K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5455–5459
5459
the reaction mixture was evaporated to dryness, the solid obtained was
neutralised with dil HCl and extracted with ethyl acetate. The combined
organic extracts were dried (Na2SO4), and concentrated in vacuo, and the
residue was further purified by column chromatography (SiO2, 1% to 2% CH3OH
in CH2Cl2 as eluant) afforded compound (13) as Yellow solid; yield: 76%; mp
218–220 °C; IR (KBr, cm1): 3455, 3018, 2906, 1582, 1360, 1216, 766; 1H NMR
(CDCl3 + CD3OD, 300 MHz) d: 8.44 (s, 1H), 8.10–8.05 (m, 2H), 7.85–7.83 (m,
3H), 7.68–7.64 (m, 4H), 7.45 (d, J = 8.4 Hz, 2H), 7.40–7.37 (m, 2H), 7.03 (d,
J = 8.5 Hz, 2H), 6.53 (s, 1H), 3.92 (s, 3H), 3.87 (s, 3H), 3.65 (s, 3H); 13C NMR
(CDCl3, 75 MHz) d: 191.1, 157.5, 156.0, 153.5, 150.3, 148.1, 146.3, 143.6, 142.2,
139.4, 135.9, 131.0, 129.5, 129.0, 128.9, 127.2, 126.1, 125.8, 125.0, 122.4, 119.3,
116.2, 115.8, 108.0, 102.0, 62.0, 62.1, 56.3; ESI-MS (m/z): 628 (M+H)+;
HRMS(m/z): calcd for C34H27Cl2N3O5 (M+H) +: 628.1406, Found: 628.1396.
(b) Ducki, S.; Rennison, D.; Woo, M.; Kendall, A.; Chabert, J. F. D.; McGown, A.
T.; Lawrence, N. J. Bioorg. Med. Chem. 2009, 17, 7698.
Singh for technical support, SAIF for NMR, IR, and Mass spectral
data. S.R.A and G.R.P. are thankful to CSIR, New Delhi, India for
financial support. This work was supported by EMPOWER grant
(OLP0007) of CSIR-CDRI to KVS. This is CSIR-CDRI communication
number 8281.
Supplementary data
Supplementary data (spectral data of all the compounds) asso-
ciated with this article can be found, in the online version, at
19. In vitro antimalarial assay: The compounds were dissolved in DMSO at 5 mg/
mL. Twofold serial dilutions of test samples were made in 96 well plates and
incubated with 1.0% parasitized cell suspension containing 0.8% parasitaemia
(Asynchronous culture with more than 80% ring stages). The plates were
incubated at 37 °C in CO2 incubator in an atmosphere of 5% CO2 and air
References and notes
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lL of lysis buffer containing 2Â concentration of SYBR
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graphic programme (EXCEL) and IC50 values were obtained by Logit regression
analysis of dose response curves. Chloroquine was used as the standard
reference drug (Singh, S.; Srivastava, R. K.; Srivastava, M.; Puri, S. K. and
Srivastava, K. In vitro culture of Plasmodium falciparum: Utility of Modified
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20. Cytotoxicity assay: Cytotoxicity of the compounds was carried out using Vero
cell line (C1008; Monkey kidney fibroblast) following the method of Mosmann
(1983) with certain modifications. The cells were incubated with different
dilutions of test agents for 72 h and MTT was used as reagent for the detection
of cytotoxicity. 50% cytotoxic concentration (CC50) was determined using non-
linear regression analysis of dose response curves. Selectivity Index (SI) was
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Immunol. Methods. 1983, 65, 55).
21. Inhibition of in vitro b-hematin formation: Male swiss mice, weighing 15–20 g
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hematin formation. The assay mixture contained 100 mM sodium acetate
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buffer pH (5.1), 50
compound/drug in
l
a
L plasma, 100
total reaction volume of 1.0 mL. The control tube
lM hemin as the substrate and 1–20 lg
contained all reagents except compound. The reaction mixture in triplicate
was incubated at 37 °C for 16 h in a rotary shaker. The reaction was stopped by
centrifugation at 10,000 rpm for 10 min at 30 °C. The pellet was suspended in
100 mM Tris–HCl buffer pH (7.4) containing 2.5% SDS. The pellet obtained after
centrifugation was washed thrice with distilled water (TDW) to remove free
hemin attached to b-hematin. The pellet was solubilized in 50 lL of 2 N NaOH
and volume was made up to 1.0 mL with TDW. Absorbance was measured at
400 nm. The 50% inhibitory concentration (IC50) was determined using non-
linear regression analysis dose response curves (Pandey, A.V.; Singh, N.;
Tekwani, B.L.; Puri, S.K.; Chauhan, V.S. Assay of beta-hematin formation by
malaria parasite. J. Pharm. Biomed. Anal. 1999, 20, 203).
22. Measurement of hydrogen peroxide-mediated hemin degradation: Hydrogen
peroxide-mediated hemin degradation was also evaluated in the presence/
absence of compounds by using the method of Loria et al. (1999) with some
modifications. The inhibition was monitored in 96 well ELISA plate with a total
16. (a) Mishra, N.; Arora, P.; Kumar, B.; Mishra, L. C.; Bhattacharya, A.; Awasthi, S.
K.; Bhasin, V. K. Eur. J. Med. Chem. 2008, 43, 1530; (b) Liu, M.; Wilairat, P.; Go,
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18. (a) Representative procedure for synthesis of (Z)-1-(4-chlorophenyl)-2-(4-((E)-(2-
(7-chloroquinolin-4-yl)hydrazono)methyl)phenoxy)-3-(2,3,4-trimethoxyphenyl)-
prop-2-en-1-one (13).
reaction volume of 200
NaOH), 180 g Bovine serum albumin (in 0.2 M Sodium acetate buffer pH 5.1),
20 mM of H2O2 and 20 of Chloroquine/Compounds. The plates were
lL for each well consisting 25 lM Hemin (in 0.1 N
l
l
M
incubated to equilibrate for 10 min. at room temperature after addition of
hemin and bovine serum albumin. The peroxidative reaction was initiated by
the addition of H2O2 and followed by measuring the decrease in absorption at
the Soret band (405 nm) after 30 min. incubation at room temperature. Control
groups (TDW instead of H2O2) were also included in each experiment. Results
were expressed as the percentage of remained hemin in the reaction mixture.
Data are the mean of three different experiments in triplicate (Loria. P.; Miller,
S.; Foley, M.; Tilley, L. Biochem. J. 1999, 339, 363).
To
a solution of 7 (449 mg, 0.001 mol) in 10% methanolic KOH, 2,3,4-
trimethoxy benzaldehyde (196 mg, 0.001 mol) was added and stirred at
room temperature for 3 h. After completion of reaction (monitored by TLC),
23. Ginsburg, H.; Ward, S. A.; Bray, P. G. Parasitol. Today 1999, 15, 357.