SI9300616A - Enzyme inhibitors - Google Patents

Abstract

Isothiourea derivatives and their use in medicine, particularly in the treatment of conditions where there is an advantage in inhibiting nitric oxide synthase, pharmaceutical formulations comprising the same and processes for the preparation thereof are disclosed.

Description

THE WELLCOME FOUNDATION LIMITED

Enzyme inhibitors

The present invention relates to isothiourea derivatives, processes for their preparation, pharmaceutical compositions containing them and their use in treatment, in particular their use as nitric oxide synthase inhibitors.

Acetylcholine-induced vascular release has been known since the early 1980s to be dependent on the presence of endothelium, and we attribute this action to a labile humoral factor called endothelium-derived relaxation factor (EDRF). Nitric oxide (NO) activity as a vasodilator has been well known for over a hundred years and NO is the active ingredient in amyl nitrite, glyceryl nitrite and other nitro vasodilators. Recent identification of EDRF as NO coincides with the discovery of a biochemical pathway through which NO is synthesized from the amino acid L-arginine by the enzyme NO synthase.

NO is an endogenous stimulator of soluble guanylate cyclase and has been implicated in a number of biological effects, in addition to endothelial-dependent release, involving phagocytic cell cytotoxicity and cell-to-cell communication in the central nervous system (see Moncada et al, Biochemical Pharmacology, 38, 1709 -1715 (1989) and Moncada et al, Pharmacological Review, 43,109-142 (1991). We now think that excess NO production can be involved in many conditions, especially conditions involving systemic hypotension, such as septic (toxic) Shock and therapy with certain cytokines.

The synthesis of NO from L-arginine can be inhibited by the L-arginine analogue of LN-monomethyl-arginine (L-NMMA) and the therapeutic use of L-NMMA for the treatment of septic shock and other types of systemic hypotension has been proposed (WO 91/04024 and GB-A2240041) . The therapeutic use of certain NO synthase inhibitors in addition to L-NMMA has been proposed for the same purpose in WO 91/04024 and in EP-A0446699.

It has recently become apparent that there are at least three types of NO synthase enzymes as follows:

(i) a constitutive Ca ⁺⁺ / calmodulin-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) Ca ⁺⁺ independent enzyme, which is induced after activation in vascular smooth muscle, macrophages, endothelial cells and many other cells, by endotoxin and cytokines. Once expressed, this inducible NO synthase synthesizes NO for a long time.

NO released by constitutive enzymes acts as a shifting mechanism underlying several physiological responses. The function of NO produced by an inducible enzyme is, as a cytotoxic molecule, to fight tumor cells and invading microorganisms (Wright et al. Card. Res. 26, 48-57 (1992) and Moncada et al, Pharmaccological Review, 43, 109 -142 (1991)). It also appears that the deleterious effects of excess NO production, especially pathological vasodilation and tissue damage, can largely result from the effects of NO synthesized by inducible NO synthase.

NO synthase inhibitors proposed so far for therapeutic use and especially L-NMMA are indiscriminate in that they inhibit both enzymes constitutive and inducible NO synthase. The use of such a non-selective NO synthase inhibitor requires great care to avoid the potentially serious consequences of over-inhibition of the constitutive NO synthase enzyme, including hypertension, thrombosis, CNS toxicity and tissue damage. In particular, in the case of the therapeutic use of L-NMMA for the treatment of septic shock, it is recommended that the patient be monitored continuously for blood pressure. Since non-selective NO synthase inhibitors have therapeutic utility, provided that appropriate precautions are taken, NO synthase inhibitors that are selective in that they inhibit the enzyme-inducible NO synthase to a much greater extent than the enzyme constitutive NO synthase would have even greater therapeutic utility and would be much easier to use.

The preparation and biological properties of isothioureas have been described in the literature (Schroeder, Chem. Revs., 1955, 55, 181; Doherty et al., J. Am.Chem. Soc., 1957, 79, 5667; and Brand and Brand, Org. Synth. ., 1942, 22, 59; Smirk et al, Brit. Med. J. 1941, 510-11; J.Physiol., 1942, 100, 474-483; Lancet, 1942, 301-303; J. Physiol., 1943 , 101, 379-388; Fastier, Brit. J. Pharmacol., 1948, 3, 198). We have now found that isothioureas are NO synthase inhibitors and are useful in the treatment of systemic hypotension, and in particular in the treatment of septic shock. In addition, many of these compounds, in comparison with the enzymatic constitutive NO synthases, have selectivity for the enzyme

NO synthase.

The present invention therefore provides a method for treating conditions that require inhibition of the nitric oxide synthase enzyme, comprising administering to the mammal in need thereof an effective amount of an isothiourea derivative having an inhibitory effect against the enzyme NO synthase, or a pharmaceutically acceptable salt thereof. In another aspect, the present invention provides the use of an isothiourea, which has an inhibitory effect against the enzyme NO synthase, for the manufacture of a medicament for the treatment of conditions where there is an advantage in inhibiting the enzyme NO synthase.

Specifically, a method for treating systemic hypotension and / or septic shock comprising administering to the mammal in need thereof an effective amount of an isothiourea derivative having an inhibitory effect against the enzyme NO synthase or a pharmaceutically acceptable salt thereof. In another aspect, the use of an isothiourea derivative having an inhibitory effect against the enzyme NO synthase is provided for the manufacture of a medicament for the treatment of systemic hypotension and / or septic shock.

Further conditions where there is an advantage in inhibiting NO production from L-arginine include treatment with cytokines such as TNF, IL-1 and IL-2 or treatment with cytokine-inducing agents, e.g. 5,6-dimethylxanthenone acetic acid, and as an adjuvant in short-term immunosuppression in transplant treatment. In addition, compounds that inhibit NO synthesis may be useful in reducing NO concentration in patients with inflammatory conditions in which excess NO contributes to the pathophysiology of the condition, e.g. Adult Respiratory Exhaustion Syndrome (ARDS) and myocarditis.

There is also evidence that the enzyme NO synthase may be involved in cartilage degeneration that occurs in autoimmune and / or inflammatory conditions such as arthritis, rheumatoid arthritis, chronic bowel disease, and systemic lupus erythematosus (SLE). It is also believed that the enzyme NO synthase may be involved in insulin-dependent diabetes melitis. Therefore, a further aspect of the present invention provides an isothiourea derivative or a salt thereof in the manufacture of a medicament for use in cytokine or cytokine-inducing therapy, as an adjuvant in short-term immunosuppression in transplant therapy, for the treatment of patients suffering from inflammatory conditions to which NO excess contributes pathophysiology of the condition, autoimmune and / or inflammatory indications and insulin-dependent diabetes mellitus.

A further aspect provides a method for treating the adverse effects associated with cytokine therapy of short-term immunosuppression in transplant therapy for patients with inflammatory conditions in which excess NO contributes to the pathophysiology of the condition, autoimmune and / or inflammatory indications, and insulin-dependent diabetes mellitus administering to the mammal in need of such treatment an effective amount of an isothiourea derivative having an inhibitory effect against the enzyme NO synthase or a pharmaceutically acceptable salt thereof.

As used herein, reference to patient treatment includes prophylaxis; the term mammal includes human or animal.

Preferred isothioureas include those of formula (I)

HN '(I) or salts thereof, where

R (1) C ₁₁₄ hydroxycarbyl group; or (2) a 5- or 6-membered heterocyclic ring; or (3) a 9-membered bicyclic heterocyclic ring system wherein each group R is optionally substituted by one or two groups independently selected from:

(a) halo;

(b) -XR \ where

X is oxygen, C (O) _m , wherein m is 1 or 2, S (O) _n , wherein O, 1 or 2, or NR ² , wherein R ^{2 is} hydrogen, C 1-6 alkyl or C ₃ - cycloalkyl, or R ² is attached to R ¹ to form a C 1-4 alkylene group;

R ^{1 is} hydrogen or C 1-6 alkyl, C ₂₆ alkenyl; _C3 ^ cycloalkyl, C _{7 9} aralkyl, C ₆ _ ₁₀ aryl or a 5- or

A 6-membered heterocyclic group, each group being optionally substituted by one or two groups independently selected from C ₁₃ alkyl, hydroxy, C ₁₃ alkoxy, amino, C ₁₃ alkylamino, halo, nitro, or C (O) _{m group} , R ^2b wherein m 'is 1 or 2 and R ^{2b is} hydrogen or ClJ (alkyl; or R ^{1 is a} group NR ³ R ⁴ wherein R ³ and R ^{4 are the} same or different and each is hydrogen or C 1-4 alkyl or is R ³ and R ^{4 are} linked to form a C _2-6 alkylene group;

NH (c) groups (Y) _w -QS -------- P ™ ^ ^{6ΠΐεΓ X} nh ₂

Y is oxygen, S (O) _n , where n is as defined above, or NR ⁵ , wherein R ^{5 is} hydrogen or C 1-6 ₍ alkyl;

w is 0 or 1;

QC _2j) hydrocarbyl or imino nitrogen is attached to group R or group Q to form a 5- or 6-membered heterocyclic ring;

(d) Group A, where A is a heterocyclic ring system, as the case may be

NH substituted with the group (Y) _w -QSv with the proviso that R is not methyl.

NH as previously defined; or (e) C ₁₆ alkyl, C _{2 6} alkenyl or alkynyl, or _C3 ^ cycloalkyl groups;

or one of the carbon atoms in R is attached to the imino nitrogen atom in the compound of formula (I) to form a 5- or 6-membered heterocyclic ring;

Corresponding to R:

(1) C 1-6 alkyl;

(2) C ₂₈ alkenyl or alkynyl;

(3) the group - (CH ₂ ) _p - ^^ - (CH ₂ ) _q CH ₃ , wherein p is 0 to 4 and q is 0 to 3; or (4) a 5- or 6-membered heterocyclic ring, each optionally substituted by one or two groups, which may be the same or different, selected from (a) halo;

(b) OR ^2b , wherein R ^2b is as previously defined;

(c) C (O) _m R ^2b , wherein m and R ^{2b are} as previously defined;

(d) S (O) _n R ⁶ , wherein n is as previously defined and R ^{6 is} C ₁₄ alkyl optionally substituted by one or two groups independently selected from amino or C (O) _m R ^2b as defined previously;

(e) NR ⁷ R ⁸ , wherein R ⁷ and R ^{8 are} each independently selected from hydrogen,

C _1-4 alkyl, C 1-4 alkenyl, C ₁₄ alkoxyalkyl;

or R ⁷ and R ^{8 are} linked together to form a 5- or 6-membered heterocyclic ring;

(f) a phenyl ring or a 5- or 6-membered heterocyclic ring, each optionally substituted with an OR ^2b group as previously defined or with a group

NH

Q-Snh ₂ wherein Q is as defined above; or imino nitrogen is attached to group Q to form a thiazole or thiazoline ring; or (g) C ₁₄ alkyl when R is a heterocyclic ring;

or one of the carbon atoms in R is attached to imino nitrogen in the compound of formula (I) to form a thiazole or thiazoline ring.

Most preferably R (1) is C 1-6 alkyl;

(2) C ₂ ^ alkenyl;

(3) the group - (CH ₂ ) ^^ - (CH ₂ ) _q CH ₃ , wherein p is 1 or 2 and q is O or 1; or (4) a 5- or 6-membered heterocyclic ring containing one or two nitrogen atoms.

each optionally substituted by one or two groups, which may be the same or different, selected from (a) halo, preferably bromo;

(b) groups OR ^2b ', wherein R ^2b ' is hydrogen or methyl;

(c) groups C (O) _m R ^2b ', where m and R ^2b ' are as previously defined (d) groups SR ⁹ , where R ^{9 is} methyl or ethyl;

(e) NR ^7b R ^8b groups, wherein R ^7b and R ^{Sb are} each independently selected from hydrogen or C ₁₄ alkyl, preferably hydrogen, methyl or ethyl;

(f) a phenyl ring optionally substituted by the group OR ^2b 'or

as previously defined;

(g) a 5- or 6-membered heterocyclic ring containing one or two heteroatoms independently selected from nitrogen or oxygen; or (h) C ^M -alkiIa, preferably methyl or one of the carbon atoms in R is linked to the imino nitrogen in the compound of formula (I), to form a thiazole or thiazoline ring.

Formula (I) includes isothiourea derivatives of formula (IA), (IB) and (IC)

(IA)

H _t N · (IB)

NH

N— ¹

(IC) wherein R 'is a C 1-4 alkylene group or a C, _g alkenylene or alkynylene group, each optionally containing a phenyl ring, a 5- or 6-membered heterocyclic ring or a group X as previously defined and dashed the line represents a double or single bond.

Formula I also includes compounds of formula (II)

SR ³

HNi

NH, (II) or salts thereof, wherein R ^{a is} C 1-6 hydrocarbyl or a 5- or 6-membered heterocyclic ring or a 9-membered bicyclic heterocyclic ring system, each optionally substituted by halo or by one or two groups-X ^a R ^1a , wherein R ^{1a is} hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, C 7 aralkyl, C 640 aryl, or a 5- or 6-membered heterocyclic group, each substituted with a C 1-3 alkyl, C 1-3 alkoxy in each case. amino, halo or nitro or R ^{1a is} a NR ^3a group R ^4a , wherein R ³³ and R ^{4a are the} same or different and each hydrogen or C 13 alkyl or R ^3a and R ^{4a are} attached to form a C 1-4 alkylene group and X is ^{a is} oxygen, C (O) ma, wherein m ^{a is} 1 or 2, S (O) na, wherein n ^{a is} 0,1 or 2 or NR ²³ , where R ^{23 is} hydrogen, C 1-6 alkyl or C 3-4 cycloalkyl or R ²³ linked to R ^1a to form a C 2 6 alkylene group, or with the group jKCH ^ UHN <^^^ NH ₂ where t is O to 4 and χν is ³ O or 1, Y ^a is oxygen, sulfur and NR ^7a wherein R ^{7a is} hydrogen or C 1-6 alkyl;

or R ^a binds the sulfur atom to one of the nitrogen atoms in the compound of formula (I) to form a 5- or 6-membered heterocyclic ring, with the proviso that R ^a is not methyl.

One preferred group of compounds are those wherein R is not methyl, ethyl, propyl or isopropyl.

Preferred compounds of formula (I) include:

S- (2-aminoethyl) isothioacetate

S- (2- (dimethylamino) propyl) isothiourea

S- (2-methyl-2-propenyl) isothiourea

S, S'-ethylenebis (isothiourea)

S, S'-pentamethylenebis (isothiourea)

S- (2- (dimethylamino) ethyl) isothiourea

2-amino-2-thiazoline

S, S′-hexamethylenebis (isothiourea)

S, S′-heptamethylenebis (isothiourea)

S-benzylisothiourea

S- (2-morpholinoethyl) isothiourea

S- (6-methyl-2- (methylthio) -4-pyrimidinyl) isothiourea

S, S '- (1,4-Phenylenebis (methylene) diisothiourea

S-tertbutylisothiourea

S- (4-ethylbenzyl) isothiourea

S - ((methylthio) methyl) isothiourea

S- (3-bromopropyl) isothiourea

S- (2-Bromoethyl) isothiourea

S- (3-methyl-2-butenyl) isothiourea

S-allyl isothiourea

S- (3-aminopropyl) isothiourea

S, S '- (1,3-phenylenebis (methylene)) diisothiourea

S, S '- (2-methylene-1,3-propanediyl) diisothiourea

S, S '- (2-Butyn-1,4-diyl) diisothiourea

S, S '- (l, 3-phenylenebis (l, 2-ethanediyl) diisothiourea

S, S '- (l, 4-phenylenebis (l, 2-ethanediyl) diisothiourea

2-amino-5-methylthiazole

S - ((2-amino-4-thiazolyl) methyl-L-cysteine

3- ((2-amino-4-thiazolyl) methyl-L-alanine

2-amino-4-methylthiazole

2-amino-4,5-dimethylthiazole

S- (2- (1H-pyrrol-1-yl) ethyl) isothiourea

S- (3-hydroxypropyl) isothiourea

S- (2- (phenyl) ethyl) isothiourea

S- (2- (3-methoxyphenyl) ethyl) isothiourea

4- ((2'amino-4-thiazolyl) methyl) -L-homoalanine

N, N-1,3-phenylenebis (methylene)) bis (S-methylisothiourea)

N, N - (-, 3-phenylenebis (methylene)) bis (S-ethylisothiourea)

5- (2- (5 - ((amidinothio) methyl) -2-thienyl) ethyl) isothiourea

S- (3- (4 - ((amidinothio) methyl) phenyl) propyl) isothiourea

S- (3- (5- (2-amidinothio) ethyl) -2-thienyl) propyl) isothiourea

S- (2- (4-fluorophenyl) ethyl) isothiourea

S- (2- (4-bromophenyl) ethyl) isothiourea

S- (2- (3-methoxyphenyl) ethyl) isothiourea

S- (2- (3-methylphenyl) ethyl) isothiourea

S- (2- (4-Ethoxyphenyl) ethyl) isothiourea

S- (2- (4-methoxyphenyl) ethyl) isothiourea

S- (2- (2-bromophenyl) ethyl) isothiourea

S- (2- (2-fluorophenyl) ethyl) isothiourea

S- (2- (3-nitrophenyl) ethyl) isothiourea

S- (3- (1H-pyrrol-1-yl) propyl) isothiourea

S- (2- (2-chlorophenyl) ethyl) isothiourea

S- (2- (2,5-dimethylphenyl) ethyl) isothiourea

S- (2- (4-Ethoxy-3-methoxyphenyl) ethyl) isothiourea and their salts.

Particularly preferred compounds include S, S '- (1,3-phenylenebis (1,2-ethanediyl) diisothiourea, S, S' - (1,4-phenylenebis (1,2-ethanediyl) diisothiourea, S- (2- ( 5-amidinothiomethyl) -2-thienyl) ethyl) isothiourea, S- (3- (5- (2-amidinothio) ethyl) -2-thienyl) propyl) isothiourea and S- (2 '- (3-methoxyphenyl) ethyl) isothiourea.

Other preferred compounds for the treatment of septic shock are S-ethylisothiourea, S-propylisothiourea and S-isopropylisothiourea, especially S-ethylisothiourea and S-isopropylisothiourea, and especially S-ethylisothiourea.

By the term hydrocarbyl group we mean a group containing only carbon and hydrogen atoms but which may contain double and / or triple bonds, and which may be cyclic or aromatic in nature.

By the term heterocyclic ring we mean a cyclic compound containing 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, and preferably nitrogen or sulfur.

By the term halo, we mean fluoro, kioro, bromo or iodine, and preferably bromo.

Compounds of formula (I) may include many asymmetric centers in a molecule, depending on the precise meaning of the various groups, and it is intended that formula (I) includes all possible isomers.

In another aspect, the present invention relates to an isothiourea of formula (I) other than a benzylisothiourea, S, S- (1,4-phenylenebis (methylene)) diisothiourea and S- (2- (dimethylamino) ethyl) isothiourea or their pharmaceutically acceptable salts , which have an inhibitory effect against the NO synthase enzyme for use in medicine.

In another aspect, the present invention relates to novel compounds of formula (IA), (IB) and (IC) as defined previously. Such compounds include:

S, S '- (l, 4-phenylenebis (l, 2-ethanediyl)) diisothiourea

S- (2 (1H-pyrrol-1-yl) ethyl) isothiourea

S - ((2-amino-4-thiazolyl) methyl) -L-cysteine

-y- (2′-amino-4-thiazolyl) -L-homoalanine

S, S '- (l, 2-phenylenebis (l, 2-ethanediyl)) diisothiourea / 3- (2'-amino-4'-thiazolyl) -L-alanine

S- (2′-amino-5 ′ - (R, S) -thiazolinesImethyl) -L-cysteine

4 - ((2-amino-4-thiazolyl) methyl) -L-homoalanine

N, N- (1,3-phenylenebis (methylene)) bis (S-methylisothiourea)

N, N- (1,3-phenylenebis (methylene)) bis (S-ethylisothiourea)

S- (3- (4 - ((amidinothio) methyl) phenyl) propyl) isothiourea

S- (2- (5 - ((amidinothio) methyl) -2-thienyl) ethyl) isothiourea

S- (3- (5- (2-amidinothio) ethyl) -2-thienyl) propyl) isothiourea

S - ((2-amino-4-thiazolyl) methyl) -D-cysteine

S - ((2-amino-4-thiazolyl) methyl) - (D, L) -homocysteine

S- (2- (2-amino-4-thiazolyl) ethyl) -L-cysteine

S- (2- (4-fluorophenyl) ethyl) isothiourea

S- (2- (4-bromophenyl) ethyl) isothiourea

S- (2- (3-methoxyphenyl) ethyl) isothiourea

S- (2- (3-methylphenyl) ethyl) isothiourea

S- (2- (4-Ethoxyphenyl) ethyl) isothiourea

S- (2- (4-methoxyphenyl) ethyl) isothiourea

S- (2- (2-bromophenyl) ethyl) isothiourea

S- (2- (2-fluorophenyl) ethyl) isothiourea

S- (2- (3-nitrophenyl) ethyl) isothiourea

S- (3- (1H-pyrrol-1-yl) propyl) isothiourea

S- (2- (4-Ethoxy-3-methoxyphenyl) ethyl) isothiourea

S- (2- (2,4,6-trimethylphenyl) ethyl) isothiourea

S- (2- (2,6-dimethoxyphenyl) ethyl) isothiourea and their salts.

The present invention includes isothioureas in the form of salts, especially acid addition salts. Suitable salts include those formed with both organic and inorganic acids. Such acid addition salts are generally pharmaceutically acceptable, although salts of non-pharmaceutically acceptable salts may be useful in the preparation and purification of the compounds in question. Thus, preferred salts include those formed from hydrogen chloride, hydrobromic, sulfuric, citric, wine, phosphorus, milk, pyruvic, acetic, trifluoroacetic, amber, oxal, fumar, maleic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, p-toluenesulfonic, acid. Isothiourea salts can be prepared by metabolizing the corresponding compound in the form of a free base with the appropriate acid.

Since it is possible for the isothioureas of the invention to be administered as a crude chemical, it is preferable to present them as a pharmaceutical preparation. In a further aspect, the present invention relates to a pharmaceutical composition comprising an isothiourea of the present invention or a pharmaceutically acceptable salt or solvate thereof, together with one or more of its pharmaceutically acceptable carriers, and optionally one or more other therapeutic ingredients, e.g. antibiotics, and / or volume of replacement fluid. The carrier (c) must be acceptable in the sense that it is compatible with the other constituents of the preparation and is not harmful to its recipient.

The preparations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration, although the most appropriate route may depend on the recipient's condition and disorder . The compositions can be conveniently presented in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All processes involve the step of contacting a compound of formula (I) or a pharmaceutically acceptable salt or solvate (active ingredient) with a carrier consisting of one or more additional ingredients. Generally, preparations are prepared by contacting the active ingredient with a single and direct contact with liquid carriers or finely ground solid carriers or both and then, if necessary, forming the product in the desired formulation.

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

The tablet may be made by compression or molding, optionally with one or more additional ingredients. Compressed tablets may be prepared by compression in a suitable machine, the active ingredients in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricant, surfactant or dispersant. The modeled tablets can be prepared by modeling in a suitable machine, a mixture of a powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or cut and can be formulated to provide a slow or controlled release of the active ingredient contained in the tablet.

Preparations for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostats and dissolved substances that make the preparation isotonic with the intended recipient's blood; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and host agents. The compositions may be presented in unit dose or multi-dose containers, e.g. sealed ampoules and vials and can be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid carrier, e.g. brine, water for injection, just before use. Alternatively, the compositions may be presented for continuous infusion.

Unprepared injectable solutions and suspensions may be prepared from sterile granule powders and tablets of the type previously described.

Rectal administration preparations can be presented as a suppository with conventional carriers such as cocoa butter or polyethylene glycol.

Topical preparations for oral administration, e.g. buccal ah sublingually include lozenges containing the active ingredient in a flavored basis, such as sucrose and acacia or tragacanth, and lozenges containing 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 of the active ingredient as indicated below, or a suitable portion thereof.

It is understood that, in addition to the ingredients specifically mentioned above, the compositions of the invention may include other agents that are customary in the art, taking into account the type of preparation in question, e.g. those suitable for oral administration may include flavoring agents.

For each of the aforementioned conditions, the compounds of the invention may be administered orally or by injection at a dose of from about 0.1 to about 250 mg / kg per day. The dosage range for adults is generally 5 mg to 17.5 g / day, preferably 5 mg to 2 g / day and most preferably 10 mg to 1 g / day. Tablets or other forms of presentation provided in discrete units may suitably contain an amount of a compound of the invention that is effective at such a dose or multiple dose, e.g. units containing 5 mg to 500 mg, usually about 10 mg to 200 mg.

The compounds of formula (I) are preferably administered orally or by injection (intravenously or subcutaneously). The exact amount of compound given to a patient is the responsibility of the treating physician. Of course, the dose used depends on many factors including the age and gender of the patient, the exact disorders being treated and their severity. So the route of administration may vary depending on the condition and its severity.

The present invention also provides processes for the preparation of novel compounds as previously defined, analogous to those known in the art for the preparation of isothiourea derivatives.

Thus, compounds of formula (I) or protected derivatives thereof may be prepared by metabolizing a thiourea with compound RL (L ') _r , wherein R is as previously defined, L and L' are both leaving groups, e.g. a halo atom such as bromo and r is 0 or 1, followed by deprotection if necessary.

Compounds of Formula (IA)

HN

(IA) as defined above can be prepared by metabolizing thiourea with compound LR'L ', with L, L' and R 'as defined above. The appropriate reaction is carried out in a polar solvent such as ethanol at a temperature of 20 ° C to the reflux temperature of the solvent.

The compounds of the formula LR'L 'are commercially available or may be prepared from a suitable diol, HO-R'-OH, suitably by reaction in a polar solvent such as dichloromethane in the presence of a halogenating agent such as carbon tetrabromide and triphenylphosphine.

Compounds of the formula HO-R'-OH are commercially available or can be prepared by methods known in the art.

(ii) Compounds of Formula (IB)

(IB) as defined above can be prepared by deprotecting a compound of formula (IB1) wherein R 'and the dashed line as defined above and P and P' being the same or different and both protecting groups as benzyl, benzoyloxycarbonyl or tert -butoxycarbonyl. The reaction may be carried out in trifluoroacetic acid at a non-extreme temperature of -20 ° C to 100 ° C, such as 0 ° C in the presence of helical molecules such as cyanisole and 1,2-ethanedithiol.

When the dashed line represents a single bond and is a substituent at position -4, compounds of formula (IB1) can be prepared from a compound of formula (IB2) (IB2) wherein R ', P and P' are as previously defined. Reduction of the azide to the amine and cyclization to the thiazoline can be carried out in tetrahydrofuran in the presence of triphenylphosphine.

Compounds of formula (IB2) can be prepared from compounds of formula (IB3)

N / 6S-

ΜΗΡ (ΙΒ3) wherein R, P and P ′ are as previously defined, by displacing tosylate with an azide anion in a polar solvent such as dimethylformamide at a non-extreme temperature of -20 ° C to 200 ° C, such as solvent reflux temperature.

Compounds of formula (IB3) can be prepared from compounds of formula (IB4)

(IB4) wherein R ', P' and P are as previously defined, with the addition of a thiocyanate anion to give a ring-open alkoxide which can be captured as a sullied derivative (IB3) with the addition of para-toluenesulfonyl chloride. The compounds of formula (IB4) can be prepared by one of ordinary skill in the art.

When the dashed line represents a single bond and is a substituent in position-5, compounds of formula (IB1) can be prepared from a compound of formula (IB5)

Ts

N, (ΊΒ5) by replacing tosylate with a thiocyanate anion (Tetrahedron Asymmetry 1992, 749-752), which can be carried out in a polar solvent such as ethanol at non-extreme temperatures, from -20 ° C to 200 ° C, such as reflux temperature solvents, followed by cyclization by methods analogous to those described for the preparation of compounds of formula (IB) from those of formula (IB2). The compounds of formula (IB5) can be prepared from a suitable epoxide by opening the ring with an azide anion, followed by trapping the alkoxide with paratoluenesulfonyl chloride in a polar solvent such as dimethylformamide at non-extreme temperatures from -20 ° C to 200 ° C (100 ° C ( Tetrahedron Lett. 1990, 31, (2), 221).

When the dashed line represents a double bond and the substituent on the thiazole ring is in position-5, compounds of formula (IB1) can be prepared by cyclization of chloroacetals of formula (IB6)

(IB6) wherein R ', P and P' are as previously defined and R ^{10 is a} C 1-6 alkyl group with a thiourea. The reaction can be carried out in a polar solvent such as acetone or ethanol at a non-ester temperature of -20 ° C to 200 ° C (Chem. Abs. 54: 14230d).

Compounds of formula (IB6) can be prepared from sulfuryl chloride and aldehydes of formula (IB7)

P— <

tOHP (IB7) wherein R ', P and P' are as previously defined (Read Indian. Acad. Sci. 1941, 14A, 630-5; Chem. Abs. 36: 410z). The compounds of formula (IB7) are commercially available or can be prepared by one of ordinary skill in the art.

When the dashed line represents a double bond and is a substituent on the thiazole ring in position-4, compounds of formula (IB1) can be prepared by methods known in the art, e.g. α-N-benzyloxycarbonyl- / 3- (2′-amino-4′-thiazolyl) alanine benzyl ester (Synthetic Communications 1990,20 (30), 3097-3102).

(iii) Compounds of Formula (IC)

(IC) can be prepared:

(a) when the dashed line represents a single bond and is a substituent in position-4, by methods analogous to those described for the preparation of compounds of formula (IB) from those of formula (IB4).

(b) When the dashed line represents a single bond and is a substituent at position-5 of the thiazoline ring, by procedures analogous to those described for the preparation of compounds of formula (IB) from those of formula (IB5).

(c) When the dashed line represents a double bond and is a substituent in position-4 of the thiazole ring by metabolizing a compound of formula (ICI)

with thiourea. Suitably, the cyclization reaction may be carried out in a polar solvent such as acetone at a non-extreme temperature of -20 ° C to 200 ° C, such as 20 ° C.

Compounds of formula (ICI) can be prepared from compounds of formula HO ₂ C-R'-CO ₂ H, wherein R 'is as previously defined, by methods known in the art (J. Chem. Soc. 1940,1304-7 Chem. Abs. 35: 113 ³ ).

(d) When the dashed line represents a double bond and is a substituent at position-5 of the thiazole ring by methods analogous to those described for the preparation of a compound of formula (IB) from a compound of formula (IB6).

The action of compounds of formula (I) as inhibitors of NO synthase enzymes has been demonstrated against NO synthase enzymes isolated from human placenta, brain and cytokine-induced cancer cells.

The present invention will be described by way of example only:

Example 1

Preparation of S.SVlAphenylenebisfl4-ethanediyl) diisothiourea dihydrobromide

A solution of 1,4-phenylenedioacetic acid (10.0 g, 51.5 mmol) in tetrahydrofuran (200 ml) was added dropwise to a stirred suspension of lithium aluminum hydride (3.91 g, 103 mmol) in tetrahydrofuran (30 ml). ). The mixture was heated to reflux for 3 hours and then cooled to 0 ° C. Excessive lithium aluminum hydride was quenched by the slow addition of water (4.0 ml), 15% sodium hydroxide (4.0 ml) and water (12 ml). The suspension was stirred with magnesium sulfate for 5 minutes, filtered and concentrated. The crude oil was purified by silica gel chromatography (ethyl acetate / hexane gradient, 50-100%) to provide the intermediate diol (7.38 g, 86%) as a clear, colorless oil. A solution of this oil in dichloromethane (200 ml) at 0 ° C was treated with carbon tetrabromide (32.4 g, 97.7 mmol) and triphenylphosphine (25.6 g, 97.7 mmol). Prior to the addition of pentane (500 ml), the mixture was stirred at 20 ° C for 4 hours. After 15 hours, the solution was decanted from a brown-colored solid, concentrated and purified by silica gel chromatography (ethyl acetate / hexane gradient 0-20%) to give 1,4phenylenebis (1,2-ethanediyl) dibromide (8.2 g , 64%) as oil. A solution of dibromide (4.0 g, 13.7 mmol) and thiourea (2.09 g, 27.4 mmol) in absolute ethanol (100 ml) was refluxed for 2 hours, cooled and concentrated to dryness. The crude solid was recrystallized from ethanol to give 1,4-phenylenebis (1,2-ethanediyl)) dithiourea dihydrobromide (2.89 g, 47%) as a white crystalline solid. Tal. = 234-236 ° C.

Ή NMR (200 MHz, D ₂ O) δ 7.26 (s, 4H), 3.38 (t, J = 7.0 Hz, 4H), 3.01 (t, J = 7.0 Hz, 4H) ).

Anal. calcd. for C24H24B3N3O: C 32.44; H, 4.54; Br 35.97; N, 12.61; S, 14.43.

Found: C, 32.45; H, 4.59; Br 35.89; N, 12.51; S, 14.35.

Example 2

Preparation of S - ((2-amino-4-thiazolyl) methyl) -L-cysteine

To a solution of 2-amino-4-chloromethylthiazole (Spraque JM et al. J. Am. Chem. Soc. 1946, 68, 2155-2159) (1.0 g, 5.4 mmol) and L-cysteine hydrochloride (804 mg , 5.1 mmol) in dimethylformamide (10 ml) was added potassium carbonate (2.5 g). The suspension was stirred for 18 hours at 22 ° C. The resulting mixture was concentrated to dryness, redissolved in water and charged to an ion exchange column (Dowex 50X8, strongly acidic). The product was eluted with a gradient of ammonium hydroxide (0.1 N to 0.5N). The combined product fractions were partially concentrated and lyophilized to give S- (2'-amino-4-triazolylmethyl) -L-cysteine (1.04 g, 87%) as a yellow-brown electrostatic solid.

1 H NMR (300 MHz, D ₂ O) δ 6.5 (s, 1H), 3.8 (m, 1H), 3.6 (s, 2H), 3.05-2.85 (m, 2H) ). Mass spectrum (CI) 234 (M + 1, 71%).

Example 3

Preparation of 7- (2'-amino-4 ^, -thiazolyl) -L-homoalanine (3 - ((2-amino-4-thiazolyl) methyl-L-alanine)

7- (2'-amino-4'-thiazolyl) -L-homoalanine was prepared from α-N-butoxycarbonyl-γ- (2'amino-4'-thiazolyl) -L-homoalanine benzyl ester (Patt et al. Synth. Commun. 1990, 20 (20), 3097-3102) in a 9.6% gain following the procedure of Example 6.

Ή NMR (D ₂ O) δ 6.5 (s, 1H), 3.9 (t, J = 6.4 Hz), 2.75 (m, 2H), 2.2 (m, 2H).

Anal. calcd. for C ₇ H _n N ₃ O ₂ SC ₂ HF ₃ O ₂ .0.3 H ₂ O: C 29.31; H, 2.89; N, 8.54; S, 6.52.

Found: C, 29.37; H, 3.02; N, 8.53; S, 6.63.

Example 4

Preparation of S.SVl.S-phenylenebisfl ^ -etandiiDdiisothiourea of dihydrobromide

S, S '- (1,3-Phenylenebis (1,2-ethanediyl)) diisothiourea dihydrobromide was prepared according to the procedure of Example 1 from 1,3-phenylenedioacetic acid (Aldrich). Recrystallization from ethanol gave a white crystalline solid (mp 194-190 ° C).

Ή NMR (200 MHz, D ₂ O) δ 7.4-7.2 (m, 4H), 3.39 (t, J = 6.9 Hz, 4H), 3.02 (t, J = 6. 9 Hz, 4H).

Anal. calcd. for C24H24B3N3O: C 32.44; H, 4.54; Br 35.97; N, 12.61; S14.43.

Found: C, 32.52; H, 4.49; Br, 36.04; N, 12.61; S, 14.35.

Example 5

Preparation of S.SVl ^ -phenylenebisfl ^ -etandiiDdiisothiourea dihydrobromide

S, S '- (1,2-Phenylenebis (1,2-ethanediyl)) diisothiourea dihydrobromide is prepared from 1,2-phenylenedioacetic acid (Aldrich) in 30% total yield as a yellow foam according to the procedure of Example 1.

¹ H NMR (300 Mhz, D ₂ O) δ 7.29 (s, 4H), 3.38 (t, J = 7.0 Hz, 4H), 3.09 (t, J = 7.0 Hz) . Recrystallization from ethanol gave an analytical sample, mp = 205-207 ° C.

Anal. for C ₁₂ H _2Q Br ₂ N ₄ S ₂ : C 32.44; H, 4.54; Br 35.97; N, 12.61; S, 14.43. Found: C, 32.47; H, 4.58; Br 35.92; N, 12.58; S, 14.43.

Example 6

Preparation of 8- (2 ^- amino-4 ^, -thiazolyl) alanine (3- (2-amino-4-thiazolyl) -L-alanine)

To a mixed solution of N-butoxycarbonyl- / 3- (2'-amino-4'-thiazolyl) alanine benzyl ester (1.47 g) (Patt et al., Synth. Commun. 1990, 20 (20), 2097-3102 ) in dichloromethane (15 ml) at -88 ° C under nitrogen atmosphere was added triethyl silane (3 ml) followed by trifluoroacetic acid (3 ml). Before concentrating, the solution was warmed to room temperature for 1 hour. The residue was treated with acetic acid (30 ml), 20% Pd / C (2.0 g) and 1,4-cyclohexanediene (20 ml). The mixture was sonicated from room temperature to 33 ° C for 2 hours and filtered through celite, washed with water. The filtrate was concentrated and redissolved in 30 ml of acetic acid and treated with 2.0 g of fresh 20% Pd / C and 2 ml of 1,4-cyclohexandiene. After sonication for two hours, the suspension was filtered through celite. This process of adding fresh catalyst was repeated 3 times before the crude product was purified by repeated semi-prep. reverse phase chromatography (C-18, elution with 10% methanol / water containing 0.1% trifluoroacetic acid. Freeze-dried product fractions gave 337 mg (18%) / 3- (2'-amino-4'-thiazolyl) - L-Alanine as a sticky glass-like solid.

1 H NMR (D ₂ O) δ 6.6 (s, 1H), 4.0 (t, J = 7.6 Hz, 1H), 3.15 (m, 2H). Mass spectrum (Cl) 188 (M + 1).

Anal. calcd. for C ₁₂ H ₁₂ N ₃ F ₉ O _g S: C 27.23; H, 2.29; N, 7.94; S, 6.06.

Found: C 27.22; H, 2.42; N, 7.8; S, 5.67.

Example 7

Preparation of S, S ^, - (2,6-pyridylenebis (methylene)) diisothiourea

To a solution of 2,6-pyridindimethanol (5.0 g, 35.9 mmol) in dichloromethane (200 ml) at 0 ° C was added carbon tetrabromide (23.83 g, 71.9 mmol) and triphenylphosphine (18.85 g, 71.9 mmol). The solution was stirred for 6 hours at warming to 20 ° C. Pentane (300 ml) was added. The solution was allowed to stand at 20 ° C for 16 hours before being filtered to remove solid impurities. Concentrate the filtrate to. of the oil purified by silica gel chromatography (hexanes followed by 10% ethyl acetate / hexanes) to give 2,6-pyridindibromide (4.47, 47%) as a gray-white powdery solid. To a solution of 2,6-pyridindibromide (3.94 g, 14.87 mmol) in ethanol (100 ml) was added thiourea (2.26 g, 29.74 mmol) and the resulting suspension was stirred at reflux for 2 hours. The solution was concentrated to dryness to give S, S '(2,6-pyridylenebis (methylene)) diisothiourea (5.3 g, 83.9%) as a white powder.

1 H NMR (200 MHz, DMSO) S 7.95 (t, J = 7.6 Hz, 1H), 7.51 (d, J = 7.8 Hz, 2H), 4.65 (s, 4H) .

Anal. calcd. for C ₉ H ₁₅ N ₅ S ₂ Br ₂ .0.6 H ₂ O: C 25.26; H, 3.81; N, 16.36; S, 14.98; Br 37.34. Found: C, 25.3; H, 3.78; N, 16.25; S, 14.94; Br 37.38.

Example 8

Preparation of S- (2 ^, -amino-5 ^, - (RS) -thiazolinylmethyl-L-cysteine

To a solution of 2-amino-5-iodomethylthiazoline (4.44 g, 12 mmol) (Creeke & Mellor, Tet. Lett. 1989, 30 (33), 4435-4438) and L-cysteine hydrochloride (1.76 g, 10 , 0 mmol) in dimethylformamide (75 ml) was added potassium carbonate (5.0 g 36 mmol). The suspension was stirred for 72 hours at 22 ° C and refluxed for 30 minutes. Acetonitrile was added and the mixture filtered. The solids recovered were washed several times with warm methanol. Dilution with methanol solutions with ethanol gives a white precipitate which is removed by filtration. The concentrated filtrate (oil) was taken up in methanol / ethanol and treated with ethanol hydrogen chloride until further precipitation was observed and the mixture filtered. The oil obtained by concentrating the filtrate was purified by ion exchange chromatography (Dowex 50X8, strongly acidic), eluting with 0, 1 N ammonium hydroxide. Ninhydrin positive fractions were combined and lyophilized to give 0.453 g of a slightly yellow-brown colored dimethylformamide-contaminated solid. This solid was purified by preparative HPLC (C18 reverse phase, methanol: water: trifluoroacetic acid / 5: 95: 0, l) to give 0.36 g of S- (2'-amino-5 '(R, S) - thiazolinylmethyl) -L-cysteine as the trifluoroacetate salt. TLC (ammonium hydroxide: methanol / l: 50) Rf = 0.5. Ή NMR (200 MHz, D ₂ O) δ 4.37-4.24 (m, 1H), 4.11-3.87 (m, 3H), 3.27-3.13 (m, 2H), 3.05-2.97 (m, 2H). Mass spectrum (FAB) 236.0 (M + 1, 48%).

Anal. calcd. for C _? H ₁₃ N ₃ O ₂ S ₂ .3 (C ₂ HF ₃ O ₂ ): C 27.04; H, 2.79; N, 7.28; S, 11.11. Found: C 27.32; H, 2.91; N, 7.41; S, 11.17.

Example 9

Preparation of 4 ((2-amino-4-thiazolyl) methyl-L-homoalanine

4 - ((2-amino-4-thiazolyl) methyl) -Na: -t-Boc-L-homoalanine t-butyl ester was prepared according to the procedure of Patt et al, (Synth. Commun. 1990, 20 (20), 3097- 3102) in 45% total yield (1.3 g) of Nt-Boc-L-2-aminoadipic acid 1-t-butyl ester (Ramsamy et al. Synthesis, 1982, 42-43). The t-Boc and t-butyl ester protecting groups are removed as follows:

To a solution of 1.68 g of 4 - ((2-amino-4-thiazolyl) methyl) -Nol-t-Boc-L-homoalanine t-butyl ester in 35 ml of dioxane was added 1.1 ml of triethylsilane and 8 ml of 4N hydrogen chloride acids in dioxane solution. After stirring for 16 hours at 22 ° C, the mixture was filtered and the solids were washed with dioxane. The crude sample NMR indicates an unfinished reaction. Re-dissolve the crude solid in 20 ml of dioxane and treat with 4N hydrochloric acid (5 ml) for 4 hours. The solids were isolated by filtration, dissolved in water, and lyophilized to give 1.23 g (80%) of 4 - ((2-amino-4-thiazolyl) methyl) -L-homoalanine as a hygroscopic white solid (bis- hydrochloride hydrated with 1.4 mol% water and solvated with 0.3 mol% dioxane). HPLC analysis; Phenomenex C 18, water / methanol / trifluoroacetic acid (95/5 / 0,1), k '= 0,34.

Ή NMR (300 MHz, DMSO-d ₆ ) δ 9.25 (broad with 2H), 8.5 (wide with 2H), 6.55 (s, 1H), 3.93 (m, 1H), 2. 6 (m, 2H), 1.8 (m, 4H).

Anal. calcd. for C _g H ₁₃ N ₃ O ₂ S · 2HC1 · 1.4 Κ, Ο · 0.3 dioxane: C 32.51, H 5.99; N, 12.36; S, 9.43; Cl, 20.86. Found: C, 32.28; H, 5.72; N, 12.71; S, 9.60; Cl, 20.86.

Example 10

Preparation of N, N'-fh3-phenylenebisfmethylene) bisfS-methylisothiourea)

7.0 ml (52 mmol) of benzoylisothiocyanate were added to a 0 ° C stirred solution of 3.30 g (25 mmol) of m-xylylenediamine (Aldrich Chemical) in 100 ml of dichloromethane. The mixture was stirred for 18 hours at 20 ° C and the solvent was removed under reduced pressure. The crude solids (pale yellow) were suspended in 100 ml of 10% sodium hydroxide solution and refluxed for 5 minutes. The mixture was acidified with concentrated hydrochloric acid while still hot, cooled and then made basic with the addition of ammonium hydroxide. The white precipitated solids were collected and dried at 60 ° C under reduced pressure to constant weight to give 4.82 g of the bis-thiourea intermediate as a gray-white solid.

Anal. calcd. for C ₁₀ H ₁₄ N ₄ S ₂ : C 47.22; H, 5.55; N, 22.03; S, 25.10. Found: C, 47.49; H, 5.50; N, 21.81; S, 25.01.

To a solution of 2.54 g (10 mmol) of the bis-urea intermediate in 25 ml of dimethylformamide was added 5.0 ml (80 mmol) of iodomethane. The solution was stirred for 65 hours at 20 ° C, the solvent was removed under reduced pressure and the residue was recrystallized from hot ethanol. The pale yellow crystals were dried under reduced pressure at 60 ° C to give 4.28 g (81%) of N, N '- (1,3-phenylenebis (methylene)) bis (S-methylisothiourea), m.p. Mp = 164-167 ° C. TLC (one site on silica gel with 1% ammonium hydroxide in methanol, Rf = 0.48). NMR (300 MHz DMSO / D ₂ O) δ 7.5-7.4 (m, 1H), 7.35-7.2 (m, 3H), 4.59 (s, 4H), 2.65 ( s, 6H).

Anal. calcd. for C ₁₂ H ₁₈ N ₄ S ₂ · 1.9 HJ: C 27.43; H, 3.82; N, 10.66; S, 12.20; J 45.89.

Found: C 27.30; H, 3.91; N, 10.57; S, 12.19; 145.92.

Example 11

Preparation of N.NVU-phenylenebisfmethylenebisfS-ethylisothiourea)

To a solution of 1.0 g (3.93 mmol) of the bis-urea prepared in Example 10 in 20 ml of ethanol was added 6.29 g (78.62 mmol) of iodoethane. The mixture was heated to reflux for 8 hours and concentrated to foam under reduced pressure. The crude product was purified by silica gel chromatography with methanol in dichloromethane (10% to 20%) to give 1.66 g (74%) of N, N '- (1,3-phenylenebis (methylene)) bis (S-ethylisothiourea). TLC (Rf = 0.30.48.20% methanol in dichloromethane). Mass spectrum (FAB) 311.2 (M + 1).

1 H NMR (200 MHz, DMSO-d ₆ ) δ 7.5-7.4 (m, 1H), 7.31-7.22 (m, 3H), 4.58 (s, 4H), 3. 20 (q, J = 7.4 Hz, 4H), 1.28 (t, J = 7.4 Hz, 6H).

Anal. calcd. for C 22 H 27 N 4 N 2 O 2: C 29.69; H, 4.27; N, 9.89; S, 11.32; J 44.82.

Found: C, 29.44; H, 4.25; N, 9.64; S 11.50; J 44.65.

Example 12

Preparation of S- (3- (4-amidinothio) methyl) phenyl) propyl isothiourea

From 3- (4-carboxyphenyl) propionic acid (Lancaster synthesis), 3- (4 ((amidinothio) methyl) phenyl) propyl) isothiourea was prepared as a white solid (2.95 g, mp = 190195 ° C) with by the procedure of Example 1. Mass spectrum (FAB) 283 (M + 1). ^X H NMR (200 MHz, D ₂ O) δ 7.38 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 8.1 Hz, 2H), 4.36 (s. 2H), 3.06 (t, J = 7.2 Hz, 2H), 2.74 (t, J = 7.4 Hz, 2H), 2.1-1.9 (m, 2H).

Anal. calcd. for C ₁₂ H ₁₈ N ₄ S ₂ · 2HBr: C 32.44; H, 4.54; N, 12.61; S, 14.43; No. 35.97. Found: C, 32.53; H, 4.58; N, 12.56; S, 14.34; No. 35.85.

Example 13

Preparation of S- (2- (5 - ((amidinothio) methyl) -2-thienyl) ethyl) isothiourea

To a solution of 21.48 g (0.19 mol) of α-dichloromethyl methyl ether (Fluka) in 300 ml of dichloromethane at 0 ° C was added 44.76 g (0.172 mol) of tin (IV) chloride (Aldrich). After 15 minutes, a solution of 24.01 g (0.14 mol) of ethyl 2-thiophenacetate (Aldrich) in 50 ml of dichloromethane was added dropwise over a few minutes. Pour the mixture into water and ice for 1 hour and stir for 30 minutes. The dichloromethane layer was washed with water, dried over sodium sulfate and concentrated. The crude product was purified by silica gel chromatography with 10% ethyl acetate in hexanes to give 22.85 g (82%) of the intermediate, 5-formyl-2-thiophenoacetic acid ethyl ester.

To a 0 ° C mixed suspension of 0.77 g (20.29 mmol) of lithium aluminum hydride (Aldrich) in 200 ml of tetrahydrofuran was added a solution of 2.0 g (10.09 mmol) of the intermediate prepared above in 50 ml of tetrahydrofuran. The suspension was stirred for 16 hours at 20 ° C, cooled to 0 ° G, and the excess hydride was quenched by carefully adding 0.8 ml of water, 0.8 ml of IN sodium hydroxide solution and 2.4 ml of water. The suspension was stirred with magnesium sulfate, filtered, concentrated and purified by silica gel chromatography with 50% ethyl acetate in hexanes to 100% ethyl acetate. 1.07 g (67%) of the diol intermediate is isolated and directly converted to dibromide as follows.

The diol intermediate (1.07 g, 6.76 mmol) in 50 ml of dichloromethane at 0 ° C was treated with 3.90 g (14.87 mmol) of triphenylphosphine and 4.93 g (14.87 mmol) of carbon tetrabromide. The solution was stirred for 1 hour before adding 200 ml of pentane. On the surface, the floating substance was decanted from the oily residue, concentrated and purified by silica gel chromatography with hexanes to give 1.10 g (57%) of the dibromide intermediate. The dibromide product (1.10 g, 3.87 mmol) in 50 ml ethanol was treated with 0.59 g (7.75 mmol) of thiourea. The solution was stirred at reflux for 2 hours. Purify the concentrated solution by preparative HPLC (Waters C18 BondaPak PrepPak Fill) with a methanol / water / trifluoroacetic acid gradient (5/95 / 0.1 to 90/10 / 0.1). The combined product fractions were concentrated, diluted with water and lyophilized to give 306 mg of S- (2- (5 - ((amidinothio) methyl) -2-thienyl) ethyl) isothiourea as a bis-trifluoroacetic acid salt and 0.1 mol of hydrate ( mp = 186-190 ° C). ^X H NMR (200 MHz, D ₂ O) δ 6.98 (d, J = 3.5 Hz, 1H), 4.57 (s, 2H), 3.38 (t, J = 6.5 Hz, 2H), 3.2 (t, J = 6.5 Hz, 2H).

Analysis of calc. for C ₉ H ₁₄ N ₄ S ₃ · 2.0 C ₂ HF ₃ O ₂ · 0.1 H ₂ O: C 30.96; H, 3.24; N, 11.11; S, 19.08. Found: C, 31.02; H, 3.19; N, 11.00; S, 18.97.

Example 14

Preparation of S- (3- (5- (2- (amidinothio) ethyl) -2-thienyl) propyl) isothiourea

5-Formyl-2-thiophenoacetic acid ethyl ester was prepared as described in Example 13. To a solution of 2.0 g (10.09 mmol) of this ester in 100 ml of tetrahydrofuran was added 3.87 g (11.10 mmol) of carboxymethylenetrophenylphosphorane. The solution was refluxed overnight and concentrated. The crude product was combined with another 2.0 g of the reaction mixture, using 10.54 g (30.27 mmol) of carboethoxymethylenetrophenylphosphorane refluxed in 100 ml of tetrahydrofuran for 3 hours. The combined crude products were purified by silica gel chromatography (10% ethyl acetate / hexane) to give 2.39 g (44%) and a diester intermediate. To a solution of 1.0 g (3.73 mmol) of this en-diester in 50 ml of ethanol was added 1.0 g of 10% palladium on carbon. The mixture was shaken for 14 hours at 20 ° C below 344.74 kPa of hydrogen. The catalyst was removed by filtration over celite and the solution concentrated to give 1.0 g of crude diester intermediate. The final three reactions at this intermediate, which include reduction with lithium aluminum hydride (82%), bromination of the resulting diol (78%) and alkylation of dibromide with thiourea (88%) are analogous to those described in Example 13. The crude alkylation reaction product was purified by preparative HPLC (Waters, 08 BondaPak PrepPak fill) eluting with a methanol / water gradient of 10% to 90% methanol over 40 minutes (spraying with 0.1% trifluoroacetic acid). The product fractions were lyophilized to give 1.53 g of S - ((3- (5- (2-amidinothio) ethyl) -2-thienyl) propyl) isothiourea as a mixed salt (1.0 HBr, 1.1 TFA) and 0 , 5 mol solvate with methanol. Tal. = 146-151 ° C. Mass spectrum (FAB) 303. (M + 1). * H NMR (200 MHz, D ₂ O) δ 6.77 (d, J = 3.3 Hz, 1H), 6.73 (d, J = 3.3 Hz, 1H), 3.37 (t. J = 6.6 Hz, 2H), 3.21-3.03 (m, 2H), 2.91 (t, J = 7.2 Hz, 2H), 2.1-1.95 (m, 2H) ).

Anal. calcd. for C _n H ₁₈ N ₄ S ₃ · 1.0 HBr · 1.1 C ₂ HF ₃ O ₂ · 0.5 H ^ O; C, 31.35; H, 4.24; Br 15.22; N, 10.68; S, 18.05. Found: C, 31.59; H, 4.01; Br 14.94; N, 10.45; S, 18.05.

Examples 15-17 were prepared according to the procedure of Example 2. The crude products were purified by preparative HPLC (Waters, 08 BondaPak PrepPak Filling). Methanol / water / trifluoroacetic acid gradient elution (5/95 / 0.1 to 90/10 / 0.1), followed by lyophilization, provides the target amino acids as the trifluoroacetic acid addition salts.

Example 15

S - ((2-amino-4-thiazolyl) methyl) -D-cysteine

Prepared from D-cysteine and 2-amino-4-chloromethylthiazole (Spraque, JM et al, J. Am. Chem.Soc. 1946, 68, 2155-2159). Analytical HPLC - phenomenex C18 water / methanol / hepentafluorobutyric acid (80/20 / 0,17), one peak, k '= 1,9. UV (pH 7.0 buffer) X _max 254 nm (log ε 3.73). ⁷ H NMR (200 MHz, DMSO-d ₆ ) δ 8.1 (broad, 2H), 8.1-7.5 (broad, 2H), 6.5 (s, 1H), 4.25 (m. 1H), 3.65 (d, J = 6 Hz, 2H), 3.0 (m, 2H).

Analysis of calc. for C ₇ H _n N ₃ O ₂ S · 2.3 C ₂ HF ₃ O ₂ · 0.1 H ₂ O; C, 27.92; H, 2.72; N, 8.43; S, 12.85. Found: C 28.00; H, 2.89; N, 8.74; S, 12.62.

Example 16

S - ((2-amino-4-thiazolyl) methyl) - (D, L) -homocysteine

Prepared from (D, L) -homocysteine and 2-amino-4-chloromethylthiazole (Spraque, JMet al. J. Am.Chem.Soc. 1946, 68, 2155-2159). Analytical HPLC - phenomenex 08, water / methanol / trifluoroacetic acid (95/5 / 0,1), one peak, k '= 2,3 UV (pH 7,0 buffer) X _max 254 nm (log ε 3,73) . 1 H NMR (200 MHz, DMSO-d ₆ ) δ 8.5-8.0 (broad, 2H), 7.27.0 (broad, 2H), 6.35 (s, 1H), 4.0 ( t, J = 4 Hz, 1H), 3.55 (s, 2H), 2.6 (m, 2H), 2.0 (m, 2H).

Anal. calcd. for C _g H ₃ N ₃ O ₂ S · 1.1 C ₂ HF ₃ O ₂ · 1.0 f ^ O; C, 31.35; H, 4.15; N, 10.75; S, 16.41. Found: C 31.30; H, 4.07; N, 10.73; S, 16.31.

Example 17

S- (2- (2-amino-4-thiazolyl) ethyl) -L-cysteine

From L-cysteine and 2-amino-4- (2-bromoethyl) thiazole (prepared from 1,5-dibromo-2-butanone according to the procedure of Spraque et at J. Am.Chem.Soc. 1946,68, 2155-2159).

Analytical HPLC - Phenomenex 08, water / methanol / trifluoroacetic acid (95/5 / 0,1), one peak, k '= 2,6. UV (pH 7.0 buffer) X _max 256 nm (log ε 3.71). ^X H NMR (200 MHz,

DMSO-d ₆ ) δ 9.2-8.8 (broad, 2H), 8.6-8.2 (broad, 2H), 6.6 (s, 1H), 4.2 (m, 1H). 3.0 (m, 2H), 2.8 (m, 4H).

Anal. calcd. for C ₈ H ₁₃ N ₃ O ₂ S ₂ · 2.0 C ₂ HF ₃ O ₂ · 2.4 H ₂ O; C, 27.79; H, 3.85; N, 8.10; S, 12.37. Found: C 27.65; H, 3.69; N, 8.02; S 12.38.

Example 18

Preparation of S- (2- (4-bromophenyl) ethyl) isothiourea hydrobromide

To a solution of 4-bromophenethyl alcohol (720 mg, 0.50 ml, 3.58 mmol) and triphenylphosphine (1.13 g, 4.30 mmol) in dichloromethane (7.0 ml) at 0 ° C was added carbon tetrabromide (1 , 42 g, 4.30 mmol). The reaction mixture was stirred for 30 minutes while warming to room temperature. The solution was poured into hexane (100 ml) and filtered through celite. The solvents were removed in vacuo, hexane was added and the solution filtered through celite. After removal of the solvent in vacuo, the crude material was distilled off (120 ° C / 93.33 Pa) to give a clear oil.

The clear oil was dissolved in 95% ethanol (7.0 ml) and thiourea (300 mg, 3.94 mmol) was added. The reaction mixture was heated to reflux for 16 hours, cooled to room temperature and the solvent removed in vacuo to give a white solid. The solid was suspended in hot acetone and filtered to give 832 mg (68% yield) of the title compound.

* H NMR (300 MHz, d-DMSO _fi); δ 9.02 (s, 4H), 7.52 (d, J = 8.3 Hz, 2H), 7.26 (d, J =

8.3 Hz, 2H), 3.43 (t, J = 7.6 Hz, 2H), 291 (t, J = 7.4 Hz, 2H).

MS (CI) for C ₉ H ₁₂ N ₂ SBr ₂ , m / z relative intensity 259 (M + -Br, 100), 183 (68), 77 (92).

Elemental analysis for C ₉ H ₁₂ N ₂ SBr ₂ , calcd: C 31.79; H, 3.56; N, 8.24; S, 9.43; Br 46.99. Found: C, 31.84; H, 3.59; N, 8.19; S, 9.34; No. 46.92.

Example 19

Preparation of S- (2- (4-fluorophenyl) ethyl) isothiourea hydrobromide

Prepared from 4-fluorophenyl alcohol according to the procedure of Example 18. The title compound was purified by recrystallization from absolute ethanol.

1 H NMR (300 MHz, d ₆ -DMSO); δ 9.10 (broad, s, 2H), 8.96 (broad s, 2H), 7.32 (m, 2H), 7.14 (m, 2H), 3.43 (t, J = 6. 8 Hz, 2H), 2.91 (t, J = 6.8 Hz, 2H).

MS (Cl) for C ₉ H ₁₂ N ₂ SFBr, m / z relative intensity 199 (M + -Br, 42), 77 (100). Elemental analysis for (C ₉ H ₁₂ N ₂ SF) (CH ₄ N ₂ S) ₀₆₃ (HBr) ₁₀₆ , calcd: C 34.84; H, 4.43; N, 13.75; S, 15.74; Nr 25.51. Found: C, 35.31; H, 4.41; N, 13.62; S, 15.51; Nr 25.92.

Example 20

Preparation of S- (2- (4-Ethoxy-3-methoxyphenyl) ethyl) isothiourea hydrobromide

Prepared from 4-ethoxy-3-methoxyphenethyl alcohol according to the procedure of Example 18. The title compound was purified by recrystallization from absolute ethanol.

Ή NMR (300 MHz, d ₆ -DMSO); δ 8.95 (broad s, 4H), 6.87 (s, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.73 (d, J = 8.2 Hz, 1H) ), 3.94 (q, J = 7.0 Hz, 2H), 3.73 (s, 3H), 3.39 (t, J =

7.3 Hz, 2H), 2.83 (t, J = 7.4 Hz, 2H), 1.28 (t, J = 7.0 Hz, 3H).

MS (Cl) for C ₁₂ H ₁₉ N ₂ O ₂ SBr, m / z relative intensity 255 (M + -Br, 56), 179 (100).

»

Elemental analysis for C ₁₂ H ₁₉ N ₂ O ₂ SBr, calculated: C 42.99; H, 5.71; N, 8.36; S, 9.56; Nr 23.83. Found: C 43.09; H, 5.73; N, 8.42; S, 9.61; Nr 23.89.

Example 21

Preparation of S- (2- (4-ethoxyphenyl) ethyl) isothiourea hydrochloride

Prepared from 4-ethoxyphenethyl alcohol according to the procedure of Example 18. The crude product was dissolved in water and 2 molar equivalents of aqueous sodium picrate were added. Light yellow precipitate was isolated by filtration and chromatographed on an AG1-X2 ion exchange resin (200-400 mesh, chloride form 10: 1 water: methanol eluent) to give the hydrochloride salt.

Ή NMR (300 MHz, d ₆ -DMSO); δ 9.19 (broad s, 4H), 7.19 (d, J = 8.1 Hz, 2H), 6.82 (d, J = 8.1 Hz, 2H), 3.9.4 (q, J = 7.0 Hz, 2H), 3.39 (t, J = 7.3 Hz, 2H), 2.83 (t, J = 7.4 Hz, 2H), 1.28 (t, J = 7 , 0 Hz, 3H).

MS (Cl) for C _n H ₁₇ N ₂ OSCl, m / z relative intensity 225 (M + -C1.37), 149 (100).

Elemental analysis for (C _U H ₁₆ N ₂ OS) (ΗΟ) _1θ8 (ΟΗ ₄ Ν ₂ 8) _θθ8 (Η ₂ 0) _θ16 , calc .: C 48.81; H, 6.55; N, 11.10; S, 12.70; Cl, 14.04. Found: C, 48.91; H, 6.41; N, 11.12; S, 12.55; Cl, 14.20.

Example 22

Preparation of S- (2- (2,6-dimethoxyphenyl) ethyl) isothiourea hydrochloride

Prepared from 2,6-dimethoxyphenethyl alcohol according to the procedure of Example 21.

1 H NMR (300 MHz, d ₆ -DMSO); δ 9.14 (broad s, 4H), 7.18 (t, J = 8.2 Hz, 1H), 6.07 (d, J = 8.2 Hz, 2H), 3.73 (s, 6H ), 3.39 (t, J = 7.3 Hz, 2H), 2.83 (t, J = 7.4 Hz, 2H).

MS (CI) for C ₁₁ H ₁₇ N ₂ O ₂ SC1, m / z relative intensity 241 (M + -C1,25), 165 (100).

Elemental analysis for C _n H ₁₇ N ₂ O ₂ SCl, calcd: C 47.74; H, 6.19; N, 10.12; S, 11.48; Cl, 12.81. Found: C, 47.77; H, 6.19; N, 10.07; S, 11.48; Cl, 12.87.

Example 23

Preparation of S- (2- (4-methoxyphenyl) ethyl) isothiourea hydrochloride

Prepared from 4-methoxyphenethyl alcohol according to the procedure of Example 21.

Ή NMR (300 MHz, d ₆ -DMSO); δ 9.19 (broad s, 4H), 7.19 (d, J = 8.0 Hz, 2H), 6.82 (d, J = 8.1 Hz, 2H), 3.73 (s, 3H ), 3.39 (t, J = 7.3 Hz, 2H), 2.83 (t, J = 7.2 Hz, 2H).

MS (CI) for C _n H ₁₇ N ₂ OSCl, m / z (relative intensity) 211 (M + -C1,22), 135 (100).

Elemental analysis for (C ₁₀ H ₁₅ N ₂ OSC1), calcd: C 48.68; H, 6.13; N, 11.36; S, 12.89; Cl, 14.45. Found: C, 48.70; H, 6.10; N, 11.36; S, 12.89; Cl, 14.45.

Example 24

Preparation of 2- (2- (3-methoxyphenyl) ethyl) isothiourea hydrochloride

Prepared from 3-methoxyphenethyl alcohol according to the procedure of Example 21.

Ή NMR (300 MHz, d ₆ -DMSO); δ 9.00 (broad s, 4H), 7.21 (t, J = 7.9 Hz, 1H), 6.80 (m, 3H), 3.72 (s, 3H), 3.43 (t , J = 7.0 Hz, 2H), 2.88 (t, J = 7.4 Hz, 2H).

MS (CI) for C ₁₀ H ₁₇ N ₂ OSCl, m / z (relative intensity) 211 (M + -C1,100), 135 (10), 77 (70).

Elemental analysis for (C ₁₀ H ₁₆ N ₂ OS) (HCl) ₁₀₅ (CH ₄ N ₂ S) ₀₂₀ (H ₂ O) _{oo 5} , calcd: C 45.39; H, 6.13; N, 12.62; S, 14.45; Cl, 13.61. Found: C, 45.28; H, 6.18; N, 12.54; S, 13.43; Cl, 13.65.

Example 25

Preparation of S- (2- (2,4,6-trimethylphenyl) ethyl) isothiourea hydrobromide

Prepared from 2,4,6-trimethoxyphenethyl alcohol according to the procedure of Example 18. The title compound was purified by recrystallization from absolute ethanol.

Ή NMR (300 MHz, d ₆ -DMSO); δ 9.00 (s, 4H), 6.80 (s, 2H), 3.24 (t, J = 6.8 Hz, 2H), 2.84 (t, J = 6.7 Hz, 2H) , 2.49 (s, 3H), 2.28 (s, 6H).

MS (CI) for C ₁₂ H ₁₉ N ₂ SBr, m / z (relative intensity) 223 (M + -Br, 50), 147 (100).

Elemental analysis for C ₁₂ H ₁₉ N ₂ SBr, Calc .: C 47.53; H, 6.32; N, 9.24; S, 10.57; Nr 26.53. Found: C, 47.26; H, 6.30; N, 9.34; S, 10.49; Br 26.48.

Example 26

Preparation of S- (2- (3-methylphenyl) ethyl) isothiourea hydrobromide

Prepared from 3-methylphenethyl alcohol according to the procedure of Example 18. The title compound was purified by recrystallization from absolute ethanol.

Ή NMR (300 MHz, d ₆ -DMSO); δ 9.08 (broad s, 2H), 9.03 (broad s, 2H), 7.20 (t, J = 7.5 Hz, 1H), 7.08 (m, 3H), 3.43 ( t, J = 7.8 Hz, 2H), 2.88 (t, J = 7.6 Hz, 2H), 2.28 (s, 3H).

MS (CI) for C ₁₀ H ₁₅ N ₂ SBr, m / z (relative intensity) 195 (m + -Br, 100), 119 (56), 77 (42).

Elemental analysis for (C _1Q H ₁₅ N ₂ SBr) (CH ₄ N ₂ S) ₀₁₄ , calcd: C 42.60, H 5.49, N 11.17; S

12.79; No. 27.95. Found: C, 42.66; H, 5.50; N, 11.20; S, 12.57; No. 27.79.

Example 27

Preparation of S- (2- (2-fluorophenyl) ethyl) isothiourea hydrochloride

Prepared from 2-fluorophenyl alcohol according to the procedure of Example 21.

Ή NMR (300 MHz, d ₆ -DMSO); δ 9.21 (broad s, 4H), 7.39 (m, 1H), 7.28 (m, 1H), 7.17 (m, 2H); 3.43 (t, J = 7.7 Hz, 2H), 2.92 (t, J = 7.8 Hz, 2H).

MS (Cl) for C ₉ H ₁₂ N ₂ SFC1, m / z (relative intensity) 199 (M + -C1,100), 123 (39).

Elemental analysis for C ₉ H ₁₂ N ₂ SFC1, calcd: C 46.05; H, 5.15; N, 11.93; S, 13.66; Cl 15.10. Found: C, 45.93; H, 5.11; N, 11.83; S, 13.59; Cl, 15.14.

Example 28

Preparation of S- (2- (3-nitrophenyl) ethyl) isothiourea hydrobromide

To a solution of 3-nitrophenethyl alcohol (500 mg, 2.99 mmol) and pyridine (35.6 mg, 36 μΐ, 0.45 mmol) in THF (10 ml) at 0 ° C was added phosphorus tribromide (298 mg, 0, 10 ml, 1.10 mmol). A white precipitate immediately forms. The reaction mixture was warmed to room temperature and stirred for 1 hour. Water and ether were added and the phases were separated. The organic phase was washed with saturated aqueous sodium bicarbonate and dried over anhydrous magnesium sulfate. The mixture was filtered and the solvents removed in vacuo to give a brown oil.

The crude bromide was dissolved in 95% ethanol (10 ml) and thiourea (251 mg, 3.30 mmol) was added. The reaction mixture was heated to reflux for 16 hours, cooled to room temperature and the solvent removed in vacuo. The crude yellow solid was suspended in acetone and the mixture heated to reflux for 10 minutes. The hot solution is filtered to give a white solid.

Ή NMR (300 MHz, d ₆ -DMSO); δ 9.12 (broad s, 2H), 8.96 (broad s, 2H), 8.22 (s, 1H), 8.14 (d, J = 7.9 Hz, 1H), 7.78 ( d, J = 7.6 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H), 3.50 (t, J = 7.0 Hz, 2H), 3.10 (t, J = 7.2 Hz, 2H).

MS (Cl) for C ₉ H ₁₂ N ₃ O ₂ SBr, m / z (relative intensity) 226 (M + -Br, 26), 136 (36), 76 (81).

Elemental analysis for C ₉ H ₁₂ N ₂ O ₂ SBr, calculated: C 35.31; H, 3.95; N, 13.72; S, 10.47; Br 26.10. Found: C, 35.22; H, 3.88; N, 13.62; S, 10.36; Num. 26,18.

Example 29

Preparation of S- (2- (1H-pyrrol-1-yl) ethyl) isothiourea

To a solution of freshly distilled N- (2-bromoethyl) pyrrole (1.00 g, 5.74 mmol) in 95% ethanol (12 ml) was added thiourea (415 mg, 5.74 mmol) at room temperature. The solution was stirred at reflux for 22 hours, cooled to room temperature, and the mixture was concentrated in vacuo to give a thick slightly beige oil. The oil was allowed to crystallize at room temperature for 16 hours and the crystals were filtered and washed with chloroform (10 ml) to give 1.078 g of the title compound as a slightly beige-like crystal (75% yield).

Ή NMR (300 MHz, d ₆ -DMSO); δ 9.06 (broad s, 4H), 6.80 (t, J = 2.10 Hz, 2H), 5.99 (t, J = 2.10 Hz, 2H), 4.12 (t, J = 6.50 Hz, 2H), 3.52 (t, J = 6.50 Hz, 2H).

MS (Cl) for C ₇ H ₁₂ N ₃ SBr, m / z relative intensity 170 (M + -Br, 100), 153 (13), 103 (14), 94 (14), 93 (10), 77 (24 ).

Elemental analysis for C _? H ₁₂ N ₃ SBr, calcd: C 33.61; H, 4.84; N, 16.80; S, 12.82; No. 31.94. Found: C 33.68; H, 4.84; N, 16.74; S, 12.74; Nr 31.99.

Tal. 94.6-95.0 ° C.

Example 30

The inhibition of NO synthase is determined by the following procedure:

Purification of NOS from the human placenta

Amnion and chorion are removed from the fresh placenta, which is then flushed with 0.9% NaCl. The tissue was homogenized in a Waring mixer in 3 volumes of HEDS buffer (20 mM Hepes pH 7,8,0,1 mM EDTA, 5 mM DTT, 0.2M sucrose) plus 0.1 mM PMSF. The homogenate was filtered through a screen cloth and then centrifuged at 1000 g for 20 min. The supernatant was centrifuged again at 27500 g for 30 min. Solid ammonium sulfate was added to the supernatant to give 32% saturation. The precipitated protein was pelleted at 25000 g and then redissolved in a minimum volume of HEDS buffer plus 0.1 mM PMSF, 10 / ig / ml leupeptin and soybean trypsin inhibitor, and 1 / ig / ml pepstatin. The dissolved pellet was centrifuged at 15000 g for 10 min. 1/20 of the volume was added to the supernatant at 2.5 'ADP agarose resin (Sigma), and the suspension was slowly stirred overnight. In the morning, fill the suspension in the column. The resin was washed successively with HEDS, 0.5M NaCl in HEDS, HEDS and then NOS eluted with 10 mM NADPH in HEDS. The enzyme can be concentrated by ultra filtration and quickly frozen and stored at -70 ° C without loss of activity for at least 3 months.

Analysis for human placental NOS

NOS were analyzed for citrulene formation, followed by the procedure of Schmidt et al (PNAS 88 365-369, 1991) with these modifications: 20 mM Hepes, pH 7.4, 10 south / ml calmodulin, 2.5 mM CaCl ₂ , 2.5 mM DTT, 125 μΜ NADPH, 10 μΜ tetrahydrobiopterin, 0.5 mg / ml BSA, and 1 μΜ L- [ ¹⁴ C] arginine (New England Nuclear). The linearity of the NOS-catalysed velocities is confirmed before kinetic studies by using a single determination of the velocity time point.

Purification of NOS from cytokine-induced human colorectal adenocarcinoma

DLD-1 cells

DLD-1 (ATCC No. CCL 221) was grown at 37 ° C, 5% CO ₂ in RPMI 1640 medium supplemented with L-glutamine, penicillin, streptomycin and 10% heat-inactivated embryonic bovine serum. Cells were cultured to confluence and then the following cocktail of cytokines was added: 100 units / ml interferon-gamma, 200 units / ml interleukin-6, 10 ng / ml tumor necrosis factor and 0.5 ng / ml interleukin1 / 3. At 18-24 hours after induction, the cells were scratched and washed with phosphate-purified physiol. diff. NaCl. Pelleted cells were stored at -70 ° C. Purification of induced NOS is performed at 4 ° C. The crude extract was prepared with three freeze / thaw cycles of cells in TDGB (20 mMtris pH 7.5, 10% glycerol, 1 mM DTT, 2 μΜ tetrahydrobiopterin). The extract was applied directly to a column of 2 ', 5' ADP Sepharose (Pharmacia). The resin was washed successively with TDGB, 0.5M NaCl in TDGB, TDGB. NOS were eluted with 2 mM NADPH in TDGB. BSA was added immediately to obtain a final concentration of 1 mg / ml. NOS can be rapidly frozen and stored at -70 ° C without loss of activity for at least 2 months.

Analysis for inducible human NOS

Citrulline formation was analyzed as described above except to include 10 μ 10 FAD and exclude calmodulinin CaCl ₂ from the assay mixture.

Purification of NOS from the human brain

Human brain NOS is prepared using variations of the procedures of Schmidt et al. (PNAS 88 365-369,1991), Mayer et al. (Fed. Eur. Biochem. Soc. 288 187-191,1991), and Bredt and Snyder, (PNAS 87 682-685, 1990). Briefly, fresh whole brain (3 with sliced myelinated tissue, 1050 g) was homogenized in cold buffer A (50 mM HEPES, pH 7.5 (pH at ST), and 0.5 mM EDTA, 10 mM DTT, 3.6 1 full volume) with a polytron. The mixture was centrifuged at 13000 g for 1 hour and the supernatant liquid (about 2050 ml) was removed. Solid ammonium sulfate (365 g, about 30% saturated) was added to the supernatant fluid and stirred slowly for 30 minutes.

Pellet the precipitate at 13,000 g for 30 minutes and resuspend the pellet in —400 ml of buffer A with 4 μ, Μ tetrahydrobiopterin, ΙμΜ FAD (Sigma), and 1 μΜ FMN (Sigma). The solution was centrifuged at 41000 g 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 swollen in buffer A) at 4 ml / min. Wash the column with 100 ml of buffer A, 200 ml of buffer A with 500 mM NaCl, 100 ml of buffer A, then 30 ml of buffer A with 5 mM NADPH. Tetrahydrobiopterin up to 10 μΜ, FAD and FMN up to 1 μΜ, and Tween up to 0.1% are added to the enzyme solution. Concentrate this solution with Centriprep 30 to a volume of approximately 500 μΐ. Enzyme activity was determined as described by Schmidt et al. 1991 except to include 10 μΜ tetrahydrobiopterin in the analysis.

The inhibition results are shown in Table 1.

Values are inhibition constants (Ki) obtained from measuring percent inhibition at three or more inhibitor concentrations and predicting competitive inhibition relative to arginine.

TABLE 1 ω

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HI

HUMAN 'BRAIN' 1.8 ± 0.1 μΜ 57 ± 5 μΜ 0.42 ± 0.09 μΜ 1.8 ± 0.1 μΜ 13 ± 0.2 μΜ 10 ± 1μΜ 0.41 ± 0.02 μΜ | 1.7 ± 0. ΙμΜ 14 ± 2μΜ 5.6 ± 0.5 μΜ 1.2 ± 0.06 μΜ | 0.62 ± 0.03 μΜ 21 ± 4 μΜ 2 d. m Oh d Ή C ' KO d 0.82 ± 0.02 μΜ | 0.59 ± 0.03 μΜ <s 2 2 2 d. oo S d. CM 2 d. S 2 d. vn o s S d. S d. 2 S 2 d. 2 X 2 d. 2 d. κη 2 d. 2 X KO H> 0 S ω ω> u o <j j X CM d. m m o +1 m who d +1 OK KO +1 o m X KO d +1 m cn d. M · CM d -H OK CM CM +1 m CM = t m wn X TJ- you d -H CM o d -H CM 00 -H r- d -H IO d +1 Oh d +1 > o cu o d 2 d. CM S d. S d. s d. S d. CM 02 μΜ 03 μΜ 4 μΜ S d. en 4 μΜ s d. 2 d. 0.1 μΜ 02 μΜ I 3 μΜ 2 d. o 2 d. 2 i r- ~ o Oh 00 o d o o o d o ** d d d o d d <J 1—1 -H +1 CM -H +) -H +1 +1 +1 -H -H +1 -H • H +1 -H -H +1 > cn m 0.59 r- Oh oo Okay KO oo OO KO Okay CM CM M o CM Tj- Okay HUMANS NDUCI three en o CM d + d 0Ω en d CM d U •> r d o 0.4 H 1 Z \ .b (U z — κ X- • 3 era H Gj with "" s L Gj Mr Rücker π c 5? .5 2 y 3 * no S • H 95 c • 3 > f 1 rt rt B prt c Q Mr (D > Q > r • Γ] «3 ert c > o 3 rn <> · * W R R > b • H • 3 Γ | U <D Oh * cd • H z-s I 1 a o t i i and | ί i 1 -P o S • M '8 H • H z — s • 9 F T s ij a i < s N • H i 3 'W-Z • 9 z-> C 1 s • r • F > 1 • 9 z— \ d t | i 4 S z— \ rd 1 Q HI 1—1 Q Q rt Q & 5 +3 <J Ie 4-> d Z-> N SPOJ noeti i ί • s-Z | j J, 1 T 1 s -g ie 7.1 x-z Z — s s S d ethyl) i • H in N Iii I w c 3 Ή 3 3 J n A 8 H 1 r \ j 1 1 v $ CM κ-τ ( 5 1 • k CM one to one 3 $ Χ-Ζ i & 1 cm CM o PS CO., LTD CO., LTD w * ? v-ž ώ M-Z cA S cA cA K .- / en s-z CM v-z 1 CO., LTD cA cA en en X CKJ w v. en cA cA cA CM m Tj- «N KO Γ · oo Okay o - CM T “* en p-K m KO »“ M e- 00

2.0 ± 0.1 μΜ 0.22 ± 0.01 μΜ | 1 0.61 ± 0.02 μΜ | 0.71 ± 0.05 μΜ 8.8 ± 0.3 μΜ 0.31 ± 0.03 μΜ | 0.25 ± 0.01 s = L o o o -H Ό Oh d 1.1 ± 0.1 μΜ 1 6.8 ± 0.5 μΜ 4.4 ± 0.3 μΜ 2 X CN +) Oh 0.38 ± 0.01 μΜ 3 ± 0.3 μΜ | 2 i m d -H 00 KO d 2 i m o • H 00 o 0.82 ± 0.03 μΜ | 0.029 ± 0.009μΜ 2 = L 00 o o d +1 m o d 2 i o d +1 m Ό d 0.1 μΜ to'0 90 0 23 ± 7 μΜ S = L CN 0.8 μΜ S = L oo o s X CN Oh Oh ro 2 s. cn o 2 X CN CN 0.8 μΜ | 0.05 μΜ 2 = L CN Oh 1 Ι0Ό 2 a. d 2 λ + 0.003μ M 0.0005μ M 2 n. <N Oh d 2.3 ± .27 ± 1.2 ± i Ή VO 6.6 ± +) o OŠ -H KO cn 3.0 ± 7.2 ± 7.7 ± .45 ± 5.6 ± +1 cn 00 -H OK +1 > .039; Tl CN CN Oh +1 r » KO o o o d d S = L KO o 10 ' S OL Oh 2 a. o 2 a. rn S a. m o 2 i cn o o S i »N o o o S = L «N o S = 1 cn o 2 -t S = L i Okay Oh 2 = L cn Oh 2 i rrt o 2 X KO Oh 2 A cn Oh 002μΜ 0007µΜ 2 a. CN Oh d o d d Oh o d d d o Oh rS d o d d d d d +1 (N +1 Ή KO -H 3 ± +1 r * +1 -H 8 ± 6 ± 6 ± -H 3 ± • H KO 2 ± +1 CN 17 ± +1 00 CN CN Tj- 00 Okay 00 00 CN «N CN o o o Oh Oh o Oh o o o o o 0 Oh d Oh d d d o d Oh 1 z—> s 4 • .1 a Λ '3 Λ A d y n ^J 8 • 3 • 3 C? • 3 T H r · d 8 Q 'S -LJ Al 00 • rt • K — z K-c a rrt • H fli 8 n η T " TJ • 3 S • 3 g ε y- ~> Λ y z — s “—I Oh n i 5 • H • rt r ~ 4 * rt 1 r- “—L m • d N »3 ' M frt / S -LJ • cape cj cj <—4 c? ΐ 11 S ( f - 4 • r r — 4 • rt M Γ Z— \ > rj C 1 i - '' 'S d 0 Mr Rücker II <2 c r-j »R · 1 1 z — s d z — S d 1 1 1 i 4 1 1 t— · Q · *> F1 r <Ά 8 · * * R4 4 8 3 4 1 J L i 5 1 d i i 40 0Q * S »^ -Z l | 1 »Rt c 1 «Rt t J »Rt v Q p, »Rt Ή Oh • »Z ~ \ CO., LTD cn m □ 1 1 ** lo • c rs Ό -J 9 * c M c p n • 3 cn q ci s — z čn i — i čn h □ ra T Q o i 2 <2 rn d 8 * Mr Rücker E iJ JS 4J cn d cn 3 * n OJ o C \ | .5 .5 J. T J. j. Ί3 Yeah δ PO rjj Ώ o 1 B- s-z S S CM y> <\ l, OJ v Ji T ⁴ čA </> <Λ S 3 en CD <u <? 5 cA <A c5 n Ji Ji Ji Ji Ji Ji Ό cn o o CN m n- cn KO l · 00 Okay o CN cn v> KO 00 (N cn CN CN CN CN CN CN CN CN cn cn cn cn cn cn cn cn cn

For

Claims (19)

