WO2002016405A1

WO2002016405A1 - S-nitrosothiols and their use as medicaments for cardiovascular diseases

Info

Publication number: WO2002016405A1
Application number: PCT/GB2001/003822
Authority: WO
Inventors: Haitham Hussein Al-Sa'doni; Albert Ferro
Original assignee: King's College London
Priority date: 2000-08-25
Filing date: 2001-08-23
Publication date: 2002-02-28
Also published as: AU2001282342A1; GB0021079D0

Abstract

S-Nitroso-N-acetyl-D-β,β-dialkylcysteinyl glycine esters for use in the treatment of cardiovascular disease.

Description

S-NITROSOTHIOLS AND THEIR USE AS MEDICAMENTS FOR CARDIOVASCULAR DISEASES

This invention relates to novel S-nitrosothiols and uses thereof.

S-Nitrosothiols are a class of chemical compounds which decompose to release nitric oxide, and show promise in the treatment of a variety of cardiovascular diseases. Some of these are present in vivo, and others have been synthesized in vitro. However, those discovered or synthesized to date have very little tissue selectivity or specificity.

S-Nitrosothiols (RSNO) [S-nitrosothiol] decompose both in vitro and in vivo by a variety of mechanisms, giving rise to nitric oxide (NO) and the corresponding disulfide (1): 2RSNO ► RSSR + 2NO

The mechanisms which have been implicated include the following: catalysis by metal ions (2-9), transnitrosation (10-11), enzymatic breakdown (12), photochemical (13-15) or thermal decomposition (13), and vitamin C-catalyzed breakdown (16,17).

S-Nitrosothiols are biologically active as vasodilators (18) and as inhibitors of platelet aggregation (19-21), and therefore show promise in the treatment of cardiovascular diseases, although as yet they are not generally available for clinical use. However, biological activity and chemical structure do not appear to correlate with NO release in vitro (22,23). Indeed, it has also been suggested that some S-nitrosothiols exert their biological action without decomposition to NO (24, 25).

In the presence of trace amounts of metal ions, light or heat, these compounds are known to be unstable. Their decomposition is catalyzed by Cu(I) ions, which themselves can be formed by the reduction of trace amounts of Cu(IT) ions by thiols which are present as impurities (5). hi addition, during evaporation of the solvent, at the last stage of their synthesis, they decompose by second order kinetics (26). Furthermore, in previously published methods of S-nitrosothiol synthesis, a certain degree of impurity was present in the reaction products, due to the presence of trace amounts of unreacted thiol and to the formation of small amounts of disulfide. In many cases, the purity of the S-nitrosothiols synthesized was not authenticated by elemental analysis or accurate mass measurement (27). All of these factors have impeded the detailed elucidation of their chemical and biological properties. Two previous studies have demonstrated that the actions of S-nitrosothiols are stereoselective (28, 29), yet most S-nitrosothiols synthesised so far have been racemic mixtures. In addition to that, S-nifroso-albu in has been shown to be unusually stable (30).

The applicants have previously synthesized a series of biologically active S-nitrosated dipeptides, which were potent vasodilators(9) but display the instability and impurities as mentioned above. The present invention aims to alleviate the difficulties associated with these and other previously available S-nitrosothiols, by synthesizing compounds specifically with one or more of the following properties: (a) increased stability, with little or no release of NO during storage but the ability to release NO in vivo; (b) increased lipophilicity, in order to increase penetrance into cells; (c) high purity, as determined by elemental analysis and accurate mass measurement; (d) increased ease of handling, achieved by producing them in solid rather than oily form; and/or (e) the presence of one stereogenic center only. The chemical synthesis of a novel series of such S-nitrosothiol compounds, and their biological effects on vascular tissue and on platelets are described herein.

The compounds according to the present invention, or for clinical use in accordance with the present invention, are S-nitroso-N-acyl-D-β,β-dialkylcysteinyl glycine alkyl esters of formulae:

wherein i , R₂ , R₃ and R_ preferably represent alkyl groups which may be the same or different and wherein when Ri, R₂ and R₃ are all methyl, ^ preferably contains 2 or more carbon atoms. Alternatively Ri , R₂ , R₃ and R₄ may each or individually be hydrogen, alkoxy, amino, aryl and/or alkenyl. The groups Ri to R may be straight or branched chain, cyclic, saturated or non-saturated, substituted or non-substituted groups containing at least one carbon atom or a plurality of carbon atoms e.g. from 1 to 4, or 1 to 6, or 1 to 8, or 1 to 12 or even higher numbers of carbon atoms, with the proviso that the groups do not create significant steric hindrance within the compound thereby causing the compound to be unstable.

Preferably, R₂ and R₃ are both methyl and Ri and R_4 each contain from 1 to 6 carbon atoms.

The N-acyl group may be N-methanoyl, N-propanoyl, N-butanoyl, N-propenoyl, N-butenoyl, N-propynyl or N-butynyl. Preferably, the N-acyl group is N-acetyl.

Preferably, the compounds are S-Νitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine C _2~ alkyl esters.

Still more preferably, the compounds are S-Νitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine methyl ester [SΝAP(D)-Gly-O-Me], S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine ethyl ester [SΝAP(D)-Gly-O-Et], S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine iso-propyl ester [SΝAP(D)-Gly-O-iso-Pr], S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine n-propyl ester [SΝAP(D)-Gly-O-n-Pr], S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine t-butyl ester [SNAP(D)-Gly-O-t-Bu], or S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine n-butyl ester [SΝAP(D)-Gly-O-n-Bu].

The present invention also relates to the use of a compound according to the present invention in therapy.

The present invention further relates to the use of a compound according to the present invention for use in the treatment of cardiovascular diseases

The present invention still further relates to the use of a compound according to the present invention as vasodilators.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising any of the compounds of the present invention, and pharmaceutically acceptable salts thereof, with any pharmaceutically acceptable carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat.

The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, rectally, nasally, buccally, vaginally or via an implanted reservoir. Preferably, the composition is administered by injection. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to tecliniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution, hi addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol. The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions, h the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are admimstered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non- irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

The compounds of the present invention may be prepared by coupling a N-acyl-D-β,β-dialkylcysteine e.g. N-acetyl-D-β,β-dimethylcysteine (N-acetyl-D-penicillamine), to a glycine alkyl ester followed by nitrosation of the resulting dipeptide. The choice of N-acetyl-D-β,β-dimethylcysteine as a sulfur-containing amino acid, in preference to cysteine, has two advantages. Firstly, the presence of the two β-methyl groups means that the thiol group does not have to be protected (and later de-protected) during the coupling procedure. Secondly, following nitrosation of the thiol, the S-nitroso compound produced is much more stable than the equivalent compound formed from cysteine. In addition, N-acetyl-D-β,β-dimethylcysteine contains one stereogenic center; this is useful in elucidating the possible enzymatic decomposition of these S-nitrosothiols in vivo, because differences in the breakdown of the two stereoisomers can be studied separately. For the coupling process between N-acetyl-D-β,β-dimethylcysteine and the alkyl ester of an amino acid, N-cyclohexyl-N'-2-[N-morpholinoethyl]carbodiimide meth-p-toluenesulfonate is preferably used as it is water soluble and the urea derivative following the coupling is separated easily by filtration. The amino acid alkyl esters used may be: glycine methyl ester, glycine ethyl ester, glycine iso-propyl ester, glycine n-propyl ester, glycine t-butyl ester, or glycine n-butyl ester. FAB-MS [fast atom bombardment-mass spectrometry] with accurate mass measurement, elemental analysis and melting point measurement are used to confirm the identity of the individual dipeptides. The identity of the corresponding S-nitrosothiols are also confirmed by FAB-MS with accurate mass measurement and elemental analysis. Spectrophotometric analysis of the decomposition of the compounds was used to demonstrate the stability of the novel S-nitrosated dipeptides and was found to be intermediate between that of SΝAP(D) [S-nitroso-N-acetyl-D-penicillamine] and GSΝO [S-nitroso-L-glutathione].

The present inventions will now be described by way of example and with reference to the accompanying Figures in which:

Figure 1 : Absorbance-time plots for reactions of (a) SΝAP(D), (b) S-nitrosated dipeptides, and (c) GSNO. Kinetics were studied for all S-nitrosothiols at an initial concentration of 1 mmol/L, in the presence of CuSO₄ (10 μmol/L) at 37°C and pH = 7.4.

