Title of Invention	A PROCESS FOR THE PREPARATION OF LONG CHIN FATTY ACIDS USEFUL AS POTENT ANTIFEEDANTS
Abstract	The long chain fatty acid esters viz. ursolic acid stearate and oleanolic acid palmitate 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 title compounds were found to exhibit exceptionally potent antifeedant activities against the tobacco caterpillar Spodoptera litura F. at 50 µg/ml concentration even after 48 hours.

Title of Invention

A PROCESS FOR THE PREPARATION OF LONG CHIN FATTY ACIDS USEFUL AS POTENT ANTIFEEDANTS

Abstract

The long chain fatty acid esters viz. ursolic acid stearate and oleanolic acid palmitate 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 title compounds were found to exhibit exceptionally potent antifeedant activities against the tobacco caterpillar Spodoptera litura F. at 50 µg/ml concentration even after 48 hours.

Full Text	The present invention relates to a process for the preparation of a long chain fatty acid ester useful as a potential antifeedant. More particularly the present invention relates to a process for the preparation of a long chain fatty acid ester selected from ursolic acid stearate and oleanolic acid palmitate useful as a potential antifeedant. Ursolic acid stearate and oleanolic acid palmitate obtained in the present process were found to exhibit exceptionally potent antifeedant activity against the tobacco caterpillar Spodoptera litura F. at 50 \|ig/ml even after 48 hours. Reference may be made to Shukla et.al. (Y.N. Shukla, A. Rani, A.K. Tripathy and S. Sharma, Phytother. Res., 1996, 10(4), 359) wherein ursolic acid isolated from the leaves and stems of Dubosia myoporoides was found to exhibit antifeedant activity under laboratory conditions against the insects, Spilosoma obliqua and Spodoptera litura. The drawbacks are : although the antifeedant activity is significant, the concentration of the test compound used is very high (5000 ppm). Reference may be made to CIMAP, Lucknow (CSIR News, 1998, 48(8), 113) wherein oleanolic acid isolated from the American ginseng (Panax quinquefolium) was reported to act as an insect antifeedant against Spilosoma obliqua and Spodoptera litura. The drawbacks are : at low concentrations (200 -100 ppm) the test compound is exhibiting very weak antifeedant activity ( The antifeedant activity of ursolic acid and oleanolic acid can be enhanced by preparing some of their lipophilic derivatives. Ursolic acid and oleanolic acid have hydroxyls at C3, double bonds at C12 - C13 and carboxyl acid functions at C17. The C3 hydroxyl group can be exploited to make new members with lipophilic side chains and enhanced solubilities. Molecules with carbon chains above C10 are fairly non polar and are good candidates for antifeedant screening. In support of our expectations, a long chain fatty acid ester viz. urs-12-ene-3ß-O-hexadecanoate (α-amyrin palmitate) isolated from the bark of Santalum album was reported to exhibit insect growth inhibition and chemosterilant activities. Structure activity studies of ursane and oleanane skeletons suggest that the C17 carboxylic acids such as ursolic acid and oleanolic acid are more active than their corresponding C17 methyl compounds such as a-amyrin and p-amyrin. The structure activity studies further suggest that the desired substituents on the lipophilic 5-ring backbone of ursolic acid and oleanolic acid can increase the selectivity and potency of a desired action. Hence some of the lipophilic derivatives of ursolic acid and oleanolic acid have been prepared and their antifeedant activity was studied. The main object of the present invention is to provide a process for the preparation of long chain fatty acid esters selected from ursolic acid stearate and oleanolic acid palmitate useful as a potential antifeedant which obviates the drawbacks as detailed above. Another object of the present invention is to utilise the major metabolites obtained in one of our programmes on Diospyros melanoxylon (Kendu) leaves i.e. ursolic acid and oleanolic acid, which are incidentally the starting materials for the title 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 oleanolic acid Figure 3 represents ursolic acid stearate Figure 4 represents oleanolic acid palmitate Figure 5 represents stearic acid Figure 6 represents palmitic acid The present invention provides a process for the preparation of ursolic acid stearate and oleanolic acid palmitate as potent antifeedants which comprises derivatisation of C3 - hydroxyl functions of ursolic acid and oleanolic acid (as shown in the drawing as fig. 1 and fig. 2) with long chain fatty acids such as stearic acid and palmitic acid (as shown in the drawing as fig. 5 and fig. 6) respectively in presence of a mild base and a powerful dehydrating agent in a low boiling chlorinated solvent at room temperature under stirring conditions and isolating the resulting products (as shown in the drawing as fig. 3 and fig. 4) by chromatographic separation and screening for antifeedant activity against the insect tobacco caterpillar Spodoptera litura F. Accordingly the present invention relates to a process for the preparation of a long chain fatty acid ester useful as a potent antifeedant which comprises reaction of a C17 carboxylic acid selected from ursolic acid and oleanolic acid with a long chain fatty acid in the presence of a mild base and a strong dehydrating agent in an aliphatic chlorinated solvent at a temperature ranging between 20-46°C under stirring condition and isolating the resulting long chain fatty acid esters by conventional chromatographic methods such as herein described. In an embodiment of the present invention the long chain fatty acid esters of ursolic acid and oleanolic acid were synthesized and screened for antifeedant activity for the first time. In another