HRP980381A2 - Nitric oxide synthase inhibitors - Google Patents

Description

The described invention relates to new amidine compounds, to the process for their production, to pharmaceutical preparations containing them and to their use in therapy, especially to their use as selective inhibitors of inducible nitric oxide synthesis.

Nitric oxide is an endogenous stimulator of the soluble guanylate cyclase enzyme that participates in numerous biological actions. Excess production of nitric oxide is thought to be associated with a number of conditions, including septic shock and many inflammatory diseases. Biochemical syntheses of nitric oxide from L-arginine are catalyzed with enzymatic NO synthesis. Many inhibitors of NO synthesis have been described and proposed for therapeutic purposes.

Recently, the object of research in this field is the provision of NO synthesis inhibitors that show selectivity either for inducible NO synthesis (iNOS) or for neuronal NO synthesis (nNOS) via endothelial NO synthesis (eNOS).

Thus, WO93/13055 describes selective NO synthesis inhibitors of the formula:

[image]

and their salts, and pharmaceutically acceptable esters and amides, where

R 1 is a C 1-6 normal or branched chain alkyl group, a C 2-6 alkenyl group, a C 2-6 alkynyl group, a C 3-6 cycloalkyl group, or a C 3-6 cycloalkyl C 1-6 alkyl group;

Q is an alkylene, alkenylene or alkynyl group having from 3 to 6 carbon atoms, and which may be optionally substituted with one or more C1-3 alkyl groups;

a group of the formula -(CH2)pX(CH2)q- where p is 2 or 3, q is 1 or 2, and X is S(O)x where x is 0, 1 or 2, O or NR 2 where R 2 is H or C1-6 alkyl; or

a group of the formula -(CH2)rA(CH2)s- where r is 0, 1 or 2, s is 0, 1 or 2, and A is a 3- to 6-membered carbocyclic or heterocyclic ring which may optionally be substituted with one or more suitable substituents, such as C 1-6 alkyl, C 1-6 alkoxy, hydroxy, halo, nitro, cyano, trifluoro C 1-6 alkyl, amino, C 1-6 alkylamino, or diC 1-6 alkylamino groups.

We have now found compounds falling within the scope of WO 93/13055, which are good as selective iNOS inhibitors, and which show advantages, including having a long half-life and being orally bioavailable when administered in vivo.

Therefore, according to the described invention, a compound of formula (I) is provided:

[image]

or a salt, solvate or physiologically functional derivative thereof.

Formula (I) includes an asymmetric center in the amino acid group, and since the natural L or (S) configuration of arginine is preferred, formula (I) is considered to include both (S) and (R) enantiomers, either substantially in pure form or in the form of a mixture in certain proportions.

Thus, alternatively, the described invention provides a compound selected from the group consisting of:

(R/S)-[2-(1-iminoethylamino)ethyl]-DL-homocysteine;

(S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine; and

(R)-[2-(1-iminoethylamino)ethyl]-D-homocysteine;

and their salts, solvates and physiologically functional derivatives.

In a preferred aspect, the described invention provides (S)-[2-(1-iminoethyl-amino)ethyl]-L-homocysteine or a salt, solvate or physiologically functional derivative thereof. In a particularly preferred aspect, the described invention provides (S)-[2-(1-imino-ethylamino)ethyl]-L-homocysteine or a salt thereof.

Salts and solvates of the compound of formula (I) which are suitable for use in medicine are those which are a counterion or a bound pharmaceutically acceptable solvent. However, salts and solvates having pharmaceutically unacceptable counterions or bound solvents are within the scope of the described invention, for example for use as intermediates in the preparation of other compounds of formula (I) and their pharmaceutically acceptable salts, solvates and physiologically functional derivatives.

The term "physiologically functional derivative" means a chemical derivative of the compound of formula (I) that has the same physiological function as the free compound of formula (I), for example, which is converted in the body into a compound of formula (I). Therefore, according to the described invention, examples of physiologically functional derivatives include esters, amides and carbamates, preferably esters and amides.

Suitable salts according to the invention include those formed with organic or inorganic acids or bases. Pharmaceutically acceptable acid addition salts include those formed with the following acids: hydrochloric, hydrobromic, sulfuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, oxalic, fumaric, maleic, oxaloacetic, methanesulfonic, ethanesulfonic, p- toluenesulfonic, benzenesulfonic and isethionic acid. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium and magnesium, and salts with organic bases such as dicyclohexyl amine and N-methyl-D-glucamine. .

Pharmaceutically acceptable esters and amides of the compound of formula (I) may have an acid group converted to a C1-6 alkyl, aryl, aryl C1-6 alkyl or amino acid ester or amide. Pharmaceutically acceptable amides and carbamates of the compound of formula (I) may have an amino group converted to a C1-6 alkyl, aryl, aryl C1-6 alkyl or amino acid amide or carbamate.

As mentioned above, compounds of formula (I) are inhibitors of NO synthesis, as shown below in NOS inhibition assays.

Therefore, the compounds of formula (I) and their pharmaceutically acceptable salts, solvates and physiologically functional derivatives are used in the prophylaxis and treatment of clinical conditions for which an inhibitor of NO synthesis, specifically an inhibitor of iNOS, is indicated. Such conditions include inflammatory conditions, shock conditions, immune disorders, and gastrointestinal motility disorders. The compounds of formula (I) and their pharmaceutically acceptable salts, solvates and physiologically functional derivatives can also be used in the prophylaxis and treatment of diseases of the central nervous system, including migraine.

Shock states are understood to mean those states resulting from excessive production of NO, such as septic shock, hemorrhagic shock, traumatic shock or shock induced with fulminant hepatic damage or with cytokine therapy, such as TNF, IL-1 and IL- 2, or with therapy with cytokine-inducing agents, for example 5,6-dimethylxanthenone acetic acid.

Examples of inflammatory conditions and immune disorders include those of the joint, particularly arthritis (for example, rheumatoid arthritis, osteoarthritis, prosthetic joint damage), or of the gastrointestinal tract (for example, ulcerative colitis, Crohn's disease, and other inflammatory bowel diseases, gastritis and mucosal inflammation resulting from infection, entropopathy induced with nonsteroidal anti-inflammatory drugs), and for the lungs (for example, respiratory distress syndrome in adults, asthma, cystic fibrosis or chronic obstructive pulmonary disease), for the heart (for example, myocarditis), for nervous tissue ( for example multiple sclerosis), for the pancreas (for example diabetes mellitus and its complications), or for the kidneys (for example glomerulonephritis), for the skin (for example dermatitis, psoriasis, eczema, urticaria), for the eyes (for example glaucoma), as and for transplanted organs (for example, rejection), and for multiorgan diseases (for example, systemic lupus erythematosus), and for inflammatory and conditions accompanying viral or bacterial infections.

Furthermore, there is evidence for excessive NO production by iNOS in atherosclerosis and in conditions accompanying hypoxic or ischemic injury (with or without reperfusion), for example in the brain or in ischemic heart disease.

Gastrointestinal motility disorders include ileus, for example post-operative ileus and ileus during sepsis.

Diseases of the central nervous system include those in which excessive production of NO is involved, for example migraine, psychosis, anxiety, schizophrenia, insomnia, cerebral ischemia, CNS trauma, epilepsy, multiple sclerosis, AIDS dementia, chronic neurodegenerative disease (for example Lewy Body Dementia , Huntington's disease, Parkinson's disease or Alzheimer's disease), and acute and chronic pain and conditions involving non-adrenergic and non-cholinergic nerves, such as priapism, obesity and hyperphagia.

Examples of acute pain include musculoskeletal pain, post-operative pain and surgical pain. Examples of chronic pain include chronic inflammatory pain (for example, rheumatoid arthritis and osteoarthritis), neuropathic pain (for example, post herpetic neuralgia, diabetic neuropathy associated with diabetes, trigeminal neuralgia, pain associated with functional bowel disease, for example, irritable bowel syndrome, non-cardiac pain in breast and sympathetically maintained pain), and pain related to cancer and fibromyalgia.

Furthermore, the inhibition of NO synthesis may be suitable in preventing the loss of lymphocytes associated with HIV infection, in increasing the radiosensitivity of tumors during radiotherapy, and in reducing tumor growth, tumor progression, angiogenesis and metastasis.

Therefore, the described invention provides a method for the prophylaxis or treatment of a clinical condition in a mammal, such as a human, for which an inhibitor of nitric oxide synthesis, for example an iNOS inhibitor, is indicated, comprising the administration of a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative. More specifically, the described invention provides a method for the prophylaxis or treatment of an inflammatory and/or immune disorder, such as arthritis or asthma. In a preferred aspect of the described invention, a method is provided for the prophylaxis or treatment of a clinical condition selected from among the following conditions: arthritis, asthma, ileus and migraine.

