Indian Patents. 272748:ANTIMICROBIAL DERIVATIVES OF ANACARDIC ACID AND PROCESS FOR PREPARING THE SAME

Title of Invention	ANTIMICROBIAL DERIVATIVES OF ANACARDIC ACID AND PROCESS FOR PREPARING THE SAME
Abstract	Derivatives of anacardic acid having antimicrobial properties and method for preparing said derivatives. The antimicrobial properties properties include bacteriostatic and bacteriocidal activity.

Full Text	THE PATENTS ACT, 1970 PROVISIONAL SPECIFICATION Section 10 Antimicrobial derivatives of anacardic acid and process for preparing the same Unichem Laboratories Ltd., an Indian company of Mahalaxmi Chambers, 22 Bhulabhai Desai Road, Mumbai - 400026, Maharashtra, India The following specification particularly describes and the nature of this invention: Field of invention: The invention discloses derivatives of anacardic acid having anti-microbial activity against microorganisms more specifically towards Stapphalococcus aureus and Methicillin resistant Stapphalococcus aureus (MRSA) and process for preparing said derivatives. Background of invention: Anacardic acid is a non-isoprenoid long chain phenolic acid primarily obtained from the nut shells of the plant Anacardium occidentale native of South America. The solvent extracted cashewnut shell liquid (CNSL) is a rich source of many long chain phenolic compounds of which anacardic acids constitutes about 60-65% of the CNSL. The other major long chain constituents being cardol a dihydric component (15-20%) and cardanol a phenolic compound (10%). The long aliphatic side chain of these phenolic constituents exists as a mixture of monoene, diene and triene, with the saturated component being present in negligible amount. Anacardic acids in CNSL exists as a mixture of monoene (38.4%), diene (17.4%) and triene (44.1 %). CNSL has for a long time being used as a raw material for the manufacture of polymers, paints, varnishes, brake linings etc. Apart from the industrial uses, the nut shell oil has also been used in the traditional medicine world wide for the treatment of inflammation, diarrhea, treatment of cracked foot etc. Anacardic acid (6-pentadecyl salicylic acid) has been screened for various biological activities. It has been shown to be an inhibitor of lipoxygenase and xanthine oxidase. The antibacterial properties of anacardic acids has also been extensively studied by various groups. Kubo et al studied the anti bacterial activity of the monoene (C15:1), diene (C15:2), triene(C15:3) and saturated (C15:0) anacardic acids against Stapphalococcus aureus and Methicillin resistant Stapphalococcus aureus. They have shown that the triene (C15:3) has a minimum inhibitory concentration (MIC) of 6.5µg/mL while the saturated anacardic acid (C15:0) has a MIC of 1600 µg/mL against 2 MRSA. They have also studied the effect of the length of the side chain on the antibacterial activity and showed that the antibacterial activity was greater as the length of the alkyl chain increased. Though a lot of study has been done to use the naturally occurring anacardic acids as such or in combination with known antibiotics to demonstrate the antimicrobial activity, there are no reports on the synthetic modifications of anacardic acids to enhance their antimicrobial activity. Antibiotic resistance is a very serious problem in the hospitals and is growing at a rapid pace. Resistant strains have been isolated for almost all the existing antibiotics. With the growing problem of antimicrobial resistance there is a dire need for an effective molecule to combat this problem. The objective of this invention is to enhance the antimicrobial activity of saturated anacardic acid (C15:0) especially against MRSA, by synthetic modifications. Summary of the invention: The present invention discloses antimicrobial derivativer of anacardic acid and process for preparing said derivative. The invention specifically relates to the process for the preparation of antimicrobial derivatives of saturated anacardic acid (C15:0) .The mixture of anacardic acids chromatographically purified from CNSL was hydrogenated using Pd-C to get the saturated anacardic acid (Cis:o). Said derivatives were prepared by adopting the techniques used in synthetic chemistry for nitration, halogenation and acetylation of various organic compounds. The synthesized derivatives were assayed for anti bacterial activity against Stapphalococcus aureus and Methicillin resistant Stapphalococcus aureus by the broth assay used for antibiotic sucesptibility tests and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determined. The derivatives showed a MIC of 1-10 µg/mL and MBC of 2-10 µg/mL against MRSA and Stapphalococcus aureus. A few of the derivatives showed a MBC of 2 µg/mL and MIC of 1 µg/mL. 3 The invention provides for process for preparation of 6-Pentadecyl-2-acetyloxybenzoic acid, 3,5-Dinitro-6-pentadecyl-2-hydroxybenzoic, 3,4-Dinitro-6-pentadecyl-2-hydroxybenzoic, 3,5-Dinitro-6-pentadecyl-2-acetyloxybenzoic, 3,4-Dinitro-6-pentadecyl-2-acetyloxybenzoic acid, 3,5-Dibromo-6-pentadecyl-2-hydroxybenzoic acid, 3-nitro-6-pentadecyl-2-hydroxybenzoic acid, 3-nitro-6-pentadecyl-2-acetyloxybenzoic acid, 5-nitro-6-pentadecyl-2-hydroxybenzoic acid, 5-nitro-6-pentadecyl-2-acetyloxybenzoic acid, 4-nitro-6-pentadecyl-2-hydroxybenzoic acid, 4-nitro-6-pentadecyl-2-acetyloxybenzoic acid and their use for preventing the growth of microorganisms including gram-negative or gram positive microorganisms such as Stapphalococcus aureus, and Methicillin resistant Stapphalococcus aureus etc. Detailed description of the invention: The following examples are provided to illustrate the different embodiments of the invention: Example-1: Preparation of 6-Pentadecyl-2-acetyloiybenzoic acid (AAA): A mixture of saturated anacardic acid (100 mg, 0.287 mmol) and acetyl chloride (5ml) was stirred at room temperature for 3 hrs. The reaction mixture was poured into ice-cold water. The product precipitated out and was extracted into ethyl acetate (25ml x 2). The ethyl acetate layer was washed with water (25 ml x 4) and dried over anhydrous Na2S04. The solvent was evaporated under reduced pressure. The residue obtained was triturated with hexane at 10°C. The solid obtained was separated by centrifugation, washed with hexane and dried under vacuum. The yield obtained was 85%( 92 mg). The product had a melting point of 62- 66 °C. Example-2: Preparation of 3,5-Dinitro-6-pentadecyl-2-bydroxybenzoic acid (DAA): A suspension of anacardic acid (200 mg, 0.574 mmol) in cone. H2SO4 (8ml) was cooled to 0°C. To the cold mixture, cone. HNO3 (2ml, 69% solution) was added slowly maintaining the temperature below 5 °C. The reaction mixture was stirred at 5 °C for 15 min and poured into crushed ice. The product precipitated out and was extracted into ethyl acetate (25ml x 2). The ethyl acetate layer was washed with water 4 (25ml x 4) and dried over anhydrous Na2SO4. Finally the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography using Hexane-ethyl acetate solvent system. The pure product obtained after concentration (oily) was triturated with hexane (5ml) to obtain a solid. The product was separated by centrifugation, washed with hexane (1ml) and dried under vacuum. The yield obtained was 80% (204 mg). The product had a melting point of 78-82°C Example-3: Preparation of 3,4-Dinitro-6-pentadecyI-2-hydroxybenzoic acid (NAA): To a suspension of anacaridic acid (200 mg, 0.574 mmol), Cone. H2SO4 (4ml), cone. HNO3 (2ml, 69% solution) was added dropwise over a period of 5min at room temperature. During the addition of nitric acid, the temperature of the reaction mixture raised to about 40 - 45 °C. The mixture was stirred for 15 min and poured into crushed ice. The product precipitated out and was extracted into ethyl acetate (25ml x 2). The ethyl acetate layer was washed with water (25ml x 4) and dried over anhydrous. Na2SO4. Finally the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography. The mobile phase used was hexane-ethylacetate system. The yield obtained was 52% (132 mg). The product obtained after column chromatography was treated with 5ml of 1:1 mixture of DCM /hexane to obtain a solid. The product was separated by centrifugation, washed with hexane and dried under vacuum. The over all yield obtained was 32% (80 mg). The product was found to decompose at 75 -80°C. Example-4: Preparation of 3,5-Dinitro-6-pentadecyl-2-acetyloxybenzoic acid (ADAA): A mixture of DAA obtained in Example-2 (80 mg, 0.23 mmol) and acetyl chloride (5ml) was refluxed for 7 hrs. The reaction mixture was poured into icecold water. The product precipitated out and was extracted into ethyl acetate (25ml x 2), washed with water (25 ml x 4) and dried over anhydrous Na2SO4 The solvent was evaporated under reduced pressure. The residue obtained was triturated with hexane (3ml) to obtain a solid. The product was separated by centrifugation, washed with 5 hexane and dried under vacuum. The yield obtained was 52%( 46 nig). The product was found to melt at 55 - 57°C, with decomposition. Example-5: Preparation of 3,4-Dinitro-6-pentadecyl-2-acetyloxybenzoic acid (ANAA): A mixture of NAA obtained in Example-3 (25 mg, 0.057 mmol) and acetyl chloride (2ml) was refluxed for 7 hrs. The reaction mixture was poured into icecold water. The product precipitated out and was extracted into ethyl acetate (15ml x 2). The ethyl acetate layer was washed with water (15 ml x 4) and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure. The residue obtained was triturated with hexane (3ml) to obtain a solid. The product was separated by centrifugation, washed with hexane and dried under vacuum for 2hrs. The yield obtained was 51%( 14 mg). Example-6: Preparation of 3,5-Dibromo-6-pentadecyl-2-hydroxybenzoic acid (BAA): A suspension of saturated anacardic acid (50 mg, 0.143 mmol) in AcOH/H2S04 (0.5ml/50 µj) mixture was heated to 50°C. To the warm mixture a solution of bromine (30µl) in AcOH (0.25 ml) was added dropwise over a period of 5 min. The mixture was heated to 100 °C for 30 min. Then water (5 ml) was added and the product that precipitated out was extracted into ethyl acetate (25ml x 2). The ethyl acetate layer was washed with water (25 ml x 4) and dried over anhydrous Na2SO4.The solvent was evaporated under reduced pressure to obtain a residue. The residue obtained was triturated with hexane (3ml). The product obtained as a solid was separated by centrifugation, washed with hexane and dried under vacuum.The yield obtained was 82%( 60 mg). The product had a melting point of 85 - 90°C. Dated this 24th day of august 2006 Abstract: This invention describes method to synthesize derivatives of anacardic acid having antimicrobial properties. These antimicrobial properties include bacteriostatic and bacteriocidal activity especially against Stapphalococcus aureus and Methicillin resistant Stapphalococcus aureus. 7

Full Text

THE PATENTS ACT, 1970
PROVISIONAL SPECIFICATION
Section 10
Antimicrobial derivatives of anacardic acid and process for
preparing the same
Unichem Laboratories Ltd., an Indian company of Mahalaxmi Chambers, 22 Bhulabhai Desai Road, Mumbai - 400026, Maharashtra, India
The following specification particularly describes and the nature of this invention:

Field of invention:
The invention discloses derivatives of anacardic acid having anti-microbial activity against microorganisms more specifically towards Stapphalococcus aureus and Methicillin resistant Stapphalococcus aureus (MRSA) and process for preparing said derivatives.
Background of invention:
Anacardic acid is a non-isoprenoid long chain phenolic acid primarily obtained from the nut shells of the plant Anacardium occidentale native of South America. The solvent extracted cashewnut shell liquid (CNSL) is a rich source of many long chain phenolic compounds of which anacardic acids constitutes about 60-65% of the CNSL. The other major long chain constituents being cardol a dihydric component (15-20%) and cardanol a phenolic compound (10%).
The long aliphatic side chain of these phenolic constituents exists as a mixture of monoene, diene and triene, with the saturated component being present in negligible amount. Anacardic acids in CNSL exists as a mixture of monoene (38.4%), diene (17.4%) and triene (44.1 %).
CNSL has for a long time being used as a raw material for the manufacture of polymers, paints, varnishes, brake linings etc. Apart from the industrial uses, the nut shell oil has also been used in the traditional medicine world wide for the treatment of inflammation, diarrhea, treatment of cracked foot etc.
Anacardic acid (6-pentadecyl salicylic acid) has been screened for various biological activities. It has been shown to be an inhibitor of lipoxygenase and xanthine oxidase. The antibacterial properties of anacardic acids has also been extensively studied by various groups. Kubo et al studied the anti bacterial activity of the monoene (C15:1), diene (C15:2), triene(C15:3) and saturated (C15:0) anacardic acids against Stapphalococcus aureus and Methicillin resistant Stapphalococcus aureus. They have shown that the triene (C15:3) has a minimum inhibitory concentration (MIC) of 6.5µg/mL while the saturated anacardic acid (C15:0) has a MIC of 1600 µg/mL against
2

MRSA. They have also studied the effect of the length of the side chain on the antibacterial activity and showed that the antibacterial activity was greater as the length of the alkyl chain increased.
Though a lot of study has been done to use the naturally occurring anacardic acids as such or in combination with known antibiotics to demonstrate the antimicrobial activity, there are no reports on the synthetic modifications of anacardic acids to enhance their antimicrobial activity.
Antibiotic resistance is a very serious problem in the hospitals and is growing at a rapid pace. Resistant strains have been isolated for almost all the existing antibiotics. With the growing problem of antimicrobial resistance there is a dire need for an effective molecule to combat this problem. The objective of this invention is to enhance the antimicrobial activity of saturated anacardic acid (C15:0) especially against MRSA, by synthetic modifications.
Summary of the invention:
The present invention discloses antimicrobial derivativer of anacardic acid and process for preparing said derivative. The invention specifically relates to the process for the preparation of antimicrobial derivatives of saturated anacardic acid (C15:0) .The mixture of anacardic acids chromatographically purified from CNSL was hydrogenated using Pd-C to get the saturated anacardic acid (Cis:o). Said derivatives were prepared by adopting the techniques used in synthetic chemistry for nitration, halogenation and acetylation of various organic compounds. The synthesized derivatives were assayed for anti bacterial activity against Stapphalococcus aureus and Methicillin resistant Stapphalococcus aureus by the broth assay used for antibiotic sucesptibility tests and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determined. The derivatives showed a MIC of 1-10 µg/mL and MBC of 2-10 µg/mL against MRSA and Stapphalococcus aureus. A few of the derivatives showed a MBC of 2 µg/mL and MIC of 1 µg/mL.
3

The invention provides for process for preparation of 6-Pentadecyl-2-acetyloxybenzoic acid, 3,5-Dinitro-6-pentadecyl-2-hydroxybenzoic, 3,4-Dinitro-6-pentadecyl-2-hydroxybenzoic, 3,5-Dinitro-6-pentadecyl-2-acetyloxybenzoic, 3,4-Dinitro-6-pentadecyl-2-acetyloxybenzoic acid, 3,5-Dibromo-6-pentadecyl-2-hydroxybenzoic acid, 3-nitro-6-pentadecyl-2-hydroxybenzoic acid, 3-nitro-6-pentadecyl-2-acetyloxybenzoic acid, 5-nitro-6-pentadecyl-2-hydroxybenzoic acid, 5-nitro-6-pentadecyl-2-acetyloxybenzoic acid, 4-nitro-6-pentadecyl-2-hydroxybenzoic acid, 4-nitro-6-pentadecyl-2-acetyloxybenzoic acid and their use for preventing the growth of microorganisms including gram-negative or gram positive microorganisms such as Stapphalococcus aureus, and Methicillin resistant Stapphalococcus aureus etc.
Detailed description of the invention:
The following examples are provided to illustrate the different embodiments of the invention:
Example-1: Preparation of 6-Pentadecyl-2-acetyloiybenzoic acid (AAA): A
mixture of saturated anacardic acid (100 mg, 0.287 mmol) and acetyl chloride (5ml) was stirred at room temperature for 3 hrs. The reaction mixture was poured into ice-cold water. The product precipitated out and was extracted into ethyl acetate (25ml x 2). The ethyl acetate layer was washed with water (25 ml x 4) and dried over anhydrous Na2S04. The solvent was evaporated under reduced pressure. The residue obtained was triturated with hexane at 10°C. The solid obtained was separated by centrifugation, washed with hexane and dried under vacuum. The yield obtained was 85%( 92 mg). The product had a melting point of 62- 66 °C.
Example-2: Preparation of 3,5-Dinitro-6-pentadecyl-2-bydroxybenzoic acid (DAA): A suspension of anacardic acid (200 mg, 0.574 mmol) in cone. H2SO4 (8ml) was cooled to 0°C. To the cold mixture, cone. HNO3 (2ml, 69% solution) was added slowly maintaining the temperature below 5 °C. The reaction mixture was stirred at 5 °C for 15 min and poured into crushed ice. The product precipitated out and was extracted into ethyl acetate (25ml x 2). The ethyl acetate layer was washed with water
4

(25ml x 4) and dried over anhydrous Na2SO4. Finally the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography using Hexane-ethyl acetate solvent system. The pure product obtained after concentration (oily) was triturated with hexane (5ml) to obtain a solid. The product was separated by centrifugation, washed with hexane (1ml) and dried under vacuum. The yield obtained was 80% (204 mg). The product had a melting point of 78-82°C
Example-3: Preparation of 3,4-Dinitro-6-pentadecyI-2-hydroxybenzoic acid
(NAA): To a suspension of anacaridic acid (200 mg, 0.574 mmol), Cone. H2SO4 (4ml), cone. HNO3 (2ml, 69% solution) was added dropwise over a period of 5min at room temperature. During the addition of nitric acid, the temperature of the reaction mixture raised to about 40 - 45 °C. The mixture was stirred for 15 min and poured into crushed ice. The product precipitated out and was extracted into ethyl acetate (25ml x 2). The ethyl acetate layer was washed with water (25ml x 4) and dried over anhydrous. Na2SO4. Finally the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography. The mobile phase used was hexane-ethylacetate system. The yield obtained was 52% (132 mg). The product obtained after column chromatography was treated with 5ml of 1:1 mixture of DCM /hexane to obtain a solid. The product was separated by centrifugation, washed with hexane and dried under vacuum. The over all yield obtained was 32% (80 mg). The product was found to decompose at 75 -80°C.
Example-4: Preparation of 3,5-Dinitro-6-pentadecyl-2-acetyloxybenzoic acid
(ADAA): A mixture of DAA obtained in Example-2 (80 mg, 0.23 mmol) and acetyl chloride (5ml) was refluxed for 7 hrs. The reaction mixture was poured into icecold water. The product precipitated out and was extracted into ethyl acetate (25ml x 2), washed with water (25 ml x 4) and dried over anhydrous Na2SO4 The solvent was evaporated under reduced pressure. The residue obtained was triturated with hexane (3ml) to obtain a solid. The product was separated by centrifugation, washed with
5

hexane and dried under vacuum. The yield obtained was 52%( 46 nig). The product was found to melt at 55 - 57°C, with decomposition.
Example-5: Preparation of 3,4-Dinitro-6-pentadecyl-2-acetyloxybenzoic acid
(ANAA): A mixture of NAA obtained in Example-3 (25 mg, 0.057 mmol) and acetyl chloride (2ml) was refluxed for 7 hrs. The reaction mixture was poured into icecold water. The product precipitated out and was extracted into ethyl acetate (15ml x 2). The ethyl acetate layer was washed with water (15 ml x 4) and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure. The residue obtained was triturated with hexane (3ml) to obtain a solid. The product was separated by centrifugation, washed with hexane and dried under vacuum for 2hrs. The yield obtained was 51%( 14 mg).
Example-6: Preparation of 3,5-Dibromo-6-pentadecyl-2-hydroxybenzoic acid (BAA): A suspension of saturated anacardic acid (50 mg, 0.143 mmol) in AcOH/H2S04 (0.5ml/50 µj) mixture was heated to 50°C. To the warm mixture a solution of bromine (30µl) in AcOH (0.25 ml) was added dropwise over a period of 5 min. The mixture was heated to 100 °C for 30 min. Then water (5 ml) was added and the product that precipitated out was extracted into ethyl acetate (25ml x 2). The ethyl acetate layer was washed with water (25 ml x 4) and dried over anhydrous Na2SO4.The solvent was evaporated under reduced pressure to obtain a residue. The residue obtained was triturated with hexane (3ml). The product obtained as a solid was separated by centrifugation, washed with hexane and dried under vacuum.The yield obtained was 82%( 60 mg). The product had a melting point of 85 - 90°C.

Dated this 24th day of august 2006

Abstract:
This invention describes method to synthesize derivatives of anacardic acid having antimicrobial properties. These antimicrobial properties include bacteriostatic and bacteriocidal activity especially against Stapphalococcus aureus and Methicillin resistant Stapphalococcus aureus.
7

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=VZDV+66HZIEHVy+7GYQBGw==&loc=vsnutRQWHdTHa1EUofPtPQ==

« Previous Patent

Next Patent »

Patent Number

272748

Indian Patent Application Number

1352/MUM/2006

PG Journal Number

18/2016

Publication Date

29-Apr-2016

Grant Date

25-Apr-2016

Date of Filing

25-Aug-2006

Name of Patentee

UNICHEM LABORATORIES LTD

Applicant Address

MAHALAXMI CHAMBERS, 22 BHULABHAI DESAI ROAD, MUMBAI-400026,

Inventors:

#	Inventor's Name	Inventor's Address
1	RADHA ACHANATH	NO.9, RAGAM, III CROSS, 13TH MAIN ROAD, MATHIKERE EXTENTION BANGALORE-560054,
2	MALLADI SRINIVAS	9/401,PADMINI ENCLAVE,7TH CROSS,VENKATESHA LAYOUT,SANJAYNAGAR,BANGALORE-560094
3	CANDADAI SESHADRI RAMADOSS	108/2,SEETHALAKSHMI APARTMENTS,19TH CROSS, 8TH MAIN,MALLESHWARAM,BANGALORE-560055

PCT International Classification Number

A61K31/192

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA