Synthesis of novel oxime-containing pyrazole derivatives and discovery of regulators for apoptosis and autophagy in A549 lung cancer cells

Article abstract of DOI:10.1016/j.bmcl.2010.06.121

A series of novel oxime-containing pyrazole derivatives were synthesized by the reaction of ethyl 3-phenyl-1H-pyrazole-5-carboxylate derivatives and 2-bromo-1-phenylethanone followed by the reaction with hydroxylamine hydrochloride. The structures were determined by IR, ¹H NMR, HRMS, and X-ray analysis. A dose- and time-dependent inhibition of proliferation was observed in A549 lung cancer cell after compound treatment. Inhibition of growth was mainly attributed to the autophagy induction.

Full text of DOI:10.1016/j.bmcl.2010.06.121

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4766–4770
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel oxime-containing pyrazole derivatives and discovery
of regulators for apoptosis and autophagy in A549 lung cancer cells
a
a
a
Liang-Wen Zheng a, , Ying Li b, , Di Ge b, Bao-Xiang Zhao a, , Ying-Rui Liu , Hong-Shui Lv , Jun Ding ,
*
Jun-Ying Miao b,c,
*
a Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China
b Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China
c The key laboratory of Experimental Teratology, Ministry of Education, Shandong University, Jinan 250012, 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 oxime-containing pyrazole derivatives were synthesized by the reaction of ethyl 3-phe-
nyl-1H-pyrazole-5-carboxylate derivatives and 2-bromo-1-phenylethanone followed by the reaction
with hydroxylamine hydrochloride. The structures were determined by IR, 1H NMR, HRMS, and X-ray
analysis. A dose- and time-dependent inhibition of proliferation was observed in A549 lung cancer cell
after compound treatment. Inhibition of growth was mainly attributed to the autophagy induction.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 2 June 2010
Revised 22 June 2010
Accepted 23 June 2010
Available online 26 June 2010
Keywords:
Synthesis
Pyrazole
Oxime
Apoptosis
Autophagy
A549 cells
Cancer is a major public health problem in the world and lung
cancer is the leading cause of death from malignancies worldwide
in both men and women. Chemotherapy is still one of the primary
modalities for the treatment of cancer. However, the use of avail-
able chemotherapeutics is often limited mainly due to undesirable
side effects and a limited choice of available anticancer drugs. This
clearly underlies the urgent need of developing novel chemother-
apeutic agents with more potent antitumor activities.
Apoptosis and autophagy have been proposed to be involved in
diverse human diseases including cancer. The regulation of autoph-
agy incancercellsis complex sinceitcan enhancetumorcellsurvival
in response to certain stresses, yet it can also act to suppress the ini-
tiation of tumor growth. Several drugs that are active on autophagy
signaling pathways are already used for the treatment of cancer or
are under preclinical development. It should, however, be noted that
in most cases it is still unclear whether the modulation of autophagy
by these compounds has a positive or a negative effect for the treat-
ment. Understanding the signaling pathways involved in the regula-
tion of autophagy as well as the autophagy process itself represents
new directions in the development of anticancer therapies.1–3
Many pyrazole derivatives are well acknowledged to possess a
wide range of anticancer bioactivities.4–6 Although the skeleton
of pyrazole plays an important role in biological effects, the type
of peripheral substituent is also crucial. Thus, pyrazole derivatives
containing oxime have also attracted considerable attention.7–12
Such modifications may be of interest in the development of new
antitumor compounds with improved characteristics.
In our effort to discover and develop apoptosis or autophagy
regulator as potential new anticancer agents, we synthesized a ser-
ies of novel pyrazole derivatives and the evaluation of biological
activity showed that these compounds can inhibit A549 lung can-
cer cell growth by inducing apoptosis or autophagy.13–23 The find-
ings encouraged further us to modify the structure of pyrazole
derivatives and identify the interaction mechanism of small mole-
cules with targets. Herein we would like to report the synthesis
and single-crystal characterization of ethyl 3-aryl-1-(2-(hydroxyi-
mino)-2-arylethyl)-1H-pyrazole-5-carboxylate derivatives as well
as their biological activities in suppressing the growth of A549 lung
cancer cells by inducing apoptosis or autophagy.
Chemistry: The synthesis of novel ethyl 1-(2-oxo-2-phenylethyl)-
3-phenyl-1H-pyrazole-5-carboxylate derivatives (3) has been per-
formed as shown in Scheme 1 from 2-bromo-1-phenylethanone
(2) and ethyl 3-phenyl-1H-pyrazole-5-carboxylate derivatives (1)
that can be synthesized as described in our previous paper.23 For
example, compound 3a was synthesized by the reaction of ethyl
* Corresponding authors. Tel.: +86 531 88366425; fax: +86 531 88564464.
E-mail addresses: bxzhao@sdu.edu.cn (B.-X. Zhao), miaojy@sdu.edu.cn,
sduzhao@hotmail.com (J.-Y. Miao).
Equal contribution.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.121