Alternatively, there is also provided a compound of formula (I) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof for medical therapy, particularly for use in the prophylaxis or treatment of a clinical condition in a mammal, such as a human, for which a nitrogenase inhibitor is indicated. oxide synthesis, for example, iNOS inhibitor. More specifically, a compound of formula (I) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof is provided for the prophylaxis or treatment of an inflammatory and/or immune disorder, such as arthritis or asthma. In a preferred aspect, a compound of formula (I) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof is provided for the prophylaxis or treatment of arthritis, asthma, ileus and migraine.

The amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate, or physiologically functional derivative thereof required to achieve a therapeutic effect will, of course, vary with the particular compound, the route of administration, the subject being treated, and the particular disorder or disease being treated. . The compounds of the invention can be administered orally or via injection at a dose of 0.1 to 1500 mg/kg, per day, preferably 0.1 to 500 mg/kg/day. The dosage range for adults is usually 5 mg to 35 g/day, preferably 5 mg to 2 g/day. Tablets or other forms presented in discrete units may typically contain an amount of a compound of the invention that is effective at such a dose or as a multiple of this dose, for example units containing 5 mg to 500 mg, usually about 10 mg to 200 mg.

Although it is possible for a compound of formula (I) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof to be administered alone, it is preferred that they are present in the form of pharmaceutical formulations.

Therefore, the described invention further provides a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof and a pharmaceutically acceptable carrier or excipient, and optionally one or more other therapeutic ingredients.

The described invention also provides the use of a compound of formula (I), or its pharmaceutically acceptable salt, solvate or physiologically functional derivative for the production of a medicament for the prophylaxis or treatment of a clinical condition for which nitric oxide synthesis is indicated, for example an iNOS inhibitor, for example, inflammatory and/or or an immune disorder, such as arthritis or asthma. In a preferred aspect, there is provided a compound of formula (I), a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof for the manufacture of a medicament for the prophylaxis or treatment of a clinical condition selected from conditions comprising arthritis, asthma, ileus and migraine.

The term "active ingredient" means a compound of formula (I) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof.

Formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, and intra-articular), inhalational (including fine particulate powders or mists that may be created using various types of metered-dose aerosols under pressure from nebulizers or sprinklers), rectal, and topical (including dermal, buccal, sublingual and intraocular) administration, although the most appropriate route of administration may depend on, for example, the condition and disorder of the recipient. The formulations may typically be present in unit dosage form and may be prepared in any manner known in the pharmaceutical art. All procedures include the step of bringing the active ingredient into association with a carrier consisting of one or more auxiliary ingredients. Typically, formulations are obtained by uniformly mixing and associating 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 described invention suitable for oral administration may be present in the form of discrete units, such as capsules, cachets or tablets, each of which contains a predetermined amount of active ingredient; as powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. The active ingredient can also be present in the form of a bolus, salve or paste.

The tablet can be obtained by pressing or casting, optionally with one or more auxiliary ingredients. Pressed tablets may be obtained by pressing in a suitable machine, the active ingredient in free liquid form from such forms as powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricants, surfactant or dispersing agent. Cast tablets may be obtained by casting in a suitable machine, a mixture of the powdered compound soaked in an inert liquid diluent. Tablets may optionally be coated or cored, and may be formulated to provide slow or controlled release of the active ingredient.

Formulations for parenteral administration include aqueous and non-aqueous sterile injectable solutions that may contain antioxidants, buffers, bacteriostats and solubilizing agents, which ensure the formulation is isotonic with the blood of the potential recipient, and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. Formulations may be present in unit dose containers or multidose containers, for example thawed ampoules and blisters, and may be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid carrier, for example saline or water-for-injection, immediately before application. Improvised (not requiring preparation) solutions and suspensions can be obtained from sterile powders, granules and tablets, some of the previously described types.

Formulations for rectal administration may be present in the form of suppositories with conventional carriers such as cocoa butter or polyethylene glycol.

Formulations for topical application in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a suitable base such as sucrose and acacia, or tragacanth and lozenges comprising the active ingredient in a base such as gelatin and glycerin or sucrose and acacia.

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

It should be understood that, in addition to the ingredients specifically mentioned above, the formulations of this invention may include other agents common in the pharmaceutical art, depending on the type of formulation given, for example those suitable for oral administration may include flavoring agents.

According to a further aspect of the invention, a process for obtaining a compound of formula (I) or its salt, solvate or physiologically functional derivative is provided, which includes:

(i) reaction of the compound of formula (II):

[image]

or its enantiomer, salt or protected derivative, with the compound of formula (III):

[image]

or with a salt thereof, where L is a leaving group, most suitably a C1-6 alkoxy group, for example an ethoxy or an alkylthio, an aralkylthio or an arylthio group, for example a benzylthio or a 1- or 2-naphthylmethylthio group; which is followed by the following stages in some order:

(ii) optionally removing protecting groups;

(iii) optionally separating enantiomers from a mixture of enantiomers;

(iv) optional conversion of the product into its corresponding salt, solvate or physiologically functional derivative.

When L is a C1-6 alkoxy group, the reaction in step (i) above can be carried out in solution at an alkaline pH, for example pH 8 to 11, conveniently pH = 10.5 and at a low temperature, for example -5 °C to 20 °C, suitable at 0 to 5 °C. When L is an alkylthio, aralkylthio or arylthio group, the reaction can be carried out in an organic solvent, for example tetrahydrofuran or a C1-4 alcohol, such as ethanol, at a moderate temperature, for example 10 to 40 °C, conveniently at room temperature .

Compounds of formula (III) and salts thereof are commercially available or can be prepared by organic chemistry procedures well known to those skilled in the art, for example as described in Shearer et al.: "Tetrahedron Letters", 38, p. 179-182, (1997).

Compounds of formula (II) and its salts and protected derivatives can be obtained from homocystine:

[image]

or its protected derivative, by breaking the disulfide bond, to form homocysteine or its protected derivative, and binding with the compound of formula (IV):

[image]

or with a protected derivative thereof, where L1 is a leaving group, for example, halo, such as bromo or an alkyl, aryl or aralkyl sulfonate ester, such as toluenesulfonyl.

Cleavage of the disulfide bond of homocysteine or its protected derivative to form homocysteine or its protected derivative can be performed using methods known to those skilled in the art, for example using sodium in liquid ammonia, dithiothreitol or sodium borohydride.

Protected derivatives of homocysteine, for example N-t-butoxycarbonyl homocysteine t-butyl ester, can be reacted with compounds of formula (IV) under conditions in a suitable organic solvent (for example toluene) in a base-promoted reaction, such as 1,8- diazobicyclo[5,4,0]undec-7-ene or a similar agent which should be known to one skilled in the art.

Homocystine compounds of formula (IV) and their protected derivatives are commercially available or can be obtained by organic chemistry procedures known to those skilled in the art.

The protecting groups used in the preparation of the compounds of formula (I) can be used in a conventional manner, for example using the procedures described in Theodora W. Green: "Protective Groups in Organic Synthesis", 2nd edition, John Wiley and Sons, (1991 .), where procedures for removing such groups are also given.

In the above reactions, the primary amines are conveniently protected using acyl groups, such as t-butoxycarbonyl or benzyloxycarbonyl groups which can be removed under acidic conditions, for example by treatment with hydrochloric or hydrobromic acid, or by hydrolysis.

As will be appreciated by one skilled in the art, the use of such protecting groups may include orthogenic protection of amino groups in compounds of formula (II) to facilitate selective removal of one group in the presence of another, thereby providing selective functionalization of one amino function. For example the benzyloxycarbonyl group can be selectively removed by hydrolysis. One skilled in the art will know of other orthogonal protection strategies, available by conventional means as described in Theodora W. Green (see above).

The enantiomeric compounds of the invention can be obtained: (a) by separating the components of the corresponding racemic mixture, for example by means of a chiral chromatographic column, enzymatic resolution procedures or by obtaining and separating the appropriate diastereoisomers; or (b) by direct synthesis from the appropriate chiral intermediates using the procedures described above.

Optional conversion of compounds of formula (I) to the corresponding salt can usually be carried out by reaction with an appropriate acid or base. Optional conversion of compounds of formula (I) into the corresponding solvate or physiologically functional derivative can usually be accomplished by methods well known to those skilled in the art.

Therefore, according to a further aspect, the described invention provides new intermediates for obtaining compounds of formula (I), for example compounds of formula (II) as defined above, or its enantiomer, salt or protected derivative, and especially compounds selected from the group:

(S)-2,7-diamino-5-thioheptanoic acid;

(S)-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid;

(R,S)-2,7-diamino-5-thioheptanoic acid;

(R,S)-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid;

(S)-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoic acid;

(S)-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid;

(S)-t-butyl-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid;

(S)-t-butyl-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoic acid;

(R,S)-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoic acid;

(R,S)-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid;

(R,S)-t-butyl-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoate; and

(R,S)-t-butyl-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoate.

