Synthesis and cytotoxic activity of novel 3-(1H-indol-3-yl)-1H-pyrazole-5- carbohydrazide derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.09.049

A series of novel 3-(1H-indole-3-yl)-1H-pyrazole-5-carbohydrazide derivatives 4Ia-n, 4IIa-b and 6 were prepared by hydrazinolysis of ethyl 3-(1H-indole-3-yl)-1H-pyrazole-5-carboxylate with hydrazine hydrate in excellent yields. These new compounds were fully characterized by spectroscopic methods, and the important intermediates 3Ie, 3IIc and 3IId were further confirmed by X-ray crystallography. All the new compounds were evaluated for their cytotoxic activity against 4 human cancer cell lines by MTT method. Some of them exhibited more potent antiproliferative activity against HepG-2, BGC823 and BT474 cell lines than the positive drug 5-fluorourcail. Flow cytometry analysis showed that 4Ik and 4Il arrested the cell cycle at S phase.

Full text of DOI:10.1016/j.ejmech.2011.09.049

European Journal of Medicinal Chemistry 46 (2011) 5868e5877
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and cytotoxic activity of novel 3-(1H-indol-3-yl)-1H-pyrazole-5-
carbohydrazide derivatives
,
a
b
b
a
Datong Zhang a , Guangtian Wang , Guilong Zhao , Weiren Xu , Lingyan Huo
*
a School of Chemistry and Pharmaceutical Engineering, Shandong Polytechnic University, Jinan 250353, Shandong, PR China
b Tianjin Key Laboratory of Molecular Design and Drug Discovery, Tianjin Institute of Pharmaceutical Research, Tianjin 300193, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 3-(1H-indole-3-yl)-1H-pyrazole-5-carbohydrazide derivatives 4Iaen, 4IIaeb and 6
were prepared by hydrazinolysis of ethyl 3-(1H-indole-3-yl)-1H-pyrazole-5-carboxylate with hydrazine
hydrate in excellent yields. These new compounds were fully characterized by spectroscopic methods,
and the important intermediates 3Ie, 3IIc and 3IId were further confirmed by X-ray crystallography. All
the new compounds were evaluated for their cytotoxic activity against 4 human cancer cell lines by MTT
method. Some of them exhibited more potent antiproliferative activity against HepG-2, BGC823 and
BT474 cell lines than the positive drug 5-fluorourcail. Flow cytometry analysis showed that 4Ik and 4Il
arrested the cell cycle at S phase.
Received 29 June 2011
Received in revised form
23 September 2011
Accepted 26 September 2011
Available online 1 October 2011
Keywords:
Indole
Pyrazole
Ó 2011 Elsevier Masson SAS. All rights reserved.
Carbohydrazide
Synthesis
Cytotoxic activity
Cell cycle
1. Introduction
synthesis and in vitro cytotoxic activity of 3-(1H-indole-3-yl)-1H-
pyrazole-5-carbohydrazide derivatives against 4 tumor cell lines,
Cancer is one of the most serious clinical problems and among
the leading causes of death worldwide [1], and considerable
attention has been paid to the discovery of new anticancer agents.
Cancer is characterized by uncontrolled cellular growth and
proliferation, and therefore finding novel compounds to inhibit the
uncontrolled proliferative pathways is believed to be an effective
strategy [2e4]. Numerous compounds, both synthetic and naturally
derived, have been screened to evaluate their cytotoxic activity.
Heterocyclic scaffold is common in many anticancer agents [5e7]
and some drugs bear different heterocycles, such as Sunitinib,
AG337 and Raltitrexed, etc [8e10] (Fig. 1). Indole derivatives
possess interesting biological activities, such as anti-inflammatory
[11], antifungal [12], antibacterial [13] and anticancer [14] activi-
ties. Meanwhile, pyrazole derivatives are known to be associated
with various biological properties, such as anticancer [15], anti-
bacterial [16], antiviral [17], analgesic [18] and anti-inflammatory
[19] activities. Recently, some pyrazole carbohydrazide derivatives
were reported to have moderate anticancer activity [20]. Therefore,
it would be of interest to combine both the indole functionality and
pyrazole carbohydrazide into one molecule. We report herein the
A549, HepG-2, BGC823 and BT474 (Fig. 1).
2. Chemistry
2.1. Synthesis
The synthesis of compounds 4Iaen and 4IIaeb was accom-
plished as outlined in Scheme 1 starting from compound 1 which
can be synthesized according to the procedure described in
previous paper [21]. The reaction of the starting material 1 with
various halides under the reaction conditions KOH/DMSO or K2CO3/
CH3CN gave a mixture of monosubstituted isomers 2Iaeg and
2IIaec (Scheme 1), which were conveniently isolated via column
chromatography on silica gel. Alkylation of 2Iaeg and 2IIaec with
different halides in the presence of NaH in DMF led to disubstituted
intermediates 3Iaem and 3IIaed, respectively. Treatment of
3Iaem and 3IIaeb with hydrazine hydrate in refluxing methanol
afforded the target compounds 4Iaem and 4IIaeb. Hydrazinolysis
of the ester intermediates 2Ig produced monosubstituted
compound 4In. Compound 6 was prepared following the procedure
described in Scheme 2.
The structures of novel 3-(1H-indol-3-yl) pyrazole-5-
carbohydrazide derivatives 4I, 4II and 6 were determined by IR,
* Corresponding author. Tel.: þ86 531 89631208; fax: þ86 531 89631207.
E-mail address: dt_zhang@yahoo.com.cn (D. Zhang).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.049

D. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5868e5877
5869
N
N
N
NH
NH
O
NH2NH2
H2O
O
N
S
N
COOEt
CONHNH2
N
H
MeOH/reflux
N
n-Pr
N
n-Pr
F
HN
H2N
N
H
6
5
N
H
O
Scheme 2. Synthetic route to compound 6.
AG337
Sunitinib
O
COOH
R1
N
peak at
moiety appeared at
d
¼ 4.50 ppm. Three aromatic proton signals in benzyl
N
O
N
N
H
COOH
d
¼ 7.34, 6.67 and 7.64 ppm as one double
CONHNH2
S
N
HN
doublet peak (J ¼ 2.0, 8.4 Hz) and two doublet peaks (J ¼ 8.4 and
N
R2
2.0 Hz), respectively. The 5- and 6-position aromatic proton signals
Raltitrexed
Target compounds
in indole moiety appeared at the range of
7.16e7.20 ppm as multiplet peaks. The 4-position and 7-position
aromatic proton signals in indole appeared at
¼ 7.50 and
8.06 ppm as doublet peaks (J ¼ 8.0 Hz). The four singlet signals
appearing at
methylene protons in benzyl group, pyrazole proton, 2-H in indole
and NH proton in carbohydrazide moiety, respectively. The n-
propyl protons had the chemical shifts of 0.85, 1.74e1.84 and
4.17 ppm in the higher field.
d
¼ 7.07e7.11 and
Fig. 1. Structures of some anticancer agents and the target compounds.
d
1H NMR and HRMS. For example, 1-(2,4-dichlorobenzyl)-3-(1-
propyl-1H-indol-3-yl)-1H-pyrazole-5-carbohydrazide 4Ie with
a quasi-molecular ion peak [M þ H]þ at m/z 442.1189 in the HRMS
was in good agreement with the molecular formula C22H22Cl2N5O.
The carbonyl group absorptions in hydrazide moiety appeared at
1653 cmꢀ1 in IR spectra and NH and NH2 bands in CONHNH2 were
observed at 3297 cmꢀ1 and 3311 cmꢀ1, respectively. The 1H NMR
spectra indicated the chemical shift of the NH2 showed a singlet
d
¼ 5.86, 7.28, 7.72, and 9.90 ppm were consistent with
Owing to the tautomerism of the pyrazole ring, compound 1
consists of two tautomers in a ratio of 7:3 based on the 1H NMR
N
NH
COOEt
HN
1
2
HN
2
N
1
NH
1
N
3
3
COOEt
COOEt
HN
HN
major isomer
minor isomer
a
2Ia, R1 = n-Pr,
2IIa, R1 = n-Pr
2IIb, R1 = 4-FBn
2IIc, R1 = 2,4-Cl2Bn
2Ib, R1 = Bn,
R1
R1
2Ic, R1 = 4-MeOBn,
2Id, R1 = 3,4-(MeO)2Bn,
2Ie, R1 = 2,4-Cl2Bn,
N
N
N
N
COOEt
COOEt
, R1 = 3,4-Cl2Bn,
, R = 4-FBn,
HN
2If
2Ig
HN
2I
2II
1
3-Ia, R1 = n-Pr,
3-Ib, R1 = Bn,
R2 = n-Pr
R2 = n-Pr
R2 = n-Pr
b
b
, R1 = 4-MeOBn,
3-Id
3-Ic
, R1 = 3,4-(MeO)2Bn, R2 = n-Bn
R1
3-Ie, R1 = 2,4-Cl2Bn,
R2 = n-Pr
R1
N
N
3-If, R1 = 3,4-Cl2Bn
3-Ig, R1 = 4-FBn,
3-Ih, R1 = n-Pr,
3-Ii, R1 = n-Pr,
R2 = n-Pr
N
N
R2 = n-Pr
c
COOEt
R2 = Bn
COOEt
N
N
R2
R2 = 4-MeOBn
R2 = 3,4-(MeO)2Bn
R2 = 2,4-Cl2Bn
R2 = 3,4-Cl2Bn
R2 = 4-FBn
R2 = n-Pr
R2
3II
3-Ij, R1= n-Pr,
3I
1
3-Ik
, R = n-Pr,
R1 = n-Pr,
3-Il
,
c
c
3-Im, R1 = n-Pr,
3-IIa, R1 = 4-FBn,
3-IIb, R1 = n-Pr,
3-IIc, R1 = 2,4-Cl2Bn,
3-IId, R1 = n-Pr,
R2 = 4-FBn
R2 = n-Pr
R2 = n-Pr
R1
N
R1
N
N
N
CONHNH2
4Ia-4In
CONHNH2
N
R2
N
R2
4IIa, 4IIb
Scheme 1. Synthetic route to compounds 4Iae4In, 4IIa and 4IIb. Reagents and conditions: a. R1X, KOH/DMSO or R1X, K2CO3/CH3CN, followed by separation by column
chromatography; b. R2X, NaH; c. NH2NH2$H2O, MeOH/reflux.

5870
D. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5868e5877
spectrum, and alkylation at N-H position of pyrazole often results in
a mixture of two regioisomers. The regioisomers were assigned
according to a known method [21], which is based on the
comparison of chemical shifts of CH2/CH3 bonded to nitrogen of
pyrazole moiety with those reported. For example, in compound
3Ie, the protons in methylene of benzyl bonded to nitrogen reso-
nated at
protons in 3IIc appeared at
d
¼ 5.83 ppm as a singlet, whereas the corresponding
d
¼ 5.54 ppm (Fig. 2).
2.2. Single-crystal structure
The single crystals of compounds 3Ie, 3IIc and 3IId were ob-
tained by slow evaporation of their solution in petroleum ether and
ethyl acetyl at room temperature for a week. The diffraction data
were collected with a Bruker SMART CCD diffractometer using
graphite-monochromated Mo-K
a
radiation
(l
¼ 0.71073 Å) at
298(2) K. The structures were solved by the direct methods with
SHELXS-97 program and the refinements on F2 were performed
with SHELXS-97 program [22] by the full-matrix least-squares
techniques with anisotropic thermal parameters for non-hydrogen
atoms. The crystal structure and crystal packing diagram of
compounds 3Ie, 3IIc and 3IId were shown in Figs. 3e5, respec-
tively, while the crystallographic data and structure refinement
details were given in Table 1.
In the molecule of 3Ie, the benzene and pyrrole rings in the
indole moiety were coplanar with each other as predicted theo-
retically, and meanwhile they were coplanar with the pyrazole ring.
The benzene ring in the benzyl moiety was essentially orthogonal
to the plane formed by the indole and pyrazole rings. However,
except for the coplanarity of the benzene and pyrrole rings in the
indole moiety, these ring orientations existing in 3Ie were not
observed in its regiomer 3IIc and 3IId. There are two intermolec-
ular hydrogen bonds each in the molecules of 3Ie and 3IIc, which
help to further stabilize the crystals.
Fig. 3. Molecular structure of compound 3Ie, with displacement ellipsoids drawn at
the 30% probability level and H atoms omitted.
The X-ray analysis proved unambiguously the regio-chemistry
of the synthesized compounds and was in good agreement with
the results by the 1H NMR analysis. As shown in Figs. 3 and 4,
compounds 3Ie and 3IIc with the same formula were clearly
assigned as the N-1 and N-2 isomers, respectively. In conclusion, all
the structures of the regioisomers in this series could be definitely
assigned by the 1H NMR combined with single-crystal X-ray
diffraction.
50% of cell growth inhibition) were determined and summaries in
Table 2. 5-Fluorourcail was taken as reference drug. The results
were expressed as the average of triplicate assays.
3.2. Cell cycle analysis
The results that 3-(1H-indol-3-yl)-1H-pyrazole-5-carbohydrazide
derivatives inhibited the proliferation of 4 human cancer cells led us
to further study the effects of them on cell cycle distribution. HepG-2
3. Pharmacology
3.1. Cytotoxic evaluation
The 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) cell proliferation assay was employed to evaluate
cytotoxic activity of the synthesized compounds against 4 human
cancer cell lines including A549, HepG-2, BGC823 and BT474. Cells
were incubated with the target compounds at various concentra-
tions for 72 h, and their IC50 values (the concentration that causes
Cl
Cl
Cl
5.83
Cl
5.54
N
N
N
N
O
O
N
N
O
O
3Ie
3IIc
Fig. 4. Molecular structure of compound 3IIc, with displacement ellipsoids drawn at
Fig. 2. The chemical shift differences between regioisomers.
the 30% probability level and H atoms omitted.

D. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5868e5877
5871
two N atoms of pyrazole and indole rings for cytotoxic activity,
various disubstituted and monosubstituted compounds were
prepared.
All the target compounds were evaluated for their cytotoxic
activity in vitro against four human tumor cell lines, representing
different tumor types, namely human alveolar adenocarcinoma cell
line (A549), human hepatocellular liver carcinoma cell line (HepG-2),
human gastric carcinoma cell line (BGC823) and breast carcinoma
cell line (BT474). The results of the cytotoxic studies are shown in
Table 2. Most of compounds showed weak cytotoxic activity against
A549 cell. When the N atom of indole was occupied by n-propyl
group, compound 6 with unsubstituted pyrazole ring showed weak
activity. The introduction of n-propyl, benzyl and monosubstituted
benzyl to the pyrazole N atom or the alternation of the substitution
position on the pyrazole did not improve the activities as exemplified
by analogs 4Iaec and 4IIa. However, keeping the n-propyl substit-
uent at 1-position of indole but incorporating a disubstituted benzyl
group at 1-position of pyrazole produced compounds 4Idef with
significantly improved activities. Compounds 4Ie, which bore
a 2,4-dichlorobenzyl at 1-position of pyrazole, inhibited the growth
of A549 with IC50 value of 17.64
mM, while compounds 4If with
a 3,4-dichlorobenzyl substituent had IC50 value of 14.16
mM against
A549. Compounds 4Ihem and 4IIb showed lower IC50 values than
those of their counterparts against A549. Nevertheless, compounds
4Ik and 4Il, carrying dichlorobenzyl groups at 1-position of indole,
were more potent than other compounds in this series. A comparison
between compounds 4Ig and 4In showed that elimination of the n-
propyl substituent at N atom of indole caused little increase in the
activity against A549.
Fig. 5. Molecular structure of compound 3IId, with displacement ellipsoids drawn at
the 30% probability level and H atoms omitted.
cells were treated with different concentrations of compounds 4Ik
and 4Il for 48 h. 0.1% DMSO was used as vehicle. 5-Fluorouracil,
a known inhibitor of thymidylate synthase, was used as the reference
drug. Cells were harvested for cell analysis by a flow cytometry.
It is noticeable that all the disubstituted compounds 4Iaem had
indistinguishable IC50 values of 1.3e4.7 mM against HepG-2 cell. In
particular, derivatives 4Ik, 4Il and 4Im, with F- or Cl-containing
benzyl group on either N atoms of indole and pyrazole, were
more potent than the reference drug 5-Fu, which implied that the
lipophilic and electron-withdrawing halobenzyl groups were
beneficial for the cytotoxic activity against the HepG-2 cell lines. In
addition, swapping the position of substituents adjacent to N-1 of
pyrazole ring and 1-position of indole ring had minor effects on the
4. Results and discussion
Seventeen novel 3-(1H-indol-3-yl) pyrazole-5-carbohydra-
zide derivatives were synthesized and identified. In order to
investigate the role of the substitution types and patterns on the
Table 1
The crystallographic data and structure refinement details of 3Ie, 3IIc and 3IId.
