U.S. patent application number 15/891433 was filed with the patent office on 2018-09-13 for 5-sulfanylhistidines and their disulfides and method of preparation thereof.
The applicant listed for this patent is TETRAHEDRON. Invention is credited to Sylvain DAUNAY, Irene ERDELMEIER.
Application Number | 20180258077 15/891433 |
Document ID | / |
Family ID | 51352623 |
Filed Date | 2018-09-13 |
United States Patent
Application |
20180258077 |
Kind Code |
A1 |
ERDELMEIER; Irene ; et
al. |
September 13, 2018 |
5-SULFANYLHISTIDINES AND THEIR DISULFIDES AND METHOD OF PREPARATION
THEREOF
Abstract
The invention relates to a compound of the
5-acylsulfanyl-histidine type and the derivatives thereof, of
general formula (I), wherein R1 to R3=H, alkyl, especially CH3;
R4=H, alkyl, especially CH3, alkyle(C=0), substituted alkyl
(C.dbd.O), aryl (C.dbd.O); .beta.-alanyl (H2NCH2CH2 (C.dbd.O);
.alpha.-amino-acyl; R5=alkyl, especially methyl, phenyl. The
invention also relates to the use of said compound for producing
compounds of the 5-sulfanyl-histidine type and the derivatives
thereof, in addition to corresponding disulfides; and to the
various methods for the production thereof.
Inventors: |
ERDELMEIER; Irene; (Paris,
FR) ; DAUNAY; Sylvain; (Paris, FR) |
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Applicant: |
Name |
City |
State |
Country |
Type |
TETRAHEDRON |
Paris |
|
FR |
|
|
Family ID: |
51352623 |
Appl. No.: |
15/891433 |
Filed: |
February 8, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15314628 |
Nov 29, 2016 |
9926300 |
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PCT/FR2015/051416 |
May 29, 2015 |
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15891433 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 403/12 20130101;
C07D 233/84 20130101 |
International
Class: |
C07D 403/12 20060101
C07D403/12; C07D 233/84 20060101 C07D233/84 |
Foreign Application Data
Date |
Code |
Application Number |
May 30, 2014 |
FR |
1454935 |
Claims
1-20. (canceled)
21. A compound of the 5-sulfanylhistidine of formula (II):
##STR00078## where: R1=H, or alkyl; R2=R3=H, or alkyl; R4=H, alkyl,
alkyl (C.dbd.O), substituted alkyl (C.dbd.O), aryl (C.dbd.O),
.beta.-alanyl (H2NCH2CH2 (C.dbd.O), or .alpha.-amino-acyl; R.sup.1
to R.sup.4 being as defined above; it being understood that when
R.sup.1.dbd.H 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
corresponding disulfides, taken separately or in a mixture; all the
salts of pharmaceutically acceptable acids of said compounds of
general formula (II).
22. The compound of claim 21, characterized in that R.sup.4
represents hydrogen, methyl, acetyl, benzoyl, or .beta.-alanyl
(H.sub.2NCH.sub.2CH.sub.2 (C.dbd.O).
23. The compound of claim 21, characterized in that it is selected
from the group consisting of: the disulfide of
L-5-sulfanyl-.alpha.,N(methyl)-histidine (Compound 22);
L-5-sulfanyl-.alpha.,N(methyl)-histidine (Compound 23); the
disulfide of L-5-sulfanyl-.alpha.,N,N(dimethyl)-histidine (Compound
24); L-5-sulfanyl-.alpha.,N,N(dimethyl)-histidine (Compound 25);
L-5-sulfanyl-.alpha.,N,N,N(trimethyl)-histidine (Compound 26); the
disulfide of L-5-sulfanyl-.alpha.,N,N,N(trimethyl)-histidine
(Compound 27); the disulfide of
L-5-sulfanyl-.alpha.,N(acetyl)-histidine (Compound 28);
L-5-sulfanyl-.alpha.,N(acetyl)-histidine (Compound 29);
L-5-sulfanylcarnosine (Compound 30); the disulfide of iso-ovothiol
A (Compound 31); iso-ovothiol A (Compound 32); the disulfide of
L-5-sulfanyl-.alpha.,N,N(dimethyl)-1-methylhistidine (Compound 33);
L-5-sulfanyl-.alpha.,N,N,N(trimethyl)-1-methylhistidine (Compound
34); L-5-sulfanyl-.alpha.,N(L-alanyl)-histidine (Compound 35); and
the disulfide of 5-sulfanyl-.alpha.,N(pentanoyl)-histidine
(Compound 36).
24. The compound of claim 21, characterized in that the
pharmaceutically acceptable acid is selected from a mineral acid,
an organic acid, an alkanesulfonic acid or an arylsulfonic acid
such as benzene- and paratoluenesulfonic acids.
25. The compound of claim 24, wherein the mineral acid is selected
from the group consisting of hydrochloric, hydrobromic, hydroiodic,
sulfuric, tartaric, and phosphoric acid.
26. The compound of claim 24, wherein the organic acid is selected
from the group consisting of formic, acetic, trifluoroacetic,
propionic, benzoic, maleic, fumaric, succinic, citric, oxalic,
glyoxylic, and aspartic acid.
27. The compound of claim 24, wherein the alkanesulfonic acid is
selected from the group consisting of methanesulfonic,
trifluoromethanesulfonic, and ethanesulfonic acid.
28. The compound of claim 24, wherein the arylsulfonic acid is
selected from the group consisting of benzenesulfonic acid and
paratoluenesulfonic acid.
29. "One-pot" method (D) for preparing the 5-sulfanyl-histidine
compounds of formula (II) as defined in claim 21, 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),
##STR00079## Where R1 to R4 are as defined for the compounds of
formula (II) and R5 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 .alpha.-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 .alpha.-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.degree. C., then with b) a
sulfur-containing reagent of the carbothioic acid type of formula
alkyl or phenyl C(.dbd.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.degree. 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.degree. 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.degree. C., then with d) a sulfur-containing reagent of the
carbothioic acid of formula alkyl or phenyl C(.dbd.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.degree. 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.degree. 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.
30. The method of claim 29, 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.
31. The method of claim 29, wherein the polar protic solvent is
water or an aqueous solution.
32. The method of claim 29, wherein the sulfur-containing reagent
of the carbothioic acid is selected from thioacetic acid,
thiobenzoic acid, potassium thioacetate, or mixtures thereof.
33. The method of claim 29, wherein the temperature in method (A)
is 0-5.degree. C.
34. The method of claim 29, wherein the thiol is selected from
mercaptopropionic acid, dithiothreitol or mixtures thereof.
35. The method of claim 29, wherein the temperature in method (B)
is between 20 and 130.degree. C.
Description
[0001] The present invention relates to: [0002] novel
5-acylsulfanyl-histidine compounds (and their derivatives); [0003]
methods for preparing same; [0004] use thereof as precursors of the
corresponding 5-sulfanylhistidines and their disulfides.
[0005] The present invention relates to novel
5-acylsulfanyl-histidine compounds and their derivatives, as well
as to methods for preparing same and to the use thereof as
precursors of the corresponding 5-sulfanylhistidines and their
disulfides. More particularly, this invention relates to the
synthesis of novel 5-acylsulfanyl-histidine compounds and their
derivatives, their salts as direct precursors of the corresponding
5-sulfanylhistidines and their disulfides. The recent IUPAC
nomenclature "sulfanyl" for the "--SH" group is used for the
compounds described in the invention instead of the different terms
used in the past in the literature, such as "thiohistidine,"
"thiolhistidine" or "mercaptohistidine."
