synthesis, spectroscopic and anti-tumor studies of polyphenol-linoleates derived from natural...

Jamal Mustafaa

Shabana I. Khana

Daneel Ferreiraa, b

Ikhlas A. Khana, b

a National Center for NaturalProducts Research (NCNPR),Research Institute ofPharmaceutical Sciences,University of Mississippi,University, USA

b Department of Pharmacognosy,School of Pharmacy,University of Mississippi,University, USA

Synthesis, spectroscopic and anti-tumor studiesof polyphenol-linoleates derived from naturalpolyphenols*

An efficient first synthesis of di-, hexa- and octa-esters (octadeca-Z-9,12-dienoates,linoleates) from natural 1,5-bis-(4’-hydroxyphenyl)penta-Z-1,4-diene (Ginkgo bilo-ba L.), (–)-epigallocatechin and (–)-epigallocatechin-3-O-gallate (jasmine tea/greentea), respectively, was developed. Using dicyclohexyl carbodiimide as an activatingreagent in the presence of a catalytic amount of 4-dimethylaminopyridine in the poly-esterification reactions gave di-, hexa- and octa-products (4), (7), and (8), respectively,in quantitative yields. A three-step efficient procedure for the synthesis of 1,5-bis-[4’-O-(octadec-Z-9”,12”-dienoate)phenyl]penta-Z-1,4-diene (4) was also developed. Thekey step in the synthesis involved a stereoselective Wittig diolefination reaction thatproduced the penta-Z-1,4-diene system of (4). 1H and 13C NMR and MS techniquesconfirmed the structures of the esters. The esterified compounds were tested for invitro anti-tumor activities against four and three human cancer cell lines at NCNPR andat NCI, respectively. They were found inactive as they failed to inhibit 50% growth ofthese cancer cells.

Keywords: Butyl Li, dicyclohexyl carbodiimide, esterification, flavonoids, 1H/13C NMR,in vitro anti-cancer activity, linoleic acid, triphenylphosphorus ylide, polyphenols, Wittigreaction.

552 DOI 10.1002/ejlt.200600235 Eur. J. Lipid Sci. Technol. 109 (2007) 552–559

1 Introduction

Chemoprevention is one of the strategies that may be usedto prevent or suppress the onset of diseases like cancer[1]. The consumption of certain edible oils, fruits andvegetables is strongly correlated with the reduction, elim-ination or even prevention of the occurrence of severalchronic medical disorders. This indicates that there areeffective mixtures of dietary components for certain dise-ase prevention since individual compounds cannot fullyexplain this action. A combination of different fatty acidsand their combination with other natural products producesynergistic effects in cancer therapies [2–4]. Recently,polyunsaturated fatty acids (PUFA) have raised interest asnovel anti-cancer agents as they possess effective tumor-icidal properties while not causing damage to normal cellsor generating harmful side effects [5–7]. It is well recog-nized that essential fatty acids could play an important rolein cancer treatment. Oxidation of such fatty acids, likelinoleic acid, by lipoxidase increases tumor cell death [8].

Fatty acids and flavonoids present in the human diet aresafe and without appreciable side effects. Several indi-vidual fatty acids [9] and flavonoids [1] are reported for

cancer prevention and treatment. However, a syntheticmolecule comprising both constituents with a view toexplore their synergistic effects has not yet been studied.Considering these facts and taking into account theinterest of the National Cancer Institute (NCI) (Bethesda,MD, USA) to evaluate the anti-cancer potential of mole-cules derived from fatty acids and flavonoids, weembarked on a program aimed at the synthesis of flavo-noid-fatty acid derivatives. Here, we report the first syn-thesis of NCI-selected di-, hexa- and octa- octadec-Z-9,12-dienoates (18:2, n-6 linoleates; Figs. 1–3) from nat-ural polyphenols, 1,5-bis-(4’-hydroxyphenyl)penta-Z-1,4-diene (1) (Ginkgo biloba L.), (–)-epigallocatechin (5) and(–)-epigallocatechin-3-O-gallate (6) (green tea), respec-tively. Compound (6) is known to inhibit various humancancer cells in nude mice while compound (5) is inactiveagainst similar cancer cells [10, 11].

Initially, 1,5-bis-(4’-hydroxyphenyl)penta-Z-1,4-diene (1)(NSC Number D731157-T) has been assayed againstthree human cancer cell lines at the NCI, and on the basisof encouraging results, it has been decided to furtherevaluate the compound (1) against a panel of 60 human

Correspondence: Ikhlas A. Khan, National Center for NaturalProducts Research, School of Pharmacy, University of Missis-sippi, University, MS 38677, USA. Phone: 11 662 9157821, Fax:11 662 9157989, e-mail: [email protected]

* Presented at the 2004 International Congress on NaturalProducts Research, A Joint Meeting of American Society ofPharmacognosy (ASP) and Phytochemical Society of Europe(PSE), July 31 – August 4, 2004 at Westin Kierland Resort &Spa, Phoenix, Arizona, USA

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

Res

earc

hP

aper

Eur. J. Lipid Sci. Technol. 109 (2007) 552–559 Polyphenol-linoleates 553

Fig. 1. Direct synthesis of 1,5-bis-[4’-O-(octa-dec-Z-9”,12”-dienoate)phenyl]penta-Z-1,4-di-ene (4) from 1,5-bis-(4’-hydroxyphenyl)penta-Z-1,4-diene (1).

cancer cell lines under their Drug Discovery Program [12].Limited bioavailability of compound (1) [13, 14] hasprompted us to develop a suitable method for the totalsynthesis of 1,5-bis-[4’-O-(octadec-Z-9”,12”-dienoate)-phenyl]penta-Z-1,4-diene (4) (Fig. 2) and also its directsynthesis from compound (1) (Fig. 1). The structures offatty acid-esterified compounds (2), (4), (7) and (8) wereelucidated by their spectroscopic data, and compounds(4), (7) and (8) were evaluated for their in vitro anti-canceractivity at the NCI and at the National Center for NaturalProducts Research (NCNPR) against a panel that isrestricted to seven human cancer cell lines.

2 Material and methods

The experimental procedures are similar to those detailedin our earlier publication [15]. Octadec-Z-9,12-dienoicacid, 4-dimethylaminopyridine (DMAP), 1,3-dibromopro-pane, butyl lithium (BuLi; 6 M solution in THF), triphenyl-phosphine, 4-hydroxybenzaldehyde, p-anisaldehyde,hexamethylphosphoramide (HMPA), and acetone (99.51)

were procured from Aldrich Chemicals (Milwaukee, WI,USA), and dicyclohexyl carbodiimide (DCC) was pur-chased from Fluka Chemical Corporation (Hauppauge,NY, USA). 1,5-Bis-(4’-hydroxyphenyl)penta-Z-1,4-dienewas isolated from Ginkgo biloba L. [13], while (–)-epi-gallocatechin and (–)-epigallocatechin-3-O-gallate [10]were isolated from green tea. THF was dried by refluxingwith sodium metal (5 g/L) and benzophenone (10 g/L) for24 h under nitrogen until the persistence of a purple color,and freshly distilled prior to use. Commercial-gradeHMPA was dried after keeping on active molecular sievesfor seven days. 1H NMR and 13C NMR spectra wererecorded on a DRX-500 Bruker NMR spectrometer.Techniques of correlating 1H and 13C resonances providedtwo-dimensional correlation spectra (2D COSY) thathelped in confirmation of different resonances. Molecularweights were determined by electron-spray ionization(ESI) on a Bruker BioApex Fourier transform mass spec-trometer. Samples were run in ESI-positive mode bydirect injection with a syringe pump spectrometer (ESI-MS). FTIR spectra were run in CHCl3 on a Genesis SeriesFTIR spectrometer.


554 J. Mustafa et al. Eur. J. Lipid Sci. Technol. 109 (2007) 552–559

Fig. 2. A simple three-step proce-dure for the total synthesis of 1,5-bis-[4’-O-(octadec-Z-9”,12”-dieno-ate) phenyl]penta-Z-1,4-diene (4)where the third step involves a Wit-tig diolefination reaction.

2.1 Chemical procedures

2.1.1 Esterification of natural polyphenols withoctadec-Z-9,12-dienoic (linoleic) acid

To stirred solutions of 1,5-bis-(4’-hydroxyphenyl)penta-Z-1,4-diene (1), (–)-epigallocatechin (5) and (–)-epigalloca-techin-3-O-gallate (6) (1 mmol each) in acetone (5 mL)under nitrogen is added octadec-Z-9,12-dienoic acid(2.20 mmol for (1), 6.20 mmol for (5) and 8.20 mmol for (6),respectively). The separate mixtures were stirred for 5 minuntil homogeneous, and then DMAP (catalytic amount)was added. The mixtures were further stirred at roomtemperature under nitrogen for 10 min before DCC(2.20 mmol for (1), 6.20 mmol for (5) and 8.20 mmol for (6),respectively) was added. The mixtures were stirred atroom temperature and reactions were completed in 2, 12and 24 h, respectively. The mixtures were filtered toremove solid dicyclohexylurea and the filtrate was evap-orated under reduced pressure at 20 7C. The semisolid

reaction mixtures were purified by flash column chroma-tography (n-hexane/ethyl acetate, 2 : 1, vol/vol) on silicagel to produce (4), (7), and (8).

2.1.2 1,5-Bis-[4’-O-(octadec-Z-9”,12”-dienoate)phenyl]penta-Z-1,4-diene (4)

Viscous colorless oil; Rf = 0.80, yield 99%. IR (cm–1):2926, 2855, 1769, 1623, 1594, 1463, 1441, 1357, 1130,1047, 1010, 909, 723; 1H NMR (dH, CDCl3): 0.93 (t,J = 6.80 Hz, 6H), 1.39–1.31 (br. m, 28H), 1.77 (m, 4H), 2.11(m, 8H), 2.60 (m, 4H), 2.82 (m, 4H), 3.42 (t, J = 7.20 Hz,2H), 5.46–5.38 (m, 8H), 5.75 (sextet, J = 11.20 Hz, 2H),6.51 (d, J = 11.60 Hz, 2H), 7.10 (d, J = 8.0 Hz, 4H), 7.30 (d,J = 8.40 Hz, 4H); 13C NMR (dC, CDCl3): 14.14, 22.63,24.96, 25.68, 27.25, 28.11, 29.20, 29.24, 29.41, 29.65,31.57, 34.42, 121.38, 127.93, 128.11, 128.97, 129.72,130.03, 130.24, 134.71, 149.41, 172.28; EI-MS found[M]1 776.5751; C53H76O4[M]1 requires 776.5743.



Fig. 3. The hydroxy groups of (–)-epigallocatechin (5) and (–)-epi-gallocatechin-3-O-gallate (6) areesterified with the carboxylic groupof octadec-Z-9,12-dienoic acid toproduce poly-fatty acid esters (7)and (8), respectively.

2.1.3 4-O(octadec-Z-9,12-dienoate)benzaldehyde (2)

Equimolar amounts of 4-hydroxybenzaldehyde and octa-dec-Z-9,12-dienoic (linoleic) acid were used to preparecompound (2) by the method described for (4). The reac-tion was completed in 30 min and the colorless oilyproduct was obtained in 99% yield after purification byflash column chromatography (n-hexane/ethyl acetate,2 : 1); Rf = 0.80. IR (cm–1, CHCl3): 2935, 2857, 1760, 1701,

1599, 1503, 1458, 1274, 1206, 1157, 1109, 853; 1H NMR(dH, CDCl3): 0.85 (t, J = 6.40 Hz, 3H), 1.32–1.27 (br. m,14H), 1.72 (m, 2H), 2.03 (m, 4H), 2.54 (t, J = 7.20 Hz, 2H),2.75 (m, 2H), 5.38–5.31 (m, 4H), 7.22 (d, J = 8.20 Hz, 2H),7.85 (d, J = 8.20 Hz, 2H), 9.92 (s, 1H); 13C NMR (dC,CDCl3): 14.07, 22.56, 24.74, 25.62, 27.14, 27.18, 29.00,29.05, 29.12, 29.33, 29.55, 31.50, 34.27, 122.31, 127.86,128.07, 129.91, 130.11, 131.11, 133.85, 155.50, 171.41,190.91; EI-MS found [M]1 384.2663; C25H36O3[M]1

requires 384.2664.



2.1.4 Synthesis of trimethylene-1,3-bis(triphenylphoshonium)dibromide (3)

A mixture of triphenylphosphine (31.5 g) and 1,3-dibromo-propane (10 g) was stirred in an oil bath with gradualincrease of the temperature to 194 7C. The reaction mixturebecame solid at 110 7C; at 180 7C, an exothermic reactionoccurred and crystals started to form. The heating wascontinued for two more hours. Total reaction time wasrecorded as 8 h and the mixture then left overnight. Thewhite solid mixture was dissolved in chloroform (200 mL),charcoal (5 g) was added and the mixture was refluxed for2 h. The charcoal was filtered off and acetone (150 mL) wasadded to the chloroform layer until crystallization occurred.Filtration gave shining white crystals (35 g), m.p. 334 7C. IR(cm–1, CHCl3,): 3013, 2866, 1583, 1481, 1435, 1328, 1190,1108, 992, 824; 1H NMR (dH, CDCl3): dH 1.71 (J = 6.0 Hz,2H), 4.44 (m, 4H), 7.42–7.72 (br. m, 30H); dC 17.63, 21.83,22.50, 117.26, 118.12, 130.41, 130.51, 130.53, 130.61,133.91, 133.97, 134.02, 134.11, 134.12, 135.10; EI-MSfound [M–Br]1 645.1466, 647.1440; C39H36P2Br [M–Br]1

requires 645.1475, [M–Br2]1 found 283.1131, 283.6155

(m/z; two charges); C39H36P2 [M–Br2]1 requires 283.1146.

2.1.5 Wittig olefination

3-Bis(triphenylphoshonium)dibromide (3) was dried at60 7C under vacuum for 24 h. The dried salt (3) (4 mmol)was suspended in dry THF (50 mL) under argon, driedHMPA (15 mL) added, the mixture cooled to –78 7C in a dryice bath and stirred at this temperature for 15 min beforeBuLi (5.63 mL, 9 mmol, 6 M solution in THF) was added.After stirring for 90 min at this temperature, a solution offreeze-dried 4-O-octadec-Z-9,12-dienoate)benzaldehyde(2) (9 mmol) in dry THF (10 mL) was injected over 30 min.The reddish brown color of the mixture turned to orange.The cooling bath was removed and the mixture was stirredfor 3 h under argon. Quenching with NH4Cl solution(100 mL, 50 g in 125 mL water), extraction with diethylether (3650 mL), drying over Na2SO4 and evaporationafforded a crude yellowish oily reaction mixture. This wassubjected to silica gel column chromatography (toluene/acetonitrile, 9 : 1) followed by reverse-phase C18 columnchromatography (MeOH/water, 4 : 6) to obtain pure 1,5-bis-[4’-O-(octadec-Z-9”,12”-dienoate)phenyl]penta-Z-1,4-diene(4) in 81% yield. Spectroscopic data fully agree with thosereported above for this compound (4).

2.1.6 3,5,7,3’,4’,5’-Hexa-O-(octadec-Z-9”,12’’-enoate)epigallocatechin (7)

The method of preparation is described above (Section2.1.1). Viscous colorless oil; Rf = 0.90 (n-hexane/ethyl

acetate, 2 : 1); yield 98%; IR (cm–1, CHCl3): 3009, 2926,2855, 1769, 1740, 1623, 1594, 1498, 1463, 1357, 1130,909; 1H NMR (dH, CDCl3): 0.90 (m, 18H), 1.37–1.22 (br. m,84H), 1.74 (m, 12H), 2.21–2.11 (br. m, 24H), 2.61–2.51 (br.m, 12H), 2.81–2.80 (br. m, 12H), 2.91 (m, 2H), 5.11 (s, 1H),5.30 (s, 1H), 5.41–5.40 (m, 24H), 6.55 (s, 1H), 6.71 (s, 1H),7.22 (s, 2H); 13C NMR (dC, CDCl3): 14.10, 22.60, 24.91,25.65, 27.21, 29.15, 29.31, 29.40, 29.71, 31.54, 33.84,34.52, 66.21, 77.93, 94.93, 94.93, 95.51, 101.82, 107.92,108.81, 109.51, 127.75, 127.81, 127.82, 127.98, 128.02,128.10, 128.11, 129.76, 129.83, 129.90, 130.01, 130.21,134.21, 135.23, 153.91, 155.50, 157.93, 159.0, 169.20,170.11 (26C), 170.95, 171.47, 172.92; EI-MS found [M]1

1879.4521; C123H194O13[M]1 requires 1879.4519.

2.1.7 5,7,3’,4’,5’-Penta-O-(octadec-Z-9”,12”-enoate)-3-O-(3,4,5-tri-O-octadec-Z-9”,12”-enoategalloyl)epigallocatechin (8)

The method of preparation is described above (Section2.1.1). Viscous colorless oil, Rf = 1.0 (n-hexane/ethylacetate, 2 : 1), yield 97.5%. IR (cm–1, CHCl3): 2927, 2855,1770, 1709, 1642, 1530, 1453, 1395, 1229, 893; 1H NMR(dH, CDCl3): 0.92 (m, 24H), 1.38–1.33 (br. m, 112H), 1.78(br. m, 16H), 2.11–2.10 (br. m, 32H), 2.59–2.50 (br. m,16H), 2.82–2.79 (br. m, 16H), 3.32 (m, 2H), 5.20 (s, 1H),5.44–5.35 (br. m, 32H), 5.70 (s, 1H), 6.62 (d, J = 2.0 Hz,1H), 6.80 (s, J = 2.0 Hz, 1H), 7.25 (s, 2H), 7.63 (s, 2H);13C NMR (dC, CDCl3): 14.12, 22.62, 24.83, 25.65, 27.23,29.20, 29.31, 29.38, 29.64, 29.68, 31.55, 33.75, 33.99,67.91, 76.61, 94.58, 95.34, 101.53, 108.03, 109.04,127.88, 127.95, 128.11, 128.13, 128.20, 129.85, 129.93,129.96, 130.03, 130.11, 130.20, 130.24, 134.340, 143.71,152.01, 153.84, 155.07, 157.11, 159.40,163.64, 168.85,169.38, 170.16 (26C), 170.34 (26C), 171.25, 171.77; EI-MS found [M]1 2555.8995; C166H258O19[M]1 requires2555.9222.

2.2 Biological procedures

Compounds (4), (7) and (8) were tested for their in vitroanti-cancer activity at NCNPR against a panel of four hu-man cancer cell lines, SK-MEL (malignant, melanoma),KB (epidermal carcinoma, oral), BT-549 (ducktail carci-noma, breast) and SK-OV-3 (ovary carcinoma). The com-pounds were also evaluated against two non-cancerouscell lines, VERO (African green monkey kidney fibroblast)and LLC-PK1 (kidney epithelial) cells as described pre-viously [15]. The anti-tumor activities of (4) (NSC Number:D732085-C), (7) (NSC Number: D732573-J) and (8) (NSCNumber: D732081-X) were also assessed at NCI(Bethesda, MD, USA), through the Developmental Ther-apeutic Program. Compounds were evaluated at one



concentration against a primary anti-cancer assay with a3-cell line panel consisting of MCF7 (breast), NCI-H460(lung), and SF-268 (CNS) [16].

3 Results and discussion

Studies on the cancer-protective effects of the con-sumption of certain edible oils, vegetables and fruitswere first carried out in the 1970s and repeatedly con-firmed since. These studies have indicated a reductionof the overall incidence of cancer by at least 20% [1].The present work describes the first synthesis of linoleicacid, an essential fatty acid, and esters of natural poly-phenols or flavonoids; both constituents are part of ourdaily diet.

3.1 Synthetic strategy

The hydroxy groups of three natural polyphenols, 1,5-bis-(4’-hydroxyphenyl)penta-Z-1,4-diene (1) (26OH,ArOH), (–)-epigallocatechin (5) (66OH, 5 ArOH and1 ROH) and (–)-epigallocatechin-3-O-gallate (6) (86OH,ArOH), were efficiently esterified with the carboxylicgroup of octadec-Z-9,12-dienoic acid to producethe corresponding poly-fatty acid esters withmethylene-interrupted Z-diene group, 1,5-bis-[4’-O-(octadec-Z-9”,12”-dienoate)phenyl]penta-Z-1,4-diene(4), 3,5,7,3’,4’,5’-hexa-O-(octadec-Z-9”,12”-enoate)epi-gallocatechin (7), and 5,7,3’,4’,5’-penta-O-(octadec-Z-9”,12”-enoate)-3-O-(3,4,5-tri-O-octadec-Z-9”,12”-enoa-tegalloyl)epigallocatechin (8), in quantitative yields. DCCis used as an activating reagent in the presence of acatalytic amount of DMAP (Fig. 3).

During the polyesterification of (5) and (6), a number ofspots on qualitative TLC representing different degrees ofester formation were observed, which gradually dis-appeared to leave a single spot corresponding to thepreesters (7) and (8), respectively.

Compound (4) is a symmetrical molecule and its 1H and13C NMR displayed the resonances of only one half of themolecule, e.g. a carbonyl resonance at dC 172.28. Two ter-minal fatty acid methyl groups resonated as one triplet at dH

0.93 (J = 6.80 Hz, 18-H, 6H) and correlated with carbons atdC 14.14 (C-18). Six methylene-interrupted Z-olefinic car-bons are present at C-9”/C-10” and C-12”/C-13”, and theremaining two at C-1/C-2 and C-4/C-5. The fatty acid ole-finic protons resonated as a broad multiplet at dH 5.46–5.38(9”-H/10”-H and 12”-H/13”-H, 8H). The remaining fourolefinic protons associated with two Z-double bonds of thepenta-Z-1,4-diene system resonated at dH 5.75 (sextet,J = 11.20 Hz, 2-H, 4-H, 2H) and 6.51 (d, J = 11.60 Hz, 1-H,

5-H, 2H). The olefinic protons correlated with olefinic car-bons at dC 127.93, 128.11, 128.97, 129.72, and 130.03 (C-9”/C-10” and C-12”/C-13”; C-1/C-2 and C-4/C-5). Thearomatic protons and carbons resonated at dH 7.10 (d,J = 8.0 Hz, 2’-H, 6’-H), dC 121.38 (C-2’, C-6’); dH 7.30 (d,J = 8.40 Hz, 3’-H, 5’-H), 130.24 (C-3’, C-5’). Two protons ofa 3-CH2 group appeared at dH 3.42 (t, J = 7.20 Hz, 3-H) andcorrelated with the carbon at dC 28.11.

3.2 Total synthesis of 1,5-bis-[4’-O-(octadec-Z-9”,12”-dienoate)phenyl]penta-Z-1,4-diene (4)

A simple three-step procedure was developed to synthe-size compound (4) (Fig. 2). The first step involves quanti-tative esterification of the 4-hydroxyl group of 4-hydro-xybenzaldehyde with octadec-Z-9,12-dienoic acid byusing a reagent system based on DCC/DMAP. The13C NMR spectrum of (4) displayed two diagnostic reso-nances in the carbonyl region at dC 171.41 (ester carbonyl)and 190.91 (aldehyde carbonyl). In the 1H NMR spectrumthe Z-olefinic protons (9-H/10-H and 12-H/13-H) reso-nated at dH 5.38–5.31 (br. m, 4H) and correlated with car-bons at dC 127.86, 128.07, 129.91 and 130.11 (C-9/C-10and C-12/C-13). The aromatic protons appeared at dH

7.22 (d, J = 8.20 Hz, 2H, 2’-H, 6’-H) and 7.85 (d,J = 8.20 Hz, 2H, 3’-H, 5’-H) and correlated with carbonsat dC 122.31 (C-2’, C-6’), and 131.11 (C-3’, C-5’). Analdehydic proton was observed as a singlet at dH 9.92.

The second step is the formation of the bis-triphenylphos-phonium salt, trimethylene-1,3-bis(triphenylphoshon-ium)dibromide (3), and comprised reaction of triphenyl-phosphine with 1,3-dibromopropane. The final and keystep in the synthesis of (4) was the stereo-selective Wittigdiolefination reaction. The conventional Wittig reactionentails the reaction of a phosphonium ylide with an alde-hyde or a ketone, and has enjoyed widespread promi-nence and recognition because of its high selectivity forZ- or E-olefins, depending on details such as the type ofylide, type of carbonyl compound or reaction conditions[17, 18]. Non-stabilized triphenylphosphorus ylides like (3)generally react with aldehydes to afford mainly Z-alkenes[17, 19]. It is known that the product of Z-configuration isproduced in maximum amount by bulky aliphatic sub-stituents and low reaction temperatures [17]. Thus, asuspension of bis-triphenylphosphonium dibromide (3) indry THF/dry HMPA was stirred under argon at –78 7C andtreated with BuLi (6 M in THF) to generate a reddish-brown colored bis-triphenylphosphonium ylide. Treat-ment of the ylide at this temperature with a dry THF solu-tion of 2 mole equivalents of 4-O-octadec-Z-9,12-die-noate)benzaldehyde (2) afforded compound (4) after suc-cessive silica gel and reverse-phase C18 columnchromatography. The 1H and 13C NMR spectra of the



product produced by the Wittig protocol reaction wassuper-imposable on the spectra of the product preparedby esterification of natural product (1).

A Wittig diolefination reaction of p-anisaldehyde (4-meth-oxybenzaldehyde) under similar reaction conditions asdescribed above gave a mixture of three stereo-isomers(ZZ, EE, ZE). This observation has given the impressionthat the presence of a linoleate at C-4’ on the benzenering may be of some significance for Z-stereoselectivityunder the prevailing reaction conditions. A systematicstudy with a variety of fatty acid ester substituents at thepara-position (C-4’) of benzaldehyde is required toexamine the possible influence of the fatty acid moietytowards Z-stereoselectivity in the Wittig dioleficationreaction under the present reaction conditions.

(–)-Epigallocatechin (5) was esterified with 6 mole equiva-lents of octadec-Z-9,12-dienoic acid as described aboveto obtain 3,5,7,3’,4’,5’-hexa-O-(octadec-Z-9”,12”-enoa-te)epigallocatechin (7). The six fatty acid ester carbonylcarbons (C-1”) resonated at dC 169.20, 170.11 (26C),170.95, 171.47, and 172.92, hence confirming completeesterification. The chemical shifts of the protons and car-bons of the twelve Z-olefinic functionalities were observedat dH 5.41–5.40 (br. m, 6 69”-H/10”-H, 12”-H/13”-H, 24H),dC 127.75, 127.81, 127.82, 127.98, 128.02, 128.10,128.11, 129.76, 129.83, 129.90, 130.01 and 130.21(6 6C-9”/C-10”, C-12”/C-13”). Six fatty acid terminalmethyl group (18”-CH3) protons appeared at 0.90 (m, 18”-H, 18H) and correlated with a carbon at dC 14.10 (C-18”).The various chemical shifts related to the epigallocatechinmoiety were: aromatic ring A, 6-H/8-H at dH 6.55 (s)/6.71 (s), dC 94.93/95.51 (C-6/C-8); aromatic ring B: 2’-Hand 6’-H dH 7.22 (s), dC 107.92 (C-2’ and C-6’); ring C: dH

5.11 (s, 2-H), dC 66.21 (C-2); dH 5.30 (s, 3-H), dC 77.93 (C-3);dH 2.91 (m, 4-H, 2H). The C-4 resonance was overlappedby the cluster of fatty acid methylene carbon resonancesranging from dC 22.60 to 34.52.

Similar esterification of compound (6) with 8 molarequivalents of octadec-Z-9,12-enoic acid afforded5,7,3’,4’,5’-penta-O-(octadec-Z-9”,12”-enoate)-3-O-(3,4,5-tri-O-octadec-Z-9”,12”-enoategalloyl)epigallocatechin(8). Complete esterification was confirmed by the pres-ence of eight fatty acid ester carbonyl carbons (C-1”) at dC

168.85, 169.38, 170.16, (26C), 170.34 (26C), 171.25 and171.77. The galloyl carbonyl carbon appeared at dC

163.64. The olefinic protons of the eight fatty acid unitsresonated in the range of dH 5.44–5.35 (8 69”-H/10”-H,12”-H/13”-H, 32H) and correlated with carbons at dC

127.88, 127.95, 128.11, 128.13, 128.13, 128.20, 129.85,129.93, 129.96, 130.03, 130.11, 130.20, 130.24 (8 6C-9”/C-10”, C-12”/C-13”). The protons of the eight terminalmethyl groups appeared at dH 0.92 (m, 24H) and corre-

lated with a single carbon signal at dC 14.12 (8 618”-C).The resonances related to the epigallocatechin-3-O-gal-late moiety appeared at: aromatic ring A, 6-H/8-H at dH

6.62 (d, J = 2.0 Hz, 1H)/6.80 (d, J = 2.0 Hz, 1H), dC 94.58/95.34 (C-6/C-8); aromatic ring B: 2’-H and 6’-H at dH 7.25(s, 2H), dC 108.03 (C-2’, C-6’); ring C: 2-H at dH 5.20 (s, 1H),C-2 at dC 67.91; 3-H at dH 5.70 (s, 1H), C-3 at dC 76.61; 4-Hat dH 3.32 (m, 2H), C-4 overlapped fatty acid methyleneresonances at dC 22.62–33.99; 3-O-gallate: 2”-H and 6”-Hat dH 7.63 (s, 2H), C-2” and C-6” at dC 109.04.

3.3 Anti-tumor evaluation

Polylinoleates (4), (7) and (8) were evaluated for their anti-tumor activities against a panel that is restricted to sevenhuman cancer cell lines. The four solid tumor cell linesagainst which these compounds were evaluated at theNCNPR are SK-MEL (malignant, melanoma), KB (epi-dermal carcinoma, oral), BT-549 (ductal carcinoma,breast) and SK-OV-3 (ovary carcinoma), up to a highestconcentration of 10 mM. The anti-tumor activities of (4)(NSC Number: D732085-C), (7) (NSC Number: D732573-J) and (8) (NSC Number: D732081-X) were also deter-mined at the NCI in a primary anti-cancer assay, involvinga 3-cell line panel consisting of MCF7 (breast), NCI-H460(lung), and SF-268 (CNS), at a concentration of 100 mM.Polyesters (4), (7), and (8) failed to inhibit 50% growth ofthe tumor cells and were considered inactive. The com-pounds were not found cytotoxic to non-cancerous VERO(kidney fibroblast) and LLC-PK1 (kidney epithelial) cells.

4 Conclusions

We have developed a facile methodology to quantitativelyesterify the phenolic functionalities of 1,5-bis-(4’-hydro-xyphenyl)penta-Z-1,4-diene (1), (–)-epigallocatechin (5),and (–)-epigallocatechin-3-O-gallate (6), with quantitativeyield of the desired poly-esterified products. This methodmay be applied to other polyphenols and to differenthighly unusual fatty acids. We have also developed amethod to stereoselectively synthesize (4), 1,5-bis-[4’-O-(octadec-Z-9”,12”-dienoate)phenyl]penta-Z-1,4-diene.However, the polyesters failed to show 50% growth inhi-bition of a panel of seven human cancer cell lines.

Acknowledgments

This investigation was supported in part by the USDA –Agricultural Research Service Specific CooperativeAgreement No. 58-6408-2-0009. We extend our thanks toNCI, Bethesda, MD, USA for the human cancer cell linepanel assay.



References

[1] M. Lopez-Lazaro: Flavonoids as anticancer agents: Struc-ture-activity relationship study. Curr Med Chem AnticancerAgents. 2000, 2, 691–714.

[2] H. Nakagawar, D. Yamamoto, Y. Kiyozuka, K. Tsuta, Y.Uemura, K. Hioki, Y. Tsutsui, A. Tsubura: Effect of genisteinand synergistic action in combination with eicosapentaenoicacid on the growth of breast cancer cell lines. J Cancer ResClin Oncol. 2000, 126, 448–454.

[3] S. Liao, T. Liang: Methods and compositions including fattyacids, catechin gallates, and their derivatives and syntheticanalogs for inhibiting 5a-reductase activity and treating dis-orders associated with androgenic activities. PCT Int Appl.1996, 136 pp, Application: WO 96-US137 19960516.

[4] T. Narisawa: An overview on chemoprevention of coloncancer. Nippon Geka Gakkai Zasshi. 1998, 99, 362–367.

[5] J. A. Menendez, S. Ropero, R. Lupu, R. Colomer: o-6 Poly-unsaturated fatty acid g-linolenic acid (18:3n-6) enhancesdocetaxel (taxotere) cytoxicity in human breast carcinomacells: Relationship to lipid peroxidation and HER-2/neuexpression. Oncol Rep. 2004, 11, 1241–1252.

[6] C. Field, P. D. Schley: Evidence for potential mechanisms forthe effect of conjugated linoleic acid on tumor metabolismand immune function: Lessons from n-3 fatty acids. Am JClin Nutr. 2004, 79, 1190S–1198S.

[7] P. Tanmahasamut, J. Liu, L. B. Hendry, N. Side II: Con-jugated linoleic acid blocks estrogen signaling in humanbreast cancer cells. J Nutr. 2004, 134, 674–680.

[8] C. H. Van Aswegen, D. J. Du Plessis: Can linoleic acid andg-linoleic acid be important in cancer treatment? MedHypotheses. 1994, 43, 415–417.

[9] I. Nobuhiko, H. Tsuji, Y. Fukuda: Anticancer agents, per-fumes or foods and drinks containing o-hydroxy fatty acids.PCT Int Appl. 2002, 41 pp, Application: WO 2001-JP966120011105 (patent written in Japanese).

[10] W. Tückmantel, A. P. Kozikowski, L. J. Romanczyk, Jr.:Studies in polyphenol chemistry and bioactivity. 1. Prepara-tion of building blocks from (1)-catechin. Procyanidin for-mation. Synthesis of the cancer cell growth inhibitor, 3-O-

galloyl-(2R,3R)-epicatechin-4b,8-[3-O-galloyl-(2R,3R)-epi-catechin]. J Am Chem Soc. 1999, 121, 12073–12081.

[11] S. Liao, Y. Umekita, J. Guo, J. M. Kokontis, R. A. Hiipakka:Growth inhibition and regression of human prostate andbreast tumors in athymic mice by tea epigallocatechin gal-late. Cancer Lett. 1995, 96, 239–243.

[12] Authors unpublished results obtained from NCI.

[13] H. Plieninger, B. Schwarz, H. Jaggy, U. Huber-Ptz, H.Rodewald, H. Imgartinger, K. Weinges: Natural productsfrom medicinal plants. XXIV. Isolation, structure determina-tion, and synthesis of (Z,Z)-4,4’-(1,4-penta-1,5-diyl)diphe-nol. An unusual natural product from the leaves of the Ginkotree (Ginkgo biloba L.). Liebigs Ann Chem. 1986, 10, 1772–1778.

[14] B. R. Barik, A. B. Kundu, A. K. Dey: Two phenolic constitu-ents from Alpinia galangal rhizomes. Phytochemistry. 1987,26, 2126–2127.

[15] J. Mustafa, S. I. Khan, G. Ma, L. A. Walker, I. A. Khan: Syn-thesis and anticancer activities of fatty acid analogs ofpodophyllotoxin Lipids. 2004, 39, 167–172.

[16] M. R. Boyd, K. D. Paull: Some practical considerations andapplications of the National Cancer Institute in vitro anti-cancer drug discovery screen. Drug Dev Res. 1995, 349, 1–109.

[17] B. E. Maryanoff, A. B. Reitz: The Wittig olefination reactionand modifications involving phosphoryl-stabilized carb-anions. Stereochemistry, mechanism, and selected syn-thetic aspects. Chem Rev. 1989, 89, 863–927.

[18] I. Gosney, A. G. Rowley: Transformations via phosphorus-stabilized anions. 1: Stereoselective syntheses of alkenesvia the Wittig reaction. In: Organophosphorus Reagents inOrganic Synthesis. Ed. J. I. G. Cardogan, Academic Press,New York, NY (USA) 1979, pp. 17–153.

[19] B. E. Maryanoff, A. B. Reitz, B. A. Duhl-Emswiler: Stereo-chemistry of the Wittig reaction. Effect of nucleophilicgroups in the phosphonium ylide. J Am Chem Soc. 1985,107, 217–226.

[Received: October 20, 2006; accepted: March 5, 2007]


synthesis, spectroscopic and anti-tumor studies of polyphenol-linoleates derived from natural...

Documents