Synthesis and Antibacterial Activity of Urea and Thiourea Derivatives at C-8 Alkyl Chain of Anacardic Acid Mixture Isolated from a Natural Product Cashew Nut Shell Liquid ( CNSL )

Synthesis and antibacterial activity of some novel urea and thiourea derivatives (7a-7k, 8a-8f) of anacardic acid prepared from commercially available anacardic acid which is obtained from natural product Cashew Nut Shell Liquid (CNSL). Compounds (7a-7k, 8a-8f) were tested for Gram positive and Gram negative bacterial cultures. Most of the compounds were showed active compared with standard drug ampicilline.


Introduction
Among the different families of plants, Anacardiaceaeshrub family is very important since this plant consist of Non-isoprenoid phenolic lipids.The cashew tree, Anacardium occidentale L., is a botanical species native of eastern Brazil and was introduced into other tropical countries such as India, Africa, Indonesia and South East Asia in the 16th century [1][2].Approximately 2 -3 cm in length kidney shaped structure is true fruit of cashew is the nut, which is attached to the end of a fleshy bulb, generally called the cashew apple.The shell consist of the raw nut (50% of the weight), the kernel (25%) and the remaining 25% consists of the natural cashew nut shell liquid (CNSL), a viscous reddish brown liquid.The CN-SL is traditionally obtained as a by-product during the isolation of the kernel by roasting the raw nuts.Crude CN-SL represents one of the major and cheapest sources of naturally occurring non-isoprenoid phenolic lipids such as anacardic acids (1), cardols (2), cardanols (3), methylcardols (4) (Figure 1) and polymeric materials.CNSL has found important commercial usage as the phenolic raw material for the manufacture of certain resins and plastics having unusual electric and frictional properties [3][4][5][6].Anacardic acid mixture (1a-d) isolated from a natural product Cashew Nut Shell Liquid (CNSL) which is a by-product of cashew nut industry and these are salicylic acid derivatives with a nonisoprenoid alk(en)yl side chain [7].Anacardic acid (pentadecyl salicylic acid) is a phenolic constituent present in Cashew Nut Shell Liquid (CNSL); (Anacardium occidentale L.) and exhibits antimicrobial properties [8][9][10][11][12][13][14], which have led to the preparation of various analogues [15][16][17][18][19][20] and soybean lipoxygenase-1 inhibitory activity [21][22] Kubo et al. [23] reported the separation of anacardic acid into monoene (15:1), diene (15:2) and triene (15:3) by preparative HPLC and tested against cancer cells, and found to show moderate cytotoxic activity on BT-20 breast and HeLa epithelioid cervix carcinoma cells.The emergence of drug resistant strains in clinical applications [24][25][26] especially to Gram positive bacteria [27][28] has created a problem of global proportions [29][30] G. C. Reddy et al. reported the synthesis of benzamide derivatives of anacardic acid [31], sildenafil analogues [32], dihydropyridine analogues [33] as calcium channel blockers, isonicotinoylhydrazones for antimycobacterial activity [34] starting from anacardic acid.Recently, a few anacardic acid derivatives exhibited various activities like affect the structure of the enzyme [35], anacardic acid is a specific activator of kinase activity of Aurora Kinase A [36], suppresses expression of nuclear factor-kB regulated gene products leading to potentiation of apoptosis [37] inhibitor of the HAT activeity of recombinant Plasmodium falciparum GCN5 [38] and as modulators of histone acetyltransferases [29].Synthesis of lasiodiplodin from the non-isoprenoid phenolic lipids of CNSL as well as the salicylate macrolactone and other derivatives were reported by santos et al. [40][41][42][43].
In the present work we wish to report to synthesize novel cell permeable urea and thiourea compounds from cheaply available anacardic acid which was a major constituent of Cashew Nut Shell Liquid (CNSL) natural source to evaluate their biological activity by various anti-bacterial strains.This report describes the synthesis, spectroscopic identification and antibacterial activity of some novel urea and thiourea derivatives at C-8 alkyl chain of anacardic acids against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes bacterial strains.

Results and Discussion
Here we described the synthesis of various biologically active novel urea and thiourea derivatives using anacardic acid mixture as starting material and various reagents in the given below conditions (Scheme 1).
The anacardic acid mixture (1a-d) was isolated from commercially available CNSL by a reported method [44,45].Accordingly CNSL was treated with calcium hydroxide, during which anacardic acid present in CNSL becomes calcium anacardate, which was isolated and hydrolyzed with dil.hydrochloric acid to generate anacardic acid ene mixture, which was a mixture of monoene, diene and triene located at (8'), (8',11') and (8',11',14') of the C15 alkyl chain respectively.Anacardic acid ene mixture was methylated using dimethyl sulphate in presence of potassium carbonate in acetonitrile to afford 2. Ozonolysis of Compound 2 resulted in the formation of 3, C8-OH.The compound 3 was converted to 4 using carbon tetra bromide in Dichloromethane.The compound 4 was reacted with sodium azide followed by reduction with Pd/C under H 2 pressure to obtaine amine 6 coupled with various isocynate or isothiocynate in chloroform to obtain compounds (7a-7k, 8a-8f, Scheme 1) of  urea and thiourea derivatives were purified by column chromatography to yield title compounds.The structure of urea and thiourea derivatives (7a-7k, 8a-8f) was determined by using different spectroscopic techniques 1 H NMR, IR, Mass.The resulting compounds are screened for their antibacterial activity.

Biological Activity
The urea and thiourea derivatives (7a-7k, 8a-8f) were screened for their antibacterial activity [27] against some of the pathogenic bacteria viz.E. coli (MTCC443), P. aeruginosa (MTCC424), S. aureus, (MTCC96) and S. pyogenes (MTCC443) using agar well diffusion method according to the literature protocol [46].The anti-bacterial activity of the analogues was compared with standard drug ampicilline and the results of investigation have been presented in Table 1 and observed that some of the compounds are showed high biological activity.Based on the test results it is evident that several of synthesized anacardic acid analogues possess moderate to good activity against the Gram +ve and Gram -ve bacteria.Of all the compounds prepared entities 7a, 7b, 7c, 7d, 7f, 7g, 8b and 8e activity against E. coli, MTCC443, 7a, 7b, 8a, 8d and 8e activity against P. aeruginosa, MT-CC424; display good to excellent activity while the remaining compounds showed moderate activity.The most active antibacterial agent against Escherichia coli found to be compound 7c, 7f, 7g, and 8e having -CN,F groups and other compounds in the series exhibited moderate to good activity.The compounds 7d, 7e, 8b and 8f showed good activity against S. aureus MTCC96 and 7a, 7b, 7e, 7h, 8b, 8c and 8f showed good activity against S. pyogenes MTCC442.This indicates chloro, dichloro, flouro, cyano and methoxy substituted compounds showed better activity when compared to other substituted groups.
Here seems to be, thiourea substituted novel compounds are exhibiting better activity than urea substituted compounds.The activity depends to some extent on the R substituent, however all the compounds showed antibacterial activity.It may be suggested that the anacardic acid derivative with a suitable R may lead to a good antibacterial agent against all the Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes bacterial strains.

Conclusions
In summary, the present study describes a convenient and efficient protocol for the synthesis of sulfonamide derivatives by using anacardic acid mixture using various reagents and different conditions.We believe that this procedure is convenient, economic and a user-friendly process for the synthesis of these various novel urea and thiourea compounds from anacardic acid mixture.All compounds structures are supported by physico chemical and IR, NMR, Mass spectral data.Urea and thiourea derivatives were screened for their antibacterial activity against few bacterial strains and observed that some of the compounds are showed more biological activity than standards used.

General Reagents and Equipment
All chemicals and solvents were obtained from Aldrich and Spectrochem., India and used without further purification.Column chromatographic separations were carried out on silica gel 60 -120 mesh size and eluting with a gradient of hexane: ethyl acetate.Analytical thin layer chromatography was performed on precoated Merck si-lica gel (60F254/0.2mm) plates using UV light, 5% ethanolic phosphomolybdic acid or iodine vapours to visualize the spots.Melting points were determined in open glass capillaries on a Mel-temp apparatus and are uncorrected.The IR spectra were recorded on a Thermo Nicolet IR 200 FT-IR spectrometer as KBr pellets and the wave numbers were given in cm −1 .The 1 H and 13 C NMR spectra of samples were recorded on a Varian EM-360, NMR spectrometer using TMS as an internal standard in CDCl 3 .The mass spectra were recorded on Jeol JMS-D 300 and Finnigan Mat b at 70 eV with an emission current of 100 µA.The oxidative cleavages were performed with a Welsbach T-408 ozonizator and the catalytic hydrogenations in a Parr apparatus.

General Procedure: Isolation of Anacardic Acids (1)
The shells (500 g) of cashew nuts from Anacardiumoccidentale were extracted in a Soxhletextractor with commercial 95% ethanol (2.0 L) during 6 h, yielding a crude extract (CNSL, 157 g, 31% by weight).Anacardic acids (1) were removed in 61% from CNSL (15.25 g) either by precipitation with lead nitrate or calcium hydroxide according to protocols described in the literature [44,45].
The spectral properties were identical to those reported in the literature [44,45].

Preparation of Methyl anacardate methyl ethers ene mixture (2):
To a solution of Compound 1 (65 g, 186.78 mmol) in acetone was added K 2 CO 3 (103.1 g, 747.12 mmol), Di methyl sulfate (44.3 mL, 466.95 mmol).The contents were heated at 65˚C for 5 h.Reaction mixture was cooled to room temperature, filtered and washed with ethyl acetate.Filtrate was distilled off, crude compound was re dissolved in ethyl acetate (300 mL).Organic layer was washed with water, brine solution and dried over anhydrous sodium sulphate and distilled off ethyl acetate.Crude compound was purified by 60 -120 silica pet ether pack column compound was eluted with 5% ethyl acetate: pet ether to get compound 2, Yield: 58 g, light yellow liquid.

Synthesis of 2-(8-Hydroxy-octyl)-6-methoxybenzoic acid methyl ester (3):
A solution of compound 2 (15 g, 40.540 mmol) in dichloro methane: methanol (1:1, 500 mL) was added a pinch of Sudan red catalyst and cooled to -78˚C.Ozone gas purged through reaction mixture until starting material was completed (8 h).Nitrogen gas was purged through reaction mixture for 30 min (to remove excess O 3 gas), dimethyl sulfide was added few drops and stirred for 20 min at -15˚C.Sodium borohydride (9.970 g, 263.51 mmol) was added portion wise over a period of 45 min.Reaction mixture was slowly bring it to room temperature and stirred at this tempera-ture for 18 h.Reaction mixture was quenched with cold water (400 mL), dichloro methane and methanol distilled off and crude compound was diluted with water and extracted with ethyl acetate (2 × 200 mL).The combined organic layer was washed with brine solution (150 mL) dried over anhydrous sodium sulphate, filtered and evaporated under vacuum, to obtaine crude compound was purified by neutral alumina pet ether packed column, compound was eluted with 20% ethyl acetate: pet ether to obtain compound (3) as yellow liquid (

Synthesis of urea and thio urea compounds:
A solution of amine (300 mg, 1.023 mmol) in dry CHCl 3 was taken in seal tube was added isocynate or iso thiocynate (1.22 mmol) at rt and stirred at rt for 3 h to 8 h and distilled off solvent and crude compound was purified by column.Antibacterial Bioassay [27]: Urea and thiourea derivatives of Anacardic acid (7a-7k, 8a-8f) were dissolved in dimethyl sulphoxide at 250 μg/mL concentration.The composition of nutrient agar medium was Bactotryptone (10 g), yeast extract (5 g), NaCl (10 g), final pH 7.4.After 18 h the exponentially growing cultures of the six bacteria in nutrient broth at 37˚C were diluted in sterile broth.From each of these diluted cultures, 1mL was added to 100 mL sterilized and cooled nutrient agar media to give a final bacterial count of 1 × 10 6 cell/ml.The plates were set at room temperature and later dried at 37˚C for 20 h.Paper discs (6 mm, punched from Whatmann no.41 paper) were ultraviolet sterilized and used for the assays.Discs were soaked in different concentration of the test solution and placed on the inoculated agar media at regular intervals of 6 -7 cm, care was taken to ensure that excess solution was not on the discs.All the samples were taken in triplicates.The plates were incubated at 37˚C in an inverted fashion.Activity was determined by zones showing complete inhibition (mm).Growth inhibition was calculated with reference to positive control.