Study on Synthesis, Characterization and Antiproliferative Activity of Novel Diisopropylphenyl Esters of Selected Fatty Acids
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Journal of Oleo Science Copyright ©2016 by Japan Oil Chemists’ Society J-STAGE Advance Publication date : December 11, 2015 doi : 10.5650/jos.ess15151 J. Oleo Sci. Study on Synthesis, Characterization and Antiproliferative Activity of Novel Diisopropylphenyl Esters of Selected Fatty Acids Yasa Sathyam Reddy1, Shiva Shanker Kaki1, Bala Bhaskara Rao2, Nishant Jain2 and Penumarthy Vijayalakshmi1,＊ 1 Centre for Lipid Research, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500007, Telangana, INDIA 2 Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500007, Telangana, INDIA Abstract: The present study describes the synthesis, characterization and evaluation of antiproliferative activity of novel diisopropylphenyl esters of alpha-linolenic acid (ALA), valproic acid (VA), butyric acid (BA) and 2-ethylhexanoic acid (2-EHA). These esters were chemically synthesized by the esterification of fatty acids with 2,6-diisopropylphenol and 2,4-diisopropylphenol (propofol). The structure of new conjugates viz. propofol-(alpha-linolenic acid) (2,6P-ALA and 2,4P-ALA), propofol-valproic acid (2,6P-VA and 2,4P-VA), propofol-butyric acid (2,6P-BA and 2,4P-BA) and propofol-(2-ethylhexanoic acid) (2,6P- 2-EHA and 2,4P-2-EHA) were characterized by FT-IR, NMR (1H, 13C) and mass spectral data. The synthesized conjugates having more lipophilic character were tested for antiproliferative in vitro studies on A549, MDA-MB-231, HeLa, Mia-Pa-Ca and HePG2 cancer cell lines. All the conjugates showed specific growth inhibition on studied cancer cell lines. Among the synthesized esters, the conjugates synthesized from BA, VA and 2-EHA exhibited prominent growth inhibition against A549, HeLa, Mia-Pa-Ca and HePG2 cancer cell lines. The preliminary results suggest that the entire novel conjugates possess antiproliferative properties that reduce the proliferation of cancer cells in vitro. Key words: propofol, fatty acids, prodrugs, synthesis, antiproliferative activity 1 INTRODUCTION therapeutic agents11, 12）. It is also reported that fatty acids Anticancer drugs despite showing progress in the treat- exhibit broad spectrum of activity which make them attrac- ment of malignant diseases are commonly associated with tive molecules for preparing various biologically active systematic toxicity and other effects1, 2）. The common ap- compounds of interest in biomedical fields. In view of these proach to overcome such difficulty and to increase the properties, various fatty acids have been combined with drug efficiency is to use the anticancer drug conjugated therapeutically active compounds to produce novel hybrid with other substitute. Hence, a molecule that shows suit- molecules anticipating enhanced biological activity13, 14）. able properties is chemically changed by coupling to To attain effective tumor-specific drug delivery, the fatty another therapeutically active compound to achieve the acids have been chemically modified to improve their spec- best combination of properties in forming the most effec- ificity, biological activity and lower toxicity against a range tive anticancer drug3, 4）. Among these chemically modified of therapeutic targets15）. Studies on the synthesis of fatty drugs are the esters of fatty acids, chosen as anticancer acid esters, where fatty acid was conjugated with propofol drug conjugates because of their low toxicity5, 6）. in order to enhance its activity, lipid-solubility, bioavailabil- It is reported that the fatty acids are taken up rapidly by ity, and reduce its side-effects are reported6, 15－19）. tumor cells7） and their hydrophobic nature facilitates their Propofol（diisopropylphenol）is one of the most widely incorporation into the lipid bilayer of cells8, 9）, resulting in accepted chemical agents used for induction of anesthesia disruption of membrane structure and fluidity10）. In view of and is reported to be non-toxic to human at high concen- these characteristics of fatty acids, they have been widely trations（3–8 μg/mL; 20–50 μM）20）. The presence of two used to enhance the anticancer activity of various chemo- isopropyl groups at the ortho position with respect to the ＊Correspondence to: Dr. P. Vijayalakshmi, Centre for Lipid Research, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, Telangana, INDIA E-mail: [email protected] Accepted October 19, 2015 (received for review June 23, 2015) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs 1 Y. S. Reddy, S. S. Kaki and B. B. Rao et al. hydroxyl group in propofol exerts a steric hindrance that have employed them to couple with propofol as esters. prevents entry of hydrophilic molecules to the hydroxyl Therefore, the present study was aimed at the synthesis, group. This makes the molecule highly hydrophobic. While characterization and antiproliferative activity for the conju- it is an intravenous sedative-hypnotic agent, propofol also gates of propofol（2,4-diisopropylphenol（2,4 P）and 2,6-di- shows preferential scavenging of organo-radical species. isopropylphenol（2,6 P））esters of ALA, VA, BA and 2-EHA. Clinically relevant concentrations of propofol are reported to decrease the metastatic potential of human cancer cells and have been shown to induce apoptosis involving both extrinsic and intrinsic pathways21, 22）. 2 EXPERIMENTAL Earlier study on propofol-fatty acid conjugates was 2.1 Chemicals mostly concentrated on the long chain fatty acids including The raw materials needed for the synthesis of propofol- poly unsaturated fatty acids and there are no studies re- fatty acid ester conjugates of selected four fatty acids, such ported on the conjugates involving short chain and medium as alpha-linolenic acid（ALA）, was extracted from eripupal chain fatty acids6, 17, 18）. In the present study, we selected oil according to a reported procedure41）, valproic acid（VA） four unusual fatty acids such as alpha-linolenic acid（ALA）, was purchased from Alfa-Aesar Chemicals（USA）. Butyric valproic acid（VA）, butyric acid（BA）and 2-ethylhexanoic acid（BA）, 2-ethylhexanoic acid（2-EHA）, 2,4-diisopropyl- acid（2-EHA）which have not been employed for such con- phenol, 2,6-diisopropylphenol and N,N’-dicyclohexylcar- jugation with propofol. Each of these fatty acids have a bodiimide（DCC）, 4-（dimethyl amino）pyridine（DMAP） wide range of applications and a brief description about were purchased from Sigma-Aldrich Chemicals（USA）. All each of these fatty acids has been provided. solvents and chemicals were of reagent grade and used di- Alpha-linolenic acid（ALA）is an essential omega-3 fatty rectly without further purification. Silica gel（60-120 mesh） acid which should be obtained through diet. ALA is report- for column chromatography was purchased from Acme ed to possess a wide range of biological activities including Synthetic Chemicals, Mumbai, India. Precoated TLC plates nutraceutical applications and potential use in cardiovas- were purchased from Merck, Darmstadt, Germany. Human cular diseases23）. ALA is also used to treat rheumatoid ar- tumor cell lines were obtained from American Type Culture thritis, multiple sclerosis, diabetes, lupus, ulcerative colitis, Collection, Manassas, VA, USA. renal disease, and Crohn’s disease24, 25）. Valproic acid（VA）, also known as dipropylacetic acid, is 2.2 Analytical methods a branched eight carbon fatty acid that is reported as a IR spectra were recorded on a Perkin Elmer（Model: 1 potent anticancer agent inhibiting cancer cells both in Spectrum BX）FT-IR spectrometer using CHCl3. All H and vitro and in vivo26）. VA’s beneficial properties were seren- 13C spectra were recorded on 300 MHz（Brucker）and 500 dipitously discovered while it was being used as a vehicle MHz（Varian）spectrometers, respectively. HRMS data were to deliver other compounds that were being tested for their recorded on a Thermo Scientific Exactive Orbitrap mass anticonvulsant activity. Researchers also observed that it spectrometer（Germany）and are given in mass units（m/z）. had substantial ability to prevent seizures27）. Valproic acid ESI-MS spectra were recorded on Waters（Model: Q STAR derivatives are also found to be potent in anticonvulsant XL, Applied Biosystems, USA）Mass Spectrometer and antioxidant properties28－30）. equipped with an Electrospray Ionization source. Elemen- Butyric acid（BA）is well known for its anticancer effects tal analyses were carried out by using Elementar Vario as it induces morphological and biochemical differentiation Micro Cube instrument（Germany）. Gas chromatography in a variety of cells leading to concomitant suppression of was performed on an Agilent 6850 gas chromatograph neoplastic properties31－33）. Butyrates are also reported to equipped with a flame ionization detector. The column help to protect colonic mucosa from oxidative stress and used was a HP-1 column having a length of 30 m, 0.25 mm inhibit its inflammation while promoting satiety34）. In view i.d and 0.25 μm film thickness. The carrier gas was nitrogen of this, several butyric acid derivatives have been designed at a flow rate of 1 mL・min－1. The oven programming was and synthesized as prodrugs of butyric acid35, 36）. BA esters as follows: 150℃ for 2 minutes, which rose to 300℃ at a such as methyl, ethyl, and amyl butyrates are also reported rate of 10℃ min－1 and held at 300℃ for 20 minutes. The to be used as fragrant and flavoring agents in beverages, injector and detector temperatures were maintained at 280 foods and cosmetic industries37, 38）. and 300℃ respectively. 2-Ethylhexanoic acid（2-EHA）, a branched aliphatic car- boxylic acid is of particular importance in material science, 2.3 Synthesis catalysis, paint industries, lubricant industries and has a Synthesis of propofol esters of ALA was carried out as variety of commercial uses39, 40）. It has been reported to shown in Fig. 1. The synthesis involves esterification of have applications in cosmetics to produce emollients. In propofol isomers i.e., 2,4-diisopropylphenol（or）2,4-propo- view of the above reported activities of the fatty acids we fol and 2,6-diisopropylphenol（or）2,6-propofol with ALA to 2 J.