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 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), (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 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. Oleo Sci. Study on Synthesis, Characterization and Antiproliferative Activity of Novel Diisopropylphenyl Esters of Selected Fatty Acids

－1 get the desired compounds 2,4P-ALA and 2,6P-ALA. Other Light yellow liquid; Yield-97.6％; FT-IR（cm , CHCl3）: fatty acids such as VA, BA and 2-EHA were also esterified 3011（s, C＝C–H symmetric mode）, 2963, 2928（s, alkyl in the similar manner with propofol to obtain desired prod- chain -C–H symmetric mode）, 2854（s, Ar–H symmetric ucts like 2,4P-VA, 2,6P-VA, 2,4P-BA, 2,6P-BA, 2,4P-2-EHA mode）, 1757（s, -C＝O symmetric mode）, 1460（s, -C–H and 2,6P-2-EHA. scissoring mode）, 1164（m, -C–O symmetric mode）, 1135 1 2.3.1 General procedure for synthesis of propofol-fatty （s, –O–C＝O asymmetric mode）; H-NMR（CDCl3）δ: 7.22- acid conjugates 7.13（3H, m, Ar–C–H）, 5.43-5.28（6H, m, vinylic H）, 2.90

A synthetic approach was designed using the reported （2H, sept, J＝6.87 Hz, Ar–CH–（CH3）2）, 2.82（4H, dd, J＝ 16） protocol . A 1 equivalent amount of the individual fatty 5.95, 5.95 Hz,（＝CH–CH2–CH＝）2）, 2.61（2H, t, J＝7.63 Hz,

acid was dissolved in dichloromethane（DCM）. To this –CO–CH2–CH2–））, 2.12-2.04（4H, m）, 1.84-1.76（2H, m）, mixture, 1.05 equivalent of coupling reagent N,N-dicyclo- 1.48-1.41（2H, m）, 1.40-1.29（6H, m）, 1.19（12H, d, J＝6.87 13 hexylcarbodiimide（DCC）was added and the reaction Hz, Ar–CH–（CH3）2）, 0.98（3H, t, J＝7.45 Hz）; C-NMR

mixture was stirred for 15 min at room temperature fol- （CDCl3）δ: 172.3, 145.5, 140.2, 131.9, 130.1, 128.2, 128.1, lowed by the addition of 1 equivalent of propofol isomer 127.7, 127.0, 126.3, 123.8, 34.1, 29.5, 29.2, 29.1 X 2, 27.4, （2,6-propofol/2,4-propofol）and 0.152 equivalents of 4-di- 27.1, 25.6, 25.5, 25.0, 23.6, 22.7, 20.5, 14.2; ESI-MS（m/z） ＋ ＋ methylaminopyridine（DMAP）and the reaction mixture was （M＋NH4）（C30H46O2＋NH4） 456.38: HRMS-ESI（m/z）cal- ＋ stirred for a period of 12 h in the dark under nitrogen at- culated 456.3836, found（M＋NH4） 456.3825, Elem. Anal:

mosphere. The progress of the reaction was monitored on calculated for C30H46O2: C, 82.14; H, 10.57. Found: C, 82.11; thin layer chromatography（TLC）and the reaction products H, 10.55. were detected in iodine vapour. Then the crude reaction 2,4-propofol-valproic acid（or）2,4-diisopropylphenyl- product was filtered and the filtrate was concentrated 2-propylpentanoate（2,4P-VA） －1 under reduced pressure. Finally, the synthesized crude Colorless liquid; Yield-98.0％; FT-IR（cm , CHCl3）: 2960, product was purified by silica gel column chromatography 2933（s, alkyl chain -C–H symmetric mode）, 2873（s, Ar–H with solvent system hexane and ethyl acetate（98:2 v/v）to symmetric mode）, 1755（s, -C＝O symmetric mode）, 1462 obtain the corresponding esters. The purity of each ester （s, -C–H scissoring mode）, 1130（m, -C–O symmetric was further monitored by GC. mode）, 1116（s, –O–C＝O asymmetric mode）; 1H-NMR

2.3.2 Spectral data of propofol-fatty acid conjugates （CDCl3）δ: 7.14（1H, d, J＝2.08 Hz, Ar–C3–H）, 7.04（1H, dd, 2,4-propofol-（alpha-linolenic acid）（ or）（ 9Z,12Z,15Z） J＝8.31, 2.08 Hz, Ar–C5–H）, 6.88（1H, d, J＝8.31 Hz, Ar–

-2,4-diisopropylphenyl octadeca-9,12,15-trienoate（2,4P- C6–H）, 3.03（1H, sept, J＝6.87 Hz, Ar–C2–CH（CH3）2）, 2.89

ALA） （1H, sept, J＝6.87 Hz, Ar–C4–CH（CH3）2）, 2.64（1H, －1 Light yellow liquid; Yield-98.0％; FT-IR（cm , CHCl3）: quintet, J＝6.80 Hz, C2-H）, 1.86-1.70（2H, m）, 1.64-1.36

3066（s, ＝C–H symmetric mode）, 2963, 2926,（s, alkyl （6H, m）, 1.24（6H, d, J＝6.87 Hz, Ar–C2–CH（CH3）2）, 1.21

chain -C–H symmetric mode）, 2854（s, Ar–H symmetric （6H, d, J＝6.87 Hz, Ar–C4–CH（CH3）2）, 0.97（6H, t, J＝7.18 13 mode）1756（s, -C＝O symmetric mode）, 1464（s, -C–H scis- Hz）; C-NMR（CDCl3）δ: 175.0, 146.3, 146.0, 139.5, 124.4, soring mode）, 1177（m, -C–O symmetric mode）, 1137（s, – 124.2, 121.7, 45.4, 34.6, 33.8, 27.1, 24.0, 22.9, 20.6, 14.0; 1 ＋ ＋ O–C＝O asymmetric mode）; H-NMR（CDCl3）δ: 7.13（1H, d, ESI-MS（ m/z）（ M ＋NH4 ）（ C 20H 32O 2 ＋NH4 ） 322; ＋ J＝2.07 Hz, Ar–C3–H）, 7.05（1H, dd, J＝8.31, 2.07 Hz, Ar– HRMS-ESI（m/z）calculated 322.2740, found（M＋NH4）

C5–H）, 6.89（1H, d, J＝8.31 Hz, Ar–C6–H）, 5.46-5.25（6H, 322.2734, Elem. Anal: calculated for C20H32O2: C, 78.90; H,

m, vinylic H）, 2.99（1H, sept, J＝6.87 Hz, Ar–C2–CH（CH3） 10.59. Found: C, 78.89; H, 10.58.

2）, 2.89（1H, sept, J＝6.87 Hz, Ar–C4–CH（CH3）2）, 2.81（4H, 2,6-propofol-valproic acid（or）2,6-diisopropylphenyl-

dd, J＝5.67, 5.67 Hz,（＝CH–CH2–CH＝）2）, 2.57（2H, t, J＝ 2-propylpentanoate（2,6P-VA） －1 7.5 Hz, –CO–CH2–CH2–））, 2.14-1.99（4H, m）, 1.84-1.70（2H, Colorless liquid; Yield-97.3％; FT-IR（cm , CHCl3）: 2962, m）, 1.48-1.29（8H, m）, 1.24（6H, d, J＝6.87 Hz, Ar–C2–CH 2934（s, alkyl chain -C–H symmetric mode）, 2873（s, Ar–H

（CH3）2）, 1.20（6H, d, J＝6.87 Hz, Ar–C4–CH（CH3）2）, 0.98 symmetric mode）, 1753（s, -C＝O symmetric mode）, 1463 13 （3H, t, J＝7.55 Hz）; C-NMR（CDCl3）δ: 172.5, 146.4, 145.9, （s, -C–H scissoring mode）, 1160（m, -C–O symmetric 139.5, 131.9, 130.2, 128.2, 128.2, 127.7, 127.0, 124.6, 124.3, mode）, 1128（s, –O–C＝O asymmetric mode）; 1H-NMR

121.8, 34.3, 33.8, 29.5, 29.1 X 5, 27.4, 27.1, 25.6, 25.5, 25.0, （CDCl3）δ: 7.23-7.11（3H, m, Ar–C–H）, 2.92（2H, sept, J＝ ＋ 24.0, 22.9, 20.5, 14.2; ESI-MS（m/z）（ M＋NH4）（C30H46O2＋ 6.87 Hz, Ar–CH（CH3）2）, 2.69（1H, quintet, J＝6.74 Hz, ＋ NH4） 456.4; HRMS-ESI（m/z）calculated 439.3571, found C2-H）, 1.90-1.75（2H, m）, 1.70-1.34（6H, m）, 1.19（12H, d, J ＋ （M＋H） 439.3574. Elem. Anal: calculated for C30H46O2: C, ＝6.87 Hz, Ar–CH–（CH3）2）, 0.98（6H, t, J＝7.18 Hz）; 13 82.14; H, 10.57. Found: C, 82.17; H, 10.56. C-NMR（CDCl3）δ: 174.6, 145.5, 140.3, 126.2, 123.7, 45.0, ＋ 2,6-propofol-（alpha-linolenic acid）（ or）（ 9Z,12Z,15Z） 33.9, 27.2, 23.7, 22.7, 20.5, 14.0, ESI-MS（m/z）（ M＋NH4） ＋ -2,6-diisopropylphenyl octadeca-9,12,15-trienoate（2,6P- （C20H32O2＋NH4） 322.25; HRMS-ESI（m/z）calculated ＋ ALA） 322.2741, found（M＋NH4） 322.2736. Elem. Anal: calcu-

3 J. Oleo Sci. Y. S. Reddy, S. S. Kaki and B. B. Rao et al.

lated for C20H32O2: C, 78.90; H, 10.59. Found: C, 78.87; H, 47.4, 33.8, 31.7, 29.6, 27.1, 25.5, 24.0, 23.0, 22.6, 13.9, 11.9; ＋ ＋ 10.58. ESI-MS（m/z）（ M＋NH4）（C 20H 32O 2＋NH4） 322.26; ＋ 2,4-propofol-butyric acid（or）2,4-diisopropylphenyl HRMS-ESI（m/z）calculated 322.2740, found（M＋NH4）

butyrate（2,4P-BA） 322.2733. Elem. Anal: calculated for C20H32O2: C, 78.90; H, －1 Colorless liquid; Yield-97.5％; FT-IR（cm , CHCl3）: 2963, 10.59. Found: C, 78.93; H, 10.61. 2928（s, alkyl chain -C–H symmetric mode）, 2873（s, Ar–H 2,6-propofol-（2-ethylhexanoic acid）（ or）2,6-diisopropyl- symmetric mode）, 1760（s, -C＝O symmetric mode）, 1495 phenyl-2-ethylhexanoate（2,6P-2-EHA） －1 （s, -C–H scissoring mode）, 1177（m, -C–O symmetric Colorless liquid; Yield-98.0％; FT-IR（cm , CHCl3）: 2963, mode）, 1151（s, –O–C＝O asymmetric mode）; 1H-NMR 2931（s, alkyl chain -C–H symmetric mode）, 2871（s, Ar–H

（CDCl3）δ: 7.13（1H, d, J＝2.14 Hz, Ar–C3–H）, 7.04（1H, dd, symmetric mode）, 1753（s, -C＝O symmetric mode）, 1462 J＝8.24, 2.14 Hz, Ar–C5–H）, 6.90（1H, d, J＝8.24 Hz, Ar– （s, -C–H scissoring mode）, 1160（m, -C–O symmetric 1 C6–H）, 2.99（1H, sept, J＝6.87 Hz, Ar–C2–CH（CH3）2）, 2.89 mode）, 1124（s, –O–C＝O asymmetric mode）; H-NMR

（1H, sept, J＝6.87 Hz, Ar–C4 –CH（CH3）2）, 2.55（2H, t, J＝ （CDCl3）δ: 7.22-7.13（3H, m, Ar–C–H）, 2.94（2H, sept, J＝

7.47 Hz, OC–CH2–CH2–）, 1.80（2H, qt, J＝7.47, 7.47 Hz, – 6.87 Hz, Ar–CH（CH3）2）, 2.60（1H, tt, J＝6.75, 6.75 Hz,

H2C–CH2–CH3）, 1.24（6H, d, J＝6.87 Hz, Ar–C2–CH（CH3）2）, C2-H）, 1.92-1.79（2H, m）, 1.74-1.61（2H, m）, 1.48-1.33（4H,

1.20（6H, d, J＝6.87 Hz, Ar–C4–CH（CH3）2）, 1.05（ 3H, t, J m）, 1.19（12H, d, J＝6.87 Hz, Ar–CH–（CH3）2）, 1.04（3H, t, J 13 13 ＝7.47 Hz）; C-NMR（CDCl3）δ: 172.4, 146.4, 145.9, 139.4, ＝7.48 Hz）, 0.94（3H, t, J＝7.17 Hz）; C-NMR（CDCl3）δ: 124.5, 124.3, 121.8, 36.2, 33.8, 27.4, 24.0, 22.9, 18.5, 13.6; 174.6, 145.5, 140.3, 126.2, 123.7, 46.9, 31.0, 29.5, 27.2, ＋ ＋ ESI-MS（ m/z）（ M ＋NH4 ）（ C 16H 24O 2 ＋NH4 ） 266; 24.7, 23.7, 22.7, 13.9, 11.8; ESI-MS（m/z）304.24, found（M ＋ ＋ ＋ HRMS-ESI（m/z）calculated 266.2114, found（M＋NH4） ＋NH4）（C20H32O2＋NH4） 322.20; HRMS-ESI（m/z）calcu- ＋ 266.2110. Elem. Anal: calculated for C16H24O2: C, 77.38; H, lated 322.2746, found（M＋NH4） 322.2743. Elem. Anal:

9.74. Found: C, 77.37; H, 9.73. calculated for C20H32O2: C, 78.90; H, 10.59. Found: C, 78.86; 2,6-propofol-butyric acid（or）2,6-diisopropylphenyl H, 10.60. butyrate（2,6P-BA） －1 Colorless liquid; Yield-97.0％; FT-IR（cm , CHCl3）: 2964, 2.4 In vitro antiproliferative activity: 2927（s, alkyl chain -C–H symmetric mode）, 2872（s, Ar–H The cell lines, human epithelial lung carcinoma（A-549）, symmetric mode）, 1758（s, -C＝O symmetric mode）, 1462 breast adenocarcinoma（MDA-MB-231）, human cervical （s, -C–H scissoring mode）, 1154（m, -C–O symmetric （HeLa）, human pancreatic carcinoma（Mia-Pa-Ca）, human mode）, 1097（s, –O–C＝O asymmetric mode）; 1H-NMR liver carcinoma（HePG2）which were used in this study

（CDCl3）δ: 7.25-7.11（3H, m, Ar–C–H）, 2.91（2H, sept, J＝ were procured from American Type Culture Collection

6.87 Hz, Ar–CH（CH3）2）, 2.60（2H, t, J＝7.55 Hz, OC–CH2– （ATCC）, United States. The synthesized test compounds

CH2–）, 1.83（2H, qt, J＝7.55, 7.36 Hz, –H2C–CH2–CH3）, 1.19 were evaluated for their in vitro antiproliferative activity

（12H, d, J＝6.87 Hz, Ar–CH–（CH3）2）, 1.07（3H, t, J＝7.36 in these five different human cancer cell lines followed by 13 42） Hz）; C-NMR（CDCl3）δ: 172.1, 145.5, 140.2, 126.3, 123.8, literature method . A protocol of 48 hrs continuous drug ＋ 36.0, 27.4, 23.6, 22.7, 18.5, 13.7; ESI-MS（m/z）（ M＋NH4） exposure was used, and a SRB cell proliferation assay was ＋ （C 16H 24O 2＋NH4） 266; HRMS-ESI（m/z）calculated used to estimate cell viability or growth. All the cell lines 249.1849, found（M＋H）＋ 249. 1849. Elem. Anal: calculated were grown in Dulbecco’s modified Eagle’s medium（con-

for C16H24O2: C, 77.38; H, 9.74. Found: C, 77.39; H, 9.72. taining 10％ FBS in a humidified atmosphere of 5％ CO2 at 2,4-propofol-（2-ethylhexanoic acid）（ or）2,4-diisopropyl- 37℃）. Cells were trypsinized when sub-confluent from T25 phenyl-2-ethylhexanoate（2,4P-2-EHA） flasks/60 mm dishes and seeded in 96-well plates in 100 μL －1 Colorless liquid; Yield-98.1％; FT-IR（cm , CHCl3）: 2962, aliquots at plating densities depending on the doubling 2931（s, alkyl chain -C–H symmetric mode）, 2872（s, Ar–H time of individual cell lines. The microtiter plates were in-

symmetric mode）, 1755（s, -C＝O symmetric mode）, 1460 cubated at 37℃, 5％ CO2, 95％ air, and 100％ relative hu- （s, -C–H scissoring mode）, 1155（m, -C–O symmetric midity for 24 hrs prior to addition of experimental drugs mode）, 1116（s, –O–C＝O asymmetric mode）; 1H-NMR and were incubated for 48 hrs with different doses（0.01,

（CDCl3）δ: 7.14（1H, d, J＝2.14 Hz, Ar–C3–H）, 7.04（1H, dd, 0.1, 1, 10, 100 μM）of prepared derivatives where DMSO J＝8.24, 2.14 Hz, Ar–C5–H）, 6.89（1H, d, J＝8.24, Ar–C6– treated cells were used as negative controls for propofol

H）, 3.04（1H, sept, J＝6.87 Hz, Ar–C2–CH（CH3）2）, 2.89 esters. After 48 hrs incubation at 37℃, cell monolayers

（1H, sept, J＝6.87 Hz, Ar–C4–CH（CH3）2）, 2.53（1H, tt, J＝ were fixed by the addition of 10％（ wt/vol）cold trichloro- 6.72, 6.72 Hz, C2-H）, 1.85-1.74（2H, m）, 1.71-1.56（2H, m）, and incubated at 4℃ for 1hr and were then 1.45-1.35（4H, m）, 1.24（6H, d, J＝6.87 Hz, Ar–C2 –CH stained with 0.057％ SRB dissolved in 1％ acetic acid for

（CH3）2）, 1.21（6H, d, J＝6.87 Hz, Ar–C4 –CH（CH3）2）, 1.03 30 min at room temperature. Unbound SRB was washed （3H, t, J＝7.48 Hz）, 0.92（3H, t, J＝7.17 Hz）; 13C-NMR with 1％ acetic acid. The protein-bound dye was dissolved

（CDCl3）δ: 175.0, 146.3, 146.0, 139.5, 124.4, 124.2, 121.7, in 10 mM Tris base solution for OD determination at 510

4 J. Oleo Sci. Study on Synthesis, Characterization and Antiproliferative Activity of Novel Diisopropylphenyl Esters of Selected Fatty Acids

nm using a micro plate reader（Enspire, Perkin Elmer, and were calculated for this parameter if the level of activity is USA）. Using the seven absorbance measurements［time reached. However, if the effect is not reached or is exceed- zero,（Tz）, control growth,（C）, and test growth in the pres- ed, the value for that parameter was expressed as greater ence of drug at the five concentration levels（Ti）］, the per- or less than the maximum or minimum concentrations centage growth was calculated at each of the drug concen- tested. trations levels. Percentage of growth inhibition was calculated as:

Ti-Tz / C-Tz 100 for concentrations for which Ti ≥ Tz ［（ ）（ ）］× 3 RESULTS AND DISCUSSION 3.1 Chemistry Ti-Tz /Tz 100 for concentrations for which Ti Tz. ［（ ） ］× ＜ In the present study, eight novel propofol-fatty acid con- The dose response parameter, growth inhibition of 50％ jugates were synthesized by a simple chemical method（Fig.

（GI50）was calculated from［（ Ti-Tz）（/ C-Tz）］× 100＝50, 1）using propofol isomers（2,4-propofol and 2,6-propofol） which is the drug concentration resulting in a 50％ reduc- and four different fatty acids（ALA, VA, BA and 2-EHA）em- tion in the net protein increase（as measured by SRB stain- ploying DCC as coupling reagent and DMAP as catalyst. ing）in control cells during the drug incubation. Values The DCC/DMAP method is a more suitable synthetic route

Fig. 1 Schematic presentation for chemical synthesis of propofol-(alpha-linolenic acid) conjugates (2,4P-ALA and 2,6P-ALA), and structures of fatty acids used in this study.

5 J. Oleo Sci. Y. S. Reddy, S. S. Kaki and B. B. Rao et al.

with mild reaction conditions and convenient product puri- 3.2 Antiproliferative evaluation fication resulting in higher yields. The isolated yields in the The antiproliferative study of novel propofol-fatty acid present study were found to be in the range of 97.0- conjugates was assessed on the five human cancer cell 98.5％. lines（A-549, MDA-MB-231, HeLa, Mia-Pa-Ca and HePG2） All the products were purified by column chromatogra- using doxorubicin as a standard drug. The concentration of phy and the pure products were characterized by IR, NMR, individual compounds at which 50％ of the growth inhibi- mass spectral and elemental analysis data. The structures tion（GI50）was observed was calculated and results are of the products are shown in Fig. 2. The IR spectra of the given in the Table 1. products showed a strong absorption at 1755 cm－1 charac- From the anti-cancer activity data, it can be observed teristic of ester carbonyl functionality. The 1H-NMR of that all the conjugates exhibited good activity against the 2,6-diisopropylphenol-butyric acid（2,6P-BA）showed a tested cell lines. Interestingly, each conjugate was showing triplet at δ 2.6 which is characteristic of protons adjacent very good activity against specific cell lines with some se- to carbonyl and isopropyl group protons were observed at lectivity. All the conjugates showed excellent activity δ 1.19 whereas the aromatic protons in the diisopropylphe- against A549, HeLa, Mia-Pa-Ca and HePG2 cell lines with a nyl moiety were observed in the range of δ 7.25 to 7.11. GI50 value of＜0.5 μM except on MDA-MB-231 cell line

Further the mass spectral data of the product showed the where the best GI50 value observed was 1.0 μM. In general, m/z at 249.18（M＋H）＋ which confirmed the structure of the 2,4-isomers exhibited slightly higher activity compared the compound. to the 2,6-isomers when the total number of cell lines was taken into consideration. It can be observed that 2,4P-ALA and 2,4P-BA isomers were found to be more potent than

Fig. 2 Molecular structures of the propofol-fatty acid conjugates (2,6P-ALA, 2,4P-ALA, 2,6P-2-EHA, 2,4P-2-EHA, 2,6P-VA, 2,4P-VA, 2,6P-BA and 2,4P-BA).

6 J. Oleo Sci. Study on Synthesis, Characterization and Antiproliferative Activity of Novel Diisopropylphenyl Esters of Selected Fatty Acids

Table 1 Antiproliferative results of novel propofol-fatty acid conjugates.

Test GI50 values (μM) compound A549 MDA-MB-231 HeLa Mia-Pa-Ca HePG2 2,4P-ALA 2.0 ±0.10 4.3 ±0.20 0.7 ±0.05 0.68±0.07 0.44±0.03 2,6P-ALA 2.9 ±0.05 1.5 ±0.05 2.6 ±0.10 1.5 ±0.20 1.8 ±0.20 2,4P-VA 0.78±0.02 6.5 ±0.20 0.77±0.04 0.6 ±0.01 0.43±0.01 2,6P-VA 1.8 ±0.06 12.1 ±1.30 0.42±0.03 0.47±0.06 0.28±0.04 2,4P-BA 2.8 ±0.20 5.5 ±0.60 5.7 ±0.50 0.48±0.04 0.21±0.01 2,6P-BA 0.19±0.01 6.7 ±0.09 0.87±0.02 0.63±0.01 0.45±0.02 2,4P-2-EHA 3.9 ±0.07 6.9 ±0.30 0.75±0.03 0.48±0.03 0.25±0.04 2,6P-2-EHA 1.2 ±0.09 1.0 ±0.02 0.62±0.01 5.7 ±0.21 6.9 ±0.06 Doxorubicin 0.07±0.01 0.09±0.01 0.03±0.01 0.08±0.01 0.06±0.01 the corresponding 2,6P-ALA and 2,6P-BA isomers. Based study. From the activity studies, it was found that all the on the structure activity relationship studies in the present synthesized propofol-fatty acid conjugates were able to study, it was found that among all the esters tested, inhibit the growth of cancer cells. Our preliminary data 2,6P-BA represented the highest activity against A549 cell strongly suggest that the synthesized conjugates possess line with a GI50 value of 0.19 μM. The standard drug, doxo- potent to moderate anticancer activity. rubicin had a GI50 value of 0.07 μM against A549 cell line. On HeLa, the conjugates 2,6P-ALA and 2,4P-BA showed One of the authors Y. Sathyam gratefully acknowledges less activity while the remaining six conjugates showed the Department of Biotechnology, New Delhi for the finan- moderate activity. It was found that 2,6P-VA exhibited cial assistance under sponsored project and Director, CSIR- highest activity against HeLa cell lines compared to other IICT for providing the facilities. conjugates with a GI50 value of 0.42 μM and doxorubicin represented a GI50 value of 0.03 μM. On the Mia-Pa-Ca cell line, 2,6 P-VA showed highest activity with a GI50 value of

0.47 μM whereas doxorubicin exhibited a GI50 value of 0.08 Supporting Information μM. On HePG2 cell line, three esters showed promising ac- This material is available free of charge via the Internet tivity. Among the three esters, it was found that, 2,4P-BA at http://dx.doi.org/jos.65.10.5650/jos.ess.15151 showed highest activity with a GI50 value of 0.21 μM whereas 2,4P-2-EHA and 2,6 P-VA exhibited GI50 values of 0.25 and 0.28 μM respectively. The standard drug, doxoru- bicin represented a GI50 value of 0.06 against HePG2 cell REFERENCES line. It is interesting to observe that there was a clear dif- 1） Miller, C. R.; McLeod, H. L. Pharmacogenomics of can- ference in activity among isomers of 2,4- and 2,6-diisopro- cer chemotherapy-induced toxicity. J. Support. On- pylphenyl esters derived from same fatty acid on the cell col. 5, 9-14（2007）. lines tested except on Mia-Pa-Ca where the difference in 2） James, S. E.; Burden, H.; Burgess, R.; Xie, Y.; Yang, T.; activity was less between two isomers. Massa, S. M.; Longo, F. M.; Lu, Q. Anti-cancer drug in- Earlier reports on the propofol-fatty acid conjugates also duced neurotoxicity and identification of Rho pathway showed similar observations on cancer cell lines where the signaling modulators as potential neuroprotectants. fatty acids used were normal long chain fatty acids6, 19）. Neurotoxicol. 29, 605-612（2008）. Thus all propofol-fatty acid conjugates in this study exhib- 3） Takahashi, M.; Fukutake, M.; Isoi, T.; Fukuda, K.; Sato, ited promising antiproliferative activity on the cell lines H.; Yazawa, K.; Sugimura, T.; Wakabayashi, K. Sup- tested. pression of azoxymethane-induced rat colon carcino- ma development by a fish oil component, docosa- hexaenoic acid（DHA）. Carcinogenesis 18, 1337-1342 （1997）. 4 CONCLUSIONS 4） Bradley, M. O.; Swindell, C. S.; Anthony, F. H.; Witman, In conclusion eight novel propofol-fatty acid conjugates P. A.; Devanesan, P.; Webb, N. L.; Baker, S. D.; Wolff, A. have been synthesized and their antiproliferative activity C.; Donehower, R.C. Tumor targeting by conjugation was tested against five cancer cell lines in the present of DHA to paclitaxel. J. Control. Rel. 74, 233-236

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