1-4 or R ^a binds the sulfur atom to one of the nitrogen atoms in the compound of formula (I) to form a 5- or 6-membered heterocyclic ring, with the proviso that R ^a is not methyl. Use of an isothiourea derivative having an inhibitory effect against the enzyme NO synthase, for the manufacture of a medicament for treating a condition where there is an advantage in inhibiting the enzyme NO synthase. 2-amino-4,5-dimethylthiazole S- (2- (1H-pyrrol-1-yl) ethyl) isothiourea S- (3-hydroxypropyl) isothiourea S- (2- (phenyl) ethyl) isothiourea S- (2- (3-methoxyphenyl) ethyl) isothiourea 2-amino-4-methylthiazole 2- amino-5-methylthiazole S - ((2-amino-4-thiazolyl) methyl-L-cysteine 2-amino-2-thiazoline S, S′-hexamethylenebis (isothioureas) S, S′-heptamethylenebis (isothioureas) S-benzylisothioureas S- (2-morpholinoethyl) isothiourea S- (6-methyl-2- (methylthio) -4-pyrimidinyl) isothiourea S, S '- (1,4-Phenylenebis (methylene)) diisothiourea S-tertbutylisothiourea S- (4-ethylbenzyl) isothiourea S - ((methylthio) methyl) isothiourea S- (3-bromopropyl) isothiourea S- (2-Bromoethyl) isothiourea S- (3-methyl-2-butenyl) isothiourea S-allyl isothioureas S- (3-aminopropyl) isothiourea S, S '- (1,3-Phenylenebis (methylene)) diisothiourea S, S '- (2-methylene-1,3-propanediyl) diisothiourea S, S '- (2-Butyn-1, 4-diyl) diisothiourea S, S '- (l, 3-phenylenebis (l, 2-ethanediyl)) diisothiourea S, S '- (l, 4-phenylenebis (l, 2-ethanediyl)) diisothiourea (2) C 1-6 alkenyl; Use according to claim 1, characterized in that the isothiourea derivative is a compound of formula (I) (I) or a salt thereof, wherein R (1) C ₁₁₄ hydrocarbyl group; or (2) a 5- or 6-membered heterocyclic ring; or (3) a 9-membered bicyclic heterocyclic ring system wherein each group R is optionally substituted by one or two groups independently selected from: (a) halo; (b) -XR ^x where X is oxygen, C (O) _m , wherein m is 1 or 2, S (O) _n , where n is 0.1 or 2, or NR ² , wherein R ^{2 is} hydrogen, C 1-4 alkyl or C 3-6 cycloalkyl, or R ² is attached to R ¹ to form a C 26 alkylene group; R ^{1 is} hydrogen; or CMalkyl, C _{2 6} alkenyl, C ₃₆ cycloalkyl, C _{? 9} aralkyl, C ₆₁₀ aryl or 5- or 3- ((2-amino-4-thiazolyl) methyl-L-alanine (3) a group - (CH,) -— C Ή - (CH ^ Ci ^ wherein p is 1 or 2 and q is 0 or 1; or (4) a 5- or 6-membered heterocyclic ring containing one or two nitrogen atoms, each optionally substituted by one or two groups, which may be the same or different, selected from (a) halo, preferably bromo; (b) groups OR ^2b ', wherein R ^2b ' is hydrogen or methyl; (c) groups C (O) _m R ^2b ', wherein m and R ^2b ' are as previously defined; (d) groups SR ⁹ , wherein R ^{9 is} methyl or ethyl; (e) NR ^7b groups R ^8b , wherein R ^7b and R ^{8b are} independently selected from hydrogen or C 1-6 alkyl, preferably hydrogen, methyl or ethyl; (f) a phenyl ring optionally substituted by the group OR ^2b 'or, the NH group Q as previously defined; “NH. ¹ (g) a 5- or 6-membered heterocyclic ring containing one or two heteroatoms independently selected from nitrogen or oxygen; or (h) C ₁ ^ alkyl, preferably methyl, or one of the carbon atoms in R is linked to the imino nitrogen in the compound of formula (I), to form a thiazole or thiazoline ring. Use according to claim 2, characterized in that R (1) is C 1-6 alkyl; 4- ((2-amino-4-thiazolyl) methyl) -L-homoalanine N, N- (1,3-phenylenebis (methylene)) bis (S-methylisothiourea) N, N- (1,3-phenylenebis (methylene)) bis (S-ethylisothiourea) 4- ((2-amino-4-thiazolyl) methyl) -L-homoalanine N, N-1,3-phenylenebis (methylene)) bis (S-methylisothiourea) N, N- (1,3-phenylenebis (methylene)) bis (S-ethylisothiourea) Use according to claims 2 or 3, with the proviso that R is not ethyl, propyl or isopropyl. 5- (3- (4 - ((amidinothio) methyl) phenyl) propyl) isothiourea S- (2- (5 - ((amidinothio) methyl) -2-thienyl) ethyl) isothiourea S- (3- (5 - ((2-amidinothio) ethyl) -2-thienyl) propyl) isothiourea S - ((2-amino-4-thiazolyl) methyl) -D-cysteine S - ((2-amino-4-thiazolyl) methyl) - (D, L) -homocysteine S- (2- (2-amino-4-thiazolyl) ethyl) -L-cysteine S- (2- (2-amino-4-thiazolyl) ethyl) -L-cysteine S- (2- (4-fluorophenyl) ethyl) isothiourea S- (2- (4-bromophenyl) ethyl) isothiourea S- (2- (3-methoxyphenyl) ethyl) isothiourea S- (2- (3-methylphenyl) ethyl) isothiourea S- (2- (4-Ethoxyphenyl) ethyl) isothiourea S- (2- (4-methoxyphenyl) ethyl) isothiourea S- (2- (2-bromophenyl) ethyl) isothiourea S- (2- (2-fluorophenyl) ethyl) isothiourea S- (2- (3'nitrophenyl) ethyl) isothiourea S- (3- (1H-pyrrol-1-yl) propyl) isothiourea S- (2- (4-Ethoxy-3-methoxyphenyl) ethyl) isothiourea S- (2- (2,4,6-trimethylphenyl) ethyl) isothiourea S- (2- (2,6-dimethoxyphenyl) ethyl) isothiourea or their salts. 5- (2- (5 - ((amidinothio) methyl) -2-thienyl) ethyl) isothiourea S- (3- (4 - ((amidinothio) methyl) phenyl) propyl) isothiourea S- (3- (5- (2-amidinothio) ethyl) -2-thienyl) propyl) isothiourea S- (2- (4-fluorophenyl) ethyl) isothiourea S- (2- (4-bromophenyl) ethyl) isothiourea S- (2- (3-methoxyphenyl) ethyl) isothiourea S- (2- (3-methylphenyl) ethyl) isothiourea S- (2- (4-Ethoxyphenyl) ethyl) isothiourea S- (2- (4-methoxyphenyl) ethyl) isothiourea S- (2- (2-bromophenyl) ethyl) isothiourea S- (2- (2-fluorophenyl) ethyl) isothiourea S- (2- (3-nitrophenyl) ethyl) isothiourea S- (3- (1H-pyrrol-1-yl) propyl) isothiourea S- (2- (2-chlorophenyl) ethyl) isothiourea S- (2- (2,5-dimethylphenyl) ethyl) isothiourea S- (2- (4-Ethoxy-3-methoxyphenyl) ethyl) isothiourea or salts thereof. Use according to claims 1 to 2, characterized in that the isothiourea derivative is a compound of formula (IA), (IB) or (IC) (IA) HN (IB) wherein R 'is C _x ^ alkylene group, _C2 ^ alkenylene or alkynylene group each optionally containing a phenyl ring, a 5- or 6-membered heterocyclic ring or a group X, as previously defined, and represents a dashed line of double or single bond. Use according to claim 1, characterized in that the isothiourea derivative is a compound of formula (II) SR ³ (II) NH, or a salt thereof, wherein R ^a C _{1g is} hydrocarbyl or a 5- or 6-membered heterocyclic ring or A 6-membered heterocyclic group wherein each group is optionally substituted by one or two groups independently selected from C _{x 3} alkyl, hydroxy, C ₁₃ alkoxy, amino, C ₁₃ alkylamino, halo, nitro, or C (O) _{m group} R ^2b wherein m 'is 1 or 2 and R ^{2b is} hydrogen or C 1-4 alkyl; or R ^{1 is a} group NR ³ R ⁴ wherein R ³ and R ^{4 are the} same or different and each is hydrogen or C 1-4 alkyl or R ³ and R ^{4 are} bonded to form a C 2-6 alkylene group; NH (c) of group (Y) _w -QS-V, where nh is ₂ Y is oxygen, S (0) _n, where n is as previously defined, s NR ^5, wherein R ⁵ is hydrogen or C _M alkyl; w is 0 or 1; QC _{2 4} hydrocarbyl or imino nitrogen is attached to group R or group Q to form a 5- or 6-membered heterocyclic ring; or (d) group A, wherein A is a heterocyclic ring system, as the case may be NH substituted with group (Y) _w -QS NH as previously defined; or (e) a C ₁₆ alkyl, C _1-6 alkenyl or alkynyl or C 1-4 cycloalkyl group; or one of the carbon atoms in R is attached to the imino nitrogen atom in the compound of formula (I) to form a 5- or 6-membered heterocyclic ring; with the proviso that R is not methyl. 7- (2′-amino-4-thiazolyl) -L-homoalanine S, S '- (l, 2-phenylenebis (l, 2-ethanediyl)) diisothiourea / 3- (2'-amino-4'-thiazolyl) -L-alanine S- (2'-amino-5 '- (R, S) -thiazolinylmethyl) -L-cysteine Use according to any one of claims 1 to 6, characterized in that the isothiourea derivative is selected from S- (2-aminoethyl) isothiourea S- (2-dimethylamino) propyl) isothiourea S- (2-methyl-2-propenyl) isothiourea S, S'-ethylenebis (isothioureas) S, S'-pentamethylenebis (isothioureas) S- (2-dimethylamino) ethyl) isothiourea Use according to any one of claims 1 to 3 or 6, characterized in that the isothiourea derivative is selected from S-Ethylisothioureas S-propylisothioureas S-isopropylisothioureas. Use of the isothiourea according to claim 1 for the treatment of systemic hypotension. A 9-membered bicyclic heterocyclic ring system, each optionally substituted by halo or by one or two groups -X ^a R ^1a , wherein R ^{1a is} hydrogen, C 1-6 alkyl, C 1-4 cycloalkyl, C? 9 aralkyl, C 640 aryl, or a 5- or 6-membered heterocyclic group, each optionally substituted by C 13 alkyl, C 13 alkoxy, amino, halo or nitro, or R ^1a is NR ^3a R ^4a , wherein R ^3a and R ^4a are the same or different and each is hydrogen or C13alkil or R ^3a and R ^4a are linked together to form a C2 6alkilensko group and X is ^a oxygen, C (O) ma, wherein m ^{a is} 1 or 2, C (O) na, wherein n ^{a is} 0,1 or 2 or NR ²³ , wherein R ^{23 is} hydrogen, C 1-6 alkyl or C 3-8 cycloalkyl or R ²³ is attached to R ^a to form a C _2-6 alkylene group, or with a group ? (CH ₂ ) _t (Y ^a ) _in nh ₂ wherein t is O to 4 and vv is ³ O or 1, Y ^a is oxygen, sulfur and NR ^7a , wherein R ^{7a is} hydrogen or C 1-6 alkyl: Use of the isothiourea according to claim 1 or 2 for the treatment of septic shock. Use of an isothiourea according to claim 9, characterized in that the systemic hypotension is induced by cytokine or cytokine-inducing therapy. Use of the isothiourea according to claim 1 for the treatment of short-term immunosuppression. Use of the isothiourea according to claim 1 for the treatment of an autoimmune disease. Use of the isothiourea according to claim 1 for the treatment of an inflammatory condition. 15. Isothiourea derivative of formula (I), characterized in that it is different from S-ethylisothiourea, S-propylisothiourea, S-isopropylisothiourea, benzylisothiourea, S, S- (1,4-phenylenebis (methylene)) diisothiosine S- (2dimethylamino) ethyl) isothioureas for medical use. 16. A new isothiourea derivative having the formula (IA), (IB) or (IC) as hereinbefore defined. An isothiourea derivative according to claim 16, selected from S, S '- (l, 4-phenylenebis (l, 2-ethanediyl)) diisothiourea S- (2- (1H-pyrrol-1-yl) ethyl) isothiourea S - ((2-amino-4-thiazolyl) methyl) -L-cystene Process for the preparation of an isothiourea derivative according to claim 16, characterized in that it comprises a) metabolising the thiourea with compound RL (L ') _r , where R is as previously defined, L and L' are both leaving groups and R is 0 or 1, followed, if necessary, by deprotection; or b) deprotecting a compound of formula (IB1) wherein R 'and the dashed line as defined previously and P and P' are the same or different and are both protecting groups. 19. Pharmaceutical preparation characterized in that it includes an isothiourea derivative as previously defined other than S-ethylisothiourea, S-propylisothiourea, S-isopropylisothiourea, S-benzylisothiourea, S, S- (1,4-phenylenebisethiothiobisethiothiobisethylene and S- (2-dimethylamino) ethyl) isothiourea or a pharmaceutically acceptable salt or solvate thereof, together with one or more of its pharmaceutically acceptable carriers, and optionally one or more other therapeutic ingredients.