Synthesis and structure-activity relationships of novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide derivatives as potential agents against A549 lung cancer cells

Article abstract of DOI:10.1016/j.bmc.2007.08.021

A series of novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide derivatives were synthesized, and the effects of all the compounds on A549 cell growth were investigated. The results showed that all the nine compounds had inhibitory effects on the growt

Full text of DOI:10.1016/j.bmc.2007.08.021

Bioorganic & Medicinal Chemistry 15 (2007) 6893–6899
Synthesis and structure–activity relationships of novel
1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide
derivatives as potential agents against A549 lung cancer cells
Yong Xia,a Zhi-Wu Dong,b Bao-Xiang Zhao,a,* Xiao Ge,b Ning Meng,b
Dong-Soo Shinc,* and Jun-Ying Miaob,*
aInstitute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China
bInstitute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China
cDepartment of Chemistry, Changwon National University, Changwon 641-773, South Korea
Received 7 July 2007; accepted 9 August 2007
Available online 21 August 2007
Abstract—A series of novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide derivatives were synthesized, and the effects of all the
compounds on A549 cell growth were investigated. The results showed that all the nine compounds had inhibitory effects on the
growth of A549 cells and induced the cell apoptosis. The study on structure–activity relationships and prediction of lipophilicities
of compounds showed that compounds with logP values in the range of 3.12–4.94 had more inhibitory effects on the growth of A549
cells.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
terial, hypoglycemic, sedative–hypnotic activity,18,19 and
anticoagulant activity.20 Recently, some arylpyrazoles
were reported to have non-nucleoside HIV-1 reverse
transcriptase inhibitory activity.21 Extensive studies
have been devoted to arylpyrazole derivatives such as
Celecoxib, a well-known cyclooxygenase-2 inhibitor.22–24
In the previous paper, we investigated the effects of
ethyl 1-(20-hydroxy-30-aroxypropyl)-3-aryl-1H-pyra-
zole-5-carboxylate derivatives on the growth of A549
lung cancer cell and found that these compounds could
suppress A549 lung cancer cell growth.25 Recently, we
described the synthesis and preliminary biological eval-
uation of ethyl 1-arylmethyl-3-aryl-1H-pyrazole-5-
carboxylate derivatives 3.26 We found that the
compounds 3a–3d promoted human umbilical vein
endothelial cell (HUVEC) apoptosis to a certain extent
at the concentrations of 5–20 lM and the effect on the
cell viability was dose-dependent. However, the com-
pounds 3 have no obvious effects on the A549 cell
growth in the test dose. Not surprisingly, the difference
of compounds 3 from ethyl 1-(20-hydroxy-30-aroxypro-
pyl)-3-aryl-1H-pyrazole-5-carboxylate derivatives in
the structures caused the different bioactivities. The
modification of pyrazole such as basic substituent
moiety should provide potential bioactivities. A few
of heterocyclic carbohydrazide derivatives have been
reported.27 However, there were no reports on the
Lung cancer is one of the leading causes of death in the
world. Since current treatment modalities are inade-
quate, novel therapies are needed to reduce the effects
of the increasing incidence in pulmonary neoplasm.1,2
An elaborate search of new anticancer agents has pri-
marily been triggered by the unveiling of new molecular
targets on which they intervene, followed by the discov-
ery of novel classes of compounds that interact with
such targets.3,4 In our effort to discover and develop
apoptosis inducers as potential new anticancer agents,
we have identified several classes of molecules as novel
apoptosis inducers, including safrole oxide, 1-alkoxy-3-
(30,40-methylenedioxy)phenyl-2-propanol, morpholi-
none
derivatives,
2,3-dihydro-3-hydroxymethyl-1,
4-benzoxazine derivatives.5–17
Many pyrazole derivatives are known to exhibit a wide
range of biological properties such as anti-hyperglyce-
mic, analgesic, anti-inflammatory, anti-pyretic, anti-bac-
Keywords: Pyrazole carbohydrazide; A549 cells; Apoptosis; Structure–
activity relationship.
