NOVEL 5-ACYLSULFANYL-HISTIDINE COMPOUNDS AS PRECURSORS OF THE CORRESPONDING 5-SULFANYLHISTIDINES AND THEIR DISULFIDES

Abstract

The invention relates to a 5-acylsulfanyl-histidine compound and the derivatives thereof, of general formula (I),

##STR00001##

wherein R1 to R3=H, alkyl, especially CH3; R4=H, alkyl, especially CH3, alkyle (C=0), substituted alkyl (CO), aryl (CO); -alanyl (H2NCH2CH2 (CO); -amino-acyl; R5=alkyl, especially methyl, phenyl. The invention also relates to the use of said compound for producing of 5-sulfanyl-histidine compounds and the derivatives thereof of formula (II)

##STR00002##

in addition to corresponding disulfides as well as these 5-sulfanylhistidines of formula (II) as novel compounds and their use as antioxidant; and to the various methods for the production thereof.

Claims

1. A 5-sulfanylhistidine compound of formula (II): ##STR00084## where: R.sup.1H, or alkyl; R.sup.2R.sup.3H, or alkyl; R.sup.4H, alkyl, alkyl (CO), substituted alkyl (CO), aryl (CO), -alanyl (H2NCH2CH2 (CO), or -amino-acyl; it being understood that when R.sup.1H then R.sup.2, R.sup.3 and R.sup.4 cannot simultaneously be H; as well as all the stereoisomers, diastereoisomers and enantiomers, in particular in terms of the carbon atom bearing the COOH group, as well as all the disulfides obtained by condensation of two 5-sulfanylhistidine compounds of formula (II), taken separately or in a mixture; and all the salts of pharmaceutically acceptable acids of said compound of formula (II).

2. The compound of claim 1, wherein R.sup.4 represents hydrogen, methyl, acetyl, benzoyl, -aminoacyl or -alanyl (H.sub.2NCH.sub.2CH.sub.2 (CO).

3. A 5-sulfanylhistidine compound selected from the group consisting of: the disulfide of L-5-sulfanyl-,N(methyl)-histidine (Compound 22); L-5-sulfanyl-,N(methyl)-histidine (Compound 23); the disulfide of L-5-sulfanyl-,N,N(dimethyl)-histidine (Compound 24); L-5-sulfanyl-,N,N(dimethyl)-histidine (Compound 25); L-5-sulfanyl-,N,N,N(trimethyl)-histidine (Compound 26); the disulfide of L-5-sulfanyl-,N,N,N(trimethyl)-histidine (Compound 27); the disulfide of L-5-sulfanyl-,N(acetyl)-histidine (Compound 28); L-5-sulfanyl-,N(acetyl)-histidine (Compound 29); L-5-sulfanylcarnosine (Compound 30); the disulfide of iso-ovothiol A (Compound 31); L-5-sulfanyl-1-methyl-histidine named iso-ovothiol A (Compound 32); the disulfide of L-5-sulfanyl-,N,N(dimethyl)-1-methylhistidine (Compound 33); L-5-sulfanyl-,N,N,N(trimethyl)-1-methylhistidine (Compound 34); L-5-sulfanyl-,N(L-alanyl)-histidine (Compound 35); and the disulfide of 5-sulfanyl-,N(pentanoyl)-histidine (Compound 36).

4. A 5-sulfanylhistidine compound of formula (II): ##STR00085## where: R.sup.1H, or alkyl; R.sup.2R.sup.3H, or alkyl; R.sup.4H, alkyl, alkyl (CO), substituted alkyl (CO), aryl (CO), -alanyl (H2NCH2CH2 (CO), or -amino-acyl; it being understood that when R.sup.1H then R.sup.2, R.sup.3 and R.sup.4 cannot simultaneously be H; as well as all the stereoisomers, diastereoisomers and enantiomers, in particular in terms of the carbon atom bearing the COOH group, as well as all the disulfides obtained by condensation of two 5-sulfanylhistidine compounds of formula (II), taken separately or in a mixture; and all the salts of pharmaceutically acceptable acids of said compound of formula (II), selected from a mineral acid, an organic acid, an alkanesulfonic acid or an arylsulfonic acid such as benzene- and paratoluenesulfonic acids.

5. The compound of claim 4, wherein the mineral acid is selected from the group consisting of hydrochloric, hydrobromic, hydroiodic, sulfuric, and phosphoric acid.

6. The compound of claim 4, wherein the organic acid is selected from the group consisting of formic, acetic, tartaric, trifluoroacetic, propionic, benzoic, maleic, fumaric, succinic, citric, oxalic, glyoxylic, and aspartic acid.

7. The compound of claim 4, wherein the alkanesulfonic acid is selected from the group consisting of methanesulfonic, trifluoromethanesulfonic, and ethanesulfonic acid.

8. The compound of claim 4, wherein the arylsulfonic acid is selected from the group consisting of benzenesulfonic acid and paratoluenesulfonic acid.

9. One-pot method (D) for preparing the 5-sulfanyl-histidine compounds of formula (II) as defined in claim 1, and their corresponding disulfides from the corresponding histidine compounds, by combining the following methods: (A) a method (A) for preparing the 5-acylsulfanyl-histidine compounds of formula (I), ##STR00086## Where R.sup.1 to R.sup.4 are as defined for the compounds of formula (II) and R.sup.5 is alkyl or phenyl; as well as all the stereoisomers, diastereoisomers and enantiomers, in particular in terms of the carbon atom bearing the COOH group, taken separately or in a mixture; and all the salts of pharmaceutically acceptable acids of said compound of general formula (I), characterized in that said method is comprising the following steps: 1) The reaction of the histidine, racemic (DL) or one of the enantiomers thereof (D or L), or one of their compounds alkylated on the nitrogen in position 1 of the imidazole ring, racemic (DL) or one of the enantiomers thereof (D or L), or one of their compounds alkylated or acylated on the nitrogen of the -amine function, racemic (DL) or one of the enantiomers thereof (D or L), or one of their compounds alkylated on the nitrogen in position 1 of the imidazole ring and alkylated or acylated on the nitrogen of the -amine function, racemic (DL) or one of the enantiomers thereof (D or L), in the presence of 1 to 2 equivalents of mineral or organic acid, with a) an agent generating halogenium ions X+ in a polar protic solvent, at temperatures of 0-25 C., then with b) a sulfur-containing reagent of the carbothioic acid type of formula alkyl or phenyl C(O)SH or one of the salts thereof in a polar protic solvent, then, 2) optionally, the purification by column liquid chromatography or any other purification method well known to the person skilled in the art. with (B) a method (B) for preparing the 5-sulfanylhistidine compounds of formula (II) obtained from the 5-acylsulfanyl-histidine compounds of formula (I) described in method (A), characterized in that said method comprising the following steps: 1) Either directly (method B1): e) by hydrolysis of the 5-acylsulfanyl-histidine compound obtained in a polar protic solvent by stirring at a temperature above 20 C. in the presence of a thiol, f) then, optionally, purification by column liquid chromatography or any other purification method well known to the person skilled in the art. 2) Or indirectly (method B2): f) by hydrolysis of the 5-acylsulfanyl-histidine compound obtained in a polar protic solvent by stirring at a temperature above 20 C. in order to obtain the corresponding disulfide, g) then reduction of the disulfide by reaction with a thiol, h) then, optionally, purification by column liquid chromatography or any other purification method well known to the person skilled in the art; or with (C) a method (C) characterized in that said method comprises the following steps: in the presence of 1 to 2 equivalents of mineral or organic acid, the reaction with: c) an agent generating halogenium ions X.sup.+ in a polar protic solvent, at a temperature of 0-25 C., then with d) a sulfur-containing reagent of the carbothioic acid of formula alkyl or phenyl C(O)SH or one of the salts thereof in a polar protic solvent, followed by 1) Either: g) the hydrolysis of the 5-acylsulfanyl-histidine compound obtained in a polar protic solvent by stirring at a temperature between 70 and 130 C. in the presence of a thiol, h) then, optionally, the purification by column liquid chromatography or any other purification method well known to the person skilled in the art. 2) Or: j) by hydrolysis of the 5-acylsulfanyl-histidine compound obtained in a polar protic solvent by stirring at a temperature between 70 and 130 C. in order to obtain the corresponding disulfide, k) then, optionally, purification by column liquid chromatography or any other purification method well known to the person skilled in the art.

10. The method of claim 9, wherein the agent generating halogenium ions X.sup.+ is selected from: bromine Br.sub.2, as commercial reagent or prepared in situ; or NBS or any N-bromo-imide and N-bromo-amide compound.

11. The method of claim 9, wherein the polar protic solvent is water or an aqueous solution.

12. The method of claim 9, wherein the sulfur-containing reagent of the carbothioic acid is selected from thioacetic acid, thiobenzoic acid, potassium thioacetate, or mixtures thereof.

13. The method of claim 9, wherein the temperature in method (A) is 0-5 C.

14. The method of claim 9, wherein the thiol is selected from mercaptopropionic acid, dithiothreitol or mixtures thereof.

15. The method of claim 9, wherein the temperature in method (B) is between 20 and 130 C.

16. A 5-sulfanylhistidine compound of formula (II): ##STR00087## where: R.sup.1H, or alkyl; R.sup.2R.sup.3H, or alkyl; R.sup.4H, alkyl, alkyl (CO), substituted alkyl (CO), aryl (CO), -alanyl (H2NCH2CH2 (CO), or -amino-acyl; under the provisos that when R.sup.1H or methyl, then R.sup.2, R.sup.3 and R.sup.4 cannot simultaneously be H; as well as all the stereoisomers, diastereoisomers and enantiomers, in particular in terms of the carbon atom bearing the COOH group, as well as all the disulfides obtained by condensation of two 5-sulfanylhistidine compounds of formula (II), taken separately or in a mixture; and all the salts of pharmaceutically acceptable acids of said compound of formula (II).

17. An antioxidant comprising or consisting essentially of a 5-sulfanylhistidine compound of formula (II): ##STR00088## where: R.sup.1H, or alkyl; R.sup.2R.sup.3H, or alkyl; R.sup.4H, alkyl, alkyl (CO), substituted alkyl (CO), aryl (CO), -alanyl (H2NCH2CH2 (CO), or -amino-acyl; it being understood that when R.sup.1H then R.sup.2, R.sup.3 and R.sup.4 cannot simultaneously be H; as well as all the stereoisomers, diastereoisomers and enantiomers, in particular in terms of the carbon atom bearing the COOH group, as well as all the disulfides obtained by condensation of two 5-sulfanylhistidine compounds of formula (II), taken separately or in a mixture; and all the salts of pharmaceutically acceptable acids of said compound of formula (II).

18. The antioxidant of claim 17, wherein R.sup.4 represents hydrogen, methyl, acetyl, benzoyl, -aminoacyl or -alanyl (H.sub.2NCH.sub.2CH.sub.2 (CO).

19. The antioxidant of claim 17, wherein said 5-sulfanylhistidine compound is selected from the group consisting of: the disulfide of L-5-sulfanyl-,N(methyl)-histidine (Compound 22); L-5-sulfanyl-,N(methyl)-histidine (Compound 23); the disulfide of L-5-sulfanyl-,N,N(dimethyl)-histidine (Compound 24); L-5-sulfanyl-,N,N(dimethyl)-histidine (Compound 25); L-5-sulfanyl-,N,N,N(trimethyl)-histidine (Compound 26); the disulfide of L-5-sulfanyl-,N,N,N(trimethyl)-histidine (Compound 27); the disulfide of L-5-sulfanyl-,N(acetyl)-histidine (Compound 28); L-5-sulfanyl-,N(acetyl)-histidine (Compound 29); L-5-sulfanylcarnosine (Compound 30); the disulfide of iso-ovothiol A (Compound 31); L-5-sulfanyl-1-methyl-histidine named iso-ovothiol A (Compound 32); the disulfide of L-5-sulfanyl-,N,N(dimethyl)-1-methylhistidine (Compound 33); L-5-sulfanyl-,N,N,N(trimethyl)-1-methylhistidine (Compound 34); L-5-sulfanyl-,N(L-alanyl)-histidine (Compound 35); and the disulfide of 5-sulfanyl-,N(pentanoyl)-histidine (Compound 36).

20. The antioxidant of claim 17, wherein the salts of pharmaceutically acceptable acids of said compound of formula (II), are selected from a mineral acid, an organic acid, an alkanesulfonic acid or an arylsulfonic acid such as benzene- and paratoluenesulfonic acids.

21. A method of use of the 5-sulfanylhistidine compound of formula (II): ##STR00089## where: R.sup.1H, or alkyl; R.sup.2R.sup.3H, or alkyl; R.sup.4H, alkyl, alkyl (CO), substituted alkyl (CO), aryl (CO), -alanyl (H2NCH2CH2 (CO), or -amino-acyl; it being understood that when R.sup.1H then R.sup.2, R.sup.3 and R.sup.4 cannot simultaneously be H; as well as all the stereoisomers, diastereoisomers and enantiomers, in particular in terms of the carbon atom bearing the COOH group, as well as all the disulfides obtained by condensation of two 5-sulfanylhistidine compounds of formula (II), taken separately or in a mixture; and all the salts of pharmaceutically acceptable acids of said compound of formula (II). as an antioxidant agent incorporated in a composition in an effective amount to minimize or impede oxidation of said composition. or any other purification method well known to the person skilled in the art.

Description

DESCRIPTION OF THE FIGURES

[0184] The invention includes 5 figures.

[0185] FIG. 1: Scheme of the method for synthesizing compounds according to general formula (I)

[0186] FIG. 2: Representative spectrum (H.sup.1 NMR, 400 MHz) of the reaction mixture obtained in Example 1, preparation of L-5-acetylsulfanyl-histidine (Compound 1)

[0187] FIG. 3: Representative spectrum (H.sup.1 NMR, 400 MHz) of the reaction mixture obtained in Example 3, preparation of L-5-acetylsulfanyl-,N,N(dimethyl)-histidine (Compound 2)

[0188] FIG. 4: Representative spectrum (H.sup.1 NMR, 400 MHz) of the reaction mixture obtained in Example 5, preparation of L-5-acetylsulfanyl-,N,N,N(trimethyl)-histidine (Compound 3)

[0189] FIG. 5: Relates to antioxidant activity tests made with several best representatives of the invention 5-sulfanylhistidines and their disulfides constituted by compounds 23, 32 and 35, as compared with the positive best recognized prior art representative potent antioxidant constituted by Ebselen. The antioxidant activity is based on well recognized by one skilled in the art method relating to GSH-Px activity of 5-sulfanyl-histidines. Reactions were initiated by addition of 1.0 mM H.sub.2O.sub.2 to the assay mixture containing 1.0 mM GSH, 0.28 mM NADPH, 1 U/ml GSSG reductase and respectively 12.5 M ebselen (.diamond-solid.); 250 M compound 32 (); 250 M compound 23 (.square-solid.); 250 M compound 35 (). Results are means of four experiments.

DESCRIPTION OF THE EXAMPLES

[0190] The following examples as well as the scheme of the method of the invention (see FIG. 1) are provided only for illustration and are in no way capable of limiting the scope of the invention.

[0191] In the examples described below, the temperature is either ambient temperature or a temperature given in degree Celsius, and the pressure is atmospheric pressure, unless otherwise indicated.

[0192] The reagents used are commercially available from international suppliers such as SAF (France), Alfa Aesar, Fisher Scientific, TCI Europe, Bachem (Switzerland, AKOS (Germany) except for the following compounds: N-methylhistidine hydrochloride, N,N-dimethylhistidine hydrochloride hydrate and L-hercynine, which were prepared according to the cited protocols.

[0193] All the experiments are carried out in the ambient atmosphere unless indicated otherwise.

[0194] The .sup.1H NMR analyses were recorded at 400 MHz or at 300 MHz in D.sub.2O or a D.sub.2O/DCl mixture, using the HOD signal (4.79 ppm) as internal reference. The chemical shifts are noted in ppm, and the multiplicity of the signals indicated by the following symbols: s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). The coupling constants are recorded in hertz (Hz). The .sup.13C NMR analyses are recorded at 75 MHz in D.sub.2O or D.sub.2O/DCl. The mass analyses are obtained by chemical ionization at atmospheric pressure (APCI-MS). The melting points were measured using an apparatus from the company Stuart Scientific. The HPLC analyses were carried out on an Acquity apparatus (Waters), using two types of columns: A. Kromasil Diol column 2504.6 (5 m). The mobile phase used is a mixture of solvent A (10/90 H.sub.2O/acetonitrile+0.05% TFA) and of solvent B (50/50 H.sub.2O/acetonitrile+0.05% TFA), with a gradient varying over 10 minutes from 90% A to 100% B and at a flow rate of 1.2 mL/min. B. Column of the Thermo Hypercarb type 1004.6 (5 m). The mobile phase used is a mixture of solvent A (100% H2O+0.2% HCOOH) and of solvent B (100% acetonitrile+0.2% HCOOH), with a gradient varying over 8 minutes from 100% A to 40% and a flow rate of 1 mL/min. The detection is carried out with a universal ELSD detector (Sedere).

IPREPARATION OF THE 5-ACYLSULFANYL-HISTIDINE DERIVATIVES AS PRECURSORS OF THE 5-SULFANYLHISTIDINES AND THEIR DISULFIDES

[0195] In the first paragraph, examples are given of the preparation of the 5-acylsulfanyl-histidine derivatives by activation with dibromine or N-bromosuccinimide (NBS) and reaction of the intermediate formed with thioacetic acid.

[0196] In the second paragraph, examples are given of the use of these 5-acylsulfanyl derivatives, generally prepared in situ, as precursor of 5-sulfanylhistidines and their derivatives.

I.1. Preparation of the 5-Acylsulfanyl-Histidine Derivatives by Activation with Dibromine or with N-Bromosuccinimide (NBS) and Reaction with Thioacetic Acid

Example 1: Preparation of L-5-acetylsulfanyl-histidine (Compound 1) by Activation with Dibromine and Reaction with Thioacetic Acid

[0197] ##STR00008##

[0198] The hydrochloride of monohydrated L-histidine (52.93 g; 250 mmoles; 1 eq.) is dissolved in 1.5 L of demineralized water, then the solution is cooled to 0 C. in 30 minutes. Under strong stirring, dibromine (16.7 mL; 51.93 g; 325 mmoles; 1.3 eq.) is added dropwise very rapidly. The solution turns red. Thioacetic acid (73.3 mL; 78.46 g; 1 mole; 4 eq.) is added very rapidly: the solution immediately becomes decolorized and changes from red to light yellow. The vigorous stirring is maintained at 0 C. for 1 h.

[0199] Compound 1 is obtained with a reaction yield of 72 mol % as calculated from the .sup.1H NMR spectrum.

[0200] .sup.1H NMR (D.sub.2O pH1, 400 MHz) of a sample of the mixture: (ppm)=2.57 (s, 3H); 3.38 (dd, J=15.6 Hz and J=6.8 Hz, 1H); 3.47 (dd, J=15.6 Hz, J=7.8 Hz, 1H); 4.34 (dd, J=7.8 Hz and J=6.8 Hz, 1H); 8.94 (s, 1H).

[0201] A singlet corresponding to the excess of thioacetic acid is detected at 2.48 ppm, as are signals of low intensity corresponding to the side products such as the acetic acid detected at 2.0 ppm. A representative spectrum is included in FIG. 2.

[0202] LCMS (APCI): 228.0 [MH].sup.