Certain protected derivatives of compounds of formula (I) are also applicable as intermediates for obtaining compounds of formula (I), and especially compounds selected from among the derivatives:

(S)-2N-t-butoxycarbonyl-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoic acid;

(S)-t-butyl-2N-t-butoxycarbonyl-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoate;

(R,S)-2N-t-butoxycarbonyl-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoic acid;

(R,S)-t-butyl-2N-t-butoxycarbonyl-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoate;

and their salts and solvates.

For a better understanding of the invention, and for the purpose of illustration, the following examples are given.

SYNTHETIC EXAMPLES

Example 1

Synthesis of (S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine or (S)-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoic acid

(i) (S)-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid

L-homocystine (3 g) was added to liquid ammonia (130 ml), cooled to -80 °C, followed by sodium metal while the blue color remained for 15 minutes (1.06 g). After this, N-benzyloxycarbonyl-ethanolamine (8.16 g) was added and the reaction was stirred at room temperature until the ammonia evaporated. The residue was dissolved in water (80 ml) and treated with 0.5 M EDTA in the form of sodium salt (2 ml). The pH of the solution was adjusted to 7.0 with 2N sulfuric acid solution, and the resulting white precipitate was filtered, washed with cold water and acetone, and dried in a vacuum desiccator to give the title compound as a white solid, 5.3 g.

Mass spectrum of M+H 313.

(ii) (S)-2,7-diamino-5-thioheptanoic acid

(S)-7N-Benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid was treated with 45% HBr in acetic acid (23 ml) for 1 hour. A hard gum is formed and ether is added to the mixture to ensure complete precipitation of the product. The liquid is decanted and the solid is dissolved in warm SVM. This warm solution is treated with pyridine until a precipitate forms and the mixture is allowed to cool to room temperature. The resulting precipitate is filtered and recrystallized from a SVM/water mixture to give the title compound as a white solid, 2.2 g, m.p. 222 °C (decomposes).

(iii)(S)-[2-(1-iminoethylaminoethyl]-L-homocysteine).

(S)-2,7-Diamino-5-thioheptanoic acid (2.17 g) was stirred in 1N NaOH (16.75 ml) to pH = 10.5 at 0-5 °C. Ethyl acetimidate hydrochloride (2.07 g) was added to this solution in portions, maintaining the pH at 10.5 with 1N NaOH solution. When the reaction is complete the pH is adjusted to 3 with 1N HCl solution and the mixture is applied to a Dowex AGX8 H+ form ion-exchange column. The column is washed to neutrality with water, then washed with 2.5 M pyridine and again to neutrality with water. Elution with 0.5 M ammonia and collection of ninhydron positive fractions is done after evaporation. The resulting residue is treated with 1N HCl to pH = 4.5 and evaporated to dryness. The residue is then treated with ethanol and evaporated to dryness, then treated with diethyl ether. Evaporate the diethyl ether to dryness to give the monohydrochloride, the title compound as a hard white foam.

Microanalysis of the product shows the presence of hydrates with 1.75 water molecules:

Found: C 33.56; H 7,11; N 13.74.

Calculated: C 33.45; H 7.49; N 14.63.

Example 2

(R/S)-[2-(1-iminoethylamino)ethyl]-D,L-homocysteine is obtained using procedures analogous to those in example 1 starting from D,L-homocysteine.

1H NMR of the product agrees with the proposed structure.

Example 2a

The racemic product from Example 2 is largely resolved into two enantiomeric components (which are identical to the (S) product in Examples 1 and 4 and the (R) product in Example 3) using a chiral (+) HPLC column and elution with aqueous trifluoroacetic acid at pH = 2.

(S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine

Microanalysis of the product corresponds to the hydrate of the trifluoroacetate salt C8H17N3O2S•(CF3CO2H)2•H2O

Found: C 31.06; H 4.53; N 9.08.

Calculated: C 30.97; H 4.55; N 9.03.

CD spectrum (0.1N aqueous HCl solution) 210 (+0.80) nm.

(R)-[2-(1-iminoethylamino)ethyl]-D-homocysteine

Microanalysis of the product corresponds to the salt form•1.67 trifluoroacetate•0.3HCl•1.5 hydrate:

C8H17N3O2S•(CF3CO2H)1.67•HCl0.3•1.5H2O

Found: C 30,18; H 4.92; N 9.53, S 7.41; Cl 1.86; F 21.36.

Calculated: C 30.40; H 4.97; N 9.41; With 7,18; Cl 2.38; F 21,28.

CD spectrum (0.1N aqueous HCl solution) 210 (-0.64) nm.

Example 3

(R)-[2-(1-iminoethylamino)ethyl]-D-homocysteine is obtained by procedures analogous to those used in Example 1, starting from D-homocysteine.

Example 4

Synthesis of (S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine

(i) (S)-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid

L-homocystine (10 g, 37.45 mmol) was added to liquid ammonia (430 ml) cooled to -80 °C. The cooling bath was removed and sodium metal (3.18 g, 138.26 mmol) was added portionwise over 25 min and allowed to rise to reflux. Stirring was continued at reflux for a further 30 minutes, after which N-benzyloxycarbonyl-ethanolamine tosylate (25 g, 74.9 mmol) was added and the reaction was stirred at room temperature overnight until the ammonia evaporated. The residue is mixed with water (250 ml) at 40 °C for 10 minutes, cooled to room temperature and filtered. The pH of the solution was adjusted to 7.0 with 2M sulfuric acid and the resulting white precipitate was filtered, washed with cold water and with acetone, and dried in a vacuum desiccator to obtain (S)-7N-benzyloxycarbonyl-2,7-diamino-5 -thioheptanoic acid in the form of a white solid, m.p. 240 °C (decomposes).

(ii) (S)-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid

(S)-7N-Benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid (15.5 g, 49.67 mmol) was added to sodium hydroxide (6.357 g, 159 mmol) in water (110 mL), then dioxane (55 ml) is added. To this mixture was added di-t-butyldicarbonate (16.26 g, 74.5 mmol) and the mixture was stirred overnight at room temperature under nitrogen. After this, the precipitated solids are filtered, toluene (300 ml) is added and the layers are separated. The aqueous layer was cooled and acidified to pH about 3 using 1N HCl. The acidified fraction was extracted with toluene (4 x 100 ml) and with ethyl acetate (3 x 100 ml) and the compounds of the organic fraction were dried over MgSO 4 . Concentration of the combined organic fractions under reduced pressure gives (S)-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid as a white gum.

Mass spectrum of M+H 413.

(iii) Formate salt of (S)-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoic acid

Platinum black (0.678 g) was added to methanol (50 ml) cooled to 5 °C under a nitrogen atmosphere, all at once. To this cooled solution was added a mixture of methanol (50 mL) and formic acid (11 mL, 196 mmol) over 1 minute, followed by (S)-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino- 5-thioheptanoic acid (2 g, 4.85 mmol) in methanol (50 mL) for 2 min. The mixture was allowed to stir overnight at room temperature, more platinum black (257 mg) was added and stirring was continued for another 3 hours. The reaction mixture is filtered through Hyflo and concentrated under reduced pressure. The residue was partitioned between water and ethyl acetate, the aqueous layer was washed with more ethyl acetate and the aqueous layer was concentrated to give the formate salt of (S)-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoic acid as a white solid substances.

Mass spectrum M+H 279 (65 %), 223 (100 %).

(iv) Hydrochloride (S)-2N-t-butoxycarbonyl-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoic acid

S-(1- naphthylmethyl)thioacetimidate hydrochloride (3.70 g, 14.75 mmol) and then ethanol (50 ml). With stirring at room temperature, the solid components dissolved after 2 hours, and the solution was stirred overnight. The reaction was concentrated in vacuo, the residue was treated with water and the aqueous fraction was washed with diethyl ether (4 x 50 ml). Concentration of the aqueous fraction in vacuo affords (S)-2N-t-butoxy-carbonyl-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoic acid hydrochloride as a white hygroscopic solid.

Mass spectrum M+H 320 (75 %), 264 (100 %), 220 (15 %).

(v) (S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine

To (S)-2N-t-butoxycarbonyl-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoic acid hydrochloride (3.086 g, 8.69 mmol) was slowly added 4N HCl/dioxane (20 ml). and the reaction mixture is stirred at room temperature overnight. The reaction was concentrated in vacuo, the residue was dissolved in water and washed with diethyl ether (3 x 20 ml). The aqueous layer was concentrated in vacuo to give the title compound as the hydrochloride as a hygroscopic solid.

Mass spectrum M+H 220;

1H NMR(D2O) �: 2.1-2.35 (5H, m); 2.76 (2H, t); 2.87 (2H, t); 3.51 (2H, t); 4.12 (1H, t).

