New Selenohydroxy Acids And Their Derivatives Applications In Nutrition, Cosmetics And Pharmaceutics
Abstract:
This invention relates to new selenohydroxy-acids (SHA) and their derivatives, their preparations and applications in nutrition, cosmetics and pharmaceutics. More particularly, this invention relates to the synthesis of 2-hydroxy-4-methylselenobutyric acid, its salts and esters and amides derived from 2-hydroxy-4-methylselenobutyric acid as selenomethionine precursors and particularly L(+)-selenomethionine precursors according to a biomimetic approach using enzymes with animal or human origin.
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Notices, Deadlines & Correspondence
FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10, Rule 13]
NEW SELENOHYDROXY ACIDS AND
THEIR DERIVATIVES APPLICATIONS IN
NUTRITION, COSMETICS AND
PHARMACEUTICS;
TETRAHEDRON, A CORPORATION ORGANIZED AND EXISTING UNDER THE LAWS OF FRANCE, WHOSE ADDRESS IS 4 BIS, ALLEE CHARLES V, F-94300 VINCENNES, FRANCE.
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.
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The purpose of mis mvention is:
- new selenohydroxy-acid compounds and their derivatives;
- their process for the preparation;
- their use as precursors of L(+)-selenomethionme and/or source of selenium in human or animal nutrition, in cosmetics and pharmaceutics;
- and nutritional, cosmetic and pharmaceutical compositions containing them.
State of the art
This invention relates to new selenohydroxy-acids (SHA) and their derivatives, their preparations and applications in nutrition, cosmetics and pharmaceutics. More particularly, this invention relates to the synthesis of 2-hydroxy-4-methylselenobutyric acid, its salts and esters and amides derived from 2-hydroxy-4-methylselenobutyric acid as selenomethionine precursors and particularly L(+)-selenomethionine precursors according to a biomimetic approach using enzymes with animal or human origin.
Selenium is a micro-nutrient essential particularly for Man and mammals (Wendel, A.; Phosphorus, Sulfur Silicon Relat Elem.; 1992; 67, 1-4, 405-415). It participates in the biosynthesis of selenoproteins such as Glutathion peroxydase, as well as Thioredoxine reductase and Selenoprotein, in the form of L(+)-selenocysteine or L(+)-selenomethionine {Mutter, S. et al; Arch. Microbiol, 1997; 168; 421). According to FDA-KDAs 10th edition 1989 {Selenium: its molecular biology and role in human health; Hatfield, D.L. Eds; 2003; Kluwer Acad. Publishers; second edition; 299-31), Man's daily needs of selenium vary from 10-30 ug for a child to 40-70 ug for an adolescent-adult, these rates being higher particularly for women during pregnancy (65 ug/day) and during breast feeding (75 ug/day). The additional amount of L(+)-selenomethionine (2,7 umoles of selenium equivalent) for breast feeding women significantly increases the concentration of selenium in their milk (McGuire, M.K. et al; Am. J. Clin. Nutr.; 1993; 58; 5; 649).
Man is auxotrophic for L(+)-selenomethionine, which means that he is incapable of synthesising it. Therefore, the only way to obtain it is through food. Ideally, selenium should be absorbed in its natural form, in other words in organic form. Nevertheless, several forms of
selenium may be used as a food complement; inorganic selenium for example such as sodium selenite; and organic selenium for example such as L(+)-selenomethionine. Knowing that more than 80% of total organic selenium in plants (particularly wheat, corn and soya) consists of ^^selenomethionine, this amino acid is the most appropriate and least toxic form of
2.
selenium, and is better than sodium selenite as an animal or human food complement (Schrauzer, G.N.; J.Am.Coll. Nutrit; 2001; 20; 1; 1-4). ^^selenomethionine has better bioavailability and is much better tolerated than sodium selenite (Mony, MC et al; J. of Trace Elem. Exp. Med.; 2000; 13; 367-380).
L(+)-selenomethionine has anti-oxidative properties due to the presence of selenium in its molecular structure (Tapiero H et al; Biomed. Pharmacother.; 2003; 57; 3-4; 134-144). It has been shown that ^^selenomethionine very effectively traps peroxynitrite, an extremely toxic metabohte generated in all inflammatory situations and for which the deleterious action causes cell death (Assman, A. et al; Arch. Biochem. Biophys.; 1998; 349; 201-203).