embodiment of the present invention the synthesized long chain fatty acid esters are highly lipophilic and soluble in most of the organic solvents. In yet another embodiment of the present invention the title compounds were found to be exhibit exceptionally potent antifeedant activity against the tobacco caterpillar Spodoptera litura F. at 50 \|Kj/ml concentration even after 48 hours. The present invention describes the derivatisation Of C3 - hydroxyl groups of ursolic acid and oleanolic acid with stearic acid and palmitic acid respectively under mild esterification conditions comprising a base, a dehydrating agent and a 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' - carbonyldiimidazole, 1- cyclo-3-(2-morpholinoethyl)carbodiimide. The chlorinated solvent used is e.g. chloroform, dichloromethane, carbontetrachloride. For several years the parent triterpenic acids i.e. ursolic and oleanolic acid have been thought of biologically inactive. Even though, some interesting biological activities have been attributed recently to these acids they could not come to limelight due to several limiting parameters like solubility and polarity. In the present invention we have demonstrated that some of their lipophilic derivatives are more potent and exhibiting exceptionally high antifeedant activities. The most significant part of the invention is that the lipophilic ester chains have been synthesized in totally mild experimental conditions. Our method has got an advantage over the conventional methods 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 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, dimethylaminopyridine, dicyclohexylcarbodiimide were wadded 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 (5.538 g) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity and collected 250 ml fractions. Fractions showing single and identical spots on TLC were combined and evaporated to give ursolic acid stearate as a colourless oil (30%). EXAMPLE -2 To oleanolic acid (1g, 1eq.) in dichloromethane (15 ml) was added palmitic acid (0.561 g,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, dimethylaminopyridine, dicyclohexylcarbodiimide were wadded 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 (5.386 g) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity and collected 250 ml fractions. Fractions showing single and identical spots on TLC were combined and evaporated to give oleanolic acid palmitate as a colourless oil (30%). EXAMPLE - 3 The 150 µg, 100 µg and 50 µg solutions of ursolic acid stearate have been prepared in acetone and were used. The percentage antifeedant activity of the test compound at these three concentrations was determined after 6, 12, 24 and 48 hrs. by employing Leaf- disk method. The results are given below in table -1. Table -1 (Table Removed) EXAMPLE - 4 The 150 µg, 100 µg and 50 µg solutions of oleanolic acid palmitate have been prepared in acetone and were used. The percentage antifeedant activity of the test compound at these three concentrations was determined after 6, 12, 24 and 48 hrs. by employing Leaf- disk method. The results are given below in table -2. Table - 2 (Table Removed) In conclusion, the long chain fatty acid esters viz. ursolic acid stearate and oleanolic acid palmitate have been synthesized and screened for antifeedant activity for the first time. The novelty of the present invention is the title compounds were synthesized under extremely mild esterification conditions and most significantly these compounds were found to exhibit exceptionally potent antifeedant activity. The main advantages of the present invention are : 1. The title compounds were synthesized under very mild experimental conditions by eliminating the intermediate acidchloride formation. 2. The title compounds, with long fatty chains, are highly lipophilic and soluble in most of the organic solvents. 3. The title compounds i.e. ursolic acid stearate and oleanolic acid palmitate are exhibiting exceptionally potent antifeedant activities and found several folds higher than their parent acids. 4. Utilisation of trivial and abundantly available natural triterpenic acids viz. ursolic acid and oleanolic acid in making some of their lipophilic derivatives with enhanced biological activities. We claim : 1. A process for the preparation of a long chain fatty acid ester useful as a potent antifeedant which comprises reaction of a C17 carboxylic acid selected from ursolic acid and oleanolic acid with a long chain fatty acid in the presence of a mild base and a strong dehydrating agent in an aliphatic chlorinated solvent at a temperature ranging between 20-46°C under stirring condition and isolating the resulting long chain fatty acid esters by conventional chromatographic methods such as herein described. 2. A process as claimed in claim 1, wherein the mild base used is pyridine, 4-pyrrolidinopyridine, 4-dimethylamino-pyridine and 4-diethylaminopyridine. 3. A process as claimed in claims 1 to 2, wherein the dehydrating agent used is selected from the group consisting of dicyclohexylcarbodiimide, N,N-carbonyldiimidazole and 1-cyclo-3-(2-morpholinoethyl) carbodiimide. 4. A process as claimed in claims 1 to 3, wherein the chlorinated solvent used is selected from the group consisting of chloroform, dichloromethane and carbontetrachloride. 5. A process as claimed in claims 1 to 4 wherein long chain fatty acid is selected from stearic acid and palmitic acid. 6. A process as claimed in claims 1 to 5, wherein the long chain fatty acid esters produced are is highly lipophilic and are soluble in most of the organic solvents. 7. A process for the preparation of a long chain fatty acid ester useful as a potent antifeedant substantially as herein described with reference to the examples and drawing accompanying the specification.

Full Text

The present invention relates to a process for the preparation of a long chain fatty acid ester useful as a potential antifeedant.
More particularly the present invention relates to a process for the preparation of a long chain fatty acid ester selected from ursolic acid stearate and oleanolic acid palmitate useful as a potential antifeedant.
Ursolic acid stearate and oleanolic acid palmitate obtained in the present process were found to exhibit exceptionally potent antifeedant activity against the tobacco caterpillar Spodoptera litura F. at 50 |ig/ml even after 48 hours.
Reference may be made to Shukla et.al. (Y.N. Shukla, A. Rani, A.K. Tripathy and S. Sharma, Phytother. Res., 1996, 10(4), 359) wherein ursolic acid isolated from the leaves and stems of Dubosia myoporoides was found to exhibit antifeedant activity under laboratory conditions against the insects, Spilosoma obliqua and Spodoptera litura. The drawbacks are : although the antifeedant activity is significant, the concentration of the test compound used is very high (5000 ppm).
Reference may be made to CIMAP, Lucknow (CSIR News, 1998, 48(8), 113) wherein oleanolic acid isolated from the American ginseng (Panax quinquefolium) was reported to act as an insect antifeedant against Spilosoma obliqua and Spodoptera litura. The drawbacks are : at low concentrations (200 -100 ppm) the test compound is exhibiting very weak antifeedant activity ( The antifeedant activity of ursolic acid and oleanolic acid can be enhanced by preparing some of their lipophilic derivatives. Ursolic acid and oleanolic acid have hydroxyls at C3, double bonds at C12 - C13 and carboxyl acid functions at
C17. The C3 hydroxyl group can be exploited to make new members with lipophilic side chains and enhanced solubilities. Molecules with carbon chains above C10 are fairly non polar and are good candidates for antifeedant screening. In support of our expectations, a long chain fatty acid ester viz. urs-12-ene-3ß-O-hexadecanoate (α-amyrin palmitate) isolated from the bark of Santalum album was reported to exhibit insect growth inhibition and chemosterilant activities. Structure activity studies of ursane and oleanane skeletons suggest that the C17 carboxylic acids such as ursolic acid and oleanolic acid are more active than their corresponding C17 methyl compounds such as a-amyrin and p-amyrin. The structure activity studies further suggest that the desired substituents on the lipophilic 5-ring backbone of ursolic acid and oleanolic acid can increase the selectivity and potency of a desired action. Hence some of the lipophilic derivatives of ursolic acid and oleanolic acid have been prepared and their antifeedant activity was studied.
The main object of the present invention is to provide a process for the preparation of long chain fatty acid esters selected from ursolic acid stearate and oleanolic acid palmitate useful as a potential antifeedant which obviates the drawbacks as detailed above.
Another object of the present invention is to utilise the major metabolites obtained in one of our programmes on Diospyros melanoxylon (Kendu) leaves i.e. ursolic acid and oleanolic acid, which are incidentally the starting materials for the title 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 oleanolic acid Figure 3 represents ursolic acid stearate Figure 4 represents oleanolic acid palmitate Figure 5 represents stearic acid Figure 6 represents palmitic acid
The present invention provides a process for the preparation of ursolic acid stearate and oleanolic acid palmitate as potent antifeedants which comprises derivatisation of C3 - hydroxyl functions of ursolic acid and oleanolic acid (as shown in the drawing as fig. 1 and fig. 2) with long chain fatty acids such as stearic acid and palmitic acid (as shown in the drawing as fig. 5 and fig. 6) respectively in presence of a mild base and a powerful dehydrating agent in a low boiling chlorinated solvent at room temperature under stirring conditions and isolating the resulting products (as shown in the drawing as fig. 3 and fig. 4) by chromatographic separation and screening for antifeedant activity against the insect tobacco caterpillar Spodoptera litura F.
Accordingly the present invention relates to a process for the preparation of a long chain fatty acid ester useful as a potent antifeedant which comprises reaction of a C17 carboxylic acid selected from ursolic acid and oleanolic acid with a long chain fatty acid in the presence of a mild base and a strong dehydrating agent in an aliphatic chlorinated solvent at a temperature ranging between 20-46°C under stirring condition and isolating the resulting long chain fatty acid esters by conventional chromatographic methods such as herein described.
In an embodiment of the present invention the long chain fatty acid esters of ursolic acid and oleanolic acid were synthesized and screened for antifeedant activity for the first time.
In another embodiment of the present invention the synthesized long chain fatty acid esters are highly lipophilic and soluble in most of the organic solvents.
In yet another embodiment of the present invention the title compounds were found to be exhibit exceptionally potent antifeedant activity against the tobacco caterpillar Spodoptera litura F. at 50 |Kj/ml concentration even after 48 hours.
The present invention describes the derivatisation Of C3 - hydroxyl groups of ursolic acid and oleanolic acid with stearic acid and palmitic acid respectively under mild esterification conditions comprising a base, a dehydrating agent and a 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' - carbonyldiimidazole,
1- cyclo-3-(2-morpholinoethyl)carbodiimide. The chlorinated solvent used is e.g. chloroform, dichloromethane, carbontetrachloride.
For several years the parent triterpenic acids i.e. ursolic and oleanolic acid have been thought of biologically inactive. Even though, some interesting biological activities have been attributed recently to these acids they could not come to limelight due to several limiting parameters like solubility and polarity. In the present invention we have demonstrated that some of their lipophilic derivatives are more potent and exhibiting exceptionally high antifeedant activities. The most significant part of the invention is that the lipophilic ester chains have been synthesized in totally mild experimental conditions. Our method has got an advantage over the conventional methods 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 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, dimethylaminopyridine, dicyclohexylcarbodiimide were wadded 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 (5.538 g) was chromatographed over a column of silica gel and
eluted with solvents of increasing polarity and collected 250 ml fractions. Fractions showing single and identical spots on TLC were combined and evaporated to give ursolic acid stearate as a colourless oil (30%).
EXAMPLE -2 To oleanolic acid (1g, 1eq.) in dichloromethane (15 ml) was added palmitic acid (0.561 g,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, dimethylaminopyridine, dicyclohexylcarbodiimide were wadded 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 (5.386 g) was chromatographed over a column of silica gel and eluted with solvents of increasing polarity and collected 250 ml fractions. Fractions showing single and identical spots on TLC were combined and evaporated to give oleanolic acid palmitate as a colourless oil (30%).
EXAMPLE - 3 The 150 µg, 100 µg and 50 µg solutions of ursolic acid stearate have been prepared in acetone and were used. The percentage antifeedant activity of the test compound at these three concentrations was determined after 6, 12, 24 and 48 hrs. by employing Leaf- disk method. The results are given below in table -1.
Table -1