Example 2: Preparation of L-5-acetylsulfanyl-histidine (Compound 1) by Activation with N-bromosuccinimide and Reaction with Thioacetic Acid

[0203] ##STR00009##

[0204] The hydrochloride of monohydrated L-histidine (10.48 g; 50 mmoles; 1 eq.) is dissolved in 300 mL of demineralized water containing a 37% concentrated hydrochloric acid solution (4.17 mL; 4.92 g; 50 mmoles; 1 eq.), then the solution is cooled to 0 C. Very strong stirring is maintained. N-Bromosuccinimide (11.56 g; 65 mmoles; 1.3 eq.) is added in a single portion: the mixture turns limpid orange after 30 seconds. The temperature rises to 1 C. After 2 minutes 30 seconds, thioacetic acid (14.7 mL; 15.69 g; 200 mmoles; 4 eq.) is added all at once: the decolorization occurs very rapidly. The temperature rises to 4 C. After cooling to 0 C., the vigorous stirring is maintained for 1 h.

[0205] Compound 1 is obtained with a reaction yield of 75 mol % as calculated from the H.sup.1 NMR spectrogram (in the reaction mixture).

[0206] The .sup.1H NMR and mass spectra are identical to those obtained in Example 1.

Example 3: Preparation of L-5-acetylsulfanyl-,N,N(dimethyl)-histidine (Compound 2) by Activation with N-bromosuccinimide and Reaction with Thioacetic Acid

[0207] ##STR00010##

[0208] The hydrochloride of monohydrated -N,N(dimethyl)-histidine (6.06 g; 25 mmoles; 1 eq.) (see V. N. Reinhold et al., J. Med. Chem. 1968, 11, 258-260) is dissolved in 135 mL of demineralized water. Then a 37% concentrated hydrochloric acid solution (2.1 mL; 2.46 g; 25 mmoles; 1 eq.) is added, and the resulting solution is cooled to 1 C. Very vigorous stirring is maintained. N-bromosuccinimide (2.31 g; 13 mmoles; 1.3 eq.) is added rapidly. After 1 minute, thioacetic acid (2.94 mL, 3.14 g; 40 mmoles; 4 eq.) is added very rapidly. Vigorous stirring is maintained at 0 C. for 30 minutes.

[0209] Compound 2 is obtained with a reaction yield of 70 mol % as calculated from the .sup.1H NMR spectrogram.

[0210] .sup.1H NMR (D.sub.2O pH1, 400 MHz) of a sample of the mixture: (ppm)=2.57 (s, 3H); 2.79 (s, 6H); 3.42 (dd, J=14.9 Hz and J=10.6 Hz, 1H); 3.49 (dd, J=14.9 Hz and J=4.4 Hz, 1H); 4.20 (dd, J=10.6 Hz and J=4.4 Hz, 1H); 8.92 (s, 1H).

[0211] A singlet corresponding to the excess of thioacetic acid is detected at 2.47 ppm, as are signals of low intensity corresponding to the side products such as the acetic acid detected at 2.0 ppm. A representative spectrum is included in FIG. 3.

[0212] Compound 2 is purified on a silica column using an ethyl acetate/ethanol gradient followed by elution with water.

[0213] .sup.1H NMR (D.sub.2O pH 2-3, 300 MHz): (ppm)=2.54 (s, 3H); 2.96 (s, 6H); 3.28 (dd, J=14.7 Hz and J=10.4 Hz, 1H); 3.39 (dd, J=14.7 Hz, J=4.4 Hz, 1H); 3.87 (dd, J=10.4 Hz and J=4.4 Hz, 1H); 8.81 (s, 1H).

[0214] A singlet of low intensity corresponding to the hydrolyzed product (compound 18b) is detected at 8.33 ppm.

[0215] .sup.13C NMR (D.sub.2O, 75 MHz): (ppm)=22.4; 30.0; 41.7; 6.84; 117.2; 134.3; 136.8; 170.9; 195.9.

[0216] LCMS (APCI): 258.9 [M+H].sup.+

Example 4: Preparation of L-5-acetylsulfanyl-,N,N(dimethyl)-histidine (Compound 2) by Activation with Dibromine and Reaction with Thioacetic Acid

[0217] ##STR00011##

[0218] The hydrochloride of monohydrated ,N,N(dimethyl)histidine (1.66 g; 6.98 mmoles; 1 eq.) (see V. N. Reinhold et al., J. Med. Chem. 1968, 11, 258-260) is dissolved in 57 mL of demineralized water, then the solution is cooled to 0 C. Under strong stirring, dibromine (470 L; 1.45 g; 9.08 mmoles; 1.3 eq.) is added dropwise in 3 minutes. The solution turns red. After 1 minute, thioacetic acid (2.56 mL; 2.74 g; 34.91 mmoles; 5 eq.) is added very rapidly: the solution immediately becomes decolorized and changes from red to clear yellow. Vigorous stirring is maintained at 0 C. for 1 h.

[0219] Compound 2 is obtained with a reaction yield of 69 mol % as calculated from the .sup.1HNMR spectrogram.

[0220] The .sup.1H NMR and mass spectra are identical to those obtained in Example 3.

Example 5: Preparation of L-5-acetylsulfanyl-,N,N,N(trimethyl)-histidine (Compound 3) by Activation with N-Bromosuccinimide and Reaction with Thioacetic Acid

[0221] ##STR00012##

[0222] L-Hercynine (2.0 g; 9.96 mmoles; 1 eq.) (see V. N. Reinhold et al., J. Med. Chem. 1968, 11, 258-260) is dissolved in 55 mL of demineralized water. Then a 37% concentrated hydrochloric acid solution (1.66 mL; 1.96 g; 19.91 mmoles; 2 eq.) is added, and cooled to 0 C. Under strong stirring, N-bromosuccinimide (2.48 g; 13.94 mmoles; 1.4 eq.) is added: the solution turns red. After 5 minutes, thioacetic acid (4.4 mL; 4.69 g; 59.74 mmoles; 6 eq.) is added very rapidly. The stirring is maintained for 40 minutes.

[0223] Compound 3 is obtained with a reaction yield of 65 mol % as calculated from the .sup.1H NMR spectrogram.

[0224] .sup.1H NMR (D.sub.2O, pH=1, 400 MHz) of a sample of the mixture: (ppm): 2.53 (s, 3H); 3.33 (s, 9H); 3.50 (m, 2H); 4.13 (m, 1H); 8.91 (s, 1H).

[0225] Two singlets corresponding to the excess of thioacetic acid and to the succinimide are at 2.44 ppm and 2.76 ppm, as are signals of low intensity corresponding to the side products such as the acetic acid detected at 2.0 ppm. A representative spectrum is included in FIG. 4.

[0226] The product is purified on a silica column (ethyl acetate/ethanol/water gradient).

[0227] .sup.1H NMR (D.sub.2O, pH 2-3, 400 MHz): (ppm)=2.53 (s, 3H); 3.30 (s, 9H); 3.37 (m, 1H); 3.44 (dd, J=14.0 Hz and J=3.8 Hz, 1H); 3.88 (dd, J=11.7 Hz and J=3.8 Hz, 1H); 8.72 (s, 1H).

[0228] .sup.13C NMR (D.sub.2O, 75 MHz): (ppm)=22.9; 30.0; 52.5; 76.5; 117.9; 133.1; 137.2; 169.7; 196.0.

[0229] LCMS (APCI): 272.1 [M+H].sup.+

Example 6: Preparation of L-5-acetylsulfanyl-,N,N,N(trimethyl)-histidine (Compound 3) by Activation with Bromine and Reaction with Thioacetic Acid

[0230] ##STR00013##

[0231] L-Hercynine (1.0 g, 5 mmoles; 1 eq.) is dissolved in 35 mL of demineralized water. Then a 37% concentrated hydrochloric acid solution (417 L; 5 mmoles; 1 eq.) is added, and the solution is cooled to 1 C. Under strong stirring, dibromine (0.33 mL; 1.03 g, 6.5 mmoles; 1.3 eq.) is added: a red gum forms in a first phase and dissolves after 30 minutes. After 4 minutes, thioacetic acid (2.20 mL; 2.68 g; 25 mmoles; 10 eq.) is added very rapidly. The stirring is continued for 30 minutes.

[0232] Compound 3 is obtained with a reaction yield of 68 mol % as calculated from the .sup.1H NMR spectrogram.

[0233] The .sup.1H NMR and mass spectra are identical to those obtained in Example 5.

Example 7: Preparation of L-5-acetylsulfanyl-,N(glycinyl)-histidine (Compound 4) by Activation with N-Bromosuccinimide and Reaction with Thioacetic Acid

[0234] ##STR00014##

[0235] ,N(Glycinyl)-histidine (212 mg, 1 mmole; 1 eq.) is dissolved in 7 mL of demineralized water and 1 mL of acetonitrile. Then a 37% concentrated hydrochloric acid solution (170 L, 2 mmoles; 2 eq.) is added, and the solution is cooled to 0 C. Under strong stirring, N-bromosuccinimide (230 mg, 1.3 mmoles; 1.3 eq.) is added. After 3 minutes, thioacetic acid (370 L, 5.0 mmoles; 5 eq.) is added very rapidly. Stirring is maintained at 0 C. for 30 minutes.

[0236] Compound 4 is obtained with a reaction yield of 62 mol % as calculated from the .sup.1H NMR spectrogram.

[0237] .sup.1H NMR (D.sub.2O, pH1, 400 MHz) of a sample of the mixture: (ppm)=2.53 (s, 3H), 3.20 (dd, J=15.3 Hz and J=8.5 Hz, 1H), 3.36 (dd, J=15.3 Hz and J=5.7 Hz, 1H), 3.79 (dd, J=16.4 Hz and J=10.7 Hz, 2H); 3.84 (m, 1H), 8.86 (s, 1H).

[0238] A singlet corresponding to the excess of thioacetic acid is detected at 2.48 ppm, a singlet at 2.78 ppm corresponding to the succinimide is detected as are signals of low intensity corresponding to the side products, such as the acetic acid detected at 2.0 ppm.

[0239] LCMS (APCI): 287.3 [M+H]+

Example 8: Preparation of the L-5-acetylsulfanyl-,N,N(dimethyl)-1-methylhistidine Derivative (Compound 5) by Activation with N-bromosuccinimide and Reaction with Thioacetic Acid

a) Preparation of ,N,N(dimethyl)-1-methyl-L-histidine

[0240] ,N,N(Dimethyl)-1-methyl-L-histidine is prepared by analogy with the protocol described for ,N,N(dimethyl)-L-histidine (V. N. Reinhold et al., J. Med. Chem. 1968, 11, 258-260) from 1-methyl-L-histidine and formaldehyde by reducing amination in the presence of palladium on activated charcoal (88%).