A selenium food complement proved to be very beneficial in many situations (nutritional deficiency, diseases, exposure to radiation, etc.). This is particularly true for children suffering from genetic diseases such as phenylcetonuria or hyperphenylalaninemia, since these children have low protein diets (Reilly, C. et al; Am. J. Clin. Nutr.; 1990; 52; 150-165). Selenium in organic form such as L(+)-selenomethionine associated with vitamins has protective effects with regard to UV radiation in man (La Ruche et al; Photodermtol Photoimmunol. Photomed.; 1991; 8; 6; 232-235). L(+)-selenomethionine protects against the deleterious biological effects of high energy ionising radiation (Kennedy, AR et al; Free Rad. Biol Med.; 2004; 36; 2; 259-266).
Furthermore, several organoselenium derivatives have been effective in the prevention of some types of cancer in Man. In this context, it has been shown that L(+)-selenomethionine causes activation of a DNA repair system, mediated by the p53 tumour suppressor, thus reducing the accumulation of mutations in somatic cells (Seo, YR et al; PNAS; 2002, 89; 22;14548). It has been shown that a complement of up to 200ug/Se/day of L(+)-selenomethionine in Man very significantly reduces the incidence of cancers such as cancer of the lungs, colorectal cancer and prostate cancer. Seven out of a total of eight clinical tests to evaluate the effect of selenium on the incidence of cancer gave positive results (Whanger, PD; Br. J. Nutr.; 2004; 91,1, 11-28). This confirms the many studies carried out on animals.
Some rare selenohydroxy-acid derivatives have already been described as synthetic intermediates in the preparation of organic derivatives. These are essentially arylselenohydroxy-acid derivatives. For example, this is the case for the methyl ester of 2-hydroxy-4-phenylselenobutyric acid (J.-G. Boiteau, Organic Letters, 2001, 3 (17), 1737 -2740). Furthermore, a selenoxide of 2-hydroxy-4-methylselenobutyric acid has been suggested as an intermediate of oxydative degradation of L(+)-selenomethionine (Gammelgaard, B. etal; Talenta; 2003; 59; 1165-1171).
Surprisingly, 2-hydroxy-4-methylselenobutyric acid itself, its salts and its ester and amide derivatives, are not known. Unlike arylselenohydroxy acid derivatives, these latter compounds may represent potential precursors of selenomethionine. After an enzymatic or
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chemical transformation, 2-hydroxy-4-methylselenobutyric acid, its salts and its ester and amide derivatives, after eventual hydrolysis, can lead to selenomethionine according to the following transformation:
Furthermore, 2-hydroxy-4-methylsulfobutyric acid is known as a methionine precursor for food (WO 9636598; 21.11.1996).
One of the purposes of this invention is to create new compounds containing selenium that, after being administered to man or to animal, may be precursors of selenomethionine, and therefore sources of selenium for the organism. Compounds according to the invention can penetrate inside tissues or cells to be biotransformed into selenomethionine or derivatives so that selenium can be incorporated into proteins of the organism.
These purposes are achieved through this invention that is based on the design of new selenohydroxy-acid derivatives and their esters and amides, which are biotransformed by enzymes present in animal or human cells to generate selenomethionine. This has been exemplified by the Applicant. Description of the invention
Therefore, the purpose of this invention is to:
1) solve the new technical problem that consists of supplying new selenohydroxy-acids, ester and amide derivatives, as selenomethionine precursors, thus forming the active constituents of nutritional, cosmetic and pharmaceutical compositions;
2) solve this new technical problem using a solution that includes a method for preparation of these new derivatives.
The technical problems mentioned above are solved simultaneously by this invention for the first time, in a very easy and economic manner, the method for preparation of the said new derivatives being high-yielding and very simple to implement.
According to the first aspect of this invention, the purpose is new selenohydroxy-acids of general formula (I):
in which
n = 0, lor2
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R, = OH, OCOR3, OP03H2, OPO(OR4XOR5), or OR*, R2 = OH, R3, orNHR7,
R3 = alkoxyl (C1-C26), ceramide 1, ceramide 2, ceramide 3, ceramide 4, ceramide 5, ceramide 6a and 6b, S-cysteinyl, or S-elutatbionyl, or
OR4 = alkoxyl (C1-C26), ceramide 1, ceramide 2, ceramide 3, ceramide 4, ceramide 5, ceramide 6a or 6b,
OR5 = alkoxyl (C1-C26), ceramide 1, ceramide 2, ceramide 3, ceramide 4, ceramide 5, ceramide 6a or 6b.
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OR* = pyruvate, lactate, citrate, fumarate, maleate, myristate, pahnitate, stearate, palmitoleate, oleate, linoleate, natural fatty acids, or 13-cis retinoate,
R7 = H, alkyl, natural amino acids, or natural amines,
it being understood that when n = 1 and R2 = OH, R\ cannot be OH.
The invention encompasses all position isomers, geometric isomers, stereo-isomers, diastereoisomers and enantiomers, particularly for the selenium and carbon atom carrying the Ri, group and for radicals Ri to R7, and all oligomers (dimers, trimers, etc.) and linear or ramified, acyclic or cyclic polymers, obtained between two or several molecules of selenohydroxy-acid derivatives described according to the invention by an esterification reaction between alcohol and carboxylic acid functions that may be present, taken separately or mixed.
It also encompasses all pharmaceutically acceptable acid and base addition salts of the said compounds of general formula (I), particularly sodium and calcium and magnesium salts.
Ammong the compounds of general formula (I), the invention has especially as object the following compounds of general formula (I):
- compounds characterized in that n is 0;
- compounds characterized in that Ri represents OH, OCOR3, OR 98% ('H-RMN, CDC13) in the form of colourless crystals.
*H-RMN (CDC13,300 MHz):
8 (ppm) = 1.18 - 2.20 (m, 22H); 2.00 (s, 3H, SeCH3); 2.38 (m, 2H); 2.97 (m, 2H); 3.94 (dd, J = 8 Hz, J = 4 Hz, 1H, a-H).
,3C-RMN (CDCI3,75.5 MHz):
8 (ppm) = 3.9; 21.5; 24.7; 25.1; 29.2; 30.9; 36.2; 52.7; 71.6; 178.6.
MS (electrospray): m/z (%) = 182 (NHafcyclo-hexylk)4).
m/z (%) = 197 (CHsSeCHaCHzCHOHCCb").
Example 3: Preparation of L-2-hvdroxy-4-methvlselenobutvric acid
L-2-hydroxy-4-methylselenobutyric acid is obtained in the form of colourless crystals by solubilisation of 750 mg (2 mmol) of the previous compound described in example 2, in 10 mL of water, acidification with concentrated HC1 until pH = 1 and extraction with 8x20 mL of diethylic ether. After drying (Na2S04), filtration and evaporation, the result is 365 mg of the required compound in the form of colourless crystals with purity > 98% ('H-RMN, CDCI3).
pF(°C): 47.4-48.0.
]H-RMN (CDCI3, 300 MHz):
8 (ppm) = 2.02 (s, 3H, SeCH3); 2.08 (m, 1H); 2.22 (m, 1H); 2.70 (m (sym.), 2H); 4.43 (dd, J = 8 Hz, J = 4 Hz, 1H, a-H).
,3C-RMN (CDCI3, 75,5 MHz):
8 (ppm) = 4.1; 20.3; 33.9; 69.9; 177.3.
MS (electrospray): m/z (%) = 197 (CHsSeGHbCHzCHOHCOz").
Rf (Si02, cyclohexane/ethyl acetate, 50/50 + 1% CF3COOH): 0.26.
[a]D = -20.5 ± 1 (c = 1, EtOH).
Example 4: Preparation of D-2-hvdroxv-4-methvlselenobutvric acid
D-2-hydroxy-4-methylselenobutyric acid is obtained using the method described in Example 1 for the « L » enantiomer with a yield of 57% in the form of colourless crystals, but
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by adding 0.76 mL (1.0 g, 9.8 mmol) of R-(+)-alpha-hydroxybutyrolactone to the solution of lithiomethylselenolate.
pF(°C): 46.0-47.0°C.
'H-RMN (CDC13,300 MHz):
8 (ppm) = 2.02 (s, 3H, SeCH3); 2.08 (m, 1H); 2.22 (m, 1H); 2.70 (m (sym.), 2H); 4.41 (dd, J = 8 Hz, J = 4 Hz, 1H, a-H).
,3C-RMN (CDCI3,75,5 MHz):
8 (ppm) = 4.1; 20.3; 34.0; 69.9; 178.6.
MS (electrospray): m/z (%) = 197 (C^SeCHzO^CHOHCCV).
[a]D=18.9±l(c=l,EtOH).
Example 5: Preparation of D.L -2-hvdroxv-4-methvlselenobutvric acid
D,L-2-hydroxy-4-methylselenobutyric acid is obtained using the method described in Example 1, but by adding 0.76 mL (1.0 g, 9.8 mmol) of racemic alpha-hydroxybutyrolactone to the solution of lithiomethylselenolate. Recrystallisation of the raw product in toluene results in D)L-2-hydroxy-4-methylselenobutyric acid with purity > 98% ('H-RMN, CDCI3) in the form of a slightly beige powder.
pF(°C): 49.3-49.9.
lH-RMN (CDCI3,300 MHz):
8 (ppm) = 2.02 (s, 3H, SeCH3); 2.08 (m, 1H); 2.22 (m, 1H); 2.70 (m (sym.), 2H); 4.41 (dd, J = 8 Hz, J = 4 Hz, 1H, a-H).
,3C-RMN (CDCI3,75,5 MHz):
8 (ppm) = 4.1; 20.3; 34.0; 69.9; 178.6.
MS (EI, 70 eV): m/z (%) = 198 (M+*, 80); 123 (40); 103 (60); 103 (60).
Rf (S1O2 cyclohexane/ethyl acetate, 50/50 + 1% CF3COOH): 0.26.
Example 6: Synthesis of D.L-2-hvdroxv-4-methvlselenobutvric acid (lOg-scale)
To a black suspension of selenium (6,45 g, 81,7 mmol) in anhydrous tetrahydrofuran (300 ml), cooled to -6°C (internal temperature) in an ice-salt bath and under an atmosphere of argon, was added an ethereal solution of methyl hlhium (1,6M; 60 ml) dropwise over 40 min, the internal temperature was maintained below 0°C during the addition. A small amount of
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18 white deposit was present on the side of the flask which was washed using additional anhydrous THF (30 ml). After 20min, 2-hydroxybutyrolactone (7,64 ml, 98,0 mmol) was added, precipitation occurred on addition forming a milky yellow mixture. After a further 10 min the ice bath was removed and the reaction vessel was sealed. After 22h stirring at room temperature the reaction mixture was heated to 35°C (internal temperature) and stirred for a further 23h. The reaction was allowed to cool to room temperature and then further cooled with an ice bath. The mixture was filtered and the solid was washed with TBME (3x100 ml). The yellow solid was dissolved in water (500 ml), the pH of the solution was adjusted to pH = 10 using an aqueous solution of sodium hydroxide (2N; ca. 1 ml), the aqueous phase was washed with TBME (200 ml) and then acidified (pH = 1) using concentrated hydrochloric acid. The organic material was extracted with TBME (4x200 ml), the organic extracts were combined, dried (Na2S04), filtered and the solvent removed under reduced pressure to leave D,L-2-hydroxy-4-methylselenobutyric acid (12,14 g, 75%) as a yellow oil, which solidified on cooling to a light yellow solid. The 'H-NMR (CDCI3) is identical to the one obtained in the previous Example 5.
Example 7: Synthesis of sodium D.L-2-hvdroxv-4-methvlseleno-butyrate
To a mixture of sodium hydride (84,6 mg; 60% in mineral oil) and anhydrous THF (2,0 ml), stirred under an atmosphere of argon, was added a solution of the DJL-2-hydroxy-4-rnethylselenobutyric acid (0,4244 g, 2,15 mmol) in anhydrous THF (2,0 ml) dropwise over 5 min. The solution bubbled vigorously during the addition. A yellow solution together with a small amount of white precipitate was present at the end of the addition. The mixture was cooled in an ice bath and cyclohexane (3 ml) was added this resulted in the formation of a yellow precipitate. The yellow solid was collected, washed with cyclohexane (3 ml) and TBME (3x3 ml) and dried under reduced pressure to leave the sodium salt of the acid (0,3780 g, 1,73 mmol, 82%).
*H-NMR (D20, 300MHz):
5(ppm) = 1,80-2,07 (m, 5H); 2,45-2,60 (m, 2H); 4,00 (dd, J = 4Hz and 8Hz, 1H; a-H).
Additional signals: 1,12(s); 3,12(s).
,3C-NMR(D20,75,5MHz):
6(ppm) = 3,3; 20,3 (CH2); 34,7 (CH2); 72,0; 180,9 (C=0).
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Example 8: Synthesis of calcium DX-2-hvdroxv-4-mefoylseleno-butvrate
To a light yellow solution of the D,L-2-hydroxy-4-methylselenobutyric acid (0,4540 g, 2,30 mmol) in water (0,9 ml) was added calcium hydroxide (81 mg, 1,09 mmol). A large amount of undissolved material was present after the addition. The mixture was diluted with water (1,1 ml), the solid was collected by filtration and washed sequentially with water (3x2 ml) and diethyl ether (3x2 ml). The solid was dried under reduced pressure to leave the desired calcium salt (87,7 mg, 0,20 mmol, 19%) as a white solid. 'H-NMR (D20,300 MHz): 5(ppm) = 1,88-2,13 (m, 5H); 2,50-2,68 (m, 2H); 4,11 (dd, J = 4Hz and 7Hz, 1H; a-H).
Additional signals: 1,12 (s); 3,12(s).
Example 9: Synthesis of elhvl-D.L-2-hvdroxv-4-methvlselenobutvrate
To a colourless solution of the DrL-2-hydroxy-4-memylselenobutyric acid (0,3225 g, 1,64 mmol) in absolute ethanol (6,5 ml), stirred under an atmosphere of argon, was added boric acid (21,1 mg, 0,34 mmol). After 25h stirring at room temperature, the reaction mixture was heated at reflux and stirred for a further 20h. TLC indicated that the reaction was not complete, additional boric acid (20,9 mg, 0,34 mmol) was added and the reaction was stirred, at reflux, for a further 4 days. The reaction mixture was allowed to cool to room temperature and the solvent was removed under reduced pressure to leave a light yellow liquid. Saturated aqueous sodium bicarbonate (20 ml) and water (20 ml) were added and the organic material was extracted with diethyl ether (3x40 ml). The extracts were combined, dried (Na2S04), filtered and the solvent removed under reduced pressure to leave the desired ethyl ester (0,3340 g, 1,48 mmol, 91%) as a light yellow liquid.
^-NMRtCDCh, 300MHz):
5(ppm) = 130 (t, J = 7Hz, 3H, CH2CH3); overlapping 2,02 (s, 3H, SeCH3) and 1,92-2,08 (m, 1H); 2,08-2,22 (m, 1H); 2,59-2,76 (m, 2H); 2,86(d, J = 5Hz, 1H, OH); overlapping 4,25 (q, J = 7Hz, 2H, Cff2CH3) and 4,22-4.32 (m, 1H, a-H).
2o
"C-NMR (CDCh, 75,5MHz): 8(ppm) = 4,1; 14,2; 20,3; 34,7; 61,9; 69,9; 174,8.
MS (EI, 70eV): m/z (%) = 226 (M+*, 23); 181 (8); 153 (7); 131 (40); 123 (9); 109 (23); 103 (27); 85 (21); 76(17); 57 (100); 41 (13).
Rf(Si02, ethyl acetate/cyclohexane, 50/50 + 1% CF3CO2H): 0,61 (stained with
phosphomolybdic acid).
Example 10: Synthesis of isopropvl-DJ^2-hvdroxv-4-memvlselenobutvrate
To a colourless solution of the D,L-2-hydroxy-4-methylselenobutyric acid (0,3284 g, 1,67 mmol) in absolute ethanol (7 ml), stirred under an atmosphere of argon, was added boric acid (42,9 mg, 0,69 mmol). The reaction mixture was heated at reflux and stirred for 3 days. TLC indicated that the reaction was not complete, additional boric acid (23,4 mg, 0,38 mmol) was added and the reaction was stirred at reflux, for a further 16h. The reaction mixture was allowed to cool to room temperature and the solvent was removed under reduced pressure to leave a light yellow liquid (0,38 g). Saturated aqueous sodium bicarbonate (20ml) and water (20 ml) were added and the organic material was extracted with diethyl ether (3x30 ml). The extracts were combined, dried (Na2S04), filtered and the solvent removed under reduced pressure to leave the desired iso-propyl ester (0,3384 g, 1,41 mmol, 85%) as a light yellow liquid.
'H-NMR (CDCI3,300MHz):
6(ppm) = overlapping 1,28 [d, J = 6Hz, 3H, CH(C#5)2] and 1,28 [d, J = 6Hz, 3H, CH(C#5)2]; overlapping 2,01 (s, 3H, SeCH3) and 1,90-2,07 (m, 1H); 2,07-2,21 (m, 1H); 2,57-2,74 (m, 2H); 2,87(d, J = 5Hz, 1H, OH); 4,21-4,27 (m, 1H, a-H); 5,10 [septet, J = 6Hz, 1H, C#(CH3)2].
l3C-NMR (CDCI3,75,5MHz):
8(ppm) = 4,2; 20,3; 21,7; 21,8; 34,8; 69,8; 70,0; 174,3.
MS (EI, 70eV): m/z (%) = 240 (M+\ 10); 103 (28); 87 (22); 71(62); 57 (73); 43 (100).
2.1
Rf (Si02, ethyl acetate/cyclohexane, 50/50 + 1% CF3CO2H): 0,63 (stained with phosphomolybdic acid).
Example 11: Synthesis of DX-2-acetoxv-4-methvlselenobutvric acid
To a colourless solution of the D,L-2-hydroxy-4-methylselenobutyric acid (0,3222 g, 1,63 mmol) in anhydrous dichloromethane (27 ml), stirred under an atmosphere of argon, was added acetic anhydride (0,62 ml, 6,57 mmol) followed by addition of a catalytic quantity of DMAP. After 6h, additional acetic anhydride (0,62 ml, 6,57 mmol) was added and the reaction was stirred overnight. TLC indicated that the reaction was complete, water (10 ml) was added and the dichloromethane was removed under reduced pressure. Saturated aqueous ammonium chloride (40 ml) was added and the organic material was extracted with diethyl ether (3x40 ml). The extracts were combined, dried (Na2S04), filtered and the solvent removed under reduced pressure to leave a crude light yellow oil (0,2954 g). The crude oil was purified by column chromatography on silica gel using a mixture of ethyl acetatexyclohexane (3:7) and 1% TFA as eluent to give the desired acetate (0,1436 g, 0.64 mmol, 40%) as a colourless liquid, and an impure fraction of the acetate [0,1068g, two spots by TLC: Rf(Si02, ethyl acetate/cyclohexane, 50/50+ 1%CF3CC>2H): 0,52 and 0,65 and additional peaks in the 'H-NMR (CDC13,300MHz): 8(ppm) = 2,17 (pseudo d); 5,14-5,22 (m)].
^-NMR (CDCI3, 300MHz):
8(ppm) = 2,01 (s, 3H); 2,16 (s, 3H); 2,19-2,27 (m, 2H); 2,54-2,69 (m, 2H); 5,16 (t, J = 6Hz, 1H; a-H).
13C-NMR (CDCI3, 75,5MHz):
8(ppm) = 4,1; 19,9; 20,5; 31,4; 71,3; 170,4 and 175,1.
MS (EI, 70eV): m/z (%) = 240 (M1", 7); 145 (8); 103 (7); 85 (10); 57 (10); 43 (100).
Rf (Si02, ethyl acetate/cyclohexane, 50/50 + 1% CF3C02H): 0,52 (stained with
phosphomolybdic acid)
Example 12: Synthesis of D.L-2-linolevloxv-4-methvlselenobutvric acid
To a colourless solution of linoleic acid (0,40 ml, 1,28 mmol) in anhydrous DMF (27 ml), stirred under an atmosphere of argon, was added 1-hydroxybenzotriazole (0,1745 g, 1,29 mmol) followed by addition of HCTU (0,5322 g, 1,29 mmol). After lh, a solution of the DJJ-2-hydroxy-4-methylselenobutyric acid (0,2545 g, 1,29 mmol) in anhydrous DMF (2,4 ml) was added, followed by addition of DffiA (0,44 ml, 2,54 mmol). After 16h, the reaction was deemed to be complete. The solvent was removed under reduced pressure to
22.
leave an orange oil (1,827 g). The crude oil was partitioned between saturated aqueous sodium bicarbonate (25 ml) and diethyl ether (40 ml). The layers were separated and the aqueous phase was washed with further diethyl ether (2x40 ml). The ethereal extracts were combined, dried (Na2S04), filtered and the solvent removed under reduced pressure to leave a crude white waxy-solid (0,719 g). The crude material was purified by column chromatography on silica gel using a mixture of ethyl acetate/cyclohexane (3/7) and 0,1% TFA as eluent to give the linoleate (0,0813 g, 0.18 mmol, 14%) as a reddish-orange oil.
'H-NMR (DMSO, 300MHz):
6(ppm) = 0,83 (t, J = 7Hz, 3H); 1,16-1,36 (m, 15H); 1,42-1,59 (m; 2H); overlapping 1,93 (s, 3H, SeCH3) and 1,91-2,10 (m, 6H); 2,28-2,36 (m, 2H); 2,47-2,59 (m, 2H); 2,69-2,78 (m, 2H); 4.91 (dd, J = 6Hz and 7Hz, 1H, oc-H); 5,21-5,39 (m, 4H).
13C-NMR (DMSO, 75,5MHz):
6(ppm) = 3,4; 13,9; 19,8; 21,9; 24,3; 25,2; 26,6; 28,3; 28,4; 28,7; 28,9; 30,8; 31,1; 33,2; 71,1; 127,7; 129,7; 170,9; 172,4.
MS (IC, NH3): m/z = 478 (M+NH4)+.
Rf (S1O2, ethyl acetate/cyclohexane, 30/70 +1% CF3CO2H): 0,53 (stained with KMn04).
Example 13: Svntiiesis of Di-DX-2-hvdroxv-4-butvric acid diselenide
A dry three-necked round-bottom flask, under an atmosphere of argon, was fitted with a thermometer and a condenser and was charged with selenium (1,3984 g; 17,7 mmol) and sodium borohydride (0,4606 g; 12,2 mmol). The flask was cooled in an ice bath and absolute ethanol (30 ml) was added, on addition an exothermic reaction occurred with vigorous bubbling. After 15 min, the ice bath was removed and the reddish-brown mixture was degassed with argon via a needle. After 20 min, the degassing was stopped and the mixture was heated at reflux. After 2h at reflux, 2-hydroxybutyrolactone (1,66 ml; 21,3 mmol) was added and the mixture was maintained at reflux for a further 39h. The orange solution was allowed to cool to room temperature and then cooled in an ice bath, a yellow precipitate formed. Diethyl ether (20 ml) was added resulting in further precipitation. The yellow solid was filtered and washed with diethyl ether (2x50 ml). The solid was dissolved in water (50 ml), the pH of the solution was adjusted to pH = 10 using aqueous sodium hydroxide (4N). A small amount of black solid remained undissolved. The mixture was filtered, the
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aqueous phase was washed with diethyl ether (2x20 ml) and acidified (pH =10) using concentrated hydrochloric acid. The organic material was extracted with diethyl ether (6x30 ml), the fractions combined, dried (Na2S04), filtered and the solvent removed under reduced pressure to leave the diselenide [1,40 g, 65%, purity ca. 95%], as a yellow oil.
'H-NMR (D20,300MHz):
8(ppm) = 1,98-2,22 (m, 2H); 2,82-2,98 (m, 2H); 4,28 (dd, J = 4Hz and 8Hz, 1H; a-H).
Additional signals: 1,04 (EtzO); 1,15(t); 3,43 (Et20); 4,10 (q).
Rf (Si02, ethyl acetate + 1% CF3CO2H): 0,57 (stained with phosphomolybdic acid).
Example 14: Preparation of compositions according to the invention.
Capsules were prepared with the following composition:
L-2-hydroxy-4-methylselenobutyric acid 0.2 mg
Excipients * and envelope ** to make a 500 mg capsule
(* corn starch, lactose, magnesium stearate, sodium lauryl sulphate, ** gelatine, titanium dioxide, colouring agents).
Capsules were prepared with the following composition:
L-2-hydroxy-4-methylselenobutyric acid 0.05 mg
Excipients * and envelope ** to make a 500 mg capsule
(* corn starch, lactose, magnesium stearate, sodium lauryl sulphate, ** gelatine, titanium dioxide, colouring agents).
Capsules were prepared with the following composition:
L-2-hydroxy-4-methylselenobutyric acid 0.1 mg
Excipients * and envelope ** to make a 500 mg capsule
(* corn starch, lactose, magnesium stearate, flavour, ** gelatine, titanium dioxide, colouring agents).
Capsules were prepared with the following composition:
Dicyclohexyl ammonium L-2-hydroxy-4-methylselenobutyrate 0.15mg
Excipients * to make a 1 g capsule
(* corn starch,, talc, magnesium stearate).
Ik
WE CLAIM
1. Organoselenium compounds of the general formula (I):
in which
n = 0, lor2,
R, = OH, OCOR3, OP03H2> OPO(OR4XOR5), or ORe,
R2 = OH,R3,orNHR7>
R3 = alkoxyl (C1-C26), ceramide 1, ceramide 2, ceramide 3, ceramide 4, ceramide 5,
ceramide 6a and 6b, S-cvsteinvl, or S-glutathionvl, or
OR4 = alkoxyl (Ci-C26), ceramide 1, ceramide 2, ceramide 3, ceramide 4, ceramide 5, ceramide 6a or 6b.
15
OR* = pyruvate, lactate, citrate, fumarate, maleate, myristate, palmitate, stearate,
palmitoleate, oleate, linoleate, natural fatty acids, or 13-cis retinoate,
R7 = H, alkyl (C1-C26), natural amino-acids, or natural amines,
it being understood that when n = 1 and R2 = OH, then Ri cannot be OH,
in their different forms including position isomers, geometric isomers, stereo-isomers,
diastereoisomers and enantiomers, taken separately or mixed, and all linear or ramified,
acyclic or cyclic oligomers and polymers,
and their pharmaceutically acceptable acid or base salts.
2. Compounds according to claim 1, characterized in that n is 0.
3. Compounds according to claim 1 or 2, characterized in that Ri represents OH, OCOR3, OR*, R3 and R§ being as defined in claim 1.
4. Compounds according to claim 1 or 2, characterized in that Ri is OH.
5. Compounds according to any one of claims 1 to 4, characterized in that R2 is chosen from the group consisting in OH, NHR7, glyceroyl, monoacylglyceroyl, diacylglyceroyl, coenzyme Q, retinoyl, cholesteroyl, alpha-tocopheroyl, carnitinoyl, sphinganine, sphingosine, phyto-sphingosine, ceramide 1, ceramide 2, ceramide 3, ceramide 4, ceramide 5, ceramide 6a and 6b, ascorbate, S-cysteinyl and S-glutathionyl, R7 being as defined in claim 1.
6. Compounds according to any one of claims 1 to 5, characterized in that R2 represents OH.
7. Compounds according to claim 1 of formula (I'), corresponding to the general formula (I) in which:
n = 0,1 or 2,
R, = OCOR3, OPO3H2, OPO(OR4)(OR5), or ORe,
R2 = R3,orNHR7,
R3 = ceramide 1, ceramide 2, ceramide 3, ceramide 4, ceramide 5, ceramide 6a and 6b,
S-cysteinyl, or S-glutathionyl, or
11
8. Compounds according to claim 1 of formula (I"), corresponding to the general
formula (I) in which:
n = 0,1 or 2,
Ri=OH,
R2 = OH,orR3,
R3 = alkoxyl (Ci-C26),
it being understood that when n = 1 and Ri = OH, then R2 cannot be OH.
9. Any one of the compounds of general formula I, as defined in claiml, with the
following name:
- L-2-hydroxy-4-methylselenobutyric acid,
- D-2-hydroxy-4-methylselenobutyric acid,
- DL-2-hydroxy-4-methylselenobutyric acid,
- dicyclohexylammonium L-2-hydroxy-4-methylselenobutyrate.
2^
10. Compounds according to any one of claims 1 to 7, in the form of sodium or calcium or magnesium salts.
11. Process for the preparation of organoselenium compounds of the general formula (I) as defined in claim 1, characterized in that it comprises at least one of the following steps:
1) the reaction of (DJL)-2-Ri-butyrolactone or one of its enantiomers (D or L), where Ri is as defined in claim 1,
- either with an alkaline methylselenolate salt of formula (Ha)
CHs-SeM*^ (Ha)
in which M represents an atom of an alkaline metal, to obtain a compound of formula (la) in the form of an alkaline salt:
in which M and Ri are as defined above;
- or with an alkaline selenium reagent of formula (lib)
HSeTvT (lib)
in which M represents an atom of an alkaline metal, to obtain a compound of formula (IS) in the form of an alkaline salt:
in which M and Ri are as defined above;
- or with an alkaline selenium reagent of formula (He)
MSeX (He)
in which M is as defined above and X represents a CN radical, or SO3M, or aryl-S02M, to obtain a compound of formula (IHa) in the form of an alkaline salt:
in which M, Ri and X are as defined above;
- or with an alkaline selenium reagent of formula (lid)
MSeSeM (Ad)
in which M is as defined above, to obtain a compound of formula (nib) in the form of an alkaline salt:
30
makes the intermediate compound of formula (Ilia) or (nib) react with a reducing agent,
to obtain a compound of formula (HI)
and then treats the compound of formula (III) with a methylation agent of formula (TV):
CH3-Y (TV)
in which Y represents a halogen atom, or an OSO2CH3, OSCVp-tolyl, or OCQ2CH3 group to obtain the compound of formula da) mentioned above.
2) if desired one or even several reactions or series of reactions described below:
- acidification of the reaction medium to obtain the acid corresponding to formula (I);
- esterification of the acid of formula (I) or its alkaline salt of formula 0a) with an alcohol or an alkyl halide to obtain the compound of general formula (I) in which R2 = R3 is as defined above;
- amidification of the acid of formula (I) or its alkaline salt of formula (la) with an appropriate amine of formula R7NH2, in which R7 is as defined above, to obtain the compound of general formula (I) in which R2 = NHR7 is as defined above;
- esterification, when Rj= OH, of the hydroxyl function by an appropriate acid to obtain the compound of general formula (I) in which Ri is different from the OH group;
- oxidation leading to the selenoxide or selenone derivative to obtain the compound of general formula (I) in which n is equal to 1 or 2;
- salification by an acid or a base.
12. Process according to claim 11, characterized in mat the nucleophile selenium reaeentis:
3