1962
L.-W. Zheng et al. / Bioorg. Med. Chem. 17 (2009) 1957–1962
1H, ArH), 7.87 (d, J = 8.7 Hz, 2H, ArH), 11.66 (s, 1H, NH), 14.22 (s,
1H, OH); ESI-MS: 512.7 (MꢀC4H9+H)+.
the apoptosis ratio.23 In brief, after cells were treated in the
presence or absence of compound 3e at 0.1, 0.5 and 1 M for
l
48 h, DNA fragmentation was detected by the DeadEndTM Fluo-
rometric TUNEL System (Promega, USA) according to the manu-
facturer0s protocol. Cells were evaluated by the laser scanning
confocal microscope (TCS-SP2, Leica, Germany). The percent
apoptosis rate was quantified according to the TUNEL-positive
rate.
4.2.7. (E)-1-(4-tert-butylbenzyl)-N0-(1-(2-hydroxy-5-
methylphenyl)ethylidene)-3-(4-methoxyphenyl)-1H-pyrazole-
5-carbohydrazide (3g)
White solid, yield 76%; mp 226–228 °C; IR (KBr)
m: 3385 (OH),
2994–2833 (NH), 1691 (C@O) cmꢀ1 1H NMR (DMSO, 400 MHz)
;
d: 1.23 (s, 9H, 3CH3), 2.28 (s, 3H, CH3), 2.48 (s, 3H, CH3), 3.80 (s,
3H, OCH3), 5.72 (s, 2H, CH2), 6.81 (d, J = 8.7 Hz, 1H, ArH), 7.02 (d,
J = 8.7 Hz, 2H, ArH), 7.13 (d, J = 8.7 Hz, 1H, ArH), 7.19 (d,
J = 8.3 Hz, 2H, ArH), 7.34 (d, J = 8.3 Hz, 2H, ArH), 7.45 (s, 1H, ArH),
7.50 (s, 1H, 4-H), 7.78 (d, J = 8.7 Hz, 2H, ArH), 11.33 (s, 1H, NH),
12.87 (s, 1H, OH); ESI-MS: 511.5 (M+H)+.
4.7. LDH assay
Lactate dehydrogenase (LDH) assay was performed on cells
treated with compound 3e at 0.1, 0.5 and 1 lM for 24 and 48 h
using a LDH kit (Nanjing Jiancheng, China) according to the manu-
facturer0s protocol. Light absorption was measured at 340 nm
using a model Cintra 5 UV–vis spectrometer (GBC, Australia).
4.2.8. (E)-1-(4-tert-butylbenzyl)-N0-(1-(5-chloro-2-
hydroxyphenyl)ethylidene)-3-(4-methoxyphenyl)-1H-pyrazole-
5-carbohydrazide (3h)
4.8. Statistical analyses
White solid, yield 87%; mp 223–225 °C; IR (KBr)
m: 3393 (OH),
3000–2834 (NH), 1694 (C@O) cmꢀ1 1H NMR (DMSO, 400 MHz)
;
Data were presented as means SE and analyzed by SPSS soft-
ware. Pictures were processed with Photoshop software. Mean val-
ues were derived from at least three independent experiments.
Differences at p < 0.05 were considered statistically significant.
d: 1.23 (s, 9H, 3CH3), 2.50 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 5.72 (s,
2H, CH2), 6.95 (d, J = 8.8 Hz, 1H, ArH), 7.02 (d, J = 8.7 Hz, 2H,
ArH), 7.18 (d, J = 8.3 Hz, 2H, ArH), 7.33–7.37 (m, 3H, ArH), 7.52 (s,
1H, 4-H), 7.67 (s, 1H, ArH), 7.78 (d, J = 8.7 Hz, 2H, ArH), 11.46 (s,
1H, NH), 13.21 (s, 1H, OH); ESI-MS: 531.3 (M+H)+.
Acknowledgments
4.2.9. (E)-1-(4-tert-butylbenzyl)-N0-(1-(3,5-dichloro-2-
hydroxyphenyl)ethylidene)-3-(4-methoxyphenyl)-1H-pyrazole-
5-carbohydrazide (3i)
This study was supported by the Science and Technology Devel-
opmental Project of Shandong Province (2008GG10002034 and
2007GG20002004) and National Natural Science Foundation of
China (90813022).
Yellow solid, yield 91%; mp 248–250 °C; IR (KBr)
m: 3486 (OH),
2966–2835 (NH), 1694 (C@O) cmꢀ1 1H NMR (DMSO, 400 MHz) d:
;
1.23 (s, 9H, 3CH3), 2.53 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 5.73 (s, 2H,
CH2), 7.02 (d, J = 8.4 Hz, 2H, ArH), 7.20 (d, J = 8.4 Hz, 2H, ArH), 7.34
(d, J = 8.4 Hz, 2H, ArH), 7.54 (s, 1H, 4-H), 7.65 (dd, J = 2.0, 18.2 Hz,
1H, ArH), 7.69 (dd, J = 2.0, 18.2 Hz,1H, ArH), 7.78 (d, J = 8.4 Hz, 2H,
ArH), 11.61 (s, 1H, NH), 14.25 (s. 1H, OH); ESI-MS: 600.6 (M+2NH4)+.
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