Identification code
3Ie
3IIc
3IId
Empirical formula
Formula weight
C24H23Cl2N3O2
456.35
C24H23Cl2N3O2
456.35
C20H25N3O2
339.43
Temperature
298(2) K
298(2) K
298(2) K
Wavelength
0.71073 Å
0.71073 Å
0.71073 Å
Crystal system, space group
Unit cell dimensions
Triclinic, P-1
a ¼ 8.362(5) Å
b ¼ 8.446(5) Å
Monoclinic, P2(1)/n
Monoclinic, P2(1)/n
a
b
¼ 82.778(10)ꢁ
¼ 76.757(10)ꢁ
a ¼ 13.895(7) Å
a
¼ 90ꢁ
a ¼ 11.992(3) Å
a
¼ 90ꢁ
b ¼ 7.737(4) Å
c ¼ 21.551(10) Å
2316(2) A3
b
¼ 91.625(9)ꢁ
b ¼ 8.873(3) Å
c ¼ 17.941(5) Å
1880.4(9) A3
4, 1.199 Mg/m3
0.079 mmꢀ1
728
b
¼ 99.934(5)ꢁ
c ¼ 16.809(10) Å
1146.0(11) A3
2, 1.323 Mg/m3
0.309 mmꢀ1
476
g
¼ 89.921 (10)ꢁ
g
¼ 90ꢁ
g
¼ 90ꢁ
Volume
Z, Calculated density
Absorption coefficient
F(000)
4, 1.309 Mg/m3
0.306 mmꢀ1
952
Crystal size
Theta range for data collection
Limiting indices
Reflections collected/unique
Completeness to
Absorption correction
Max. and min. transmission
Refinement method
0.21 ꢂ 0.16 ꢂ 0.12 mm
0.22 ꢂ 0.14 ꢂ 0.08 mm
0.23 ꢂ 0.14 ꢂ 0.12 mm
2.43e25.05ꢁ
1.72e25.05ꢁ
1.90e25.05ꢁ
ꢀ9 ꢃ h ꢃ 7, ꢀ9 ꢃ k ꢃ 0, ꢀ19 ꢃ l ꢃ 20
5844/3910 [R(int) ¼ 0.0304]
96.6%
ꢀ16 ꢃ h ꢃ 16, ꢀ9 ꢃ k ꢃ 8, ꢀ23 ꢃ l ꢃ 25
11,626/4098 [R(int) ¼ 0.0430]
99.8%
ꢀ14 ꢃ h ꢃ 7, ꢀ10 ꢃ k ꢃ 10, ꢀ21 ꢃ l ꢃ 21
9499/3330 [R(int) ¼ 0.0261]
99.9%
q
¼ 25.05
Semi-empirical from equivalents
0.9639 and 0.9380
Semi-empirical from equivalents
0.9760 and 0.9358
Semi-empirical from equivalents
0.9906 and 0.9822
Full-matrix least-squares on F2
3910/0/281
Full-matrix least-squares on F2
4098/0/281
Full-matrix least-squares on F2
3330/0/227
Data/restraints/parameters
Goodness-of-fit on F2
1.657
1.015
1.016
Final R indices [I > 2
R indices (all data)
Extinction coefficient
Largest diff. peak and hole
s
(I)]
R1 ¼ 0.1516, wR2 ¼ 0.4388
R1 ¼ 0.1871, wR2 ¼ 0.4597
0.025(13)
R1 ¼ 0.0456, wR2 ¼ 0.1040
R1 ¼ 0.0803, wR2 ¼ 0.1221
0.0040(7)
R1 ¼ 0.0364, wR2 ¼ 0.0896
R1 ¼ 0.0509, wR2 ¼ 0.0999
0.0322(19)
1.110 and ꢀ0.401 e. Åꢀ3
0.245 and ꢀ0.203 e. Åꢀ3
0.152 and ꢀ0.136 e. Åꢀ3

5872
D. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5868e5877
Table 2
The characteristics of the target compounds and their IC50 values on human cancer lines.
Compd
R1
R2
R
1 substitution position
IC50
(
m
M)a
A549
HepG-2
BGC823
BT474
4Ia
4Ib
4Ic
4Id
4Ie
4If
4Ig
4Ih
4Ii
n-propyl
benzyl
n-propyl
n-propyl
n-propyl
n-propyl
n-propyl
n-propyl
n-propyl
benzyl
4-MeO-benzyl
3,4-Di-MeO-benzyl
2,4-Di-Cl-benzyl
3,4-Di-Cl-benzyl
4-F-benzyl
H
n-propyl
4-F-benzyl
n-propyl
N-1
N-1
N-1
N-1
N-1
N-1
N-1
N-1
N-1
N-1
N-1
N-1
N-1
N-1
N-2
N-2
169.7 ꢄ 10.34
e
3.91 ꢄ 0.28
4.71 ꢄ 0.42
3.12 ꢄ 0.03
2.72 ꢄ 0.27
3.14 ꢄ 0.14
4.07 ꢄ 0.84
1.65 ꢄ 0.10
3.59 ꢄ 0.80
3.14 ꢄ 0.75
2.95 ꢄ 0.47
1.32 ꢄ 0.15
1.58 ꢄ 0.32
2.41 ꢄ 0.67
17.23 ꢄ 1.89
50.53 ꢄ 3.69
106.8 ꢄ 27.70
32.86 ꢄ 4.31
2.70 ꢄ 0.72
1.94 ꢄ 0.46
5.15 ꢄ 1.54
1.89 ꢄ 0.24
6.53 ꢄ 0.45
12.80 ꢄ 0.54
11.06 ꢄ 0.59
0.71 ꢄ 0.14
1.88 ꢄ 0.43
1.49 ꢄ 0.27
8.78 ꢄ 1.45
4.90 ꢄ 0.67
10.22 ꢄ 0.31
7.23 ꢄ 0.61
8.79 ꢄ 1.27
153.0 ꢄ 26.95
49.16 ꢄ 5.15
e
3.78 ꢄ 0.76
3.70 ꢄ 0.05
5.54 ꢄ 0.55
4.57 ꢄ 1.01
5.77 ꢄ 0.72
6.49 ꢄ 1.13
2.33 ꢄ 0.37
4.68 ꢄ 0.56
5.44 ꢄ 0.78
6.24 ꢄ 2.19
4.42 ꢄ 1.75
3.80 ꢄ 1.07
1.39 ꢄ 0.18
14.26 ꢄ 3.58
83.27 ꢄ 16.92
10.48 ꢄ 1.48
17.59 ꢄ 5.67
72.90 ꢄ 13.34
4-MeO-benzyl
3,4-Di-MeO-benzyl
2,4-Di-Cl-benzyl
3,4-Di-Cl-benzyl
4-F-benzyl
n-propyl
n-propyl
n-propyl
n-propyl
n-propyl
69.90 ꢄ 11.86
20.37 ꢄ 4.57
17.67 ꢄ 1.42
14.16 ꢄ 1.52
51.80 ꢄ 14.02
151.0 ꢄ 18.08
130.9 ꢄ 26.44
52.67 ꢄ 8.36
29.40 ꢄ 2.79
24.69 ꢄ 3.68
54.26 ꢄ 3.78
46.07 ꢄ 2.65
63.13 ꢄ 11.12
e
4Ij
4Ik
4Il
4Im
4In
4IIa
4IIb
6
n-propyl
4-F-benzyl
4-F-benzyl
n-propyl
H
51.18 ꢄ 8.65
0.42 ꢄ 0.04
5-Fub
6.15 ꢄ 0.59
a
Each experiment was independently performed three times and expressed as means ꢄ SD. “e” means IC50 values > 250
mM.
b
5-Fu is used as reference drug.
activity. Altering the substitution position on pyrazole ring resulted
1 mM and 2 mM of 5-Fluorourcail arrested the cell cycle at S phase
in a precipitous loss in potency, which could be found in the
comparison among 4IIa and 4IIb with their counterparts 4Ig and
4Im. The poor inhibitory activity of the two monosubstituted
derivatives 4In and 6 indicated that the disubstitution pattern plays
an important role for the activity against HepG-2.
(11.1% and 15.5% increase compared with the blank group, respec-
tively). After the cells were treated with compound 4Ik at the
concentrations of 2 mM and 4 mM for 48 h, there caused 15% and
16.9% increased in the percentage of the cells in S phase in
comparison with the blank group, respectively. Upon incubation
Compounds 4Iae4Ic, 4Ige4Ii and 4Ik were found to be more
active against BGC823 cell line than the reference drug 5-Fu. When
the N atom of indole was attached by n-propyl group, compound 4Ig
with 4-F-benzyl group at position-1 of pyrazole displayed the
with compound 4Il (with 2 and 4 mM) there was an increase of cells
in the S phase of about 7.1% and 10.1%, respectively, in comparison
with the blank group. The increase in the S phase cell population
was accompanied by a decreased in the G1 and G2 phase cell
populations. According to these findings, compounds 4Ik and 4Il
made the HepG-2 cell arrested at S phase.
highest antiproliferative activity with IC50 value of 0.71 mM which
was 8.6-fold more potent than that of 5-Fu. This finding suggested
that small and electron-withdrawing group on the benzyl at the N-1
position of pyrazole ring played an important role for the activity.
The antiproliferative activity of 4Ic with a 4-methoxybenzyl group
on N-1 of pyrazole was 3-fold higher than that of 5-Fu and better
than that of most of the other benzyl-containing derivatives. These
results indicated that 4-methoxybenzyl was a more beneficial
functional group for the antiproliferative activity against BGC823
cell line. In the series with the n-propyl substituent at position-1 of
pyrazole and various benzyl groups at position-1 of indole,
compound 4Ii with the electron-donating methoxy group at the 4-
position of the benzyl exhibited the best activity against BGC823
5. Conclusion
The synthesis and characterization of a new series of 3-(1H-indol-
3-yl)pyrazole-5-carbohydrazide derivatives were carried out and all
the compounds were evaluated for their cytotoxic activity against 4
human cancer cell lines. The majority of the tested compounds
possessed antiproliferative activities against all of the tested cancer
cell lines, while the HepG-2, BGC823 and BT474 cell lines were more
sensitive in response to the growth inhibition than A549 cell line. It is
of notice that mostof the compoundsexhibited more potent cytotoxic
activitythanthedrug5-FuagainstBT474cellline. Compound4Imwas
the mostactive and52-fold more potent than5-Fu against BT474 with
with IC50 value of 1.49 mM.
