Synthesis and biological evaluation of chalcones and their derived pyrazoles as potential cytotoxic agents

Article abstract of DOI:10.1016/j.bmcl.2005.03.121

A series of substituted chalcones and their corresponding pyrazoles were synthesized and evaluated for in vitro cytotoxic activity against a panel of human cancer cell lines. Out of 93 compounds screened, 8 compounds, 1s, 3i,j,n, 4i,j,n and 4s, showed marked activity. Compounds 4j,n and 4s were found to be the most promising in this study. SAR is also discussed.

Full text of DOI:10.1016/j.bmcl.2005.03.121

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3177–3180
Synthesis and biological evaluation of chalcones and their derived
pyrazoles as potential cytotoxic agents
B. A. Bhat,a K. L. Dhar,a,* S. C. Puri,a A. K. Saxena,b M. Shanmugavelb and G. N. Qazib
aDivision of Natural Product Chemistry, Regional Research Laboratory (CSIR) Jammu 180001, India
bDivision of Pharmacology, Regional Research Laboratory (CSIR) Jammu 180001, India
Received 3 November 2004; revised 1 March 2005; accepted 3 March 2005
Available online 12 May 2005
Abstract—A series of substituted chalcones and their corresponding pyrazoles were synthesized and evaluated for in vitro cytotoxic
activity against a panel of human cancer cell lines. Out of 93 compounds screened, 8 compounds, 1s, 3i,j,n, 4i,j,n and 4s, showed
marked activity. Compounds 4j,n and 4s were found to be the most promising in this study. SAR is also discussed.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
group placements in the two molecules has led to the
synthesis and subsequent evaluation of a large number
of chalcones.14
Microtubules are essential components of cell structure
and are involved in many cellular processes, including
mitosis, morphogenesis, intracellular transport and
secretion.1 They are hollow tubes consisting of a- and
b-tubulin heterodimers that polymerize parallel to the
cylindrical axis. Tubulin binding molecules interfere
with the dynamic instability of microtubules and thereby
disrupt microtubule inducing cell cycle arrest in the M-
phase, forming abnormal spindles and finally leading
to apoptotic cell death.2 A variety of natural compounds
such as paclitaxol, vinblastin, combretastatin A-4 and
colchicine attack microtubules by interfering with the
dynamics of tubulin polymerization and depolymeriza-
tion, resulting in mitotic arrest.3
As a part of our drug discovery programme, we have
been actively involved in determining the features
important for anti-mitotic activity and in searching no-
vel anti-cancer agents that show strong growth inhibi-
tory activities against a panel of human cancer cell
lines. Herein, we report the synthesis of four series of
organic compounds, viz., chalcones, epoxy-chalcones,
3,5-diphenyl-4-hydroxy-4,5-dihydro,1H-pyrazoles and
3,5-diphenyl,1H-pyrazoles, and we determine their cyto-
toxic potential in an in vitro cell culture system. The
cytotoxic potential of 3,5-diphenyl-4-hydroxy-4,5-dihy-
dro,1H-pyrazoles and 3,5-diphenyl,1H-pyrazoles ap-
pears to be an unexplored field and needs attention.
SAR studies revealed that the introduction of pyrazole
nucleus between two aryl rings of chalcones played an
integral role for the increase in cytotoxic potential.
For a structurally simple group of compounds, chal-
cones have displayed an impressive array of biological
activities, among which anti-malarial,4 anti-protozoal,5
anti-inflammatory,6 immunomodulatory,7 nitric oxide
inhibition,8 tyronase inhibition,9 cytotoxic10 and anti-
cancer11 activities have been cited in the literature. These
compounds obtained by convenient synthetic methods
strongly inhibit the polymerization of tubulin by binding
to the colchicine-binding site.12,13 The relatively simple
structure and high affinity of chalcones for the colchi-
cine-binding site because of similarity of the two-aryl
2. Chemistry
Chalcones were synthesized by a base catalyzed Claisen–
Schmidt condensation reaction15 of appropriately
substituted acetophenones and aldehydes. The method
is attractive since it specifically generates (E)-isomer.
1
From H NMR spectra, all chalcones were geometri-
cally pure and with trans-configuration (J Ha–
Hb = 15.50–15.60 Hz). Reacting chalcones with hydro-
gen peroxide in alkaline conditions at 0 ꢁC afforded
Keywords: Chalcone analogues; In vitro cytotoxicity; Cell lines.
*
Corresponding author. Tel.: +91 0191 2549051(O); fax: +91 0191
2548607; e-mail: dharkl@yahoo.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.121

