Synthesis, Characterization and Cytotoxic Studies of Benzamide Derivatives of Anacardic Acid using Human Liver Cancer Cells

Naturally occurring anacardic acid based benzamides were reported to show anti-inflammatory and anticancer activities, where we synthesized and characterized by NMR and HRMS analysis and also tested a series of anacardic acid based benzamides and evaluated against the proliferation of human liver cancer cells (HepG2). Among the tested compounds, 6j-m showed good inhibitory activity against HepG2 cells with IC 50 values ranging from 78.2-91.9 μM. In conclusion, we herein reported the newer series of an academic acid benzamides for the first time.


Introduction
Cancer is a deadly disease in the world with the highest mortal rate [1]. Based on the source and origin of the cancer cells, they are classified into carcinoma, sarcoma, lymphoma or leukemia [2]. Uncontrolled cell proliferation, anti-apoptosis, angiogenesis, metastasis, and genomic instability are the most common features of all types of cancers [3]. There are many ways to treat cancer such as chemotherapy, radiation therapy, surgery, stem cell transplant, immunotherapy, precision medicine etc. Among them, in chemotherapy small molecules are used to treat cancer via different mechanisms.

I Chemistry
Initially, anacardic acid was isolated according to reported protocol. Further, the liquid was catalytically hydrogenated and purified using column chromatography to obtain pure anacardic acid. Anacardic acid 1 was converted into its corresponding ester 3 by treatment with dimethyl sulphate (2a), diethyl sulphate (2b) and isopropyl chloride (2c). Meanwhile, phenolic-OH was alkylated by these reagents into respective alkoxy groups according to reported methods [6,28,29]. Later, alkylated carboxylic acid group was selectively cleaved into carboxylic acid 4 by heating with potassium tertiary butoxide in DMSO. Coupling of carboxylic acid 4 with various amines 5 in the presence of 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC . HCl), hydroxyl benzotriazole (HOBt) and base triethylamine furnished final products: amides of anacardic acid 6. The structures and yields of all title compounds are given in (Table 1). The schematic representation of the synthesis of title compounds were shown in Scheme 1. Further, these compounds were completely characterized by 1 H NMR, 13 C NMR and IR spectroscopic techniques.
Scheme 1: Synthesis of amides of anacardic acid. Using a basic MTT colorimetric assay, we carried out the antiproliferative effect of anacardic acid derivatives on Human liver cancer cells (HepG2) and it was found that the compounds 6j, 6k, 6l and 6m significantly decreased the proliferation of HepG2 cells with an IC50 values of 86.8, 78.2, 81.6 and 91.9 μM respectively ( Table 1). The structure activity relationship studies indicated that anacardic acids with methoxy substituents on benzene ring are inactive against HepG2 cells (<100μM). While, their analogues with ethoxy substituent are active (6jm) with aforementioned IC50 values. While isobenzofuranone, phenylthiadiazole, 3-brommopyridine and 3-cyano-pyrrole were inactive (<100μM). Finally, anacardic acid derivative with isopropyloxy group is also inactive (<100μM).

Experimental Section I General Methods/ Materials/Instrumentation
CNSL was purchased from commercial source and all other reagents are of analytical grade purchased from Aldrich. The compounds were analysed and confirmed through 1H and 13C NMR spectra using CDCl3/DMSO-d6 as a sovlent at 400 & 100MHz respectively on a Bruker A.G Spectrometer. Chemical Shifts were recorded using tetra methyl silane (TMS) as an internal standard. Nicolet avatar 320 FT-IR spectrometer was used to record IR spectra. Acro Steel Pvt. Ltd. melting point apparatus was used to determine melting points.

II General Procedure for the Preparation of Final Compounds 6
2-alkoxy-6-pentadecylbenzoic acid 4 (0.6 mmol) was dissolved in dichloromethane (5 mL) using a magnetic stirrer and cooled to 0°C . To that EDC.HCl (0.6 mmol), HOBt (0.6 mmol) and amine 5 (0.6 mmol) were added. The completion of the reaction was monitored through TLC and neutralized with dilute HCl (25 mL) extracted with ethyl acetate (25 mL x 3), washed with water and brine (25 mL) and dried over sodium sulphate and concentrated. The obtained crude title compounds were purified by column chromatography using silica gel (60-120 mesh) and 0-5% ethyl acetate in petroleum ether as an effluent to procure white solids of pure title compounds.

XVII In Vitro Cytotoxicity Assay
The recently synthesized derivatives were assessed for toxicity against hepatocellular carcinoma (HepG2) cell lines as reported earlier [30][31][32]. Briefly, the cells (1 X 104/ml) were transferred to 96-well microtiter plate in with or without novel compounds of different concentration of 0.15ml and incubated at 37oC for 72h. After incubation, 20 μl of MTT (5 mg/ml in PBS) dye was transferred to the wells and incubated for 2h at 37oC. The incubation was continued for 24h after the addition of 0.1ml lysis buffer (20% SDS, 50%DMF) and their obsorbance was measured using a microplate reader at a wavelength of 570nm.

Conclusion
In conclusion, the toxicity of newly synthesized benzamide derivatives of anacardic acid were tested against human liver cancer cell lines (HepG2). Amongst, 6j-m showed good inhibitory activity with IC50 values 78.2-91.9 μM.

Consent for Publication
All the authors given consent for this publication.

Conflicts of Interest
None.

Funding
None.

Supplementary Material
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