As shown in Table 2, all the disubstituted compounds 4Iae4Im
with substituent situated at position-1 of pyrazole were 11- to 50-
fold and the monosubstituted compounds were w5-fold more
potent than the reference drug 5-Fu against BT474 cells. Compounds
4Ig and 4Im that contain a 4-fluorobenzyl group exhibited high
IC50 value of 1.39
cells. Compound 4Ig was the most effective against BGC823 cell with
IC50 value of 0.71 M, 8.6-fold more potent than 5-Fu against HepG-2
cell, but showed weak activity against A549. Compounds 4Ik showed
the highest potency against HepG-2 with IC50 value of 1.32 M, while
mM, but was less potent against other three cancer
m
growth inhibitory activity with IC50 values of 2.32 and 1.39
mM,
m
respectively. This was to say that a small and electron-withdrawing
group at 4-position of benzyl was favorable for the antiproliferative
activity. Swapping the position of substituents adjacent to N-1 of
pyrazole and N-1 of indole had minor effects on the activity.
To understand the mechanism of the target compounds, cell
cycle analysis was performed. The HepG-2 cells were treated with
compounds 4Ik and 4Il at indicated concentration for 48 h. The
eukaryotic cell cycle consists of alternating rounds of DNA repli-
cation (S phase) and cell division (M phase) separated by the gap
phase G0, G1 and G2. The final results in Fig. 6 suggested that both
compounds interfere with the normal cell cycle distribution of this
cell line. The result showed that treatment of HepG-2 cells with
it showed modest activity toward A549, BGC823 and BT474 cells.
These differences suggested that our compounds displayed selective
cytotoxicity in contrast to being a general broad cytotoxic agent. The
cell cycle analysis revealed that compounds 4Ik and 4Il arrested the
HepG-2 cell cycle at S phase in a dose-dependent manner. These
preliminary results are beneficial for further lead optimization.
6. Experimental
All reagents were used as purchased from commercial suppliers
without further purification unless otherwise noted. Melting points
were determined by using
a WRS-1B Digital Melting-Point

D. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5868e5877
5873
Fig. 6. (a) DNA contents obtained by flow cytometry. (b) FACS of cell cycle distribution of HepG-2 cells after treatment with compounds 4Ik and 4Il for 48 h. 5-Fu was used as
reference drug and 0.1% DMSO was used as vehicle control.
apparatus and are uncorrected. Thin-layer chromatography (TLC)
was performed on Silica Gel F254 plates with visualization by UV or
iodine vapor. 1H NMR spectra of DMSO-d6 solutions (TMS as an
internal standard) were recorded on Bruker AVANCE II 400 spec-
trometers. The IR spectra were measured on a Bruker Vector FT-IR
spectrophotometer as KBr pellets or film. The high resolution mass
spectra were obtained with an Agilent-6510-Q-TOF spectrometer.
Abbreviations: DMF, dimethylformamide; DMSO, dime-
thylsulfoxide; PE, petroleum ether; EtOAc, ethyl acetate.
OCH2CH3), 5.83 (s, 2H, CH2Ph), 7.02e7.06 (m, 1H, ArH), 7.10e7.14
(m, 1H, ArH), 7.35e7.44 (m, 2H, ArH, 4-H), 7.51e7.54 (m, 1H, ArH),
7.66e7.68 (m, 2H, ArH), 7.92 (d, J ¼ 2.4 Hz, 1H, IndH), 8.08 (d,
J ¼ 7.6 Hz, 1H, ArH); IR (KBr,
n
/cmꢀ1) 3350, 3011, 2985, 2897, 1645,
1510, 1416, 1341, 1286, 1096, 746.
6.1.2. Ethyl 1-(3, 4-dichlorobenzyl)-3-(1H-indol-3-yl)-1H-pyrazole-
5-carboxylate (2If)
Yellow solid, yield: 71%; mp: 129.5e130.8 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
: 1.28 (t, J ¼ 7.2 Hz, 3H, OCH2CH3), 4.29 (q, J ¼ 7.2 Hz, 2H,
OCH2CH3), 5.76 (s, 2H, CH2Ph), 7.09e7.20 (m, 2H, ArH), 7.27e7.30
(m, 2H, ArH, 4-H), 7.48e7.57 (m, 3H, ArH), 7.90 (d, J ¼ 2.4 Hz, 1H,
6.1. General procedure for the preparation of compounds 2Iae2If,
2IIa and 2IIc
IndH), 8.14 (d, J ¼ 7.6 Hz, 1H, ArH); IR (KBr,
n
/cmꢀ1) 3310, 3080,
2986, 1696, 1580, 1485, 1273, 1085, 760.
Compounds 2Iaed and 2IIa were reported in Ref. [21].
Compounds 2Ieef and 2IIc were prepared in a similar way.
As an example, to a stirred mixture of 1 (5.1 g, 20 mmol), KOH
(1.23 g, 22 mmol) and KBr (2.6 g, 22 mmol) in dried DMSO (15 ml)
under nitrogen was added 2,4-dichlorobenzyl chloride (4.3 g,
22 mmol) at 0 ꢁC and the resulting mixture was stirred at 0 ꢁC for
1.5 h. The mixture was then poured into water, and extracted with
CH2Cl2 (3 ꢂ 50 ml). The combined organic layers were washed with
water and brine, dried (Na2SO4), and concentrated. The yellow
residue was purified by flash column chromatography (silica gel,
PE/EtOAc) to give the desired product. For compound 2Ie, its
regioisomer (compound 2IIc) was also obtained.
6.1.3. Ethyl 1-(2, 4-dichlorobenzyl)-5-(1H-indol-3-yl)-1H-pyrazole-
3-carboxylate (2IIc)
Light yellow oil, yield: 10%. 1H NMR (400 MHz, DMSO-d6)
d: 1.29
(t, J ¼ 7.0 Hz, 3H, OCH2CH3), 4.30 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 5.54
(s, 2H, CH2Ph), 6.78 (d, J ¼ 8.0 Hz, 1H, ArH), 6.94 (s, 1H, 4-H),
7.09e7.13 (m, 1H, ArH), 7.19e7.23 (m, 1H, ArH), 7.33e7.36 (m, 1H,
ArH), 7.50e7.58 (m, 4-H, ArH, IndH); IR (KBr, n
/cmꢀ1) 3345, 3030,
2980, 2900, 1640, 1515, 1415, 1250, 1011, 785.
6.2. The procedure for compounds 2Ig and 2IIb
6.1.1. Ethyl 1-(2, 4-dichlorobenzyl)-3-(1H-indol-3-yl)-1H-pyrazole-
5-carboxylate (2Ie)
Compound 2Ig was prepared according to the procedure in Ref.
[21] and its regioisomer (compound 2IIb) was obtained as a minor
product.
Yellow solid, yield: 68%; mp: 138.1e139.1 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d: 1.25 (t, J ¼ 7.2 Hz, 3H, OCH2CH3), 4.27 (q, J ¼ 7.2 Hz, 2H,

5874
D. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5868e5877
6.2.1. Ethyl 1-(4-fluorobenzyl)-5-(1H-indol-3-yl)-1H-pyrazole-3-
(m, 2H, ArH), 7.18 (s, 1H, 4-H), 7.21e7.31 (m, 5H, ArH), 7.50 (d,
J ¼ 8.4 Hz, 1H, ArH), 8.03 (s, 1H, IndH), 8.14 (d, J ¼ 7.8 Hz, 1H, ArH);
carboxylate (2IIb)
Light yellow oil, yield: 10%. 1H NMR (400 MHz, DMSO-d6)
d
: 1.30
IR (KBr, n
/cmꢀ1) 3103, 2951, 2910, 1700, 1586, 1515, 1480, 1465,
(t, J ¼ 7.0 Hz, 3H, OCH2CH3), 4.30 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 5.50
(s, 2H, CH2Ph), 6.92 (s, 1H, 4-H), 7.00e7.03 (m, 2H, ArH), 7.08e7.13
(m, 3H, ArH), 7.16e7.20 (m, 1H, ArH), 7.45e7.52 (m, 3H, ArH); IR
1320, 1260, 745.
6.3.5. Ethyl 3-[1-(2,4-dichlorobenzyl)-1H-indol-3-y]-1-propyl-1H-
(KBr,
747.
n
/cmꢀ1) 3266, 3068, 2977, 1712, 1578, 1509, 1459, 1263, 1216,
pyrazole-5-carboxylate (3Ik)
Light yellow oil, yield: 88%. 1H NMR (400 MHz, DMSO-d6)
d: 0.87
(t, J ¼ 7.0 Hz, 3H, CH2CH2CH3), 1.32 (t, J ¼ 7.0 Hz, 3H, OCH2CH3),
1.81e1.85 (m, 2H, CH2CH2CH3), 4.32 (q, J ¼ 7.2 Hz, 2H, OCH2CH3),
4.50 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 5.51 (s, 2H, CH2Ph), 7.06e7.14
(m, 2H, ArH), 7.18 (s,1H, 4-H), 7.39e7.45 (m, 2H, ArH), 7.49e7.53 (m,
2H, ArH), 7.64 (d, J ¼ 2.0 Hz, 1H, ArH), 7.94 (s, 1H, IndH), 8.14 (d,
6.3. General procedure for compounds 3I and 3II
Compounds 3Ia, 3Ic, 3Id, 3Ig, 3Ii, 3Ij and 3Im were reported in
Ref. [21]. Compounds 3Ib, 3Ie, 3If, 3Ih, 3Ik, 3Il and 3IIaed were
prepared in a similar way.
J ¼ 7.6 Hz, 1H, ArH); IR (KBr,
n
/cmꢀ1) 3015, 2980, 2903, 1720, 1595,
As an example, to a solution of compound 2Ib (0.345 g, 1 mmol)
in dried DMF (1 ml), NaH (60%, 44 mg, 1.1 mmol) was added in
portions. After stirring for 10 min below 10 ꢁC, 1-bromoproane
(135 mg, 1.1 mmol) was added and stirred for another 1 h at r.t.
The mixture was poured into water and extracted with CH2Cl2
(3 ꢂ10 ml). The combined organic layers were then washed with
water, dried over Na2SO4, and the solvent was removed by rotary
evaporation. The crude product was purified by silica gel column
chromatography to give compound 3Ib.
1487, 1275, 1213, 1093, 765.
6.3.6. Ethyl 3-[1-(3,4-dichlorobenzyl)-1H-indol-3-y]-1-propyl-1H-
pyrazole-5-carboxylate (3Il)
Light yellow oil, yield: 91%. 1H NMR (400 MHz, DMSO-d6)
d: 0.88
(t, J ¼ 7.4 Hz, 3H, CH2CH2CH3), 1.32 (t, J ¼ 7.4 Hz, 3H, OCH2CH3),
1.81e1.86 (m, 2H, CH2CH2CH3), 4.32 (q, J ¼ 7.2 Hz, 2H, OCH2CH3),
4.50 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 5.44 (s, 2H, CH2Ph), 7.11e7.20
(m, 4-H, ArH, 4-H), 7.52 (d, J ¼ 8.4 Hz, 1H, ArH), 7.55e7.57 (m, 3H,
ArH), 8.05 (s,1H, IndH), 8.14 (d, J ¼ 7.8 Hz,1H, ArH); IR (KBr,
n
/cmꢀ1
)
6.3.1. Ethyl 1-benzyl-3-(1-propyl-1H-indol-3-yl)-1H-pyrazole-5-
carboxylate (3Ib)
2967, 2903, 1687, 1610, 1585, 1461, 1371, 1216, 764.
White solid, yield: 87%; mp: 104.4e105.1 ꢁC. 1H NMR (400 MHz,
6.3.7. Ethyl 1-(4-fluorobenzyl)-5-(1-propyl-1H-indol-3-yl)-1H-
pyrazole-3-carboxylate (3IIa)
DMSO-d6)
d
: 0.84 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3), 1.28 (t, J ¼ 7.2 Hz,
3H, OCH2CH3), 1.75e1.84 (m, 2H, CH2CH2CH3), 4.14 (t, J ¼ 7.2 Hz, 2H,
CH2CH2CH3), 4.29 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 5.78 (s, 2H, CH2Ph),
7.08e7.12 (m, 1H, ArH), 7.16e7.27 (m, 5H, ArH, 4-H), 7.30e7.34 (m,
2H, ArH), 7.50 (d, J ¼ 7.6 Hz, 1H, ArH), 7.93 (s, 1H, IndH), 8.14 (d,
Light yellow oil, yield: 86%. 1H NMR (400 MHz, DMSO-d6)
d: 0.78
(t, J ¼ 7.2 Hz, 3H, CH2CH2CH3), 1.29, (t, J ¼ 7.2 Hz, 3H, OCH2CH3),
1.73e1.78 (m, 2H, CH2CH2CH3), 4.15 (t, J ¼ 7.0 Hz, 2H, CH2CH2CH3),
4.29 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 5.50 (s, 2H, CH2Ph), 6.92 (s, 1H, 4-
H), 6.99e7.13 (m, 4-H, ArH), 7.22 (t, J ¼ 7.4 Hz, 2H, ArH), 7.50e7.59
J ¼ 8.0 Hz, 1H, ArH); IR (KBr,
n
/cmꢀ1) 2985, 2937, 1715, 1580, 1480,
1456, 1255, 1097, 740.
(m, 3H, ArH, IndH), 9.47 (brs, 1H, NH); IR (KBr,
2866, 1720, 1585, 1513, 1456, 1286, 1215, 744.
n
/cmꢀ1) 3015, 2934,
6.3.2. Ethyl 1-(2, 4-dichlorobenzyl)-3-(1-propyl-1H-indol-3-yl)-
1H-pyrazole-5-carboxylate (3Ie)
6.3.8. Ethyl 5-[1-(4-fluorobenzyl)-1H-indol-3-yl]-1-propyl-1H-
pyrazole-3-carboxylate (3IIb)
Light yellow solid, yield: 78%; mp: 126.3e126.9 ꢁC. 1H NMR
(400 MHz, DMSO-d6)
d
: 0.85 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3), 1.26 (t,
Light yellow oil, yield: 86%. 1H NMR (400 MHz, DMSO-d6)
d: 0.72
J ¼ 7.2 Hz, 3H, OCH2CH3), 1.78e1.83 (m, 2H, CH2CH2CH3), 4.13 (t,
J ¼ 7.0 Hz, 2H, CH2CH2CH3), 4.27 (q, J ¼ 7.0 Hz, 2H, OCH2CH3), 5.83
(s, 2H, CH2Ph), 6.71 (d, J ¼ 8.4 Hz, 1H, ArH), 7.05e7.07 (m, 1H, ArH),
7.15e7.18 (m, 1H, ArH), 7.31 (s, 1H, 4-H), 7.36 (dd, J ¼ 1.2, 8.0 Hz, 1H,
ArH), 7.50 (d, J ¼ 7.6 Hz, 1H, ArH), 7.66 (d, J ¼ 1.2 Hz, 1H, ArH), 7.96
(t, J ¼ 7.4 Hz, 3H, CH2CH2CH3), 1.29 (t, J ¼ 7.2 Hz, 3H, OCH2CH3),
1.71e1.75 (m, 2H, CH2CH2CH3), 4.18 (t, J ¼ 6.8 Hz, 2H, CH2CH2CH3),
4.28 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 5.50 (s, 2H, CH2Ph), 6.84 (s, 1H,
ArH), 7.10e7.24 (m, 4-H, ArH), 7.32e7.36 (m, 2H, ArH), 7.50 (d,
J ¼ 8.0 Hz, 1H, ArH), 7.57 (d, J ¼ 8.0 Hz, 1H, ArH), 7.91 (s, 1H, IndH);
(s, 1H, IndH), 8.07 (d, J ¼ 7.6 Hz, 1H, ArH); IR (KBr,
n
/cmꢀ1) 3045,
IR (KBr,
1095, 742.
n
/cmꢀ1) 2989, 2945, 1718, 1585, 1515, 1460, 1275, 1215,
2960, 2935, 2872, 1713, 1580, 1460, 1265, 1215, 1132, 740.
6.3.3. Ethyl 1-(3, 4-dichlorobenzyl)-3-(1-propyl-1H-indol-3-yl)-
1H-pyrazole-5-carboxylate (3If)
6.3.9. Ethyl 1-(2, 4-dichlorobenzyl)-5-(1-propyl-1H-indol-3-yl)-
1H-pyrazole-3-carboxylate (3IIc)
Off-white solid, yield: 75%; mp: 118.4e119.6 ꢁC. 1H NMR
Light yellow solid, yield: 85%; mp: 91.0e91.4 ꢁC. 1H NMR
(400 MHz, DMSO-d6)
d
:
1H NMR (400 MHz, DMSO-d6)
d
: 0.84 (t,
(400 MHz, DMSO-d6)
d
: 0.76 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3), 1.30 (t,
J ¼ 7.2 Hz, 3H, CH2CH2CH3), 1.28 (t, J ¼ 7.2 Hz, 3H, OCH2CH3),
1.77e1.82 (m, 2H, CH2CH2CH3), 4.14 (t, J ¼ 7.0 Hz, 2H, CH2CH2CH3),
4.29 (q, J ¼ 7.0 Hz, 2H, OCH2CH3), 5.76 (s, 2H, CH2Ph), 7.08e7.12 (m,
2H, ArH), 7.14e7.19 (m, 1H, ArH), 7.26 (s, 1H, 4-H), 7.34 (dd, J ¼ 2.0,
8.4 Hz, 1H, ArH), 7.47e7.50 (m, 1H, ArH), 7.60 (d, J ¼ 8.0 Hz, 1H,
J ¼ 7.0 Hz, 3H, OCH2CH3), 1.70e1.78 (m, 2H, CH2CH2CH3), 4.15 (t,
J ¼ 6.8 Hz, 2H, CH2CH2CH3), 4.29 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 5.54
(s, 2H, CH2Ph), 6.79 (d, J ¼ 8.4 Hz, 1H, ArH), 6.95 (s, 1H, 4-H),
7.09e7.13 (m, 1H, ArH), 7.20e7.24 (m, 1H, ArH), 7.35 (dd, J ¼ 1.6,
8.4 Hz, 1H, ArH), 7.51 (d, J ¼ 8.0 Hz, 1H, ArH), 7.55 (s, 1H, IndH),
ArH), 7.94 (s, 1H, IndH), 8.13 (d, J ¼ 7.6 Hz,1H, ArH); IR (KBr,
n
/cmꢀ1
)
7.57e7.58 (m, 1H, ArH); IR (KBr, n
/cmꢀ1) 3035, 2990, 2850, 1650,
3013, 2986, 2922, 1713, 1610, 1580, 1466, 1260, 1217, 741.
