Synthesis of novel pyrazole carboxamide derivatives and discovery of modulators for apoptosis or autophagy in A549 lung cancer cells

Article abstract of DOI:10.1016/j.bmcl.2009.07.131

A series of novel 3-aryl-1-arylmethyl-1H-pyrazole-5-carboxamide derivatives 3a-l, were synthesized by the reaction of 3-aryl-1-arylmethyl-1H-pyrazole-5-carbonyl chloride with substituted amine in excellent yields. The compounds 3e-h could suppress A549 lu

Full text of DOI:10.1016/j.bmcl.2009.07.131

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5325–5328
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel pyrazole carboxamide derivatives and discovery
of modulators for apoptosis or autophagy in A549 lung cancer cells
a
a
Xiao-Ling Ding a,c, , Hai-Yan Zhang b, , Lei Qi b, Bao-Xiang Zhao a, , Song Lian , Hong-Shui Lv ,
*
Jun-Ying Miao b,
*
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 College of Advanced Professional Technology, Qingdao University, Qingdao 266061, 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-aryl-1-arylmethyl-1H-pyrazole-5-carboxamide derivatives 3a–l, were synthesized by
the reaction of 3-aryl-1-arylmethyl-1H-pyrazole-5-carbonyl chloride with substituted amine in excellent
yields. The compounds 3e–h could suppress A549 lung cancer cell growth. More interestingly, com-
pounds 3e and 3f might inhibit the A549 cell growth by inducing apoptosis; whereas compounds 3g
and 3h with fluorine group might inhibit the A549 cell growth by inducing autophagy.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 16 June 2009
Revised 25 July 2009
Accepted 29 July 2009
Available online 3 August 2009
Keywords:
Pyrazole
Carboxamide
A549 cells
Apoptosis
Autophagy
Lung cancer is one of the leading causes of death worldwide.1
The discovery and development of new, more active, more selec-
tive, and less toxic compounds for the treatment of malignancy
are one of the most important goals in medicinal chemistry. The
understanding of the biology of cancer has undoubtedly improved
in recent years and has strongly impacted both experimental and
clinical tumor therapy.2 Apoptosis, or programmed cell death, is
a critical cellular process in normal development and homeostasis
of multicellular organisms. Inappropriate regulation of apoptosis is
associated with many human diseases, including cancer, and it is
now recognized that one hallmark of cancer cells is their compro-
mised ability to undergo apoptosis.3,4 However, non-apoptotic
forms of cell death, such as autophagy and extrinsic senescence,
have also been shown to contribute to the overall tumor response.
Non-apoptotic forms of programmed cell death are targets for no-
vel approaches in anticancer therapy. Autophagy is a process in
which subcellular membranes undergo dynamic morphological
changes that lead to the degradation of cellular proteins and cyto-
plasmic organelles. Recently, interest in autophagy has been re-
newed among oncologists, because different types of cancer cells
undergo autophagy after various anticancer therapies.5–9 Targeting
critical apoptosis or autophagy regulators with a goal to promote
apoptosis or autophagy in cancer cells is an attractive new cancer
therapeutic strategy.
Many pyrazole derivatives are known to exhibit a wide range of
biological properties such as cannabinoid type-1 (CB1) receptor
antagonists;10,11 inhibitors of coactivator associated arginine
methyltransferase 1 (CARM1) that has been shown to be up-regu-
lated during the progression of prostate cancer;12,13 inhibitor of
CDK2, with good activity against a range of human tumor cell
lines;14 EP1 receptor antagonists;15 trypsin-like serine protease
factor Xa inhibitors;16,17 HIV-1 integrase inhibitors;18 inhibitors
of tissue-nonspecific alkaline phosphatase (TNAP);19 non-steroidal
selective glucocorticoid receptor (GR) agonists and potent PPARa
activators.20,21 In our effort to discover and develop apoptosis or
autophagy inducers as potential new anticancer agents, we
reported a series of novel pyrazole and fused-pyrazole derivatives
such as ethyl 1-(20-hydroxy-30-aroxypropyl)-3-aryl-1H-pyrazole-
5-carboxylate derivatives, ethyl 1-arylmethyl-3-aryl-1H-pyrazole-
5-carboxylate derivatives, 1-arylmethyl-3-aryl-1H-pyrazole-5-
carbohydrazide derivatives, and 1-arylmethyl-3-aryl-1H-pyra-
zole-5-carbohydrazide hydrazone derivatives.22–26 The evaluation
of biological activity showed that these compounds can inhibit
A549 lung cancer cell growth. Thus, it is important to modify the
structure of pyrazole derivatives and identify the interaction
mechanism of small molecules with targets. Herein, we would like
* Corresponding authors. Tel.: +86 531 88366425; fax: +86 531 88564464
(B.-X.Z.).
E-mail addresses: bxzhao@sdu.edu.cn, sduzhao@hotmail.com (B.-X. Zhao),
miaojy@sdu.edu.cn (J.-Y. Miao).
Equal contribution.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.131

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X.-L. Ding et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5325–5328
to report the synthesis of 3-aryl-1-arylmethyl-1H-pyrazole-5-car-
boxamide and the discovery of their biological activities in sup-
pressing the growth of A549 lung cancer cells by inducing
apoptosis or autophagy.
The compounds induced the changes of A549 cell morphology
concomitant with cell growth inhibition induced by them
(Fig. 2). When A549 cells were treated with compounds 3e and
3f, the cells became round and detached from the bottom of cell
culture dish, indicating that the compounds might induce A549
cell death by apoptosis. When treated with compound 3g and 3h,
the cells vacuolated gradually as the concentration increased and
the time elongated, indicating that the compounds might induce
A549 cell death by autophagy. It was confirmed by the analysis
of nuclear and acidic compartment morphology as well as LDH
activity assay.
Modulation of the compounds 3e–h for apoptosis or autophagy in
A549 lung cancer cells. DNA fragmentation, chromatin condensa-
tion, cell shrinkage, and membrane blebbing are the characteristics
of apoptotic cells. The chromatin condensation and DNA fragmen-
tation in the cells were observed by Hoechst 33258 staining under
a Nikon fluorescence microscope. The results showed that nuclear
Chemistry. The synthesis of 3-aryl-1-arylmethyl-1H-pyrazole-5-
carboxamide derivatives (3) has been accomplished as outlined in
Scheme 1 starting from ethyl 3-aryl-1-arylmethyl-1H-pyrazole-5-
carboxylate (1) that can be synthesized as described in our previ-
ous paper.26 Briefly, 3-aryl-1-arylmethyl-1H-pyrazole-5-carbonyl
chloride (2) was synthesized by alkali hydrolysis of ethyl 3-aryl-
1-arylmethyl-1H-pyrazole-5-carboxylate (1) followed by the
reaction with thionyl chloride. 3-Aryl-1-arylmethyl-1H-pyrazole-
5-carbonyl chloride (2) reacts with substituted amine in the pres-
ence of triethylamine in dichloromethane at room temperature to
afford 3-aryl-1-arylmethyl-1H-pyrazole-5-carboxamide (3) in 86–
97% yield. The structures of 3a–i were determined by IR, 1H NMR
and HRMS spectroscopy.27
Inhibition of the compounds on the growth of A549 lung cancer
cells. In order to evaluate the inhibitory effects on the growth of
A549 cells we carried out the MTT assay with synthesized com-
pounds. The compounds 3a–h have suitable solubility in cell cul-
ture medium, however, compounds 3i–l are difficulty to dissolve
in cell culture medium. The data obtained by MTT assay showed
that compounds 3e–h had inhibitory effects on the growth of
A549 cells in dosage- and time-dependent manners. As typically
DNA fragmentation occurred in the cells treated with 20 lM of
compounds 3e and 3f for 48 h (Fig. 3). In the case of 3g and 3h,
the volume of the cellular acidic compartment, as a marker of
autophagy,25,28,29 was visualized by acridine orange staining. As
shown in Figure 4 compounds 3g and 3h at 20 lM promoted
autophagy dramatically. The data suggested that the compounds
3e and 3f might inhibit A549 cell growth through modulating
apoptosis while the compounds 3g and 3h might inhibit A549 cell
growth through modulating autophagy.
shown in Figure 1, exposure of cells to 3e–f at 10 lM for 24 h re-
sulted in cell viability decrease from 100% to 59.32%–45.52%. When
the exposure continued on to 48 h, compared with the control
group, the cell viability reduced more significantly from 100% to
To detect whether compounds resulted in necrosis of A549
cells, LDH activity in cell culture medium was measured. As shown
in Figure 5, there was no significant difference (p >0.05) in LDH re-
lease between the cells of control group and the cells treated with
44.46%–24.07%. Further, exposure of cells to 3e–g at 20 lM for
48 h, the cell viability reduced more significantly from 100% to
40.44–10.43%, respectively. Taken together, compound 3f was
the most effective compound in suppressing A549 cell growth.
the compounds 3e–h at 10 and 20 lM for 48 h. The results indi-
cated that the compounds at the test range of concentration did
not cause necrosis in A549 cells.
Scheme 1. Synthesis of 3-aryl-1-arylmethyl-1H-pyrazole-5-carboxamide derivatives 3a–l. Reagents and conditions: (i) KOH/EtOH, reflux, 4 h, then HCl aq; (ii) SOCl2, reflux,
3 h; (iii) CH2Cl2, Et3N, ArCH2CH2NH2, rt, 2 h.

X.-L. Ding et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5325–5328
5327
Figure 1. Effects of the compounds 3a–h on cell viability at 24 h (black bar) and 48 h (white bar).
Figure 2. Morphological changes induced by compounds 3e–h at 20
lM. Microscopic photographs (200ꢀ) were taken under an inverted phase contrast microscope (Nikon).
Figure 3. The compounds 3e and 3f induced apoptosis in A549 cells at 48 h (p <0.01
vs control, n = 3). Microscopic photographs (200ꢀ) were taken under a fluorescent
microscope (Nikon).
Figure 4. The compounds 3g and 3h increased the acidic vesicle level in the cells at
24 h (p <0.01 vs control, n = 3). Microscopic photographs (200ꢀ) were taken under a
fluorescent microscope (Nikon).
Structure–activity relationships. In our previous papers, we re-
ported that 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide
and 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide hydra-
zone derivatives could inhibit the A549 cell growth obviously
and the cytotoxic potency was highly dependent on the substitu-
tion types and patterns on the aryl ring, for example, replacing
the hydrogen at the 4-position of 1-aryl ring with a bulkier tert-bu-
tyl group resulted in a significant activity increasing. In present
study, we observed that the nature and the position of substituent
on the molecule improve biological functions. Indeed, compounds
3e–h with tert-butylbenzyl in N-1 position and p-chlorophenyl
group in 3-position were proved to be the most active member
with a unique antitumor potency against A549 cell lines. More
interestingly, compounds 3e and 3f in which R3 is hydrogen or
methoxyl group might inhibit the A549 cell growth by inducing
apoptosis, whereas compounds 3g and 3h with fluorine group
might inhibit the A549 cell growth by inducing autophagy.
In summary, we have described that a series of novel 1-aryl-
methyl-3-aryl-1H-pyrazole-5-carboxamide derivatives were read-

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ily synthesized from ethyl 1-arylmethyl-3-aryl-1H-pyrazole-5-car-
boxylate and substituted amine. The biological evaluation of these
compounds on A549 lung cancer cell growth showed that the com-
pounds 3e–h with tert-butylbenzyl in N-1 position and p-chloro-
phenyl group in 3-position inhibited much more proliferation of
A549 cell. More interestingly, compounds 3e and 3f might inhibit
the A549 cell growth by inducing apoptosis; whereas compounds
3g and 3h might inhibit the A549 cell growth by inducing autoph-
agy. The novel compounds certainly deserve further careful investi-
gation in terms of their possible mechanism. Currently,
investigations are underway to elucidate the mechanism and the re-
sults will be published in due course.
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Acknowledgments
phenethyl-1H-pyrazole-5-carboxamide 3e: mp 116–118 °C; IR (KBr)
m: 1641
This study was supported by National Natural Science Founda-
tion of China (90813022) and the Science and Technology Develop-
mental Project of Shandong Province (2008GG10002034).
(C@O) cmꢁ1 1H NMR (400 MHz, CDCl3) d: 1.27 (s, 9H), 2.89 (t, 2H, J = 6.8 Hz),
;
3.72 (q, 2H, J = 6.8 Hz), 5.76 (s, 2H), 6.59 (t, 1H, J = 5.6 Hz), 7.18–7.33 (m, 10H),
7.39 (d, 2H, J = 8.4 Hz), 7.78 (d, 2H, J = 8.4 Hz); HRMS (ESI) calcd for [M+Cl]+
C29H30Cl2FN3O: 506.1766, found: 506.1768.
1-(4-tert-Butylbenzyl)-3-(4-chlorophenyl)-N-(3-methoxyphenethyl)-1H-pyrazole-
Supplementary data
5-carboxamide 3f: mp 107–109 °C; IR (KBr) m ;
: 1640 (C@O) cmꢁ1 1H NMR
(400 MHz, CDCl3) d: 1.26 (s, 9H), 2.87 (t, 2H, J = 6.8 Hz), 3.71 (q, 2H, J = 6.8 Hz),
3.78 (s, 3H), 5.76 (s, 2H), 6.61 (t, 1H, J = 5.6 Hz), 6.76–6.79 (m, 3H), 7.21 (t, 1H,
J = 8.0 Hz), 7.26 (s, 1H), 7.27–7.33 (m, 4H), 7.40 (d, 2H, J = 6.8 Hz), 7.78 (d, 2H,
J = 6.8 Hz); HRMS (ESI) calcd for [M+Cl]+ C30H32Cl2N3O2: 536.1872, found:
536.1866.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.131.
1-(4-tert-Butylbenzyl)-3-(4-chlorophenyl)-N-(4-fluorophenethyl)-1H-pyrazole-5-
carboxamide 3g: mp 126–128 °C; IR (KBr)
References and notes
m: ;
1641 (C@O) cmꢁ1 1H NMR
(400 MHz, CDCl3) d: 1.27 (s, 9H), 2.83 (t, 2H, J = 6.4 Hz), 3.61 (q, 2H, J = 6.4 Hz),
5.75 (s, 2H), 5.93 (t, 1H, J = 5.6 Hz), 6.61 (s, 1H), 6.97 (t, 2H, J = 8.4 Hz), 7.08 (dd,
1H, J = 5.6, 8.4 Hz), 7.25 (d, 2H, J = 8.4 Hz)), 7.32 (d, 2H, J = 8.4 Hz), 7.35 (d, 2H,
J = 8.4 Hz), 7.71 (d, 2H, J = 8.4 Hz); HRMS (ESI) calcd for [M+Cl]+ C29H29Cl2N3O:
524.1672, found: 524.1667.
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(400 MHz, CDCl3) d: 1.27 (s, 9H), 2.94 (t, 2H, J = 6.8 Hz), 3.72 (q, 2H, J = 6.8 Hz),
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