Example 5

Synthesis of (S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine

(i) (S)-t-butyl-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoate

N-benzyloxycarbonyl ethanolamine tosylate (349 mg, 1 mmol) and 1,8-diazobicyclo[5,4,0]undec-7-ene (150 µL, 1 mmol) and the mixture was stirred vigorously overnight at room temperature under nitrogen. The mixture was distributed between 50 ml each of ethyl acetate and 1N aqueous HCl solution. The organic layers are combined, and these extracts are washed with an aqueous solution of sodium bicarbonate, with water and with a saline solution, and then dried and evaporated. Purification by column chromatography gives the title compound.

Mass spectrum M+H 469 (25 %), 369 (100 %).

In an alternative procedure, conversion of the product of Example 4, step (ii) to its t-butyl ester using either N,N-dimethylformamide di-O-t-butyl acetal or O-t-butyl 1,1,1-trichloroacetimidate gives the title compound in in the form of a white crystalline solid.

(ii) Formate salt of (S)-t-butyl-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoic acid

Palladium hydroxide on carbon is added to a solution of (S)-t-butyl-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoate (1 g, 2.1 mmol) in ethanol (50 ml) (20 %, 0.5 g) and ammonium formate (1.34 g). The suspension is refluxed for 2.5 hours, cooled and filtered through a layer of silica which is washed well with a mixture of 1:1 ethanol/water, and evaporated to obtain the title compound in the form of a formate salt.

Mass spectrum of M+H 335.

(iii) Hydrochloride (S)-t-butyl-2N-t-butoxycarbonyl-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoate

The crude formate salt of (S)-t-butyl-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoic acid from step (ii) is suspended with 50 ml of tetrahydrofuran, and the liquid is decanted and mixed with (1- naphthylmethyl)thioacetimidate hydrochloride (0.5 g, 2 mmol), and stirred for 24 hours at room temperature. The solvent is evaporated and the residue is distributed between 25 ml each of ether and water, then washed twice with ether. The obtained aqueous extracts are combined and evaporated to obtain a white paste. This is freeze-dried twice to obtain the title compound as a hygroscopic solid.

Mass spectrum M+H 376 (100 %), 320 (15 %), 276 (12 %).

(iv) (S)-S-[2-(1-iminoethylamino)ethyl]-L-homocysteine

Deprotection of (S)-t-butyl-2N-t-butoxycarbonyl-7N-(1-iminoethyl)-2,7-diamino-5-thioheptanoate hydrochloride using 4N HCl in dioxane, using procedures analogous to those used in Example 4 step (v) gives (S)-S-[2-(1-iminoethylamino)ethyl]-L-homocysteine.

Characteristic data for the title compound agree with those for the product from example 4.

BIOLOGICAL ACTIVITY

1. Inhibition of eNOS and iNOS in rat aortic rings

Inhibition of eNOS and iNOS in situ in rat aortic rings was examined by measuring the increase in ring pressure induced by inhibition of NO synthesis. For studies of basal tone (related to eNOS), endothelium-intact thoracic aortic rings were obtained as described previously (Rees et al.: "Br. J. Pharmol.", 96, pp. 418-424, (1989 .)). Cumulative concentration curves were obtained for the inhibitors in the presence of an initial concentration of phenylephrine (ED10 about 10 nM). For studies of induced smooth muscle tone (related to iNOS), endothelium-denudid rings were exposed to LPS (0.1 µg/ml from S. Typhos) in the presence of phenylephrine at about ED90 for 6 hours as described previously (Rees et al.: "Biochem. Biophys. Res. Commun.", 173, pp. 541-547, (1990)). During this time, a progressive loss of tone occurs due to iNOS induction. Cumulative concentration curves for inhibitors were then obtained.

The results are given in the following table:

[image]

In contrast, 2-(1-iminoethylamino)ethyl cysteine hydrochloride (Example 4 of WO93/13055) is only 33-fold more selective for iNOS over eNOS in the same assay.

2. Inhibition of nNOS in rat cortical lobes

Compound effects on nNOS in rat cerebral lobes were determined as described in Furfine et al.: "J. Biol. Chem.", 269, p. 26677-26683, (1994); and Lizasoain et al.: "J. Neurochem.", 64, p. 636-642, (1995).

KCl (54 mM)-stimulated NO synthesis was measured by the conversion of 14C-arginine to 14C-citrulline over a 2-hour period at 37 °C in Mcllwain's minced (0.2 mm × 0.2 mm) rat cerebral cortical slices, with the use of a pre-incubation period of 1 hour in the absence of compound or KCl.