1510, 1415, 1215, 1120, 744.
6.3.4. Ethyl 3-(1-benzyl-1H-indol-3-yl)-1-propyl-1H-pyrazole-5-
6.3.10. Ethyl 1-propyl-5-(1-propyl-1H-indol-3-yl)-1H-pyrazole-3-
carboxylate (3IId)
carboxylate (3Ih)
Colorless oil, yield: 93%. 1H NMR (400 MHz, DMSO-d6)
d: 0.87 (t,
Colorless solid, yield: 78%; mp: 104.9e105.5 ꢁC. 1H NMR
J ¼ 7.4 Hz, 3H, CH2CH2CH3), 1.32 (t, J ¼ 7.0 Hz, 3H, OCH2CH3),
1.79e1.87 (m, 2H, CH2CH2CH3), 4.30 (q, J ¼ 7.2 Hz, 2H, OCH2CH3),
4.50 (t, J ¼ 7.0 Hz, 2H, CH2CH2CH3), 5.42 (s, 2H, CH2Ph), 7.09e7.15
(400 MHz, DMSO-d6)
d
: 0.72 (t, J ¼ 7.4 Hz, 3H, CH2CH2CH3), 0.84 (t,
J ¼ 7.2 Hz, 3H, CH2CH2CH3), 1.67e1.74 (m, 2H, CH2CH2CH3),
1.78e1.86 (m, 2H, CH2CH2CH3), 4.17 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3),

D. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5868e5877
5875
4.22 (t, J ¼ 6.8 Hz, 2H, CH2CH2CH3), 4.29 (q, J ¼ 7.2 Hz, 2H,
OCH2CH3), 6.81 (s, 1H, ArH), 7.10e7.14 (m, 1H, ArH), 7.21e7.25 (m,
1H, ArH), 7.49 (d, J ¼ 8.0 Hz, 1H, ArH), 7.59 (d, J ¼ 8.0 Hz, 1H, ArH),
HRMS (ESI) calcd for [M þ H]þ C24H28N5O3: 434.2192, found:
434.2184.
7.73 (s, 1H, IndH); IR (KBr,
n
/cmꢀ1) 3085, 2950, 2915, 2860, 1700,
6.4.5. 1-(2,4-Dichlorobenzyl)-3-(1-propyl-1H-indol-3-yl)-1H-
pyrazole-5-carbohydrazide (4Ie)
1550, 1530, 1440, 1210, 1100, 760.
Light yellow solid, yield: 93%; mp: 161.3e162.3 ꢁC. 1H NMR
6.4. General procedure for the synthesis of 3-(1H-indol-3-yl)-1H-
pyrazole-5-carbohydrazide derivatives (4I, 4II and 6)
(400 MHz, DMSO-d6)
d
:
0.85 (t, J ¼ 7.4 Hz, 3H, CH2CH2CH3),
1.74e1.84 (m, 2H, CH2CH2CH3), 4.17 (t, J ¼ 7.0 Hz, 2H, CH2CH2CH3),
4.50 (s, 2H, NH2), 5.86 (s, 2H, CH2Ph), 6.67 (d, J ¼ 8.4 Hz, 1H, ArH),
7.07e7.11 (m, 1H, ArH), 7.16e7.20 (m, 1H, ArH), 7.28 (s, 1H, 4-H),
7.34 (dd, J ¼ 2.0, 8.4 Hz, 1H, ArH), 7.50 (d, J ¼ 8.0 Hz, 1H, ArH), 7.64
(d, J ¼ 2.0 Hz, 1H, ArH), 7.72 (s, 1H, IndH), 8.06 (d, J ¼ 8.0 Hz, 1H,
For example, to a stirred solution of compound 3Ia (0.339 g,
1 mmol) in methanol (5 ml), 1 ml of 80% hydrazine monohydrate
was added. After maintained under reflux until TLC indicated
consumption of starting materials, the mixture was quenched with
water, and extracted with EtOAc. The combined organic phase was
washed with water and brine, dried (Na2SO4), and concentrated.
The residue was purified by flash column chromatography to give
the target compound 4Ia in high yield.
ArH), 9.90 (s, 1H, NH); IR (KBr, n
/cmꢀ1) 3311, 3297, 3043, 2964,
2931, 2872, 1653, 1618, 1585, 1535, 1471, 1448, 1313, 1217, 742;
HRMS (ESI) calcd for [M þ H]þ C22H22Cl2N5O: 442.1201, found:
442.1189.
6.4.6. 1-(3,4-Dichlorobenzyl)-3-(1-propyl-1H-indol-3-yl)-1H-
pyrazole-5-carbohydrazide (4If)
6.4.1. 1-Propyl-3-(1-propyl-1H-indol-3-yl)-1H-pyrazole-5-
carbohydrazide (4Ia)
Light yellow solid, yield: 92%; mp: 173.3e173.5 ꢁC. 1H NMR
Off-white solid, yield: 95%; mp: 136.5e136.9 ꢁC. 1H NMR
(400 MHz, DMSO-d6)
d
:
0.84 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3),
(400 MHz, DMSO-d6)
d: 0.85 (m, 6H, CH2CH2CH3), 1.76e1.83 (m, 4-
1.74e1.83 (m, 2H, CH2CH2CH3), 4.17 (t, J ¼ 6.8 Hz, 2H, CH2CH2CH3),
4.55 (s, 2H, NH2), 5.77 (s, 2H, CH2Ph), 7.09e7.13 (m, 1H, ArH),
7.16e7.20 (m, 2H, ArH), 7.23 (s,1H, 4-H), 7.46 (d, J ¼ 2.0 Hz,1H, ArH),
7.51 (d, J ¼ 8.0 Hz, 1H, ArH), 7.59 (d, J ¼ 8.0 Hz, 1H, ArH), 7.72 (s, 1H,
H, CH2CH2CH3), 4.16 (t, J ¼ 7.0 Hz, 2H, CH2CH2CH3), 4.48 (t,
J ¼ 7.0 Hz, 2H, CH2CH2CH3), 4.52 (s, 2H, NH2), 7.08e7.12 (m, 2H,
ArH, 4-H), 7.18(dt, J ¼ 1.2, 7.2 Hz, 1H ArH), 7.49 (d, J ¼ 8.4 Hz, 1H,
ArH), 7.69 (s,1H, IndH), 8.08 (d, J ¼ 7.6 Hz,1H, ArH), 9.78 (s,1H, NH);
IndH), 8.01 (d, J ¼ 8.0 Hz, 1H, ArH), 9.91 (s, 1H, NH); IR (KBr,
n )
/cmꢀ1
IR (KBr,
n
/cmꢀ1) 3307, 3271, 3207, 2964, 2933, 2873, 1664, 1615,
3263, 3045, 2960, 2931, 2872, 1643, 1616, 1581, 1531, 1469, 1446,
1332, 1313, 1213, 1132, 740; HRMS (ESI) calcd for [M þ H]þ
C22H22Cl2N5O: 442.1201, found: 442.1190.
1583, 1521, 1448, 1315, 1219, 748; HRMS (ESI) calcd for [M þ H]þ
C18H24N5O: 326.1981, found: 326.1983.
6.4.2. 1-Benzyl-3-(1-propyl-1H-indol-3-yl)-1H-pyrazole-5-
carbohydrazide (4Ib)
6.4.7. 1-(4-Fluorobenzyl)-3-(1-propyl-1H-indol-3-yl)-1H-pyrazole-
5-carbohydrazide (4Ig)
White solid, yield: 94%; mp: 130.0e130.5 ꢁC. 1H NMR (400 MHz,
Light yellow solid, yield: 94%; mp: 122.9e123.6 ꢁC. 1H NMR
DMSO-d6)
d
: 0.83 (t, J ¼ 7.4 Hz, 3H, CH2CH2CH3), 1.76e1.81 (m, 2H,
(400 MHz, DMSO-d6)
d
:
0.83 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3),
CH2CH2CH3), 4.16 (t, J ¼ 6.8 Hz, 2H, CH2CH2CH3), 4.53 (s, 2H, NH2),
5.79 (s, 2H, CH2Ph), 7.08e7.12 (m, 1H, ArH), 7.15e7.25 (m, 5H, ArH,
4-H), 7.28e7.32 (m, 2H, ArH), 7.50 (d, J ¼ 8.0 Hz, 1H, ArH), 7.71 (s,
1.73e1.83 (m, 2H, CH2CH2CH3), 4.16 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3),
4.55 (s, 2H, NH2), 5.76 (s, 2H, CH2Ph), 7.09e7.20 (m, 5H, ArH, 4-H),
7.30 (dd, J ¼ 1.6, 5.6 Hz, 2H, ArH), 7.50 (d, J ¼ 8.0 Hz, 1H, ArH), 7.71
1H, IndH), 8.07 (d, J ¼ 7.6 Hz, 1H, ArH), 9.86 (s, 1H, NH); IR (KBr,
n/
(s,1H, IndH), 8.08 (d, J ¼ 7.6 Hz,1H, ArH), 9.88 (s,1H, NH); IR (KBr,
n/
cmꢀ1) 3305, 3271, 3211, 3142, 3032, 2962, 2931, 2873, 1643, 1620,
1533, 1452, 1323, 1217, 740; HRMS (ESI) calcd for [M þ H]þ
C22H24N5O: 374.1981, found: 374.1971.
cmꢀ1) 3296, 3211, 3047, 2962, 2926, 2872, 2854, 1662, 1639, 1616,
1583, 1553, 1450, 1327, 1222, 748; HRMS (ESI) calcd for [M þ H]þ
C22H23FN5O: 392.1886, found: 392.1867.
