Synthesis and characterization of some novel fatty acid analogues: A preliminary investigation on their activity against human lung carcinoma cell line

Background Preparation of some novel heterocyclic compounds with long alkyl and alkenyl chain of cytotoxic activity. Methods Gamma linolenic acid, a poly unsaturated fatty acid and stearic acid, a saturated fatty acid were isolated from the microalga Spirulina platensis. Some novel gamma linolenic acid and stearic acid analogues having 1,3,4-oxadiazole and 1,2,4-triazole were synthesized and characterized by IR, 1H NMR, 13C NMR and mass spectral analysis. Cytotoxicity of these compounds was evaluated by the growth inhibition of A-549 cells in-vitro. Results Compound 1 and 3 showed comparable cytotoxicity against the human lung carcinoma A-549 cell lines.


Background
Spirulina platensis, blue green microalgae is being widely studied, not only for nutritional reasons but also for its reported medicinal properties [1]. It is a potential source of GLA (Gamma linolenic acid), an essential polyunsaturated fatty acid of excellent economic interest [2]. In-vitro and in-vivo studies have shown GLA to selectively kill tumor cells without harming normal cells [3]. Natural sources of GLA contain variable amounts of this acid which rarely exceed 25%; hence there has been a keen interest in producing higher concentrates of GLA. The commercial methods for producing GLA concentrates include winterization, fractional distillation, urea-inclusion, high performance liquid chromatography and argentated silica gel chromatography [4]. GLA concentrations mentioned above are sufficient for most applications; however several uses, particularly pharmaceutical applications require higher concentrations of GLA, often in excess of 90% [5]. Recently Centrifugal chromatography system was shown to play a vital role in extracting phyto constituents from natural sources.
Although billions of dollars have been spent on research and development on anticancer drugs, the disease remains uncontrolled. A number of investigations have demonstrated that, a variety of modified fatty acid analogues are promising molecules in cancer prevention and have potential in the treatment of cancer [6,7]. During the last two decades, the chemistry of 1,2,4-triazole, 1,3,4-oxadiazole and their derivatives have received considerable attention owing to their anticancer activities [8][9][10].
Based on these findings and continuation of our previous work [11], the present work has been aimed to develop some novel 1,2,4-triazoles and 1,3,4 oxadiazoles synthesized from GLA which is isolated from Spirulina platensis, and evaluate their anticancer activity. GLA was isolated from Spirulina platensis by a novel method using Cyclograph centrifugal chromatography system.

Results and Discussion
Isolation of GLA methyl ester Fatty acid methyl esters (FAME) were prepared from freeze dried biomass of Spirulina platensis, which was then subjected to urea fractionation for getting gamma linolenic acid methyl ester by extraction with n-hexane. The n-hexane fraction was quantified by HPTLC and was found to contain 57.62% w/w of GLA methyl ester. The GLA methyl ester was separated by Cyclograph Centrifugal Chromatography System, which combines the advantages of both preparative TLC and column chromatography. It delivers fast and efficient separations. Centrifugal action combined with the use of a solvent pump to apply the mobile phase allows complete control of solvent velocity profile.
In-vitro cytotoxicity screening Compounds 1, 2, 3 and 4, were tested for cytotoxic potential in A549 (lung adenocarcinoma) cells by determination of CTC 50 (concentration of the sample required to kill 50% of the cells) by SRB assay. The experiments were carried out in triplicate and the results are presented in Table 1. The compounds showed dosedependent destruction of the cell. The 1,3,4-oxadiazole substituted fatty acid analogues 1 and 3 showed maximum cytotoxic activity. The presence of toxophoric -N=C-O-linkage in 1,3,4-oxadiazole nucleus may be responsible for the activity. Further, 1,3,4-oxadiazole is a good bioisostere of amide and ester functionalities with substantial improvement in biological activity in hydrogen bonding interactions with different receptors. It is also observed that the length of the fatty acids play a vital role in anti-tumor activity. The 1,2,4-triazole substituted fatty acid analogues 2 and 4 displayed promising cytotoxicity.

Materials
All chemicals used were purchased from Fluka chemicals. Their purity was checked by GC. All solvents were purified by distillation and if necessary residual water was removed. The composition of solvents and eluents are given in volume ratios of the components. Fresh cultures of Spirulina platensis was obtained from Antenna Research Foundation Pvt Ltd., Madurai, Tamilnadu, India. The cell paste was lyophilized and stored at −20°C for further use. Stearic acid ester was isolated by previously reported method [11]. GLA ester was isolated by using Cyclograph Centrifugal Chromatography System (Analtech inc). Products were purified by the column chromatography and identified using different spectral techniques. Melting points were taken in glass capillary tubes on a Veego VMP-1 apparatus and are uncorrected. The 1 H NMR and 13 C NMR were recorded on Bruker DRX-300 (300 MHZ FT-NMR) using deuterated chloroform as solvent and TMS as internal standard. The mass spectra of compounds were recorded on JEOL GC MATE II GC-MS.
General procedure for isolation of GLA methyl ester The cyclograph system is a centrifugally accelerated device for performing preparative chromatographic separations. The device spins a layer of adsorbent material coated as a flat ring on a glass backing. A solvent pump is used to apply the sample and mobile phase to the centre of the spinning adsorbent ring. The centrifugal action accelerates the flow of the mobile phase through the adsorbent, separating the sample components as circular bands. The mobile phase elutes continuously into a specially shaped collection channel inside the body of the instrument. Component bands are collected manually in test tubes or optionally by an automated fraction collector (Figure 1).

Preparation of fatty acid methyl esters (FAME)
Freeze dried biomass of Spirulina platensis (50 g) was extracted by reflux for 4 hours using a mixture of methanol and acetyl chloride (95:5, 800 ml). The extract obtained was diluted with water and extracted thrice with equal volume of n-hexane containing 0.01% butylated hydroxyl toluene. The combined n-hexane layer was evaporated to get the fatty acid methyl ester (FAME).

Enrichment of FAME for GLA methyl ester
The FAME obtained was subjected to urea complexation as described previously to remove the saturated fatty acids from the polyunsaturated fatty acids. For the urea complexation, methanol (9 ml) and urea (3 g) were added to FAME. The mixture was heated to get a clear solution. It was cooled at room temperature and stored at 0°C overnight. Then it was filtered to remove the crystals settled at bottom. The filtrate was extracted with n-hexane containing 0.01% butylated hydroxyl toluene. The n-hexane fraction was evaluated for the presence of GLA methyl ester by HPTLC.
Hydrazine hydrate (99%, 5 mmol) was placed in a round bottom flask fitted with a reflux condenser. To this solution methyl stearste/methyl gamma linolenate (5 mmol) was added dropwise and heated gently for 15 min. Then absolute ethanol (5 ml) was added through the condenser to produce clear solution and refluxed for 4-5 h. The ethanol was distilled off and cooled. To this stearic acid hydrazide/gamma linolenic acid hydrazide solution, a mixture of potassium hydroxide (5 mmol), carbon disulphide (5.51 mmol) and ethanol (20 ml) was added and further refluxed for 10 h. After concentration of the solution to small volume, the residue was dissolved in 10 ml of water. The precipitate was obtained by adding the solution to ice containing conc.HCl.  To a mixture of appropriate oxadiazole 1/3 (10 mmol) in absolute ethanol (10 ml), hydrazine hydrate (10 mmol) was added and the reaction mixture was refluxed for 5 h. On completion of reaction potassium hydroxide (10 mmol) was added to the reaction mixture and the precipitate formed was filtered. The solid obtained was acidified with conc. HCl to pH-3 and washed with water. The resultant solid was recrystallised from ethanol.

Cell lines and culture medium
A-549 cell cultures used in the experiments were procured from National Centre for Cell Sciences, Pune, India. A-549 cells were grown in Earl's Minimal Essential Medium supplemented with 2 mmol L-glutamine, 10%