Synthesis and biological evaluation of chalcone-linked pyrazolo[1,5-: A] pyrimidines as potential anticancer agents

Article abstract of DOI:10.1039/c7md00193b

A series of pyrazolo[1,5-a]pyrimidines substituted at C5 with 1-phenylprop-2-en-1-one (6a-q) and 3-phenylprop-2-en-1-one (7a-k) was synthesized and evaluated for antiproliferative activity. Among them, 6h was found to be the most active compound against the MDA-MB-231 cell line with an IC₅₀ of 2.6 μM. The antiproliferative activity of this series of compounds ranged from 2.6 to 34.9 μM against A549 (lung cancer), MDA-MB-231 (breast cancer) and DU-145 (prostate cancer) cell lines. FACS analysis revealed that these hybrids arrest the cell cycle at the subG1 phase. Western blot analysis and an immunofluorescence assay showed the inhibition of the EGFR and STAT3 axis, which plays an important role in cell survival and apoptosis. Western blot and RT-PCR analyses that displayed an increase in apoptotic proteins such as p53, p21 and Bax and a decrease in the anti-apoptotic proteins Bcl-2 and procaspase-9 confirmed the ability of these hybrids to trigger cell death by apoptosis. Molecular docking studies described the binding of these hybrids to the ATP binding site of EGFR.

Full text of DOI:10.1039/c7md00193b

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Synthesis and biological evaluation of Chalcone-linked
pyrazolo[1,5-a]pyrimidines as potential anticancer agents
Received 00th January 20xx,
Accepted 00th January 20xx
a,†
c,†
c
Chandrakant Bagul, Garikapati Koteswara Rao, V,ae,bnkata Krishna Kanth Makani, Jaki R.
b
,c
Tamboli, Manika Pal-Bhadra,* and Ahmed Kamal*
DOI: 10.1039/x0xx00000x
A series of pyrazolo[1,5-a]pyrimidines substituted at C5 with 1-phenylprop-2-en-1-one (6a-q) and 3-phenylprop-2-en-1-
one (7a-k) was synthesized and evaluated for its antiproliferative activity. Among them, 6h was found to be the most
active compound with IC50 2.6 μM against the MDA-MB-231 cell line. Antiproliferative activity of this series of compounds
ranged from 2.6 – 34.9 μM against A549 (lung cancer), MDA-MB-231 (breast cancer) and DU-145 (prostate cancer) cell
lines. FACS analysis revealed that these hybrids arrest cell cycle at subG1 phase. Western blot analysis and immu
nofluorescence assay showed the inhibition of EGFR and STAT3 axis which plays an important role in the cell survival and
apoptosis. Western blot and RT-PCR analysis that displayed an increase in apoptotic proteins such as p53, p21 and Bax,
and decrease in the antiapoptotic protein Bcl-2 and procaspase-9, confirmed the ability of these hybrids to trigger cell
death by apoptosis. Molecular docking studies described the binding of these hybrids to the ATP binding site of EGFR.
www.rsc.org/
progression of several types of cancers. EGFR is overexpressed in
1
6-
Introduction
many solid tumors and has been associated with poor prognosis.
1
8
It plays a central role in the development of non-small-cell lung
Cancer has become a leading cause of mortality which solely
accounted for 8.5 million deaths (17% of total deaths) in 2012 and is
1
9,20
21,22
cancer (NSCLC),
as well as breast cancer.
Therefore the
1
,2
molecules which are inhibiting EGFR are effective in the treatment
of cancer. Since, EGFR has become a fascinating and validated
target for developing new anticancer agents, exploration of EGFR
with newly synthesized hybrids is likely to be a fruitful research
area.
estimated to cause 13.1 million deaths in 2030. Despite the
advance in diagnosis and treatment the burden of cancer is
increasing every year. Despite the fact that, enormous efforts are
being carried out for developing novel molecules and newer
chemotherapeutic strategies to treat different types of cancers, this
3
,4
remains a key concern across the globe.