[0241] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=2.91 (s, 6H); 3.18 (d, J=6.4 Hz, 2H); 3.66 (s, 3H); 3.85 (t, J=6.4 Hz, 1H); 6.96 (s, 1H); 7.57 (s, 1H).

b) Preparation of the L-5-acetylsulfanyl-,N,N(dimethyl)-1-methylhistidine Derivative

[0242] ##STR00015##

[0243] ,N,N(dimethyl)-1-methylhistidine (604 mg, 3 mmoles; 1 eq.) is dissolved in 22 mL of demineralized water. 37% Concentrated hydrochloric acid (250 L, 3 mmoles; 1 eq.) is added, then the solution is cooled to 0 C. Very strong stirring is maintained. N-Bromosuccinimide (700 mg, 3.9 mmoles; 1.3 eq.) is added rapidly. After 3 minutes, thioacetic acid (1.1 mL, 15 mmoles; 5 eq.) is added very rapidly. Vigorous stirring is maintained at 0 C. for 30 minutes.

[0244] Compound 5 is obtained with a reaction yield of 65 mol % as calculated from the .sup.1H NMR spectrogram.

[0245] .sup.1H NMR (D.sub.2O, pH1,400 MHz) of a sample of the mixture: (ppm)=2.58 (s, 3H), 3.00 (s, 6H), 3.38 (dd, J=14.9 Hz and J=10.7 Hz, 1H), 3.46 (dd, J=14.9 Hz and J=4.3 Hz, 1H), 3.77 (s, 3H), 4.12 (dd, J=10.7 Hz and J=4.3 Hz, 1H), 8.97 (s, 1H)

[0246] A singlet corresponding to the excess of thioacetic acid is detected at 2.48 ppm, a singlet at 2.78 ppm corresponding to the succinimide is detected as are signals of low intensity corresponding to the side products such as acetic acid detected at 2.0 ppm.

[0247] The product is purified on a silica column using an 2/2/1 ethyl acetate/ethanol/water gradient followed by elution with a 1/1 ethanol/water mixture. Compound 5 (48%) is obtained in the form of a transparent oil.

[0248] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=2.56 (s, 3H); 2.95 (s, 6H); 3.25 (dd, J=15.0 Hz and J=9.0 Hz, 1H); 3.31 (dd, J=15.0 Hz and J=5.4 Hz, 1H); 3.69 (s, 3H); 3.86 (dd, J=9.0 Hz and J=5.4 Hz, 1H); 8.53 (s, 1H).

[0249] The signals of ethanol are detected at 1.18 ppm and 3.65 ppm.

[0250] LCMS (APCI): 272.3 [M+H]+

Example 9: Preparation of L-5-acetylsulfanyl-,N,N,N(trimethyl)-1-methylhistidine (Compound 6) by Activation with N-bromosuccinimide and Reaction with Thioacetic Acid

a) Preparation of 1-methyl-hercynine

[0251] 1-Methyl-hercynine is prepared by analogy with the protocol described for hercynine (V. N. Reinhold et al., J. Med. Chem. 1968, 11, 258-260) from 1-methyl-dimethyl-L-histidine and iodomethane by quaternization in methanol (89%).

[0252] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=3.19 (m, 2H); 3.28 (s, 9H); 3.67 (s, 3H); 3.89 (dd, J=10.6 Hz and 4.5 Hz, 1H); 6.94 (s, 1H); 7.57 (s, 1H).

b) Preparation of L-5-acetylsulfanyl-,N,N,N(trimethyl)-1-methylhistidine (Compound 6)

[0253] ##STR00016##

[0254] 1-Methyl-hercynine (430 mg, 2 mmoles; 1 eq.) is dissolved in 15 mL of demineralized water. 37% concentrated hydrochloric acid (170 L, 2 mmoles; 1 eq.) is added, then the solution is cooled to 0 C. Under strong stirring, N-bromosuccinimide (465 mg, 2.6 mmoles; 1.3 eq.) is added. After 3 minutes, thioacetic acid (740 L, 10 mmoles; 5 eq.) is added very rapidly. The stirring is maintained at 0 C. for 30 minutes.

[0255] Compound 6 is obtained with a reaction yield of 67 mol % as calculated from the .sup.1H NMR spectrogram.

[0256] .sup.1H NMR (D.sub.2O, pH1, 400 MHz) of a sample of the mixture: (ppm)=2.57 (s, 3H), 3.32 (s, 9H), 3.53 (m, 2H), 3.75 (s, 3H), 4.08 (dd, J=11.9 Hz and J=3.7 Hz, 1H), 8.98 (s, 1H).

[0257] A singlet corresponding to the excess of thioacetic acid is detected at 2.48 ppm, a singlet at 2.78 ppm corresponding to succinimide is detected as are signals of low intensity corresponding to the side products, such as acetic acid detected at 2.0 ppm.

[0258] LCMS (APCI): 286.0 [M+H]+

Example 10: Preparation of L-5-acetylsulfanyl--N(L-alanyl)-histidine (Compound 7) by Activation with N-bromosuccinimide and Reaction with Thioacetic Acid

[0259] ##STR00017##

[0260] -N(L-alanyl)-histidine (500 mg, 2.2 mmoles; 1 eq.) is dissolved in 15 mL of demineralized water containing a 37% concentrated hydrochloric acid solution (370 L, 4.4 mmoles; 2 eq.), then the solution is cooled to 0 C. N-Bromosuccinimide (510 mg, 2.9 mmoles; 1.3 eq.) is added in one portion: the mixture turns limpid orange after 30 seconds. After 3 minutes, thioacetic acid (820 L, 11.0 mmoles; 5 eq.) is added very rapidly. Vigorous stirring is maintained at 0 C. for 30 minutes.

[0261] Compound 7 is obtained with a reaction yield of 65 mol % as calculated from the .sup.1H NMR spectrogram of a sample.

[0262] The reaction mixture is washed with 225 mL of ethyl acetate, then the compound is purified on a silica column (ethyl acetateethanol/water 2/2/1). Compound 7 (410 mg, 54%, purity 88%) is obtained in the form of a transparent oil.

[0263] .sup.1H NMR (D.sub.2O, acid pH, 400 MHz): (ppm)=1.49 (d, J=7.2 Hz, 3H); 2.53 (s, 3H); 3.20 (dd, J=15.3 Hz and J=8.9 Hz, 1H); 3.36 (dd, J=15.3 Hz and J=5.8 Hz, 1H), 4.01 (q, J=7.2 Hz, 1H); 4.77 (m superposed over HOD signal); 8.86 (s, 1H).

[0264] A singlet corresponding to the succinimide is detected at 2.68 ppm.

[0265] LCMS (APCI): 301.1 [M+H]+

Example 11: Preparation of the 5-acetylsulfanyl-,N(pentanoyl)-histidine Derivative (Compound 8) by Activation with N-bromosuccinimide and Reaction with Thioacetic Acid

[0266] ##STR00018##

[0267] ,N(pentanoyl)-histidine (450 mg, 1.43 mmoles; 1 eq.) is dissolved in 10 mL of demineralized water containing a 37% concentrated hydrochloric acid solution (120 L, 1.43 mmoles; 1 eq.), then the solution is cooled to 0 C. Very strong stirring is maintained. N-Bromosuccinimide (330 mg, 1.86 mmoles; 1.3 eq.) is added. After 3 minutes, thioacetic acid (530 L, 7.15 mmoles; 5 eq.) is added very rapidly. Vigorous stirring is maintained at 0 C. for 30 minutes.

[0268] Compound 8 is obtained with a reaction yield of 67 mol % as calculated from the .sup.1H NMR spectrogram of a sample.

[0269] The product is purified on a silica column (mixture 90% of ethyl acetate/ethanol 3/1 and 10% of water). 5-Acetylsulfanyl-,N(pentanoyl)-histidine (compound 8) is obtained in the form of a transparent oil (320 mg, 64%, purity 90%).

[0270] .sup.1H NMR (D.sub.2O1, 400 MHz): (ppm): 0.85 (t, J=7.3 Hz, 3H); 1.17 (h, J=7.4 Hz, 2H); 1.47 (p, J=7.4 Hz, 2H); 2.22 (t, J=7.4 Hz, 2H); 2.55 (s, 3H); 3.17 (dd, J=15.2 Hz and J=9.6 Hz, 1H); 3.37 (dd, J=15.2 Hz and J=5.2 Hz, 1H); 4.79 (m superposed over HOD signal); 8.88 (s, 1H).

[0271] LCMS (APCI): 314.1 [M+H]+

[0272] To illustrate the invention, compounds 9-17 are prepared (Examples 12-21) by analogy with the preceding examples. The results, as well as the spectral characteristics, are summarized in Table 1 below.

TABLE-US-00001 TABLE 1 Examples 12-21 describing the preparation of Compounds 9-17 according to the invention. The compound formulas follow the table. % conversion .sup.1H NMR: by to Characteristic signals LC- Ex. analogy desired MS No. Formed product with Reagent product H-2 -H AcS [M + H]+ Ex. 12 Compound 9 Ex. 2 NBS 78% 8.91 4.13 (dd, 2.55 244.1 J = 7.7 Hz, J = 5.6 Hz) Ex. 13 Compound 10 Ex. 2 NBS 65% 8.83 4.73 (m) 2.51 272.1 (1 eq. HCl) Ex. 14 Compound 11 Ex. 2 NBS 58% 8.85 4.93 (dd, 2.56 334.1 (1 eq. J = 9.3 HCl) Hz, J = 4.7 Hz) Ex. 15 Compound 12 Ex. 2 NBS 63% 8.84 4.66 (m) 2.51 301.0 Ex. 16 Compound 13 Ex. 2 NBS 63% 9.00 4.31 (dd, 2.58 244.6 Ex. 17 Ex. 1 Br2 55% J = 7.6 Hz, J = 6.9 Hz) Ex. 18 Compound 14 Ex. 2 NBS 31% 8.98 4.37 292.1 (water/ CH3CN) Ex. 19 Compound 15 Ex. 2 NBS 12% 8.95 4.28 320.9 (water/ CH3CN) Ex. 20 Compound 16 Ex. 2 NBS 12% 9.06 4.32 334.1 (water/ CH3CN) Ex. 21 Compound 17 Ex. 2 NBS 40% 8.89 4.36 2.58 377.2

##STR00019## ##STR00020##

II. APPLICATION EXAMPLES

[0273] II.1 Transformation of the 5-Acylsulfanyl Derivatives Prepared In Situ into Corresponding 5-Sulfanylhistidine Derivatives by Hydrolysis

[0274] To illustrate the application of the 5-acylsulfanyl-histidine derivatives according to the invention, in a nonlimiting manner, application examples are given in this paragraph of the novel 5-acylsulfanyl-histidine derivatives, generally prepared in situ, as precursor of 5-sulfanylhistidines and their derivatives.

[0275] These examples illustrate the usefulness of the novel 5-acylsulfanyl derivatives described in the invention for easily preparing 5-sulfanylhistidine compounds and their derivatives such as the disulfides, which, furthermore, are very difficult to prepare and require multistep syntheses.

[0276] In order to obtain better yields of 5-sulfanylhistidine derivatives, the 5-acylsulfanyl compounds are prepared in situ, then hydrolyzed directly thereafter, by stirring the reaction medium, preferably while heating the reaction medium. The presence of a thiol, such as mercaptopropionic acid or dithiothreitol, proves to be useful for the easy isolation of the 5-sulfanylhistidine derivatives, but it is not needed at all for the hydrolysis itself, as demonstrated in the follow-up examples 18d, 19b and 19c.

Example 22: One Pot Preparation of L-5-sulfanylhistidine Via In-Situ Preparation of 5-acetylsulfanylhistidine Followed by Hydrolysis (Compound 18)

[0277] ##STR00021##

[0278] The hydrochloride of monohydrated L-histidine (10.48 g; 50 mmoles; 1 eq.) is dissolved in 300 mL of demineralized water and hydrochloric acid concentrated at 37% (4.17 mL; 4.92 g; 50 mmoles; 1 eq.), then the solution is cooled to 0 C. Very strong stirring is maintained. N-Bromosuccinimide (11.56 g; 65 mmoles; 1.3 eq.) is added in a single portion: the mixture becomes limpid orange. Thioacetic acid (14.7 mL; 15.69 g; 200 mmoles; 4 eq.) is added all at once. Vigorous stirring is maintained at 0 C. for 1 h. 3-Mercaptopropionic acid (26 mL; 32.2 g; 300 mmoles; 6 eq.) is added, then the slightly yellow solution is heated at 90 C. for 18 h. The solution is extracted with three times 300 mL of ethyl acetate. After neutralization and crystallization in the presence of dithiothreitol (231 mg; 1.5 mmoles; 0.03 eq.), the desired compound 18 crystallizes. The solid is filtered and dried under a vacuum to yield 2.97 g (31%; 41% with respect to the quantity of the intermediate SAc) of L-5-sulfanylhistidine (Compound 18) in the form of an off-white solid.

[0279] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=3.18 (dd, J=15.8 Hz, J=7.3 Hz, 1H); 3.26 (dd, J=15.8 Hz and J=5.1 Hz, 1H); 4.33 (dd, J=7.3 Hz, J=5.1 Hz, 1H); 8.25 (s, 1H).

[0280] .sup.1H NMR (D.sub.2O+DCl, 400 MHz): (ppm)=3.11 (dd, J=15.1 Hz, J=6.5 Hz, 1H); 3.19 (dd, J=15.1 Hz and J=6.6 Hz, 1H); 4.12 (t, J=7.0 Hz, 1H); 8.37 (s, 1H).

[0281] .sup.13C NMR (D.sub.2O+DCl, 75 MHz): (ppm)=26.3; 55.2, 122.1; 130.1; 135.5; 173.6.

[0282] LC-MS (AP): 186.0 [MH].sup.

[0283] [].sub.D+7.4 (c=0.1; 1N HCl)

[0284] Elemental analysis: C.sub.6H.sub.9N.sub.3O.sub.2S; Theoretical: C, 38.49%; H, 4.84% N, 22.44; Measured: C, 38.0%; H, 4.96%; N, 22.06.

Example 23: One Pot Preparation of D-5-sulfanylhistidine Via In-Situ Preparation of 5-acetylsulfanyl-histidine Followed by Hydrolysis (Compound 19)

[0285] ##STR00022##

[0286] D-Histidine (3.92 g; 25 mmoles; 1 eq.) is dissolved in 150 mL of demineralized water and a hydrochloric acid solution concentrated at 37% (4.17 mL; 4.92 g; 50 mmoles; 2 eq.), then the solution is cooled to 0 C. Very strong stirring is maintained. N-Bromosuccinimide (5.78 g; 32.5 mmoles; 1.3 eq.) is added all at once: the solution turns limpid orange. Thioacetic acid (7.33 mL; 7.85 g; 200 mmoles; 4 eq.) is added all at once. Vigorous stirring is maintained at 0 C. for 1 h. 3-Mercaptopropionic acid (13 mL; 16.1 g; 150 mmoles; 6 eq.) is added, then the solution is heated at 100 C. for 18 h. After cooling, the solution is extracted with three times 150 mL of ethyl acetate. Dithiothreitol (13 mL; 16.1 g; 150 mmoles; 6 eq.) is added to the aqueous phase. After recrystallization in the presence of activated charcoal, the yield consists of 1.25 g of D-5-sulfanylhistidine (Compound 19) (26%; 35% with respect to the quantity of the intermediate SAc) in the form of a beige solid.

[0287] The .sup.1H NMR, .sup.13C NMR and mass spectra are identical to those obtained in Example 13 for Compound 9.

[0288] [].sub.D: 7.1 (C=0.1; 1N HCl)

Example 24: One Pot Preparation of D,L-5-sulfanylhistidine Via In-Situ Preparation of 5-acylsulfanyl-histidine Followed by Hydrolysis (Compound 20)

[0289] ##STR00023##

[0290] The hydrochloride of monohydrated DL-histidine (3.21 g; 15 mmoles; 1 eq.) is dissolved in 100 mL of demineralized water and a 37% concentrated hydrochloric acid solution (1.25 mL; 1.48 g; 15 mmoles; 1 eq.), then the solution is cooled to 0 C. Under very strong stirring, N-bromosuccinimide (3.47 g; 19.5 mmoles; 1.3 eq.) is added all at once. After 2 minutes, thioacetic acid (4.4 mL; 4.71 g; 60 mmoles; 4 eq.) is added all at once. Stirring is continued at 0 C. for 1 h. 3-Mercaptopropionic acid (8.0 mL; 9.65 g; 90 mmoles; 6 eq.) is added, then the solution is heated at 100 C. for 18 h. A precipitate corresponding to the disulfide of thioacetic acid and of mercaptopropionic acid is eliminated by filtration. The filtrate is washed with two times 100 mL of ethyl acetate. After neutralization and crystallization in the presence of dithiothreitol (233 mg; 1.5 mmoles; 0.1 eq.), 650 mg of D,L-5-sulfanylhistidine (Compound 20) (23%, 29% with respect to the quantity of the intermediate SAc) are obtained in the form of a white solid.

[0291] The .sup.1H NMR, .sup.13C NMR and mass spectra are identical to those obtained in Example 22 for Compound 18.

II.2 Transformation of the 5-acylsulfanyl Derivatives Prepared In Situ into Corresponding 5,5-disulfane-diyl-bis-histidine Derivatives (Disulfides) by Hydrolysis

Example 25: One Pot Preparation of the Disulfide of L-5-sulfanylhistidine Via In-Situ Preparation of 5-acylsulfanyl-histidine Followed by Hydrolysis and Oxidation (Compound 21)

[0292] ##STR00024##

[0293] The hydrochloride of monohydrated L-histidine (14.82 g; 70 mmoles; 1 eq.) is dissolved in 126 mL of demineralized water, then the solution is cooled to 0 C. Under strong stirring, dibromine (4.32 mL; 13.42 g; 84 mmoles; 1.2 eq.) is added dropwise very rapidly. The solution turns red. Thioacetic acid (18.0 mL; 19.2 g; 245 mmoles; 3.5 eq.) is added very rapidly. Vigorous stirring is maintained at 0 C. for 20 minutes. 3-Mercaptopropionic acid (25 mL; 29.71 g; 280 mmoles; 4 eq.) is added, and the solution is heated at 80 C. overnight. The solution is cooled, then extracted with 3 times 150 mL of ethyl acetate. After oxidation with a 30% oxygenated water solution (3.5 mL; 3.97 g; 35 mmoles; 0.5 eq.), followed by a purification on Dowex WX2 resin, the disulfide of L-5-sulfanylhistidine hydrated hydrochloride (Compound 21) (4.66 g; 24%; 37% with respect to the quantity of the intermediate SAc) is obtained in the form of a light gray powder.

[0294] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=3.27 (m, 21H); 3.32 (m, 21H); 4.17 (dd, J=8.0 Hz, J=6.6 Hz, 21H); 8.87 (s, 21H).