PRIOR ART
[0006] The 5-sulfanyl-imidazole group is rarely found in nature
(Caroll A. and Avrey V. M.; J. Nat. Prod.; 2009; 72; 696-699). Very
few natural products comprising a 5-sulfanylhistidine backbone
(methylated or not in position 3 of the imidazole ring) have been
found to date (Hand C. E. and Honek J. F.; J. Nat. Prod.; 2005; 68;
293-308). Most are of bacterial or marine origin. A first example
consists of the group of the adenochromines A, B and C (Ito S. and
Prota G.; JCS Chem. Comm.; 1977; 251-252; Rossi F., Nardi G.,
Palumbo A. and Prota G.; Comp. Biochem. Physiol. 1985, 80b,
843-845) and the seco-adenochromines A, B and C (Ito S., Nardi G.
and Prota G.; JCS Chem. Comm.; 1976; 1042). Imbricatine, produced
by Dermasterias imbricate, constitutes a second example (Pathirana
C. and Andersen R. J.; J. Am. Chem. Soc.; 1986; 108, 8288-8289).
The ovothiols A, B and C. (Turner E., Klevit R. E. and Shapiro B.
M.; J. Biol. Chem.; 1986; 261; 13056) constitute a third example of
the even more restricted group of natural products comprising a
5-sulfanylhistidine backbone methylated in position 3 (It should be
noted that the position of the methyl group was initially
incorrectly located on the N1 nitrogen of histidine, as
demonstrated in Holler et al. JOC 1987, 4421-4423 vs. Palumbo et
al., THL 1982, 3207-3208). Very recently, a new indole alkaloid
containing a 5-sulfanylhistidine backbone, leptoclinidamine C, was
found (Caroll A. and Avrey V. M.; J. Nat. Prod.; 2009; 72;
696-699).
[0007] The biosynthesis of the ovothiols A, B and C has been
described (Vogt R. N., Spies H. S. C. and Steenkamp D. J.; Eur. J.
Biochem.; 2001, 268, 5229-5241). The introduction of sulfur, in
position 5 of the imidazole ring of L-histidine, is carried out in
the presence of the sulfoxide synthase enzyme (OvoA) as well as of
ferrous iron (Fe.sup.2+) and oxygen O.sub.2. L-cysteine is used as
sulfur donor leading to a sulfoxide intermediate (Braunshausen A.
and Seebeck F.; JACS; 2011; 133, 1757). The latter is then
transformed into ovothiol A, B or C in the presence of the
sulfoxide lyase enzyme and pyridoxal phosphate, its cofactor
(Mashabela G. and Seebeck F.; JCS Chem. Comm.; 2013,
7714-7716).
[0008] The preparation of 2-sulfanylhistidine and their derivatives
by chemical synthesis has already been documented by the applicant
(patent U.S. Ser. No. 13/121,891 and patent U.S. Ser. No.
13/500,887 A1).
[0009] The preparation of 5-sulfanylhistidine and of their
derivatives by chemical synthesis turned out to be much more
difficult than that of their 2-sulfanylhistidine isomers. Several
synthesis strategies were considered and tested without success. To
date, only 2 access routes have led only to the series of the
5-sulfanyl-3-methylhistidines. The first approach consisted of the
de novo synthesis of the 5-sulfanylimidazole ring (Hopkins P. et
al.; JOC; 1987, 52, 2977 and 4420) in the context of the synthesis
of the ovothiols A and C in 10 to 12 steps. The second approach
consisted of a nucleophilic substitution of a 5-bromoimidazole ring
activated by a CHO carboxaldehyde electron-withdrawing group (Ohba
M., Nishimura Y., Kato M. and Fujii T.; Tetrahedron; 1999, 55,
4999-5016) in the context of the synthesis of imbricatine.
Currently, there is no known non-enzymatic chemical method for
directly introducing a sulfur atom into the histidine or one of its
derivatives in position 5 of the imidazole ring.
[0010] The article by SPALTENSTEIN in "The Journal of Organic
Chemistry, Vol. 52, No. 14, pp. 2977-2979 discloses a method for
preparing a compound 8 (p. 2978) obtained by the cyclization of the
corresponding thionoamide, but the latter cannot correspond to any
compound of the invention in view of the technical elements that
follow.
[0011] In the same way, the article by Heng Song in Organic
Letters, Vol. 15, No. 18, Sep. 20, 2013, pp. 4854-4857, entitled
"Regioselectivity of the oxidative C--S Bond Formation in
Ergothioneine and Ovothiol Biosyntheses," discloses a compound
ovothiol (8) (p. 4855, scheme 1) which does not correspond to any
compound of the invention in view of the technical elements that
follow.
[0012] Indeed, as mentioned above, page 2, lines 2-5, the initial
structure of the ovothiols A, B and C was defined incorrectly in
these two articles by SPALTENSTEIN and Song with regard to the
positioning of the methyl group on the nitrogen of the imidazole
ring of histidine. Initially located incorrectly on the N1 nitrogen
of histidine, this methyl group was "repositioned" on the N3
nitrogen, as demonstrated in Holler et al. JOC 1987, 20, 4421-4423
vs. Palumba et al., THL 1982, 3207-3208).
[0013] Thus, the structure of the ovothiols A, B and C is well
established following the publication by Holler et al. (JOC 1987,
20, 4421-4423), already cited in the application and accepted by
the scientific community according to the following examples:
[0014] Ovothiol C: see Bailly et al., Bioorg. Med. Chem., 2003, 11,
4623-4630, FIG. 1, p. 4624;
[0015] Ovothiols A, B and C: see De Luna et al., J. Phys.
Chemistry, 2013, DOI: 10.1021/jp402514w;
[0016] Ovothiol A: see Mashabela et al., Chem. Comm., 2013, 49,
7714-7716.
[0017] Since the SPALTENSTEIN and Song documents are based on the
erroneous location of the methyl in position N1, when it should be
correctly located in position N3, the result is that the compound 8
of SPALTENSTEIN or Song (with the corrected structure) corresponds
to the condition of formula (II) of the invention described
below.
[0018] The 5-acylsulfanyl-histidine compounds and their derivatives
would constitute very good precursors of 5-sulfanylhistidine and
their derivatives. Since these 5-acylsulfanyl-histidine compounds
and their derivatives are not known, it would therefore be
necessary to have a synthesis method that makes it possible to
directly introduce an acylsulfanyl group in position 5 of a
histidine. To our knowledge, no such method has been described to
date. This novel method for the direct introduction of an
acylsulfanyl group in position 5 of histidine or of one of its
derivatives would be all the more advantageous since it could be
carried out without protective group and in water as a reaction
solvent.
OBJECTS OF THE INVENTION
[0019] One of the objects of the present invention is thus to
provide novel 5-acylsulfanyl-histidine compounds and their
derivatives that are capable of being precursors of the
corresponding 5-sulfanylhistidines and their disulfides.
[0020] Another object of the present invention is a method for
preparing these novel 5-acylsulfanyl-histidine compounds and their
derivatives using a novel method of direct introduction of an
acylsulfanyl group in position 5 of a histidine or one of its
derivatives without protective group and in water as solvent.
[0021] Another object of the present invention is the use of these
novel 5-acylsulfanyl-histidine compounds and their derivatives as
precursors of the corresponding 5-sulfanylhistidines and their
disulfides.
[0022] These objects are achieved by the present invention which is
based on the design and the preparation of novel
5-acylsulfanyl-histidine compounds and their derivatives, which are
shown to be excellent precursors of the corresponding
5-sulfanylhistidines and their disulfides, by using a novel method
for introducing an acylsulfanyl group. This has been exemplified by
the applicant.
DESCRIPTION OF THE INVENTION
[0023] Thus, the object of the present invention is:
[0024] 1) to solve the technical problem consisting in providing
novel 5-acylsulfanyl-histidine compounds and their derivatives,
thus constituting precursors of the corresponding
5-sulfanylhistidines and their disulfides;
[0025] 2) to solve this technical problem according to a solution
that includes a method for preparing these novel
5-acylsulfanyl-histidine derivatives using a novel method for
directly introducing an acylsulfanyl group in position 5 of the
imidazole ring of a histidine without protective group and in water
as reaction solvent.
[0026] The technical problems stated above are solved for the first
time simultaneously by the present invention, in a very easy and
economic manner, the method for preparing said novel
5-acylsulfanyl-histidine derivatives being very simple to carry out
while producing good yields.