The compound of Example 1 was found to have IC50 = 220 µM, suggesting about 300-fold selectivity for iNOS over nNOS.

3. Procedure for determining the oral bioavailability of iNOS inhibitory compounds

Working with animals:

Mice (three animals per time point) were dosed intravenously (10 mg/kg) and orally (50 mg/kg) with the test compound in aqueous solution. Blood samples were taken at time intervals after administration, and plasma was obtained by centrifugation. The samples were stored at -20 °C until analysis.

Analysis of the compound in plasma:

Plasma (50 µl) was deproteinized and the compound was derivatized with a quaternary ammonium reagent. The samples were then injected into the HPLC system, and the concentration of the compound was determined using mass spectrometric detection.

Pharmacokinetic analyses:

Plasma concentrations obtained using the above procedure were entered into a pharmacokinetic software package (PKCAL, v 1.2 s) and data were fitted using a non-compartmental procedure. The oral bioavailability of the compound was determined by comparing the area under the curve (AUC) values calculated using the software for the oral profile with the AUC for the intravenous profile. Half-lives were obtained by fitting the time points of the terminal phase of the intravenous profile.

(S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine has been shown to have an oral bioavailability of 55% and a half-life of 5.7 hours.

When the procedure was repeated with intravenous and oral doses of 10 mg/kg in rats, (S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine had a bioavailability of 92%.

Claims (11)

1. Compound of formula (I): [image] or a salt, solvate or physiologically functional derivative thereof. 2. Compound of formula (I), characterized in that it is selected from the group comprising: (R/S)-[2-(1-iminoethylamino)ethyl]-DL-homocysteine; (S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine; and (R)-[2-(1-iminoethylamino)ethyl]-D-homocysteine; and their salts, solvates and physiologically functional derivatives. 3. The compound of formula (I), characterized in that it is (S)-[2-(1-iminoethylamino)ethyl]-L-homocysteine or its salt, solvate or physiologically functional derivative. 4. A method for the prophylaxis or treatment of a clinical condition in a mammal, such as a human, for which a nitric oxide synthesis inhibitor is indicated, characterized in that it comprises the administration of a therapeutically effective amount of the compound of formula (I) as defined in any of claims 1 to 3, its pharmaceutically acceptable salt, solvate or physiologically functional derivative. 5. The method according to claim 4, characterized in that the clinical condition is selected from the group comprising arthritis, asthma, ileus and migraine. 6. A compound of formula (I) as defined in one of claims 1 to 3 or its pharmaceutically acceptable salt, solvate or physiologically functional derivative, indicated by the fact that it is used in medical therapy. 7. Pharmaceutical formulation, characterized in that it comprises a compound of formula (I) as defined in one of claims 1 to 3 or its pharmaceutically acceptable salt, solvate or physiologically functional derivative and a pharmaceutically acceptable carrier or excipient and optionally one or more other therapeutic ingredients. 8. A compound of formula (I) as defined in one of claims 1 to 3 or its pharmaceutically acceptable salt, solvate or physiologically functional derivative, characterized in that it is used in the production of a medicament for the prophylaxis or treatment of a clinical condition for which a nitrogenase inhibitor is indicated oxide syntheses. 9. Use according to claim 8, characterized in that the clinical condition is selected from the group consisting of arthritis, asthma, ileus and migraine. 10. A process for obtaining a compound of formula (I) or its salt, solvate or physiologically functional derivative, characterized in that it includes: (i) reaction of the compound of formula (II): [image] or its enantiomer, salt or protected derivative, with the compound of formula (III): [image] or with a salt thereof, where L is a leaving group which is followed by the following steps in some order: (ii) optionally removing protecting groups; (iii) optionally separating enantiomers from a mixture of enantiomers; (iv) optional conversion of the product into its corresponding salt, solvate or physiologically functional derivative. 11. Compound of formula (I), characterized in that it is selected from the group comprising: (S)-2,7-diamino-5-thioheptanoic acid; (S)-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid; (R,S)-2,7-diamino-5-thioheptanoic acid; (R,S)-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid; (S)-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoic acid; (S)-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid; (S)-t-butyl-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid; (S)-t-butyl-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoic acid; (R,S)-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoic acid; (R,S)-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoic acid; (R,S)-t-butyl-2N-t-butoxycarbonyl-7N-benzyloxycarbonyl-2,7-diamino-5-thioheptanoate; and (R,S)-t-butyl-2N-t-butoxycarbonyl-2,7-diamino-5-thioheptanoate.