*
Corresponding authors. Tel.: +86 531 88366425; fax: +86 531
88564464; e-mail addresses: bxzhao@sdu.edu.cn; miaojy@sdu.
edu.cn
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.021

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Y. Xia et al. / Bioorg. Med. Chem. 15 (2007) 6893–6899
synthesis and biological evaluation of 1-arylmethyl-3-
aryl-1H-pyrazole-5-carbohydrazide derivatives. We thus
tried to replace the ester group of compounds 3 with
hydrazine group to give 1-arylmethyl-3-aryl-1H-pyra-
zole-5-carbohydrazide derivatives 4. We herein would
like to report the synthesis of 4 and the findings of their
biological activities in suppressing the growth of A549
lung cancer cells.
pared with the control group, the cell viability reduced
more significantly from 100% to 32%–26% (p < 0.01).
Further, exposure of cells to 4b, 4e, and 4h at 80 lM
for 48 h, the cell viability reduced more significantly from
100% to 15%, 13%, and 6% (p < 0.01), respectively.
2.2.2. Structure–activity relationships and prediction of
lipophilicity. Growth inhibitory properties (IC50) for the
compounds 4a–4i are listed in Table 1. The data sug-
gested that compounds 4b–4f and 4h could inhibit the
cell growth obviously in the concentrations of 20–
80 lM after 48 h of the treatment. From these results
we could propose that the hydrazine group possessing
basicity plays a more important role than the ester
group in molecule. Furthermore, the compounds with
tert-butyl group 4b, 4e, and 4h were more effective to in-
hibit A549 cell growth, particularly compound 4e was
the most effective compound in suppressing A549 cell
growth. By comparing the cytotoxic activities of the
compounds 3a–3i and 4a–4i, it was shown that the cyto-
toxic potency was highly dependent, as expected, on the
substitution types and patterns on the aryl ring.
Replacement of the hydrogen at the 4-position of 3-aryl
ring with electron-donating methoxy group and chlorine
did not significantly alter the cytotoxicities against the
cancer cells tested. However, replacing the hydrogen at
the 4-position of 1-aryl ring with a bulkier tert-butyl
group resulted in significant activity increasing (com-
pounds 4b, 4e, and 4h), which was probably caused by
favorable steric interactions of the bulky hydrophobic
alkyl group at 4-position of 1-aryl ring with the binding
site. In another variation, we replaced the substituted
benzene ring with 2-chloropyridine ring such as 4c and
4f and found that it significantly affected the activity
compared with benzene and 4-chlorobenzene.
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 4 has been accomplished as
outlined in Scheme 1 starting from compounds 3 that
can be synthesized from 1 and the commercially avail-
able 2 as described in previous paper.26 For example,
compound 4a was synthesized by the reaction of com-
pound 3a with hydrazine hydrate in the condition of
refluxing (4 h) in methanol in yield 93%. The structures
1
of 4a–4i were determined by IR, H NMR, mass spec-
troscopy, and elemental analyses. Thus, for example
4f, obtained as yellow crystal, gave a [M+H]-ion peak
at m/z 362.3 in the ESI-MS, in accord with the molecular
formula C16H13Cl2N5O. The carbonyl group absorp-
tions in hydrazide moiety were observed in the
1675 cmꢀ1 in IR spectra and NH and NH2 bands in
CONHNH2 were observed in the 3203 cmꢀ1 and
3300–3260 cmꢀ1 regions, respectively. The 1H NMR
spectra indicated the chemical shift of the NH2 at
d = 4.11 ppm in the form of singlet broad peak. Two
ortho-aromatic proton signals in pyridine moiety ap-
peared at the range of d = 7.27 and 7.70 ppm as doublet
peaks (J = 8.4 Hz). Two ortho-aromatic proton signals
in benzene moiety appeared at the range of d = 7.39
and 7.72 ppm as doublet peaks (J = 8.7 Hz). Four sin-
glet signals appearing at d = 5.78, 6.78, 7.25, and
8.45 ppm are consistent with methylene protons, pyra-
zole proton, NH proton in carbohydrazide moiety and
ArH in pyridine moiety, respectively. The elemental
analyses shows satisfactory accord. Furthermore, the
structure was confirmed by the X-ray diffraction as
shown in Figure 1.
Lipophilicity is an important indicator of how easily
molecules can cross cell membranes. It is generally ac-
cepted that the more lipophilic a molecule, the more
favorably it will interact with the fatty acid tails of the
lipid bilayer, thus allowing the molecule to traverse the
membrane more easily. The partition coefficient logP
is a parameter which describes the manner in which a
drug partitions between polar and non-polar phases,
and it has been demonstrated to be an indispensable tool
in predicting the transport and activity of drugs.28–31
Prediction of lipophilicity (logP) and aqueous solubility
(logS) of compounds 4a–4i was calculated using
ALOGPS 2.1 software.32,33 The compounds 4 showed
lipophilicity with logP values in the range of 2.52–4.94
(Table 2) and aqueous solubility with logS values in
the range of 1.50–41.20 mg/L. These studies indicate
that compounds possessing the lipophilicity with logP
values in the range of 3.12–4.94 have more inhibitory ef-
fects on the growth of A549 cells.
2.2. Evaluation of biological activity
2.2.1. Effects of the compounds on the viability of A549
lung cancer cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) cell proliferation
assay is widely accepted as a reliable way to measure
the cell proliferation rate, and conversely when meta-
bolic events lead to apoptosis or necrosis. The data ob-
tained by MTT assay showed that compounds 3a–3i
have no effects on the growth of A549 cells in dosage-
and time-dependent manners in the test dosages (data
not shown), however, all the nine compounds 4a–4i
had inhibitory effects on the growth of A549 cells in dos-
age- and time-dependent manners as indicated by the re-
sults in Figure 2. As typically shown in Figure 3,
exposure of cells to 4b, 4e, and 4h at 40 lM for 24 h re-
sulted in cell viability decrease from 100% to 61%–52%
(p < 0.01). When the exposure continued onto 48 h, com-
2.2.3. Effects of the compounds on the morphology of
A549 cells. Morphological changes are associated with
the physiological and pathological processes in A549
cells. The morphology of the cells treated with com-
pounds 4b, 4e, and 4h was studied to verify whether
the cells underwent apoptosis. The obviously morpho-
logical changes of the cells treated with compounds 4b,

Y. Xia et al. / Bioorg. Med. Chem. 15 (2007) 6893–6899
R2 R2
6895
H
CO2Et
+
X
X
N
R2
N
NH2NH2/H2O
MeOH/reflux
K2CO3 / CH3CN
reflux
X
N
N
N
N
CO2Et
CONHNH2
Cl
R1
1
R1
R1
4
3
2
1a: R1 = H
2a: R2 = H, X = C
2b: R2 = t-Bu, X = C
2c: R2 = Cl, X = N
1b: R1 = Cl
1c: R1 = OMe
3 and 4
a: R1 = H, R2 = H, X = C
b: R1 = H, R2 =
t-Bu, X = C
c: R1 = H, R2 = Cl, X = N
d: R1 = Cl, R2 = H, X = C
e: R1 = Cl, R2 =
t-Bu, X = C
f: R1 = Cl, R2 = Cl, X = N
g: R1 = OMe, R2 = H, X = C
h: R1 = OMe, R2 =
t
-Bu, X = C
i: R1 = OMe, R2 = Cl, X = N
Scheme 1. Synthesis of compounds 4a–4i.
Figure 1. X-ray crystal structures of 4b (a) and 4f (b). Displacement ellipsoids are drawn at 50% probability level.
Figure 2. Effects of the compounds 4a–4i on the viability of A549 lung cancer cells. Control (Ctr), the viability of the cells cultured in the medium
without any compounds. DMSO, the viability of the cells cultured in the medium containing DMSO 0.1% (v/v) used as a vehicle control. Other bars
show the viability of the cells treated with the compounds 4a–4i at the concentration of 40 lM for 24 h.

6896
Y. Xia et al. / Bioorg. Med. Chem. 15 (2007) 6893–6899
40 lM for 24 h became shrinked, and the shrinkage is
more remarkable in 48 h. Such morphological changes
were not apparent in the control cells. The results told
us the apoptosis should take place. It was confirmed
by the analysis of DNA fragmentation and LDH activ-
ity assay.
2.2.4. Analysis of DNA fragmentation and LDH activity
assay. DNA fragmentation, chromatin condensation,
cell shrinkage, and membrane blebbing are the charac-
teristics of apoptotic cells. The chromatin condensation
and DNA fragmentation in the cells were observed by
acridine orange (AO) staining under laser scanning con-
focal microscope. The results showed that obvious nu-
clear DNA fragmentation occurred in the cells treated
with compounds 4b, 4e, and 4h, respectively (Fig. 5).
Such morphological changes were not apparent in the
control cells. To confirm the mode of cell death induced
by compounds 4b, 4e, and 4h typically, LDH assay were
performed on cells treated with or without 4b, 4e and 4h.
As shown in Figure 6, there were no obvious differences
in LDH release between the cells in the control group
(normal group) and the compound treatment groups.
The results indicated that the compounds at the test
range of concentrations did not cause necrosis in A549
cells. Thus, our results suggested that compounds 4b,
4e, and 4h induced A549 cell apoptosis.
3. Conclusion
All of the compounds 4 could inhibit the growth of
A549 cells in dosage- and time-dependent manners, typ-
ically compounds 4b, 4e, and 4h induced A549 cells to
apoptosis but did not cause necrosis in the cells. The
study on structure–activity relationships and prediction
of lipophilicities of compounds 4 showed that com-
pounds with logP values in the range of 3.12–4.94 had
more inhibitory effects on the growth of A549 cells.
Figure 3. Effects of the compounds 4b, 4e, and 4h on the viability of
A549 lung cancer cells. Control (Ctr), the viability of the cells cultured
in the medium without any compounds. DMSO, the viability of the
cells cultured in the medium containing DMSO 0.1% (v/v) used as a
vehicle control. Other bars show the viability of the cells treated with
the compounds 4b, 4e, and 4h at the concentrations of 10–80 lM for 24
and 48 h, respectively.
4. Experimental
Thin-layer chromatography (TLC) was conducted on
silica gel 60 F254 plates(Merck KGaA). 1H NMR spectra
were recorded on a Bruker Avance 300 (300 MHz) spec-
trometer, using CDCl3 or DMSO as solvents and tetra-
methylsilane (TMS) as internal standard. Melting points
were determined on an XD-4 digital micro-melting point
apparatus and are uncorrected. IR spectra were re-
4e, and 4h for 24 and 48 h were observed under a con-
trast phase microscope, as shown in Figure 4A and B.
We found that the cells treated with compound 4e at
Table 1. Growth inhibitory properties IC50 (lM) for the compounds 4a–4i at 24 and 48 h
Compound
4a
4b
4c
4d
4e
4f
4g
4h
4i
24 h
48 h
440.2
—
66.15
26.55
223.9
67.71
232.0
38.72
68.33
18.52
121.3
30.16
550.0
—
49.85
23.86
240.3
—
Table 2. Lipophilicity (logP) and aqueous solubility (logS) of compounds 4a–4i
Compound
4a
2.73
41.20
4b
4c
2.52
30.53
4d
3.24
11.66
4e
4f
4g
4h
4i
logP
logS
4.49
3.06
4.94
1.50
3.12
15.60
2.87
26.80
4.60
3.07
2.76
28.78

Y. Xia et al. / Bioorg. Med. Chem. 15 (2007) 6893–6899
6897
Figure 5. Effect of compounds 4b, 4e, and 4h on A549 nuclear
fragmentation. AO staining was performed after A549 cells were
incubated with compounds 4b, 4e, and 4h for 48 h. Then photographs
were taken by a laser scanning confocal microscope (Leica, Germany).
The pictures are representatives of three independent experiments. (a)
Control, (b) compound 4b, (c) compound 4e, (d) compound 4h.
Figure 6. Effect of compounds 4b, 4e, and 4h on LDH activities of
A549 cells. The culture medium was collected as samples for LDH
assay after 48 h treatment at the concentration of 40 lM. (#p > 0.05 vs
control group, n = 3).