[0027] According to a first aspect, the present invention relates
to novel 5-acylsulfanyl-histidine compounds and their derivatives
having the following general formula (I):
##STR00001##
[0028] Where:
[0029] R.sup.1 H, alkyl, in particular CH.sub.3;
[0030] R.sup.2.dbd.R.sup.3.dbd.H, alkyl, in particular
CH.sub.3;
[0031] R.sup.4.dbd.H, alkyl, in particular CH.sub.3, alkyl
(C.dbd.O), substituted alkyl (C.dbd.O), aryl (C.dbd.O),
.beta.-alanyl (H.sub.2NCH.sub.2CH.sub.2 (C.dbd.O);
.alpha.-amino-acyl;
[0032] R.sup.5=alkyl, in particular methyl, phenyl;
[0033] The invention includes all the stereoisomers,
diastereoisomers and enantiomers, in particular in terms of the
carbon atom that bears the group COOH, taken alone or in a
mixture.
[0034] It also includes all the salts of pharmaceutically
acceptable acids of said compounds of general formula (I).
[0035] Among the compounds of general formula (I), the invention
relates in particular to:
[0036] those characterized in that R.sup.4 represents hydrogen, or
the methyl group, or the acetyl group, or the benzoyl group, or the
.beta.-alanyl (H.sub.2NCH.sub.2CH.sub.2 (C.dbd.O) group;
[0037] those prepared in the experimental part, in particular
[0038] 1. L-5-acetylsulfanyl-histidine (Compound 1);
[0039] 2.
L-5-acetylsulfanyl-histidine-.alpha.,N,N(dimethyl)-histidine
(Compound 2);
[0040] 3. L-5-acetylsulfanyl-.alpha.,N,N,N(trimethyl)-histidine
(Compound 3);
[0041] 4. L-5-acetylsulfanyl-.alpha.,N(glycinyl)-histidine
(Compound 4);
[0042] 5.
L-5-acetylsulfanyl-.alpha.,N,N(dimethyl)-1-methylhistidine
(Compound 5);
[0043] 6.
L-5-acetylsulfanyl-.alpha.,N,N,N(trimethyl)-1-methylhistidine
(Compound 6);
[0044] 7. L-5-acetylsulfanyl-.alpha.,N(L-alanyl)-histidine
(Compound 7);
[0045] 8. L-5-acetylsulfanyl-.alpha.,N(pentanoyl)-histidine
(Compound 8);
[0046] 9. L-5-acetylsulfanyl-.alpha.,N(methyl)-histidine (Compound
9);
[0047] 10. L-5-acetylsulfanyl-.alpha.,N(acetyl)-histidine (Compound
10);
[0048] 11. L-5-acetylsulfanyl-.alpha.,N(benzoyl)-histidine
(Compound 11);
[0049] 12. L-5-acetylsulfanyl-.alpha.,N(.beta.-alanyl)-histidine
(Compound 12);
[0050] 13. L-1-methyl-5-acetylsulfanyl-histidine (Compound 13);
[0051] 14. L-5-benzoylsulfanyl-histidine (Compound 14);
[0052] 15. L-5-benzoylsulfanyl-.alpha.,N,N(dimethyl)-histidine
(Compound 15);
[0053] 16. L-5-benzoylsulfanyl-.alpha.,N,N,N(trimethyl)-histidine
(Compound 16);
[0054] 17. L-5-acetylsulfanyl-.alpha.,N(L-phenylalanyl)-histidine
(Compound 17).
[0055] Among the pharmaceutically acceptable acids, one can cite,
in a nonlimiting manner, the mineral acids such as the
hydrochloric, hydrobromic, hydroiodic, sulfuric, tartaric,
phosphoric acids, or the organic acids such as the formic, acetic,
trifluoroacetic, propionic, benzoic, maleic, fumaric, succinic,
citric, oxalic, glyoxylic, aspartic acids and alkanesulfonic acids
such as methanesulfonic, trifluoromethanesulfonic, ethanesulfonic
acids, and arylsulfonic acids such as benzene- and
paratoluenesulfonic acids.
[0056] In the above formula (I):
[0057] alkyl radical denotes a linear or cyclic, possibly branched,
group comprising 1 to 6 carbon atoms,
[0058] substituted alkyl radical denotes an alkyl group substituted
with one or more fluorine atoms, or substituted with an alkenyl
group comprising one or more carbon-carbon double bonds, or
substituted with one or more OH or SH or NH.sub.2 or COOH
functions, as well as the enantiomers thereof, and the
diastereoisomers thereof.
[0059] aryl radical denotes an optionally fluorinated or
polyfluorinated phenyl group, and comprising optionally one or more
OH or SH or NH.sub.2 or COOH functions
[0060] .alpha.-amino-acyl radical denotes the acyl radical of any
proteogenic amino acid, that is to say any amino acid entering into
the composition of proteins found in the plant or animal world,
including man.
[0061] According to a second aspect, the invention also relates to
a method A for preparing novel 5-acylsulfanyl-histidine compounds
and their derivatives of general formula (I), given explicitly in
the accompanying FIG. 1, and characterized in that it includes the
following steps:
[0062] 1) The reaction of the histidine, racemic (DL) or one of the
enantiomers thereof (D or L), or
[0063] one of their derivatives alkylated on the nitrogen in
position 1 of the imidazole ring, racemic (DL) or one of the
enantiomers thereof (D or L), or
[0064] one of their derivatives alkylated or acylated on the
nitrogen of the .alpha.-amine function, racemic (DL) or one of the
enantiomers thereof (D or L), or
[0065] one of their derivatives alkylated on the nitrogen in
position 1 of the imidazole ring and alkylated or acylated on the
nitrogen of the .alpha.-amine function, racemic (DL) or one of the
enantiomers thereof (D or L),
[0066] in the presence of 1 to 2 equivalents of mineral or organic
acid, with
[0067] a) an agent generating halogenium ions X.sup.+ in a polar
protic solvent, at temperatures of 0-25.degree. C., then with
[0068] b) a sulfur-containing reagent of the carbothioic acid type
of formula alkyl C(.dbd.O)SH or one of the salts thereof in a polar
protic solvent,
[0069] 2) then, optionally, the purification by column liquid
chromatography or any other purification method well known to the
person skilled in the art.
[0070] According to a particular embodiment of the method A
according to the invention: the agent generating halogenium ions
X.sup.+ can be:
[0071] a) bromine Br.sub.2 (as commercial reagent or prepared in
situ); or
[0072] b) NBS or any N-bromo-imide and N-bromo-amide derivative
[0073] According to another particular embodiment of this method A
according to the invention, the polar protic solvent can be water
or an aqueous solution.
[0074] According to yet another particular embodiment of the method
A according to the invention, the sulfur-containing reagent of the
carbothioic acid type can be, for example, thioacetic acid; or
thiobenzoic acid, or mixtures thereof.
[0075] According to another particular embodiment of the method A
according to the invention, the sulfur-containing reagent of the
carbothioic acid salt type can be, for example, potassium
thioacetate, optionally in a mixture with an above-mentioned
carbothioic acid.
[0076] According to yet another particular embodiment of this
method A according to the invention, the temperature will be
0-5.degree. C.
[0077] The innovating character of this method A is based on a
novel reaction of direct introduction of an acylsulfanyl
RC(.dbd.O)S group in position 5 of the imidazole ring of the
histidine or of one of its derivatives, without the use of a
protective group and in water as reaction solvent. This is
particularly surprising given that, under the same operating
conditions, the use of cysteine instead of carbothioic acid leads
to an introduction of sulfur in position 2 of the imidazole ring,
as shown in the patent U.S. Ser. No. 13/121,891 and the patent U.S.
Ser. No. 13/500,887 A1.
[0078] According to a third aspect, the present invention relates
to the use of the above-mentioned 5-acylsulfanyl compounds of
formula (I) or their derivatives, for the preparation of
corresponding 5-sulfanylhistidine compounds and their disulfides
described below.
[0079] According to a fourth aspect, the invention covers novel
5-sulfanylhistidine compounds and their derivatives having the
following general formula (II):
##STR00002##
[0080] Where:
[0081] R.sup.1 to R.sup.4 are as defined for the radicals R.sup.1
to R.sup.4 of formula (I), in particular: R.sup.1.dbd.H, alkyl, in
particular CH.sub.3; R.sup.2.dbd.R.sup.3.dbd.H, alkyl, in
particular CH.sub.3;
[0082] R.sup.4.dbd.H, alkyl, in particular CH.sub.3, alkyl
(C.dbd.O), substituted alkyl (C.dbd.O), aryl (C.dbd.O),
.beta.-alanyl (H.sub.2NCH.sub.2CH.sub.2 (C.dbd.O);
.alpha.-amino-acyl;
[0083] being understood that when R.sup.1.dbd.H then R.sup.2,
R.sup.3 and R.sup.4 cannot be H simultaneously.
[0084] The invention includes all the stereoisomers,
diastereoisomers and enantiomers, in particular in terms of the
carbon atom bearing the COOH group, as well as all the
corresponding disulfides, taken separately or in a mixture.
[0085] It also includes all the salts of pharmaceutically
acceptable acids of said compounds of general formula (II).
[0086] Among the compounds of general formula (H), the invention
relates in particular to:
[0087] those characterized in that R.sup.4 represents hydrogen, or
the methyl group, or the acetyl group, or the benzoyl group, or the
.beta.-alanyl (H.sub.2NCH.sub.2CH.sub.2 (C.dbd.O) group;
[0088] those prepared in the experimental part, in particular:
[0089] 1. the disulfide of L-5-sulfanyl-.alpha.,N(methyl)-histidine
(Compound 22);
[0090] 2. L-5-sulfanyl-.alpha.,N(methyl)-histidine (Compound
23);
[0091] 3. the disulfide of
L-5-sulfanyl-.alpha.,N,N(dimethyl)-histidine (Compound 24);
[0092] 4. L-5-sulfanyl-.alpha.,N,N(dimethyl)-histidine (Compound
25);
[0093] 5. L-5-sulfanyl-.alpha.,N,N,N(trimethyl)-histidine (Compound
26);
[0094] 6. the disulfide of
L-5-sulfanyl-.alpha.,N,N,N(trimethyl)-histidine (Compound 27);
[0095] 7. the disulfide of L-5-sulfanyl-.alpha.,N(acetyl)-histidine
(Compound 28);
[0096] 8. L-5-sulfanyl-.alpha.,N(acetyl)-histidine (Compound
29);
[0097] 9. L-5-sulfanylcamosine (Compound 30);
[0098] 10. the disulfide of iso-ovothiol A (Compound 31);
[0099] 11. iso-ovothiol A (Compound 32);
[0100] 12. the disulfide of
L-5-sulfanyl-.alpha.,N,N(dimethyl)-1-methylhistidine (Compound
33);
[0101] 13. L-5-sulfanyl-.alpha.,N,N,N(trimethyl)-1-methylhistidine
(Compound 34);
[0102] 14. L-5-sulfanyl-.alpha.,N(L-alanyl)-histidine (Compound
35);
[0103] 15. the disulfide of
5-sulfanyl-.alpha.,N(pentanoyl)-histidine (Compound 36).
[0104] Among the pharmaceutically acceptable acids, one can cite,
in a nonlimiting manner, the mineral acids such as hydrochloric,
hydrobromic, hydroiodic, sulfuric, tartaric, phosphoric acids or
the organic acids such as the formic, acetic, trifluoracetic,
propionic, benzoic, maleic, fumaric, succinic, citric, oxalic,
glyoxylic, aspartic acids, alkanesulfonic acids such as
methanesulfonic, trifluoromethanesulfonic, ethanesulfonic acids,
the arylsulfonic acids such as benzene- and paratoluenesulfonic
acids.
[0105] In formula (II) above:
[0106] alkyl radical denotes a linear or cyclic, optionally
branched, group comprising 1 to 6 carbon atoms
[0107] substituted alkyl radical denotes an alkyl group substituted
with one or more fluorine atoms, or substituted with an alkenyl
group comprising one or more carbon-carbon double bonds, or
substituted with one or more OH or SH or NH, or COOH functions, as
well as the enantiomers thereof, and the diastereoisomers
thereof.
[0108] aryl radical denotes an optionally fluorinated or
polyfluorinated phenyl group, and comprising optionally one or more
OH or SH or NH, or COOH functions
[0109] .alpha.-amino-acyl radical denotes the acyl radical of any
proteogenic amino acid, that is to say any amino acid entering into
the composition of the proteins found in the plant or animal world,
including man.
[0110] disulfide denotes any compound obtained by oxidation between
two identical molecules of derivatives of the 5-sulfanylhistidine
type described in the invention.
[0111] The novel 5-sulfanylhistidine compounds and their
derivatives having the general formula (II) as well as their
disulfides could prove to be nutritional, cosmetic or medicinal
active substances.
[0112] According to a fifth aspect, the invention furthermore
relates to a method B for preparing the 5-sulfanylhistidine
compounds and their derivatives of general formula (H) obtained
from 5-acylsulfanyl-histidine compounds and their derivatives of
general formula (I) described in the method A above, and
characterized in that it includes the following steps:
[0113] 1) Either directly (method B1):
[0114] a) by hydrolysis of the 5-acylsulfanyl-histidine derivatives
obtained according to the invention in a polar protic solvent by
stirring at a temperature above 20.degree. C. in the presence of a
thiol,
[0115] b) then, optionally, purification by column liquid
chromatography or any other purification method well known to the
person skilled in the art.
[0116] 2) Or indirectly (method B2):
[0117] a) by hydrolysis of the 5-acylsulfanyl-histidine derivatives
obtained according to the invention in a polar protic solvent by
stirring at a temperature above 20.degree. C. in order to obtain
the corresponding disulfide,
[0118] b) then reduction of the disulfide by reaction with a
thiol,
[0119] c) then, optionally, purification by column liquid
chromatography or any other purification method well known to the
person skilled in the art.
[0120] According to a particular implementation of this method B
according to the invention, the polar protic solvent can be water
or an aqueous solution.
[0121] According to another particular implementation of the method
B according to the invention, the thiol can be, for example,
mercaptopropionic acid or dithiothreitol, or mixtures thereof.
[0122] According to yet another particular implementation of this
method B according to the invention, the temperature can be between
20 and 130.degree. C.
[0123] By this aspect, the applicant demonstrates the ability of
the compounds of general formula (I) to be precursors of
5-sulfanylhistidine compounds and their derivatives of general
formula (II) after hydrolysis.
[0124] According to a sixth aspect, the invention also relates to a
method C for preparing disulfides of the 5-sulfanylhistidines and
of their derivatives:
[0125] 1) either directly from the 5-acylsulfanyl-histidine
compounds and their derivatives of general formula (I),
characterized in that it includes the following steps:
[0126] a) hydrolysis of the 5-acylsulfanyl-histidine derivatives of
general formula (I) obtained according to the invention in a polar
protic solvent by stirring in air and at a temperature above
20.degree. C. in order to obtain the corresponding disulfide,
[0127] b) then, optionally, purification by column liquid
chromatography or any other purification method well known to the
person skilled in the art;
[0128] 2) or from 5-acylsulfanyl-histidines and their derivatives
of general formula (II), characterized in that it includes the
following steps:
[0129] a) oxidation by oxygen or dimethyl sulfoxide or any other
oxidation method well known to the person skilled in the art,
[0130] b) then, optionally, purification by column liquid
chromatography or any other purification method well known to the
person skilled in the art.
[0131] By this aspect, the applicant demonstrates the ability of
the 5-acylsulfanyl-histidine compounds of general formula (I) to be
precursors of disulfides of 5-sulfanylhistidines and of their
derivatives after hydrolysis and oxidation.