[0295] LCMS (APCI): 373.0 [M+H].sup.+

[0296] [].sub.D: +23.6 (c=0.1; 1N HCl)

Example 26

a) One Pot Preparation of the Disulfide of L-5-sulfanyl-,N(methyl)-histidine (Compound 22) Via In-Situ Preparation of 5-acylsulfanyl-histidine Followed by Hydrolysis and Oxidation (Compound 22)

[0297] ##STR00025##

[0298] The hydrochloride of ,N(methyl)-L-histidine (1.05 g; 5 mmoles; 1 eq.) (V. N. Reinhold et al., J. Med. Chem. 1968, 11, 258-260) is dissolved in 35 mL of demineralized water containing a 37% concentrated hydrochloric acid solution; 420 L (5 mmoles; 1 eq.), then the solution is cooled to 1 C. Very strong stirring is maintained. N-bromosuccinimide (1.17 g; 6.5 mmoles; 1.3 eq.) is added rapidly. Then thioacetic acid (2.57 mL; 2.74 g; 35 mmoles; 7 eq.) is added very rapidly. Vigorous stirring is maintained at 0 C. for 30 minutes. The solution is extracted with 40 mL of ethyl acetate, then 3-mercaptopropionic acid (2.2 mL; 2.65 g; 25 mmoles; 5 eq.) is added to the aqueous phase. The hydrolysis is carried out by heating at 100 C. for 20 h. After cooling of the solution, the reaction medium is extracted with 4 times 35 mL of ethyl acetate. After oxidation and purification on DOWEX 50WX2-400 resin, the disulfide of L-5-sulfanyl-,N(methyl)-histidine (Compound 22) (620 mg, 61%, 75% with respect to the quantity of the intermediate SAc) is obtained in the form of a brown powder.

[0299] .sup.1H NMR (MeOD/D.sub.2O 20/1, 400 MHz): (ppm)=2.69 (s, 23H); 2.94 (dd, J=14.0 Hz, J=7.0 Hz, 21H); 2.99 (dd, J=14.0 Hz, J=5.0 Hz, 21H); 3.92 (dd, J=7.0 Hz, J=5.0 Hz, 21H); 7.79 (s, 21H).

[0300] LCMS (APCI): 401.0 [M+H].sup.+

b) Preparation of L-5-sulfanyl-,N(methyl)-histidine by Reduction of the Disulfide (Compound 23)

[0301] ##STR00026##

[0302] The disulfide of L-5-sulfanyl-,N(methyl)-histidine (620 mg; 1.52 mmoles, 1 eq.) (Compound 22) is dissolved in 50 mL of water. The dithiothreitol (473 mg; 3.03 mmoles; 2 eq.) and the activated charcoal (300 mg) are added. The mixture is stirred for 4 h at ambient temperature. After filtration and crystallization in absolute ethanol, L-5-sulfanyl-,N(methyl)-histidine (Compound 23) (351 mg, 56%) is obtained in the form of a beige powder.

[0303] .sup.1H NMR (120, 400 MHz): (ppm)=2.80 (s, 3H); 3.21 (dd, J=15.9 Hz, J=6.4 Hz, 1H); 3.28 (dd, J=15.9 Hz, J=5.2 Hz, 1H); 3.92 (m, 1H); 8.25 (s, 1H).

[0304] LCMS (APCI): 202.1 [M+H].sup.

Example 27

a) One Pot Preparation of the Disulfide of L-5-sulfanyl-,N,N(dimethyl)-histidine Via In-Situ Preparation of 5-acylsulfanyl-histidine Followed by Hydrolysis and Oxidation (Compound 24)

[0305] ##STR00027##

[0306] The hydrochloride of monohydrated ,N,N(dimethyl)-histidine (2.43 g; 10 mmoles; 1 eq.) is dissolved in 54 mL of demineralized water containing a 37% concentrated hydrochloric acid solution (835 L; 985 mg; 10 mmoles; 1 eq.), then the solution is cooled to 1 C. Very strong stirring is maintained. N-Bromosuccinimide (2.31 g; 13 mmoles; 1.3 eq.). is added rapidly. After 2 minutes, thioacetic acid (3.0 mL; 3.14 g; 40 mmoles; 4 eq.) is added very rapidly. Vigorous stirring is maintained at 0 C. for 30 minutes. The slightly yellow solution obtained is extracted with 2 times 120 mL of ethyl acetate. After hot hydrolysis, oxidation and purification on DOWEX 50WX2-400 resin, the hydrated hydrochloride of the disulfide of L-5-sulfanyl-,N,N(dimethyl)-histidine (Compound 244HClH2O), 1.2 g, 41%) is obtained in the form of a beige powder.

[0307] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=3.01 (s, 26H); 3.37 (dd, J=14.6 Hz, J=11.2 Hz, 21H); 3.51 (dd, J=14.6 Hz, J=4.0 Hz, 21H); 4.09 (dd, J=11.2 Hz, J=4.0 Hz, 21H); 8.86 (s, 21H).

[0308] LCMS (APCI): 429.2 [M+H].sup.+

b) Preparation of the Compound 24 Free Base

[0309] ##STR00028##

[0310] The hydrated hydrochloride of the disulfide of L-5-sulfanyl-,N,N(dimethyl)-histidine (3.6 g; 5.89 mmoles; 1 eq.) is dissolved in 53 mL of demineralized water. The resin Amberlite IRA-410 (8 g) in hydrogen carbonate form (according to K. A. Piez et al., J. Biol. Chem. 194, 669-672 (1952)) is added. The suspension is stirred under a vacuum for 30 minutes, then filtered. The filtrate is evaporated leading to the disulfide of L-5-sulfanyl-,N,N(dimethyl)-histidine free base (Compound 24) (2.47 g, 84%) in the form of a yellow solid.

[0311] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=2.88 (s, 26H); 2.92 (m, 22H); 3.70 (m, 21H); 8.17 (s, 21 H).

c) Invention of Compound 25 by Reduction of Compound 184HClH.SUB.2.O)

[0312] ##STR00029##

[0313] The hydrated hydrochloride of the disulfide of L-S-sulfanyl-,N,N(dimethyl)-histidine (1.2 g; 2.07 mmoles; 0.5 eq.) is dissolved in 40 mL of demineralized water. The resin Amberlite IRA-410 (2 g) in hydrogen carbonate form is added. The suspension is stirred under a vacuum for 30 minutes and then filtered. After reduction with dithiothreitol (967 mg; 6.20 mmoles; 1.5 eq.) and crystallization with absolute ethanol, under nitrogen, L-5-sulfanyl-,N,N(dimethyl)-histidine (Compound 25) (450 mg, 58%) is obtained in the form of a white solid.

[0314] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=3.00 (s, 6H); 3.23 (dd, J=15.5 Hz and J=7.5 Hz, 1H); 3.31 (dd, J=15.5 Hz and J=5.8 Hz, 1H); 4.00 (dd, J=7.5 Hz and J=5.8 Hz, 1H); 8.28 (s, 1H).

[0315] .sup.13C NMR (D.sub.2O, 75 MHz): (ppm)=22.7; 41.8, 67.3; 124.5; 129.6; 131.7; 171.0.

[0316] LCMS (APCI): 216.1 [M+H]+

d) Analytical Monitoring of the Hydrolysis of the 5-acylsulfanyl Compound (Compound 2) into the 5-sulfanylhistidine Compound 25

[0317] ##STR00030##

[0318] Compound 2 is prepared and purified by column as described in Example 3, using an ethyl acetate/ethanol gradient followed by elution with water. The aqueous fraction containing the pure compound 2 is placed in a water bath at 40 C. and heated under stirring for 8 h. Samples are collected every 60 minutes and the mixture is analyzed by HPLC.

[0319] The hydrolysis of compound 2 is nearly complete after 8 h, and compound 19 is obtained with a yield of 70%.

TABLE-US-00002 TABLE 2 Monitoring of the formation of compound 25 by hydrolysis of compound 2: t (h) 0 1 2 3 5 8 % compound 25 0% 14% 23% 34% 49% 70%

Example 28

a) One Pot Preparation of L-5-sulfanyl-,N,N,N(trimethyl)-histidine Via In-Situ Preparation of 5-acylsulfanyl-histidine Followed by Hydrolysis (Compound 26)

[0320] ##STR00031##

[0321] L-Hercynine (5.02 g; 25 mmoles; 1 eq.) is dissolved in 135 mL of demineralized water and a 37% concentrated hydrochloric acid solution (4.17 mL; 4.93 g; 50 mmoles; 2 eq.) is added; then the solution is cooled to 0 C. Under strong stirring, N-bromosuccinimide (5.78 g; 32.5 mmoles; 1.3 eq.) is added. After 5 minutes, thioacetic acid (18.33 mL; 19.61 g; 250 mmoles; 10 eq.) is added very rapidly. Stirring is maintained for 40 minutes. The solution is extracted with 2 times 135 mL of ethyl acetate. 3-Mercaptopropionic acid (11.07 ml: 13.4 g; 125 mmoles; 5 eq.) is added to the aqueous phase, then the solution is heated at 130 C. for 3 h. After extraction, neutralization and crystallization in the presence of dithiothreitol (1.95 g; 12.5 mmoles; 0.5 eq.), L-5-sulfanyl-,N,N,N(trimethyl)-histidine (Compound 26) (2.22 g; 38%; 58% with respect to the quantity of the intermediate SAc) is obtained in the form of a white powder (to be stored under an inert atmosphere).

[0322] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=3.29 (s, 9H); 3.19 (m, 1H); 3.35 (m, 1H); 4.00 (dd, J=10.6 Hz, J=3.9 Hz, 1H); 8.22 (s, 1H).

[0323] LCMS (APCI): 230.0 [M+H]+

b) Analytical Monitoring by .SUP.1.H NMR of the Hydrolysis of the 5-acylsulfanyl Compound 3 into the Compound L-5-sulfanyl-,N,N,N(trimethyl)-histidine (Compound 26) in the Presence of a Thiol

[0324] ##STR00032##

[0325] Compound 3 is prepared and purified by column as described in Example 5. 100 mg (0.33 mmoles, 1 eq.) of compound 3 are dissolved in 2.4 mL of D.sub.2O. 172 mg of 3-mercaptopropionic acid (142 L, 5 equivalents) are added, and the solution is heated at 40 C. The conversion is monitored by .sup.1H NMR and by HPLC-ELSD. The yield of hydrolysis of compound 3 is 90% after 3 h (monitored by .sup.1H NMR). Compound 26 is formed after 3 h 30 with a yield of 97% (HPLC-ELSD).

c) Preparation of the Compound L-5-sulfanyl-,N,N,N(trimethyl)-histidine (Compound 26) by Hydrolysis of the 5-acylsulfanyl Compound 3

[0326] ##STR00033##

[0327] Compound 3 is prepared and purified by column as described in Example 5. 170 mg (0.6 mmoles) of compound 3 are dissolved in 10 mL of water, and the solution is heated at 90 C. in air for 7 h. The conversion is monitored by HPLC. The hydrolysis of compound 3 is complete after 7 h. The solution is evaporated to dryness. The residue is dissolved in a mixture of 5 mL of methanol and 93 mg (0.6 mmol) of dithiothreitol. After stirring for 4 h under an inert atmosphere, 2 mL of ethanol are added. A precipitate forms immediately, which is filtered and washed with ethanol (22 mL), then with ethyl ether (22 mL). After drying, 104 mg (72%) of L-5-sulfanyl-,N,N,N(trimethyl)-histidine are obtained in the form of a beige powder.

[0328] The .sup.1H NMR and mass spectra are identical to those obtained in Example 28a.

d) Preparation of the Disulfide of L-5-sulfanyl-,N,N,N(trimethyl)-histidine (Compound 27)

[0329] ##STR00034##

[0330] L-5-Sulfanyl-,N,N,N(trimethyl)-histidine (Compound 26, 300 mg, 1.29 mmoles, 1 eq.) is dissolved in 50 mL of demineralized water. The colorless solution is stirred at ambient temperature for 4 days. After filtration and lyophilization of the filtrate, the disulfide of L-5-sulfanyl-,N,N,N(trimethyl)-histidine (Compound 27) (263 mg; 89%) is obtained in the form of a yellow powder.

[0331] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=2.68 (dd, J=13.5 Hz, J=11.0 Hz, 21H); 2.75 (dd, J=13.5 Hz, J=4.3 Hz, 21H); 3.19 (s, 29H); 3.68 (dd, J=11.0 Hz, J=4.3 Hz, 21H); 7.97 (s, 21H).

[0332] LCMS (APCI): 457.1 [M+H]+.

e) Analytical Monitoring by HPLC of the Hydrolysis of the 5-acylsulfanyl Compound 3 and Oxidation In Situ into Compound 27

[0333] ##STR00035##

[0334] Compound 3 is prepared and purified by column as described in Example 5, using an ethyl acetate/ethanol gradient, followed by elution with water. The aqueous fraction containing the pure compound 3 is placed in a water bath at 40 C. and heated under stirring for two days. Samples are collected every hour, and the mixture is analyzed by HPLC.

[0335] The hydrolysis of compound 3 is nearly complete after 2 days, and compound 27 is obtained with a yield of 80%.

TABLE-US-00003 TABLE 3 Monitoring of the hydrolysis of compound 3: t 0 h 2 h 4 h 6 h 8 h 18 h 2 j % hydrolysis 0% 7% 21% 38% 60% 85% 95%

f) Analytical Monitoring by .SUP.1.H NMR of the Hydrolysis of the 5-acylsulfanyl Compound 3 and Oxidation In Situ into Compound 27

[0336] ##STR00036##

[0337] Compound 3 is prepared and purified by column as described in Example 5. 30 mg of compound 3 are dissolved in 600 L of D.sub.2O, the solution is transferred to an NMR tube, which is stored at ambient temperature. The conversion is monitored by .sup.1H NMR. The hydrolysis of compound 3 is nearly complete after 2 days, and a mixture which contains the disulfide 27 and the thiol 26 (3:1) is obtained.

TABLE-US-00004 TABLE 4 Monitoring of the hydrolysis of compound 3: t 0 h 2 h 10 h 2 j % hydrolysis 0% 19% 26% 86%

Example 29

a) One Pot Preparation of the Disulfide of L-5-sulfanyl--N(acetyl)-histidine (Compound 28 Hydrochloride) Via In-Situ Preparation of 5-acylsulfanyl-histidine Followed by Hydrolysis and Oxidation

[0338] ##STR00037##

[0339] Monohydrated -N(acetyl)-L-histidine (2.15 g, 10 mmoles; 1 eq.) is dissolved in 63 mL of demineralized water containing 37% concentrated hydrochloric acid (1.67 mL, 1.97 g; 20 mmoles; 2 eq.); then the solution is cooled to 0 C. Dibromine (668 L, 2.07 g; 13 mmoles; 1.3 eq.) is added. Thioacetic acid (3.67 mL; 3.92 g; 50 mmoles; 5 eq.) is added all at once. Stirring is maintained at 0 C. for 45 minutes. The solution is reheated at ambient temperature. 3-Mercaptopropionic acid (5.26 mL, 6.36 g; 60 mmoles; 6 eq.) is added, then the solution is heated at 80 C. overnight. The solution is cooled at ambient temperature, then extracted with 4 times 50 mL of ethyl acetate. The aqueous phase is purified on silica in order to obtain the hydrochloride of the disulfide of L-5-sulfanyl-,N(acetyl)-histidine (compound 28) in the form of an orange oil (520 mg, 17%; 36% with respect to the quantity of the intermediate SAc).

[0340] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=1.86 (s, 23H); 2.92 (dd, J=15.0 Hz, J=8.0 Hz, 21H); 3.03 (dd, J=15.0 Hz, J=5.5 Hz, 21H); 4.47 (dd, J=8.0 Hz, J=5.5 Hz, 21H); 8.73 (s, 21H).

[0341] LCMS (APCI): 457.4 [M+H]+

b) Preparation of L-5-sulfanyl-,N(acetyl)-histidine (Compound 29)

[0342] ##STR00038##

[0343] The hydrochloride of the disulfide of L-5-sulfanyl-,N(acetyl)-histidine (Compound 28) (520 mg; 834 moles, 1 eq.) is dissolved in 50 mL of water, then the pH of the brown colored solution is adjusted to 4.5 by adding NH.sub.4OH. 3-Mercaptopropionic acid (4.38 mL; 5.31 g; 50 mmoles; 5 eq.) is added. The solution is heated at 70 C. for 2 h. The solution is extracted with 4 times 50 mL of ethyl acetate. The aqueous phase is evaporated to dryness yielding L-5-sulfanyl-,N(acetyl)-histidine (Compound 29) (390 mg; 86%) in the form of a beige solid.

[0344] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=1.97 (s, 3H); 3.01 (dd, J=15.2 Hz, J=8.6 Hz, 1H); 3.16 (dd, J=15.2 Hz, J=4.8 Hz, 11H); 4.50 (dd, J=8.6 Hz, J=4.8 Hz, 1H); 8.22 (s, 1H).

[0345] LCMS (APCI): 230.0 [M+H].sup.+

Example 30: One Pot Preparation of L-5-sulfanylcarnosine Via In-Situ Preparation of 5-acylsulfanyl-histidine Followed by Hydrolysis (Compound 30)

[0346] ##STR00039##

[0347] L-Carnosine (425 mg; 1.88 mmoles; 1 eq.) is dissolved in 12 mL of demineralized water containing a 37% concentrated hydrochloric acid solution (370 mg; 3.75 mmoles; 2 eq.), then the solution is cooled to 0 C. N-Bromosuccinimide (440 mg; 2.44 mmoles; 1.3 eq.) is added in one portion: the solution turns limpid orange. Thioacetic acid (960 L; 1.03 g, 13.14 mmoles; 7 eq.) is added. The mixture is stirred at 0 C. for 1 h. The solution is extracted with 4 times 12 mL of ethyl acetate. After neutralization and purification on silica in the presence of dithiothreitol (290 mg; 1.88 mmoles; 1 eq.), L-5-sulfanylcarnosine (Compound 30) (70 mg; 14%; 22% with respect to the quantity of the intermediate SAc) is obtained in the form of a colorless lacquer.

[0348] .sup.1H NMR (120, 400 MHz): (ppm)=2.69 (t, J=6.7 Hz, 2H); 3.00 (m, 1H); 3.12 (m, 1H); 3.23 (t, J=6.7 Hz, 2H); 4.43 (dd, J=8.5 Hz, J=4.2 Hz, 1H); 8.20 (s, 1H).

[0349] LCMS (APCI): 259.1 [M+H]+

Example 31: Preparation of Compounds 31 and 32

a) One Pot Preparation of the Disulfide of iso-ovothiol a Via In-Situ Preparation of 5-acetylsulfanyl-1-methylhistidine Followed by Hydrolysis and Oxidation (Compound 31)

[0350] ##STR00040##

[0351] 1-Methyl-L-histidine (0.84 g; 5 mmoles; 1 eq.) is dissolved in 35 mL of demineralized water, and a 37% concentrated hydrochloric acid solution (835 L (10 mmoles; 2 eq.) is added; then the solution is cooled to 1 C. Very strong stirring is maintained. N-Bromosuccinimide (1.17 g; 6.5 mmoles; 1.3 eq.) is added rapidly. After 3 minutes, thioacetic acid (2.57 mL; 2.74 g; 35 mmoles; 7 eq.) is added very rapidly. Vigorous stirring is maintained at 0 C. for 30 minutes. The solution is extracted with 40 mL of ethyl acetate, then 3-mercaptopropionic acid (2.2 mL; 2.65 g; 25 mmoles; 5 eq.) is added to the aqueous phase. The hydrolysis is carried out by heating at 100 C. for 20 h. After cooling of the solution, the reaction medium is extracted with 4 times 35 mL of ethyl acetate. After oxidation and purification with DOWEX 50WX2-400 resin, the disulfide of L-1-methyl-L-5-sulfanylhistidine (Compound 31) (740 mg, 65%, 90% with respect to the quantity of the intermediate SAc) is obtained in the form of a brown powder.

[0352] .sup.1H NMR (D.sub.2O+DCl, 400 MHz): (ppm)=3.14 (m, 22H); 3.85 (s, 23H); 4.17 (m, 21H); 8.89 (s, 21H).

[0353] LCMS (APCI): 401.1 [M+H]+

b) Preparation of iso-ovothiol A (Compound 32)

[0354] ##STR00041##

[0355] The disulfide of L-5-sulfanyl-1-methylhistidine (Compound 25) (427 mg; 0.52 mmoles, 1 eq.) is suspended in 25 mL of methanol. The mixture is heated to 50 C., then dithiothreitol (299 mg; 1.92 mmoles; 2 eq.) is added. After stirring for 1 h at ambient temperature and precipitation with ethyl ether, L-5-sulfanyl-1-methylhistidine (iso-ovothiol A, Compound 32) (295 mg; 69%) is obtained in the form of a slightly grayish powder.

[0356] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=3.19 (dd, J=15.7 Hz, J=7.2 Hz, 1H); 3.29 (dd, J=15.7 Hz, J=5.2 Hz, 1H); 3.66 (s, 3H); 4.09 (dd, J=7.1 Hz, J=5.2 Hz, 1H); 8.33 (s, 1H).

[0357] LCMS (APCI): 202.1 [M+H]+

Example 32: Preparation of the Disulfide of L-5-sulfanyl-,N,N(dimethyl)-1-methylhistidine Via Hydrolysis of the 5-acetylsulfanyl-,N,N(dimethyl)-1-methylhistidine Derivative Followed by Air Oxidation (Compound 33)

[0358] ##STR00042##

[0359] Compound 5 is prepared and purified by column as described in Example 8. 180 mg (0.63 mmoles, 1 eq.) of compound 5 are dissolved in 20 mL of water. The limpid solution is stirred in the presence of oxygen for 20 h at ambient temperature. After lyophilization, the disulfide of L-5-sulfanyl-,N,N(dimethyl)-1-methylhistidine (Compound 33, 98%) is obtained in the form of a greenish amorphous solid.

[0360] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=2.97 (s, 26H); 3.17 (m, 21H); 3.28 (dd, J=15.8 Hz and J=4.3 Hz, 21H); 3.69 (s, 23H); 4.00 (m, 21H); 8.44 (s, 21H).

[0361] LCMS (APCI): 457.2 [M+H]+

Example 33: One Pot Preparation of the Disulfide of L-5-sulfanyl-,N,N,N(trimethyl)-1-methylhistidine (Compound 34 Dihydrochloride) Via In-Situ Preparation of L-5-acetylsulfanyl-,N,N,N(trimethyl)-1-methylhistidine Followed by Hydrolysis and Oxidation

[0362] ##STR00043##

[0363] 1-Methyl-hercynine (510 mg, 2 mmoles; 1 eq.) is dissolved in 15 mL of demineralized water containing a 37% concentrated hydrochloric acid solution (170 L, 2 mmoles; 1 eq.), then the solution is cooled to 0 C. Very strong stirring is maintained. N-Bromosuccinimide (465 mg, 2.6 mmoles; 1.3 eq.) is added rapidly. After 3 minutes, thioacetic acid (740 ALL, 10 mmoles; 5 eq.) is added very rapidly. Vigorous stirring is maintained at 0 C. for 30 minutes. The mixture is extracted with 220 mL of ethyl acetate, then diluted in 160 mL of an ethyl acetate/ethanol mixture (3/1) for purification on a silica column (ethyl acetate/ethanol/water 2/2/1). The slightly pink oil obtained is oxidized with dimethyl sulfoxide (140 L, 2 mmoles, 1 eq.) in a solution of glacial acetic acid. The solution is heated for one hour at 80 C. The dihydrochloride of the disulfide of L-5-sulfanyl-,N,N,N(trimethyl)-1-methylhistidine (compound 34) is obtained after purification on a silica column (ethyl acetate/ethanol/water 2/2/1, followed by elution with 0.5M hydrochloric acid) in the form of a slightly yellow oil (110 mg, 10%).

[0364] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=3.30 (s, 29H); 3.61 (dd, J=14.1 Hz and 3.4 Hz, 21H); 3.72 (m, 21H); 3.73 (s, 23H); 4.09 (dd, J=12.2 Hz and 3.4 Hz, 21H); 8.98 (s, 21H).

[0365] LCMS (APCI): 485.1 [M+H]+

Example 34: Preparation of the L-5-sulfanyl-,N(L-alanyl)-histidine Derivative (Compound 35) by Hydrolysis of the 5-acylsulfanyl Compound

[0366] ##STR00044##

[0367] Compound 7 is prepared and purified by column as described in Example 10. 340 mg (1 mmole, 1 eq.) of compound 7 are dissolved in 20 mL of water. The limpid solution is stirred with protection from oxygen for 6 days at ambient temperature. After evaporation to dryness, L-5-sulfanyl-,N(L-alanyl)-histidine (Compound 35, 92%) is obtained in the form of a beige amorphous solid.

[0368] .sup.1H NMR (D.sub.2O, 400 MHz): (ppm)=1.42 (d, J=7.2 Hz, 3H); 3.12 (dd, J=15.2 and J=8.0 Hz, 1H); 3.22 (dd, J=15.2 Hz and J=6.2 Hz, 1H); 4.05 (q, J=7.2 Hz, 1H); 4.65 (m, 1H); 8.71 (s, 1H).

[0369] LCMS (APCI): 258.9 [M+H]+

Example 35: Preparation of the Disulfide of 5-sulfanyl-,N(pentanoyl)-histidine (Compound 36) by Hydrolysis and Oxidation of the Compound 5-acetylsulfanyl-,N(pentanoyl)-histidine

[0370] ##STR00045##

[0371] The 5-acetylsulfanyl-,N(pentanoyl)-histidine derivative (compound 8) is prepared and purified as described in Example 1. 320 mg (0.9 mmole; 1 eq.) of compound 8 are dissolved in 8.0 mL of demineralized water. 3-Mercaptopropionic acid (400 L, 4.60 mmoles; S eq.) is added. The solution is heated at 90 C. for 3 h. The reaction mixture is extracted with 410 mL of ethyl acetate, then the aqueous phase is evaporated to dryness. The residue is dissolved in 10 mL of water. The solution is heated at 90 C. under stirring for 2 hours, then at ambient temperature for 18 hours. After evaporation to dryness, the disulfide of L-5-sulfanyl-,N(pentanoyl)-histidine (Compound 36) is obtained in the form of an orange lacquer (44%).

[0372] LCMS (APCI): 541.2 [M+H]+

Example 36-Activity Testing of 5-sulfanyl-histidine Derivatives as Antioxidant Involving Measure of GSH-Px Activity as Compared with a Positive Control Constituted by Ebselen

Protocol:

[0373] GSH-Px activity is measured by the reduction of GSSG formed via the NADPH-glutathione reductase system as a continuous indicator system. Loss of NADPH was monitored continuously at 366 nm using a UVIKON 932 spectrophotometer and a molar absorption coefficient of 3300/M/cm. Experiments were conducted at room temperature (22 C.) in 50 mM phosphate-buffered saline pH 7.6 containing 0.1 mM EDTA and 1.0 mM NaN.sub.3. Test compound (12.5 M for ebselen used as reference standard and 250 M for 5-sulfanyl-histidine derivatives), glutathione (1.0 mM), NADPH (280 M) and GSSG reductase (1 U/ml) were incubated for 4 min to estimate the sample blank and reactions were initiated with H.sub.2O.sub.2 (1.0 mM). Appropriate blanks were also run in the absence of added test compound and in the presence of H.sub.2O.sub.2. The GSH-Px activity of 5-sulfanyl-histidine derivatives results from two successive reactions: oxidation of the 5-sulfanyl-histidine derivatives to the corresponding disulphides in presence of H.sub.2O.sub.2 and regeneration of the 5-sulfanyl-histidine derivatives by exchange reaction of the disulphides with glutathione. 5-sulfanyl-histidine disulphides (100 M) and glutathione (1.0 mM) were incubated for 2 min at room temperature. NADPH (200 M) and GSSG reductase (1 U/ml) were subsequently added. The absorbance decrease at 366 nm was measured and compared to that obtained in presence of glutathione disulphide alone (100 M). The ratio of both NADPH disappearances gave the amount of glutathione/disulphide exchange.

Results:

[0374] The consumption of NADPH upon addition of H.sub.2O.sub.2 in absence of test substance (control) was 131 M NADPH/min. In the catalysed reactions, the following NADPH consumptions (M/min) were recorded: ebselen [895], L-5-sulfanyl-histidine (compound 18) [392], L-5-sulfanyl-,N(methyl)-histidine (compound 23) [483], L-5-sulfanyl-,N,N(dimethyl)-histidine (compound 25) [443], L-5-sulfanyl-,N,N,N(trimethyl)-histidine (compound 26) [422], L-5-sulfanyl-,N(acetyl)-histidine (compound 29) [393], L-5-sulfanyl-1-methyl-histidine or iso-Ovothiol A (compound 32) [544], L-5-sulfanyl-,N(L-alanyl)-histidine (compound 35) [414].

[0375] These antioxidant Experiments revealed that all the 5-sulfanyl-histidine derivatives exhibit significant GSH-Px activity (see FIG. 5). As encountered for ovothiols, 5-sulfanyl-histidine disulphides are readily reduced by glutathione. At equilibrium, the fraction of exchanged disulphides ranges from 21 to 88%.

TABLE-US-00005 TABLE 5 Summary of the examples: Compound Example prepared Structure 1 2 1 1 [00046] 3 4 2 2 [00047] 5 6 3 3 [00048] 7 4 [00049] 8 5 [00050] 9 6 [00051] 10 7 [00052] 11 8 [00053] 12 9 [00054] 13 10 [00055] 14 11 [00056] 15 12 [00057] 16 17 13 13 [00058] 18 14 [00059] 19 15 [00060] 20 16 [00061] 21 17 [00062] 22 18 [00063] 23 19 [00064] 24 20 [00065] 25 21 x 4HCl x 2H2O [00066] 26a 22 [00067] 26b 23 [00068] 27a 24 x 4HCl x 2H2O [00069] 27b 24 [00070] 27c 27d 25 25 [00071] 28a 28b 26c 26 26 26 [00072] 28d 28e 28f 27 27 27 [00073] 29a 28 x 2HCl [00074] 29b 29 [00075] 30 30 [00076] 31a 31 [00077] 31b 32 [00078] 32 33 [00079] 33 34 x 2HCl [00080] 34 35 [00081] 35 36 [00082] [00083]