Figure 2: Example of the effect of a S-nitrosated dipeptide on platelet aggregation, (a) Effect of SNAP(D)-Gly-O-n-Bu at different concentrations, as marked, on U46619-induced aggregation of platelets from one subject, (b) Corresponding concentration-effect curve.

Figure 3: Example of the effect of an S-nitrosated dipeptide on relaxation of rat mesenteric arterial branches, following NE-induced contraction, (a) Effect of SNAP(D)-Gly-O-Me at different concentrations, as marked, on vessels from two different rates, (b) Corresponding concentration-effect curve, plotted as mean of effect in both vessels.

Materials: All chemicals used were laboratory grade and purchased from Sigma, Aldrich or Lancaster. Ultra-pure water was used for all experiments (Milli-Q_pius). All solvents were Analar grade. Dichloromethane was carefully purified before use. For all buffers and physiological salt solution (PSS) Aristar grade materials were used. Stock solutions of all S-nitrosothiols (0.01 mol/L for myography experiments, 0.05 mol/L for platelet aggregation studies) were prepared in dimethyl sulfoxide (DMSO), and serial log molar dilutions were subsequently prepared in PSS (for vasorelaxation experiments) or in 0.9% saline (for platelet aggregometry). All solutions were kept in the dark, on ice, during all experiments.

Synthesis of S-nitrosothiols:

Purification of Dichloromethane: Dichloromethane was carefully purified before use as described by Nogel (31), as follows. The commercial grade was purified by washing with portions of concentrated sulfuric acid until the acid layer remained colourless, and then with water, sodium carbonate solution and water once again. It was dried initially over calcium chloride and then distilled from calcium hydride before use. The fraction b.p. 40-41°C was collected.

Coupling Reaction for Dipeptide Synthesis (9): A suspension of N-acetyl-D-β, β

-dimethylcysteine (20 mmol) in dichloromethane (50 mL) was cooled in an ice bath, treated with N-cyclohexyl-N'-2-[N-morpholinoethyl]carbodiimide metho-_/?-toluenesulfonate (20 mmol) and subsequently with a solution of the appropriate amino acid alkyl ester hydrochloride (21 mmol) and triethylamine (20 mmol) in dichloromethane (50 mL). The mixture was stirred at 0°C for 1 h and then at room temperature for 2 days. The precipitate was filtered off and washed with dichloromethane (40 mL). The combined filtrate and washing were washed in sequence with saturated citric acid solution (60 mL), saturated potassium hydrogen carbonate solution (60 mL), and water (60 mL). The organic solution was dried over anhydrous MgSO4 and evaporated to dryness in vacuo.

The residue was dissolved in dry diethyl ether. The resulting crystallized solid was collected by filtration, washed with cold diethyl ether, and dried. This solid was identified by elemental analysis and accurate FAB-MS as being the corresponding dipeptide. This was then used as the starting material for nitrosation. All dipeptides synthesized had sharp melting points.

Nitrosation Reactions: (a) S-Nitrosated amino acid: S-Nitroso-N-acetyl-D- β, β-dimethylcysteine [SΝAP(D)] was prepared by the method of Fields et al. (32) using NaNO₂. NaNO₂ (0.345g; 5 mmol) in 5 mL of water was added to N-acetyl-D-penicillamine (0.478g; 2.5 mmol) dissolved in methanol-lΝ HC1 (5 mL each) with 0.5 mL of concentrated H₂SO , over the course of 20 min with vigorous stirring at 25°C. After a further 15 min, SΝAP(D) was separated off, washed well with water, and air-dried, to give deep green crystals with red reflections: 0.33g, 1.5 mmol, 60%.

(b) S-Νitrosated dipeptides.- S-Νitrosated dipeptides were prepared as previously published (5), except that in the last stage of preparation faster evaporation of the solvent was achieved in vacuo, to avoid decomposition of the synthesized S-nitrosothiol. Briefly, the appropriate dipeptide (1 mmol) was dissolved in dichloromethane (8 mL) and t-butyl nitrite (1 mL) added. After 1 h the solvent was removed by evaporation to give a green solid. After washing with diethyl ether the solid was dried in vacuo.

(c) S-Νitrosated tripeptide: S-Νitroso-L-glutathione (GSΝO) was prepared by the method of Hart (33), using ΝaΝO . During the last stage, this compound was washed and dried extensively, in order to prepare it in an anhydrous form. Previous procedures for synthesizing GSΝO have reported the presence of 0.25 H2O (1.3%) (33), and it has not been synthesized previously in pure anhydrous form.

Briefly, to a stirred ice-cold solution of L-glutathione (1.53 g, 5 mmol) in water (8 mL) containing 2Ν HC1 (2.5 mL) was added NaNO₂ (0.345g; 5 mmol). After 40 minutes at 5°C the red solution was treated with acetone (10 mL) and stirred for a further 10 minutes. The resulting fine pale red solid powder was filtered off and washed successively with ice-cold water (5 x 1 mL), acetone (3 x 10 mL) and ether (3 x 10 mL) to yield GSNO (1.16g, 3.4 mmol, 69%).

Kinetic Studies:

Chemical stability was determined spectrophotometrically as previously described (5), with the modification that, for solubility reasons, the solvent used was 10% aqueous DMSO. Briefly, kinetic studies were all carried out in phosphate-buffered solution (composition: 50 mmol/L KH₂PO₄, 39.1 mmol/L NaOH, pH 7.4) at 37° C, in the presence of CuSO₄ 10 mM and in the absence of EDTA. Reactions were followed by noting the decrease in absorbance at 340 nm (A₃₄₀) of the S-nitrosothiol at an initial concentration of 1 mmol/L, in a recording spectrophotometer.

Platelet aggregometry:

Platelet aggregation studies were performed as previously described (34). Briefly, 60mL venous blood was obtained from healthy, normotensive, non-smoking subjects, and collected into trisodium citrate (0.38%) final concentration). Platelet rich plasma (PRP) and platelet poor plasma (PPP) was prepared by differential centrifugation. A Payton 600B dual channel aggregometer was calibrated using the difference in light transmission of PRP and PPP to represent 100% aggregation.

PRP was equilibrated at 37°C for one minute, followed by the addition of an appropriate concentration of S-nitrosothiol. After a further 5 min incubation, the thromboxane-mimetic U46619 2 μmol/L was added, and aggregation was measured for the subsequent 3 min. The effect of each S-nitrosothiol was expressed as percentage inhibition of aggregation in the control sample.

Small vessel myograph assay:

Female Sprague-Dawley rats (286.7 ± 8.4 g, n= 5) were killed by cervical dislocation. Small arteries were mounted on a small vessel myograph as previously described (35). Briefly, second-order branches (294.58 ± 10.38 μm, n=30) of the mesenteric arterial tree were dissected free of connective tissue and mounted on fine wires in pairs as ring preparations, for the measurement of isometric tension. Arteries were bathed in PSS of the following composition (mmol/L): NaCl 119, KC1 4.7, CaCl₂ 2.5, MgSO₄ 1.17, NaHCO₃ 25, KHPO₄ 1.18, EDTA 0.025, glucose 6.0, pH 7.4. Vascular rings were maintained at 37°C and gassed with 95% O₂ / 5% CO₂. The passive tension-internal circumference characteristics of the arteries were determined, by sfretching to achieve an internal circumference equivalent to 90% of that which would be attained when relaxed in situ under a fransmural pressure of 100 mmHg. To confirm viability of the arteries, four contractions (4 min duration) were performed to 5 mmol/L norepinephrine (NE), KPSS (PSS containing 125 mmol/L KC1) or a combination of both. Arteries failing to produce active tension equivalent to 100 mmHg were rejected. Following submaximal preconstriction with 5 mmol/L NE, cumulative concentration-response curves were constructed for each S-nitrosothiol, with log molar increments added every 2 minutes.

5) Statistical Analysis:

All data are expressed as mean ± SEM. Statistical comparisons were by one-way ANOVA, with statistical significance taken as P < 0.05 (two-tailed).

RESULTS

Chemical synthesis and characterization of S-nitrosothiols

A number of thiols and S-nitrosothiols were prepared and characterized. All the S-nitrosothiols produced were stable as solids, and in solution in the absence of copper ions, heat or light and the presence of EDTA. FAB-MS, accurate mass measurements, and elemental analysis were used for the identification of these drugs, hi addition, the melting point was measured for each of the thiols only. The analytical data for the dipeptides synthesised are given below.

N-acetyl-D-β,β-dimethylcysteinyl glycine methyl ester [ΝAP(D)-Gly-O-Me] was obtained as a white solid powder (mp 148-149°C with dec); m/z (FAB) 263 (M++1). N-acetyl-D-β,β-dimethylcysteinyl glycine ethyl ester [ΝAP(D)-Gly-O-Et] was obtained as a white solid powder (mp 162-163°C with dec); m/z (FAB) 277 (M++1).

N-acetyl-D-β,β-dimethylcysteinyl glycine iso-propyl ester [ΝAP(D)-Gly-O-iso-Pr] was obtained as a white solid powder (mp 145-146°C with dec); m/z (FAB) 291 (M⁺+l). N-acetyl-D-β,β-dimethylcysteinyl glycine n-propyl ester [ΝAP(D)-Gly-O-n-Pr] was obtained as a white solid powder (mp 137-138°C with dec); m/z (FAB) 291 (M++1). N-acetyl-D-β,β-dimethylcysteinyl glycine t-butyl ester [ΝAP(D)-Gly-O-t-Bu] was obtained as a white solid powder (mp 130-131°C with dec); m/z (FAB) 305 (M++1).

N-acetyl-D-β,β-dimethylcysteinyl glycine n-butyl ester [ΝAP(D)-Gly-O-n-Bu] was obtained as a white solid powder (mp 123-124°C with dec); m/z (FAB) 305 (M++1). The resulting S-containing dipeptides were nitrosated with t-butyl nitrite (8). The new S-nitrosothiols prepared are displayed in Scheme 1 and the analytical data are detailed below.

(a) S-Nitrosated amino acid:

S-Nitroso-N-acetyl-D-β,β-dimethylcysteine [SΝAP(D)] was obtained as a green solid powder; m/z (FAB) 221 (M++1).

(b) S-Nitrosated dipeptides: S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine methyl ester [SΝAP(D)-Gly-O-Me] was obtained as a green solid powder; m/z (FAB) 292 (M⁺+l).

S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine ethyl ester [SΝAP(D)-Gly-O-Et] was obtained as a green solid powder; m/z (FAB) 306 (M⁺+l).

S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine iso-propyl ester [SΝAP(D)-Gly-O-iso-Pr] was obtained as a green solid powder; m/z (FAB) 320 (M⁺+l).

S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine n-propyl ester [SΝAP(D)-Gly-O-n-Pr] was obtained as a green solid powder; m/z (FAB) 320 (M⁺+l).

S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine t-butyl ester [SΝAP(D)-Gly-O-t-Bu] was obtained as a green solid powder; m z (FAB) 334 (M+H-l). S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine n-butyl ester [SΝAP(D)-Gly-O-n-Bu] was obtained as a green solid powder; m/z (FAB) 334 (M⁺+l).

(c) S-Nitrosated tripeptide:

S-Nitroso-L-glutathione [GSNO] was obtained as a fine pale red precipitate; m z (FAB) 337 (M++1).

Chemical stability

The biological effects of the novel S-nitrosothiols, as detailed below, were examined within a few weeks of synthesis, and subsequently after storage at 0°C for 12 months; during that time, no diminution in biological activity was observed, as assessed by effects on vasorelaxation and on inhibition of platelet aggregation (data not shown). Additionally, in solution the rate of decomposition of the S-nitrosothiols at 37° C was examined by monitoring changes in A₃₄₀ with time. An approximate measure of the catalytic effect of Cu(I) ions on decomposition of different S-nitrosated dipeptides was obtained, by examining the rate of decomposition (at an initial concentration of concentration of 1 mmol/L) in the presence of CuSO₄ 10 iM, at pH 7.4 and in the absence of EDTA. Under these conditions, copper is present as Cu(I) ions so long as thiol, produced by hydrolysis of S-nitrosothiol or present as an impurity, is present at a concentration greater than that of copper (5). We found that all the novel S-nitrosated dipeptides decomposed at a rate approximately tenfold less than SNAP(D), although considerably faster than GSNO (Figure 1, Table I)._

Effects of novel S-nitrosothiols on platelet aggregation

The anti-aggregatory effects of the newly-synthesized S-nitrosothiols were determined, in platelets from healthy human subjects, by inhibition of U46619-induced platelet aggregation. All the compounds produced inhibited platelet aggregation in a concentration-dependent fashion (Figure 2), with a maximal inhibitory effect of 100% in all cases. The anti-aggregatory potency for all the S-nitrosated dipeptides was similar to that of SNAP(D) and GSNO (Table 1).