L.-W. Zheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4766–4770
4767
Scheme 1. Synthesis of ethyl 1-(2-oxo-2-phenylethyl)-3-phenyl-1H-pyrazole-5-carboxylate and ethyl 1-(2-(hydroxyimino)-2-phenylethyl)-3-phenyl-1H-pyrazole-5-
carboxylate derivatives.
3-phenyl-1H-pyrazole-5-carboxylate (1a) with 2-bromo-1-phenyl-
ethanone (2a) in the presence of potassium carbonate in acetonitrile
over a 2 h reflux period. After flash chromatography on silica gel,
ethyl 1-(2-oxo-2-phenylethyl)-3-phenyl-1H-pyrazole-5-carboxyl-
ate (3a) was obtained in 71% yield. Compound 3 reacted with
hydroxylamine hydrochloride in the mixture of ethanol and sodium
acetateanhydrousovera2–4 hrefluxperiodtoaffordethyl3-aryl-1-
(2-(hydroxyimino)-2-arylethyl)-1H-pyrazole-5-carboxylate deriv-
atives (4) in 85–99% yields.
are shown in Figure 1, and important hydrogen bonds are given
in Table 1. Both 4c and 4d consist of three ring systems and an
oxime frame in the molecular structures. In the two compounds,
the carbonyl groups occupy an almost planar position in relation
The structures of ethyl 1-(2-oxo-2-arylethyl)-3-aryl-1H-pyra-
zole-5-carboxylate derivatives (3) and ethyl 3-aryl-1-(2-(hydroxyi-
mino)-2-arylethyl)-1H-pyrazole-5-carboxylate derivatives (4) were
determined by IR, 1H NMR, and HRMS spectroscopy. For example,
the compound3a showed bands at 1711and 1271 cmÀ1 arising from
C@O stretching and asymmetric C–O–C stretching vibrations in IR
spectrum, respectively. In 1H NMR spectra, signal of two ortho-aro-
matic protons in 2-oxo-2-phenylethyl moiety appeared at the range
of d = 8.00 and 8.02 ppm (J = 7.4 Hz). While one triplet signal of the
para-aromatic protons upfielded and resonated at the range of
d = 7.62 and 7.65 ppm (J = 7.4 Hz). Two protons of benzene bonded
to pyrazole ring appeared as doublet peaks at rang of d = 7.81 and
7.83 ppm (J = 7.4 Hz). In the same benzene moiety, signals of other
two protons appeared at rang of d = 7.39, 7.41, and 7.43 ppm
(J = 7.4 Hz) as triplet peaks. Singlet signal of pyrazole proton ap-
peared at d = 7.25 ppm. In the HRMS, in accordance with the molec-
ular structureof C20H18N2O3, compound3a gave a [M+H]-ion peakat
m/z 335.1378. In the case of compound 4, for example, in the HRMS,
compound 4a gave a [M+H]-ion peak at m/z 350.1516 (calcd
350.1505) in accordance with the molecular structure of
C
20H19N3O3. It is well known that Z/E isomerism is obvious while
synthesizing the oximes from unsymmetrical ketones.24–26 In the
present study, indeed, the mixtures of Z/E isomers were achieved
in all cases, and the ratios of Z/E were around 2.6:1–4.1:1 according
to 1H NMR spectroscopy. The signals of methylene bonded to pyra-
zole moiety in Z isomer were deshielded and the deshielding magni-
tude was about 0.29 ppm comparing with E isomer. The difference
may be attributed to the interaction of N–O and C–H of methylene.
More interestingly, a purified Z isomer determined by X-ray,
such as compound 4d, was converted to a mixture of Z/E isomers
(Z/E = 13.2:1) in deuteriochloroform due to equilibrium of isomeri-
zation at room temperature.
Crystallography: Suitable crystals of 4c and 4d for X-ray diffrac-
tion were grown by slow crystallization from ethanol. Novel com-
pounds of 4c and 4d crystallized in the same space group, P21/c.
The two molecular structures with the atom-numbering scheme
Figure 1. View of the molecules 4c (a) and 4d (b) with the atomic labeling.
Displacement ellipsoids are drawn at the 30% probability level.

4768
L.-W. Zheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4766–4770
Table 1
Table 2
Hydrogen-bond geometry for structures of 4c and 4d (Å, °)
In vitro cytotoxicity of synthesized compounds (4a–i) at 48 h
Compound
D–HÁ Á ÁA
D–H
HÁ Á ÁA
DÁ Á ÁA
\D–HÁ Á ÁA
Compounds 4a
4b
4c
4d
4e
4f
4g
4h
4i
4c
O1–H1Á Á ÁN1a
C6–H6Á Á ÁO3b
C8–H8AÁ Á ÁO1
0.82
0.93
0.97
2.11
2.55
2.28
2.8059(18)
3.407(2)
2.665(2)
143
154
103
IC50 M) 29.8 31.4 42.7 26.3 14.5 16.3 29.1 17.8 36.5
(l
4d
O3–H3Á Á ÁN2c
0.82
0.97
0.97
2.04
2.24
2.26
2.8335(18)
2.623(2)
2.982(2)
164
102
130
determinant factor of the response of tumors to chemotherapeutic
agents. The morphological features of apoptosis include chromatin
condensation, cell shrinkage, and nuclear fragmentation.29 To fur-
ther investigate the induction of apoptosis, we examined the mor-
phological changes induced by compounds 4a–i in A549 cells
using Hoechst 33258 staining. The A549 cells were exposed to
C13–H13AÁ Á ÁO3
C13–H13BÁ Á ÁO1
a
b
c
Symmetry codes: 1 À x, 2 À y, 2 À z.
x + 1, y, z.
1 À x, 1 À y, 2 À z.
10, 20 and 40 lM for 48 h. As illustrated in Figure 3, compared
to the mean plane of the pyrazole rings. Torsion angles C8–C1–N1–
O1 [2.0(2)°] in 4c and C13–C14–N3–O3 [À1.8(2)°] in 4d revealed
the two compounds adopt Z configuration about the C@N double
bonds in the crystals.
Inhibitory effects of compounds 4a–i on the proliferation of A549
lung cancer cells: All the compounds synthesized were evaluated
for their cytotoxicity against A549 lung cancer cell line. After incu-
with vehicle (0.1% DMSO), most of which contained intact genomic
DNA, cells cultured with compounds 4a–f had some cells with con-
densed chromatin. Granulation of the nucleus appeared as fluores-
cent blue in the detail of the Hoechst 33258 stained cells (those
from which vehicle was absent). Since nucleus granulation is a fea-
ture of the morphological change in apoptosis, these results sug-
gest that 4a–f may induce partly apoptosis in A549 cells.
However, the apoptosis magnitude of cells seems hardly to be deci-
sive for inhibiting growth of cells. In addition, in the case of com-
pounds 4g–i, the morphological features of apoptosis were less
found.
bated with compounds at the concentration of 10, 20 or 40 lM for
48 h, respectively, the cells were treated with SRB to measure their
growth/viability (% of the untreated control) using a spectropho-
tometer as described previously.14,27,28 The reading of SRB staining
is known to accurately reflect the levels of total cellular macromol-
ecules.27 Figure 2 indicates the cytotoxic effects of the compounds
tested at different concentration on the viability of A549 lung can-
cer cells that were incubated for 48 h. The viability of A549 cells
was significantly dose-dependently suppressed by these com-
pounds. For example, exposure of cells to compound 4e, at 10, 20
Compounds 4b, 4d–f, 4h, and 4i induced autophagic cell death in
A549 cells: In order to determine if the growth inhibitory effects
were due to necrosis that is believed to be an unwanted side effect
of cancer-fighting agents, we performed the LDH assay and the re-
sults showed that 40 lM of compounds 4 at 48 h did not cause a
significant (P >0.05) increase in LDH release as shown in Figure 4.
Like apoptosis, autophagy is also believed to be a frequent cell
death mechanism and a number of studies have reported that
autophagy is activated in response to various anticancer thera-
pies.30,31 Growing evidence indicates that nonapoptotic pro-
grammed cell death is principally attributed to autophagy (type
II programmed cell death).32 Considering the majority of com-
pounds 4 treated A549 cells do not primarily display typical fea-
tures of apoptosis, we therefore assessed whether compounds 4
induces autophagy in lung cancer.
Autophagy is the process of sequestering cytoplasmic proteins
into lytic components and is characterized by the formation and
promotion of acidic vesicular organelles (AVO). The volume of
the cellular acidic compartment, as a marker of autophagy, can
be visualized by acridine orange (AO) staining.33,34 To assess the
occurrence of acidic vesicular organelles, we treated A549 cells
and 40 lM for 48 h, the cell viability reduced significantly from
100% to 58.6%, 51.9% and 10.3%, respectively. The IC50 concentra-
tion for each compound was calculated with reference to a stan-
dard curve (control cells), which represents the concentration
that results in a 50% decrease in cell growth after 48 h of incuba-
tion. For each compound, the 50% growth inhibition (IC50) was re-
ported in Table 2.
From these data, it is possible to relate the structural character-
istics of the compounds to their antitumor activity. When substitu-
ent R2 in benzene bonded to oxime moiety is chlorine, the
inhibition effect of growth is stronger. Taken together, compound
4e was the most effective compound in suppressing A549 cell
growth.
Apoptosis assays of morphological changes and DNA fragmentation
in compound (4a–i) treated A549 cells: Apoptosis is an important
Figure 2. Effects of the compounds on A549 cell viability. A549 cells were treated with compounds 4a–i at concentrations of 10, 20 or 40
lM or left untreated (control) for
*
**
48 h. Cell viability was analyzed by SRB assay and illustrated in column figures. Results are presented as mean SE; (n = 3. , P <0.05 vs control; , P <0.01 vs control).

L.-W. Zheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4766–4770
4769
Figure 3. Compounds 4a–i induces A549 cell apoptosis significantly. (A) Fluorescent micrographs of Hoechst 33258 staining (400Â). (B) Ratio of apoptotic cells vs control.
Results are presented as mean SE; (n = 3. *, P <0.05 vs control; **, P <0.01 vs control).
Figure 4. Effects of the compounds on LDH release in A549 cells. A549 cells were treated with compounds 4a–i at the concentration of 40
48 h. LDH release was determined. Results are presented as mean SE; n = 3.
lM or left untreated (control) for
with compounds 4 for 48 h and then stained them with acridine
orange. As shown in Figure 5, compounds 4b, 4d–f, and 4h–i treat-
ment resulted in the appearance of AVO when cells were stained
with acridine orange after 48 h treatment. It is acceptable that
LC3 is the credible marker of the autophagosome in mammalian
cells. Enhancement of LC3-II level occurs when autophagy is in-
duced. Thus, induction or suppression of autophagy can be easily
monitored by examining the levels of LC3-II by immunoblot.35 To
further identify autophagosome, we determined the protein level
of LC3-II in the A549 cells treated with compounds 4b and 4d–i,
which were chosen based acridine orange (AO) staining, at 24 h
by Western blot analysis.36 It can be seen from Figure 6 that pro-
tein levels of LC3-II in the A549 cells treated with compounds at
24 h, except 4g, were elevated (P <0.05).
In summary, a series of novel oxime-containing pyrazole deriv-
atives were synthesized and characterized by X-ray analysis. A
dose- and time-dependent inhibition of proliferation was observed
in A549 lung cancer cell after compound treatment. The morpho-
logical feature of apoptosis was demonstrated by Hoechst 33258
staining and the induction of autophagy was determined by acri-
dine orange (AO) staining and enhancement of LC3-II level. Inhibi-
tion of growth was mainly attributed to the autophagy induction.

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L.-W. Zheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4766–4770
Figure 5. Compounds 4a–i increased the acidic vesicle level in the cells at 48 h (one representative result of three independent experiments n = 3). Microscopic photographs
(400Â) were taken under a fluorescent microscope (Nikon).
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*
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