thin bed of alumina using hexane–ethylacetate (9.7 : 0.3) to give
methyl-3-(3,4-dioxymethylenephenyl)-propenoate (15b, 0.081 g,
64% yield).
diphenyltetrazolium bromide (MTT) (5 mg mL-1 in 1¥ phosphate-
buffered saline) was added at 20 mL per well, and incubated for
another 4 h. After spinning the plate at 1500 RPM for 5 min,
supernatant was removed and 100 mL of the stop agent DMSO was
added to each well. Formation of formazon, an index of growth,
was read at 570 nm and IC50 values were determined by analysis
of dose–response curves. Therapeutic index was calculated as IC50
mammalian cell/IC50 Pf 3D7.
Methyl-3-(3,4-dioxymethylenephenyl)propenoate (15b, Table 3).
White solid, m.p. 67–68 ◦C, 1H NMR d (CDCl3, 300 MHz), 7.59
(1H, d, J = 15.9 Hz), 6.99 (1H, s), 6.95 (1H, d, J = 8.0 Hz), 6.78 (1H,
d, J = 8.0 Hz), 6.26 (1H, d, J = 15.9 Hz), 5.97 (2H, s), 3.77 (3H, s);
13C NMR d (75.4 MHz, CDC13), 167.9, 150.0, 148.7, 144.9, 129.2,
124.8, 116.1, 108.9, 106.9, 101.9 and 51.9. HRMS-ESI: m/z [M +
H]+ for C11H10O4, calculated 207.0651; observed 207.0673.
The above procedure was also followed for synthesis of various
other cinnamic esters (Table 3, entries 16–22). The structures of
the corresponding products were confirmed by NMR (1H and 13C)
and HRMS (see the ESI for details†).
Acknowledgements
We graciously acknowledge the IHBT project MLP 0025 and
the Department of Science and Technology, New Delhi, for
the financial support (Vide Grant No. SR/S1/OC-16/2008).
The authors gratefully acknowledge the Director(s) of I.H.B.T.,
C.S.I.R., Palampur and I.C.G.E.B, New Delhi for their kind
encouragement as well as providing infrastructure for conducting
the above work. Authors (AS, NS & RK) are indebted to CSIR,
New Delhi, for Senior Research fellowship. DM and DS thank
MR4 who generously provided the chloroquine resistant Dd2 and
INDO strains used in the study. Thanks to David Walliker and X
Su who deposited these strains with MR4. IHBT Communication
No: 2125.
Measurement of inhibition of P. falciparum growth in culture
In this study, chloroquine sensitive 3D7 and chloroquine resistant
Dd2 and INDO strains of P. falciparum were used in in vitro
culture. Parasite strains were cultivated by the method of Trager
and Jensen19 with minor modifications. Cultures were maintained
in fresh O+ human erythrocytes at 4% hematocrit in complete
medium (RPMI 1640 with 0.2% sodium bicarbonate, 0.5%
Albumax, 45 mg L-1 hypoxanthine and 50 mg L-1 gentamicin)
at 37 ◦C under reduced O2 (gas mixture 5% O2, 5% CO2, and
90% N2). Stock solutions of chloroquine were prepared in water
(milliQ grade) and test compounds were dissolved in DMSO.
All stocks were then diluted with culture medium to achieve
the required concentrations (in all cases the final concentration
contained 0.4% DMSO, which was found to be non-toxic to the
parasite). Drugs and test compounds were then placed in 96-well
flat-bottom tissue culture grade plates to yield triplicate wells with
drug concentrations ranging from 0 to 10-4 M in a final well volume
of 100 mL. Chloroquine was used as a positive control in all exper-
iments. Parasite culture was synchronized at ring stage with 5%
sorbitol. Synchronized culture was aliquoted to a drug-containing
96-well plate at 2% hematocrit and 1% parasitemia. After 48 h
of incubation under standard culture conditions, plates were har-
vested and read by the SYBR Green I fluorescence-based method20
using a 96-well fluorescence plate reader (Victor, Perkin Elmer),
with excitation and emission wavelengths at 497 and 520 nm,
respectively. The fluorescence readings were plotted against drug
concentration, and IC50 values obtained by visual matching of the
drug concentration giving 50% inhibition of growth.
Notes and References
1 (a) M. Eissen, J. O. Metzger, E. Schmidt and U. Schneidewind, Angew.
Chem., Int. Ed., 2002, 41, 414–436; (b) B. Kamm, Angew. Chem., Int.
Ed., 2007, 46, 5056–5058; (c) P. K. Vemula and G. John, Acc. Chem.
Res., 2008, 41, 769–782; (d) A. L. Marshall and P. J. Alaimo, Chem.–Eur.
J., 2010, 16, 4970–4980.
2 (a) D. E. Fogg and E. N. Santos, Coord. Chem. Rev., 2004, 248, 2365–
2379; (b) S. J. Broadwater, S. L. Roth, K. E. Price, M. Kobaslija and D.
T. McQuade, Org. Biomol. Chem., 2005, 3, 2899–2906.
3 (a) J. B. Harborne and H. Baxter, Phytochemical Dictionary:A Hand-
book of Bioactive Compounds from Plants, Taylor & Francis Ltd.
Washington, DC, 1993; (b) R. A. Dixon and N. L. Paiva, Plant Cell,
1995, 7, 1085–1097; (c) J. Harmatha and L. Dinan, Phytochem. Rev.,
2003, 2, 321–330.
4 (a) A. M. Hernandez, L. Lopez, G. Chamorro and F. T. Mendoza,
Planta Med., 1993, 59, 121–124; (b) E. C. Miller, A. B. Swanson, D. H.
Phillips, T. L. Fletcher, A. Liem and J. A. Miller, Cancer Res., 1983, 43,
1124–1134.
5 (a) R. J. K. Taylor, M. Reid, J. Foot and S. A. Raw, Acc. Chem. Res.,
2005, 38, 851–869; (b) R. A. Periana, O. Mironov, D. Taube, G. Bhalla
and C. J. Jones, Science, 2003, 301, 814–818; (c) S. A. Raw, C. D. Wilfred
and R. J. K. Taylor, Org. Biomol. Chem., 2004, 2, 788–796; (d) M. Davi
and H. Lebel, Org. Lett., 2009, 11, 41–44; (e) J. Brioche, G. Masson
and J. Zhu, Org. Lett., 2010, 12, 1432–1435.
6 (a) J. H. Delcamp and M. C. White, J. Am. Chem. Soc., 2006, 128,
15076–15077; (b) D. Crich and Y. Zou, Org. Lett., 2004, 6, 775–777;
(c) J. Eames and M. Watkinson, Angew. Chem., Int. Ed., 2001, 41,
3567–3571.
7 (a) N. Yayli, S. O. Sivrikaya, A. Yasar, O. Ucuncu, C. Gulec, S. Kolaylı,
M. Kucuk and E. Celik, J. Photochem. Photobiol., A, 2005, 175, 22–
28; (b) C. Ruzie, M. Krayer and J. S. Lindsey, Org. Lett., 2009, 11,
1761–1764.
Measurement of cytotoxic activity against mammalian cell lines in
culture
Animal cell lines (HeLa and fibroblast L929) were used to
determine drug toxicity by using MTT assay for mammalian
21
cell viability assay as described by Mosmann
using HeLa
8 (a) K. Zeitler, Org. Lett., 2006, 8, 637–640; (b) S. Gobec, M. Sova, K.
Kristanb and T. L. Rizner, Bioorg. Med. Chem. Lett., 2004, 14, 3933–
3936; (c) M. Sova, A. Perdih, M. Kotnik, K. Kristan, T. L. Rizner, T.
Solmajerb and S. Gobec, Bioorg. Med. Chem., 2006, 14, 7404–7418.
9 (a) J. B. Baell, R. W. Gable, A. J. Harvey, N. Toovey, T. Herzog, W.
Hansel and H. Wulff, J. Med. Chem., 2004, 47, 2326–2336; (b) D.
Batovska, S. Parushev, A. Slavova, V. Bankova, I. Tsvetkova, M. Ninova
and H. Najdenski, Eur. J. Med. Chem., 2007, 42, 87–92; (c) D. S. Black,
N. Kumar and P. S. R. Mitchell, J. Org. Chem., 2002, 67, 2464–2473;
(d) C. Ruzie, M. Krayer and J. S. Lindsey, Org. Lett., 2009, 11, 1761–
1764; (e) Y. Xin, Z. H. Zang and F. L. Chen, Synth. Commun., 2009,
and fibroblast L929 cells cultured in complete RPMI containing
10% fetal bovine serum, 0.2% sodium bicarbonate, 50 mg ml-1
gentamycin. Briefly, cells (104 cells/200 mL/well) were seeded
into 96-well flat-bottom tissue-culture plates in complete culture
medium. Drug solutions were added after overnight s◦eeding and
incubated for 24 h in a humidified atmosphere at 37 C and 5%
CO2. DMSO (final concentration 10%) was added as +ve control.
An aliquot of a stock solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-
5218 | Org. Biomol. Chem., 2011, 9, 5211–5219
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