6.4.3. 1-(4-Methoxybenzyl)-3-(1-propyl-1H-indol-3-yl)-1H-
pyrazole-5-carbohydrazide (4Ic)
6.4.8. 3-(1-Benzyl-1H-indol-3-yl)-1-propyl-1H-pyrazole-5-
carbohydrazide (4Ih)
White powder, yield: 96%; mp: 161.0e161.3 ꢁC. 1H NMR
White solid, yield: 95%; mp: 172.8e173.3 ꢁC. 1H NMR (400 MHz,
(400 MHz, DMSO-d6)
d
:
0.84 (t, J ¼ 7.4 Hz, 3H, CH2CH2CH3),
DMSO-d6)
d
: 0.85 (t, J ¼ 7.4 Hz, 3H, CH2CH2CH3), 1.76e1.85 (m, 2H,
1.76e1.81 (m, 2H, CH2CH2CH3), 3.69 (s, 3H, OCH3), 4.16 (t, J ¼ 7.0 Hz,
2H, CH2CH2CH3), 4.53 (s, 2H, NH2), 5.70 (s, 2H, CH2Ph), 6.86 (d,
J ¼ 8.4 Hz, 2H, ArH), 7.09e7.23 (m, 5H, ArH, 4-H), 7.49 (d, J ¼ 8.0 Hz,
1H, ArH), 7.69 (s, 1H, IndH), 8.08 (d, J ¼ 7.6 Hz, 1H, ArH), 9.82 (s, 1H,
CH2CH2CH3), 4.49 (t, J ¼ 6.8 Hz, 2H, CH2CH2CH3), 4.52 (s, 2H, NH2),
5.46 (s, 2H, CH2Ph), 7.09e7.17 (m, 3H, ArH, 4-H), 7.23e7.32 (m, 5H,
ArH), 7.49 (d, J ¼ 7.6 Hz, 1H, ArH), 7.82 (s, 1H, IndH), 8.10 (d,
J ¼ 7.2 Hz, 1H, ArH), 9.78 (s, 1H, NH); IR (KBr,
n
/cmꢀ1) 3313, 3271,
NH); IR (KBr,
n
/cmꢀ1) 3311, 3259, 2958, 2931, 2872,1651,1616,1583,
3201, 3115, 3026, 2962, 2924, 2877, 1658, 1616, 1577, 1533, 1469,
1450, 1321, 1211, 1170, 948, 723; HRMS (ESI) calcd for [M þ H]þ
C22H24N5O: 374.1981, found: 374.1971.
1521, 1448, 1315, 1247, 744; HRMS (ESI) calcd for [M þ H]þ
C23H26N5O2: 404.2086, found: 404.2082.
6.4.4. 1-(3,4-Dimethoxybenzyl)-3-(1-propyl-1H-indol-3-yl)-1H-
pyrazole-5-carbohydrazide (4Id)
6.4.9. 3-[1-(4-Methoxybenzyl)-1H-indol-3-yl]-1-propyl-1H-
pyrazole-5-carbohydrazide (4Ii)
Light yellow solid, yield: 97%; mp: 162.9e163.1 ꢁC. 1H NMR
Lacteous oil, yield: 91%. 1H NMR (400 MHz, DMSO-d6)
d: 0.85 (t,
(400 MHz, DMSO-d6)
d
:
0.84 (t, J ¼ 7.4 Hz, 3H, CH2CH2CH3),
J ¼ 7.4 Hz, 3H, CH2CH2CH3), 1.75e1.84 (m, 2H, CH2CH2CH3), 3.70 (s,
3H, OCH3), 4.48 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 4.51 (s, 2H, NH2),
5.36 (s, 2H, CH2Ph), 6.87 (dd, J ¼ 2.0, 6.8 Hz, 2H, ArH), 7.08e7.17 (m,
3H, ArH, 4-H), 7.22 (d, J ¼ 8.4 Hz, 1H, ArH), 7.51 (d, J ¼ 7.6 Hz, 1H,
ArH), 7.78 (s, 1H, IndH), 8.07e8.09 (m, 1H, ArH), 9.78 (s, 1H, NH); IR
1.74e1.83 (m, 2H, CH2CH2CH3), 3.69 (s, 3H, OCH3), 3.71 (s, 3H,
OCH3), 4.16 (t, J ¼ 7.0 Hz, 2H, CH2CH2CH3), 4.54 (s, 2H, NH2), 5.68
(s, 2H, CH2Ph), 6.75 (dd, J ¼ 2.0, 8.4 Hz, 1H, ArH), 6.86 (d, J ¼ 8.4 Hz,
1H, ArH), 7.00 (d, J ¼ 2.0 Hz, 1H, ArH), 7.08e7.13 (m, 3H, ArH, 4-H),
7.49 (d, J ¼ 8.4 Hz, 1H, ArH), 7.69 (s, 1H, IndH), 8.10 (d, J ¼ 7.6 Hz,
(KBr, n
/cmꢀ1) 3311, 3047, 3007, 2962, 2933, 2873, 2835, 1658, 1614,
1H, ArH), 9.83 (s, 1H, NH); IR (KBr,
n
/cmꢀ1) 3278, 3049, 2993,
1585, 1514, 1465, 1452, 1309, 1249, 1176, 746; HRMS (ESI) calcd for
2958, 2937, 2872, 1645, 1620, 1585, 1519, 1450, 1317, 1263, 732;
[M þ H]þ C23H26N5O2: 404.2086, found: 404.2076.

5876
D. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5868e5877
6.4.10. 3-[1-(3,4-Dimethoxybenzyl)-1H-indol-3-yl]-1-propyl-1H-
pyrazole-5-carbohydrazide (4Ij)
6.89 (s, 1H, 4-H), 7.02e7.13 (m, 5H, ArH), 7.22 (t, J ¼ 7.4 Hz, 1H, ArH),
7.55e7.56 (m, 3H, ArH, IndH), 9.47 (brs, 1H, NH); IR (KBr,
/cmꢀ1
3311, 3209, 2966, 2933, 2875, 1660, 1618, 1510, 1442, 1338, 1224,
746; HRMS (ESI) calcd for [M þ H]þ C22H23FN5O: 392.1886, found:
392.1867.
n
)
White solid, yield: 93%; mp: 159.3e161.1 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
: 0.85 (t, J ¼ 7.6 Hz, 3H, CH2CH2CH3), 1.75e1.84 (m, 2H,
CH2CH2CH3), 3.68 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 4.48 (t,
J ¼ 7.2 Hz, 2H, CH2CH2CH3), 4.51 (s, 2H, NH2), 5.34 (s, 2H, CH2Ph),
6.74 (dd, J ¼ 2.0, 8.4 Hz, 1H, ArH), 6.86 (d, J ¼ 8.4 Hz, 1H, ArH), 7.02
(d, J ¼ 2.0 Hz, 1H, ArH), 7.08e7.18 (m, 3H, ArH, 4-H), 7.53 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.78 (s, 1H, IndH), 8.08 (d, J ¼ 7.6 Hz, 1H, ArH),
6.4.16. 5-[1-(4-Fluorobenzyl)-1H-indol-3-yl]-1-propyl-1H-
pyrazole-3-carbohydrazide (4IIb)
Light yellow oil, yield: 91%. 1H NMR (400 MHz, DMSO-d6)
d: 0.73
977 (s, 1H, NH); IR (KBr,
n
/cmꢀ1) 3317, 3298, 3091, 3047, 2960, 2931,
(t, J ¼ 7.4 Hz, 3H, CH2CH2CH3), 1.69e1.79 (m, 2H, CH2CH2CH3), 4.14
(t, J ¼ 6.8 Hz, 2H, CH2CH2CH3), 4.41 (brs, 2H, NH2), 5.49 (s, 2H,
CH2Ph), 6.80 (s, 1H, 4-H), 7.10e7.22 (m, 4-H, ArH), 7.32e7.36 (m, 2H,
ArH), 7.54e7.58 (m, 2H, ArH), 7.88 (s, 1H, IndH), 9.34 (s, 1H, NH); IR
2872, 2835, 1641, 1616, 1516, 1465, 1450, 1313, 1257, 1234, 1138,
1026, 740; HRMS (ESI) calcd for [M þ H]þ C24H28N5O3: 434.2192,
found: 434.2181.
(KBr, n
/cmꢀ1) 3313, 3051, 2964, 2933, 2875, 1662, 1618, 1510, 1463,
6.4.11. 3-[1-(2,4-Dichlorobenzyl)-1H-indol-3-yl]-1-propyl-1H-
pyrazole-5-carbohydrazide (4Ik)
1440, 1340, 1222, 1157, 744; HRMS (ESI) calcd for [M þ H]þ
C22H23FN5O: 392.1886, found: 392.1880.
White solid, yield: 94%; mp: 91.4e91.9 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
: 0.85 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3), 1.76e1.85 (m, 2H,
6.4.17. 3-(1-Propyl-1H-indol-3-yl)-1H-pyrazole-5-carbohydrazide
(6) (major/minor ¼ 60:40)
CH2CH2CH3), 4.49 (t, J ¼ 7.2 Hz, 2H, CH2CH2CH3), 4.52 (s, 2H, NH2),
5.54 (s, 2H, CH2Ph), 6.73 (d, J ¼ 8.4 Hz, 1H, ArH), 7.10e7.19 (m, 3H,
ArH, 4-H), 7.33 (dd, J ¼ 0.8, 8.4 Hz, 1H, ArH), 7.41 (d, J ¼ 8.0 Hz, 1H,
ArH), 7.69 (s, 1H, ArH), 7.75 (s, 1H, IndH), 8.13e8.15 (m, 1H, ArH),
Compound 5 was prepared according to the procedure in Ref.
[21] and subsequent hydrazinolysis produced compound 6.
White solid, yield: 94%; mp: 194.1e195.5 ꢁC. 1H NMR (400 MHz,
9.79 (s, 1H, NH); IR (KBr,
n
/cmꢀ1) 3315, 3163, 3088, 3055, 2962,
DMSO-d6) d: 0.84e0.87 (m, 3H, CH2CH2CH3), 1.77e1.84 (m, 2H,
2931, 2872,1660,1627,þ1587,1527,1469,1448,1311,1174, 740; HRMS
CH2CH2CH3), 4.16e4.19 (m, 2H, CH2CH2CH3), 4.40/4.50 (s/s, 2H,
NH2), 6.94 (s, 1H, ArH, the proton signal of the minor isomer was
merged in 7.11e7.24 ppm), 7.11e7.24 (m, 2H, ArH), 7.48e7.51/
7.55e7.57 (m/m, 1H, ArH), 7.67e8.08 (m, 2H, ArH), 8.35/8.75 (brs/
(ESI) calcd for [M þ H] C22H22Cl2N5O: 442.1201, found: 442.1186.
6.4.12. 3-[1-(3,4-Dichlorobenzyl)-1H-indol-3-yl]-1-propyl-1H-
pyrazole-5-carbohydrazide (4Il)
brs, 1H, NH); IR (KBr, n
/cmꢀ1) 3302, 3278, 3176, 3062, 2960, 2924,
White powder, yield: 95%; mp: 86.5e87.8 ꢁC. 1H NMR (400 MHz,
2852, 1660, 1631þ, 1543, 1469, 1328, 1286, 1193, 734; HRMS (ESI)
DMSO-d6)
d
: 0.85 (t, J ¼ 7.2 Hz, 3H, CH2CH2CH3), 1.76e1.85 (m, 2H,
calcd for [M þ H] C15H18N5O: 284.1511, found: 284.1511.
CH2CH2CH3), 4.49 (t, J ¼ 6.8 Hz, 2H, CH2CH2CH3), 4.52 (s, 2H, NH2),
5.48 (s, 2H, CH2Ph), 7.11e7.19 (m, 4-H, ArH, 4-H), 7.50 (d, J ¼ 8.0 Hz,
1H, ArH), 7.55e7.57 (m, 2H, ArH), 7.87 (s, 1H, IndH), 8.11 (d,
6.5. MTT assay for cell viability
J ¼ 7.2 Hz, 1H, ArH), 9.80 (s, 1H, NH); IR (KBr,
n
/cmꢀ1) 3288, 3053,
Cell viability was determined by 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT) assay. Cancer cells were
purchased from American Type Culture Collection (ATCC, USA). The
cells were cultured in RPMI-1640 medium, supplemented with 10%
fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin
in a humidified environment with 5% CO2 at 37 ꢁC.
2960, 2929, 2872, 1651, 1616, 1585, 1527, 1476, 1450, 1334, 1313,
1203, 1176, 744; HRMS (ESI) calcd for [M þ H]þ C22H22Cl2N5O:
442.1201, found: 442.1196.
6.4.13. 3-[1-(4-Fluorobenzyl)-1H-indol-3-yl]-1-propyl-1H-
pyrazole-5-carbohydrazide (4Im)
Cells in logarithmic growth were plated in 96-well microtiter
plates at following densities of 3500 cells/well for A549, 3500 cells/
well for HepG-2, 3500 cells/well for BGC823 and 6000 cells/well for
BT474, respectively. After 24 h incubation, cells were treated with
vehicle alone (0.4% DMSO) or compounds (drugs were dissolved in
DMSO previously) at the concentrations indicated. After three days
Yellow solid, yield: 97%; mp: 113.1e114.2 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
: 0.85 (t, J ¼ 7.0 Hz, 3H, CH2CH2CH3), 1.76e1.85 (m, 2H,
CH2CH2CH3), 4.49 (t, J ¼ 6.8 Hz, 2H, CH2CH2CH3), 4.52 (s, 2H, NH2),
5.44 (s, 2H, CH2Ph), 7.10e7.18 (m, 5H, ArH, 4-H), 7.20e7.31(m, 2H,
ArH), 7.50 (d, J ¼ 8.0 Hz, 1H, ArH), 7.83 (s, 1H, IndH), 8.08e8.10 (m,
1H, ArH), 9.79 (s, 1H, NH); IR (KBr,
n
/cmꢀ1) 3294, 3113, 3049, 2962,
of incubation (37 ꢁC, 5% CO2 in a humid atmosphere) 20
ml of MTT
2931, 2872,1653,1614,1585,1527,1510,1465,1450,1313,1222,1157,
742; HRMS (ESI) calcd for [M þ H]þ C22H23FN5O: 392.1886, found:
392.1885.
(5 mg/ml in PBS) was added to each well and the plate was incu-
bated for a further 3 h (37 ꢁC). To dissolve the resulting MTT-
formazan, 150 ml of DMSO was added to each well, followed by
thorough mixing with a mechanical plate mixer. Absorbance at
492 nm was measured on a microplate reader, and the data were
analyzed by Prism Graphpad software. In all of these experiments,
three replicate wells were used to determine each point.
6.4.14. 1-(4-Fluorobenzyl)-3-(1H-indol-3-yl)-1H-pyrazole-5-
carbohydrazide (4In)
Yellow powder, yield: 95%; mp: 198.5e200.7 ꢁC. 1H NMR
(400 MHz, DMSO-d6) d: 4.54 (s, 2H, NH2), 5.76 (s, 2H, CH2Ph),
7.05e7.06 (m, 4-H, ArH), 7.20(s, 1H, 4-H), 7.28e7.31 (m, 2H, ArH),
7.41 (d, J ¼ 8.0 Hz, 1H, ArH), 7.66 (d, J ¼ 2.4 Hz, 1H, IndH), 8.7 (d,
6.6. Flow cytometric analysis of cell cycle distribution
J ¼ 7.6 Hz, 1H, ArH), 9.79 (s, 1H, NH); IR (KBr,
n
/cmꢀ1) 3311, 3288,
The effects of compounds 4Ik and 4Il on cell cycle distribution
were studied on HepG-2 cells by flow cytometric analysis after
staining with propidium iodide. HepG-2 cells were exposed to 0.1%
DMSO, active compounds or 5-Fluorouracil for 48 h. After treat-
ment, cells were harvested by trypsinization, washed twice with
ice-cold phosphate buffered saline and fixed in ethanol (75%) at
4 ꢁC for 2 h. Fixed cells were pelleted by centrifugation, washed
2956, 2900, 1641, 160þ4, 1531, 1510, 1450, 1313, 1222, 748; HRMS
(ESI) calcd for [M þ H] C19H17FN5O: 350.1417, found: 350.1413.
6.4.15. 1-(4-Fluorobenzyl)-5-(1-propyl-1H-indol-3-yl)-1H-
pyrazole-3-carbohydrazide (4IIa)
Light yellow oil, yield: 96%. 1H NMR (400 MHz, DMSO-d6)
d: 0.80
(t, J ¼ 7.8 Hz, 3H, CH2CH2CH3), 1.71e1.81 (m, 2H, CH2CH2CH3), 4.15
once with PBS, and treated with 500
m
g/ml Rnase in PBS for 30 min
g/ml propidium
(t, J ¼ 7.0 Hz, 2H, CH2CH2CH3), 4.41 (s, 2H, NH2), 5.50 (s, 2H, CH2Ph),
at 37 ꢁC. Then the cells were stained with 25
m

D. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 5868e5877
5877
iodide in PBS at 4 ꢁC in the dark for 30 min. The DNA content was
analyzed by FACScan flow cytometer (Becton Dickinson, Mountain
View, CA) and CELLQUEST software (Becton Dickinson).
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Acknowledgments
This research was supported by the Foundation for Outstanding
Young Scientist in Shandong Province (BS2009SW032) and the
Natural Science Foundation of Shandong Province (2009ZRB01128).
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