Patent Grant
6852759
The present invention relates to a process for he preparation of trans-alkenoic acids of general formula CH3—(CH2)n—CH═CH—(CH2)mCO2H where n=4 to 9 m=8 to 16 16. More particularly, the present invention relates to a process for the preparation of trans-tetracos-15-enoic acid, which is a bioactive constituent possessing dose-related hepatoprotective activity.
Trans-alkenoic acids possess hepatoprotective activity comparable to/better than know formulations. Herbal preparations in use as antihepatotoxic/hepatoprotectives are non-standard both from biological and chemical aspects. The first report about natural occurrence of an acid of this type viz, trans-tetracos-15-enoic acid [Wang Huiying, Yu Xuetian, Yi Yuanfen & Ding Jingksi, Yuman Zhiwu Yanjiu, 1989, 11 (1), 60-4 (Ch)] as a constituent of Jojoba oil ex Simmondsia chinensis seeds (0.62-1.11%) was based on GLC analysis.
Both nervonic acid from the brain cerebrosides of cattle and man [Klenk, L. Physiol Chem. 1925, 145,244; 1926, 157, 283; 1927, 166, 268] and selacholeic acid from shark and ray-liver oils [Tsujimoto, J. Soc. Chem. Ind., Japan 1927, 30, 868] were formulated as cis-tetracos-15-enoic acids. The two identical natural products have been synthesized by malonate chain extension of cis-docos-13-enoic acid [J. B. Hale, W. H. Lycan and Roger Adams, J. Am. Chem. Soc., 1930, 52, 4536; Muller & Benzer, Ber., 1939, 72, 615]. In the process tetracos-15-enoic acid was prepared in six steps i.e., (a) Esterification of erucic yield (yield 93%). (b) Reduction of methylerucate with Na metal in n-butyl alcohol (yield 55%), (c) Conversion of erucyl alcohol to erucyl bromide by the action of PBr3 (yield 50%) (d) Condensation of erucyl bromide with malonic ester over a period of forty eight hours (yield 78%). (e) Hydrolysis and decarboxylation of erucyl malonic ester to tetracos-15-enoic acid (yield 50%) (f) Isomerization of cis product to trans form (yield) 90%), in an overall yield of 9.05%.
D. G. Bounds, R. P. Linstead and B. C. L. Weedon, [Journal of Chemical Society, 1954, 448] report synthesis of cis and trans-tetracos-15-enoic acid by anodic chain extension of oleic and claidic acids respectively. This publication discloses the electrolysis of oleic and elaidic acids in presence of excess of methyl hydrogen substrate to give an expected mixture of three products by both symmetrical and unsymmetrial coupling of the two compounds. By distillation and hydrolysis of the unsymmetrical products, mixture of cis and trans-tetracos-15-enoic acids was obtained in 30-35% overall yield.
All the reported synthetic procedures for trans-alkenoic acids till date are lengthy, non-specific i.e., yielding mixture of cis and trans isomers and end up with very poor yields.
The main object of the present invention is to provide a synthetic method for the production of trans-alkenoic acids, hepatoprotective constituents of natural origin.
Another object is to report an economic high yielding synthesis, where the end product i.e., trans-alkenoic acids are obtained up to 70% in overall yield.
General formula of t-alkenoic acid has been described and when n=7 and m=13 it represents t-tetracos 15-enoic acid.
Accordingly the present invention provides a process for preparation of a trans-alkenoic acid of the formula CH3—(CH2)n—CH═CH—(CH2)mCOOH wherein n is 4 to 9, m is 8 to 16, said process comprising (i) reducing a bis noralkenoic acid to the corresponding alkenol in the presence of metal hydride (ii) reacting the alkenol obtained above with a brominating agent at a temperature in the range of −10 to 25° C. for 1 to 4 hours and recovering the bromoalkene so formed, (iii) condensing the bromoalkene obtained above with dialkyl malonate, ethyl acetoacetate or ethyl cyanoacetate in he presence of an alkali metal alkoxide to give dicarbalkoyalkene followed by hydrolysis to give corresponding dicarboxylic acid, (iv) decarboxylating the dicarboxylic acid obtained above partially to trans-alkenoic acid.
In another embodiment of the invention the process relates to the preparation of trans-tetracos-15-enoic acid.
In yet another embodiment of the invention trans-tetracos-15-enoic acid shows dose-related hepatoprotective activity.
In yet another embodiment of the present invention the bis noralkenoic acid used is selected from the group consisting of trans-henicos-12-enoic acid, trans-docos-14-enoic acid, trans-icos-12-enoic acid, trans-tetradec-8-enoic acid and trans docos-13-enoic acid.
In a further embodiment of the invention, an organic acid is used in step (i), and said organic acid is selected from Cl3CCOOH, F3CCOOH and the like.
In another embodiment of the invention, the metal hydride used in step (i) is selected from LiAlH4, NaBH4 and the like.
In another embodiment of the invention, the brominating agent used is selected from PBr3 and Ph3P-Br2 complex.
In yet another embodiment the decarboxylation is carried out of heating the dicarboxylic acid in a dry stat or by heating in wet dimethyl sulphoxide containing NaCl, Na3PO4 or any other simple salt.
In yet another embodiment of the invention, the ether medium employed in step (i) is selected from diethyl either, tetrahydrofuran, dioxane and methyl cellosolve.
In another embodiment of the invention, the alkyl malonate used in step (iii) is selected from dimethylmalonate and diethylmalonate.
In another embodiment of the invention, the acetoacetate used in step (iii) is selected from ethyl acetoacetate and methyl acetoacetate.
In another embodiment of the invention the cyanoacetate used in step (iii) is selected from ethyl cyanoacetate and methyl cyanoacetate.
In another embodiment of the invention, the alkali metal alkoxide employed in (iii) is selected from Na/K methoxide and ethoxide.
In another embodiment of the invention, the hydrolysis of diester in step (iv) is carried out using aqueous or methanolic NaOH or KOH or (CH3)3COK (1.5M-2.5M).
In another embodiment of the invention, the partial decarboxylation in step (v) is carried out by dry heating of the diacid at 170-200° C. in an oil bath or by heating in wet dimethyl sulphoxide containing NaCl, Na3PO4 or any other simply salt at 120-130° C.
In another embodiment of the invention, the solvent employed by crystallization of the product is selected from ethanol, ethanol, isopropanol and ethyl acetate.
The present invention also relates to the use of trans-alkenoic acids of general formula CH3—(CH2)n—CH═(CH2)mCO2H where n=4 to 9 m=8 to 16 for dose related hepatoprotection.
In another embodiment of the invention, the dose of trans-alkenoic acids of general formula CH3—(CH2)n—CH═CH—(CH2)mCO2H where n=4 to 9 m=8 to 16 is in the range of 12.5 to 100 mg per kg body weight of the patient.
The present invention also relates to a method for hepatoprotection comprising administering to a patient a trans-alkenoic acid of general formula CH3—(CH2)n—CH═CH—(CH2)mCO2H where n=4 to 9 m=8 to 16 in an amount of 12.5 to 100 mg per kg body weight of the patient.
The present invention also relates to a process for preparing a pharmaceutical composition for hepatoprotection comprising mixing a trans-alkenoic acid of general formula CH3—(CH2)n—CH═(CH2)m CO2H where n=4 to 9 m=8 to 16 in a pharmaceutically acceptable carrier.
In one embodiment of the invention, the trans-alkenoic acid of general formula CH2—(CH2)n—CH═CH—(CH2)mCO2H where n=4 to 9 m=8 to 16 is mixed in the pharmaceutically acceptable carrier to provide a dose of 12.5 to 100 mg per kg body weight of the patient.
The present invention provides an improved high yielding process for the synthesis of trans-alkenoic acids particularly trans-tetracos-15-enoic acid, bioactive constituents possessing dose related hepatoprotective activity, which is based on malonate, acetoacetate or cyanoacetate chain extension of bis noralkenoic acids, comprising:
Complex metal hydride used in step (i) is preferably chosen from LiAlH4, NaBH4 or the like. Organic acid used in step (i) is preferably chosen from Cl3CCOOH, F3CCOOH and the like. Ether medium employed in step (i) is preferably chosen from diethyl ether, tetrahydrofuran, dioxane or methyl cellosolve. Conversion of enol to bromocompound in step (ii) is preferably carried out with PBr3 or PH3P—Br2 complex.
Alkyl malonate used in (iii) is preferably chosen from dimethylmalonate and diethylmalonate, acetoacetate is preferably chosen from ethyl acetoacetate or methyl acetoacetate and cyanoacetate is preferably chosen form ethyl cyanoacetate or methyl cyanoacetate. Alkali metal alkoxide employed in (iii) is preferably selected from Na/K methoxide and ethoxide.
Hydrolysis of diester in step (iv) is preferable carried out using aqueous or methanolic NaOH or KOH or (CH3)3COK (1.5M-2.5M). Partial decarboxylation in step (v) is carried out by dry heating of the diacid at 170-200° C. in an oil bath or by heating in wet dimethyl sulphoxide containing NaCl, Na3PO4 or some other simple salt at 120-130° C. The solvent employed for crystallization of the final product is preferably chosen from methanol, ethanol, isopropanol or ethyl acetate.
The invention is described with reference to the examples given below which should not, however, be construed to limit the scope of present invention.
a. Reduction of docos-13-enoic acid to docos-13-enol
Lithium Aluminium Hydride (LAH, 6.9 g) was suspended in absolute diethyl ether in a three necked R.B. flask provided with a dropping funnel, a reflux condenser and a magnetic stirring paddle. To stirred suspension (placed in an ice bath), docos-13-enoic acid (50 g) dissolved in absolute diethyl ether (300 mL) was added dropwise, the operation being completed in one hour. The reaction mixture was further stirred for another half an hour at room temperature (22±3° C.). The reaction was quenched by adding ethyl acetate (100 mL) and 10% aq. H2SO4 (100 mL). Ether layer was separated, washed with water (5×50 mL) and dried over anhydrous Na2SO4. Ether was removed by distillation and reaction product recovered by vacuum distillation (200-203° C., 1 torr). Product (43 g, 90% yield) was characterized by 1H-NMR.
b. Conversion of docos-13-enol to bromodocos-13-ene
Docos-13-enol (50 g) was dissolved in dry toluene (160 g) and the solution was placed in a three necked R.B. flask fitted with a reflux condenser and a dropping funnel. The solution was cooled to −152 C. (cryostat) and PBr3 (20 g) was added to it dropwise. After the addition, the reaction mixture was heated on a steam bath for four hours (Progress monitored on TLC). Toluene was removed from the reaction mixture by distillation and the residue was dissolved in n-hexane (200 mL), the solution was washed with aq. KOH (20%, 50 mL), aq. NaCl (10%, 50 mL), finally with water and then dried over anhydrous MgSO4. Hexane was removed by distillation under atmospheric pressure and the product under diminished pressure (203-205° C. 1 torr), yield (55 g, 93%).
c. Conversion of bromodocos-13-ene to 1,1-dicarbalkoxytricos-14-ene
Absolute ethanol (15 mL) was taken in a three necked R.B. flask fitted with a dropping funnel and a reflux condenser. Sodium metal (600 mg) was added in ethanol, in small pieces, with constant stirring followed by dropwise addition of diethyl malonate (6 g) over fifteen minutes. This was followed by dropwise addition of bromoalkene (10 g). The reaction mixture was refluxed on a steam bath for four hours. Ethanol was removed from the reaction mixture on a rotavapour and to the residue HCl (1%, 100 mL) was added. The mixture was extracted with diethyl ether (5×100 mL). The ether extract was dried over anhydrous Na2SO4. Ether extract was concentrated to 50 ml, and then filtered through a small column (packed with neutral Al2O3) to get 1,1-dicarbalkoxytricos-14-ene, yield (11 g, 88%).
d. Hydrolysis of the 1,1-dicarbalkoxytricos-14-ene to 1,1-dicarboxytricos-14-ene
Enediester (10 g) was suspended in 60% q. ethanolic KOH (6 g, 40 mL) and refluxed on a water bath for one hour. Ethanol was removed from the reaction mixture by distillation and the residual aqueous solution was acidified (pH=3) with 10% aqueous H2SO4 and extracted with CHCl3 (5×50 mL). CHCl3 extract was dried over anhydrous CaCl2 and then distilled. The residue on cooling gave 1,1-dicarboxytricos-14-ene, a solid mass, yield (8 g, 91%).
e. Thermal decomposition of the 1,1-dicarboxytricos-14-ene to t-tetracos-15-enoic acid
Diacid (5.5 g) was taken in a R.B. flask (50 mL) and heated at 178° in a Woods metal bath for one hour. The product was recovered by distillation under reduced pressure and the distillate crystallized from methanol by chilling at −20° C., yield (3.92 g, m.p. 61° C., 80%).
a. Reduction of docos-13-enoic acid to docos-13-enol
To a suspension of BaBH4 (0.76 g, 0.01 mole) in dry THF (20 mL), docos-13-enoic acid (3.38 g, 0.01 mole) in dry THP (20 mL) was added. CF3COOH (1.14 g, 0.01 mole) was added in 15 minutes at 0° C. and stirred for four hours at r.t. (22±3° C.). The reaction mixture was quenched with 3N-HCl (5 mL) and extracted with diethyl ether (3×10 mL). Ether extract was washed with 1N-aq. NaOH (2×5 mL), water (2×5 mL), brine (2×5 mL) and dried over anhydrous MgSO4 Ether was removed by distillation and docos-13-enol recovered by vacuum distillation (200-203° C., 1 torr), yield (2.6 g, 81%).
b. Conversion of docos-13-enol to bromodocos-13-ene
To an ice-cold solution of enol (1.86 g), 5.76 m mole) and pyridine (0.72 g, 9.21 mmole) in CH3CN (12mL), solid pH3P-Br2 (3.16 g, 7.48 mmole) was added over 10 minutes. After stirring at r.t. (22±3° C.) for one hour (Progress monitored on TLC), the reaction mixture was washed with water (3×5 mL), dried over anhydrous Na2SO4 and distilled under diminished pressure (203-205° C., 1 torr), to get bromodocos-13-ene (2.18 g, 95%).
c. Conversion of bromodocos-13-ene to 1,1-dicarbalkoxytricos-14-ene
Absolute ethyl alcohol (7.5 mL) was taken in a three necked R.B. flask fitted with a dropping funnel and a reflux condenser. Cleaned Sodium metal (330 mg) was added in ethanol, in small pieces. When whole of the sodium metal was reacted, diethyl malonate (3 g) was added dropwise, with constant stirring over a period of 15 minutes. Following this bromodocos-13-ene (5 g) was added similarly. Reaction mixture was further stirred for four hours. The ethanol was completely removed from the reaction mixture and to the residue HCl (1%, 50 mL) was added. The mixture was extracted with diethyl ether (5×50 mL). The ether layer was dried over anhydrous MgSO4. The ether extract was concentrated to 25 mL and then filtered through a small column (packed with neutral Al2O3) to get 1,1-dicarbalkoxytricos-14-ene, yield (5.4 g, 86%).
d. Hydrolysis of the 1,1-dicarbalkoxytricos-14-ene to 1,1-dicarboxytricos-14-ene
The enediester (5 g) was suspended in 60% aq ethanolic potassium hydroxide (3 g, 20 mL) and refluxed on a water bath for 50 minutes. The ethyl alcohol was removed from the reaction mixture by distillation ad residual aqueous solution was acidified (pH=3-4) with 10% aqueous sulphuric acid and extracted with CHCl3 (5×25 mL). The chloroform extract was dried over anhydrous CaCl2 and then distilled. The residue on cooling gave 1,1-dicarboxytricos-14-ene, yield (3.9 g, 89%).
e. Thermal decomposition of the 1,1-dicarboxytricos-14-ene to t-tetracos-15-enoic acid
The diacid (2.75 g) was taken in a R.B. flask (25 mL) and heated at 178° in a Woods metal bath for one hour. The product was recovered by distillation under reduced pressure and the distillate crystallized from methanol by chilling at −25° C. yield (19 g, m.p. 61° C., 79%).
a. Reduction of tetradec-8-enoic acid to tetradec-8-enol
To a suspension of NaBH4 (0.3 g, 0.005 mole) in dry methyl cellosolve (15 mL), tetradec-8-enoic acid (1.13 g, 0.005 mole) in dry methyl cellosolve (15 mL) was added. CF3COOH (0.57 g, 0.005 mole) was added in 10 minutes at 0° C. and stirred for 3 hours at r.t. (22±3° C.). 2N-HCl (4.0 mL) was added to quench the reaction.
The quenched reaction mixture was extracted with diethyl ether (2×10 mL). Ether extract was washed with 1N-aq. NaOH (1×5 mL), water (1×5 mL), brine (1×5 mL) and dried over anhydrous MgSO4. Ether was removed by distillation and product recovered by distillation under diminished pressure (180-185° C., 1 torr), to yield tetradec-8-enol (0.958, 93%).
b. Conversion of tetradec-8-enol to bromotetradec-8-ene
A mixture of the tetradec-8-enol (0.61 g), pyridine (0.36 g, 4.60 mmole) in CH3CN (8 mL) was cooled in an ice bath and solid PH3P-Br2 complex (1.60 g, 3.75 mmole) was added over 20 minutes. Reaction mixture was stirred at r.t. (22±32 C.) for two hours and washed with water (2×5 mL), dried over anhydrous Na2SO4 and subjected to vacuum distillation (178-180°, 1 torr), yield (0.72 g, 91%).
c. Condensation of bromotetradec-8-ene with dimethyl sodium malonate
Absolute methyl alcohol (10 mL), was placed in three necked R.B. flask fitted with dropping funnel and a reflux condenser. Sodium metal (450 mg) was added in methyl alcohol, in small pieces, accompanied by constant stirring. To clear solution of sodium methoxide, dimethyl malonate (4.2 g) was added dropwise over a period of ten minutes. After this bromotetradec-8-ene (6.5 g) was added in a similar manner. Reaction mixture was refluxed over a steam bath for two hours.
Methyl alcohol was distilled on a rotavapour and residue was treated with 70 mL of 1% aq. HCl. The mixture was extracted with diethyl ether (3×100 ml). Ether extract was dried over anhydrous Na2SO4 and concentrated to 50 mL and then filtered through a SiO2 gel (50 g) column to get 1,1-dicarbalkoxypentadec-9-ene, yield (7.01 g, 91%).
d. Hydrolysis of the 1,1-dicarbalkoxypentadec-9-ene to 1,1-dicarboxypentadec-9-ene
The enediester (10 g) was suspended in 60% aq. ethanolic KOH (6 g, 40 mL) and refluxed on a water bath for one hour. Ethanol was removed from the reaction mixture by distillation and the residual aqueous solution was acidified (pH=3) with 10% aqueous H2SO4 and extracted with CHCl3 (5×50 mL). The CHCl3 extract was dried over anhydrous CaCl2 and then distilled. The residue on cooling gave 1,1-dicarboxypentadec-9-ene, yield (8 g, 91%)
e. Thermal decomposition of 1,1-dicarboxypentadec-9 -ene to t-hexadecen-10-oic acid
The diacid (5.5 g) was taken in a R.B. flask (50 mL) and heated at 178° in a Woods metal bath for one hour. The product was recovered by distillation under reduced pressure and the distillate crystallized from methanol by chilling at −20° C., yield (3.92 g, m.p. 61° C., 80%).
Pharmacological Activity
t-Tetracos-15-enoic acid (TCA) has been evaluated for antihepatotoxicity/hepato-protective activity both on prophylactic and curative aspects in vivo using CCl4, Paracetamol, Galactosamine and alcohol as hepatotoxins employing silymarin as positive standard. In almost all the aspects TCA has shown better protection and reversal of biochemical parameters better then those obtained with silymarin. The results of the pharmacological activity are given in Tables 1 to 9 below.
aValues represent the mean ± SE of six animals in each group.
bDifference in relation to vehicle treated control group.
cDifference in relation to CCl4 control group.
aValues represent the mean ± SE and within parentheses hepatoprotective activity percent mean ± SE of six animals in each group.
bis μmole of p-nitrophenol formed/min/L,
NS > 0.05 (Dunnett's t 0 test).
aValues represent the mean ± SE and within parentheses hepatoprotective activity percent mean ± SE of six animals in each group.
bis μmole of p-nitrophenol formed/min/L,
NS>0.05 (Durrett's t 0 test).
aValues represent the mean ± SE and within parentheses hepatoprotective activity percent mean ± SE of six animals in each group.
bis μmole of p-nitrophenel formed/min/L,
NS>0.05 (Dunnett's t 0 test).
aValues represent the mean ± SE and within parentheses hepatoprotective activity percent mean ± SE of six animals in each group.
bis μmole of p-nitophenol formed/min/L,
NS>0.05 (Dunnett's t 0 test).
aValues represent the mean ± SE and within parentheses hepatoprotective activity percent mean ± SE of six animals in each group.
bis μmole of p-nitrophenol formed/min/L,
NS>0.05 (Dunnett's t 0 test).
aValues represent the mean ± SE and within parentheses hepatoprotective activity percent mean ± SE of six animals in each group.
bis μmole of p-niotrophenol formed/min/L,
NS>0.05 (Dunnett's t 0 test).
aValues represent the mean ± SE of six animals in each group.
1Each unit is μmole pyruvate/min/L.
2is μmole p-nitrophenol formed/min,
bis μmole p-nitrophenol formed/min/L,
aValues represent the mean ± SE of six animals in each group.
2is μmole K3 Fe(CN)6 , utilised/min,
4is μmole of phosphate formed/min,
5Represent only change and not protection.
| Number | Date | Country | |
|---|---|---|---|
| 20040024232 A1 | Feb 2004 | US |