(Table Removed)
EXAMPLE - 4
The 150 µg, 100 µg and 50 µg solutions of oleanolic acid palmitate have been prepared in acetone and were used. The percentage antifeedant activity of the test compound at these three concentrations was determined after 6, 12, 24 and 48 hrs. by employing Leaf- disk method. The results are given below in table -2.
Table - 2

(Table Removed)
In conclusion, the long chain fatty acid esters viz. ursolic acid stearate and oleanolic acid palmitate have been synthesized and screened for antifeedant activity for the first time. The novelty of the present invention is the title compounds were synthesized under extremely mild esterification conditions and most significantly these compounds were found to exhibit exceptionally potent antifeedant activity.
The main advantages of the present invention are :
1. The title compounds were synthesized under very mild experimental conditions by eliminating the intermediate acidchloride formation.
2. The title compounds, with long fatty chains, are highly lipophilic and soluble in most of the organic solvents.
3. The title compounds i.e. ursolic acid stearate and oleanolic acid palmitate are exhibiting exceptionally potent antifeedant activities and found several folds higher than their parent acids.
4. Utilisation of trivial and abundantly available natural triterpenic acids viz. ursolic acid and oleanolic acid in making some of their lipophilic derivatives with enhanced biological activities.

We claim :
1. A process for the preparation of a long chain fatty acid ester useful as a potent antifeedant which comprises reaction of a C17 carboxylic acid selected from ursolic acid and oleanolic acid with a long chain fatty acid in the presence of a mild base and a strong dehydrating agent in an aliphatic chlorinated solvent at a temperature ranging between 20-46°C under stirring condition and isolating the resulting long chain fatty acid esters by conventional chromatographic methods such as herein described.
2. A process as claimed in claim 1, wherein the mild base used is pyridine, 4-pyrrolidinopyridine, 4-dimethylamino-pyridine and 4-diethylaminopyridine.
3. A process as claimed in claims 1 to 2, wherein the dehydrating agent used is selected from the group consisting of dicyclohexylcarbodiimide, N,N-carbonyldiimidazole and 1-cyclo-3-(2-morpholinoethyl) carbodiimide.
4. A process as claimed in claims 1 to 3, wherein the chlorinated solvent used is selected from the group consisting of chloroform, dichloromethane and carbontetrachloride.
5. A process as claimed in claims 1 to 4 wherein long chain fatty acid is selected from stearic acid and palmitic acid.
6. A process as claimed in claims 1 to 5, wherein the long chain fatty acid esters produced are is highly lipophilic and are soluble in most of the organic solvents.
7. A process for the preparation of a long chain fatty acid ester useful as a potent antifeedant substantially as herein described with reference to the examples and drawing accompanying the specification.

Documents:

1182-del-2001-abstract.pdf

1182-del-2001-claims.pdf

1182-del-2001-complete specification (granded).pdf

1182-del-2001-correspondence-others.pdf

1182-del-2001-correspondence-po.pdf

1182-del-2001-description(complete).pdf

1182-del-2001-drawings.pdf

1182-del-2001-form-1.pdf

1182-DEL-2001-Form-2.pdf

1182-del-2001-form-3.pdf

1182-del-2001-form-4.pdf

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Patent Number

231039

Indian Patent Application Number

1182/DEL/2001

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

28-Feb-2009

Date of Filing

26-Nov-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	UPPULURI VENKATA MALLAVADHANI	REGIONAL RESEARCH LABORATORY, BHUBANESWAR-751013, ORISSA, INDIA.
2	ANITA MAHAPATRA	REGIONAL RESEARCH LABORATORY, BHUBANESWAR-751013, ORISSA, INDIA.
3	YERRAAMILLI RAMACHANDRA	REGIONAL RESEARCH LABORATORY, BHUBANESWAR-751013, ORISSA, INDIA.

PCT International Classification Number

A61K-31/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA