Title of Invention	''A PROCESS FOR THE PREPARATION OF FATTY ACID ESTERS OF URSOLIC ACID AS ANTIMICROBIAL AGENTS''
Abstract	The long chain fatty acid esters Viz. ursolic acid laurate, ursolic acidmyristate, ursolic acid palmitate and ursolic acid stearate have now been synthesized and screened for the first time. A very effective and mild esterification conditions have been used for their synthesis. Significantly the test compounds were found to exhibit antimicrobial activities several folds more against the gram -ve and gram +ve bacteria than the controls tetracycline and benzyl penicillin sodium.

Title of Invention

''A PROCESS FOR THE PREPARATION OF FATTY ACID ESTERS OF URSOLIC ACID AS ANTIMICROBIAL AGENTS''

Abstract

The long chain fatty acid esters Viz. ursolic acid laurate, ursolic acidmyristate, ursolic acid palmitate and ursolic acid stearate have now been synthesized and screened for the first time. A very effective and mild esterification conditions have been used for their synthesis. Significantly the test compounds were found to exhibit antimicrobial activities several folds more against the gram -ve and gram +ve bacteria than the controls tetracycline and benzyl penicillin sodium.

Full Text	The present invention relates to a process for the preparation of fatty acid esters of ursolic acid as antimicrobial agents. The long chain fatty acid esters of ursolic acid obtained in the present process were found to exhibit potent antimicrobial activity in vitro against the gram - ve bacteria Pseudomonas syringae ( ATCC # 13457) and the gram + ve bacteria Bacillus sphaericus ( ATCC # 14577 ) and Bacillus subtilis ( ATCC # 6051 ) Reference may be made to Collins et.al. ( M.A. Collins and H. P. Charles, Food Microbiol., 1987, 4(4), 311) wherein ursolic acid isolated as one of the anti- oxidant constituents of Rosemary, was tested against the growth of some food associated bacteria. The drawbacks are : No significant activity is reported to ursolic acid. Reference may be made to Zaletova et. al. (N.I. Zaletova, A.N. Shchavlinskii, 0. N. Tolkachev, S. A. Vichkanova, T. V. Fateeva, N. M. Krutikova, I. V. Yartseva and N. A. Klyuev, Khim - Farm. Zh., 1986, 20(5), 568) wherein some 3 - keto and 11 - keto derivatives of ursolic acid were prepared and screened for antimicrobial activity against Staphylococous aureus. These compounds were found to be active only at 250 ug/ml concentrations. The drawbacks are : the concentrations of the test compounds are very high ( 250 ug/ml and above). The enhanced antimicrobial activity of ursolic acid can be achieved by preparing some of its lipophilic derivatives. The low acute toxicity of ursolic acid and structure activity studies, which suggest that the desired substituents on the lipophilic 5 - ring backbone can increase the selectivity and potency of a desired action, indicate that ursolic acid provide a rich source for natural lead compounds for drug development. The limiting parameter with ursolic acid is its low solubility. Ursolic acid has a hydroxyl at C3 position, a double bond between C12 and C13 and a carboxylic acid group at C17. The C3 hydroxyl group can be exploited to make new members with enhanced lipophilic properties. Lipophilicity is an important parameter in drug development and it increases with the increase of the side carbon chain. Molecules with carbon chains above C10 are fairly lipophilic and good candidates for antimicrobial screening. The main object of the present invention is to provide a process for the preparation of long chain fatty acid esters of ursolic acid as potent antimicrobial agents which obviates the drawbacks as detailed above. Another object of the present invention is to utilise the major metabolite obtained in one of our programmes on Diospyros melanoxylon (kendu) leaves i.e. ursolic acid, which is incidentally the starting material for the test compounds. Still another object of the present invention is to make 'New Molecules' with enhanced biological activities from abundantly available inactive natural products. In the drawing accompanying this specification Figure 1 represents ursolic acid Figure 2 represents esters of ursolic acid Figure 2a represents ursolic acid laurate, R = CH3(CH2)10 Figure 2b represents ursolic acid myristate, R = CH3(CH2)12 Figure 2c represents ursolic acid palmitate, R = CH3(CH2)14 Figure 2d represents ursolic acid stearate, R = CH3(CH2)16 Figure 3 represents fatty acid esters Figure 3a represents lauric acid, n = 10 Figure 3b represents myristic acid, n = 12 Figure 3c represents palmitic acid, n = 14 Figure 3d represents stearic acid, n = 16 The process for the preparation of long chain fatty acid esters of ursolic acid as potent antimicrobial agents comprises derivatisation of the C3 - hydroxyl function of ursolic acid (as shown in the drawing as fig.1) with long chain fatty acids such as lauric acid , myristic acid, palmitic acid and stearic acid (as shown in the drawings as figs. 3a - 3d) in presence of mild base and a powerful dehydrating agent in a low boiling chlorinated solvent at room temperature under stirring and isolating the resulting products( as shown in the drawing as figs. 2a - 2d) by chromatographic separation and screening for antimicrobial activity in vitro against the gram -ve bacteria Pseudomonas syringae and gram +ve bacteria, Bacillus sphaericus and Bacillus subtilis. The long chain fatty acid esters of ursolic acid as prepared by the process of present invention are synthesised and screened for antimicrobial activity for the first time. Accordingly, the present present invention relates to a process for the preparation of fatty acid esters of ursolic acid of formula 2 wherein R is alkyl group which comprises reacting ursolic acid of formula 1 with fatty acids of formula 3 as shown in the drawing wherein n is 10 to 16 carbons in presence of a mild organic base and a dehydrating agent in chlorinated solvent at room temperature under stirring for at least a period of 72 hrs., recovering the esters of ursolic acid and purifying the esters by conventional methods In another embodiment of the present invention, the mild base used may be such as pyridine, 4 - pyrrolidinopyridine, 4 - dimethylaminopyridine, 4 - diethyl- aminopyridine. In another embodiment of the present invention, the dehydrating agent used may be such as dicyclohexyl carbodiimide, N,N' - carbonyldiimidazol, 1-cyclo-3- 2-(2-morpholinoethyl)carbodiimde. In another embodiment of the present invention the synthesised long chain fatty acid esters are found highly lipophilic and soluble in most of the organic solvents such as dichloromethane, chloroform, ethyl acetate, acetone. In yet another embodiment of the present invention the fatty acid esters of ursolic acid were found to exhibit potent antimicrobial activity in vitro against gram - ve bacteria Pseudomonas syringae and gram + ve bacteria Bacillus sphaericus and Bascillus subtilis. In still another embodiment of the present invention the fatty acid esters of ursolic acid were found to be highly active than tetracycline and benzyl penicillin sodium against the gram - ve bacteria Pseudomonas syringae. In still another embodiment of the preset invention the fatty acid esters of ursolic acid were found to be highly active than benzyl penicillin sodium against the gram + ve bacteria Bacillus sphaericus and Bacillus subtilis. The present invention describes the drivatisation of C3 hydroxyl group of ursolic acid with the long chain fatty acids (C10 - C-16) under mild esterification conditions comprising a base, a dehydrating agent and a low boiling chlorinated solvent. The base used is e.g. pyridine, 4 - pyrrolidinopyridine, 4 -dimethylaminopyridine, 4 - diethylaminopyridine. The dehydrating agent used is e.g. carbodiimides such as dicyclohexylcarbodiimide, N,N' - cabonyldiimidazol, 1- cycle - 3 - (2 - morpholinoethyl)carbodiimide. The chlorinated solvent used is e.g. chloroform, dichloromethane, carbon tetrachloride. For several years ursolic acid has been thought of biologically inactive. Even though some interesting biologically activities have been attributed recently to ursolic acid, it could not come to limelight due to several limiting parameters like solubility and polarity. In the present invention we have demonstrated that some of the lipophilic derivatives of ursolic acid are more potent and exhibiting high antimicrobial activities than the parent acid. The most significant part of the present invention is that lipophilic ester chains have been synthesised in totally mild experimental conditions. Our method has got an advantage over the conventional methods for the preparation of esters in eliminating the intermediate acid chloride formation, which is very cumbersome. The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention. EXAMPLE -1 To ursolic acid (1g. 1eq.) in dichloromethane(15 ml.) was added lauric acid (0.438g,1eq.), dimethylaminopyridine (0.268g,1eq.) and dicyclohexylcarbodiimide (0.452g, 1eq.) and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC. As the reaction found incomplete, some more equivalents of lauric acid (0.438g, 1eq), dimethylaminopyridine (1.072g, 4eq) and dicyclohexylcarbodiimide (1.808g, 4eq) were added and stirring was continued. After the reaction is over, the reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The resulting residue (4.48g.) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity (n-hexane, n-hexane - ethyl acetate : 98-2) and collected 250 ml fractions. Fractions (n-hexane - ethyl acetate : 98-2) showing single and identical spots on TLC were combined and evaporated to give ursolic acid laurate as a colourless oil (30%). EXAMPLE-2 To ursolic acid(1g. 1eq.) in chloroform(15 ml.) was added lauric acid (0.438g,1eq.), dimethylaminopyridine (0.268g,1eq.) and dicyclohexylcarbodiimide (0.452g, 1eq.) and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC. As the reaction found incomplete, some more equivalents of lauric acid (0.438g, 1eq.), dimethylaminopyridine (1.072g, 4eq.) and dicyclohexylcarbodiimide (1.808g, 4eq.) were added and stirring was continued. After the reaction is over, the reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The resulting residue (4.48g.) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity (n-hexane, n-hexane - ethyl acetate : 98-2) and collected 250 ml fractions. Fractions (n-hexane - ethyl acetate : 98-2) showing single and identical spots on TLC were combined and evaporated to give ursolic acid laurate as a colourless oil (20%). The reaction is not only low yielding but also gave some undesirable side products. EXAMPLE - 3 To ursolic acid(1g. 1eq.) in dichloromethane(15 ml.) was added lauric acid (0.438g,1eq.), pyridine (0.172g,1eq.) and dicyclohexylcarbodiimide (0.452g, 1eq.) and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC. As the reaction found incomplete, some more equivalents of lauric acid (0.438g, 1eq.), pyridine (0.688g, 4eq.) and dicyclohexylcarbodiimide (1.808g, 4eq.) were added and stirring was continued. Even after 120 hrs. most of the starting material was found unconverted. The reaction mixture was filtered and the filtrate was evaporated under reduced pressure.The resulting residue (4.0 g.) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity (n-hexane, n-hexane - ethyl acetate : 98-2) and collected 250 ml fractions. Fractions (n-hexane - ethyl acetate : 98-2) showing single and identical spots on TLC were combined and evaporated to give ursolic acid laurate as a colourless oil (14%). EXAMPLE-4 To ursolic acid (1g. 1eq.) in dichloromethane(15 ml.) was added myristic acid (0.499g,1eq.), dimethylaminopyridine (0.268g,1eq.) and dicyclohexylcarbodiimide (0.452g, 1eq.) and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC. As the reaction found incomplete, some more equivalents of myristic acid (0.499g,1eq.), dimethylaminopyridine (1.072g, 4eq) and dicyclohexylcarbodiimide (1.808g, 4eq) were added and stirring was continued. After the reaction is over, the reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The resulting residue (4.48g.) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity (n-hexane, n-hexane - ethyl acetate : 98-2) and collected 250 ml fractions. Fractions (n-hexane - ethyl acetate : 98-2) showing single and identical spots on TLC were combined and evaporated to give ursolic acid myristate as a colourless oil (30%). EXAMPLE - 5 To ursolic acid (1g. 1eq.) in dichloromethane(15 ml.) was added palmitic acid (0.561g,1eq.), dimethylaminopyridine (0.268g,1eq.) and dicyclohexylcarbodiimide (0.452g, 1eq.) and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC. As the reaction found incomplete, some more equivalents of palmitic acid (0.56g, 1eq), dimethylaminopyridine (1.072g, 4eq) and dicyclohexylcarbodiimide (1.808g, 4eq) were added and stirring was continued. After the reaction is over, the reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The resulting residue (3.44g.) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity (n-hexane, n-hexane - ethyl acetate : 98-2) and collected 250 ml fractions. Fractions (n-hexane - ethyl acetate : 98-2) showing single and identical spots on TLC were combined and evaporated to give ursolic acid myristate as a colourless oil (30%). EXAMPLE - 6 To ursolic acid (1g. 1eq.) in dichloromethane(15 ml.) was added stearic acid (0.62g,1eq.), dimethylaminopyridine (0.268g,1eq.) and dicyclohexylcarbodiimide (0.452g, 1eq.) and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC. As the reaction found incomplete, some more equivalents of stearic acid (0.62g, 1eq), dimethylaminopyridine (1.072g, 4eq) and dicyclohexylcarbodiimide (1.808g, 4eq) were added and stirring was continued. After the reaction is over, the reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The resulting residue (5.538g) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity (n-hexane, n-hexane - ethyl acetate : 98-2) and collected 250 ml fractions. Fractions (n-hexane - ethyl acetate : 98-2) showing single and identical spots on TLC were combined and evaporated to give ursolic acid stearate as a colourless oil (30%). EXAMPLE - 7 The test compounds (2a - 2d) and the two controls viz; tetracycline and benzyl penicillin sodium were screened for antimicrobial activity against the gram -ve bacteria P. syringae as per the Broth Macro Dilution method. The compounds shown in the drawing as fig. 2a - 2d are found highly potent with several fold more active than tetracycline and benzyl pencillin sodium.The Minimum Ihibitory Concentration (MIC) values of the test compounds and the controls are given below in table -1. Table-1 (Table Removed) Example - 8 The test compounds (2b & 2d) and the two controls viz. tetracycline and benzyl pencillin sodium were screened for antimicrobial activity against the gram +ve bacteria, B. sphaericus as per Broth Macro Dilution method. The compounds shown in the drawing as fig. 2b & fig. 2d are found to be highly active and exhibiting about 1.4 times more activity than benzyl pencillinsodium. The results are given below in table - 2. Table-2 (Table Removed) EXAMPLE - 9 The test compounds (2a & 2d) and the two controls viz. tetracycline and benzyl pencillin sodium were screened for antimicrobial activity against the gram +ve bacteria, B. subtilis as per the Broth Macro Dilution method. The compounds shown in the drawing as fig. 2a & fig. 2d are found to be highly active and exhibiting about 1.3 times more activity than benzyl penicillin sodium. The results are given below in table - 3. Table – 3 (Table Removed) In conclusion, for the first time the long chain fatty acid esters viz. ursolic acid laurate, ursolic acid myristate, ursolic acid palmitate and ursolic acid stearate have been synthesised and screened for antimicrobial activity. The novelty of the present invention is the long chain fatty acid esters were synthesised under extremely mild esterification conditions and most significantly these compounds were found to exhibit very high antimicrobial activity. The main advantages of the present invention are : 1. The test compounds are synthesised under very mild experimental conditions by eliminating the intermediate acid chloride formation. 2. The test compounds with long fatty chains are highly lipophilic and soluble in most of he organic solvents. 3. The test compounds are exhibiting high antimicrobial activities and found several folds higher activities than the parent acid. 4. Utilisation of trivial and the abundantly available natural triterpenic acid viz. ursolic acid in making some of its lipophilic derivatives with enhanced biological activity. We Claim: 1. A process for the preparation of fatty acid esters of ursolic acid of formula 2 wherein R is alkyl group which comprises reacting ursolic acid of formula 1 with fatty acids of formula 3 as shown in the drawing wherein n is 10 to 16 carbons in presence of a mild organic base and a dehydrating agent in chlorinated solvent at room temperature under stirring for at least a period of 72 hrs., recovering the esters of ursolic acid and purifying the esters by conventional methods. 2. A process as claimed in 1 wherein a base used is selected from pyridine, 4 pyrrolidinopyridine, 4 - dimethylaminopyridine, 4 diethylaminopyridine. 3. A process as claimed in 1 wherein a dehydrating agent used is selected from dicyclohexylcarbodiimide, N,N' - carboxyldiimidazol, 1 - cycio - 3 - (-2-morpholinoethyl) carbodiimide. 4. A process as claimed in 1 wherein a low boiling chlorinated solvent used is selected from chloroform, dichloromethane, carbontetrachloride. 5. A process as claimed in 1 - 4 wherein ursolic acid fatty esters shown in the drawing as figs. 2a - 2d consists of screening of the said compounds against the gram -ve and gram +ve bacteria by employing the Broth Macro Dilution method. 6. A process as claimed in claims 1-5 wherein the fatty acid esters of ursolic acid are exhibiting very high antimicrobial activity than the controls tetracycline and benzyl penicillin sodium against the gram - ve bacteria, P.syringae. 7. A process as claimed in claims 1 • 6 wherein the fatty acid esters of ursolic acid as shown in the drawing as fig. 2b & 2d and fig. 2a & 2d are exhibiting enhanced antimicrobial activity than the control benzyl pencillin sodium against the gram + ve bacteria, B.sphearicus and B.subtilis respectively. 8. A process for preparation of esters of ursolic acid as potent antimicrobial agents substantially as herein described with reference to the examples and drawings accompanying this specification.

Full Text

The present invention relates to a process for the preparation of fatty acid esters of ursolic acid as antimicrobial agents.
The long chain fatty acid esters of ursolic acid obtained in the present process were found to exhibit potent antimicrobial activity in vitro against the gram - ve bacteria Pseudomonas syringae ( ATCC # 13457) and the gram + ve bacteria Bacillus sphaericus ( ATCC # 14577 ) and Bacillus subtilis ( ATCC # 6051 )
Reference may be made to Collins et.al. ( M.A. Collins and H. P. Charles, Food Microbiol., 1987, 4(4), 311) wherein ursolic acid isolated as one of the anti- oxidant constituents of Rosemary, was tested against the growth of some food associated bacteria. The drawbacks are : No significant activity is reported to ursolic acid.
Reference may be made to Zaletova et. al. (N.I. Zaletova, A.N. Shchavlinskii, 0. N. Tolkachev, S. A. Vichkanova, T. V. Fateeva, N. M. Krutikova, I. V. Yartseva and N. A. Klyuev, Khim - Farm. Zh., 1986, 20(5), 568) wherein some 3 - keto and 11 - keto derivatives of ursolic acid were prepared and screened for antimicrobial activity against Staphylococous aureus. These compounds were found to be active only at 250 ug/ml concentrations. The drawbacks are : the concentrations of the test compounds are very high ( 250 ug/ml and above).
The enhanced antimicrobial activity of ursolic acid can be achieved by preparing some of its lipophilic derivatives. The low acute toxicity of ursolic acid and
structure activity studies, which suggest that the desired substituents on the
lipophilic 5 - ring backbone can increase the selectivity and potency of a desired
action, indicate that ursolic acid provide a rich source for natural lead compounds
for drug development. The limiting parameter with ursolic acid is its low solubility.
Ursolic acid has a hydroxyl at C3 position, a double bond between C12 and C13
and a carboxylic acid group at C17. The C3 hydroxyl group can be exploited to
make new members with enhanced lipophilic properties. Lipophilicity is an
important parameter in drug development and it increases with the increase of
the side carbon chain. Molecules with carbon chains above C10 are fairly
lipophilic and good candidates for antimicrobial screening.
The main object of the present invention is to provide a process for the
preparation of long chain fatty acid esters of ursolic acid as potent antimicrobial
agents which obviates the drawbacks as detailed above.
Another object of the present invention is to utilise the major metabolite
obtained in one of our programmes on Diospyros melanoxylon (kendu) leaves
i.e. ursolic acid, which is incidentally the starting material for the test compounds.
Still another object of the present invention is to make 'New Molecules' with
enhanced biological activities from abundantly available inactive natural
products.
In the drawing accompanying this specification
Figure 1 represents ursolic acid
Figure 2 represents esters of ursolic acid
Figure 2a represents ursolic acid laurate, R = CH3(CH2)10
Figure 2b represents ursolic acid myristate, R = CH3(CH2)12
Figure 2c represents ursolic acid palmitate, R = CH3(CH2)14
Figure 2d represents ursolic acid stearate, R = CH3(CH2)16
Figure 3 represents fatty acid esters
Figure 3a represents lauric acid, n = 10
Figure 3b represents myristic acid, n = 12
Figure 3c represents palmitic acid, n = 14
Figure 3d represents stearic acid, n = 16
The process for the preparation of long chain fatty acid esters of ursolic acid as
potent antimicrobial agents comprises derivatisation of the C3 - hydroxyl
function of ursolic acid (as shown in the drawing as fig.1) with long chain fatty
acids such as lauric acid , myristic acid, palmitic acid and stearic acid (as shown
in the drawings as figs. 3a - 3d) in presence of mild base and a powerful
dehydrating agent in a low boiling chlorinated solvent at room temperature under
stirring and isolating the resulting products( as shown in the drawing as figs. 2a
- 2d) by chromatographic separation and screening for antimicrobial activity in
vitro against the gram -ve bacteria Pseudomonas syringae and gram +ve
bacteria, Bacillus sphaericus and Bacillus subtilis.
The long chain fatty acid esters of ursolic acid as prepared by the process of
present invention are synthesised and screened for antimicrobial activity for the
first time.
Accordingly, the present present invention relates to a process for the
preparation of fatty acid esters of ursolic acid of formula 2 wherein R is alkyl
group which comprises reacting ursolic acid of formula 1 with fatty acids of
formula 3 as shown in the drawing wherein n is 10 to 16 carbons in presence of a
mild organic base and a dehydrating agent in chlorinated solvent at room
temperature under stirring for at least a period of 72 hrs., recovering the esters of
ursolic acid and purifying the esters by conventional methods
In another embodiment of the present invention, the mild base used may be
such as pyridine, 4 - pyrrolidinopyridine, 4 - dimethylaminopyridine, 4 - diethyl-
aminopyridine.
In another embodiment of the present invention, the dehydrating agent used
may be such as dicyclohexyl carbodiimide, N,N' - carbonyldiimidazol, 1-cyclo-3-
2-(2-morpholinoethyl)carbodiimde.
In another embodiment of the present invention the synthesised long chain
fatty acid esters are found highly lipophilic and soluble in most of the organic
solvents such as dichloromethane, chloroform, ethyl acetate, acetone.
In yet another embodiment of the present invention the fatty acid esters of
ursolic acid were found to exhibit potent antimicrobial activity in vitro against
gram - ve bacteria Pseudomonas syringae and gram + ve bacteria Bacillus
sphaericus and Bascillus subtilis.
In still another embodiment of the present invention the fatty acid esters of
ursolic acid were found to be highly active than tetracycline and benzyl penicillin
sodium against the gram - ve bacteria Pseudomonas syringae.
In still another embodiment of the preset invention the fatty acid esters of ursolic acid were found to be highly active than benzyl penicillin sodium against the gram + ve bacteria Bacillus sphaericus and Bacillus subtilis. The present invention describes the drivatisation of C3 hydroxyl group of ursolic acid with the long chain fatty acids (C10 - C-16) under mild esterification conditions comprising a base, a dehydrating agent and a low boiling chlorinated solvent. The base used is e.g. pyridine, 4 - pyrrolidinopyridine, 4 -dimethylaminopyridine, 4 - diethylaminopyridine. The dehydrating agent used is e.g. carbodiimides such as dicyclohexylcarbodiimide, N,N' - cabonyldiimidazol, 1- cycle - 3 - (2 - morpholinoethyl)carbodiimide. The chlorinated solvent used is e.g. chloroform, dichloromethane, carbon tetrachloride.
For several years ursolic acid has been thought of biologically inactive. Even though some interesting biologically activities have been attributed recently to ursolic acid, it could not come to limelight due to several limiting parameters like solubility and polarity. In the present invention we have demonstrated that some of the lipophilic derivatives of ursolic acid are more potent and exhibiting high antimicrobial activities than the parent acid. The most significant part of the present invention is that lipophilic ester chains have been synthesised in totally mild experimental conditions. Our method has got an advantage over the conventional methods for the preparation of esters in eliminating the intermediate acid chloride formation, which is very cumbersome.
The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention.
EXAMPLE -1
To ursolic acid (1g. 1eq.) in dichloromethane(15 ml.) was added lauric acid (0.438g,1eq.), dimethylaminopyridine (0.268g,1eq.) and dicyclohexylcarbodiimide (0.452g, 1eq.) and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC. As the reaction found incomplete, some more equivalents of lauric acid (0.438g, 1eq), dimethylaminopyridine (1.072g, 4eq) and dicyclohexylcarbodiimide (1.808g, 4eq) were added and stirring was continued. After the reaction is over, the reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The resulting residue (4.48g.) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity (n-hexane, n-hexane - ethyl acetate : 98-2) and collected 250 ml fractions. Fractions (n-hexane - ethyl acetate : 98-2) showing single and identical spots on TLC were combined and evaporated to give ursolic acid laurate as a colourless oil (30%).
EXAMPLE-2
To ursolic acid(1g. 1eq.) in chloroform(15 ml.) was added lauric acid (0.438g,1eq.), dimethylaminopyridine (0.268g,1eq.) and dicyclohexylcarbodiimide (0.452g, 1eq.) and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC. As the reaction found incomplete, some more equivalents of lauric acid (0.438g, 1eq.), dimethylaminopyridine (1.072g, 4eq.) and dicyclohexylcarbodiimide (1.808g, 4eq.) were added and stirring was continued. After the reaction is over, the reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The resulting residue (4.48g.)
was chromatographed over a column of silica gel and eluted with solvents of increasing polarity (n-hexane, n-hexane - ethyl acetate : 98-2) and collected 250 ml fractions. Fractions (n-hexane - ethyl acetate : 98-2) showing single and identical spots on TLC were combined and evaporated to give ursolic acid laurate as a colourless oil (20%). The reaction is not only low yielding but also gave some undesirable side products.
EXAMPLE - 3
To ursolic acid(1g. 1eq.) in dichloromethane(15 ml.) was added lauric acid (0.438g,1eq.), pyridine (0.172g,1eq.) and dicyclohexylcarbodiimide (0.452g, 1eq.) and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC. As the reaction found incomplete, some more equivalents of lauric acid (0.438g, 1eq.), pyridine (0.688g, 4eq.) and dicyclohexylcarbodiimide (1.808g, 4eq.) were added and stirring was continued. Even after 120 hrs. most of the starting material was found unconverted. The reaction mixture was filtered and the filtrate was evaporated under reduced pressure.The resulting residue (4.0 g.) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity (n-hexane, n-hexane - ethyl acetate : 98-2) and collected 250 ml fractions. Fractions (n-hexane - ethyl acetate : 98-2) showing single and identical spots on TLC were combined and evaporated to give ursolic acid laurate as a colourless oil (14%).
EXAMPLE-4
To ursolic acid (1g. 1eq.) in dichloromethane(15 ml.) was added myristic acid (0.499g,1eq.), dimethylaminopyridine (0.268g,1eq.) and dicyclohexylcarbodiimide
(0.452g, 1eq.) and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC. As the reaction found incomplete, some more equivalents of myristic acid (0.499g,1eq.), dimethylaminopyridine (1.072g, 4eq) and dicyclohexylcarbodiimide (1.808g, 4eq) were added and stirring was continued. After the reaction is over, the reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The resulting residue (4.48g.) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity (n-hexane, n-hexane - ethyl acetate : 98-2) and collected 250 ml fractions. Fractions (n-hexane - ethyl acetate : 98-2) showing single and identical spots on TLC were combined and evaporated to give ursolic acid myristate as a colourless oil (30%).
EXAMPLE - 5
To ursolic acid (1g. 1eq.) in dichloromethane(15 ml.) was added palmitic acid (0.561g,1eq.), dimethylaminopyridine (0.268g,1eq.) and dicyclohexylcarbodiimide (0.452g, 1eq.) and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC. As the reaction found incomplete, some more equivalents of palmitic acid (0.56g, 1eq), dimethylaminopyridine (1.072g, 4eq) and dicyclohexylcarbodiimide (1.808g, 4eq) were added and stirring was continued. After the reaction is over, the reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The resulting residue (3.44g.) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity (n-hexane, n-hexane - ethyl acetate : 98-2) and collected 250 ml fractions. Fractions (n-hexane - ethyl acetate : 98-2) showing single and
identical spots on TLC were combined and evaporated to give ursolic acid myristate as a colourless oil (30%).
EXAMPLE - 6
To ursolic acid (1g. 1eq.) in dichloromethane(15 ml.) was added stearic acid (0.62g,1eq.), dimethylaminopyridine (0.268g,1eq.) and dicyclohexylcarbodiimide (0.452g, 1eq.) and the reaction mixture was stirred at room temperature. The reaction was monitored by TLC. As the reaction found incomplete, some more equivalents of stearic acid (0.62g, 1eq), dimethylaminopyridine (1.072g, 4eq) and dicyclohexylcarbodiimide (1.808g, 4eq) were added and stirring was continued. After the reaction is over, the reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The resulting residue (5.538g) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity (n-hexane, n-hexane - ethyl acetate : 98-2) and collected 250 ml fractions. Fractions (n-hexane - ethyl acetate : 98-2) showing single and identical spots on TLC were combined and evaporated to give ursolic acid stearate as a colourless oil (30%).
EXAMPLE - 7
The test compounds (2a - 2d) and the two controls viz; tetracycline and benzyl penicillin sodium were screened for antimicrobial activity against the gram -ve bacteria P. syringae as per the Broth Macro Dilution method. The compounds shown in the drawing as fig. 2a - 2d are found highly potent with several fold more active than tetracycline and benzyl pencillin sodium.The Minimum Ihibitory
Concentration (MIC) values of the test compounds and the controls are given below in table -1.
Table-1
(Table Removed)
Example - 8
The test compounds (2b & 2d) and the two controls viz. tetracycline and benzyl pencillin sodium were screened for antimicrobial activity against the gram +ve bacteria, B. sphaericus as per Broth Macro Dilution method. The compounds shown in the drawing as fig. 2b & fig. 2d are found to be highly active and exhibiting about 1.4 times more activity than benzyl pencillinsodium. The results are given below in table - 2.
Table-2 (Table Removed)
EXAMPLE - 9
The test compounds (2a & 2d) and the two controls viz. tetracycline and benzyl pencillin sodium were screened for antimicrobial activity against the gram +ve bacteria, B. subtilis as per the Broth Macro Dilution method. The compounds shown in the drawing as fig. 2a & fig. 2d are found to be highly active and exhibiting about 1.3 times more activity than benzyl penicillin sodium. The results are given below in table - 3.
Table – 3 (Table Removed)
In conclusion, for the first time the long chain fatty acid esters viz. ursolic acid laurate, ursolic acid myristate, ursolic acid palmitate and ursolic acid stearate have been synthesised and screened for antimicrobial activity. The novelty of the present invention is the long chain fatty acid esters were synthesised under extremely mild esterification conditions and most significantly these compounds were found to exhibit very high antimicrobial activity.
The main advantages of the present invention are :
1. The test compounds are synthesised under very mild experimental
conditions by eliminating the intermediate acid chloride formation.
2. The test compounds with long fatty chains are highly lipophilic and soluble
in most of he organic solvents.
3. The test compounds are exhibiting high antimicrobial activities and found
several folds higher activities than the parent acid.
4. Utilisation of trivial and the abundantly available natural triterpenic acid viz.
ursolic acid in making some of its lipophilic derivatives with enhanced
biological activity.

We Claim:
1. A process for the preparation of fatty acid esters of ursolic acid of formula
2 wherein R is alkyl group which comprises reacting ursolic acid of
formula 1 with fatty acids of formula 3 as shown in the drawing wherein n
is 10 to 16 carbons in presence of a mild organic base and a dehydrating
agent in chlorinated solvent at room temperature under stirring for at least
a period of 72 hrs., recovering the esters of ursolic acid and purifying the
esters by conventional methods.
2. A process as claimed in 1 wherein a base used is selected from pyridine,
4 pyrrolidinopyridine, 4 - dimethylaminopyridine, 4
diethylaminopyridine.
3. A process as claimed in 1 wherein a dehydrating agent used is selected
from dicyclohexylcarbodiimide, N,N' - carboxyldiimidazol, 1 - cycio - 3 -
(-2-morpholinoethyl) carbodiimide.
4. A process as claimed in 1 wherein a low boiling chlorinated solvent used
is selected from chloroform, dichloromethane, carbontetrachloride.
5. A process as claimed in 1 - 4 wherein ursolic acid fatty esters shown in
the drawing as figs. 2a - 2d consists of screening of the said compounds
against the gram -ve and gram +ve bacteria by employing the Broth
Macro Dilution method.
6. A process as claimed in claims 1-5 wherein the fatty acid esters of
ursolic acid are exhibiting very high antimicrobial activity than the controls
tetracycline and benzyl penicillin sodium against the gram - ve bacteria, P.syringae.
7. A process as claimed in claims 1 • 6 wherein the fatty acid esters of
ursolic acid as shown in the drawing as fig. 2b & 2d and fig. 2a & 2d are
exhibiting enhanced antimicrobial activity than the control benzyl pencillin
sodium against the gram + ve bacteria, B.sphearicus and B.subtilis
respectively.
8. A process for preparation of esters of ursolic acid as potent antimicrobial
agents substantially as herein described with reference to the examples
and drawings accompanying this specification.

Documents:

1058-del-2001-abstract.pdf

1058-del-2001-claims.pdf

1058-del-2001-correspondence-others.pdf

1058-del-2001-correspondence-po.pdf

1058-del-2001-description (complete).pdf

1058-del-2001-drawings.pdf

1058-del-2001-form-1.pdf

1058-del-2001-form-18.pdf

1058-del-2001-form-2.pdf

1058-del-2001-form-3.pdf

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Patent Number

220524

Indian Patent Application Number

1058/DEL/2001

PG Journal Number

30/2008

Publication Date

25-Jul-2008

Grant Date

29-May-2008

Date of Filing

16-Oct-2001

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	ANITA MAHAPATRA
2	YERRAMILLI RAMACHANDRA RAO
3	UPPULURI VENKATA MALLAVADHANI

PCT International Classification Number

A61K 9/19

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA