48% @25 μM 4.8 36% @ 25 μM 9.4 0.32 ± 0.02 A 0 0 ll%@25μM B 2.4 1.8 0.45 C 10 6.5 1.13 ±0.02 D 25 7.7 0.33 E 3.2 28 1.1 ±0.01 F 22 1.8 1.4 ±0.01 G 2.9 1.8 0.17 H 1.4 1.6 0.17 ±0.02 1 1.9 1.0 0.18 ±0.04 J 47% @ 25 μM 6.9 1.0 K 17 8.2 4.2 L 20% 6% 40% M 23 6.9 1.0 ±0.2 N 22 16 0.8 ±0.2
4% 14% 24% A 12% 35% 1.9 ±0.1
3.3 5.5 A 11 21
2.3 17 0.5
45 22 3.2
0.034a 0.07b 0.001c
0.028b 0.030a 0.0005c A 1.7 4.1 0.09 B 0.69 3.3 3.0 C 0.15 2.1 0.9 D 22.6 23.2 28 E 5.3 14 5.5 F 79 61 G 3.6 3.7 0.2 9H 5.3 1.2 0.4
91 15 11 11
9J 7.0 1.3 0.56
9K 40 30 61
9L 19 23 108
9M 13 19.5
9N 99 61
10c(i) 0.22b 0.12b 0.10b lθc(ii) 0.10b 0.006a 0.002c
12 1.7 4.1 0.09
13 0.69 3.3 3.0
13A 0.15 2.1 0.9
14 22.6 23.2 28
14A 5.3 14 5.5
aThe progress curve was an exponential decay followed by a linear steady state rate. Inhibition constant was calculated by dividing the steady state inhibited rate by the control uninhibited rate; percent inhibition was then used to calculate the inhibition constant assuming competitive inhibition with respect to arginine.
alue obtained from measuring percent inhibition at three or more concentrations of inhibitor at a single time point and assuming competitive inhibition with respect to arginine.
cThe progress curve was an exponential decay of the rate. Value is a K^ determined by measuring association and dissociation rate constants for the slow onset of inhibition, as previously described (Furfine, E.S., Harmon, M.F., Paith, J.E., and Garvey, E.P. (1993) Biochemistry 32, 8512-8517).

Claims

1. The use of an N-substituted urea derivative or a salt, ester or amide thereof other than N-(2,6-dimethylphenyl)-5,6-dihydro-4H-l,3-thiazin-2-amine for the manufacture of a medicament for the treatment of a condition where there is an advantage in inhibiting the neuronal NO synthase enzyme.
2. The use of a N-substituted urea derivative or a salt, ester or amide thereof, other than N-(2,6-dimethylphenyl)-5,6-dihydro-4H-l,3-thiazin-2-amine for the manufacture of a medicament for the treatment of a disease of the nervous system due to over production of the neuronal nitric oxide synthase enzyme.
3. The use according to claim 1 or 2 wherein the N-substituted urea derivative is a compound of formula (I)
Figure imgf000038_0001
wherein
Q is oxygen or sulphur
R is hydrogen or C \ _g hydrocarbyl;
R2 is a mono- or bi-cyclic heterocyclic ring system, a C\.\4 hydrocarbyl group which may optionally contain an oxygen atom, a group S(O)n wherein n is 0, 1 or 2, or a group NR^ wherein R^ is a C .Q aliphatic group, each group R2 optionally being substituted by one to five groups independently selected from
(i) C\. alkyl or C3_6 cycloalkyl each optionally substituted by one to three halo atoms; (ii) a group OR^ wherein R^ is hydrogen, C\. alkyl, phenyl or benzyl; (iii) a halo atom; (iv) a group CO2R^ wherein R^ is hydrogen or C 1.4 alkyl; (v) a group NR6R7 wherein R^ and R are independently selected from hydrogen, C]_4 alkyl or a group
Figure imgf000039_0001
wherein Q and R are as hereinbefore defined:
(vi) nitro; or
(vii) cyano;
or R! may be linked to the imino nitrogen to form a monocyclic heterocyclic ring; with the exception ofN-(2,6-dimethylphenyl)-5, 6-dihydro-4H, 1,3- thiazin-2-amine.
4. The use according to any one of claims 1, 2 or 3 wherein the N-substituted urea derivative is a compound of formula (I A), (IB), (IC), (ID), (IE) or (IF)
Figure imgf000039_0002
Figure imgf000039_0003
Figure imgf000040_0001
Figure imgf000040_0002
wherein Z is oxygen or sulphur; R1 is as hereinbefore defined; X is a C2-10 hydrocarbyl group which may optionally contain an oxygen atom, a group S(O)n as hereinbefore defined, or a group NRJ as hereinbefore defined; T is a Cj.g hydrocarbyl group optionally containing a phenylene ring or a 5- or 6- membered heterocyclic ring; and Ar is a mono- or bicyclic aromatic ring system optionally substituted by one to five groups selected from
(i) C g alkyl or C3.6 cycloalkyl each optionally substituted by one to three halo atoms; (ii) a group OR^ wherein R^ is hydrogen, C\. alkyl, phenyl or benzyl; (iii) a halo atom;
(iv) a group CO2R^ wherein R^ is hydrogen or C 1.4 alkyl; (v) a group NR >R7 wherein R^ and R7 are independently selected from hydrogen,
Figure imgf000040_0003
wherein Q and R are as hereinbefore defined; (vi) nitro; or (vii) cyano.
The use according to any one of claims 1 to 4 wherein the N-substituted urea derivative is a compound of formula (I) other than N^-(aminothiocarbonyl)-L- ornithine, N6-(aminothiocarbonyl)-L-lysine, N^-(aminothiocarbonyl)-D- ornithine and N^-(aminothiocarbonyl)-D-lysine.
6. The use according to any one of claims 1 to 5 wherein the N-substituted urea derivative is a compound of formula (IA) wherein X is a C2-10 hydrocarbyl group which contains an oxygen atom, a group S(O)n or NR-3 wherein n and R3 are as hereinbefore defined, or of formulae (IB) to (IF) as hereinbefore defined.
7. The use according to any one of claims 1 to 6 wherein the N-substituted urea derivative is selected from
S-Ethyl-N-(4-phenoxyphenyl)isothiourea
S-ethyl-N-(3-methoxyphenyl)isothiourea
S-ethyl-N-[4-(benzyloxy)phenyl]isothiourea
S-ethyl-N-[4-(ethoxycarbonyl)phenyl]isothiourea
S-ethyl-N-(4-carboxyphenyl)isothiourea
S-ethyl-N-(3-carboxyphenyl)isothiourea
S-ethyl-N-(2-bromophenyl)isothiourea
S-ethyl-N-(4-dimethylaminophenyl)isothiourea
S-ethyl-N-(4-cyclohexylphenyl)isothiourea
S-ethyl-N-(4-hydroxyphenyl)isothiourea
S-ethyl-N-(4-methoxyphenyl)isothiourea
S-ethyl-N-(2-pyridyl)isothiourea
S-Ethyl-N-[4-trifluoromethyl)phenyl]isothiourea
S-Benzyl-N-[4-(trifluoromethyl)phenyl]isothiourea
S-Ethyl-N-(3-chlorophenyl)isothiourea
S-Ethyl-N-(2-isopropylphenyl)isothiourea
S-Ethyl-N-(4-isopropylphenyl)isothiourea
S-Ethyl-N-[3-(trifluoromethyl)phenyl]isothiourea
S-Ethyl-N-[2-(trifluoromethyl)phenyl]isothiourea
S-Ethyl-N-[2-(chlorophenyl)isothiourea
S-Ethyl-N-(2-methoxyphenyl)isothiourea
S-Ethyl-N-(4-methylphenyl)isothiourea
S-Ethyl-N-(3-pyridyl)isothiourea
S-Ethyl-N-(4-chloro-3-(trifluoromethyl)phenyl) isothiourea
S-Ethyl-N-(2-chloro-5-(trifluoromethyl)phenyl) isothiourea
S-Ethyl-N-(3-pyridyl)isothiourea S-Ethyl-N-(4-pyridyl)isothiourea
O-Methyl-N-(4-(trifluoromethyl)phenyl)isourea
O-Ethyl-N-(4-(trifluoromethyl)phenyl)isourea
S-Ethyl-N-[4-(trifluoromethoxy)phenyl]isothiourea
N5 -(2-thiazolin-2-yl)-L-orni thine
N6-(2-thiazolin-2-yl)-L-lysine
N, N'-((methylthio)iminomethyl)-m-xylylenediamine
N, N-((methylthio)iminomethyl)-p-xylylenediamine
N5-(imino(methylthio)methyl-L-ornithine
N5-((ethylthio)iminomethyl)-L-omithine
N6-(imino(methylthio)methyl)-L-lysine
N6-((ethylthio)iminomethyl)-L-lysine
N5-(imino( 1 -methylethylthio)methyl)-L-ornithine
N6-(imino( 1 -methylethylthio)methyl)-L-lysine
N5-(imino(2-memylpropylthio)meΛyl)-L-oπnt__ine
N6-(imino(2-methylpropylthio)methyl)-L-lysine
N5-((methylthio)iminomethyl)-D-ornithine
N6-((methylthio)iminomethyl)-D-lysine
N5-((ethylthio)iminomethyl)-D-ornithine
N6-((ethylthio)iminomethyl)-D-lysine
N5-(imino( 1 -memylethylthio)methyl)-D-ornithine
N6-(( 1 -methylethylthio)iminomethy l)-D-lysine
N5-((2-memylpropyltl_io)iιninomemyl)-D-oπ_ithine
N6-((methylpropylthio)iminomethyl)-D-lysine
N5 -(iminomethoxymethyl)-L-ornithine
N5-(ethoxyiminomethyl)-L-ornithine
N5-(iminoisopropoxymethyl)-L-ornithine
N6-iminomethoxymethyl)-L-lysine
N6-(ethoxyiminomethyl)-L-lysine l-(3-(Aminomethyl)benzyl)-O-ethylisourea l-(3-(Aminomethyl)benzyl)-S-methylisothiourea l-(3-(_ minomethyl)benzyl)-S-ethylisothiourea l-(4-(Aminomethyl)benzyl)-S-methylisothiourea l-(4-(Aminomethyl)benzyl)-S-ethylisothiourea
S-ethyl-N-(4-diethylamino)phenyl) isothiourea S-ethyl-N-(5-chloro-2-pyridyl)isothiourea S-ethyl-N-(4-nitrophenyl) isothiourea S-ethyl-N-(4-chlorophenyl) isothiourea S-ethyl-N-(3, 4-dichlorophenyl) isothiourea S-benzyl-N-phenyl isothiourea S-ethyl-N-phenyl isothiourea
and salts, esters or amides thereof thereof.
8. The use according to anyone of claims 1 to 6 of a compound of formula (I) with the proviso that when Q is sulphur and R1 is hydrogen or C 1.5 alkyl, R2 is not an ornithine or lysine derivative optionally substituted by a C\. alkyl group on either the α-, β- or γ- carbon atoms, or a tautomer thereof.
9. The use of a compound of formula (I) as defined in claim 3 other than 5- methyl-2-(2-thiazolylamino)phenol and S-ethyl-N-phenylisothiourea for the manufacture of a medicament of the treatment of a condition requiring inhibition of the inducible nitric oxide synthase enzyme.
10. .An N-substituted urea derivative of formula (I) other than S-ethyl-N- phenylisothiourea, S-ethyl-N-(2-chlorophenyl)isothiourea, S-ethyl-N-(2- trifluoromethylphenyl) isothiourea, 2-propenylthiourea, and 5-methyl-2-(2- thiazolylamino)phenol, or a pharmaceutically acceptable salt, ester or amide thereof for use in medicine.
11. A N-substituted urea derivative of formula (I) or a salt, ester or amide thereof, with the proviso that:
(a) when Q is sulphur and
(i) R is methyl, R2 is not a phenyl ring substituted by 3-chloro, 2- ethyl, 2-chloro-5-trifluoromethyl, 3-trifluoromethyl, 3-methyl, 3-bromo, 4-nitro, 4-chloro, 3,4-dichloro or CO2H; or R2 is not a group 5-chloro-2-pyridyl; (ii) R! is ethyl, R2 is not a phenyl ring or a phenyl ring substituted by 4-methoxy, 2-chloro, 4-hydroxy, 2-methoxy, 4-methyl, 2- trifluoromethyl or 3-trifluoromethyl; or (b) the compound of formula (I) is not
2-propenylthiourea
N-(2,6-dimethylphenyl)-5,6-dihydro-4H- 1 ,3-thiazin-2-amine
5-methyl-2-(2-thiazolylamino)phenol.
12. A pharmaceutical formulation comprising a compound of formula (I) other than S-ethyl-N-phenylisothiourea, S-ethyl-N-(2-chlorophenyl)isothiourea, S- ethyl-N-(2-trifluoromethylphenyl)isothiourea, 2-propenylthiourea, and 5- methyl-2-(2-thiazolylamino)phenol or a pharmaceutically acceptable salt, ester or amide thereof, together with one or more pharmaceutically acceptable carriers therefor and optionally one or more other therapeutic ingredients.
13. A process for the preparation of a compound of formula (I) as defined in claim 11, which comprises:
(i) when Q is S and R1 is other than hydrogen, the S-alkylation of a compound of formula (II)
S
X R2 H2N^N"K (II)
2 H
or a protected derivative thereof, wherein R2 is as hereinbefore defined, and thereafter deprotection if required;
(ii) when Q is O and RI is other than hydrogen, the acid catalysed addition of an alcohol of formula R -OH wherein R is as hereinbefore defined other than hydrogen, to a compound of formula (III)
Figure imgf000044_0001
wherein R2 is as hereinbefore defined. 12. A method of treatment of a condition where there is an advantage in inhibiting the neuronal NO synthase enzyme comprising administering to a mammal in need thereof a therapeutically effective amount of an N-substituted urea derivative or a salt, ester or amide thereof other than N-(2,6-dimethylphenyl)- 5,6-dihydro-4H- 1 ,3-thiazin-2-amine.
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WO1996009286A1 (en) * 1994-09-20 1996-03-28 Astra Aktiebolag Isothiourea derivatives as no synthase inhibitors
WO1996018608A1 (en) * 1994-12-12 1996-06-20 Chugai Seiyaku Kabushiki Kaisha Aniline derivatives having nitrogen monoxide synthase inhibitory activity
EP0750906A1 (en) * 1994-02-25 1997-01-02 Chugai Seiyaku Kabushiki Kaisha Delta-(s-methylisothioureido)-l-norvaline and cerebrovascular disorder remedy having nitrogen monoxide synthase inhibitor activity
WO1997017344A1 (en) * 1995-11-07 1997-05-15 Astra Aktiebolag Amidine and isothiourea derivatives as inhibitors of nitric oxide synthase
EP0776886A1 (en) * 1994-08-18 1997-06-04 Chugai Seiyaku Kabushiki Kaisha Amino acid derivative having nitrogen monoxide synthetase inhibitor activity
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US6331553B1 (en) 1996-12-24 2001-12-18 Chugai Seiyaku Kabushiki Kaisha Aromatic amine derivatives having NOS inhibiting action
US6489322B1 (en) 1997-05-05 2002-12-03 Astrazeneca Ab Amidine derivatives as inhibitors of nitric oxide synthase
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US7456220B2 (en) 1999-04-06 2008-11-25 Genzyme Corporation Immunodulatory compositions and methods of use thereof
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