3178
B. A. Bhat et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3177–3180
the corresponding a,b-epoxide derivatives.16 These epoxy
derivatives were condensed with hydrazine hydrate to
corresponding 4-hydroxy pyrazoles and pyrazoles.17,18
In addition, compounds 1i–l and 1s were brominated
with Br2 to corresponding 2,3-dibromochalcones, and
1e and 1s reduced with H2/Pd to 2,3-dihydrochalcones
in order to observe the effect of double bond on activity
(Scheme 1).
noncancerous cell line CV-1 (derived from African mon-
key kidney) and were found devoid of any cytotoxicity.
Compound 4s, which contained a 3,4,5-trimethoxy
moiety at ring A and 3,4-dimethoxy moiety at ring B
linked with pyrazole nucleus, showed significant cyto-
toxic activity, especially selective against HCT-15 and
SW-620 cell lines.
By comparing the cytotoxic potential of compounds
with different substitution as well as at different posi-
tions, the following conclusions were drawn: (a) Pres-
ence of 3,4,5-trimethoxy moiety on either ring is
essential; introducing it at ring A (1j,t) led to enhanced
cytotoxic activity than when attached to ring B (1d,i).
Acetylation of –OH in 1e and 1g to 1f and 1h, respec-
tively, also increased the activity. (b) Introducing the
40-methoxy, 30,40-dimethoxy, 30,40-methylenedioxy and
30,40,50-trimethoxy (1j,k,l,s) led to enhanced cytotoxic
activity compared with B ring unsubstituted compound
(1t) and introducing 40-flouro (1n) also enhanced the
activity in comparison with 40-chloro- (1m). (c) Convert-
ing a,b-unsaturated ketones (1a–d,i–u) to corresponding
epoxides (2a–d,i–u) dramatically reduced the cytotoxic-
ity (IC50 value P100 lM), in spite of the fact that the
3. Biology
Chalcones and their derived analogues were assayed for
in vitro cytotoxicity against 12 human cancer cell lines,
HT-29, HCT-15, SW-620 (colon) A-549, HOP-62 (lung),
Hep-2 (liver), SiHa (cervix), SKOV-3, OVCAR-5
(ovary), PC-3, DU-145 (prostate) and SNB-78 (CNS),
using sulforhodamine B.19 The cells were allowed to
proliferate in presence of test material for 48 h and re-
sults are reported in terms of IC50 values (Table 1).
From the IC50 values, it is clear that the compounds
4j,n and 4s showed significant cytotoxic activity
especially against HT-29, HCT-15 and SW-620 cell
lines. These three compounds were also evaluated for
Scheme 1. Reagents and conditions: (a) KOH/EtOH, 0 ꢁC; (b) H2O2, 5% NaOH, 0–4 ꢁC; (c) NH2NH2ÆH2O/EtOH reflux; (d) AcOH/H2SO4; (e)
Ac2O/pyridine; (f) H2/5% Pd/C; (g) Br2/CCl4.

B. A. Bhat et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3177–3180
3179
Table 1. IC50 values (lM) of 1d–n,s,t, 3i–n,s, 4i–n,s, 8s and 5k
Compound HT-29 HCT-15 SW-620 A-549 Hop-62 Hep-2 SiHa
PC-3
DU-145 SKOV-3 OVCAR-5 SNB-78
1d
1e
1f
14.41
20.15
10.50
12.01
8.37
21.31
7.08
NT
8.77
7.75
16.01
NTa
NT
21.3
19.15
15.6
52.01
12.03
NT
20.49.
20.25
9.2
7.55
6.01
6.95
36.5
40.4
41.12 25.4
21.01
NT
8.35
NT
NT
NT
NT
NT
NT
NT
NT
6.09
56.51
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
39.06
8.9
NT
22.8
NT
NT
NT
NT
NT
NT
7.75
26.00
7.75
33.04 NT
15.61 NT
13.30 NT
NT
NT
NT
NT
1g
1h
1i
NT
NT
160.41
56.15
13.75
10.07
9.61
NT
13.06
8.62
13.71
10.1
7.75
12.01
10.02
6.15
11.86
NT
NT
NT
22.23
4.90
NT
NT
64.60
10.91
12.83
13.43 25.4
11.62 14.93
11.68 19.8
25.01
9.00
5.65
9.23
16.81
18.32
8.3
28.70
10.01
16.73
NT
1j
8.07
NT
NT
1k
1l
23.71
6.55
NT
NT
NT
NT
10.00
26.94
24.72
8.12
8.55 NT
NT
1m
1n
1s
1t
24.00
11.73
7.95
NT
NT
NT
NT
40.02
7.92
9.35
24.06 28.05
11.65 13.32
25.41
13.78
22.81
NT
NT
6.64
12.01
5.95
0.96
5.89
7.41
11.08
6.01
8.43
2.95
0.62
6.01
5.62
8.09
4.75
0.47
10.44
15.64
5.80
8.45
7.35
2.67
6.75
13.58
8.41
5.85
8.63
5.15
3.41
9.25
10.01
4.96
5.9
6.90
8.96
7.2
7.85
9.21
NT
15.80
7.85
0.98
16.64
8.1
0.94
20.81 NT
NT
3i
NT
20.13
43.03
28.04 NT
15.17 NT
34.52 34.7
7.05
13.01
6.81
20.55
8.65
7.15
8.76
9.54
7.85
33.01
9.95
8.61
6.45
4.40
16.71
NT
NT
NT
3j
1.07
6.74
15.55
15.08
5.72
10.04
4.35
1.01
8.85
7.58
15.82
3.8
7.81
40.00
NT
3k
3l
NT
20.27
20.45
20.41
6.55
53.68
NT
NT
8.35
9.45
83.00 108.01 94.07
34.55 NT
3m
3n
3s
4i
NT
NT
56.41
NT
NT
8.45
NT
NT
NT
NT
NT
NT
12.01
10.12
0.46
10.71
4.81
0.65
NT
12.45
0.81
34.01
6.43
NT
15.91
14.47
10.01
25.04
NT
15.81 24.00
11.07 26.99
21.03
50.03
74.04
22.04
14.04
22.45
NT
4j
4k
4l
15.55
6.01
6.45
13.45
NT
9.55
13.35 35.08
NT NT
8.93 3.87
2.64 NT
NT
18.05 48.03
8.56
4m
4n
4s
8s
5k
12.34
0.56
0.93
0.48
0.25
3.81
8.41
NT
1.08
3.63
0.23
16.05
NT
0.87
10.04
NT
16.00
8.90
22.05
18.45
54.81 NT
58.10
NT
32.97
61.60
a NT = Not tested.
epoxy group might also act as an alkylating moiety20 at
the same time it disturbs the conformation of two aryl
rings. In addition, hydrogenation and bromination
across carbon–carbon double bond also reduced the
activity markedly (IC50 value P60 lM). This result
demonstrated that the conformation of the two aryl
rings in the molecule has an essential role to play for
the activity of chalcones and the double bond seems
to be a crucial moiety as reported earlier.10e (d) Con-
verting epoxides to hydroxy pyrazoles resulted in the
re-appearance of the activity significantly (3i–m,n,s)
compared with the corresponding epoxy chalcones. On
dehydration to corresponding pyrazoles, the cytotoxic
activity was enhanced to its maximum, in fact more
than the corresponding chalcones (4i–m,n,s). This result
demonstrated that introduction of pyrazole nucleus be-
tween two aryl rings enhanced the rigidity besides polar-
ity and solubility in the molecule which played an
integral role for their increase in cytotoxic potential.
Pyrazoles with appropriate substitution (4i,j,l and 4s)
are more active than their hydroxy pyrazoles, confirm-
ing that –OH at position-400 in pyrazole nucleus has
either no or minimal role to play. When these hydrox-
ypyrazoles and pyrazoles were converted into corre-
sponding diacetates and acetates, respectively, the
activity was markedly reduced (8s). This result demon-
strated that the hydroxy pyrazoles and pyrazoles are
also reacting via N-100 of pyrazole nucleus and it
might be the active binding site which gets deactivated
by acylation. Finally, by comparing the activity of
chalcones, 2,3-dibromochalcones, 2,3-dihydrochalcones,
epoxy-chalcones, 4-hydroxy pyrazoles and pyrazoles, it
is concluded that pyrazoles are more active than hydro-
xy pyrazoles, which in turn, are generally more active
than their corresponding chalcones while the activity
of other derivatives was insignificant.
In conclusion, we have discovered a novel class of chal-
cone-derived pyrazoles such as 3,5-diphenyl,1H-pyraz-
oles as potential cytotoxic agents. The position and
size of substituents, rigidity and solubility of the mole-
cule and finally the conformation of ring A and ring B
seems to be crucial for the cytotoxicity. Compounds
4j,n and 4s were found to be most promising in this
study. SAR studies and evaluation of more potent ana-
logues are continuing.
Acknowledgements
The authors are grateful to the instrumentation division
for elemental and spectral analysis and one of the
authors, B. A. Bhat, is thankful to CSIR (India) for
fellowship.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2005.03.121.

3180
B. A. Bhat et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3177–3180
14. (a) 10d–e; (b) Ducki, S.; Forrest, R.; Hadfield, J. A.;
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1
1
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