Vasorelaxant effects of novel S-nitrosothiols

The vasorelaxant effects of the newly-synthesized S-nitrosothiols were determined in rat mesenteric arteries by small vessel myography, following preconsfriction with NE 5 μmol/L. All compounds elicited full vasorelaxation in this vessel, in a concentration-dependent manner (Figure 3). All S-nitrosated dipeptides had similar vasorelaxant potencies (as reflected by log EC₅o) [where EC50 is the concentration of S-nitrosothiol eliciting 50% of maximal effect] but in all cases their potency was significantly greater than that of either GSNO or SNAP(D), by approximately one log molar unit (Table 2). Furthermore, all of the S-nitrosated dipeptides, possessed vasorelaxant potency which was significantly greater than their anti-aggregatory potency, again by approximately one log molar unit (Table 2).

To determine the effects of the novel compounds in elements of the vascular system in vitro their effects on relaxation of rat mesenteric arterial branches, and on inhibition of aggregation of human platelets was examined. We found that all compounds elicited full vasorelaxation and inhibition of platelet aggregation. Interestingly, all the novel S-nitrosated dipeptides were approximately tenfold more potent in eliciting vasorelaxation than either SNAP(D) or GSNO in our system, and were also approximately tenfold more potent in causing vasorelaxation than inhibition of platelet aggregation. By contrast, the effects of the S-nitrosated dipeptides, SNAP(D) and GSNO on inhibition of platelet aggregation were not different. The reasons for this difference in behaviour is unclear. All of the S-nitrosated dipeptides synthesized in accordance with the present invention are more lipophilic than either SNAP(D) or GSNO, by virtue of the absence of free carboxylic acid or amino groups in these compounds, unlike SNAP(D) and GSNO. As a consequence, cell penetrance should be greater with the S-nitrosated dipeptides of the present invention. For example these compounds should penetrate more easily into vascular smooth muscle cells than do SNAP(D) and GSNO, whereas penetrance of all these compounds into platelets may not be different.

In the solid state, the novel S-nitrosated dipeptides were found to be stable at 0°C for 12 months, and possibly longer. In solution, the catalytic effect of copper ions on the release of NO was approximately more than tenfold slower than their effect on SNAP(D). All the S-nitrosated dipeptides decomposed in the presence of CuSO₄ with similar reaction kinetics to each other. In contrast, the decomposition of GSNO under these conditions was much slower than that of the S-nitrosated dipeptides. A recent study (36) has shown that the kinetics of GSNO decomposition are complex, with a long induction period during which there is no sign of reaction. It has previously been demonstrated that both the spontaneous release of NO (37) and Cu-catalyzed decomposition (2) are responsible for the vasodilator action of S-nitrosothiols, and the anti-aggregatory effect of GSNO has been shown to be diminished by the presence of a selective complexing agent for Cu(I), bathocuproine disulphonic acid (38). Although less stable than GSNO, the novel S-nitrosated dipeptides exhibit a degree of tissue selectivity which may be useful therapeutically.

It has been found previously that the chemical stability of the S-nitrosothiols in solution dose not correlate with their ability to induce relaxation of vascular smooth muscle (9). hi accord with this, Mathews and Kerr (22) and Kowaluk and Fung (23) found that, for a number of S-nitrosothiols, no correlation exists between vasodilator^' effects in biological systems and spontaneous liberation of NO in solution. It appears that the breakdown of S-nitrosothiols to generate NO in vivo or in vitro may involve a whole variety of mechanisms (1), which operate quite independently from the spontaneous breakdown of these compounds in solution. Clinical application

The compounds of this invention may be administered intravenously at doses ranging from 50 to 250 micrograms/min.

NO donor drugs are already in clinical use, especially the organic nitrates (glyceryl trinitrate, isosorbide dinitrate, isosorbide mononitrate) for the treatment of ischaemic heart disease, and sodium nitroprusside for the treatment of severe hypertensive crises. However, the use of both types of drugs carries drawbacks:

(a) Patients often develop tolerance to the action of organic nitrates so that, with time, they become less effective in treatment of cardiac ischaemia. (b) Sodium nitroprusside use is associated with accumulation of cyanide in the body, so it must not be used for prolonged periods in patients with severe hypertension.