[0132] According to a seventh aspect, the invention also relates to
a "one-pot" method D for preparing the 5-sulfanylhistidine
derivatives and the corresponding disulfides thereof from
corresponding histidine derivatives, by combining the methods A
with B or with C, and characterized in that it includes the
following steps:
[0133] in the presence of 1 to 2 equivalents of mineral or organic
acid, the reaction with:
[0134] a) an agent generating halogenium ions X.sup.+ in a polar
protic solvent, at a temperature of 0-25.degree. C., then with
[0135] b) a sulfur-containing reagent of the carbothioic acid type
of formula alkyl C(.dbd.O)SH or one of the salts thereof in a polar
protic solvent,
[0136] followed by
[0137] 1) Either:
[0138] c) the hydrolysis of the 5-acylsulfanyl-histidine
derivatives obtained in a polar protic solvent by stirring at a
temperature between 70 and 130.degree. C. in the presence of a
thiol,
[0139] d) then, optionally, the purification by column liquid
chromatography or any other purification method well known to the
person skilled in the art.
[0140] 2) Or:
[0141] d) by hydrolysis of the 5-acylsulfanyl-histidine derivatives
obtained in a polar protic solvent by stirring at a temperature
between 70 and 130.degree. C. in order to obtain the corresponding
disulfide,
[0142] e) then, optionally, purification by column liquid
chromatography or any other purification method well known to the
person skilled in the art.
DESCRIPTION OF THE FIGURES
[0143] The invention includes 4 figures.
[0144] FIG. 1: Scheme of the method for synthesizing compounds
according to general formula (I)
[0145] 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)
[0146] FIG. 3: Representative spectrum (H.sup.1 NMR, 400 MHz) of
the reaction mixture obtained in Example 3, preparation of
L-5-acetylsulfanyl-.alpha.,N,N(dimethyl)-histidine (Compound 2)
[0147] FIG. 4: Representative spectrum (H.sup.1 NMR, 400 MHz) of
the reaction mixture obtained in Example 5, preparation of
L-5-acetylsulfanyl-.alpha.,N,N,N(trimethyl)-histidine (Compound
3)
DESCRIPTION OF THE EXAMPLES
[0148] 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.
[0149] 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.
[0150] 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.
[0151] All the experiments are carried out in the ambient
atmosphere unless indicated otherwise.
[0152] 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 250.times.4.6 (5
.mu.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 100.times.4.6 (5 .mu.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).
I--Preparation of the 5-acylsulfanyl-histidine Derivatives as
Precursors of the 5-sulfanylhistidines and their disulfides.
[0153] 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.
[0154] 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
##STR00003##
[0156] 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.degree. 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.degree. C.
for 1 h.
[0157] Compound 1 is obtained with a reaction yield of 72 mol % as
calculated from the .sup.1H NMR spectrum.
[0158] .sup.1H NMR (D.sub.2O pH.about.1, 400 MHz) of a sample of
the mixture: .delta. (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).
[0159] 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.
[0160] LCMS (APCI): 228.0 [M-H].sup.-
EXAMPLE 2
Preparation of L-5-acetylsulfanyl-histidine (Compound 1) by
Activation with N-bromosuccinimide and Reaction with thioacetic
Acid
##STR00004##
[0162] 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.degree.
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.degree.
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.degree. C. After cooling
to 0.degree. C., the vigorous stirring is maintained for 1 h.
[0163] Compound 1 is obtained with a reaction yield of 75 mol % as
calculated from the H.sup.1 NMR spectrogram (in the reaction
mixture).
[0164] The .sup.1H NMR and mass spectra are identical to those
obtained in Example 1.
EXAMPLE 3
Preparation of L-5-Acetylsulfanyl-.alpha.,N,N(dimethyl)-histidine
(Compound 2) by Activation with N-bromosuccinimide and Reaction
with thioacetic Acid
##STR00005##
[0166] The hydrochloride of monohydrated
.alpha.-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.degree. 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.degree. C. for 30 minutes.
[0167] Compound 2 is obtained with a reaction yield of 70 mol % as
calculated from the .sup.1H NMR spectrogram.
[0168] .sup.1H NMR (D.sub.2O pH.apprxeq.1, 400 MHz) of a sample of
the mixture: .delta. (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).
[0169] 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.
[0170] Compound 2 is purified on a silica column using an ethyl
acetate/ethanol gradient followed by elution with water.
[0171] .sup.1H NMR (D.sub.2O pH.apprxeq.2-3, 300 MHz): .delta.
(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).
[0172] A singlet of low intensity corresponding to the hydrolyzed
product (compound 18b) is detected at 8.33 ppm.
[0173] .sup.13C NMR (D.sub.2O, 75 MHz): .delta. (ppm)=22.4; 30.0;
41.7; 6.84; 117.2; 134.3; 136.8; 170.9; 195.9.
[0174] LCMS (APCI): 258.9 [M+H].sup.-
EXAMPLE 4
Preparation of L-5-acetylsulfanyl-.alpha.,N,N(dimethyl)-histidine
(Compound 2) by Activation with dibromine and Reaction with
thioacetic Acid
##STR00006##
[0176] The hydrochloride of monohydrated
.alpha.,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.degree. C. Under strong stirring, dibromine (470 .mu.t; 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.degree. C. for 1 h.
[0177] Compound 2 is obtained with a reaction yield of 69 mol % as
calculated from the .sup.1HNMR spectrogram.
[0178] The .sup.1H NMR and mass spectra are identical to those
obtained in Example 3.
EXAMPLE 5
Preparation of
L-5-acetylsulfanyl-.alpha.,N,N,N(trimethyl)-histidine (Compound 3)
by Activation with N-bromosuccinimide and Reaction with thioacetic
Acid
##STR00007##
[0180] 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.degree. 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.
[0181] Compound 3 is obtained with a reaction yield of 65 mol % as
calculated from the .sup.1H NMR spectrogram.
[0182] .sup.1H NMR (D.sub.2O, pH=1, 400 MHz) of a sample of the
mixture: .delta. (ppm): 2.53 (s, 3H); 3.33 (s, 9H); 3.50 (m, 2H);
4.13 (m, 1H); 8.91 (s, 1H).
[0183] 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.
[0184] The product is purified on a silica column (ethyl
acetate/ethanol/water gradient).
[0185] .sup.1H NMR (D.sub.2O, pH 2-3, 400 MHz): .delta. (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).
[0186] .sup.13C NMR (D.sub.2O, 75 MHz): .delta. (ppm)=22.9; 30.0;
52.5; 76.5; 117.9; 133.1; 137.2; 169.7; 196.0.
[0187] LCMS (APCI): 272.1 [M+H].sup.+
EXAMPLE 6
Preparation of
L-5-acetylsulfanyl-.alpha.,N,N,N(trimethyl)-histidine (Compound 3)
by Activation with bromine and Reaction with thioacetic Acid
##STR00008##
[0189] 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 .mu.L; 5 mmoles; 1 eq.) is added, and the solution is
cooled to 1.degree. 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.
[0190] Compound 3 is obtained with a reaction yield of 68 mol % as
calculated from the .sup.1H NMR spectrogram.
[0191] The .sup.1 H NMR and mass spectra are identical to those
obtained in Example 5.
EXAMPLE 7
Preparation of L-5-acetylsulfanyl-.alpha.,N(glycinyl)-histidine
(Compound 4) by Activation with N-bromosuccinimide and Reaction
with thioacetic Acid
##STR00009##
[0193] .alpha.,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 .mu.L, 2
mmoles; 2 eq.) is added, and the solution is cooled to 0.degree. C.
Under strong stirring, N-bromosuccinimide (230 mg, 1.3 mmoles; 1.3
eq.) is added. After 3 minutes, thioacetic acid (370 .mu.L, 5.0
mmoles; 5 eq.) is added very rapidly. Stirring is maintained at
0.degree. C. for 30 minutes.
[0194] Compound 4 is obtained with a reaction yield of 62 mol % as
calculated from the .sup.1H NMR spectrogram.
[0195] .sup.1H NMR (D.sub.2O, pH.about.1, 400 MHz) of a sample of
the mixture: .delta. (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).
[0196] 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.
[0197] LCMS (APCI): 287.3 [M+H]+
EXAMPLE 8
Preparation of the
L-5-acetylsulfanyl-.alpha.,N,N(dimethyl)-1-methylhistidine
Derivative (Compound 5) by Activation with N-bromosuccinimide and
Reaction with thioacetic Acid
[0198] a) Preparation of
.alpha.,N,N(dimethyl)-1-methyl-L-histidine
[0199] .alpha.,N,N(Dimethyl)-1-methyl-L-histidine is prepared by
analogy with the protocol described for
.alpha.,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%).
[0200] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (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-.alpha.,N,N(dimethyl)-1-methylhistidine
Derivative
##STR00010##
[0201] .alpha.,N,N(dimethyl)-1-methylhistidine (604 mg, 3 mmoles; 1
eq.) is dissolved in 22 mL of demineralized water. 37% Concentrated
hydrochloric acid (250 .mu.L, 3 mmoles; 1 eq.) is added, then the
solution is cooled to 0.degree. 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.degree. C. for 30 minutes.
[0202] Compound 5 is obtained with a reaction yield of 65 mol % as
calculated from the .sup.1H NMR spectrogram.
[0203] .sup.1H NMR (D.sub.2O, pH.about.1, 400 MHz) of a sample of
the mixture: .delta. (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)
[0204] 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.
[0205] 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.
[0206] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (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).
[0207] The signals of ethanol are detected at 1.18 ppm and 3.65
ppm.
[0208] LCMS (APCI): 272.3 [M+H]+
EXAMPLE 9
Preparation of
L-5-acetylsulfanyl-.alpha.,N,N,N(trimethyl)-1-methylhistidine
(Compound 6) by Activation with N-bromosuccinimide and Reaction
with thioacetic Acid
[0209] a) Preparation of 1-methyl-hercynine
[0210] 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%).
[0211] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (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-.alpha.,N,N,N(trimethyl)-1-methylhistidine
(Compound 6)
##STR00011##
[0212] 1-Methyl-hercynine (430 mg, 2 mmoles; 1 eq.) is dissolved in
15 mL of demineralized water. 37% concentrated hydrochloric acid
(170 .mu.L, 2 mmoles; 1 eq.) is added, then the solution is cooled
to 0.degree. C. Under strong stirring, N-bromosuccinimide (465 mg,
2.6 mmoles; 1.3 eq.) is added. After 3 minutes, thioacetic acid
(740 .mu.L, 10 mmoles; 5 eq.) is added very rapidly. The stirring
is maintained at 0.degree. C. for 30 minutes.
[0213] Compound 6 is obtained with a reaction yield of 67 mol % as
calculated from the .sup.1H NMR spectrogram.
[0214] .sup.1H NMR (D.sub.2O, pH.about.1, 400 MHz) of a sample of
the mixture: .delta. (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).
[0215] 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.
[0216] LCMS (APCI): 286.0 [M+H]+
EXAMPLE 10
Preparation of L-5-acetylsulfanyl-.alpha.-N(L-alanyl)-histidine
(Compound 7) by Activation with N-bromosuccinimide and Reaction
with thioacetic Acid
##STR00012##
[0218] .alpha.-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 .mu.L, 4.4 mmoles; 2
eq.), then the solution is cooled to 0.degree. 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 .mu.L, 11.0 mmoles; 5 eq.) is added
very rapidly. Vigorous stirring is maintained at 0.degree. C. for
30 minutes.
[0219] Compound 7 is obtained with a reaction yield of 65 mol % as
calculated from the .sup.1H NMR spectrogram of a sample.
[0220] The reaction mixture is washed with 2.times.25 mL of ethyl
acetate, then the compound is purified on a silica column (ethyl
acetate/ethanol/water 2/2/1). Compound 7 (410 mg, 54%, purity 88%)
is obtained in the form of a transparent oil.
[0221] .sup.1H NMR (D.sub.2O, acid pH, 400 MHz): .delta. (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).
[0222] A singlet corresponding to the succinimide is detected at
2.68 ppm.
[0223] LCMS (APCI): 301.1 [M+H]+
EXAMPLE 11
Preparation of the 5-acetylsulfanyl-.alpha.,N(pentanoyl)-histidine
derivative (Compound 8) by Activation with N-bromosuccinimide and
Reaction with thioacetic Acid
##STR00013##
[0225] .alpha.,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 .mu.L, 1.43 mmoles; 1
eq.), then the solution is cooled to 0.degree. C. Very strong
stirring is maintained. N-Bromosuccinimide (330 mg, 1.86 mmoles;
1.3 eq.) is added. After 3 minutes, thioacetic acid (530 .mu.L,
7.15 mmoles; 5 eq.) is added very rapidly. Vigorous stirring is
maintained at 0.degree. C. for 30 minutes.
[0226] Compound 8 is obtained with a reaction yield of 67 mol % as
calculated from the .sup.1H NMR spectrogram of a sample.
[0227] The product is purified on a silica column (mixture 90% of
ethyl acetate/ethanol 3/1 and 10% of water).
5-Acetylsulfanyl-.alpha.,N(pentanoyl)-histidine (compound 8) is
obtained in the form of a transparent oil (320 mg, 64%, purity
90%).
[0228] .sup.1H NMR (D.sub.2O.about.1, 400 MHz): .delta. (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).
[0229] LCMS (APCI): 314.1 [M+H]+
[0230] 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. analog y desired .delta. .delta. MS No. Formed
product with Reagent product H-2 .delta. .alpha.-H AcS [M + H]+ Ex.
Compound 9 Ex. 2 NBS 78% 8.91 4.13 (dd, 2.55 244.1 12 J = 7.7 Hz, J
= 5.6 Hz) Ex. Compound 10 Ex. 2 NBS 65% 8.83 4.73 (m) 2.51 272.1 13
(1 eq. HCl) Ex. Compound 11 Ex. 2 NBS 58% 8.85 4.93 (dd, 2.56 334.1
14 (1 eq. J = 9.3 Hz, HCl) J = 4.7 Hz) Ex. Compound 12 Ex. 2 NBS
63% 8.84 4.66 (m) 2.51 301.0 15 Ex. Compound 13 Ex. 2 NBS 63% 9.00
4.31 (dd, 2.58 244.6 16 J = 7.6 Hz, Ex. Ex. 1 Br2 55% J = 6.9 Hz)
17 Ex. Compound 14 Ex. 2 NBS 31% 8.98 4.37 -- 292.1 18 (water/
CH3CN) Ex. Compound 15 Ex. 2 NBS 12% 8.95 4.28 -- 320.9 19 (water/
CH3CN) Ex. Compound 16 Ex. 2 NBS 12% 9.06 4.32 -- 334.1 20 (water/
CH3CN) Ex. Compound 17 Ex. 2 NBS 40% 8.89 4.36 2.58 377.2 21
##STR00014## ##STR00015##
II. Application Examples:
[0231] II.1 Transformation of the 5-acylsulfanyl Derivatives
Prepared In Situ into Corresponding 5-sulfanylhistidine Derivatives
by Hydrolysis
[0232] 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.
[0233] 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.
[0234] 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)
##STR00016##
[0236] 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.degree. 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.degree. 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.degree. 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.
[0237] .sup.1H NMR (D.sub.2O. 400 MHz): .delta. (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).
[0238] .sup.1H NMR (D.sub.2O+DCl, 400 MHz): .delta. (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).
[0239] .sup.13C NMR (D.sub.2O+DCl, 75 MHz): .delta. (ppm)=26.3;
55.2, 122.1; 130.1; 135.5; 173.6.
[0240] LC-MS (AP-): 186.0 [M-H].sup.-
[0241] [.alpha.].sub.D=+7.4.degree. (c=0.1; 1N HCl)
[0242] Elemental analysis: C.sub.6H.sub.9N.sub.3O2S; 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)
##STR00017##
[0244] 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.degree. 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.degree. 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.degree. 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.
[0245] The .sup.1H NMR, .sup.13C NMR and mass spectra are identical
to those obtained in Example 13 for Compound 9.
[0246] [.alpha.].sub.D: -7.1.degree. (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)
##STR00018##
[0248] 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.degree. 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.degree. 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.degree. 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.
[0249] 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)
##STR00019##
[0251] 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.degree. 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.degree. 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.degree. 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.
[0252] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (ppm)=3.27 (m,
2.times.1H); 3.32 (m, 2.times.1H); 4.17 (dd, J=8.0 Hz, J=6.6 Hz,
2.times.1H); 8.87 (s, 2.times.1H).
[0253] LCMS (APCI): 373.0 [M+H].sup.-
[0254] [.alpha.].sub.D:+23.6.degree. (c=0.1; 1N HCl)
EXAMPLE 26
[0255] a) "One Pot" Preparation of the disulfide of
L-5-sulfanyl-.alpha.,N(methyl)-histidine (Compound 22) via In-Situ
Preparation of 5-acylsulfanyl-histidine Followed by Hydrolysis and
Oxidation (Compound 22)
##STR00020##
[0256] The hydrochloride of .alpha.,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 .mu.L (5 mmoles; 1
eq.), then the solution is cooled to 1.degree. 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.degree. 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.degree. 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-.alpha.,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.
[0257] .sup.1H NMR (MeOD/D.sub.2O 20/1, 400 MHz): .delta.
(ppm)=2.69 (s, 2.times.3H); 2.94 (dd, J=14.0 Hz, J=7.0 Hz,
2.times.1H); 2.99 (dd, J=14.0 Hz, J=5.0 Hz, 2.times.1H); 3.92 (dd,
J=7.0 Hz, J=5.0 Hz, 2.times.1H); 7.79 (s, 2.times.1H).
[0258] LCMS (APCI): 401.0 [M+H].sup.+
b) Preparation of L-5-sulfanyl-.alpha.,N(methyl)-histidine by
Reduction of the disulfide (Compound 23)
##STR00021##
[0259] The disulfide of L-5-sulfanyl-.alpha.,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-.alpha.,N(methyl)-histidine
(Compound 23) (351 mg, 56%) is obtained in the form of a beige
powder.
[0260] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (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).
[0261] LCMS (APCI): 202.1 [M+H].sup.-
EXAMPLE 27
[0262] a) "One Pot" Preparation of the disulfide of
L-5-sulfanyl-.alpha.,N,N(dimethyl)-histidine via In-Situ
Preparation of 5-acylsulfanyl-histidine Followed by Hydrolysis and
Oxidation (Compound 24)
##STR00022##
[0263] The hydrochloride of monohydrated
.alpha.,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 .mu.L; 985 mg; 10
mmoles; 1 eq.), then the solution is cooled to 1.degree. 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.degree. 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-.alpha.,N,N(dimethyl)-histidine (Compound
24.times.4HCl.times.H2O), 1.2 g, 41%) is obtained in the form of a
beige powder.
[0264] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (ppm)=3.01 (s,
2.times.6H); 3.37 (dd, J=14.6 Hz, J=11.2 Hz, 2.times.1H); 3.51 (dd,
J=14.6 Hz, J=4.0 Hz, 2.times.1H); 4.09 (dd, J=11.2 Hz, J=4.0 Hz,
2.times.1H); 8.86 (s, 2.times.1H).
[0265] LCMS (APCI): 429.2 [M+H].sup.-
b) Preparation of the Compound 24 Free Base
##STR00023##
[0267] The hydrated hydrochloride of the disulfide of
L-5-sulfanyl-.alpha.,N,N(dimethyl)-histidine (3.6 g; 5.89 mmoles; 1
eq.) is dissolved in 53 mL of demineralized water. The resin
Amberlite.RTM.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-.alpha.,N,N(dimethyl)-histidine free base (Compound
24) (2.47 g, 84%) in the form of a yellow solid.
[0268] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (ppm)=2.88 (s,
2.times.6H); 2.92 (m, 2.times.2H); 3.70 (m, 2.times.1H); 8.17 (s,
2.times.1H).
c) Obtention of Compound 25 by Reduction of Compound
18.times.4HCl.times.H.sub.2O)
##STR00024##
[0270] The hydrated hydrochloride of the disulfide of
L-5-sulfanyl-.alpha.,N,N(dimethyl)-histidine (1.2 g; 2.07 mmoles;
0.5 eq.) is dissolved in 40 mL of demineralized water. The resin
Amberlite.RTM.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-.alpha.,N,N(dimethyl)-histidine (Compound 25) (450 mg,
58%) is obtained in the form of a white solid.
[0271] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (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).
[0272] .sup.13C NMR (D.sub.2O, 75 MHz): .delta. (ppm)=22.7; 41.8,
67.3; 124.5; 129.6; 131.7; 171.0.
[0273] 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
##STR00025##
[0274] 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.degree. C. and heated
under stirring for 8 h. Samples are collected every 60 minutes and
the mixture is analyzed by HPLC.
[0275] 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
[0276] a) "One Pot" Preparation of
L-5-sulfanyl-.alpha.,N,N,N(trimethyl)-histidine via In-Situ
Preparation of 5-acylsulfanyl-histidine Followed by Hydrolysis
(Compound 26)
##STR00026##
[0277] L-Hereynine (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.degree. 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.degree. 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-.alpha.,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).
[0278] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (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).
[0279] LCMS (APCI): 230.0 [M+H]+
b) Analytical monitoring by .sup.1H NMR of the hydrolysis of the
5-acylsulfanyl compound 3 into the compound
L-5-sulfanyl-.alpha.,N,N,N(trimethyl)-histidine (Compound 26) in
the presence of a thiol
##STR00027##
[0280] 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 D2O. 172 mg of 3-mercaptopropionic acid (142
.mu.L, 5 equivalents) are added, and the solution is heated at
40.degree. 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-.alpha.,N,N,N(trimethyl)-histidine (compound 26) by
Hydrolysis of the 5-acylsulfanyl Compound 3
##STR00028##
[0281] 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.degree. 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
(2.times.2 mL), then with ethyl ether (2.times.2 mL). After drying,
104 mg (72%) of L-5-sulfanyl-.alpha.,N,N,N(trimethyl)-histidine are
obtained in the form of a beige powder.
[0282] The .sup.1H NMR and mass spectra are identical to those
obtained in Example 28a.
d) Preparation of the disulfide of
L-5-sulfanyl-.alpha.,N,N,N(trimethyl)-histidine (Compound 27)
##STR00029##
[0283] L-5-Sulfanyl-.alpha.,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-.alpha.,N,N,N(trimethyl)-histidine (Compound 27) (263
mg; 89%) is obtained in the form of a yellow powder.
[0284] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (ppm)=2.68 (dd,
J=13.5 Hz, J=11.0 Hz, 2.times.1H); 2.75 (dd, J=13.5 Hz, J=4.3 Hz,
2.times.1H); 3.19 (s, 2.times.9H); 3.68 (dd, J 11.0 Hz, J=4.3 Hz,
2.times.1H); 7.97 (s, 2.times.1H).
[0285] 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
##STR00030##
[0286] 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.degree. C. and heated
under stirring for two days. Samples are collected every hour, and
the mixture is analyzed by HPLC.
[0287] 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.1H NMR of the Hydrolysis of the
5-acylsulfanyl Compound 3 and Oxidation In Situ into Compound
27
##STR00031##
[0288] Compound 3 is prepared and purified by column as described
in Example 5. 30 mg of compound 3 are dissolved in 600 .mu.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 (.about.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
[0289] a) "One Pot" Preparation of the disulfide of
L-5-sulfanyl-.alpha.-N(acetyl)-histidine (Compound 28
hydrochloride) via In-Situ Preparation of 5-acylsulfanyl-histidine
Followed by Hydrolysis and Oxidation
##STR00032##
[0290] Monohydrated .alpha.-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.degree. C.
Dibromine (668 .mu.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.degree. 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.degree. 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-.alpha.,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).
[0291] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (ppm)=1.86 (s,
2.times.3H); 2.92 (dd, J=15.0 Hz, J=8.0 Hz, 2.times.1H); 3.03 (dd,
J=15.0 Hz, J=5.5 Hz, 2.times.1H); 4.47 (dd, J=8.0 Hz, J=5.5 Hz,
2.times.1H); 8.73 (s, 2.times.1H).
[0292] LCMS (APCI): 457.4 [M+H]+
b) Preparation of L-5-sulfanyl-.alpha.,N(acetyl)-histidine
(Compound 29)
##STR00033##
[0293] The hydrochloride of the disulfide of
L-5-sulfanyl-.alpha.,N(acetyl)-histidine (Compound 28) (520 mg; 834
.mu.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.degree. 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-.alpha.,N(acetyl)-histidine (Compound 29) (390 mg;
86%) in the form of a beige solid.
[0294] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (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,
1H); 4.50 (dd, J=8.6 Hz, J=4.8 Hz, 1H); 8.22 (s, 1H).
[0295] 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)
##STR00034##
[0297] 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.degree. C. N-Bromosuccinimide (440 mg; 2.44
mmoles; 1.3 eq.) is added in one portion: the solution turns limpid
orange. Thioacetic acid (960 .sub.1AL; 1.03 g, 13.14 mmoles; 7 eq.)
is added. The mixture is stirred at 0.degree. 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.
[0298] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (ppm)=2.69 (t,
J=6.7 Hz, 2H); 3.00 (m, 1H); 3.12 (in, 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).
[0299] LCMS (APCI): 259.1 [M+H]+
EXAMPLE 31
Preparation of Compounds 31 and 32
[0300] 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)
##STR00035##
[0301] 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 .sub.tit (10 mmoles; 2 eq.) is
added; then the solution is cooled to 1.degree. 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.degree. 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.degree. 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.
[0302] .sup.1H NMR (D.sub.2O+DCl, 400 MHz): .delta. (ppm)=3.14 (m,
2.times.2H); 3.85 (s, 2.times.3H); 4.17 (m, 2.times.1H); 8.89 (s,
2.times.1H).
[0303] LCMS (APCI): 401.1 [M+H]+
b) Preparation of iso-ovothiol A (Compound 32)
##STR00036##
[0304] 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.degree. 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.
[0305] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (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).
[0306] LCMS (APCI): 202.1 [M+H]+
EXAMPLE 32
Preparation of the disulfide of
L-5-sulfanyl-.alpha.,N,N(dimethyl)-1-methylhistidine via Hydrolysis
of the 5-acetylsulfanyl-.alpha.,N,N(dimethyl)-1-methylhistidine
Derivative Followed by Air Oxidation (Compound 33)
##STR00037##
[0308] 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-.alpha.,N,N(dimethyl)-1-methylhistidine (Compound 33,
98%) is obtained in the form of a greenish amorphous solid.
[0309] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (ppm)=2.97 (s,
2.times.6H); 3.17 (m, 2.times.1H); 3.28 (dd, J=15.8 Hz and J=4.3
Hz, 2.times.1H); 3.69 (s, 2.times.3H); 4.00 (m, 2.times.1H); 8.44
(s, 2.times.1H).
[0310] LCMS (APCI): 457.2 [M+H]+
EXAMPLE 33
"One Pot" Preparation of the disulfide of
L-5-sulfanyl-.alpha.,N,N,N(trimethyl)-1-methylhistidine (Compound
34 dihydrochloride) via In-Situ Preparation of
L-5-acetylsulfanyl-.alpha.,N,N,N(trimethyl)-1-methylhistidine
Followed by Hydrolysis and Oxidation
##STR00038##
[0312] 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 .mu.L, 2 mmoles; 1 eq.), then the
solution is cooled to 0.degree. C. Very strong stirring is
maintained. N-Bromosuccinimide (465 mg, 2.6 inmates; 1.3 eq.) is
added rapidly. After 3 minutes, thioacetic acid (740 .mu.L, 10
mmoles; 5 eq.) is added very rapidly. Vigorous stirring is
maintained at 0.degree. C. for 30 minutes. The mixture is extracted
with 2.times.20 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 .mu.L, 2 mmoles,
1 eq.) in a solution of glacial acetic acid. The solution is heated
for one hour at 80.degree. C. The dihydrochloride of the disulfide
of L-5-sulfanyl-.alpha.,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%).
[0313] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (ppm)=3.30 (s,
2.times.9H); 3.61 (dd, J=14.1 Hz and 3.4 Hz, 2.times.1H); 3.72 (m,
2.times.1H); 3.73 (s, 2.times.3H); 4.09 (dd, J=12.2 Hz and 3.4 Hz,
2.times.1H); 8.98 (s, 2.times.1H).
[0314] LCMS (APCI): 485.1 [M+H]+
EXAMPLE 34
Preparation of the L-5-sulfanyl-.alpha.,N(L-alanyl)-histidine
Derivative (Compound 35) by Hydrolysis of the 5-acylsulfanyl
Compound
##STR00039##
[0316] 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-.alpha.,N(L-alanyl)-histidine (Compound 35,
92%) is obtained in the form of a beige amorphous solid.
[0317] .sup.1H NMR (D.sub.2O, 400 MHz): .delta. (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).
[0318] LCMS (APCI): 258.9 [M+H]+
EXAMPLE 35
Preparation of the disulfide of
5-sulfanyl-.alpha.,N(pentanoyl)-histidine (Compound 36) by
Hydrolysis and Oxidation of the Compound
5-acetylsulfanyl-.alpha.,N(pentanoyl)-histidine
##STR00040##
[0320] The 5-acetylsulfanyl-.alpha.,N(pentanoyl)-histidine
derivative (compound 8) is prepared and purified as described in
Example 11. 320 mg (0.9 mmole; 1 eq.) of compound 8 are dissolved
in 8.0 mL of demineralized water. 3-Mercaptopropionic acid (400
.mu.L, 4.60 mmoles; 5 eq.) is added. The solution is heated at
90.degree. C. for 3 h. The reaction mixture is extracted with
4.times.10 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.degree. C. under stirring for 2 hours,
then at ambient temperature for 18 hours. After evaporation to
dryness, the disulfide of
L-5-sulfanyl-.alpha.,N(pentanoyl)-histidine (Compound 36) is
obtained in the form of an orange lacquer (44%).
[0321] LCMS (APCI): 541.2 [M.alpha.H]+
TABLE-US-00005 TABLE 5 Summary of the examples: Compound Example
prepared Structure 1 1 ##STR00041## 2 1 3 2 ##STR00042## 4 2 5 3
##STR00043## 6 3 7 4 ##STR00044## 8 5 ##STR00045## 9 6 ##STR00046##
10 7 ##STR00047## 11 8 ##STR00048## 12 9 ##STR00049## 13 10
##STR00050## 14 11 ##STR00051## 15 12 ##STR00052## 16 13
##STR00053## 17 13 18 14 ##STR00054## 19 15 ##STR00055## 20 16
##STR00056## 21 17 ##STR00057## 22 18 ##STR00058## 23 19
##STR00059## 24 20 ##STR00060## 25 21 x 4HCl x 2H2O ##STR00061##
26a 22 ##STR00062## 26b 23 ##STR00063## 27a 24 x 4HCl x 2H2O
##STR00064## 27b 24 ##STR00065## 27c 25 ##STR00066## 27d 25 28a 26
##STR00067## 28b 26 28c 26 28d 27 ##STR00068## 28e 27 28f 27 29a 28
x 2HCl ##STR00069## 29b 29 ##STR00070## 30 30 ##STR00071## 31a 31
##STR00072## 31b 32 ##STR00073## 32 33 ##STR00074## 33 34 x 2HCl
##STR00075## 34 35 ##STR00076## 35 36 ##STR00077##
* * * * *