Chalcones (1,3-diarylprop-2-en-1-one) represents one of the largest
class of plant metabolites important precursors in the biosynthesis
Protein tyrosine kinase inhibitors are of tremendous interest due to
their potential as therapeutic agents for the treatment of a variety
2
3
of flavonoids and related compounds. It remain an attractive
scaffold for researchers due to their abundance in plants, ease of
synthesis, availability of various ways to introduce diversity in the
core structure and ability to confer drug-like properties to the
synthesized compounds. Derivatives of chalcone are known to
possess broad spectrum of biological activities, such as antidiabetic,
antihypertensive, antihistaminic, antiretroviral, anti-inflammatory,
5
-7
of diseases, particularly cancer.
Among the protein tyrosine
kinases, epidermal growth factor receptor (EGFR) has emerged as a
key and validated target for the development of anticancer
8
-10
11
agents. Over ten drugs including erlotinib 1, gefitinib, lapatinib,
icotinib, vandetanib etc., which act on EGFR have been approved by
the United States Food and Drug Administration (US FDA) in the
2
4-26
1
2,13
antioxidant, antibacterial, antituberculosis, and anticancer.
past two decades and several molecules, such as BMS-690514
are currently in clinical trials. EGFR transduces the signals across the
Moreover, chalcones are successfully employed as tubulin inhibitors
2
7
28,29
1
0,14
(2) and EGFR inhibitors (3, 4)
towards the development of
membrane important for cellular functions such as proliferation,
anticancer agents, which highlights the importance of the chalcone
pharmacophore in the development of therapeutic agents.
Similarly, pyrazolo[1,5-a]pyrimidine derivatives demonstrate a wide
angiogenesis, invasiveness, decreased apoptosis and
1
5
differentiation, through which it regulates the development and
3
0, 31
variety of promising biological activities, such as antitumor,
a.
32,33
34,35
Department of Medicinal Chemistry, National Institute of Pharmaceutical
Education and Research (NIPER), Hyderabad-500 037, India
anxiolytic,
antimicrobial,
6
antifungal, antiobesity and anti-
3
b.
inflammatory. They are effectively employed in the development
Medicinal Chemistry & Pharmacology, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007, India; Email: ahmedkamal@iict.res.in
Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500
3
0,37,38
of tyrosine kinase inhibitors (CDK).
Compound SCH727965
c.
39
(
5) with pyrazolo[1,5-a]pyrimidine scaffold in it, was found to be a
J. Name., 2013, 00, 1-3 | 1
007, India; Email: manika@iict.res.in
These authors contributed equally to this work
†
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selective CDK inhibitor and is undergoing clinical trials. Also, our carbaldehydes to afford the corresponding chalcone-linked
previous efforts towards the synthesis of pyrazolo[1,5-a]pyrimidine pyrazolo[1,5-a]pyrimidines (6a-q)
.
40
37
scaffold coupled with anthranilamide, aminobenzothiazole led
to several compounds with promising anticancer activity, thus
substantiating the potential of pyrazolo[1,5-a]pyrimidine scaffold
further.
The amalgamation of two pharmacophores into a single chemical
entity is an effective and commonly used strategy for finding novel
potential molecules, the resulting molecules being known as
41,42
hybrids or chimeric molecules.
The selection of two
pharmacophores is based on their anticipated additive or
synergistic effect. Hybrids are designed to synergise biological
activity, minimizing resistance, effecting multiple targets and/or
4
3-45
reducing known side effects.
Thus, owing to the importance of
chalcone, pyrazolo[1,5-a]pyrimidine pharmacophores and our
interest in the synthesis of biologically active molecules, herein we
have combined both pharmacophores into a single chemical entity
by anticipating their better pharmacological activity. The resulting
series of chalcone-linked pyrazolo[1,5-a]pyrimidines were
evaluated for their antiproliferative activity.
Scheme 1 Synthesis of pyrazolo[1,5-a]pyrimidines (6a-q); Reagents and conditions: a)
a) diethyl oxalate, NaOEt, EtOH, rt, 12 h, (yield 80-85%); b) 3-amino-5-phenyl-pyrazole,
2 2
conc. HCl (cat.), EtOH, reflux, 2-4 h, (yield 90-95%); c) DIBAL-H, CH Cl , -78 °C, 2 h, (yield
7
8-80%); d) Ba(OH) , MeOH, rt, 4-6 h, (yield 71-91%).
2
Synthesis of 7a-k hybrids
a-k were synthesized as shown in Scheme 2. Substituted
7
pyrazolo[1,5-a]pyrimidine-5-carbaldehydes 11a, b subjected with
Weinreb amide in presence of trimethylaluminum gives
corresponding amides (13a, b) which were further subjected to
Grignard reaction with methylmagnesium bromide to give the
corresponding 1-(pyrazolo[1,5-a]pyrimidin-5-yl)ethan-1-ones (14a,
b). Different aldehydes (15a-f) were condensed with 1-
(
pyrazolo[1,5-a]pyrimidin-5-yl)ethan-1-one to give chalcone-linked
pyrazolo[1,5-a]pyrimidines (7a-k).
Fig. 1 Chemical structures of EGFR inhibitors: 1 (erlotinib), chalcone scaffold containing
compounds 2 (MDL-27048), 3 (butein), 4 (flavokavain B), pyrazolo[1,5-a]pyrimidine
containing scaffold compound
5 (SCH-727965) and designed chalcone-linked
pyrazolo[1,5-a]pyrimidines (6a-q, 7a-k).
Results and discussion
Chemistry
Synthesis of 6a-q hybrids
6a-q were synthesized as shown in Scheme 1. Different
acetophenones 8a-c were oxalylated by diethyl oxalate in
presence of sodium ethoxide in ethanol to give 9a-c. These
were condensed with 3-amino-5-phenyl-1H-pyrazole in
Scheme 2 Synthesis of pyrazolo[1,5-a]pyrimidines (7a-k); Reagents and conditions: a)
3 3
N,O-dimethylhydroxylamine hydrochloride, Al(CH ) , dry dichloromethane, 0-25 °C, 2 h,
presence of a catalytic amount of conc. HCl in ethanol to yield (yield 77-79%); b) methylmagnesium bromide, dry tetrahydrofuran, 0 °C, 1 h, (yield 85-
The 92%); c) barium hydroxide, methanol, rt, 6 h, (yield 79-92%).
ethyl pyrazolo[1,5-a]pyrimidine-5-carboxylates 10a-c
.
ester functionality of ethyl pyrazolo[1,5-a]pyrimidine-5-
carboxylate was then reduced with DIBAL-H in
dichloromethane to give the corresponding pyrazolo[1,5-
a]pyrimidine-5-carbaldehydes 11a-c. Different acetophenones
12a-g were condensed with pyrazolo[1,5-a]pyrimidine-5-
2
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ARTICLE
a
1 Cytotoxicity (IC50 values in µM) of chalcone-linked pyrazolo[1,5-
Table
Anti-proliferative activity MTT assay was performed to a]pyrimidines 6a-q and 7a-k on selected cell lines
evaluate the cytotoxic effect of newly synthesized chalcone-
linked pyrazolo[1,5-a]pyrimidine hybrids against the selected
cell lines viz A549 (lung cancer), MDA-MB-231 (breast cancer)
and DU-145 (prostate cancer) using erlotinib as a reference
standard, the results obtained are summarized in Table 1. All
the hybrids in this series possess moderate to good
antiproliferative potential against selected cell lines. Hybrid
b
c
d
e
A549
MDA-MB-231
DU-145
HEK293
6
6
6
6
6e
6f
a
b
c
8.6 ± 0.4
2.9 ± 0.3
7.4 ± 0.2
10.7 ± 0.3
17.2 ± 0.4
9.3 ± 0.6
11.9 ± 0.3
3.9 ± 0.4
9.9 ± 0.2
6.3 ± 0.3
8.7 ± 0.4
11.8 ± 0.4
19.8 ± 0.3
11.5 ± 0.2
13.9 ± 0.5
2.6 ± 0.6
13.7 ± 0.3
8.5 ± 0.4
16.4 ± 0.4
10 ± 0.3
26.8 ± 0.4
12.1 ± 0.6
14.6 ± 0.5
7.2 ± 0.4
53.2 ± 1.9
36.1 ± 0.9
48.5 ± 0.5
51.2 ± 0.6
62.6 ± 0.4
46.2 ± 0.7
40.0 ± 0.3
32.5 ± 0.7
35 ± 1.2
d
6h with 3,4-dimethoxy substitutions on C7 phenyl ring and
phenylprop-2-en-1-one on C5 of pyrazolo[1,5-a]pyrimidine
6g
6
6
6
6
6
6
6
6
6
6
7
7
7
7d
7e
7f
7
7
7
7
7
h
i
scaffold was found to be the most active against MDA-MB-
7.2 ± 0.4
4.7 ± 0.3
8.3 ± 0.3
2
31 (IC50 2.6 μM) cell line. The same hybrid showed effective
potential against A549 and DU-145 cell lines with IC50 values
.9 μM and 7.2 μM respectively. In a SAR study, it was very
j
15.4 ± 0.5
18.2 ± 0.4
19.4 ± 0.3
10.6 ± 0.4
14.6 ± 0.5
17.8 ± 0.3
20.5 ± 0.6
15.5 ± 0.4
18.62±1.04
22.19±2.86
19.27±1.91
15.44±2.11
22.01±2.15
17.97±0.57
20.03±2.71
21.23±1.91
20.47±1.87
25.86±2.8
23.83±2.4
17.1 ± 0.4
15.8 ± 0.4
23.9 ± 0.6
13.5 ± 0.6
13.2 ± 0.4
18.3 ± 0.4
24.3 ± 0.5
32.5 ± 0.4
23.32±0.8
22.59±1.87
21.98±0.92
16.73±0.67
23.74±0.91
19.15±1.11
18.64±1.12
16.5±1.46
14.19±2.16
18.37±1.8
21.62±.92
14.74±2.5
21.3 ± 0.4
34.3 ± 0.5
27.1 ± 0.3
15.4 ± 0.6
25.2 ± 0.6
29.9 ± 0.4
34.9 ± 0.4
29.3 ± 0.2
15.57±1.65
19.49±1.31
14.93±0.78
14.74±1.41
22.4±1.87
16.31±1.41
21.89±1.03
16.91±0.53
15.33±0.9
17.91±1.2
19.51±2.4
18.4±1.4
44.1 ± 0.8
≥ 100
k
l
3
50.4 ± 1.9
72.5 ± 1.2
79.8 ± 0.7
81.7 ± 1.6
≥ 100
interesting to evaluate the effect of substitutions on C5
phenylprop-2-en-1-one and C7 phenyl ring. It was observed
that the series 6a-q having superior antiproliferative activity
than the series 7a-k except for DU-145 cell line. In both series,
the only difference is in the orientation of the enone bond. In
the case of 6a-q series, the double bond is near to the
pyrazolo[1,5-a]pyrimidine scaffold whereas in the case of 7a-
m
n
o
p
q
a
b
c
≥ 100
64.1 ± 1.4
57.4 ± 0.9
50.7 ± 1.2
46.8 ± 1
39.1 ± 0.7
44.8 ± 1.3
56.1 ± 0.8
46.6 ± 1
41.0 ± 1.9
41.0 ± 0.3
53.9 ± 1.6
30.3 ± 2.1
k
series the carbonyl group is near to pyrazolo[1,5-
a]pyrimidine scaffold. The probable reason for this activity
difference might be due to the extra hydrogen bonding
formed by the carbonyl group of the hybrids of 6a-q series
with Cys773 amino acid of EGFR, similar hydrogen bond was
not possible for the hybrids in 7a-k series (explained with
docking poses in molecular modeling section). Further
structure-activity relationship is discussed for 6a-q series.
Hybrids with 4-methoxy and 3,4-dimethoxy substitutions on
C7 phenyl ring displayed superior antiproliferative potential
with IC50 ranging from 2.6 to 19.8 μM against MDA-MB-231
cell line. Hybrids with 3,4,5-trimethoxy substitution on C7
phenyl ring showed lower antiproliferative potential with IC50
ranging from 13.2-32.5 μM against MDA-MB-231 cell line. The
activity order for C7 phenyl ring substitutions were 3,4-
dimethoxy ≥ 4-methoxy > 3,4,5-trimethoxy. Furthermore, the
hybrids with electron withdrawing substitution on C5
phenylprop-2-en-1-one showed lower cytotoxic potential than
the hybrids with electron donating substituent. Hybrids with
meta-, para- disubstitution has more cytotoxic potential than
meta-, para-, meta- trisubstitution followed by hybrids with no
substitution. However hybrids with only para- substitution
exhibit least cytotoxic potential except for 6b. The activity
order for C5 phenylprop-2-en-1-one substitutions was 3,4-
dimethoxy > 3,4,5-trimethoxy > 3,4-dimethyl > no substitution
g
h
i
j
k
Erlo 10.39±1.6
a
Note: Cell lines were treated with different concentrations of compounds.
Cell viability was measured by employing the MTT assay. The concentration
required for 50% inhibition of cell growth was calculated and the values
represent means ± S.D. of three different experiments performed in
triplicates, lung cancer, breast cancer, prostate cancer, and Human
embryonic kidney cells.
b
c
d
e
Effect on Cell cycle distribution Anticancer molecules exert
their action either by arresting the cell cycle at a checkpoint or
by induction of apoptosis or a combined effect of both cycle
block and apoptosis. Furthermore, regulation of the cell cycle
and apoptosis are considered to be effective strategies in the
development of cancer therapeutics. To shed light on the
effects of active hybrids 6b, 6h, and 6i on cell cycle
progression, cell cycle analysis was performed on A549 cells.
These cells were treated with 2 μM concentration of the test
compounds for 24 h. Cell cycle analysis performed with PI
staining showed accumulation of cells at G2/M phase (Fig. 2).
Untreated cells showed 20.4% cells in G2/M phase. In contrast,
>
4-methoxy > 4-chloro > 4-methyl. Thus it could be concluded
that 3,4-dimethoxy and 4-methoxy substitutions on C7 phenyl
ring were optimum for antiproliferative potential and 3,4-di
substitution on C5 phenylprop-2-en-1-one was enhancing the
activity. In contrast, 3,4,5-trimethoxy substitution on C7
phenyl ring was detrimental for activity. Based on the results
obtained with the MTT assay, four of the most potent hybrids
compounds 6b, 6h, and 6i-treated cells showed accumulation
of 29.6%, 29.5%, and 33.11% respectively. Wherein erlotinib
was used aspositive control, it showed 24.5% in G2/M
population. Thus, compounds of this investigation showed a
significant effect in restricting A549 cells to G2/M phase, which
in itself is a strong evidence of apoptotic cell death. Also, the
effect of the compounds was validated in a non-cancerous HEK
(
6b, 6h, and 6i) were considered for further mechanistic
studies.
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cell line, which revealed the distribution of treated cells similar Effect on the co-localization of EGFR and STAT3 To further
to the untreated cells (Supplementary Fig 1).
clarify the deregulation of the EGRF/STAT axis in the treated
A549 cells, the cellular protein levels and the localization of p-
EGFR
and
STAT3
proteins
were
identified
by
immunofluorescence method. Cells after 24h treatment were
fixed, stained with EGFR/STAT3 tagged with Cy3-conjugated
secondary antibody fluorophore were analyzed with aid of
laser scanning confocal microscopy. Hybrids 6b, 6h and 6i
treated cells showed a significant downregulated of EGFR as
well as STAT3, in comparison to the positive control (erlotinib)
treated cells (Fig. 4). This clearly shows the reduced co-
localization of EGFR and STAT3 upon treatment with chalcone-
Fig. 2. Flow cytometric analysis displaying G2/M arrest in cells post-treatment. A549
cells subjected to compound treatment for 24h at 2 μM concentration were collected,
fixed and stained for FACS analysis. Untreated cells were employed as negative control.
Effect on EGFR/STAT3 pathway To investigate the pathway
responsible for cell death and arrest, A549 cells were treated
with hybrids 6b, 6h, and 6i at 2 μM concentration for 24 h,
followed by total protein lysate extraction. Protein lysate was
subjected to western blot analysis. Western blot analysis
revealed elevated levels of EGFR and P-EGFR upon treatment
with hybrids in comparison to erlotinib-treated cells (Fig. 3).
STAT3 is a well-known transcription factor responsible for cell
survival and proliferation, and is commonly over expressed in
most cancer types. Interestingly, significant down regulation of
STAT3 and P-STAT3 upon treatment was observed. Further,
AKT, a key component of cellular survival pathways was linked pyrazolo[1,5-a]pyrimidine hybrids.
observed to be down-regulated. Both, 6h and 6i have shown
Fig. 4 Regulation of EGFR and STAT3 in cells 24 h post-treatment. A549 cells were fixed,
the significant downregulation compared to the untreated
cells. Among the compounds studied, 6b 6h and 6i
stained and observed for 24 h post treatment with compounds 6b, 6h and 6i at 2 μM
,
final concentration under confocal microscope (FLOW VIEW FV 1000 series). Cy3 dye
demonstrated a significant effect on the modulation of the was used to stain both EGFR and STAT3, whereas DAPI as nuclear stain. Compounds 6h
EGFR/STAT3 axis and AKT pathway (Fig. 3). They also show a and 6i showed profound inhibitory effect on levels of EGFR and STAT3.
high degree of correlation with the erlotinib-treated cells. Also,
a non-cancerous HEK cell line was employed to study the Effect on pro-apoptotic and anti-apoptotic proteins To
deleterious effects on normal cells and much to our interest, understand the role of chalcone-linked pyrazolo[1,5-
there was no significant alteration in the expression of a]pyrimidine hybrids in regulating apoptosis, A549 cells were
EGFR/STAT3 pathway (Supplementary Fig 2A).
treated and extracted protein lysate was subjected to analysis
at both transcriptional and translation level of key apoptotic
proteins like p53, p21, BAX, caspase-3 and Bcl-2.The test
compounds significantly increased the levels of p53, p21 and
BAX and decreased the expression of Bcl2 (Fig. 5a, 5b). The
increase in the levels of many of these proapoptotic proteins
also showed a concomitant decrease in the levels of
antiapoptotic protein Bcl-2 upon compound treatment. This
regulation of apoptosis at both transcriptional and
translational level shows the prominent effect of chalcone-
linked pyrazolo[1,5-a]pyrimidine hybrids in inducing apoptosis.
To understand the effect on normal cells, non-cancerous HEK
cell line was studied with the same compounds and there was
no significant alteration in the expression pattern in
Fig. 3 Identification of signaling pathway effected through Western blot analysis. A549
cells were treated with 6b, 6h and 6i at 2 μM final concentration and collected 24 h
post-treatment. Total protein was isolated and analyzed by Western blotting using
corresponding antibody. Compounds 6b and 6h revealed significantly reduced levels of comparison to the untreated control cells (Supplementary Fig
p-EGFR, p-STAT3 and AKT. Erlotinib was utilized as positive control, whereas untreated
2B).
cells were considered as negative control.
4
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bonds with Met769. On the other hand, 4-methoxy substituted
hybrids were forming only one hydrogen bond (Supporting
information Fig 3a). The superimposed pose of 3,4-dimethoxy
and 4-methoxy explain the higher activity of 3,4-dimethoxy
substituted hybrids but it does not explain the least activity of
3,4,5-trimethoxy substituted hybrids. However, most of the
drugs acting on EGFR having dialkoxy substitution supports the
dialkoxy substitution is optimum for the activity (structures of
drugs having dialkoxy substitution are given in supplementary
information Fig 4). In the series, least active hybrid is 6p with
3,4,5-trimethoxy substitution on both C7 phenyl ring and C5
phenylprop-2-en-1-one. The docking pose shows that carbonyl
group of 6p was not forming the hydrogen bond with the
Cys773 which supports the least activity of 6p (Supporting
information Fig 3b). Docking studies showed that these hybrids
bind well in the ATP binding site of EGFR with the required
hydrophobic interactions in the specificity pocket and
Fig. 5 Analysis of pro-apoptotic and anti-apoptotic proteins post 24h treatment. (a)
Total lysate was isolated from A549 cells after 24h treatment with compounds 6b, 6h
and 6i at
2 μM final concentration and analyzed by Western blotting using
corresponding antibody. Compounds 6b and 6i show clear up-regulation of apoptotic
proteins including Bax, p53, p21and caspase-3, whereas down-regulation of anti- hydrogen bonding with Met769 amino acid in the hinge region
apoptotic protein Bcl2 was observed. Erlotinib was employed as positive control and
and explained the SAR of the series.
untreated as negative control. (b) RNA was isolated from cells using Trizol reagent and
cDNA was synthesized. End point RT-PCR results showing p53 and Bax upregulation in
cells treated with compounds 6b and 6h and 6i. Significant reduction in levels of anti-
apoptotic protein, Bcl2.
Molecular Docking studies
Molecular docking studies were performed to get the insight
of interactions between chalcone-linked pyrazolo[1,5-
a]pyrimidine hybrids and EGFR. Molecular docking studies
were carried out for all hybrids (6a-q, and 7a-k). Docking pose
of 6j displayed that these hybrids bind well at the ATP binding
site of EGFR (Fig. 6B). The C2 phenyl ring was buried in
specificity pocket where it was surrounded by Val702, Ala719,
Ile720, Lys721, Met742, Leu764, Ile765 and Thr766 amino
acids. The superimposed pose of 6j with cocrystal ligand
Fig.
6 Docking poses on EGFR A) 2D structure of chalcone linked-pyrazolo[1,5-
erlotinib showed that C2 phenyl ring of 6j was overlapping on
the acetylphenyl group of erlotinib (Fig. 6C). The C7 phenyl ring
is oriented towards the hinge region and surrounded by
a]pyrimidine with observed interactions with EGFR B) Binding pose of 6j (yellow color,
EGFR in green color and hydrogen bonds in red dotted lines) C) Superimposed pose of
6
j and erlotinib D) Superimposed pose of 6j and 7a showing the difference in hydrogen
Leu694, Val702, Ala719, Gln767, Leu768, Met769, Pro770, bonding between carbonyl carbon and Cys773.
Gly772, and Leu820 amino acids. 3-Methoxy and 4-methoxy
groups were forming hydrogen bonds with –NH of Met769.
Conclusion
This hydrogen bond is crucial for EGFR inhibitors and is present
A series of chalcone-linked pyrazolo[1,5-a]pyrimidines (6a-q
in all the drug molecules and ATP as well. The C5 phenylprop-
-en-1-one was oriented towards the ‘A loop’ and forms a
and 7a-k) was synthesized and evaluated for its cytotoxic
activity. Most of the hybrids exhibited prominant anti-
proliferative potential with IC50 ranging from 2.6 μM to 34.9
2
close contact with Cys773, Arg817 and Asn818 amino acids
and the carbonyl carbon was forming a hydrogen bond with
Cys773. In MTT assay it was observed that 6a-q series has
superior activity than the 7a-k series. In the superimposed
pose for the hybrids of 6a-q series, hydrogen bonding was
observed between the carbonyl carbon and Cys773 amino
acid, whereas, for hybrids in 7a-k series such hydrogen
bonding was not present (Fig. 6D). This is due to the difference
in the geometrical orientation of the enone group in 6a-q and
μM. In the series, 6b, 6h and 6i showed promising cytotoxicity
and were advanced for further mechanistic studies. Cell cycle
analysis displayed a significant accumulation of cells at G2/M
phase of cell cycle. Western blot analysis and immunostaining
assay suggested the downregulation of EGFR, p-EGFR, STAT3,
and p-STAT3 with a stronger downregulation of EGFR/ STAT3
axis. Western blot analysis and RT-PCR also showed the up-
regulation of apoptotic proteins like p53, p21 and BAX
whereas downregulation of antiapoptotic proteins such as Bcl-
7
a-k and may be a reason for the superior activity of
compounds 6a-q over 7a-k. The activity order for C7 phenyl
substitutions was 3,4-dimethoxy 4-methoxy 3,4,5-
2
and caspase-3 at both the transcriptional and translation
>
>
level. Our study establishes the role of chalcone-linked
trimethoxy. From the docking studies it was observed that 3,4-
dimethoxy substituted hybrids were forming two hydrogen
pyrazolo[1,5-a]pyrimidine hybrids in lung carcinoma cell line,
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Journal Name
A459 in inducing cellular arrest and activation of apoptosis. 16. S. Temam, H. Kawaguchi, A. K. El-Naggar, J. Jelinek, H. Tang, D.
Molecular docking studies provided insight into the binding
mode of these molecules at the APT binding site of EGFR and
D. Liu, W. Lang, J.-P. Issa, J. J. Lee and L. Mao, J. Clin. Oncol.,
2007, 25, 2164-2170.
shed light on the superior activity of compounds 6a-q in 17. E. Tanaka, Y. Hashimoto, T. Ito, T. Okumura, T. Kan, G.
comparison to those of 7a-k series. These studies suggest the
potential of synthesized hybrid molecules for development
into promising anticancer agents.
Watanabe, M. Imamura, J. Inazawa and Y. Shimada, Clin. Cancer
Res., 2005, 11, 1827-1834.
18. M. Kurai, T. Shiozawa, H.-C. Shih, T. Miyamoto, Y.-Z. Feng, H.
Kashima, A. Suzuki and I. Konishi, Hum. Pathol., 2005, 36, 1281-
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Acknowledgements
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2
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9. A. Jemal, R. Siegel, E. Ward, T. Murray, J. Xu and M. J. Thun, CA
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The author C.B acknowledges the Department of
Pharmaceuticals (Ministry of Chemicals and Fertilizers, Govt. of
India) for the financial support & the CSIR-IICT, Hyderabad for
providing facilities. K.R.G thanks UGC for his fellowship. This
work was financially supported by the CSIR 12th FYP CSC0111
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Conflict of Interest
2
The authors declare no competing interests.
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Synthesis and biological evaluation of chalcone-linked
pyrazolo[1,5-a]pyrimidines as anticancer agents
Chandrakant Bagul, Garikapati Koteswara Rao, Venkata Krishna Kanth Makani, Jaki R. Tamboli,
Manika Pal-Bhadra and Ahmed Kamal