S-nitrosothiols do not share these drawbacks. In general, in the setting of ischaemic heart disease, both vasodilatory and antiplatelet actions are desirable therapeutically; indeed, in addition to nitrates, such patients are also often treated with aspirin, for antiplatelet effect. However, under certain conditions, vasodilatation may be desired without antiplatelet effect, e.g. if the patient has a bleeding disorder, active peptic ulcer, recent haemorrhagic stroke. In addition, in the context of treatment of acute severe hypertension, antiplatelet action is disadvantageous, due to the high risk of intracerebral bleeding. In such situations, the S-nitrosated dipeptides of the present invention will have a great advantage over existing S-nitrosothiols such as SNAP [S-nitroso-N-acetyl-D,L-penicillamine] and GSΝO, as they have an approximate tenfold selectivity for causing vasodilatation as compared with inhibition of platelet aggregation. Given at an appropriate dose, they will dilate blood vessels, with minimal effect on platelets, unlike other previously described S-nitrosothiols, a property of considerable interest in the treatment of acute severe hypertension (in place of sodium nitroprusside).

Since these drugs are peptide derivatives, they are broken down in the gut, so initially at least will have to be given intravenously (other routes are theoretically possible, e.g. sublingual, transdermal) rather than orally. It is also contemplated that their administration may be in the form of an oral delivery system which will allow their absorption from the gut. In conclusion, the present invention discloses a novel series of S-nitrosothiols, all of which are highly pure, contain one stereogenic center and are stable in solid form. It has been found that, for a family of S-nitrosated dipeptides built on the base structure of the S-nitrosated amino acid SNAP(D), where the chemical environment of the NO group remains unchanged, chemical stability in the presence of copper ions is greater than for SNAP(D), further, alterations in chain length and branching do not give rise to detectable differences in stability between these compounds. All the S-nitrosated dipeptides described are lipophilic, by virtue of the absence of free carboxylic acid or amino groups. Despite greater chemical stability in solution, however, the vasorelaxant potency of the S-nitrosated dipeptides of the present invention is significantly greater than that of SNAP(D) in rat mesenteric artery branches, although the anti-aggregatory potency on human platelets was not different between SNAP(D) and the S-nitrosated dipeptides. This indicates that factors other than spontaneous chemical decomposition are operating in these biological systems, and that these factors may be different between platelets and blood vessels. This may reflect differences in cell penetrance and/or in intracellular mechanisms for releasing NO from these compounds. The precise nature of these differences remains to be determined, but the results demonstrate a degree of tissue selectivity for these compounds of considerable clinical potential.

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Novel .S-nitrosated dipeptides synthesized, with the structure of GSNO and SNAP(D) (also newly synthesized) depicted for comparison.

1- S-Nitrosated amino-acid

2- S-Nitrosated dipeptides

3- S-Nitrosated tripeptide

GSNO TABLE 1

Kinetic data for the decomposition of .S-nitrosothiols., in the presence of

CuS0₄ 10 μmol/L.

Compound ltf K s ¹

SNAP(D) 11.5

SNAP(D)-0-Me 1.3

SNAP(D)-0-Et 1.3

SNAP(D)-0-n-Pr 1.2

SNAP(D)-0-iso-Pr 1.1

SNAP(D)-0-£-Bu 0.9

SNAP(D)-0-n-Bu 1.0

GSNO Very slow

The actual concentration of copper ions may be slightly higher than 10 μmol/L, due to copper present as an impurity.

Rate = k_obs x [RSNO], k_ohs = λr_Cu x [Cu^*]. k_Cu is the rate constant of copper- catalyzed decomposition. TABLE 2

Vasorelaxant and anti-aggregatory potencies of S-nitrosothiols

COMPOTJTO -log EC₅₀ (mol/L) -log EC₅₀ (mol/L)

(vasorelaxant) (anti-aggregatory)

SNAP(D) 6.0 + 0.1 (n=9) 5.4 + 0.3 (n=6)

SNAP(D)-0-Me 6.6 + 0.1 (n=6)^*# 5.3 + 0.1 (n=6)

SNAP(D)-0-Et 6.5 + 0.1 (n=6) " 5.5 + 0.1 ^"(n=6)

SNAP(D)-0-n-Pr . ^' 6.7 + 0.2 (n=6)^*# 5.8 + 0.2 (n*=6)

SNAP(D)-0-iso~Pr 6.8 + 0.2 (n=6)^*# 5.5 + 0.1 (n=6)

SNAP(D)-0-/-Bu 6.3 + 0.2 (n=6)^*# 5.5 + 0.0 (n=6)

SNAP(D)-0-n-Bu 6.8 + 0.2 (n=6)^{*# •'} 5.5 + 0.1 (n=6)

GSNO 5.8 + 0.1 (n=9) 5.4 + 0.1 (n=6)

EC₅₀: concentration of S-nitrosothiol eliciting 50% of maximal effect. ^* P < 0.05 as compared with SNAP(D). P < 0.05 as compared with -log EC₅₀ for anti-aggregatory action.

Claims

1. S-nitroso-N-acyl-D-β,β-dialkylcysteinyl glycine alkyl esters of formula:

wherein Ri , R₂ , R₃ and R4 represent hydrogen, alkyl, alkoxy, amino, aryl and/or alkenyl groups which may be the same or different and wherein when Ri, R₂ and R₃ are all methyl, R4 preferably contains 2 or more carbon atoms.

2. Compounds according to claim 1, in which any or all of Ri to R4 are straight or branched chain, cyclic, saturated or non-saturated, substituted or non-substituted alkyl groups.

3. Compounds according to claim 1 or claim 2 in which the alkyl groups comprise from 1 to 4 carbon atoms.

4. Compounds according to claim 1 or claim 2 in which the alkyl groups comprise from 1 to 6 carbon atoms.

5. Compounds according to claim 1 or claim 2 in which the alkyl groups comprise from 1 to 8 carbon atoms.

6. Compounds according to claim 1 or claim 2 in which the alkyl groups comprise from 1 to 12 carbon atoms.

7. Compounds according to any one of the preceding claims, in which R₂ and R₃ are both methyl and Ri and R4 each contains from 1 to 6 carbon atoms.

Compounds according to any one of claims 1 to 7, in which the N-acyl group is N-acetyl, N-methanoyl, N-propanoyl, N-butanoyl, N-propenoyl, N-butenoyl, N-propynyl or N-butynyl.

Compounds according to any one of the preceding claims in which the compounds are S-Νitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine C ₂- alkyl esters.

10. Compounds according to any one of the preceding claims in which the compounds are: S-Νitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine methyl ester [SΝAP(D)-Gly-O-Me], S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine ethyl ester [SΝAP(D)-Gly-O-Et], S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine iso-propyl ester [SΝAP(D)-Gly-O-iso-Pr], S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine n-propyl ester [SΝAP(D)-Gly-O-n-Pr], S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine t-butyl ester [SΝAP(D)-Gly-O-t-Bu], or S-Nitroso-N-acetyl-D-β,β-dimethylcysteinyl glycine n-butyl ester [SΝAP(D)-Gly-O-n-Bu].

11. Pharmaceutical composition comprising a compound according to any one of the preceding claims.

12. Use of a compound according to any one of claims 1 to 10 in therapy.

13. Use of a compound according to any one of claims 1 to 10 in the treatment of cardiovascular diseases.

14. Use of a compound according to any one of claims 1 to 10 as a vasodilator.

15. Use of a compound according to any one of claims 1 to 10 in the manufacture of a composition for the treatment of cardiovascular diseases.

16. A process for producing compounds according to any of claims 1 to 10, comprising coupling an N-acyl-D-β,β-dialkylcysteine to a glycine alkyl ester followed by nitrosation of the resulting dipeptide.

17. A process according to claim 16, in which the resulting dipeptide is: N-acetyl-D-β,β-dimethylcysteinyl glycine methyl ester [ΝAP(D)-Gly-O-Me], N-acetyl-D-β,β-dimethylcysteinyl glycine ethyl ester [ΝAP(D)-Gly-O-Et],

N-acetyl-D-β,β-dimethylcysteinyl glycine iso-propyl ester [ΝAP(D)-Gly-O-iso-Pr], N-acetyl-D-β,β-dimethylcysteinyl glycine n-propyl ester [ΝAP(D)-Gly-O-n-Pr], N-acetyl-D-β,β-dimethylcysteinyl glycine t-butyl ester [ΝAP(D)-Gly-O-t-Bu], or N-acetyl-D-β,β-dimethylcysteinyl glycine n-butyl ester [ΝAP(D)-Gly-O-n-Bu].