Design, synthesis and biological evaluation of novel pyrazolochalcones as potential modulators of PI3K/Akt/mTOR pathway and inducers of apoptosis in breast cancer cells

Article abstract of DOI:10.1016/j.ejmech.2017.07.056

Cancer has been established as the “Emperor of all maladies”. In recent years, medicinal chemistry has focused on identifying novel anti-cancer compounds; though discovery of these compounds appears to be a herculean task. In present study, we synthesized

Full text of DOI:10.1016/j.ejmech.2017.07.056

Accepted Manuscript
Design, synthesis and biological evaluation of novel pyrazolochalcones as potential
modulators of PI3K/Akt/mTOR pathway and inducers of apoptosis in breast cancer
cells
Anver Basha Shaik, Garikapati Koteswara Rao, G. Bharath Kumar, Nibeditha Patel,
Vangala Santhosh Reddy, Irfan Khan, Sunitha Rani Routhu, C. Ganesh Kumar,
Immadi Veena, Kunta Chandra Shekar, Madan Barkume, Shailesh Jadhav, Aarti
Juvekar, Jyoti Kode, Manika Pal-Bhadra, Ahmed Kamal
PII:
S0223-5234(17)30578-0
10.1016/j.ejmech.2017.07.056
EJMECH 9619
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 March 2017
Revised Date: 25 June 2017
Accepted Date: 23 July 2017
Please cite this article as: A.B. Shaik, G.K. Rao, G.B. Kumar, N. Patel, V.S. Reddy, I. Khan, S.R.
Routhu, C.G. Kumar, I. Veena, K. Chandra Shekar, M. Barkume, S. Jadhav, A. Juvekar, J. Kode, M.
Pal-Bhadra, A. Kamal, Design, synthesis and biological evaluation of novel pyrazolochalcones as
potential modulators of PI3K/Akt/mTOR pathway and inducers of apoptosis in breast cancer cells,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.056.
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ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Novel Pyrazolochalcones as
Potential Modulators of PI3K/Akt/mTOR Pathway and Inducers of Apoptosis
in Breast Cancer Cells
1
2
1
2
Anver Basha Shaik, Garikapati Koteswara Rao, G. Bharath Kumar, Nibeditha Patel, Vangala
1
1
1
1
2
Santhosh Reddy, Irfan Khan, Sunitha Rani Routhu, C. Ganesh Kumar, Immadi Veena, Kunta
Chandra Shekar, Madan Barkume, Shailesh Jadhav, Aarti Juvekar, Jyoti Kode,* Manika Pal-
Bhadra,* Ahmed Kamal*
1
3
3
3
3
2
1
1
2
Medicinal Chemistry and Pharmacology, Centre for Chemical Biology, CSIR - Indian Institute of
3
Chemical, Technology, Hyderabad, 500007, Anti-Cancer Drug Screening Facility (ACDSF), Advanced
Centre for Treatment, Research & Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi
Mumbai 410210, India.
Abstract: Cancer has been established as the “Emperor of all maladies”. In recent years,
medicinal chemistry has focused on identifying novel anti-cancer compounds; though discovery
of these compounds appears to be a herculean task. In present study, we synthesized forty
pyrazolochalcone conjugates and explored their cytotoxic activity against a panel of sixty cancer
cell lines. Fifteen conjugates of the series showed excellent growth inhibition (13b-e, 13h-j, 14c-
d, 15 a, 15 c-d, 16b, 16d and 18f; GI for MCF-7: 0.4 – 20 µM). Conjugates 13b, 13c, 13d, 16b
5
0
and 14d were also evaluated for their cytotoxic activity in human breast cancer cell line (MCF-
). The promising candidates induced cell cycle arrest, mitochondrial membrane depolarization
7
and apoptosis in MCF-7 cells at a 2 µM concentration. Furthermore, inhibition of
PI3K/Akt/mTOR pathway-regulators such as PI3K, p-PI3K, p-AKT, and mTOR were observed;
as well as upregulation of p-GSK3β and tumor-suppressor protein, PTEN. Our study indicates
that pyrazolochalcone conjugates could serve as potential leads in the development of tailored
cancer therapeutics.
Key Words: Apoptosis, cytotoxicity, PI3K/Akt/mTOR signaling pathway, molecular modeling,
Pyrazolochalcones.
1

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1. Introduction
There has been considerable development in the synthesis of antiproliferative drugs that inhibit
the function of various proteins. Phosphatidyl-inositol-3-kinases (PI3Ks) are a family of lipid
kinases characterized by their ability to phosphorylate the 3′-OH group in the ring of inositol
[
1]
phospholipids. PI3Ks are involved in a number of cellular processes including cell growth,
proliferation, nutrient uptake, differentiation, motility, survival, and intracellular trafficking. The
well characterized members of this family are class I PI3Ks (α, β, χ and δ isoforms) that link
PI3K activity to a large variety of cell-surface receptors, including growth factor receptors and G
protein-coupled receptors (GPCRs). Class I PI3Ks are heterodimers composed of a catalytic
subunit (p110) and an adaptor/regulatory subunit (p85). There is significant evidence that the
PI3K/Akt/mTOR pathway is crucial to the survival of many human cancer cells and regulates
[
2-3]
cell metabolism as well as proliferation.
Aberrant activation of this pathway is considered
important in the initiation and maintenance of human tumors. This signaling cascade includes a
[
4]
number of proteins and therefore provides numerous targets for inhibition.
PF-04691502 (1) bearing pyridopyrimidinone acts as a dual inhibitor of PI3K/mTOR
[
5]
kinase resulting in induced apoptosis and anti-proliferation of cancer cells. Wortmannin (2) and
LY294002 (3a) (Figure 1) are potent nonselective inhibitors of PI3Ks which also demonstrate
[
6-8]
inhibitory effects on targets not related to PI3K family.
Quercetin (3b) and tangeretin (3c)
(
Figure 1) flavonoids reported to exhibit anti-carcinogenic and chemopreventive activities by
[
9-11]
inducing apoptosis apart from impeding the function of critical growth factors.
Moreover,
adriamycin (4) (Figure 1) a well-known chemotherapeutic drug, inhibits cell growth by
[
12]
interacting with DNA to induce apoptosis.
Pyrazoles are well recognized conjugates of biological importance and are considered
[
13]
versatile building blocks in the fields of organic and medicinal chemistry. Recently, some
scaffolds like pyrazole restricted CA-4 (5a) dihydropyridopyrazoles (5b) and flavokawain B
(
FKB) (6) have shown potential antiproliferative action, as well as apoptosis inducing properties
[
14-15]
apart from antitubulin activity.
Notably, pyrazole derivatives received attention as
chemotherapeutic agents due to their remarkable effect on cellular targets like p38α MAP kinase
[
16]
[17]
[18]
(5c)
p53−MDM2 and NF-κB, topoisomerase I, STAT3 phosphorylation, sirtuin (SIRT1
[
19]
[20]
and SIRT2)
and PI3K.
Additionally, pyrazole-based pyrrolidinyl pyridopyrimidinone
[
21]
derivatives have been reported as dual inhibitors of PI3Kα and mTOR. It is noteworthy to
2

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mention that the pyrazole containing NSC-45410 is currently considered for preclinical
[
22-25]
reassessment as a cytotoxic agent.
Chalcones are also important bioactive molecules known
[
26]
[27]
to exhibit cytotoxicity by modulating cellular targets like EGFR,
ODC,
NF-κB,
[
28]
[29]
proteasome, and Nrf2/ARE. SD400 (7) inhibited the growth of P388 murine leukemia cell
[
30-33]
line at a low concentration (IC , 2.6 nM).
Despite the numerous efforts in developing novel
5
0
antitumor agents that modulate apoptotic markers, many drugs currently under clinical
investigation are still limited by critical restrictions. Specifically, occurrence of peripheral
neuropathy and acute toxicity are major limitations in the development of PI3K/Akt/mTOR
[
34-37]
modulators and apoptosis inducers as cancer drugs.
Therefore, novel chemotherapeutic
design must still overcome the challenges posed by multi-drug resistance, neuropathies and acute
[
38-39]
toxicity.
Structural combinations of two pharmacophores significantly impart antiproliferative activity
[
40]
to the designed molecules. In light of these facts and to achieve some innovative molecules
with remarkable cytotoxicity, this study illustrates the synthesis of novel pyrazolochalcone
hybrids as chemotherapeutic agents that profoundly induce apoptosis. Thus, a set of conjugates
3 2
with different groups like OCH , OCH O, Cl, and F as phenyl ring substituents were generated
and their structure-activity relationships (SAR) evaluated. It is noteworthy to mention that this
forms the first report wherein pyrazolochalcone derivatives have been systematically synthesized
and were examined for their cytotoxic potency against a panel of 60 cancer cell lines (NCI
[
41]
developmental therapeutic program), (International Patent: WO2015029051 A1).
Significantly, conjugates 13b, 13c, 13d, 14d and 16b exhibited promising activity which
prompted us to assess their prospective role in controlling cell proliferation and apoptosis in
human breast cancer cells (MCF-7). We considered evaluating their mode of action on cell cycle
progression as well as cell survival. Fascinatingly, cell signaling pathway demonstrated that they
are potential modulators of PI3K/AKT/mTOR targets leading to induced apoptosis. To validate
the mechanism, we evaluated the effect on mitochondrial membrane potential as a measure of
early induction of intrinsic apoptosis. PI3K, docking simulations depicted that they occupy ATP
binding site of PI3Kγ. To gain more insight into these findings, the promising conjugates were
studied for their acute toxicity (data not shown). These findings led to a new paradigm for the
pharmacological use of pyrazolochalcones in the treatment of human breast cancer.
<Insert figure 1 here>
3

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2
. Results and discussion
2
.1. Chemistry: Synthesis of pyrazolochalcones is outlined in Schemes 1, 2 and 3, wherein the
final step has been carried out by the application of Claisen–Schmidt condensation between
equimolar mixtures of 3-phenyl-1H-pyrazole-5-carbaldehydes (12a-d) and substituted
acetophenones/indanones in the presence of base in ethanol. The key intermediates 3-substituted
phenyl-1H-pyrazole-5-carbaldehydes (12a-d) were individualy prepared in four sequential steps.
Initially, the substituted acetophenones (8a-d) were oxylated in precence of sodium ethanolate in
ethanol yielded ethyl 2,4-dioxo-4-(substituted phenyl)butanoates (9a-d). These diketo esters
were further cyclised with NH
phenyl-1H-pyrazole-5-carboxylates (10a-d) and these carboxylates were reduced to
corresponding pyrazole alcohols 11a-d by LiAlH . These were selectively oxidized to 3-
substituted phenyl-1H-pyrazole-5-carbaldehydes (12a-d) by IBX in DMSO.
2 2
-NH .2HCl in refluxing ethanol to result ethyl 3-substituted
4
4
2-43
The final target
1
13
conjugates obtained were characterized by IR, H NMR, C NMR, mass and HRMS spectral
data, wherein the α,β-unsaturated carbonyl protons (trans H) were confirmed by coupling
constant (J = 14-18 Hz). All the synthesized compounds have >95% purity as determined by
high-performance liquid chromatography (HPLC).
<Insert scheme 1-3>
3
. Biological results
3
.1.1. Cytotoxicity
The newly synthesized conjugates of pyrazolochalcones bearing various substituents on
the A and C-rings were initially evaluated for their cytotoxicity in a panel of sixty human cancer
cell lines (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and
breast cancer) as per NCI protocol. Our data suggest that the A-ring decorated with electron
3 3 3 2 3 2
donating substituents (OCH ) , (OCH ) , (OCH ), and 3,4(-OCH O-) whereas the C-ring
contains both electron donating as well as withdrawing groups (OCH , 3,4(-OCH O-), NH , H,
3
2
2
Cl, Cl and F ) (Figure 1). Among them, 27 conjugates were taken up for single dose (10 µM)
2
2
screening and the conjugates 13 (b-e, h-j), 14(c-d), 15 (a, c-d), 16 (b, d) and 18f showed
significant activity in the preliminary screening. When subjected to five dose screening (0.01,
0
.1, 1.0, 10, 100 µM), conjugates like 13b-d, 14c-d, 15a, 15c-d, 16b and 16d showed broad
cytotoxic activity against cancer cell lines with GI50 values of 0.18-5.5 µM. Interestingly, SR and
RPMI 8226 cells of leukemia type were particularly affected by these conjugates with GI value
5
0
4

ACCEPTED MANUSCRIPT
<
1 µM. Moreover, 13b, 13c,13d, 13e and 13j with a trimethoxy phenyl pharmacophore as A-
2 2
ring and electropositive substituents (OCH3, OCH O and NH ) on the C-ring elicit pronounced
cytotoxicity with the GI values of 0.34 µM (SR), 0.18 µM (NCI522), 0.31 µM (RPMI 8226),
5
0
0
.55 µM (SR) and 0.32 µM (SR), respectively. In comparison, conjugates 13h and 13i
functionalized with same unit on the A-ring and electronegative (Cl and F ) substitutions on the
2
2
C-ring exhibited less potency (GI = 0.39 µM and 2.92 µM in SR cells). In addition, conjugates
5
0
1
5a, 15c and 15d decorated with dimethoxy units on A-ring and similar electropositive groups
on their C-ring demonstrated potential activity in SR cells with the GI values of 0.20 µM, 0.25
5
0
µM and 0.58 µM, respectively. However, 3,4-(methylenedioxy) (14c-d) and monomethoxy (16b,
6d) substitution on the A-ring and a common electropositive group on the D-ring also
1
established significant cytotoxicity in sub-micromolar levels; however, in lesser magnitude when
compared to 13b-d.
<Insert Table 1 here>
A total 40 compounds were also evaluated for their antiproliferative activity against
human breast cancer cell lines, MCF-7 and MDA-MB-231 (Table 2) employing adriamycin and
CA-4 as standards. Most of them exhibited potential cytotoxicity in both the breast cancer cells
with IC values of <1 µM. Conjugates 13b, 13c and 13d preferentially inhibited the growth of
5
0
MCF-7 cells with an IC of 0.81 µM, 0.51 µM and 0.43 µM, respectively, indicating that
5
0
potency had been increased with an increase in the number of methoxy units on the C-ring.
Similarly, conjugates 13e, 14c-d, 15a-b and 16b possessing 3,4-(methylenedioxy), di- and tri-
methoxy substitutions on their C-ring also demonstrated pronounced growth inhibition. Whereas,
conjugates 13h (IC = 3.2 µM) and 13i (IC = 2.5 µM) containing electronegative chloro and
5
0
50
fluoro groups on the C-ring, suffered from a reduction in observed potency. Therefore, the
optimal activity order of substitutions on the C-ring is (OCH ) > (OCH ) = 3,4-(OCH O-) >
3
3
3 2
2
OCH > NH > H > F > Cl > 3-F, 4-OCH > Cl (Figure 1). Additionally, conjugates 17, 18 (a-f)
3
2
2
2
3
with cyclic acetones have shown moderate to good cytotoxicity. Interestingly, 17a-f anchorage
with trimethoxy substitutions on A-ring displayed enhanced potency when compared to 18a-f
that contain 3,4-(methylenedioxy) group on the A-ring. Wherein, the presence of methoxy
substitutions on the C-ring 17e-17f (2.1 µM, and 2.0 µM), 18e-18f (2.7 µM and 1.7 µM) showed
considerable IC values in MCF-7 cells. Overall, the conjugates 13b-d, 14c-d, 15a, 15c-d, 16b
5
0
5

ACCEPTED MANUSCRIPT
and 16d that demonstrated potential cytotoxicity in the NCI 60 cell line study also showed
similar effects in MTT assay.
<Insert Table 2 here>
3
.1.2. Effect on intrinsic apoptotic pathway
To examine the potential role of these conjugates in triggering early apoptosis, MCF-7
cells were treated with 13c, 13d, 16b and 14d for 18 h at 2 µM and then stained with DiOC6.
Intrinsic apoptosis was detected as early as 5 h (3.4% for 14d and 7.6% for CA-4) which
increased after 18 h incubation of MCF-7 cells with conjugates that were 26% for 14d and 16b.
Moreover, 13c exhibited extensive level of induction of intrinsic apoptosis with 42%, while the
positive control CA-4 with 46% cells with depolarized mitochondria (Figure 2A). However,
diminished effect on mitochondrial membrane potential was observed in response to treatment
with 13d. As evident in Figure 2 (panels A-C) at 18 h, the conjugates 13b, 16b and 14d induced
significant mitochondrial membrane depolarization in MCF-7 cells, whereas, 13d significantly
induced hyperpolarisation at 18 h. It is reported that hyperpolarisation is a step prior to
destabilization and progresses eventually to depolarization later. Therefore, this data suggests
that these conjugates play a crucial role in the alteration of mitochondrial membrane potential
resulting in the initiation of intrinsic apoptosis (Figure 2).
<
Insert figure 2>
3
.1.3. Effect on cell cycle progression
To gain further evidence regarding the role of these conjugates in the induction of
apoptosis, we measured the cellular arrest in different phases of the cell cycle. Thus, MCF-7 cells
were treated with 13c, 13d, 16b and 14d at 2 µM for 24 h and 48 h. Normal lymphocyte DNA
was used to set diploid G0-G1 phase and the untreated cells exhibited significant arrest in
synthetic S-phase. Flow cytometric analysis of synchronous cells showed that the majority of
cells were arrested in G2-M phase of the cell cycle at 24 h ranging from 15.3% to 39.9% (Figure
3
A (e-h) and 3B). Additionally, the conjugates 13c and 14d at 24 h induced a profound block in
G0-G1 phase by 30.8% and 61.9%, respectively. Wherein, at 48 h, the conjugates 13b, 13d, 16b
and 14d demonstrated a distinct tendency to block cell growth at G0-G1 (39% to 62%).
Intriguingly, 13d with trimethoxy groups on both phenyl rings showed a pronounced cell arrest
at G0-G1 phase by 62% as compared to that of positive control ADR (59.1%) at 48 h. The
6

ACCEPTED MANUSCRIPT
induced apoptosis was determined by measuring the percent of cells stalled in the sub-G1 peak:
3
1 and 44% of cells were found in the sub-G1 peak after a 48 h exposure of significant
conjugates. Whereas the positive controls CA-4 and ADR at 24 h accumulate the cells at G2-M
and G0-G1 stages, respectively, which continued at 48 h incubation (Figure 3A and 3C-c,d).
<
Insert figure 3>
3
1
.1.4. Detection of apoptosis by Annexin V-FITC and Propidium iodide staining
Therefore, extrinsic as well as intrinsic apoptosis in cells was induced by conjugates 13c,
3d, 16b and 14d were also evaluated by Annexin V-FITC and PI staining. In this study MCF-7
cells were incubated with conjugates at 2 µM concentration for 24 h. Interestingly, all the
conjugates induced early apoptosis in MCF-7 at 24 h, while the promising conjugates 13d (11%)
and 14d (8%) preferentially showed enhanced late apoptotic induction over the control. In
addition, these conjugates induced necrosis in MCF-7 cells to a maximum percent, whereas 13c
displayed a diminished effect on necrosis. Moreover, the apoptotic cells (range 5.5%-14.3%)
were noticed in all the treatments of conjugates and supported our hypothesis that theses
conjugates inhibited the cell growth at 24 h. Cumulatively, 13d induced apoptosis with 14.3%,
whereas 14d with 9.3% took the second place in effect and the results are compared with CA-4
(
Figure 4). Wherein, the conjugates 13c and 16b exhibited a reduced effect in the induction of
apoptotic cell death with 5.5% and 5.6%, respectively.
Insert figure 4>
<
3
.1.5. Effect on PI3K/Akt/mTOR pathway
To unravel the molecular mechanism as to how these conjugates induced apoptosis in
cancer cells and to identify the implicated signaling pathway targets, Western blotting
experiments were performed after isolating the total proteins from both untreated and treated
MCF-7 cells with 13b, 13c, 13d, 16b and 14d for 24 h. These blots were hybridized with
antibodies for known PI3K/Akt/mTOR pathway regulators that include PI3K, Akt, p-Akt,
mTOR, and GSK-3β. A concordant decrease in the expression of PI3K, Akt (Figure 5A), p-Akt
and mTOR proteins was observed in the treatments as compared to untreated cells, whereas a
significant increase in the expression of p-GSK-3β (ser-9) and PTEN were observed upon
treatment (Figure 5B) was noticed. In a separate experiment, conjugate treated MCF-7 cells
were stained with anti-Akt antibody conjugated with Alexa dye and visualized via
immunofluorescence microscopy and the data corroborated with western blotting results. Akt
7

ACCEPTED MANUSCRIPT
expression in conjugate-treated MCF-7 cells varied based on potency of the conjugates. In
conclusion, both studies indicated that 13c, 16b and 14d demonstrated significant
downregulation of PI3K, Akt and mTOR, whereas p-GSK-3β (ser 9) and PTEN protein levels
were significantly increased. Further to investigate the mRNA levels of PI3K(110a), Akt1,
PTEN of PI3K Akt axis and Bcl-2, BAX, TP53 of apoptosis related genes, we performed qRT-
PCR and results indicated that the clear decrease in PI3K, Akt1 and Bcl-2 mRNA levels and
significant increase of PTEN, BAX and TP53 mRNA levels upon compound treatment (Figure
5
C).
<
Insert figure 5>
3
.2. Molecular modeling
In order to have a more precise picture of the interaction mode of these promising conjugates as
phosphatidylinositol 3-kinase (PI3K) inhibitors, we performed molecular docking simulations for
[
44]
1
3c, 13d, 14d and 16b with the PI3K catalytic subunit (PDB 1E7V) as a template. Docking
analysis revealed that these conjugates occupied the same site corresponding to specific PI3K
inhibitor LY294002 at ATP binding position of PI3Kγ. Generally, the ATP binding site of PI3K
is positioned in a cleft between smaller N- and the larger C-terminal lobes of the catalytic
domain. Most of the PI3K inhibitors have shown potential hydrogen bonding interactions with
hinge residues, Val883 and Tyr867. In the case of LY294002, the morpholine ring hydrogen-
bonds to hinge residue Val882 and the edge of the morpholine ring interacts with the face of
Tyr867 (Toledo et al., 1999). In the present study, all the docked conjugates encompass potential
amino acid residues of both N, C-lobes, like Val882, Ala885, Lys883, Asp884, Ser806, Glu880,
Tyr867, Ala805, Leu848, Lys800, Lys808, Asp964, Gly958, Asn951, Asn954, Val941, Ile1002,
Met953 and showed maximum interactions with the receptor pocket. 13c with a trimethoxy
group on the A-ring and dimethoxy substituent on the C-ring showed more molecular
interactions explaining pronounced inhibition of kinase function. A strong hydrogen bonding
was observed between C3 methoxy O of C-ring and OH of Ser806 (O---HO, distance: 3.0 Å). In
addition, C3 methoxy O of A-ring develops hydrogen bonding with NH of Asp884 (O---HN,
distance: 3.0 Å) as well as NH of Lys883 (O---HN). Moreover, the dipole interactions were
observed between the carbonyl O of Val882 and pyrazole N atom (N---O=C). Further, a weak
hydrogen bond between carbonyl O of enone unit and NH of Asp964 (C=O---HN) was also
noticed. However, the S of Met953 formed weak electrostatic interactions with NH of pyrazole
8

ACCEPTED MANUSCRIPT
ring (S---HN). Additionally, 13c also exhibited some hydrophobic interactions with Ile963,
Ile828 and Phe783 apart from π−π stalking of Trp812 with trimethoxyphenyl A-ring. The
probable interactions of 13c with the catalytic domain harbouring significant residues (stick
model) are visualized in mesh representation and the superimposition of 13c on LY294002 also
showed a reference site (Figure 6 Panel 1). Furthermore, some crystal structure poses of 13c in
catalytic domain of PI3K revealed that the docking simulations at catalytic site are identical to
the known inhibitor (Figure 6 Panel 1).
<
Insert figure 6>
Docking of 13d with trimethoxy substituent on the A and C-rings develops a strong
hydrogen bonding between pyrazole NH and carbonyl O of Val882 (NH---O). Additionally, the
N atom of pyrazole showed a dipole interaction with O of Ala885 (N---O); however, another
dipole interaction was found between carbonyl O of enone and carbonyl O of Glu880 (C=O---
O=C). Moreover, both C3 and C4 methoxy O atoms formed weak hydrogen bonds with NH of
Asp964. (O---NH---O) and the C5 methoxy O of same ring developed a strong hydrogen bond
with phenolic H of Tyr867 (H---O). These significant hydrogen bonds at C ring of 13d probably
made the moiety to deviate from the active catalytic site demonstrating a reduced inhibitory
effect. The docking pose of 14d showed potential hydrogen bonding between pyrazole NH and
carbonyl O of Val882 (NH---O), methylenedioxy O of A-ring and NH of Asp964 (O---HN) as
well as dipole interactions between carbonyl O of enone and carbonyl O of Ala885 (C=O---
O=C). Additionally, the hydrophobic interactions with Val941, Asn954, Ile1002 include a
π−π stacking between Phe1009 and trimethoxyphenyl group of C-ring. However, 16b formed
two hydrogen bonding interactions between carbonyl O of Val882 and pyrazole NH (O---HN),
and NH of same residue with O of enone carbonyl unit (NH---O). However, a weak hydrogen
bond was noticed between NH of Asp964 and C4 methoxy O of C-ring. Interestingly, the dipole
interaction between pyrazole N and O of Ala885 (N---O) include some hydrophobic interactions
with Lys800, Glu880 and Asn951. Notably no significant interactions were noticed at A-ring of
1
6b suggesting a minimum activity. Therefore, these findings reveal that pyrazolochalcones
specifically occupy the ATP binding site of PI3Kγ and exhibit significant molecular docking
simulations with adjoining amino acids (Figure 6 Panel 2).
<
Insert figure 7>
4. Conclusion
9

ACCEPTED MANUSCRIPT
In summary, a library of pyrazolochalcones functionalized with various methoxy substituents on
the phenyl ring were successfully synthesized and screened for their cytotoxicity on sixty cancer
cell lines. Among them a total of fifteen compounds have shown remarkable potency towards
tested cell lines with IC50 values <1 µM. Most active compounds (13c, 13d, 16b and 14d) were
further evaluated for their mechanistic action using assays in MCF-7 cells. These conjugates
evidently exhibited G -M arrest At 24 h, while after 48 h the cells were found to show G -G
1
2
0
growth arrest. Furthermore, Annexin-PI staining and western bolt analysis revealed that both
intrinsic as well as extrinsic apoptosis could be induced by these conjugates. Additionally, 13d
and 14d also demonstrated increased early as well as late apoptotic cells though to a lesser
extent. The promising conjugates showed distinct downregulation of PI3K, Akt and mTOR
proteins along with significant upregulation of p-GSK-3β (ser 9) and PTEN proteins. In
consistency with these findings, we also observed activation of caspase-3 and caspase-9 involved
in mitochondrial membrane disruption. To support this hypothesis, docking at the ATP binding
site of PI3Kγ exhibited significant interactions. This forms the first study where
pyrazolochalcone conjugates have been synthesized and found to be efficient inducers of pro-
apoptotic signalling, and inhibitors of the PI3K/Akt/mTOR pathway. Further exploration of these
conjugates, possessing pyrazole and chalcone moieties, for chronic toxicity and efficacy in-vivo,
may help to advance the development of tailored cancer therapeutics.
5
. Experimental methods
5
.1. General experimental conditions
All chemicals and reagents were obtained from Aldrich (Sigma–Aldrich, St. Louis, MO, USA),
Lancaster (Alfa Aesar, Johnson Matthey Company, Ward Hill, MA, USA), or Spectrochem Pvt.
Ltd. (Mumbai, India) and were used without further purification. Reactions were monitored by
TLC performed on silica gel glass plates containing 60 GF-254, and visualization was achieved
by UV light or iodine indicator. Column chromatography was performed with Merck 60–120
1
mesh silica gel. H NMR spectra were recorded on Bruker UXNMR/XWIN-NMR (300 MHz) or
1
3
Inova Varian-VXR-unity (400, 500 MHz) instruments. C NMR spectra were recorded on
Bruker UXNMR/XWIN-NMR (75 MHz) instrument. Chemical shifts (d) are reported in ppm
downfield from an internal TMS standard. Purity was evaluated by analytical HPLC using a LC-
2
4
0AD-SHIMADZU. Mobile phase: Methanol–water (80:20, v/v) (buffer: ammonium acetate, pH
.2) PDA detector. The column used was Phenomenex Luna (C-18) 250 x 4.6 mm particle size.
1
0

ACCEPTED MANUSCRIPT
Flow rate: 0.9 mL min−1; injection volume: 5 µL; retention times are given in minutes. ESI
spectra were recorded on a Micro mass Quattro LC using ESI+ software with capillary voltage
3
.98 kV and ESI mode positive ion trap detector. High-resolution mass spectra (HRMS) were
recorded on a QSTAR XL Hybrid MS–MS mass spectrometer. Melting points were determined
with an Electro thermal melting point apparatus and are uncorrected.
5
.2. Synthetic Methods: Synthesis of (E)-1-substitutedphenyl-3-(3-(3,4,5-trimethoxyphenyl)-1H-
pyrazol-5-yl)prop-2-en-1-one (13a-j):
The different pyrazole carbaldehydes (12a-d) used in this study were taken from our previous
[
45]
report.
To the 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde (12a) prepared in the previous
step was added corresponding substituted acetophenones and catalytic amount of sodium
hydroxide in ethanol. The reaction mixture was stirred at room temperature for 3-4 h and
progress of the reaction was monitored by TLC. After completion, ethanol was evaporated under
vacuum and the residue was neutralized with dilute HCl solution. Finally the chalcones were
extracted with ethyl acetate followed by purification was done by using column chromatography
to obtain pure compounds of 13a-j with good yields (70-85%).
5
.2.1. (E)-1-phenyl-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-1-one (13a):
This compound was prepared employing the above procedure by the addition of 3-(3,4,5-
trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde (12a) (262 mg 1.0 mmol) and acetophenone
(120 mg 1.0 mmol). The compound obtained as brown colored solid Yield: 265 mg (73%); mp:
1
1
2
1
5
90-192 °C; H NMR (300 MHz, DMSO-d ); δ 3.89 (s, 9H, -OCH ), 6.85 (s, 1H, ArH), 6.92 (s,
6
3
1
H, ArH), 7.46 (m, 2H, J = 7.4 Hz, ArH), 7.52-7.62 (m, 2H, ArH, -transH, J = 15.7 Hz), 7.7 (d,
1
3
H, J = 15. 8 Hz, -transH,) 7.98 (d, 2H, J = 7.7 Hz, ArH); C NMR (75 MHz, DMSO-d ): δ
6
5.9, 60.8, 123.0, 126.0, 128.4, 128.6, 132.7, 133.0, 137.4, 138.3, 144.9, 149.1, 153.1, 189.8
-1
ppm; IR (KBr) (νmax/cm ): ν = 3230, 2927, 1681, 1590, 1506, 1472, 1424, 1348, 1281, 1242,
-
1
1
189, 1125,1089 cm ; MS (ESI) m/z 365[M+H]; HR-MS (ESI) m/z for C21
m/z: 365.1501, found m/z: 365.1499. Purity (HPLC): 97.80%.
.2.2. (E)-1-(4-methoxyphenyl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-1-one
13b):
This compound was prepared employing the above described procedure by the addition of 12a
262 mg 1.0 mmol) and 1-(4-methoxyphenyl)ethanone (150 mg 1.0 mmol). Yellow colored solid
21 2 4
H N O calculated
5
(
(
1
1

ACCEPTED MANUSCRIPT
1
Yield: 248 mg (63%); mp: 189-191 °C; H NMR (300 MHz, CDCl +DMSO-d ); δ 3.89 (s, 6H, -
3
6
3 3
OCH ), 3.91 (s, 6H, -OCH ) 6.87 (s, 1H, ArH), 6.91-7.01 (m, 4H, ArH), 7.58 (m, 2H, J = 15.8
1
3
Hz, transH ), 8.02 (d, 2H, J = 9.0 Hz, ArH); C NMR (75 MHz, DMSO-d ): δ 54.1, 55.5, 59.9,
6
-
1
1
2
02.3, 113.3, 121.7, 130.1, 152.8, 162.8, 187.1 ppm; IR (KBr) (νmax/cm ): ν = 3245, 3004, 2932,
-1
834, 1659, 1588, 15557, 1515, 1476, 1457, 1424, 1390, 1337, 1312, 1230, 1182, 1127, cm ;
MS (ESI) m/z 395[M+H]; HR-MS (ESI) m/z for C22
m/z: 395.1599. Purity (HPLC): 97.64%.
23 2 5
H N O calculated m/z: 395.1601, found
5
.2.3. (E)-1-(3,4-dimethoxyphenyl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-1-
one (13c):
This compound was prepared using the procedure described above by the addition of 12a (262
mg 1.0 mmol) and 1-(3,4-dimethoxyphenyl)ethanone (180 mg 1.0 mmol). Pale yellow colored
1
solid, yield: 275.8 mg (65%); mp: 176-177 °C; H NMR (300 MHz, DMSO-d ); δ 3.70 (s, 3H, -
6
OCH ), 3.87 (s, 12H, -OCH ), 7.12-7.16 (m, 3H, ArH), 7.39 (d, 1H, J = 14.9 Hz, transH), 7.54-
3
3
1
3
7
.69 (m, 2H, ArH), 7.77-7.89 (m, 1H, ArH), 7.92 (d, 1H, J = 15.8 Hz, trasH); C NMR (75
MHz, DMSO-d ): δ 55.5, 55.7, 55.8, 55.9, 60.0, 101.6,102.7, 103.6, 110.5, 110.8, 122.1, 123.0,
24.2, 129.0, 130.1, 130.3, 135.7, 137.5, 140.2, 143.5, 149.2, 151.4, 153.0, 186.7 ppm; IR (KBr)
6
1
-1
(
νmax/cm ): ν = 3273, 2934, 2836, 1644, 1592, 1568, 1509, 1470, 1421, 1331, 1266,
-1
1
25 6 2
198,1156,1016, 915 cm ; MS (ESI) m/z 425 [M+H]; HR-MS (ESI) m/z for C23H O N
calculated m/z: 425.1707, found m/z: 425.1696. Purity (HPLC): 98.31%.
5
1
.2.4. (E)-1-(3,4,5-trimethoxyphenyl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-
-one (13d):
This compound was prepared using the procedure described above by the addition of 12a (262
mg 1.0 mmol) and 1-(3,4,5-trimethoxyphenyl)ethanone (210 mg 1.0 mmol). Yellow colored
1
solid Yield: 263 mg (58%); mp: 208-210 °C; H NMR (300MHz, DMSO-d
6
); δ 3.78-3.99 (m,
1
1
6
3
8H, -OCH ), 6.85 (s, 1H, ArH), 6.90 (s, 2H, ArH), 7.19-7.40 (m, 3H, ArH), 7.55 (d, 1H, J =
3
1
3
5.8 Hz transH), 7.77 (d, 1H, J = 15.8 Hz, transH); C NMR (75 MHz, DMSO-d ): δ 55.9, 56.2,
6
-1
0.0, 60.1, 102.7, 106.1, 122.1, 132.7, 152.8, 153.2, 166.8, 187.6 ppm; IR (KBr) (νmax/cm ): ν =
-1
281, 2938, 2834, 1662, 1584, 1504, 1464, 1413, 1338,1235, 1189, 1126, 1000, cm ; MS (ESI)
m/z 455[M+H]; HR-MS (ESI) m/z for C H O N calculated m/z: 455.1812, found m/z:
2
4
27
7
2
4
55.1810. Purity (HPLC): 97.10%.
1
2

ACCEPTED MANUSCRIPT
5
.2.5. (E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-
en-1-one (13e):
This compound was prepared using the procedure described above by the addition of 12a (262
mg 1.0 mmol) and 1-(benzo[d][1,3]dioxol-5-yl)ethanone (164 mg 1.0 mmol). Brown red colored
1
solid Yield: 277.4 mg (68%); mp: 218-220 °C; H NMR (300MHz, DMSO-d ); δ 3.81 (s, 3H, -
6
OCH ), 3.81 (s, 6H, -OCH ), 6.13 (s, 2H, OCH2O), 6.95 (dd, 1H, J = 8.1 Hz, ArH), 6.99 (d, 1H,
3
3
J = 13.7 Hz, transH), 7.01-7.09 (m, 1H, ArH), 7.50-7.56 (m, 1H, ArH) 766-7.79 (m, 2H, J =
1
3
1
6.8 Hz, transH, ArH), 7.81-7.91 (m, 1H, ArH), 13.27 (brs, 1H, NH); C NMR (75 MHz,
DMSO-d ): δ 55.3, 59.9, 101.1, 102.0, 107.1, 107.4, 121.3, 123.9, 131.8, 147.4, 150.9, 152.6,
6
-
1
1
1
86.8 ppm; IR (KBr) (νmax/cm ): ν = 3420, 3239, 2928, 1657, 1578, 1508, 1457, 1385, 1314,
-1
22 6 2
243, 1126, 1035, 993 cm ; MS (ESI) m/z 409[M+H]; HR-MS (ESI) m/z for C23H O N
calculated m/z: 409.1394, found m/z: 409.1388. Purity (HPLC): 97.67%.
.2.6. (E)-1-(3-fluoro-4-methoxyphenyl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-
5
en-1-one (13f):
This compound was prepared using the procedure described above by the addition of 12a (262
mg 1.0 mmol) and 1-(3-fluoro-4-methoxyphenyl)ethanone (168 mg 1.0 mmol). The compound
1
obtained as yellow colored crystal Yield: 267.8 mg (67.8%); mp: 198-200 °C; H NMR
(
400MHz, DMSO-d ); δ 3.86 (s, 3H, -OCH ), 3.96 (s, 6H, -OCH ), 4.0 (s, 3H, -OCH ), 6.89 (s,
6 3 3 3
1
7
5
H, ArH), 7.04-7.07 (m, 1H, ArH), 7.12 (d, 1H, J= 8.4 Hz, ArH), 7.49-7.57 (m, 2H, ArH), 7.67-
13
.97 (m, 3H, J= 15.8 Hz, J= 8.3 Hz, transH, ArH); C NMR (75 MHz, DMSO-d
6
): δ 55.0, 54.9,
9.3, 101.6, 11.5, 114.5, 114.7, 120.4, 124.6, 129.8, 148.9, 150.5, 152,1, 184.8, 185.6 ppm; IR
-1
(
KBr) (νmax/cm ): ν = 3329, 2937, 1680, 1587, 1503, 1467, 1421, 1240, 1128, 1030, 1002, 830
-1
cm ; MS (ESI) m/z 413[M+H]; HR-MS (ESI) m/z for C23
found m/z: 413.1498. Purity (HPLC): 96.91%.
5 2
H22FO N calculated m/z: 413.1497,
5
.2.7.
(E)-1-(4-chlorophenyl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-1-one
(13g):
This compound was prepared using the procedure described above by the addition of 12a (262
mg 1.0 mmol) and 1-(4-chlorophenyl)ethanone (154 mg 1.0 mmol). The compound obtained as
1
brown colored solid Yield: 260 mg (65.1%); mp: 208-210 °C; H NMR (300 MHz, DMSO-d
6
); δ
3
.8-3.95 (m, 9H, -OCH ), 6.83 (s, 1H, ArH), 6.89 (d, 1H, J = 15.0 Hz, transH), 6.94 (d, 1H,
3
1
3
ArH), 7.32-7.54 (m, 3H, J = 16.0 Hz, ArH, transH), 7.01-8.04 (m, 3H, ArH); C NMR (75
1
3

ACCEPTED MANUSCRIPT
MHz, DMSO-d ): δ 55.4, 60.0, 99.5, 102.1, 134.5, 138.1, 138.6, 152.6, 196.6 ppm; IR (KBr)
6
-1
(νmax/cm ): ν = 3322, 2933, 1731, 1682, 1589, 1502, 1466, 1402, 1363, 1237, 1125, 1090, 1001
-1
cm ; MS (ESI) m/z 399 [M+H]; HR-MS (ESI) m/z for C21
found m/z: 399.1105. Purity (HPLC): 97.42%.
4 2
H20ClO N calculated m/z: 399.1106,
5
.2.8. (E)-1-(3,4-dichlorophenyl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-1-
one (13h):
This compound was prepared using the procedure described above by the addition of 12a (262
mg 1.0 mmol) and 1-(3,4-dichlorophenyl)ethanone (189 mg 1.0 mmol). Yellow colored solid
1
Yield: 280 mg (65%); mp: 203-205 °C; H NMR (300 MHz, DMSO-d ); δ 3.85 (s, 3H, -OCH ),
6
3
3
1
1
.94 (s, 3H, -OCH ), 3.99 (s, 3H, -OCH ), 6.90 (s, 1H, ArH), 7.03-7.07 (m, 1H, ArH), 7.12 (dd,
3 3
H, J = 8.4 Hz, ArH), 7.53 (s, 1H, ArH), 7.67 (d, 1H, J = 14.16 Hz, transH), 7.76 (d, 1H, J =
1
3
5.4 Hz, transH), 7.84 (dd, 1H, J = 8.4 Hz, ArH), 7.92 (dd, 1H, J = 8.8 Hz, ArH); C NMR (75
MHz, DMSO-d ): δ 55.53, 60.03, 102.19, 112.08, 115.19, 115.44, 121.01, 125.35, 130.42,
6
-
1
1
1
37.18, 151.01, 152.78, 186.42 ppm; IR (KBr) (νmax/cm ): ν = 3315, 2930, 2886, 1665, 1623,
-1
545, 1506, 1474, 1444, 1315, 1296, 1236, 1145, 1103, 1087, 1015 cm ; MS (ESI) m/z 434
[
M+H]; HR-MS (ESI) m/z for C H O N Cl calculated m/z: 434.0643, found m/z: 434.0644.
21 19 4 2 2
Purity (HPLC): 97.00%.
5
.2.9.
(E)-1-(3,4-difluorophenyl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-1-
one (13i):
This compound was prepared using the procedure described above by the addition of 12a (262
mg 1.0 mmol) and 1-(3,4-difluorophenyl)ethanone (156 mg 1.0 mmol). Yellow colored solid,
1
yield: 240 mg (59.9%); mp: 191-193 °C; H NMR (500 MHz, DMSO-d ); δ 3.84 (s, 9H, -
6
3
OCH ), 5.90 (s, 1H, ArH), 6.19 (s, 1H, ArH), 6.50-6.52 (m, 1H, ArH), 6.85-7.05 (m, 2H, J = 8.3
Hz, J = 16.0 Hz, transH, ArH), 7.10 (d, 1H, J = 8.4 Hz, ArH), 7.35-7.56 (m, 2H J = 8.3Hz, J =
1
3
1
1
6.5 Hz, transH, ArH); C NMR (75 MHz, DMSO-d ): δ 54.4, 55.1, 59.7, 100.0, 101.8, 113.4,
6
13.6, 115.6, 115.8, 116.6, 120.0, 120.2, 124.4, 134.5, 136.6, 141.6, 144.3, 152.3, 187.5 ppm; IR
-1
(
KBr) (νmax/cm ): ν = 3345, 2925, 2853, 1659, 1608, 1516, 1468, 1425, 1282, 1741, 1127, 1159,
-
1
1
4
039 cm ; MS (ESI) m/z 401[M+H]; HR-MS (ESI) m/z for C H O N F calculated m/z:
21 19 4 2 2
01.1307, found m/z: 401.1322. Purity (HPLC): 97.89%.
1
4

ACCEPTED MANUSCRIPT
5
.2.10. (E)-1-(4-aminophenyl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-1-one
(13j):
This compound was prepared using the procedure described above by the addition of 12a (262
mg 1.0 mmol) and 1-(4-aminophenyl)ethanone (135 mg 1.0 mmol). Yellow colored solid, yield:
1
2
80 mg (73.8%); mp: 197-199 °C; H NMR (500 MHz, DMSO-d ); δ 3.78 (s, 3H, -OCH ), 3.86
6
3
(
s, 6H, -OCH ), 4.74 (brs, 1H, -NH2), 6.62 (d, 2H, J = 8.4Hz, ArH), 6.73-6.75 (m, 1H, ArH),
3
6
2
1
2
.97 (s, 2H, ArH), 7.58 (d, 1H, J = 15.6Hz, transH), 7.64 (d, 1H, J = 15.6 Hz, transH), 7.85 (dd,
1
3
H, J = 8.6 Hz, ArH); C NMR (75 MHz, DMSO-d ): δ 55.2, 59.8, 101.4, 101.8, 112.5, 121.8,
6
-1
25.8, 130.1, 136.7, 152.4, 152.0, 186.1 ppm; IR (KBr) (νmax/cm ): ν = 3448, 3368, 3243, 2923,
-1
853, 1729, 1647, 1584, 1461, 1338, 1310, 1274, 1227, 1172, 1119, 1025, 981 cm ; MS (ESI)
calculated m/z: 380.1606, found m/z:
22 4 3
m/z 380 [M+H]; HR-MS (ESI) m/z for C21H O N
3
5
2
80.1605. Purity (HPLC): 97.47%.
.3. Synthesis of (E)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-1-substitutedphenylprop-
-en-1-one 14(a-j):
To the 3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde (12b) prepared in the previous
step was added corresponding substituted acetophenones and catalytic amount of sodium
hydroxide in ethanol. The reaction mixture was stirred at room temperature for 3-4 h and
progress of the reaction was monitored by TLC. After completion, ethanol was evopourated
under vacuum and the residue was neutralised with dilute HCl solution. Finally the chalcones
were extracted with ethyl acetate followed by purification was done by using column
chromatography to obtain pure compounds of 14a-j with good yields (60-75%).
5
.3.1. (E)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-1-phenylprop-2-en-1-one (14a):
This compound was prepared using the procedure described above by the addition of 3-
benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 12b (216 mg 1.0 mmol) and
(
acetophenone (120 mg 1.0 mmol). Brown colored solid, yield: 245 mg (76%); mp: 190-192 °C;
1
H NMR (300MHz, DMSO-d
6
2
); δ 6.03 (s, 2H, -OCH O-), 6.87 (d, 1H, J = 7.9 Hz, ArH), 6.93 (s,
1
7
H, ArH), 7.25-7.33 (m, 2H, ArH), 7.53 (d, 1H, J = 12.8 Hz, transH), 7.52-7.54 (m, 1H, ArH),
1
3
.58 (d, 1H, J = 14.8 Hz, transH), 7.69-7.72 (m, 2H, ArH), 8.06 (d, 2H, J = 7.1 Hz, ArH); C
NMR (75 MHz, DMSO-d ): δ 100.1, 101.4, 104.9, 107.5, 118.2, 121.2, 127.3, 127.6, 131.8,
6
-
1
1
1
33.0, 136.8, 146.4, 146.9, 188.7 ppm; IR (KBr) (νmax/cm ): ν = 3296, 3120, 2996, 1690, 1638,
-1
596, 1541, 1436, 1412, 1396, 1311, 1274, 1156, 1100, 1026 cm ; MS (ESI) m/z 319 [M+H];
1
5

ACCEPTED MANUSCRIPT
15 2 3
HR-MS (ESI) m/z for C19H N O calculated m/z: 319.1004, found m/z: 319.1003. Purity
(HPLC): 96.32%.
5
.3.2.
(E)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-1-(4-methoxyphenyl)prop-2-en-1-
one (14b):
This compound was prepared using the procedure described above by the addition of 12b (216
mg 1.0 mmol) and 1-(4-methoxyphenyl)ethanone (150 mg 1.0 mmol). Yellow colored solid,
1
yield: 265 mg (76.1%); mp: 196-198 °C; H NMR (300 MHz, DMSO-d
6
); δ 3.98 (s, 3H, -
OCH ), 6.00 (s, 2H, -OCH O-), 6.77 (d, 1H, J = 8.6 Hz, ArH), 6.87 (m, 1H, J = 8.5 Hz, ArH),
3
2
7
1
.01 (m, 1H, J = 8.6 Hz, ArH), 7.24-7.32 (m, 1H, ArH) 7.35 (d, 2H, J = 7.7 Hz, ArH) 7.65 (d,
1
3
H, J = 16.7 Hz, transH), 7.76 (d, 1H, J = 15.8 Hz, transH), 8.08 (m, 2H, J = 8.5 Hz, ArH); C
NMR (75 MHz, DMSO-d ): δ 54.0, 99.7, 104.4, 107.0, 112.4, 117.7, 120.7, 129.2, 145.9, 146.5,
6
-
1
1
1
61.9, 186.9 ppm; IR (KBr) (νmax/cm ): ν = 3237, 2909, 1659, 1608, 1587, 1549, 1503, 1470,
-1
342, 1294, 1256, 1226, 1174, 1037 cm ; MS (ESI) m/z 349 [M+H]; HR-MS (ESI) m/z for
C H O N calculated m/z: 349.1195, found m/z: 349.1194. Purity (HPLC): 97.50%.
20
17
4
2
5
1
.3.3. (E)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-1-(3,4-dimethoxyphenyl)prop-2-en-
-one (14c):
This compound was prepared using the procedure described above by the addition of 12b (216
mg 1.0 mmol) and 1-(3,4-dimethoxyphenyl)ethanone (180 mg 1.0 mmol). Pale yellow colored
1
solid, yield: 285 mg (75.3%); mp: 190-192 °C; H NMR (300 MHz, DMSO-d
6
); δ 3.95 (s, 3H, -
OCH ), 3.97 (s, 3H, -OCH ), 6.02 (s, 2H, OCH O), 6.83-7.06 (m, 3H, J = 16.4 Hz, transH,
3
3
2
ArH), 7.29 (d, 2H, J = 8.1 Hz, ArH), 7.60-7.73 (m, 2H, J = 14.8Hz, transH, ArH), 7.78 (d, 1H, J
1
3
=
6.8 Hz, ArH) 7.82 (s, 1H, ArH); C NMR (75 MHz, DMSO-d
6
): δ 54.0, 54.2, 99.4, 104.1,
1
06.7, 108.8, 109.0, 117.3, 120.2, 121.4, 145.6, 146.2, 147.2, 151.8, 185.6 ppm; IR (KBr)
-1
(
νmax/cm ): ν = 3274, 2923, 2852, 1654, 1598, 1573, 1501, 1457, 1419, 1394, 1346, 1297, 1272,
-1
1
236, 1167, 1031 cm ; MS (ESI) m/z 379[M+H]; HR-MS (ESI) m/z for C H O N calculated
21 19 5 2
m/z: 379.1289, found m/z: 379.2900. Purity (HPLC): 98.11%.
5
.3.4. (E)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (14d):
This compound was prepared using the procedure described above by the addition of 12b (216
mg 1.0 mmol) and 1-(3,4,5-trimethoxyphenyl)ethanone (210 mg 1.0 mmol). Pale yellow colored
1
solid, yield: 270 mg (66%); mp: 208-210 °C; H NMR (300MHz, DMSO-d
6
); δ 3.90 (s, 3H, -
1
6

ACCEPTED MANUSCRIPT
3 3 2
OCH ), 3.96 (s, 6H, -OCH ), 6.01 (s, 2H, -OCH O-), 6.82-6.92 (m, 2H, ArH), 7.27-7.37 (m, 4H,
13
J = 17.0 Hz, transH, ArH), 7.66-7.67 (m, 2H, J = 16.0 Hz, transHArH); C NMR (75 MHz,
DMSO-d ): δ 55.6, 60.0, 100.4, 102.0, 105.3, 107.8, 118.6, 121.1, 124.8, 141.6, 146.7, 147.2,
6
-
1
1
1
52.3, 197.8 ppm; IR (KBr) (νmax/cm ): ν = 3255, 3156, 2924, 1653, 1574, 1499, 1464, 1415,
-1
335, 1270, 1239, 1159, 1125, 1044, 1004 cm ; MS (ESI) m/z 409[M+H]; HR-MS (ESI) m/z for
C H O N calculated m/z: 409.1403, found m/z: 409.1402. Purity (HPLC): 97.68%.
22
21
6
2
5
.3.5. (E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)prop-2-
en-1-one (14e):
This compound was prepared using the procedure described above by the addition of 12b (216
mg 1.0 mmol) and 1-(benzo[d][1,3]dioxol-5-yl)ethanone (164 mg 1.0 mmol). Brown red colored
1
solid, yield: 280 mg (77.3%); mp: 218-220 °C; H NMR (300MHz, DMSO-d
6
); δ 6.03 (s, 2H, -
OCH O-), 6.13 (s, 2H, -OCH O-), 6.89 (d, J = 7.9 Hz, 1H, ArH), 6.97 (s, 1H, ArH), 7.02 (d, 1H,
2
2
J = 12.6 Hz, TransH), 7.24-7.36 (d, 1H, J = 12.6 Hz, transH) 7.56 (s, 1H, ArH), 7.62 (d, 1H, J =
1
3
8
.0 Hz, ArH), 7.69-7.80 (m, 2H, ArH), 8.02-8.05 (m, 1H, ArH); C NMR (75 MHz, DMSO-d ):
6
δ 99.3, 100.2, 103.8, 106.0, 106.1, 106.7, 117.1, 120.2, 122.9, 130.4, 145.4, 146.0, 146.3, 149.8,
-
1
1
1
84.9 ppm; IR (KBr) (νmax/cm ): ν = 3252, 2921, 2852, 1659, 1606, 1585, 1555, 1500, 1468,
-
1
15 5 2
346, 1289, 1115, 1036, 972 cm ; MS (ESI) m/z 363[M+H]; HR-MS (ESI) m/z for C20H O N
calculated m/z: 363.0990, found m/z: 363.0988. Purity (HPLC): 96.58%.
5
2
.3.6. (E)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-1-(3-fluoro-4-methoxyphenyl)prop-
-en-1-one (14f):
This compound was prepared using the procedure described above by the addition of 12b (216
mg 1.0 mmol) and 1-(3-fluoro-4-methoxyphenyl)ethanone (168 mg 1.0 mmol). Yellow colored
1
crystal Yield: 260 mg (71%); mp: 199-200 °C; H NMR (400MHz, DMSO-d
6
); δ 3.99 (s, 3H, -
OCH ), 6.02 (s, 2H, -OCH O), 6.87 (dd, 1H, J= 11.1 Hz, ArH), 7.07 (d, 1H, J= 16.8 Hz,
3
2
1
3
transH), 7.25-7.35 (m, 1H, ArH), 7.63 (s, 5H, ArH), 7.88 (d, 1H, J= 16.8 Hz, transH); C NMR
(
75 MHz, DMSO-d ): δ 54.7, 99.6, 104.3, 106.9, 111.3, 117.2, 117.6, 120.0, 124.4, 129.2, 145.8,
6
-
1
1
1
46.4, 151.1, 185.1 ppm; IR (KBr) (νmax/cm ): ν = 3242, 2922, 2851, 1661, 1607, 1516, 1465,
-1
276, 1239, 1139, 1037, 970, 930 cm ; MS (ESI) m/z 367 [M+H]; HR-MS (ESI) m/z 367 for
C H O N Fcalculated m/z: 367.1099, found m/z: 367.1090. Purity (HPLC): 97.80%.
20
16
4
2
5
.3.7. (E)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-1-(4-chlorophenyl)prop-2-en-1-one
(14g):
1
7

ACCEPTED MANUSCRIPT
This compound was prepared using the procedure described above by the addition of 12b (216
mg 1.0 mmol) and 1-(4-chlorophenyl)ethanone (154 mg 1.0 mmol). Brown colored solid, yield:
1
2
40 mg (68%); mp: 209-211 °C; H NMR (300 MHz, DMSO-d ); δ 5.97 (s, 2H, -OCH O-), 6.29
6
2
(s, 1H, ArH), 6.81 (d, 1H, J= 16.8 Hz, transH), 7.10-7.15 (d, 1H, J= 16.0 Hz, transH), 7.26 (s,
-
1
1
3
H, ArH) 7.42 (d, 3H, J= 7.9 Hz, ArH), 7.90 (d, 3H, J= 8.9 Hz, ArH); IR (KBr) (νmax/cm ): ν =
-1
421, 2899, 1682, 1589, 1492, 1463, 1401, 1360, 1239, 1093, 1038, 1012, 983 cm ; MS (ESI)
m/z 353 [M+H]; HR-MS (ESI) m/z for C H O N Cl calculated m/z: 353.1448, found m/z:
1
9
14
3
2
3
53.1448. Purity (HPLC): 95.85%.
.3.8. (E)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-1-(3,4-dichlorophenyl)prop-2-en-1-
5
one (14h):
This compound was prepared using the procedure described above by the addition of 12b (216
mg 1.0 mmol) and 1-(3,4-dichlorophenyl)ethanone (189 mg 1.0 mmol). Yellow colored solid
1
Yield: 182 mg (80%); mp: 196-198 °C; H NMR (300 MHz, DMSO-d
6
); δ 6.06 (s, 2H, -OCH
2
O-
)
, 6.84-6.88 (m, 1H, ArH), 6.88-6.95 (m, 1H, ArH), 6.98 (d, 1H, J= 16.0 Hz, transH), 6.08-6.18
m, 1H, ArH) 7.22 (d, 1H, J= 7.9 Hz, ArH), 7.40 (d, 1H, J= 16.1 Hz, transH), 7.57-7.66 (m, 1H,
(
1
3
-1
ArH), 7.82 (m, 2H, ArH); C NMR (75 MHz, DMSO-d
149, 3120, 2939, 2845, 1687, 1589, 1529, 1513, 1436, 1354, 1285, 1196, 1235, 1065 cm ; MS
ESI) m/z 388 [M+H]; HR-MS (ESI) m/z for C H O N Cl calculated m/z: 388.0195, found
6
): δ ppm; IR (KBr) (νmax/cm ): ν =
-
1
3
(
1
9
13
3
2
2
m/z: 388.0196. Purity (HPLC): 96.94%.
5
.3.9.
(E)-1-(3,4-difluorophenyl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-1-
one (14i):
This compound was prepared using the procedure described above by the addition of 12b (216
mg 1.0 mmol) and 1-(3,4-difluorophenyl)ethanone (156 mg 1.0 mmol). Yellow colored solid,
1
yield: 193 mg (77%); mp: 198-199 °C; H NMR (500 MHz, DMSO-d ); δ 6.01 (s, 2H, -OCH O-
6
2
), 6.86 (d, 1H, J = 7.9 Hz, ArH), 6.91 (s, 1H, ArH), 7.07-7.18 (m, 1H, ArH), 7.20-7.32 (m, 1H,
ArH), 7.68-7.72 (m, 2H, ArH), 7.80 (s, 1H, ArH), 7.84 (d, 1H, J = 12.2 Hz, transH), 7.89 (d,
1
3
1
H, J = 16.0 Hz, transH); C NMR (75 MHz, DMSO-d ): δ 100.8, 105.5, 108.3, 115.1, 116.7,
6
-1
1
16.9, 118.8, 121.7, 136.0, 143.6, 146.9, 147.6, 163.6, 191.5 ppm; IR (KBr) (νmax/cm ): ν =
-1
3
120, 3066, 3011, 2856, 1638, 1620, 1545, 1496, 1491, 1432, 1379, 1246 cm ; MS (ESI) m/z
55 [M+H]; HR-MS (ESI) m/z for C H O N F calculated m/z: 355.0816, found m/z: 355.0816.
3
1
9
13
3
2 2
Purity (HPLC): 97.66%.
1
8

ACCEPTED MANUSCRIPT
5
.3.10. (E)-1-(4-aminophenyl)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)prop-2-en-1-one
(14j):
This compound was prepared using the procedure described above by the addition of 12b (216
mg 1.0 mmol) and 1-(4-aminophenyl)ethanone (135 mg 1.0 mmol). Yellow colored solid, yield:
1
2
80 mg (84%); mp: 185-187 °C; H NMR (500 MHz, DMSO-d ); δ 5.51 (brs, 2H, -NH ), 6.02
6
2
(
s, 2H, -OCH O-), 6.69 (d, 2H, J = 8.6 Hz, ArH), 6.8-6.94 (m, 2H, ArH), 7.24-7.36 (m, 2H,
2
ArH), 7.60 (d, 1H, J = 15.8 Hz, transH), 7.70 (d, 1H, J = 15.8 Hz, transH), 7.81 (s, 1H ArH),
1
3
7
1
1
.88 (d, 1H, J = 8.6 Hz, ArH); C NMR (75 MHz, DMSO-d ): δ 55.2, 59.8, 101.4, 101.8, 112.5,
6
-1
21.8, 125.8, 130.1, 136.7, 152.0, 186.1 ppm; IR (KBr) (νmax/cm ): ν = 3155, 2926, 1650, 1550,
-
1
466, 1460, 1410, 1365, 1280, 1250, 1180, 1125, 1100, 1065, 985 cm ; MS (ESI) m/z 334
calculated m/z: 334.1190, found m/z: 334.1190.
[
M+H]; HR-MS (ESI) m/z for C19
H
16
O
3
N
3
Purity (HPLC): 96.85%.
5
1
.4. Synthesis of (E)-1-(substitutedphenyl)-3-(3-(substitutedphenyl)-1H-pyrazol-5-yl)prop-2-en-
-one (15-16a-d)
To the 3-subtitutedphenyl-1H-pyrazole-5-carbaldehydes (12c&d) prepared in the previous step
was added corresponding substituted acetophenones and catalytic amount of sodium hydroxide
in ethanol. The reaction mixture was stirred at room temperature for 3-4 h and progress of the
reaction was monitored by TLC. After completion, ethanol was evaporated under vacuum and
the residue was neutralised with dilute HCl solution. Finally the chalcones were extracted with
ethyl acetate followed by purification was done by using column chromatography to obtain pure
compounds of 17a-j with good yields (60-75%).
5
.4.1. (E)-3-(3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-
one (15a):
This compound was prepared using the procedure described above by the addition of 3-(3,4-
dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 12c (232 mg
1.0 mmol) and 1-(3,4,5-
trimethoxyphenyl)ethanone (210 mg 1.0 mmol). Yellow colored solid, yield: 310 mg (73.1%);
1
mp: 197-199 °C; H NMR (400MHz, DMSO-d ); δ 3.92 (s, 3H, -OCH ), 3.93 (s, 3H, -OCH ),
6
3
3
3
7
.97 (s, 6H, -OCH ), 6.86 (s, 1H, ArH), 6.94 (d, 1H, J= 8.3 Hz, ArH ), 7.31-7.42 (m, 3H, ArH),
3
1
3
.42-7.53 (m, 1H, ArH) 7.65 (d, 1H, J= 15.8 Hz, transH) 7.79 (d, 1H, J= 15.8 Hz, transH); C
NMR (75 MHz, DMSO-d
6
): δ 54.4, 54.9, 59.2, 101.0, 104.6, 105.3, 107.5, 110.2, 116.7, 120.4,
-1
1
31.8, 140.8, 147.5, 147.6, 151.6, 186.7 ppm; IR (KBr) (νmax/cm ): ν = 3253, 2923, 2852, 1735,
1
9

ACCEPTED MANUSCRIPT
1
687, 1654, 1568, 1509, 1464, 1411, 1388, 1337, 1250, 1225, 1184, 1155, 1118, 1065, 1019, 980
-1
cm ; MS (ESI) m/z 425 [M+H]; HR-MS (ESI) m/z for C H25O N calculated m/z: 425.1707,
23
6
2
found m/z: 425.1699. Purity (HPLC): 98.79%.
.4.2. (E)-1-(3,4-dimethoxyphenyl)-3-(3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-1-
5
one (15b):
This compound was prepared using the procedure described above by the addition of 12c (232
mg 1.0 mmol) and 1-(3,4-dimethoxyphenyl)ethanone (180 mg 1.0 mmol). Yellow solid, yield:
1
3
00 mg (76.1%); mp: 208-210 °C; H NMR (300MHz, DMSO-d ); δ 3.82 (s, 3H, -OCH ), 3.89
6
3
3
(s, 9H, -OCH ), 6.73-7.00 (m, 3H, ArH), 7.21-7.41 (m, 2H, ArH), 7.42-7.54 (dd, 1H, J = 13.5
1
3
Hz, transH), 7.56-7.61 (s, 1H, ArH), 7.62-7.80 (m, 2H, J = 14.9 Hz, transH, ArH); C NMR (75
MHz, DMSO-d ): δ 55.2, 108.4, 109.9, 110.2, 111.1, 117.5, 121.3, 122.6, 130.3, 148.3, 148.4,
6
-
1
1
1
52.7, 186.9 ppm; IR (KBr) (νmax/cm ): ν = 3251, 2930, 2838, 1655, 1590, 1570, 1513, 1465,
-1
418, 1388, 1331, 1266, 1163, 1023, 968 cm ; MS (ESI) m/z 395 [M+H]; HR-MS (ESI) m/z for
C H O N calculated m/z: 395.1601, found m/z: 395.1599. Purity (HPLC): 97.45%.
22
23
5
2
5
1
.4.3. (E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-
-one (15c):
This compound was prepared using the procedure described above by the addition of 12c (232
mg 1.0 mmol) and 1-(benzo[d][1,3]dioxol-5-yl)ethanone (164 mg 1.0 mmol). Yellow colored
1
solid, yield: 285 mg (75.3%); mp: 196-197 °C; H NMR (300 MHz, DMSO-d
6
); δ 3.85 (s, 3H, -
OCH ), 3.89 (s, 3H, -OCH ), 6.14 (s, 2H, -OCH O-), 6.77-7.22 (m, 3H, ArH), 7.27-7.48 (m, 2H,
3
3
2
J = 13.7 Hz, transH, ArH), 7.57-7.71 (m, 2H, ArH), 7.75 (dd, 1H, J = 8.1 Hz, ArH), 8.12 (d, 1H,
1
3
6
J = 16.2 Hz, transH); C NMR (75 MHz, DMSO-d ): δ 54.7, 100.7, 106.7, 106.9, 110.4, 116.9,
-
1
1
2
20.8, 123.5, 131.4, 147.0, 147.9, 150.5, 165.7, 186.2 ppm; IR (KBr) (νmax/cm ): ν = 3237,
922, 2844, 1769, 1657, 1607, 1581, 1524, 1455, 1373, 1323, 1252, 1165, 1131, 1028, 971, 939
-1
cm ; MS (ESI) m/z 379 [M+H]; HR-MS (ESI) m/z for C H O N calculated m/z: 379.1285,
2
1
19
5
2
found m/z: 379.1280. Purity (HPLC): 97.95%.
.4.4. (E)-1-(4-aminophenyl)-3-(3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-1-one
15d):
5
(
This compound was prepared using the procedure described above by the addition of 12c (232
mg 1.0 mmol) and 1-(4-aminophenyl)ethanone (135 mg 1.0 mmol). Yellow solid Yield: 245 mg
1
(
70.2%); mp: 218-220 °C; H NMR (400 MHz, DMSO-d
6
); δ 3.91 (s, 3H, -OCH
3
), 3.97 (s, 3H,
2
0

ACCEPTED MANUSCRIPT
OCH3), 4.93 (brs, 2H, NH
2
), 6.71 (d, 2H, J = 8.4 Hz, ArH), 6.81 (s, 1H, ArH), 7.29-7.43 (m,
2
H, ArH), 7.50 (s, 2H, ArH), 7.65 (d, 1H, J = 14.3 Hz, transH), 7.72 (d, 2H, J = 16.1 Hz,
1
3
transH), 7.93 (d, 2H, J = 6.9 Hz, ArH); C NMR (75 MHz, DMSO-d ): δ 54.5, 110.3, 107.7,
6
-
1
1
3
11.8, 116.7, 121.2, 124.7, 129.6, 147.5, 147.7, 152.0, 185.1 ppm; IR (KBr) (νmax/cm ): ν =
-
1
428, 3129, 3018, 2875, 1675, 1545, 1501, 1498, 1452, 1350, 1265, 1135, 1072, 945 cm ; MS
(
ESI) m/z 350 [M+H]; HR-MS (ESI) m/z for C H O N calculated m/z: 350.1499, found m/z:
20 20 3 3
3
5
50.1504. Purity (HPLC): 97.19%.
.4.5. (E)-3-(3-(4-methoxyphenyl)-1H-pyrazol-5-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(16a):
This compound was prepared employing above procedure by the addition of 3-(4-
methoxyphenyl)-1H-pyrazole-5-carbaldehyde 12d (202 mg 1.0 mmol) and 1-(3,4,5-
trimethoxyphenyl)ethanone (210 mg 1.0 mmol). Yellow solid, yield: 300 mg (75.9%); mp: 190-
1
1
6
92 °C; H NMR (500MHz, DMSO-d
6
); δ 3.75 (s, 3H, -OCH
3
), 3.80 (s, 3H, -OCH
3
), 3.87 (s,
H, -OCH ), 6.81 (s, 1H, ArH), 6.86 (d, 1H, J = 8.4 Hz, ArH), 7.22-7.27 (s, 3H, ArH), 7.55-7.69
3
1
3
(
m, 4H, J = 15.8 Hz, tranH, ArH); C NMR (75 MHz, DMSO-d ): δ 55.1, 56.1, 60.1, 105.9,
6
-
1
1
3
14.2, 121.8, 126.5, 132.7, 152.8, 159.1, 175.2, 187.5 ppm; IR (KBr) (νmax/cm ): ν = 3254,
-1
008, 2934, 2837, 1655, 1596, 1569, 1511, 1452, 1416, 1392, 1251, 1165, 1059, 964 cm ; MS
(
ESI) m/z 395 [M+H]; HR-MS (ESI) m/z for C H N O calculated m/z: 395.1601, found m/z:
22 23 2 5
3
5
95.1607. Purity (HPLC): 96.81%.
.4.6. (E)-1-(3,4-dimethoxyphenyl)-3-(3-(4-methoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-1-one
(16b):
This compound was prepared using the above procedure by the addition of 12d (202 mg 1.0
mmol) and 1-(3,4-dimethoxyphenyl)ethanone (180 mg 1.0 mmol). Yellow solid, yield: 270 mg
1
(
74.1%); mp: 177-179 °C; H NMR (300 MHz, DMSO-d ); δ 3.76 (s, 3H, -OCH ), 3.86 (s, 3H, -
6
3
OCH ), 3.87 (s, 3H, -OCH ), 6.78-6.90 (m, 3H, ArH), 6.95 (d, 1H, J = 12.3 Hz, transH), 7.53 (s,
3
3
13
1
H, ArH), 7.58-7.75 (m, 4H, J = 12.9 Hz, tranH, ArH), 7.83 (s, 1H, ArH); C NMR (75 MHz,
DMSO-d ): δ 54.7, 55.3, 55.4, 101.6, 110.1, 110.3, 113.7, 121.4, 122.7, 126.2, 130.3, 148.5,
6
-
1
1
1
52.8, 158.9, 186.9 ppm; IR (KBr) (νmax/cm ): ν = 3260, 2957, 1581, 1600, 1346, 1370, 1244,
-1
438, 1821, 1340, 1169, 1306, 1215, 1189, 1040 cm ; MS (ESI) m/z 365 [M+H]; HR-MS (ESI)
calculated m/z: 365.1495, found m/z: 365.1489. Purity (HPLC): 98.59%.
21 4 2
m/z for C21H O N
2
1

ACCEPTED MANUSCRIPT
5
.4.7.
(E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(3-(4-methoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-1-
one (16c):
This compound was prepared using the above procedure by the addition of 12d (202 mg 1.0
mmol) and 1-(benzo[d][1,3]dioxol-5-yl)ethanone (164 mg 1.0 mmol). Yellow colored solid,
1
yield: 260 mg (74.7%); mp: 208-210 °C; H NMR (300 MHz, DMSO-d ); δ 3.82 (s, 3H, -
6
OCH ), 6.14 (s, 2H, -OCH O), 6.85-7.13 (m, 4H, J = 15.8 Hz, transH, ArH), 7.43-7.80 (m, 5H,
3
2
1
3
J = 15.6 Hz, transH, ArH), 8.09 (s, 1H, ArH), 13.38 (brs, 1H, -NH); C NMR (75 MHz,
DMSO-d ): δ 54.3, 100.6, 107.6, 109.0, 109.4, 110.3, 116.5, 120.4, 121.3, 121.6, 129.3, 147.4,
6
-
1
1
1
47.6, 151.8, 186.0 ppm; IR (KBr) (νmax/cm ): ν = 3236, 2928, 1676, 1628, 1577, 1542, 1502,
-1
476, 1342, 1320, 1296, 1265, 1140, 1020, 944 cm ; MS (ESI) m/z 349 [M+H]; HR-MS (ESI)
calculated m/z: 349.1195, found m/z: 349.1194. Purity (HPLC): 97.22%.
.4.8. (E)-1-(4-aminophenyl)-3-(3-(4-methoxyphenyl)-1H-pyrazol-5-yl)prop-2-en-1-one (16d):
17 4 2
m/z for C20H O N
5
This compound was prepared using the procedure described above by the addition of 12d (202
mg 1.0 mmol) and 1-(4-aminophenyl)ethanone (135 mg 1.0 mmol). Pale yellow colored solid
1
Yield: 255 mg (79.9%); mp: 190-192 °C; H NMR (300 MHz, DMSO-d ); δ 3.84 (s, 3H, -
6
OCH ), 5.72 (brs, 2H, NH ), 6.67 (d, 2H, J = 8.6 Hz, ArH), 6.80-7.01 (m, 3H, ArH), 7.65-7.76
3
2
1
3
(
m, 3H, J = 13.6 Hz, transH, ArH), 7.82-8.02 (m, 3H, J = 13.4 Hz, transH, ArH); C NMR (75
MHz, DMSO-d ): δ 53.8, 111.8, 112.7, 121.1, 124.8, 125.3, 129.5, 152.0, 157.1, 185.1 ppm; IR
6
-1
(
KBr) (νmax/cm ): ν = 3448, 3363, 3203, 3003, 2923, 1660, 1609, 1632, 1526, 1464, 1335, 1294,
-
1
1
3
5
244, 1177 cm ; MS (ESI) m/z 320 [M+H]; HR-MS (ESI) m/z for C H O N calculated m/z:
19 18 2 3
20.1398, found m/z: 320.1393. Purity (HPLC): 97.27%.
.5. Synthesis of (E)-1-substitutedphenyl-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-
en-1-one 17(a-f)
To the 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde (12a) prepared in the
above step was added different ketones and catalytic amount of sodium hydroxide (1.0 ml) in
ethanol. The reaction mixture was heated to reflux for 1-2 h at 85 °C. Completion of the reaction
was determined by TLC followed by extracted the crude compounds with ethyl acetate thrice and
combined organic layers dried on anhydrous sodium sulphate. The crude compounds were
purified by means of column chromatography using ethyl acetate and hexane as solvent system
to provide pure compounds of 17a-f in good yields.
5
.5.1. (E)-4-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)but-3-en-2-one (17a):
2
2

ACCEPTED MANUSCRIPT
This compound was prepared using the procedure described above by the addition of 3-(3,4,5-
trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde (12a) (262 mg 1.0 mmol) and propan-2-one (58
1
mg 1.0 mmol). Yellow colored solid, yield: 210 mg (69.4%); mp: 193-195 °C; H NMR (500
MHz, DMSO-d
.77 (s, 1H, ArH), 6.89 (s, 2H, ArH), 7.48 (d, 1H, J = 16.4 Hz transH); C NMR (75 MHz,
DMSO-d ): δ 41.9, 57.4, 58.6, 98.3, 99.6, 104.3, 107.0, 109.6, 117.5, 127.4, 128.2, 138.9, 146.4,
6 3
); δ 2.35 (s, 3H, CH3), 3.89 (s, 9H, -OCH ), 6.71 (d, 1H, J = 16.4 Hz transH),
13
6
6
-
1
1
1
95.7 ppm; IR (KBr) (νmax/cm ): ν = 3229, 3131, 2940, 2832, 1734, 1665, 1629, 1591, 1504,
-1
19 4
472, 1416, 1327, 1125, 1039 cm ; MS (ESI) m/z 303 [M+H]; HR-MS (ESI) m/z for C16H O
N calculated m/z: 303.1339, found m/z: 303.1339. Purity (HPLC): 97.80%.
2
5
.5.2. (E)-2-((3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-l)methylene)cyclohexanone (17b):
This compound was prepared using the procedure described above by the addition of 12a (262
mg 1.0 mmol) and cyclohexanone (98 mg 0.0659 mmol). Yellow colored solid, yield: 205 mg
1
(
59.8%); mp: 181-183 °C; H NMR (500 MHz, DMSO-d ); δ 1.85-1.92 (m, 2H, -CH ), 1.92-
6
2
2
.00 (m, 2H, -CH
2
), 2.57 (t, J = 5.9 Hz, 2H, -CH
2
), 2.89-2.97 (m, 2H, -CH
2
), 3.89 (s, 3H, -
OCH ), 3.94 (s, 6H, -OCH ), 6.71 (s, 1H, ArH), 7.0 (s, 2H, ArH), 7.50 (s, 1H, ArH); IR (KBr)
3
3
-1
(
νmax/cm ): ν = 3144, 3060, 2927, 2854, 1680, 1588, 1503, 1565, 1423, 1312, 1245, 1191, 1036,
-
1
1
3
5
1
004 cm ; MS (ESI) m/z 343[M+H]; HR-MS (ESI) m/z for C19
43.1653, found m/z: 343.1653. Purity (HPLC): 97.15%.
23 4 2
H O N calculated m/z:
.5.3.
(E)-2-((3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)methylene)-3,4-dihydronaphthalen-
(2H)-one (17c):
This compound was prepared using the procedure described above by the addition of 12a (262
mg 1.0 mmol) and 3,4-dihydronaphthalen-1(2H)-one (146 mg 1.0 mmol). Yellow colored solid,
1
yield: 245 mg (62.8%); mp: 199-200 °C; H NMR (500 MHz, DMSO-d
6
); δ 3.08 (t, 2H, J = 6.7
Hz -CH ), 3.30 (t, 2H, J = 6.7 Hz, -CH ), 3.90 (s, 6H, -OCH ), 3.98 (s, 3H, -OCH ), 6.79 (s, 1H,
2
2
3
3
ArH), 7.0 (s, 3H, ArH), 7.30 (d, 1H, J = 7.5 Hz, ArH), 7.39 (dd, 1H, J = 7.5 Hz, ArH), 7.70-7.75
1
3
(
6
m, 1H, ArH), 8.13 (d, 1H, J = 7.5 Hz, ArH); C NMR (75 MHz, DMSO-d ): δ 26.5, 56.0,
-1
6
0.0, 102.8, 126.8, 126.9, 127.2, 128.4, 133.4, 143.5, 153.1, 186.9 ppm; IR (KBr) (νmax/cm ): ν
-1
=
3275, 2937, 2837, 1652, 1587, 1517, 1468, 1428, 1313,1234, 1119, 1030, 1006 cm ; MS (ESI)
m/z 391 [M+H]; HR-MS (ESI) m/z for C H O N calculated m/z: 391.1652, found m/z:
23
23
4
2
3
91.1652. Purity (HPLC): 96.62%.
2
3

ACCEPTED MANUSCRIPT
5
.5.4. (E)-2-((3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)methylene)-2,3-dihydro-1H-inden-1-
one (17d):
This compound was prepared using the procedure described above by the addition of 12a (262
mg 1.0 mmol) and 2,3-dihydro-1H-inden-1-one (132 mg 1.0 mmol). The compound obtained as
1
yellow colored solid Yield: 275 mg (73%); mp: 188-190 °C; H NMR (500 MHz, DMSO-d ); δ
6
2
.60 (s, 2H, -CH ), 3.88 (s, 3H, -OCH ), 3.98 (s, 6H, -OCH ), 6.84 (s, 1H, ArH), 7.08 (dd, 1H, J
2 3 3
=
10.0 Hz, ArH), 7.47 (s, 4H, ArH), 7.67 (d, 1H, J = 10.0 Hz ArH), 7.88 (d, 1H, J = 7.9 Hz
-1
ArH); IR (KBr) (νmax/cm ): ν = 3217, 2939, 1682, 1626, 1594, 1504, 1468, 1403, 1331, 1308,
-1
1
249, 1126, 1043, 1004 cm ; MS (ESI) m/z 377 [M+H]; HR-MS (ESI) m/z for C H O N Cl
20 17 3 3
calculated m/z: 377.1477, found m/z: 377.1478. Purity (HPLC): 97.53%.
5
1
.5.5.
(E)-5-methoxy-2-((3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)methylene)-2,3-dihydro-
H-inden-1-one (17e):
This compound was prepared using the procedure described above by the addition of 12a (262
mg 1.0 mmol) and 5-methoxy-2,3-dihydro-1H-inden-1-one (162 mg 1.0 mmol). Yellow colored
1
solid, yield: 280 mg (68.9%); mp: 196-197 °C; H NMR (500 MHz, DMSO-d ); δ 1.26 (s, 2H,
6
CH
2
3 3 3
), 3.86 (s, 3H, -OCH ), 3.94 (s, 3H, -OCH ), 3.96 (s, 6H, -OCH ), 6.80 (s, 1H, ArH), 6.97
(d, 1H, J = 8.1 Hz, ArH), 7.06-7.09 (m, 2H, ArH), 7.54 (s, 1H, ArH), 7.60 (s, 1H, ArH), 7.79
1
3
(
d, 1H, J = 8.3 Hz, ArH); C NMR (75 MHz, DMSO-d ): δ 27.1, 33.8, 54.2, 58.2, 101.0, 113.4,
6
-
1
1
1
23.4, 146.5, 151.3, 163.1, 189.7 ppm; IR (KBr) (νmax/cm ): ν = 3241, 3120, 2925, 2847, 1686,
-1
634, 1603, 1517, 1467, 1426, 1444, 1253, 1127, 1023, 1000 cm ; MS (ESI) m/z 407 [M+H];
HR-MS (ESI) m/z for C H O N calculated m/z: 407.1609, found m/z: 407.1610. Purity
23
23
5
2
(HPLC): 95.91%.
5
.5.6.
(E)-5,6-dimethoxy-2-((3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)methylene)-2,3-
dihydro-1H-inden-1-one (17f):
This compound was prepared using the procedure described above by the addition of 3-(3,4,5-
trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde (12a) (262 mg 1.0 mmol) and 5,6-dimethoxy-
2
,3-dihydro-1H-inden-1-one (192 mg 0.0659 mmol). Yellow colored solid, yield: 290 mg
1
(
66.4%); mp: 184-185 °C; H NMR (500 MHz, DMSO-d
6
); δ 2.59 (s, 2H, CH
2
), 3.87 (s, 3H, -
OCH ), 3.96 (s, 6H, -OCH ), 4.01 (s, 6H, -OCH ), 6.79 (s, 1H, ArH), 7.08 (s, 2H, ArH), 7.317
3
3
3
1
3
(
s, 1H, ArH), 7.46-7.65 (m, 2H, ArH); C NMR (75 MHz, DMSO-d ): δ 30.84, 59.27, 60.34,
6
1
03.08, 104.26, 123.78, 126.92, 127.93, 135.30, 137.77, 149.99, 153.45, 193.51 ppm; IR (KBr)
2
4

ACCEPTED MANUSCRIPT
-1
(
νmax/cm ): ν = 3258,2924, 2850, 1681, 1631, 1589, 1502, 1469, 1428, 1311, 1233, 1129, 1098,
-
1
1
4
5
033, 916 cm ; MS (ESI) m/z 437 [M+H]; HR-MS (ESI) m/z for C24
37.1696, found m/z: 437.1696. Purity (HPLC): 96.54%.
24 6 2
H O N calculated m/z:
.6. Synthesis of (E)-1-substitutedphenyl-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)prop-2-
en-1-one 18(a-f):
To the 3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde (12b) prepared in the previous
step was added corresponding substituted acetophenones and catalytic amount of sodium
hydroxide in ethanol. The reaction mixture was stirred at room temperature for 3-4 h and
progress of the reaction was monitored by TLC. After completion, ethanol was evopourated
under vacuum and the residue was neutralised with dilute HCl solution. Finaly the chalcones
were extracted with ethyl acetate followed by purification was done by using column
chromatography to obtain pure compounds of 18a-f with good yields (60-75%).
5
.6. 1. (E)-4-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)but-3-en-2-one (18a):
This compound was prepared using the procedure described above by the addition of 3-
(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 12b (216 mg 1.0 mmol) and propan-2-
1
one (58 mg 1.0 mmol). Yellow colored solid, yield: 191 mg (76%); mp: 201-203 °C; H NMR
(500 MHz, DMSO-d
6 3 2
); δ 2.33 (s, 3H, CH ), 6.02 (s, 2H, OCH O), 6.69 (d, 1H, J = 16.2 Hz
transH), 6.82 (s, 1H, ArH), 6.86 (d, 1H, J = 7.9 Hz, ArH), 7.08-7.17 (m, 1H, ArH), 7.21-7.26
1
3
(
m, 1H, ArH), 7.46 (d, 1H, J = 16.2 Hz transH); C NMR (75 MHz, DMSO-d ): δ 27.5, 99.4,
6
-
1
1
3
04.0, 106.7, 117.3, 123.8, 125.6, 126.3, 145.5, 146.1, 195.8 ppm; IR (KBr) (νmax/cm ): ν =
-
1
220, 2928, 2872, 1764, 1685, 1630, 1581, 1462, 1424, 1353, 1326, 1287, 1045, 960 cm ; MS
calculated m/z: 257.0917, found m/z:
(ESI) m/z 257 [M+H]; HR-MS (ESI) m/z for C14
H
13
O
3
N
2
2
5
57.0917. Purity (HPLC): 95.70%.
.6. 2. (E)-2-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methylene)cyclohexanone (18b):
This compound was prepared using the procedure described above by the addition of 12b (216
mg 1.0 mmol) and cyclohexanone (98 mg 1.0 mmol). Pale yellow colored solid, yield: 210 mg
1
(
70.9%); mp: 193-195 °C; H NMR (300 MHz, DMSO-d
6
); δ 1.76-1.98 (m, 4H, -CH
2
-), 2.42-
2
.61 (m, 2H, -CH -), 2.91 (s, 2H, -CH -), 6.01 (s, 2H, OCH O), 6.64 (s, 1H, ArH), 6.85 (d, 1H, J
2 2 2
1
3
=
7.9 Hz ArH), 7.28-7.34 (m, 1H, ArH), 7.45 (s, 1H ArH), 7.54 (s, 1H, ArH); C NMR (75
MHz, DMSO-d
6
): δ 20.7, 21.0, 26.5, 31.6, 99.2, 101.7, 103.9, 106.5, 117.1, 133.1, 145.2, 145.9,
-1
1
97.3 ppm; IR (KBr) (νmax/cm ): ν = 3197, 3096, 2932, 2883, 1699, 1662, 1578, 1463, 1434,
2
5

ACCEPTED MANUSCRIPT
-1
1
250, 1203, 1153, 969 cm ; MS (ESI) m/z 297 [M+H]; HR-MS (ESI) m/z for C17
calculated m/z: 297.1232, found m/z: 297.1233. Purity (HPLC): 97.30%.
.6. 3. (E)-2-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methylene)cyclohexanone (18c):
17 3 2
H O N
5
This compound was prepared using the procedure described above by the addition of 12b (216
mg 1.0 mmol) and 3,4-dihydronaphthalen-1(2H)-one (146 mg 1.0 mmol). Yellow colored solid
1
Yield: 245 mg (71.2%); mp: 189-190 °C; H NMR (500 MHz, DMSO-d ); δ 2.58 (s, 2H, -CH -),
6
2
4
7
.03 (s, 2H, -CH
2 2
-), 6.01 (s, 2H, OCH O), 6.77 (s, 1H, =CH), 6.87 (d, 1H, J = 7.8 Hz, ArH),
.28-7.36 (m, 1H, ArH), 7.40-7.50 (m, 3H, ArH), 7.58-7.68 (m, 2H ArH), 7.86 (d, 1H, J = 7.8
1
3
Hz, ArH); C NMR (75 MHz, DMSO-d ): δ 26.1, 27.1, 100.1, 102.5, 105.1, 107.5, 118.3,
6
-
1
1
3
24.3, 125.9, 127.3, 132.2, 133.5, 142.4, 146.7, 146.9, 186.0 ppm; IR (KBr) (νmax/cm ): ν =
-
1
229, 3145, 3028, 2882, 1658, 1605, 1584, 1487, 1457, 1411, 1243, 1132, 1037, 977 cm ; MS
(
ESI) m/z 345 [M+H]; HR-MS (ESI) m/z for C H O N calculated m/z: 345.1235, found m/z:
21 17 3 2
3
5
45.1235. Purity (HPLC): 96.58%.
.6.4. (E)-2-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methylene)-2,3-dihydro-1H-inden-1-
one (18d):
This compound was prepared using the procedure described above by the addition of 12b (216
mg 1.0 mmol) and 2,3-dihydro-1H-inden-1-one (132 mg 1.0 mmol). Yellow colored solid, yield:
1
2
2
7
70 mg (81%); mp: 177-178 °C; H NMR (500 MHz, DMSO-d ); δ 4.05 (s, 2H, -CH -), 6.02 (s,
6
2
H, -OCH
2
O-), 6.80 (s, 1H, =CH), 6.88 (d, 1H, J = 7.7 Hz, ArH), 7.28-7.38 (m, 1H, ArH), 7.40-
.50 (m, 1H, ArH), 7.56 (s, 2H ArH), 7.60-7.71 (m, 2H, ArH), 7.85 (d, 1H, J = 7.5 Hz, ArH);
-1
IR (KBr) (νmax/cm ): ν = 3238, 2920, 1697, 1636, 1606, 1496, 1463, 1325, 1243, 1201, 1105,
-
1
1
3
5
1
034 cm ; MS (ESI) m/z 331 [M+H]; HR-MS (ESI) m/z for C H O N calculated m/z:
20 15 3 2
31.1085, found m/z: 331.1085. Purity (HPLC): 97.44%.
.6.5. (E)-2-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methylene)-5-methoxy-2,3-dihydro-
H-inden-1-one (18e):
This compound was prepared using the procedure described above by the addition of 12b (216
mg 1.0 mmol) and 5-methoxy-2,3-dihydro-1H-inden-1-one (162 mg 1.0 mmol). Yellow colored
1
solid, yield: 260 mg (72.2%); mp: 184-186 °C; H NMR (400 MHz, DMSO-d
6
); δ 3.94 (s, 3H, -
OCH ), 3.99 (s, 2H, -CH ), 6.02 (s, 2H, OCH O), 6.77 (s, 1H, ArH), 6.85-6.92 (m, 1H, ArH),
3
2
2
6
.97 (d, 1H, J = 7.9 Hz, ArH), 7.06 (s, 1H, ArH), 7.28-7.36 (m, 1H ArH), 7.54-7.66 (m, 2H,
1
3
6
ArH), 7.78 (d, 1H, J = 8.9 Hz, ArH); C NMR (75 MHz, DMSO-d ): δ 27.7, 55.1, 101.8, 102.7,
2
6

ACCEPTED MANUSCRIPT
-
1
1
1
23.6, 126.5, 134.1, 152.3, 199.0 ppm; IR (KBr) (νmax/cm ): ν = 3293, 2920, 2851, 1687, 1632,
-1
598, 1546, 1512, 1464, 1338, 1294, 1140, 1111, 966 cm ; MS (ESI) m/z 361 [M+H]; HR-MS
(
ESI) m/z for C H O N calculated m/z: 361.1195, found m/z: 361.1195. Purity (HPLC):
21 17 4 2
9
5
6.78%.
.6.6.
(E)-2-((3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methylene)-5,6-dimethoxy-2,3-
dihydro-1H-inden-1-one (18f):
This compound was prepared using the above procedure by the addition of 12b (216 mg 1.0
mmol) and 5,6-dimethoxy-2,3-dihydro-1H-inden-1-one (192 mg 0.0659 mmol). Yellow colored
1
solid, yield: 296 mg (76%); mp: 190-192 °C; H NMR (500 MHz, DMSO-d ); δ 1.24 (s, 2H, -
6
2 3 3 2
CH ), 3.93 (s, 3H, -OCH ), 4.00 (s, 3H, -OCH ), 6.03 (s, 2H, -OCH O-), 6.79 (s, 1H, =CH),
6
.84-6.94 (m, 1H, J = 7.9 Hz ArH), 7.01-7.18 (m, 1H, ArH), 7.29-7.39 (m, 1H ArH), 7.48-7.51
1
3
(
m, 1H, ArH), 7.75-7.78 (m, 2H, ArH); C NMR (75 MHz, DMSO-d ): δ 28.0, 55.5, 60.0,
6
-1
1
02.2, 103.1, 124.0, 126.9, 134.5, 137.1, 142.5, 148.0, 152.7, 199.4 ppm; IR (KBr) (νmax/cm ): ν
=
3290, 3136, 2928, 2811, 1680, 1620, 1573, 1480, 1445, 1414, 1362, 1240, 1215, 1155, 1115,
-
1
1
3
5
045, 992 cm ; MS (ESI) m/z 391 [M+H]; HR-MS (ESI) m/z for C H O N calculated m/z:
22 19 5 2
91.1285, found m/z: 391.1287. Purity (HPLC): 97.94%.
.7. Cell culture and reagents All the cell lines used in this study were obtained from the
:
American Type Culture Collection (ATCC). MCF-7 and MDA-MB-231 (human breast
carcinoma cell lines) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing
non essential amino acids and 10% FBS. All the cells maintained under humidified atmosphere
°
of 5% CO at 37 C. Cells were trypsinized when sub confluent from T75 flasks/90mm dishes and
2
4
seeded on to 96 well test plates at a concentration of 1×10 cells/mL in complete medium, treated
with compounds at desired concentrations and harvested as required.
5
.8. Ananlysis of antiproliferative activity: Cell proliferation and viability was determined by 3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The pale yellow
colored tetrazolium salt (MTT) reduces to a dark blue water-insoluble formazan by metabolically
active cells and that is measured quantitatively after soluble in DMSO. The absorbance of the
soluble formazan is directly proportional to the number of viable cells. Cells were seeded at a
4
density of 1×10 cells in 200 µL of medium per well of 96-well plate. The 96-well microtiter
plates were incubated for 24 h prior to addition of the experimental compounds. Cells were
treated with vehicle alone (0.4% DMSO) or compounds (drugs were dissolved in DMSO
2
7

ACCEPTED MANUSCRIPT
previously) at different concentrations (1, 10 and 25µM) of test compounds for 48 hours. The
0
assay was completed with the addition of MTT (5 %, 10µL) and incubated for 60 min at 37 C.
The supernatant was aspirated and plates were air dried and the MTT-formazon crystals
dissolved in 100 µL of DMSO. The optical density (O.D) was measured at 560 nm using
TECAN multimode reader. The growth percentage of each treated well of 96 well plate have
been calculated based on test wells relative to control wells. The cell growth inhibition was
calculated by generating dose response curves as a plot of the percentage of surviving cells
versus drug concentration. Antiproliferative activity of the cancer cells to the test compounds
was expressed in terms of IC50 value, which defines as a concentration of compound that
produced 50% absorbance reduction relative to control.
5
.9. Cell cycle distribution analysis: MCF-7 Cells (NCI, USA) were grown to 70% confluency in
0
RPMI medium containing 10% serum at 37 C with 5% CO in humidified incubator. Cells were
2
then washed with 1X PBS and incubated in medium containing 0.05% fetal bovine serum (FBS,
Invitrogen, USA) for 24 hours for cell cycle synchronization. Later, culture media was
replenished with 10% FBS and cells were further incubated for 24 hours in media alone or in
presence of conjugates 13c, 13d, 16b, 14d , CA-4 at 2 µM concentration and/ or adriyamycin at
1
µM concentration. After incubation, cells were washed once with 1X PBS and fixed with 70%
0
cold ethanol (S.D. Fine Chemicals, Mumbai) overnight at 4 C. Fixed cells were washed with 1X
0
PBS and incubated with RNase A (40µg/ml) and Propidium Iodide (40µg/ml) for 1hour at 37 C.
Cells were passed through 26gauge needle 2-3 times to get rid of aggregates. These cells were
acquired on FACS Calibre (BD Biosciences, San Vose, USA) and analyzed by ModFit LT 4.1
Verity Software House, Country.
5
.8. Detection of mitochondrial membrane potential by DiOC6 staining: MCF-7 cells were
6
cultured in complete medium in a 6- well tray at density of 0.3x10 cells /ml/ well at 37 °C in
humidified air with 5% CO Cells were treated with compounds 13c, 13d, 16b, 14d and CA-4 at
2
.
2
µM concentration for 18 hours. In another set, cells were CA-4 and 14d at 5 hours separately.
After treatment, cells were harvested. For positive control for mitochondrial membrane
6
destabilization 1x10 cells were suspended in 4% freshly prepared paraformaldehyde (Sigma-
Aldrich, MO, USA) and incubated at room temperature for 10 min and then washed with
®
prewarmed 1ml PBS three times. DiOC6 ((Life Technologies, Molecular Probes ,USA) dye was
6
0
added to a concentration of 0.1 µM to the 1x10 cells per ml. Cells were incubated at 37 C for 15
2
8

ACCEPTED MANUSCRIPT
min and washed three times by centrifugation at 1000 rpm for 5 min. Cells were resuspended in
5
00 µl PBS and acquired on FACSCalibre (BD Biosciences, USA). Data was analyzed on
CellQuest Software (BD Biosciences, USA). Data was represented as histogram overlay, percent
cells with depolarized mitochondrial membrane, and mean fluorescence intensity change
observed before and after treatment with compounds.
5
.9. Detection of apoptosis by Annein V-FITC and propidium iodide staining: MCF-7 cells were
cultured in presence and absence of compounds for 24 hours and then resuspended in 1X
6
Annexin Binding Buffer at a density of 1x10 cells per ml. 100µl of this suspension was
transferred to flow cytometry tubes and mixed with 5µl of Annexin V FITC (BD Biosciences,
USA) and 1µl of Propidium Iodide (40ug/ml; Sigma-Aldrich, USA). Cells were gently vortexed
and incubated for 15 min at RT in the dark. After incubation 400ul of 1X Annexin Binding
Buffer was added to each tube and samples were acquired on FACSCalibre (BD Biosciences,
USA). Data was analyzed on FlowJo Software (www.flowjo.com). For compensation MCF-7
cells treated with etoposide 50µM for 24 hours stained with only PI or only annexin and stained
dually with annexin/PI were used.
5
.10. Immunofluoresence: MCF-7 cells were seeded on glass cover slips, incubated for 24 h in
the presence or absence of test compounds at a final concentration of 2µM. Cells grown on cover
slips were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) pH 7.4 for 20 min at
room temperature. Cells were permeablized for 5 min in PBS containing 0.5% Triton X-100
(
Sigma) and 0.05% Tween-20 (Sigma). The permeablized cells were blocked with 1% BSA
Sigma) in PBS for 1 h. Later, the cells were incubated with primary antibody diluted in blocking
(
solution for 4 h at room temperature. Subsequently the antibodies were removed and the cells
were washed thrice with PBST. Cells were then incubated with secondary antibody for 1 h at
room temperature. Cells were washed thrice with PBST and mounted in medium containing
DAPI (vecta shield). Images were captured using the Olympus confocal microscope FLOW
VIEW FV 1000 series and analyzed with FV10ASW 1.7 series software.
5
.11. Western blot analysis: Cells were lysed and lysate was extracted using RIPA lysis buffer
after treatment with compounds for 48h at 1uM final drug concentration. Protein was quantitated
using the Bradford assay and 20mg of total protein was loaded per well, and resolved on 8.0%,
1
0.0%, or 12% SDS-polyacrylamide gels. The gels were then transferred to Immoblion-P, PVDF
(Millipore, Billerica, Massachusetts) using semidry transfer technique and probed with the
2
9