Figure 4. Effect of compounds 4b, 4e, and 4h on A549 cell morphology
for 24 h (A) and 48 h (B) at the concentration of 40 lM. The
photographs were obtained by a phase contrast microscope (·800). (a)
Control, (b) compound 4b, (c) compound 4e, (d) compound 4h.
the solid was recrystallized from ethanol. As a result
of this process the following compounds were prepared
in yields of 72–93% (4a–4i).
corded with an IR spectrophotometer Avtar 370 FT-IR
(Termo Nicolet). Elemental analyses were performed on
a Vario EL III (Elementar Analysensysteme GmbH)
spectrometer. MS spectra were recorded on a Trace
DSQ mass spectrograph. logP and logS were calculated
using ALOGPS 2.1 software.
4.1.1. 1-Benzyl-3-phenyl-1H-pyrazole-5-carbohydrazide
(4a). White solid, yield: 93%; mp: 132–133 ꢁC; IR
(KBr) m: 3302–3031 (NH, NH2), 1681 (C@O) cmꢀ1
;
1H NMR (300 MHz, CDCl3) d (ppm): 4.03 (s, 2H,
NH2), 5.80 (s, 2H, CH2), 6.80 (s, 1H, 4-H), 7.24–
7.43 (m, 9H, ArH, NH), 7.79 (d, J = 7.2 Hz, 2H,
ArH); ESI-MS: 293.5 (M+H)+; Anal. Calcd for
C17H16N4O: C, 69.85; H, 5.52; N, 19.17. Found: C,
69.50; H, 5.25; N, 19.06.
4.1. General procedure for the synthesis of 1-arylmethyl-
3-aryl-1H-pyrazole-5-carbohydrazide derivatives (4a–4i)
To a stirred solution of 1 mmol of 3a–3i in methanol
(5 ml), 1.2 ml of 80% hydrazine monohydrate was
added. The reaction mixture was maintained under re-
flux for 1–5 h, until TLC indicated the end of reaction.
After this time, the reaction mixture stood overnight
and the solid formed was collected by filtration. Then
4.1.2.
1-(4-tert-Butylbenzyl)-3-phenyl-1H-pyrazole-5-
carbohydrazide (4b). White solid, yield: 76%; mp: 148–
149 ꢁC; IR (KBr) m: 3329–2865 (NH, NH2), 1663

6898
Y. Xia et al. / Bioorg. Med. Chem. 15 (2007) 6893–6899
(C@O) cmꢀ1
;
1H NMR (300 MHz, CDCl3) d (ppm):
4.1.8.
1-(4-tert-Butylbenzyl)-3-(4-methoxyphenyl)-1H-
1.27 (s, 9H, 3CH3), 4.01 (s, 2H, NH2), 5.77 (s, 2H,
CH2), 6.79 (s, 1H, 4-H), 7.24–7.43 (m, 8H, ArH, NH),
7.79 (d, J = 7.8 Hz, 2H, ArH); ESI-MS: 349.4
(M+H)+; Anal. Calcd for C21H24N4O: C, 72.39; H,
6.94; N, 16.08. Found: C, 72.16; H, 6.78; N, 15.96.
pyrazole-5-carbohydrazide (4h). White solid, yield: 72%;
mp: 142–144 ꢁC; IR (KBr) m: 3325–2867 (NH, NH2),
1656 (C@O) cmꢀ1 1H NMR (300 MHz, CDCl3) d
;
(ppm): 1.27 (s, 9H, 3CH3), 3.84 (s, 3H, OCH3), 4.01
(s, 2H, NH2), 5.75 (s, 2H, CH2), 6.71 (s, 1H, 4-H),
6.93 (d, J = 8.4 Hz, 2H, ArH), 7.26–7.32 (m, 5H, ArH,
NH), 7.72 (d, J = 8.4 Hz, 2H, ArH); ESI-MS: 379.6
(M+H)+; Anal. Calcd for C22H26N4O2: C, 69.82; H,
6.92; N, 14.80. Found: C, 69.29; H, 6.55; N, 14.71.
4.1.3. 1-((6-Chloropyridin-3-yl)methyl)-3-phenyl-1H-pyr-
azole-5-carbohydrazide (4c). White solid, yield: 86%; mp:
152–154 ꢁC; IR (KBr) m: 3423–2944 (NH, NH2), 1676
(C@O) cmꢀ1 1H NMR (300 MHz, CDCl3) d (ppm):
;
4.03 (s, 2H, NH2), 5.79 (s, 2H, CH2), 6.82 (s, 1H, 4-
H), 7.26 (d, J = 8.1 Hz, 1H, PyH), 7.33–7.44 (m, 4H,
ArH, NH), 7.68 (dd, J = 2.1, 8.1 Hz, 1H, PyH), 7.77
(d, J = 7.2 Hz, 2H, ArH), 8.45 (d, J = 2.1 Hz, 1H,
PyH); ESI-MS: 328.5 (M+H)+; Anal. Calcd for
C16H14ClN5O: C, 58.63; H, 4.31; N, 21.37. Found: C,
58.46; H, 4.39; N, 21.51.
4.1.9. 1-((6-Chloropyridin-3-yl)methyl)-3-(4-methoxyphe-
nyl)-1H-pyrazole-5-carbohydrazide (4i). Yellow solid,
yield: 86%; mp: 157–159 ꢁC; IR (KBr) m: 3410–2834
1
(NH, NH2), 1669 (C@O) cmꢀ1; H NMR (300 MHz,
CDCl3) d (ppm): 3.84 (s, 3H, OCH3), 4.01 (s, 2H, NH2),
5.76 (s, 2H, CH2), 6.74 (s, 1H, 4-H), 6.94 (d, J = 8.4 Hz,
2H, ArH), 7.25 (d, J = 7.8 Hz, 1H, PyH), 7.42 (s, 1H,
NH), 7.67 (d, J = 7.8 Hz, 1H, PyH), 7.70 (d, J = 8.4 Hz,
2H, ArH), 8.44 (s, 1H, PyH); ESI-MS: 358.5 (M+H)+;
Anal. Calcd for C17H16ClN5O2: C, 57.07; H, 4.51; N,
19.57. Found: C, 57.24; H, 4.62; N, 19.42.
4.1.4. 1-Benzyl-3-(4-chlorophenyl)-1H-pyrazole-5-carbo-
hydrazide (4d). White solid, yield: 89%; mp: 180–
182 ꢁC; IR (KBr) m: 3266–2958 (NH, NH2), 1630
(C@O) cmꢀ1 1H NMR (300 MHz, CDCl3) d (ppm):
;
3.98 (s, 2H, NH2), 5.79 (s, 2H, CH2), 6.77 (s, 1H, 4-
H), 7.28–7.29 (m, 6H, ArH, NH), 7.37 (d, J = 8.4 Hz,
2H, ArH), 7.72 (d, J = 8.4 Hz, 2H, ArH); ESI-MS:
327.3 (M+H)+; Anal. Calcd for C17H15ClN4O: C,
62.48; H, 4.63; N, 17.15. Found: C, 62.52; H, 4.72; N,
17.06.
4.2. Biological activity assay
4.2.1. Materials. RPMI 1640 was obtained from Gibco
BRL Co. (Grand Island, USA) and bovine calf serum
was from Beijing DingGuo Biotechnology Co. (China).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) was purchased from Amresco. Lactate
dehydrogenase (LDH) assay kit was from ZhongSheng
Co. (Beijing, China). Acridine orange (AO) from Fluka.
4.1.5. 1-(4-tert-Butylbenzyl)-3-(4-chlorophenyl)-1H-pyra-
zole-5-carbohydrazide (4e). White solid, yield: 79%; mp:
122–124 ꢁC; IR (KBr) m: 3319–2860 (NH, NH2), 1654
(C@O) cmꢀ1
;
1H NMR (300 MHz, CDCl3) d (ppm):
4.2.2. Cell culture. A549 lung cancer cells were cultured
in RPMI 1640 medium, supplemented with 10% (v/v)
newborn calf serum at 37 ꢁC in 5% CO2 and 95% air.
The cells were routinely seeded at the density of 1000/
cm2 into 96-well plates or other appropriate dishes con-
taining the medium.
1.28 (s, 9H, 3CH3), 4.02 (s, 2H, NH2), 5.75 (s, 2H,
CH2), 6.76 (s, 1H, 4-H), 7.24–7.31 (m, 3H, ArH, NH),
7.32 (d, J = 8.4, 2H, ArH), 7.37 (d, J = 8.4, 2H, ArH),
7.73 (d, J = 8.4 Hz, 2H, ArH); ESI-MS: 383.5
(M+H)+; Anal. Calcd for C21H23ClN4O: C, 65.87; H,
6.05; N, 14.63. Found: C, 65.66; H, 5.92; N, 14.32.
4.2.3. MTT assay for cell viability. The compounds were
dissolved in DMSO. The final concentration of DMSO
was below 0.1% in the culture medium (v/v) (DMSO
at these final concentrations did not affect the viability
of the cells). Cells were seeded in 96-well plates and trea-
ted with compounds 4a–4i 20–80 lM for 24 and 48,
respectively. The cell viability was determined by the
MTT assay following the procedure described by Price
and McMillan.34 The light absorptions were measured
at 570 nm using SpectraMAX 190 microplate spectro-
photometer (GMI Co., USA).
4.1.6. 3-(4-Chlorophenyl)-1-((6-chloropyridin-3-yl)methyl)-
1H-pyrazole-5-carbohydrazide (4f). Yellow solid, yield:
84%; mp: 192–193 ꢁC; IR (KBr) m: 3299-2936 (NH,
1
NH2), 1675 (C@O) cmꢀ1; H NMR (300 MHz, CDCl3)
d (ppm): 4.11 (s, 2 H, NH2), 5.78 (s, 2H, CH2), 6.78 (s,
1H, 4-H), 7.25 (s, 1H, NH), 7.27 (d, J = 8.7 Hz, 1H,
PyH), 7.39 (d, J = 8.7 Hz, 2H, ArH), 7.70 (d, J = 8.7
Hz, 1H, PyH), 7.72 (d, J = 8.7 Hz, 2H, ArH), 8.45 (s,
1H, PyH); ESI-MS: 362.3 (M+H)+; Anal. Calcd for
C16H13Cl2N5O: C, 53.05; H, 3.62; N, 19.33. Found: C,
52.96; H, 3.72; N, 19.41.
4.2.4. LDH assay for drug toxicity. The detection was
performed as described previously.35
4.1.7. 1-Benzyl-3-(4-methoxyphenyl)-1H-pyrazole-5-car-
bohydrazide (4g). White solid, yield: 75%; mp: 142–
144 ꢁC; IR (KBr) m: 3306–2835 (NH, NH2), 1673
4.2.5. Nuclear fragmentation assay. Nuclear fragmenta-
tion was detected with AO staining and observed under
a fluorescence microscope. Briefly, the cells were cul-
tured in fresh medium with 0, 40 lM compounds 4b,
4e, and 4h, respectively, for 48 h and stained with
5 lg/ml AO at room temperature. Then the cells were
observed and photographed under an Olympus BH-2
fluorescence microscope.
(C@O) cmꢀ1 1H NMR (300 MHz, CDCl3) d (ppm):
;
3.84 (s, 3H, OCH3), 4.01 (s, 2H, NH2), 5.78 (s, 2H,
CH2), 6.72 (s, 1H, 4-H), 6.94 (d, J = 8.4 Hz, 2H, ArH),
7.23-7.28 (m, 6H, ArH, NH), 7.72 (d, J = 8.4 Hz, 2H,
ArH); ESI-MS: 323.4 (M+H)+; Anal. Calcd for
C18H18N4O2: C, 67.07; H, 5.63; N, 17.38. Found: C,
66.86; H, 5.71; N, 17.26.

Y. Xia et al. / Bioorg. Med. Chem. 15 (2007) 6893–6899
6899
18. Cottineau, B.; Toto, P.; Marot, C.; Pipaud, A.; Chenault,
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Differences at p < 0.05 were considered statistically
significant.
s
t
Acknowledgments
21. Genin, M. J.; Biles, C.; Keiser, B. J.; Poppe, S. M.;
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J. Med. Chem. 2000, 43, 1034.
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This study was supported by the Foundation of the
Ministry of Education (104112) and the Natural Science
Foundation of Shandong Province (Y2005B12).
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