Synthesis of (Z)-1-(1,3-diphenyl-1 H -pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-one derivatives as potential anticancer and apoptosis inducing agents

Article abstract of DOI:10.1016/j.ejmech.2016.03.051

A series of (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-one derivatives were synthesized and characterized by ¹H and ¹³C NMR, ESI-MS and HRMS. All the synthesized compounds were evaluated for their anticancer activity against HT-29, PC-3, A549 and U87MG human tumor cell lines. Most of the synthesized compounds displayed potent growth inhibition selectively of A549 cancer cells and did not show significant toxicity to the non-cancerous HaCaT cells. Some of the representative compounds, particularly, 16, 22 and 28 exhibited potent growth inhibition with IC₅₀ values in the range of 1.25-3.98 μM, which are comparable or even better than the standard chemotherapeutic drug 5-fluorouracil. Preliminary mechanistic studies revealed that these compounds could effectively inhibit the migration ability of A549 cells. Flow-cytometry analysis revealed that the compounds treatment led to G2/M cell cycle arrest. Moreover, the compounds induced apoptosis in A549 cells through depolarization of mitochondrial membrane potential (DΦm) and increased reactive oxygen species (ROS) production, suggesting their potential to act as promising lead compounds for the development of cancer chemotherapeutics.

Full text of DOI:10.1016/j.ejmech.2016.03.051

Accepted Manuscript
Synthesis of (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-one
derivatives as potential anticancer and apoptosis inducing agents
T. Srinivasa Reddy, V. Ganga Reddy, Hitesh Kulhari, Ravi Shukla, Ahmed Kamal,
Vipul Bansal
PII:
S0223-5234(16)30233-1
10.1016/j.ejmech.2016.03.051
EJMECH 8476
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 January 2016
Revised Date: 17 March 2016
Accepted Date: 18 March 2016
Please cite this article as: T. Srinivasa Reddy, V. Ganga Reddy, H. Kulhari, R. Shukla, A. Kamal, V.
Bansal, Synthesis of (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-one derivatives
as potential anticancer and apoptosis inducing agents, European Journal of Medicinal Chemistry (2016),
doi: 10.1016/j.ejmech.2016.03.051.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis of (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-one
derivatives as potential anticancer and apoptosis inducing agents
T. Srinivasa Reddy,a,b,c V. Ganga Reddy,a Hitesh Kulhari,a,b,c Ravi Shukla,c,d Ahmed
Kamal,a,b* Vipul Bansal c*
b
aMedicinal Chemistry and Pharmacology, IICT-RMIT Research Centre, CSIR-Indian
Institute of Chemical Technology, Hyderabad, 500007, India
cIan Potter NanoBioSensing Facility, Nano Biotechnology Research Laboratory, School of
Science, RMIT University, Melbourne, VIC 3000, Australia
dCentre for Advanced Materials and Industrial Chemistry, School of Science, RMIT
University, Melbourne, VIC 3000, Australia

ACCEPTED MANUSCRIPT
Synthesis of (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-one
derivatives as potential anticancer and apoptosis inducing agents
T. Srinivasa Reddy,a,b,c V. Ganga Reddy,a Hitesh Kulhari,a,b,c Ravi Shukla,c,d Ahmed
Kamal,a,b* Vipul Bansal c*
b
aMedicinal Chemistry and Pharmacology, IICT-RMIT Research Centre, CSIR-Indian
Institute of Chemical Technology, Hyderabad, 500007, India
cIan Potter NanoBioSensing Facility, Nano Biotechnology Research Laboratory, School of
Science, RMIT University, Melbourne, VIC 3000, Australia
dCentre for Advanced Materials and Industrial Chemistry, School of Science, RMIT
University, Melbourne, VIC 3000, Australia
*Corresponding authors:
Prof. Vipul Bansal
School of Science, RMIT University
GPO Box 2476, Melbourne, VIC 3000 Australia
E-mail: vipul.bansal@rmit.edu.au
Dr. Ahmed Kamal
CSIR-Indian Institute of Chemical Technology,
Hyderabad, 500007, India
E-mail: ahmedkamal@iict.res.in
1

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Abstract
series
A
of
(Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-one
derivatives were synthesized and characterized by 1H and 13C NMR, ESI-MS and HRMS. All
the synthesized compounds were evaluated for their anticancer activity against HT-29, PC-3,
A549 and U87MG human tumor cell lines. Most of the synthesized compounds displayed
potent growth inhibition selectively on A549 cancer cells and did not show significant
toxicity to the non-cancerous HaCaT cells. Some of the representative compounds,
particularly, 16, 22 and 28 exhibited potent growth inhibition with IC50 values in the range of
1.25-3.98 µM, which are comparable or even better than the standard chemotherapeutic drug
5-fluorouracil. Preliminary mechanistic studies revealed that these compounds could
effectively inhibit the migration ability of A549 cells. Flow-cytometry analysis revealed that
the compounds treatment led to G2/M cell cycle arrest. Moreover, the compounds induced
apoptosis in A549 cells through depolarization of mitochondrial membrane potential (DΨm)
and increased reactive oxygen species (ROS) production, suggesting their potential to act as
promising lead compounds for the development of cancer chemotherapeutics.
Keywords:
Pyrazole; anti-cancer activity; cell cycle; apoptosis
2

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1.0 Introduction
Cancer is a global health issue, representing the second leading cause of deaths [1]. Although
current cancer treatments help to reduce or eliminate cancerous cells from the body, the
current therapies are not always effective, efficient and may often cause adverse effects. The
available chemotherapeutic drugs face several limitations such as drug resistance, high
systemic toxicity, complex syntheses, and isolation procedures [2]. Therefore, there is a need
for the development of new anticancer agents which can overcome these problems.
Most of the chemotherapeutic agents act primarily by activating the cell death signaling
pathways including apoptosis [3]. Apoptosis is a programmed cell death that plays a crucial
role in the maintenance of tissue development and homeostasis. Generally, it is considered
that apoptosis is a multi-step process and is mainly regulated under the control of pro- and
anti-apoptotic proteins, such as the Bcl-2 and inhibitor of apoptosis protein (IAP) family
members, and executed through caspases, death receptors and mitochondria-mediated
apoptosis pathway [4]. Thus, deregulation of these processes results in several diseases
including cancer. Therefore, development of novel chemotherapeutic agents that induce
apoptosis in cancer cells has emerged as an attractive strategy in cancer drug discovery [5].
Recently there has been growing interest in investigations on pyrazole ring systems because
of their diverse pharmacological activities including antibacterial, antifungal, anti-
inflammatory, antioxidant and antitumor activities [6-11]. These derivatives occupy an
unique position in drug discovery for the synthesis of pharmaceutically active compounds
with good safety profiles [12]. Several studies have shown that pyrazoles owe their anticancer
activity either to inhibition of the enzymes or to the generation of reactive oxygen species
(ROS), leading to cellular apoptosis [13-14].
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On the other hand, a large number of small molecules that contain arylaminoprop-2-en-1-
ones have been reported for their anticancer activity [15-17]. Earlier, our group also reported
the
synthesis
of
2-ailinopyridine-arylpropenones
[15]
and
(Z)-(arylamino)-
pyrazolyl/isoxazolyl-2-propenones as anticancer and apoptosis inducing agents [16]. Further,
other studies have demonstrated that incorporation of propen-1-one moiety in to the
molecules result in compounds with promising anticancer activity. For instance, (Z)-1-aryl-3-
arylamino-2-propen-1-one’s derivatives displayed a remarkable cytotoxicity towards various
cancer cell lines with potent anti-tubulin activity by arresting the cells at G2/M phase of cell
cycle [17].
Based on these observations and our ongoing interest on pyrazole ring systems [18-19] in the
search for new compounds with potent anti-cancer activities, it seemed interesting to
synthesize compounds incorporating pyrazole ring and arylaminoprop-2-en-1-ones to explore
their anticancer activities. Therefore in this work, we synthesized a series of (Z)-1-(1,3-
diphenyl-1H-pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-one derivatives and evaluated their
anticancer activity.
2.0 Result and Discussions
2.1 Chemistry
The synthesis route for (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-
one derivatives is illustrated in Scheme 1. The intermediate compounds, 1-(1,3-diphenyl-1H-
pyrazol-4-yl)prop-2-yn-1-one derivatives 6 (a-e) were synthesized according to our
previously reported literature methods [18]. Briefly, acetophenones 1 (a-e) on condensation
with phenyl hydrazine (2) in ethanol at refluxing condition for 3 h gave the corresponding
acetophenone phenyl hydrazones 3 (a-e) which underwent cyclization into corresponding 1,3
di-phenyl pyrazole carboxaldehydes 4 (a-e) on Vilsmeir–Haack reaction with DMF-POCl3
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complex via the two step attack process by the complex; first at the CH3 group of the
substrate with the subsequent cyclization into 3-arylpyrazoles and then at the C4 atom of the
resulting heterocycles. These aldehyde intermediates 4 (a-e) were reacted with
ethynylmagnesium bromide in dry tetrahydrofuran (THF) at 0 °C to room temperature to
produce alcohol substituted derivatives 5 (a-e). The secondary alcohols on oxidation with 2-
iodoxybenzoic acid (IBX) in the presence of dimethyl sulfoxide (DMSO) gave 1-(1,3-
diphenyl-1H-pyrazol-4-yl)prop-2-yn-1-one derivatives 6 (a-e). The final target compounds
(Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-one
(7-36)
were
synthesized according to a previously reported method [17] which involves reaction of
terminal alkyne substituted pyrazoles 6 (a-e) with different substituted aryl amines in ethanol
1
13
at room temperature. All the compounds were characterized by H NMR, C, ESI-MS and
HRMS (supplementary information).
< Scheme 1 >
2.2 In vitro anticancer activity
All the synthesized compounds were investigated for their in vitro cytotoxicity against four
human tumor cells HT-29 (colon), PC-3 (prostate), A549 (lung) and U87MG (glioblastoma)
using standard MTT assay [20]. 5-fluorouracil (5-FU) was used as a standard reference
compound and the results are summarized in Table 1. Close observation of results from
Table 1 indicated that almost all the synthesized derivatives were selectively cytotoxic to the
A549 lung cancer cells. For instance, some of the compounds such as 10, 11, 16, 20, 22, 25,
26, 28, 34 displayed potent growth inhibition on A549 cells with IC50 values in the range of
1.25-3.98 µM, which are comparable or even better than the standard 5-FU. The potent
growth inhibitory activity was observed with the compound 16 (IC50 - 1.5±0.45 ꢀM),
followed by the compounds 22 (IC50 - 1.91±0.21 ꢀM) and 28 (IC50 - 2.77±0.24 ꢀM) against
5

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A549 cancer cells. The other three remaining tumor cell lines also displayed maximum
sensitivity towards these compounds with IC50 values not more than 4.71 ꢀM. Compound 11
was highly active on HT-29 colon cancer cells (IC50 - 1.56±0.32 ꢀM), while the compounds
28 and 34 showed selective potency against PC-3 prostate cancer cells (IC50 - 1.98±0.16 ꢀM)
and U87MG brain cancer cells (IC50-1.8±0.57 ꢀM), respectively. When all the synthesized
compounds were further tested on non-cancerous human HaCaT cells, most of these did not
exhibit cytotoxicity, which indicates the selectivity of these compounds towards cancer cells.
The most active compounds 16, 22 and 28 displayed moderate growth inhibition of HaCaT
cells with IC50 in the range of 16.2-34.6 µM. These are about 10-12 times less toxic than that
of A549 lung cancer cell growth inhibition values.
< Table 1 >
Structure-activity relationship (SAR) could be observed by examining the effect of
substituents such as fluorine, chlorine, trifluoromethyl and methoxy in A and B phenyl rings
of these derivatives on cell growth inhibition. It was noted that strong electron donating
group (OH) on the aromatic B ring produced the compounds (10, 16, 22, 28 and 34) with
potent and broad spectrum of activity on all the tested cancer cell lines with IC50 values in the
range of 1.35-3.21 µM. Further, the other electron donating groups such as methoxy (11, 17,
23, 29 and 35) and 3,4,5-methoxy (12, 18, 24, 30 and 36) substitutions also resulted in
compounds with remarkable cytotoxicity. In contrast, the compounds with electron
withdrawing substitutions chloro (8, 14, 20, 26 and 32) and trifluoromethyl (9, 15, 21, 27 and
33) on B ring displayed no cytotoxicity on cancer cells. It should be noted that different
substitutions on the aryl A ring didn’t have any significant role in activity. The interesting
biological activity of compounds 16, 22 and 28 prompted us to investigate their effects at
cellular level and in particular, the mechanisms responsible for cell death.
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2.3 In vitro cell migration assay
The mortality and prognosis in cancer patients correlate with metastatic potential of tumors.
As cell motility and migration associate with the metastatic behavior of cancer cells, we
investigated the effect of selected potent compounds on A549 cell migration capability using
wound healing assay [21]. To achieve this, A549 cells were independently treated with
compounds 16, 22 and 28 after creating the wounds/scratch in the confluent monolayers of
cultured cells. Images of wounded areas were captured after 0 h, 24 h and 48 h of compound
treatments. Figure 1 shows that almost complete healing of wounded area was observed in
control cells after 48 h, whereas the healing was strongly inhibited in the cells treated with
compounds, thereby reflecting the ability of these compounds to inhibit the migration
capability of the cells. The compound 22 displayed strongest cellular migration inhibition
ability, followed by compounds 28 and 16.
< Figure 1 >
2.4 Effect on actin polymerisation
Directional migration or motility of cancer cells is initiated by changes in cell polarity,
remodelling of cytoskeleton and regulation of microtubules and actin [22]. It is established
that the localized polymerisation of actin filaments is the driving force for cellular motility
[23]. As tested compounds inhibited the migration of A549 cancer cells, it was of interest to
further investigate their effect on actin polymerisation. The formation of actin cytoskeleton
was assessed by staining the cells with rhodamine-phalloidin [24], which specifically binds to
F-actin. The results from the Figure 2 demonstrate that all the three compounds disrupted the
actin fibre assembly which is more prominent with the compound 22. The cells treated with
the compounds displayed disappearance of F-actin extensions. This loss of F-actin extensions
at the periphery of the cell membrane resulted in loss of migration ability of the cancer cells.
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Collectively, these results demonstrate that the compounds 16, 22 and 28 inhibit the
migration potential of A549 lung cancer cells, at least in part through disrupting F-actin
assembly.
< Figure 2 >
2.5 Cell cycle analysis
To further investigate the mechanism of cell growth inhibition, we examined the effect of
these compounds on cell cycle progression using propidium iodide staining [25]. Cell cycle
phase distribution analysis, as studied using flow cytometry, demonstrated that treatment of
A549 cells with compounds 16, 22 and 28 resulted in significant increase in the DNA content
in G2/M phase and subsequent reduction in G1 phase of the cell cycle (Figure 3). The
percentage of cells in the G2/M phase after the treatment with the compounds 16, 22 and 28
were 41%, 49.2% and 43.1%, respectively, in comparison to the control (27.2%). These
results demonstrate that the compound-induced G2/M phase of cell cycle arrest may be one
of the possible mechanisms of cell growth inhibition.
< Figure 3 >
2.6 Apoptosis studies
After G2/M arrest, many of the tumor cells, for instance lung cancer cells, exhibit
morphologic changes consistent with apoptosis, i.e., membrane blebbing, the appearance of a
rounded morphology, and eventually detachment from the surface of the tissue culture dish
[26]. It has been previously suggested that G2/M cell cycle arrest leads to apoptosis in a
number of cancer cell lines [27-28]. Therefore it was considered of interest to examine the
apoptosis inducing effect of these active compounds in A549 cells.
2.6.1 Hoechst Staining
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To investigate the apoptosis inducing effect of these compounds on the basis of
morphological changes of the cellular nucleus, we carried out a Hoechst staining assay.
Hoechst 33242 is a cell permeable, fluorescent nucleus staining dye that stains apoptotic cells
as bright blue because of nuclear condensation [29]. A549 cells were incubated with the
compounds 16, 22 and 28 for 24 h and stained with Hoechst 33242. Figure 4 shows that the
compound-treated cells reflect distinct pattern of morphological features such as chromatin
condensation and nuclear fragmentation (as indicated by arrowheads), which are typical
hallmarks of apoptosis; whereas the untreated control cells displayed uniform rounded
nucleus. Compounds 16, 22 and 28 induced varying degrees of apoptosis with compounds 22
being the strongest inducer of morphologic changes characteristic of apoptosis in A549 cells.
< Figure 4 >
2.6.2 Effect on mitochondrial membrane potential (DΨm)
Maintenance of mitochondrial membrane potential is essential for mitochondrial integrity and
cellular bioenergetics [30]. Loss of mitochondrial membrane functions results in release of
cytochrome C into the cytosol, which leads to apoptosis. Therefore, to investigate the effect
of these compounds on DΨm, we carried out rhodamine 123 staining [31]. Mitochondria that
maintain normal DΨm retain the rhodamine fluorescence, whereas depolarised mitochondria
are unable to retain the rhodamine that results in loss of rhodamine green fluorescence. Thus,
the loss of DΨm can be monitored by a shift in green fluorescence intensity. As shown in
Figure 5, cells treated with the compounds 16, 22 and 28 displayed a significant 30-46% loss
of DΨm in comparison to the untreated cells. Among these, compounds 22 and 28 induced
45.8 and 43.5% loss of DΨm, respectively, followed by the compound 16 (30%). This
suggests that the compounds are able to induce apoptosis through the collapse of
mitochondrial membrane potential in A549 lung cancer cells.
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< Figure 5 >
2.6.3 Effect on intracellular reactive oxygen species (ROS) levels
Reactive oxygen species are reported to be involved in early stages of apoptosis in many
cellular systems. Further, decrease in DΨm typically damages mitochondrial electron
transport chain, which results in elevation of intracellular ROS [32]. To determine whether
this event occurs during apoptosis induction by our compounds, we examined intracellular
ROS production using DCFDA. DCFDA is a cell permeable non-fluorescent dye, which upon
oxidation by intracellular esterases, converts into a green fluorescent DCF. Therefore, the
increase in green fluorescence intensity represents the increased production of ROS. Figure 6
indicates that cells treated with the compounds 16, 22 and 28 display significantly higher
green fluorescence intensity compared to the untreated cells. Quantitative analysis by
fluorescence spectrofluorimetry revealed that the compounds treatment resulted in 2.1-2.8
fold increase in green fluorescence intensity, which suggests that elevated intracellular ROS
levels may also play an important role in the cytotoxicity and apoptosis induced by these
compounds.
< Figure 6 >
3.0 Conclusion
In the present work, a series of (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-(phenylamino)prop-2-
en-1-one derivatives were synthesized and evaluated for their anticancer activity against four
human tumor cells and an additional non-cancerous cell. The compounds 16, 22 and 28
displayed potent and broad spectrum of growth inhibition on all the tested cancer cells. The
SAR study demonstrated that electron donating groups at para position of aromatic B ring is
essential for the activity of these derivatives, which led to the selection of the most active
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compounds in the series. The compounds inhibit the in vitro cellular migration of A549 lung
cancer cells through disruption of cytoskeleton. Further biological assessments of 16, 22 and
28 revealed that these compounds trigger cell cycle arrest in G2/M phase and induce
apoptosis through depolarization of mitochondrial membrane potential and through enhanced
production of ROS in lung cancer cells. These findings suggest that (Z)-1-(1,3-diphenyl-1H-
pyrazol-4-yl)-3-(phenylamino)prop-2-en-1-one derivatives offer promising leads for the
further development of anticancer agents.
4.0 Experimental Section
All the reagents and solvents were of commercial grade and used as received. The progress of
the reactions was monitored by thin layer chromatography (TLC) performed on silica gel
glass plates containing 60 F-254, and visualization on TLC was achieved by UV light or
iodine indicator. Melting points were measured with an Electro-thermal melting point
1
apparatus, and are uncorrected. H and 13C NMR spectra were recorded on INOVA (400
MHz) or Gemini Varian-VXR-Unity (200 MHz) or Bruker UXNMR/XWIN-NMR (300
MHz) instruments. ESI spectra were recorded on Micro mass, Quattro LC using ESI+
software with capillary voltage of 3.98 kV and ESI mode positive ion trap detector.
4.1 General procedure for the synthesis of 1-(1,3-diphenyl-1H-pyrazol-4-yl)prop-2-yn-1-
one derivatives (6a-e)
The 1,3-diphenyl-1H-pyrazole-4-carboxaldehydes (4a-e), 1-(1,3-diphenyl-1H-pyrazol-4-
yl)prop-2-yn-1-ol derivatives (5a-e), and their corresponding oxidation products 1-(1,3-
diphenyl-1H-pyrazol-4-yl)prop-2-yn-1-one derivatives (6a-e) were synthesized according to
our previously reported methods [14, 15].
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4.2
General
synthesis
of
(Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-((4-
fluorophenyl)amino)prop-2-en-1-ones
To a solution of ethynylketones 6a-e (1 mmol) in absolute ethanol, different substituted
arylamines (1 mmol) were added at room temperature and the reaction was continued for 4 h
at room temperature. After the completion of reaction (checked by TLC), the mixture was
diluted with water and the products were filtered. The obtained crude products (7-36) were
purified through column chromatography using Hexane/Ethyl acetate as eluent system.
4.2.1 (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-((4-fluorophenyl)amino)prop-2-en-1-one (7)
Light Brown solid, yield 74%, mp: 164-166 ºC; 1H NMR (300 MHz, CDCl3) δ 11.92 (d, J =
12.0 Hz, 1H), 8.45 (s, 1H), 7.87 – 7.67 (m, 4H), 7.56 – 7.43 (m, 4H), 7.41 – 7.30 (m, 2H),
13
7.28 (s, 1H), 7.21 (dd, J = 12.0, 7.8 Hz, 1H), 7.00 (m, 3H), 5.51 (d, J = 7.7 Hz, 1H). C
NMR (75 MHz, CDCl3) δ 186.2, 160.7, 157.5, 152.6, 144.3, 139.4, 136.6, 133.0, 130.0,
129.5, 128.5, 128.2, 127.1, 123.9, 119.4, 117.7, 116.6, 116.3, 96.7. MS (ESI): 384 [M+H]+;
HRMS (ESI) calculated for C24H19ON3F [M+H]+; 384.15067; found: 384.15110.
4.2.2
(8)
(
Z)-3-((4-chlorophenyl)amino)-1-(1,3-diphenyl-1H-pyrazol-4-yl)prop-2-en-1-one
1
White solid, yield 77%, mp: 196-198 ºC; H NMR (300 MHz, CDCl3) δ 11.89 (d, J = 12.1
Hz, 1H), 8.45 (s, 1H), 7.87 – 7.72 (m, 4H), 7.55 – 7.44 (m, 5H), 7.36 (t, J = 7.4 Hz, 1H), 7.32
13
– 7.25 (m, 2H), 7.19 (s, 1H), 7.02 – 6.94 (m, 2H), 5.53 (d, J = 7.9 Hz, 1H). C NMR (75
MHz, CDCl3) δ 186.4, 152.7, 143.4, 139.4, 139.0, 133.0, 130.0, 129.7, 129.5, 129.5, 128.6,
128.4, 128.2, 127.2, 124.0, 119.4, 117.2, 97.3. MS (ESI): 400 [M+H]+; HRMS (ESI)
calculated for C24H19ON3Cl [M+H]+; 400.12112; found: 400.12131.
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4.2.3 (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-((4-(trifluoromethyl)phenyl)amino)prop-2-
en-1-one (9)
1
Pale yellow solid, yield 68%, mp: 145-148 ºC; H NMR (300 MHz, CDCl3) δ 11.83 (d, J =
12.0 Hz, 1H), 8.36 (s, 1H), 7.76-7.63 (m, 4H), 7.48 (d, J = 8.5 Hz, 2H), 7.45 – 7.32 (m, 5H),
7.28 (d, J = 7.3 Hz, 1H), 7.18 (dd, J = 7.1, 4.8 Hz, 1H), 7.00 (d, J = 8.4 Hz, 2H), 5.50 (d, J =
13
8.0 Hz, 1H). C NMR (75 MHz, CDCl3) δ 186.7, 152.8, 143.2, 142.5, 139.3, 132.9, 130.2,
129.5, 128.7, 128.2, 127.3, 127.1, 125.9, 125.1, 124.7, 123.8, 119.5, 115.5, 98.3. MS (ESI):
434 [M+H]+; HRMS (ESI) calculated for C25H19ON3F3 [M+H]+; 434.14747 found:
434.14636.
4.2.4 (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-((4-hydroxyphenyl)amino)prop-2-en-1-one
(8)
1
Brown solid, yield 81%, mp: 157-159 ºC; H NMR (300 MHz, CDCl3) δ 11.89 (d, J = 12.2
Hz, 1H), 8.41 (s, 1H), 7.81-7.69 (m, 4H), 7.45 (t, J = 7.6 Hz, 2H), 7.31 (t, J = 7.4 Hz, 1H),
7.19 (dd, J = 11.9, 7.6 Hz, 1H), 7.00 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 6.77 (d, J =
8.7 Hz, 2H), 5.49 (d, J = 7.8 Hz, 1H). 13C NMR (75 MHz, CDCl3+DMSO-d6) δ 184.4, 153.2,
151.3, 144.3, 138.4, 132.2, 131.5, 129.1, 128.6, 128.4, 127.1, 126.1, 123.0, 118.1, 116.9,
115.5, 94.6. MS (ESI): 382 [M+H]+; HRMS (ESI) calculated for C24H20O2N3 [M+H]+;
382.15500; found: 382.15557.
4.2.5 (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-((4-methoxyphenyl)amino)prop-2-en-1-one
(11)
1
Brown solid, yield 82%, mp: 139-141 ºC; H NMR (300 MHz, CDCl3) δ 11.84 (d, J = 11.8
Hz, 1H), 8.36 (s, 1H), 7.78-7.62 (m, 4H), 7.51-7.38 (m, 5H), 7.30 – 7.02 (m, 2H), 6.92 (d, J =
13
8.4 Hz, 2H), 6.79 (d, J = 8.2 Hz, 2H), 5.36 (d, J = 7.5 Hz, 1H), 3.70 (s, 3H). C NMR (75
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MHz, CDCl3) δ 185.8, 156.2, 152.5, 145.0, 139.4, 133.8, 133.1, 129.8, 129.5, 128.5, 128.2,
127.1, 124.2, 119.4, 117.8, 114.9, 96.0, 55.5. MS (ESI): 396 [M+H]+; HRMS (ESI)
calculated for C25H22O2N3 [M+H]+; 396.17065; found: 396.17065.
4.2.6 (Z)-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-((3,4,5-trimethoxyphenyl)amino)prop-2-en-
1-one (12)
1
Brown solid, yield 75%, mp: 152-154 ºC; H NMR (300 MHz, CDCl3) δ 11.93 (d, J = 12.1
Hz, 1H), 8.46 (d, J = 7.8 Hz, 1H), 7.89-7.76 (m, 4H), 7.57 – 7.41 (m, 5H), 7.36 (t, J = 7.3 Hz,
1H), 7.27 – 7.20 (m, 1H), 6.28 (s, 2H), 5.51 (d, J = 7.8 Hz, 1H), 3.87 (s, 6H), 3.83 (s, 3H).
13C NMR (75 MHz, CDCl3) δ 186.0, 154.1, 152.7, 144.1, 139.4, 136.5, 134.5, 133.1, 129.8,
129.5, 128.7, 128.3, 127.2, 124.0, 119.4, 96.5, 93.8, 61.0, 56.1. MS (ESI): 456 [M+H]+;
HRMS (ESI) calculated for C27H26O4N3 [M+H]+; 456.19178; found: 456.19088.
4.2.7
(Z)-1-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((4-
fluorophenyl)amino)prop-2-en-1-one (13)
1
Pale yellow, yield 77%, mp: 196-198 ºC; H NMR (500 MHz, CDCl3) δ 11.89 (d, J = 12.0
Hz, 1H), 8.42 (s, 1H), 7.81-7.69 (m, 4H), 7.57-7.48 (m, 2H), 7.41 (d, J = 8.2 Hz, 2H), 7.39 (t,
J = 7.3 Hz, 1H), 7.23 – 7.18 (m, 1H), 6.99 (dd, J = 10.6, 6.4 Hz, 4H), 5.48 (d, J = 7.8 Hz,
1H). 13C NMR (75 MHz, CDCl3) δ 185.8, 161.9, 157.4, 151.4, 144.6, 139.2, 136.5, 134.6,
131.5, 130.7, 130.1, 128.4, 127.5, 123.9, 119.4, 117.7, 116.6, 116.3, 96.4. MS (ESI): 402
[M+H]+; HRMS (ESI) calculated for C24H18ON3F2 [M+H]+; 402.14125; found: 402.14100.
4.2.8
(Z)-3-((4-chlorophenyl)amino)-1-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-
yl)prop-2-en-1-one (14)
Light yellow solid, yield 70%, mp: 157-159 ºC; 1H NMR (300 MHz, CDCl3) δ 11.87 (d, J =
12.1 Hz, 1H), 8.42 (s, 1H), 7.86 – 7.72 (m, 3H), 7.51 (t, J = 7.8 Hz, 2H), 7.37 (t, J = 7.4 Hz,
14

ACCEPTED MANUSCRIPT
1H), 7.29 (dt, J = 8.5, 5.2 Hz, 4H), 7.21 – 7.13 (m, 2H), 7.08-7.01 (m, 2H), 5.55 (d, J = 7.9
Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 185.9, 151.8, 143.5, 139.3, 138.9, 131.3, 131.2,
130.0, 129.7, 129.6, 129.0, 128.5, 127.3, 123.7, 119.4, 117.3, 115.3, 115.0, 97.0. MS (ESI):
418 [M+H]+; HRMS (ESI) calculated for C24H18ON3ClF [M+H]+; 418.11169; found:
418.11105.
4.2.9
(Z)-1-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((4
(trifluoromethyl)
phenyl) amino) prop-2-en-1-one (15)
1
Brown solid, yield 66%, mp: 168-171 ºC; H NMR (300 MHz, CDCl3) δ 11.89 (d, J = 12.0
Hz, 1H), 8.43 (s, 1H), 7.85–7.66 (m, 5H), 7.57-7.43 (m, 3H), 7.18 (d, J = 8.6 Hz, 2H), 7.07-
6.99 (m, 2H), 6.81 (d, J = 8.7 Hz, 2H), 5.59 (d, J = 7.9 Hz, 1H). 13C NMR (75 MHz, CDCl3)
δ 187.7, 160.3, 153.3, 152.7, 142.3, 139.1, 135.3, 130.7, 129.5, 127.9, 127.2, 127.1, 123.9,
122.3, 119.8, 119.4, 115.5, 113.6, 81.6. MS (ESI): 452 [M+H]+; HRMS (ESI) calculated for
C25H18ON3F4 [M+H]+ 452.13824; found: 452.13828.
4.2.10
(Z)-1-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((4-
hydroxyphenyl)amino)prop-2-en-1-one (16)
1
Brown solid, yield 71%, mp: 191-193 ºC; H NMR (300 MHz, CDCl3) δ 11.91 (d, J = 12.1
Hz, 1H), 8.43 (s, 1H), 7.78 – 7.76 (m, 4H), 7.47 (t, J = 8.0 Hz, 2H), 7.36 (t, J = 7.3 Hz, 1H),
7.17-7.03 (m, 3H), 6.78 (d, J = 8.9 Hz, 2H), 6.63-6.54 (m, 2H) 5.51 (d, J = 7.8 Hz, 1H). 13C
NMR (75 MHz, CDCl3+DMSO-d6) δ 184.1, 163.3, 160.1, 153.1, 150.3, 144.3, 138.2, 131.3,
130.1, 129.0, 128.5, 128.3, 126.0, 122.6, 117.9, 116.8, 115.4, 113.9, 113.7, 94.2. MS (ESI):
400 [M+H]+; HRMS (ESI) calculated for C24H19O2N3F [M+H]+; 400.14558; found:
400.14553
15

ACCEPTED MANUSCRIPT
4.2.11
(Z)-1-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((4-
methoxyphenyl)amino)prop-2-en-1-one (17)
1
Yellow solid, yield 73%, mp: 149-151 ºC; H NMR (300 MHz, CDCl3) δ 11.92 (d, J = 12.3
Hz, 1H), 8.42 (s, 1H), 7.85 – 7.73 (m, 5H), 7.51 (t, J = 7.8 Hz, 2H), 7.36 (t, J = 7.4 Hz, 1H),
7.17 (t, J = 8.7 Hz, 2H), 7.03 (d, J = 9.0 Hz, 2H), 6.89 (d, J = 8.9 Hz, 2H), 5.48 (d, J = 7.7
Hz, 1H), 3.81 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 185.6, 164.7, 156.3, 151.6, 145.0, 139.4,
133.8, 131.3, 131.2, 129.8, 129.5, 127.2, 123.9, 119.4, 117.8, 115.3, 115.0, 95.7, 55.5. MS
(ESI): 414 [M+H]+; HRMS (ESI) calculated for C25H21O2N3F [M+H]+ 414.16103; found:
414.16103.
4.2.12
(Z)-1-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((3,4,5-
trimethoxyphenyl)amino)prop-2-en-1-one (18)
1
Yellow solid, yield 79%, mp: 147-149 ºC; H NMR (300 MHz, CDCl3) δ 11.90 (d, J = 12.2
Hz, 1H), 8.42 (s, 1H), 7.82-7.75 (m, 4H), 7.51 (t, J = 7.8 Hz, 2H), 7.37 (t, J = 7.4 Hz, 1H),
7.33 – 7.23 (m, 1H), 7.16-7.07 (m, 2H), 6.29 (s, 2H), 5.53 (d, J = 7.8 Hz, 1H), 3.87 (s, 6H),
13
3.83 (s, 3H). C NMR (75 MHz, CDCl3) δ 185.8, 154.1, 151.8, 144.3, 139.3, 136.4, 134.5,
131.3, 129.8, 129.5, 129.1, 127.3, 123.7, 119.4, 115.3, 115.0, 96.2, 93.9, 61.0, 56.2. MS
(ESI): 474 [M+H]+; HRMS (ESI) calculated for C27H25O4N3F [M+H]+; 474.18236; found:
474.18135.
4.2.13
(Z)-1-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((4-
fluorophenyl)amino)prop-2-en-1-one (19)
1
Yellow solid, yield 68%, mp: 153-155 ºC; H NMR (500 MHz, CDCl3) δ 11.87 (d, J = 12.2
Hz, 1H), 8.40 (s, 1H), 7.78-7.61 (m, 4H), 7.52-7.47 (m, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.36 (t,
J = 7.4 Hz, 1H), 7.25–7.21 (m, 1H), 7.04 (dd, J = 10.6, 6.4 Hz, 4H), 5.52 (d, J = 7.8 Hz, 1H).
16

ACCEPTED MANUSCRIPT
13C NMR (75 MHz, CDCl3) δ 185.8, 160.8, 157.5, 151.5, 144.6, 139.3, 136.5, 134.6, 131.4,
130.7, 129.9, 129.6, 128.4, 127.4, 123.9, 119.4, 117.7, 116.6, 116.3, 96.5. MS (ESI): 418
[M+H]+; HRMS (ESI) calculated for C24H18ON3ClF [M+H]+; 418.11169; found: 418.11110.
4.2.14
(Z)-1-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((4-
chlorophenyl)amino)prop-2-en-1-one (20)
1
Pale yellow solid, yield 71%, mp: 176-178 ºC; H NMR (500 MHz, CDCl3) δ 11.85 (d, J =
12.2 Hz, 1H), 8.40 (s, 1H), 7.79–7.71 (m, 4H), 7.55–7.46 (m, 2H), 7.46–7.42 (m, 2H), 7.36 (t,
J = 7.4 Hz, 1H), 7.30–7.28 (m, 2H), 7.24 (s, 1H), 6.99 (dd, J = 9.2, 2.4 Hz, 2H), 5.54 (d, J =
13
7.9 Hz, 1H). C NMR (75 MHz, CDCl3) δ 185.8, 151.1, 144.3, 139.5, 136.6, 134.9, 131.4,
130.7, 129.8, 129.6, 128.2, 127.6, 127.4, 123.9, 119.8, 117.7, 116.5, 116.3, 95.8. MS (ESI):
434 [M+H]+; HRMS (ESI) calculated for C24H18ON3Cl2 [M+H]+ 434.08214; found:
434.08242.
4.2.15
(Z)-1-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((4-
(trifluoromethyl)phenyl)amino)prop-2-en-1-one (21)
1
Pale yellow solid, yield 78%, mp: 183-185 ºC; H NMR (300 MHz, CDCl3) δ 11.87 (d, J =
12.1 Hz, 1H), 8.42 (s, 1H), 7.81–7.68 (m, 4H), 7.57-7.43 (m, 2H), 7.38-7.28 (m, 2H), 7.19 (d,
J = 8.6 Hz, 2H), 7.11-7.03 (m, 2H), 6.83 (d, J = 8.6 Hz, 2H), 5.56 (d, J = 7.8 Hz, 1H). 13C
NMR (75 MHz, CDCl3) δ 187.9, 152.8, 142.3, 139.2, 135.3, 130.9, 129.8, 128.1, 127.8,
127.5, 123.9, 122.3, 119.8, 119.4, 115.5, 113.6, 81.9. MS (ESI): 468 [M+H]+; HRMS (ESI)
calculated for C25H18ON3F3Cl [M+H]+ 468.10900; found: 468.10911.
4.2.16
(Z)-1-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((4-
hydroxyphenyl)amino)prop-2-en-1-one (22)
17

ACCEPTED MANUSCRIPT
1
Yellow solid, yield 69%, mp: 158-160 ºC; H NMR (500 MHz, CDCl3) δ 11.89 (d, J = 12.1
Hz, 1H), 8.42 (s, 1H), 7.89–7.78 (m, 2H), 7.55–7.53 (m, 3H), 7.47–7.41 (m, 2H), 7.37 (t, J =
7.4 Hz, 1H), 7.18–7.09 (m, 2H), 7.01 (s, 1H), 6.53 (d, J = 8.2 Hz, 2H), 5.57 (d, J = 7.8 Hz,
1H). 13C NMR (75 MHz, CDCl3+DMSO-d6) δ 184.1, 153.2, 150.2, 144.5, 138.3, 133.1,
131.4, 130.8, 129.8, 129.1, 128.6, 127.1, 126.2, 122.8, 118.2, 116.9, 115.5, 94.3. MS (ESI):
416 [M+H]+; HRMS (ESI) calculated for C24H19O2N3Cl [M+H]+ 416.11603; found:
416.11562.
4.2.17
(Z)-1-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((4-
methoxyphenyl)amino)prop-2-en-1-one (23)
1
Pale yellow solid, yield 71%, mp: 178-180 ºC; H NMR (300 MHz, CDCl3) δ 11.89 (d, J =
12.0 Hz, 1H), 8.42 (s, 1H), 7.81–7.69 (m, 4H), 7.53 (t, J = 7.8 Hz, 2H), 7.38 (d, J = 7.6 Hz,
2H), 7.36 –7.28 (m, 4H), 7.00 (t, J = 8.4 Hz, 2H), 5.51 (d, J = 7.9 Hz, 1H), 3.87 (s, 3H). 13C
NMR (75 MHz, CDCl3) δ 186.4, 153.4, 143.5, 143.2, 139.4, 139.1, 138.9, 130.6, 129.9,
129.7, 129.3, 128.6, 127.2, 125.3, 123.7, 119.4, 117.4, 117.2, 113.5, 97.5, 55.3. MS (ESI):
430 [M+H]+; HRMS (ESI) calculated for C25H21O2N3Cl [M+H]+ 430.13168; found:
430.13107.
4.2.18
(Z)-1-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((3,4,5-
trimethoxyphenyl)amino)prop-2-en-1-one (24)
1
Pale yellow solid, yield 76%, mp: 171-173 ºC; H NMR (500 MHz, CDCl3) δ 11.88 (d, J =
12.2 Hz, 1H), 8.40 (s, 1H), 7.81–7.69 (m, 4H), 7.50 (t, J = 7.9 Hz, 2H), 7.44 (d, J = 8.4 Hz,
2H), 7.36 (t, J = 7.4 Hz, 1H), 7.28 (dd, J = 9.8, 5.3 Hz, 1H), 6.28 (s, 2H), 5.51 (d, J = 7.8 Hz,
1H), 3.85 (s, 6H), 3.81 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 185.8, 154.1, 151.6, 144.4,
139.3, 136.4, 134.6, 134.5, 131.4, 130.7, 129.9, 129.5, 128.5, 127.4, 123.8, 119.4, 96.2, 93.8,
18

ACCEPTED MANUSCRIPT
61.0, 56.2. MS (ESI): 490 [M+H]+; HRMS (ESI) calculated for C27H25O4N3Cl [M+H]+
490.15236; found: 490.15258.
4.2.19
(Z)-3-((4-fluorophenyl)amino)-1-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)prop-2-en-1-one (25)
Yellow solid, yield 82%, mp: 169-171 ºC; 1H NMR (300 MHz, CDCl3) δ 11.91 (d, J = 12.2
Hz, 1H), 8.43 (s, 1H), 7.80 (d, J = 7.8 Hz, 2H), 7.72 (dd, J = 9.2, 2.3 Hz, 2H), 7.50 (t, J = 7.9
Hz, 2H), 7.36 (t, J = 7.4 Hz, 1H), 7.22 (dd, J = 12.3, 7.9 Hz, 1H), 7.07-6.97 (m, 6H), 5.54 (d,
J = 7.8 Hz, 1H), 3.89 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 185.2, 158.9, 151.3, 143.5,
138.4, 135.8, 129.7, 129.3, 128.6, 126.2, 124.4, 122.5, 118.2, 116.7, 115.6, 115.3, 112.6,
95.7, 54.3. MS (ESI): 414 [M+H]+; HRMS (ESI) calculated for C25H21O2N3F [M+H]+
414.16123; found: 414.16093.
4.2.20 (Z)-3-((4-chlorophenyl)amino)-1-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)prop-2-en-1-one (26)
1
Yellow solid, yield 78%, mp: 187-189 ºC; H NMR (300 MHz, CDCl3) δ 11.88 (d, J = 12.1
Hz, 1H), 8.43 (s, 1H), 7.79 (d, J = 7.6 Hz, 2H), 7.74 – 7.69 (m, 2H), 7.50 (t, J = 7.9 Hz, 2H),
7.36 (d, J = 7.4 Hz, 2H), 7.32–7.26 (m, 4H), 7.00 (t, J = 8.6 Hz, 2H), 5.56 (d, J = 7.9 Hz,
1H), 3.88 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 186.5, 160.0, 152.5, 143.4, 143.1, 139.4,
139.0, 138.8, 130.7, 129.9, 129.7, 129.5, 128.3, 127.2, 125.3, 123.7, 119.4, 117.2, 117.1,
113.6, 97.3, 55.3. MS (ESI): 430 [M+H]+; 452 [M+Na]+; HRMS (ESI) calculated for
C25H20O2N3ClNa [M+Na]+ 452.11363; found: 452.11365.
4.2.21
(Z)-1-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((4-
(trifluoromethyl)phenyl)amino)prop-2-en-1-one (27)
19

ACCEPTED MANUSCRIPT
1
Brown solid, yield 67%, mp: 148-150 ºC; H NMR (300 MHz, CDCl3) δ 11.93 (d, J = 12.0
Hz, 1H), 8.45 (s, 1H), 7.90 (d, J = 8.8 Hz, 2H), 7.86–7.68 (m, 4H), 7.54-7.41 (m, 2H), 7.11
(d, J = 8.4 Hz, 2H), 7.01 (t, J = 8.9 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 5.63 (d, J = 7.9 Hz,
1H), 3.89 (s, 3H).13C NMR (75 MHz, CDCl3) δ 188.0, 169.3, 160.4, 160.1, 153.2, 152.7,
142.4, 138.9, 135.3, 130.7, 129.5, 127.9, 127.2, 127.0, 123.9, 122.3, 119.8, 119.4, 115.5,
113.6, 81.7, 55.4. MS (ESI): 464 [M+H]+; HRMS (ESI) calculated for C26H21O2N3F3 [M+H]+
411.1014; found: 464.15874.
4.2.22 (Z)-3-((4-hydroxyphenyl)amino)-1-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-
yl)prop-2-en-1-one ( 28)
1
Brown solid, yield 79%, mp: 151-153 ºC; H NMR (300 MHz, CDCl3) δ 11.88 (d, J = 12.3
Hz, 1H), 8.40 (s, 1H), 7.73 (dd, J = 16.2, 8.2 Hz, 4H), 7.47 (t, J = 7.8 Hz, 2H), 7.33 (t, J = 7.4
Hz, 1H), 7.20 (dd, J = 12.0, 7.8 Hz, 1H), 7.00 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H),
6.80 (d, J = 8.8 Hz, 2H), 5.46 (d, J = 7.7 Hz, 1H), 3.87 (s, 3H). 13C NMR (75 MHz, CDCl3) δ
186.0, 159.9, 152.9, 152.4, 145.4, 139.4, 133.3, 130.7, 129.9, 129.5, 127.1, 125.4, 123.8,
119.4, 118.0, 116.6, 113.7, 95.9, 55.3. MS (ESI): 412 [M+H]+; 434 [M+Na]+; HRMS (ESI)
calculated for C25H21O3N3Na [M+Na]+ 434.14751; found: 434.14745.
4.2.23
(Z)-1-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((4-
methoxyphenyl)amino)prop-2-en-1-one (29)
1
Yellow solid, yield 82%, mp: 159-161 ºC; H NMR (300 MHz, CDCl3) δ 11.93 (d, J = 12.2
Hz, 1H), 8.42 (s, 1H), 7.76 (dd, J = 18.5, 8.0 Hz, 4H), 7.50 (t, J = 7.5 Hz, 2H), 7.41 – 7.17
(m, 2H), 7.01 (d, J = 7.1 Hz, 4H), 6.89 (d, J = 8.6 Hz, 2H), 5.49 (d, J = 7.6 Hz, 1H), 3.89 (s,
3H), 3.81 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 186.0, 160.0, 156.1, 152.3, 144.7, 139.4,
133.8, 130.6, 129.7, 129.5, 127.0, 125.5, 124.0, 119.3, 117.7, 114.9, 113.6, 96.0, 55.5, 55.3.
20

ACCEPTED MANUSCRIPT
MS (ESI): 426 [M+H]+ and 448 [M+Na]+ ; HRMS (ESI) calculated for C26H24O3N3 [M +H]+
426.18122; found: 426.18042
4.2.24
(Z)-1-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3-((3,4,5-
trimethoxyphenyl)amino)prop-2-en-1-one (30)
1
Yellow solid, yield 75%, mp: 148-150 ºC; H NMR (300 MHz, CDCl3) δ 11.91 (d, J = 12.2
Hz, 1H), 8.42 (s, 1H), 7.78 (d, J = 7.9 Hz, 2H), 7.72 (d, J = 8.6 Hz, 2H), 7.49 (t, J = 7.8 Hz,
2H), 7.41 – 7.20 (m, 1H), 7.01 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.6 Hz, 1H), 6.28 (s, 2H),
5.54 (d, J = 7.8 Hz, 1H), 3.88 (s, 3H), 3.86 (s, 6H), 3.81 (s, 3H). 13C NMR (75 MHz, CDCl3)
δ 186.2, 159.9, 154.1, 152.5, 144.0, 139.4, 136.5, 134.3, 130.7, 129.8, 129.5, 127.1, 125.4,
123.7, 119.4, 113.6, 96.5, 93.7, 61.0, 56.1, 55.3. MS (ESI): 486 [M+H]+; 508 [M+Na]+;
HRMS (ESI) calculated for C28H27O5N3Na [M+Na]+ 508.18429; found: 508.18409.
4.2.25
(Z)-3-((4-fluorophenyl)amino)-1-(1-phenyl-3-(3,4,5-trimethoxyphenyl)-1H-
pyrazol-4-yl)prop-2-en-1-one (31)
1
Pale yellow solid, yield 71%, mp: 169-171 ºC; H NMR (300 MHz, CDCl3) δ 11.87 (d, J =
12.2 Hz, 1H), 8.36 (s, 1H), 7.71 (d, J = 7.8 Hz, 2H), 7.41 (t, J = 7.8 Hz, 2H), 7.27 (t, J = 7.3
Hz, 1H), 7.17 (d, J = 5.9 Hz, 1H), 7.15 (d, J = 4.4 Hz, 1H), 7.11 (s, 1H), 7.01 – 6.81 (m, 4H),
5.45 (d, J = 7.8 Hz, 1H), 3.83 (s, 9H). 13C NMR (75 MHz, CDCl3) δ 186.1, 160.8, 157.6,
153.0, 152.4, 144.3, 139.3, 138.5, 136.6, 130.0, 129.5, 128.3, 127.3, 124.0, 119.4, 117.6,
116.6, 116.3, 106.9, 96.8, 61.0, 56.2. MS (ESI): 474 [M+H]+; HRMS (ESI) calculated for
C27H25O4N3F [M+H ]+ 474.18236; found: 474.18130.
4.2.26
(Z)-3-((4-chlorophenyl)amino)-1-(1-phenyl-3-(3,4,5-trimethoxyphenyl)-1H-
pyrazol-4-yl)prop-2-en-1-one (32)
21

ACCEPTED MANUSCRIPT
1
Pale yellow solid, yield 68%, mp: 185-187 ºC; H NMR (300 MHz, CDCl3) δ 11.82 (d, J =
12.1 Hz, 1H), 8.33 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H), 7.39 (t, J = 7.8 Hz, 2H), 7.25 (t, J = 7.4
Hz, 1H), 7.21 – 7.08 (m, 3H), 6.89 (s, 3H), 6.85 (s, 1H), 5.45 (d, J = 7.9 Hz, 1H), 3.80 (s,
13
9H). C NMR (75 MHz, CDCl3) δ 186.4, 152.9, 152.5, 143.5, 139.3, 138.9, 138.5, 130.1,
129.7, 129.5, 128.5, 128.3, 127.3, 124.0, 119.4, 117.2, 106.8, 97.4, 60.9, 56.2. MS (ESI): 490
[M+H]+; HRMS (ESI) calculated for C27H25O4N3Cl [M+H ]+ 490.15281; found: 490.15275.
4.2.27
(Z)-1-(1-phenyl-3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-4-yl)-3-((4-
(trifluoromethyl)phenyl)amino)prop-2-en-1-one (33)
1
Pale yellow solid, yield 79%, mp: 191-193 ºC; H NMR (500 MHz, CDCl3) δ 11.97 (d, J =
12.0 Hz, 1H), 8.46 (s, 1H), 7.80 (dd, J = 10.4, 9.6 Hz, 2H), 7.57 (t, J = 9.1 Hz, 2H), 7.50 (t, J
= 7.9 Hz, 2H), 7.37 (d, J = 7.4 Hz, 1H), 7.36 – 7.29 (m, 1H), 7.11 (d, J = 8.4 Hz, 2H), 6.99 (s,
2H), 5.62 (d, J = 8.0 Hz, 1H), 3.92 (s, 3H), 3.91 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 186.7,
153.1, 152.6, 143.1, 142.5, 139.2, 138.6, 130.2, 129.6, 128.1, 127.4, 127.1, 123.8, 119.5,
115.5, 106.8, 98.4, 60.9, 56.2. MS (ESI): 524 [M+H]+; HRMS (ESI) calculated for
C28H25O4N3F3 [M+H]+ 524.17917; found: 524.17888.
4.2.28
(Z)-3-((4-hydroxyphenyl)amino)-1-(1-phenyl-3-(3,4,5-trimethoxyphenyl)-1H-
pyrazol-4-yl)prop-2-en-1-one (34)
1
White solid, yield 73%, mp: 171-173 ºC; H NMR (300 MHz, CDCl3) δ 11.95 (d, J = 12.4
Hz, 1H), 8.45 (s, 1H), 7.78 (d, J = 7.7 Hz, 2H), 7.49 (t, J = 7.8 Hz, 2H), 7.36 (t, J = 7.4 Hz,
1H), 7.22 (dd, J = 9.0, 3.2 Hz, 1H), 7.00 (s, 2H), 6.92 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 8.8 Hz,
2H), 5.48 (d, J = 7.7 Hz, 1H), 3.92 (s, 3H), 3.91 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 185.7,
152.9, 152.3, 145.3, 139.3, 138.4, 133.3, 130.0, 129.5, 128.4, 127.2, 124.1, 119.5, 118.0,
116.5, 106.8, 95.9, 60.9, 56.2. MS (ESI): 472 [M+H]+; HRMS (ESI) calculated for
C27H26O5N3 [M+H]+ 472.18670; found: 472.18583.
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4.2.29
(Z)-3-((4-methoxyphenyl)amino)-1-(1-phenyl-3-(3,4,5-trimethoxyphenyl)-1H-
pyrazol-4-yl)prop-2-en-1-one (35)
1
White solid, yield 81%, mp: 178-180 ºC; H NMR (300 MHz, CDCl3) δ 12.00 (d, J = 12.3
Hz, 1H), 8.45 (s, 1H), 7.80 (d, J = 7.9 Hz, 2H), 7.50 (t, J = 7.7 Hz, 2H), 7.36 (t, J = 7.3 Hz,
1H), 7.31 – 7.18 (m, 2H), 7.01 (d, J = 5.0 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 5.50 (d, J = 7.7
Hz, 1H), 3.90 (d, J = 16.7 Hz, 9H), 3.80 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 185.7, 156.3,
153.0, 152.3, 144.9, 139.42, 138.4, 133.8, 129.9, 129.5, 128.5, 127.2, 124.3, 119.4, 117.7,
115.0, 106.9, 96.1, 60.9, 56.2, 55.5. MS (ESI): 486 [M+H]+; HRMS (ESI) calculated for
C28H28O5N3 [M+H]+ 486.20235; found: 486.20118.
4.2.30
(Z)-1-(1-phenyl-3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-4-yl)-3-((3,4,5-
trimethoxyphenyl)amino)prop-2-en-1-one (36)
1
White solid, yield 84%, mp: 169-171 ºC; H NMR (500 MHz, CDCl3) δ 11.96 (d, J = 12.2
Hz, 1H), 8.45 (s, 1H), 7.79 (d, J = 7.9 Hz, 2H), 7.49 (dd, J = 17.0, 9.1 Hz, 2H), 7.35 (t, J =
7.2 Hz, 1H), 7.29 (t, J = 8.9 Hz, 1H), 6.98 (s, 2H), 6.28 (s, 2H), 5.52 (d, J = 7.8 Hz, 1H), 3.91
(s, 9H), 3.86 (s, 6H), 3.80 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 185.8, 158.7, 153.9, 151.9,
143.8, 139.7, 136.6, 134.4, 130.8, 129.7, 129.5, 127.1, 125.2, 123.7, 119.4, 113.5, 96.7, 93.2,
60.9, 56.3, 55.1. MS (ESI): 546 [M+H]+; HRMS (ESI) calculated for C30H32O7N3 [M+H]+
546.22348; found: 546.22283.
4.3 Cell culture
HT-29 (colon), PC-3 (prostate), A549 (lung) and U87MG (brain) cancer cells were purchased
from ATCC (Manassas, VA) and keratinocytes (HaCaT) were procured from Life
Technologies. PC-3, A549 and HaCaT cells were grown in Roswell Park Memorial Institute
(RPMI-1640) medium (GIBCO-Invitrogen, NY) with 10% fetal bovine serum (FBS)
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supplemented with glutamine (2 mmol/L), penicillin G (100 µg/mL)/ streptomycin (100
µg/mL) at 37 ºC under 5% CO2. HT-29 and U87MG cells were grown in Dulbecco’s
modified Eagle’s (DMEM) medium containing 10% FBS, 1% penicillin/ streptomycin at 37
ºC under 5% CO2. The growth medium was changed every two days. After reaching 80-90%
confluency, cells were treated with 0.25% trypsin-EDTA for further passage.
4.3.1 MTT assay
HT-29, U87MG, A549, PC-3 and HaCaT cells were used to test the cytotoxicity of the
synthesized pyrazolo-triazole compounds (7-36). The cells under study were collected with
0.25% trypsin-EDTA, counted and seeded into a 96 well plate at a density of 3,000-5,000
cells per well depending on their doubling times. The cells were allowed to grow for 24 h at
37 °C and 5% CO2. The culture media was removed after 24 h incubation and treated with
100 µL of test compounds. Then, the cells were further incubated with the compounds for 48
h under the same conditions. The culture medium containing compounds was removed after
48 h and 100 µL of culture media containing 5 mg/mL MTT (Thiazoyl blue tetrazolium
bromide) was added to each well and cells were incubated for 4 hours. After 4 h incubation,
the media containing MTT was aspirated off and 100 µL DMSO was added to each well to
solubilize the crystallized formazan product. The plates were then read on a microplate reader
at 570 nm and at a reference wavelength of 630 nm. The absorbance readings for 630 nm
were subtracted from the 570 nm readings and the results were adjusted by dividing the
average by control to adjust for any toxicity that may have occurred in this control treatment
set. The percentage inhibition was calculated as 100-[(Mean OD of treated cell x 100)/Mean
OD of vehicle treated cells (DMSO)]. The IC50 Values were calculated using Probit Software.
All the tests were repeated in at least three independent experiments, while each experiment
was performed in quadruplets.
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4.3.2 In vitro migration assay
The wound-healing assay is a proven method to study cell migration. A549 cells (5x105
cells/well) were grown in 6 well plates as confluent monolayers for 24 hours. Then the
monolayers were scratched with 200 µL pipette tip and washed twice with PBS to remove
non-adherent cells. The media containing IC50 concentrations of the test compounds 16, 22
and 28 were added to each well. Then, cells which migrated across the wound area were
photographed under the phase contrast microscope (Nikon) immediately after scratch (0 h),
and at 24 h and 48 time interval after treatment in three or more randomly selected fields.
4.3.3 F-actin staining
A549 cells (1x106 cells/well) were grown on coverslips in 6 well plates for 24 h and then
incubated with the compounds 16, 22 and 28 for 12 h. After the compound treatment, cells
were washed with PBS and fixed with 4% para-formaldehyde in PBS. Cells were incubated
with rhodamine phalloidin for actin staining and Hoechst 33242 for nucleus staining. After
washing thrice with PBS, cells were mounted with ProLong Gold anti-fade reagent
(Molecular Probes, Eugene, OR) on microscopic slide and were observed by confocal
microscopy (Nikon). Images were captured using 20X objective lenses.
4.3.4 Cell cycle analysis
A549 cells (1x106 cells /well) in 6 well plate were treated with IC50 concentrations of the
compounds 16, 22 and 28 for 24 h. Cells were collected by trypsininsation, washed with 150
mM PBS and were fixed with 70 % ethanol for 30 min at 4 ºC. After fixing, cells were
washed with PBS and stained with 400 µL of Propidium Iodide staining buffer [PI (200 µg),
Triton X (100 µL), DNAse-free RNAse A (2 mg) in 10 mL PBS] for 15 min at room temp in
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dark. The samples were then analyzed for propidium iodide fluorescence from 15,000 events
by flow cytometry using BD Accuri C6 flow-cytometer.
4.3.5 Hoechst staining
The nuclear morphology of A549 cells after treatment with the compounds was analyzed
using Hoechst 33242. Cells (1x106 cells/well) at their growth phase were grown on cover
slips in 6 well plates and were incubated with IC50 concentrations of the compounds 16, 22
and 28 for 24 h. After 24 h incubation, cells were washed with 150 mM PBS and fixed with
4% para-formaldehyde solution at 4 ºC for 10 min. Then, cells were washed twice with PBS
and stained with Hoechst 33242 (5 µg/mL) for 20 min at room temp and again cells were
washed twice with PBS to remove excess dye. Cells were examined for morphological
changes under confocal microscope using 350 nm excitation and 460 nm emission (Nikon,
magnification 20X).
4.3.6 Measurement of mitochondrial membrane potential (DΨm)
Intracellular DΨm in A549 cells was investigated using mitochondria specific rhodamine 123
dye. In this assay, A549 cells were cultured in 6 well plates at a density of 5x105 cells/mL
and allowed to adhere overnight. The cells were treated with the IC50 concentrations of the
test compounds 16, 22 and 28 for 24 h. After 24 h treatment, the cells were harvested by
trypsinsation, washed with PBS and resuspended in solution of PBS containing rhodamine-
123 (10 µg/mL) and further incubated for 30 min at room temp. Cells were washed twice
with PBS to remove excess dye and resuspended in PBS. The samples were analyzed for
rhodamine fluorescence using spectrofluorometer with an excitation and emission
wavelengths of 480 and 530 nm.
4.3.7 Measurement of intracellular reactive oxygen species (ROS) levels
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For analysis of ROS, A549 cells (2x105 cells/well) were plated in 24 well plates and allowed
to grow for 24 h for attachment and then further incubated with the IC50 concentrations of the
compounds 16, 22 and 28 for 24 h. After treatment with the compounds, cells were washed
with PBS and fixed with 4% para-formaldehyde. After fixing, cells were washed with PBS
and incubated with carboxy-DCFDA (10 µM) and images were capture using fluorescence
microscope with an excitation 485 nm and emission 535 nm.
Acknowledgements
T.S.R. and H.K. are thankful to IICT-RMIT Research Centre for the award of research
fellowships towards their Ph.D. V.B. acknowledges the Australian Research Council (ARC)
for a Future Fellowship (FT140101285). V.B. and R.S. acknowledge the ARC for research
support through the ARC-Linkage (LP130100437) scheme. R.S. acknowledges the support of
Maxwell Eagle Endowment for cancer research. V.B. acknowledges the Ian Potter
Foundation for supporting the establishment of the Ian Potter NanoBioSensing Facility at
RMIT University and providing equipment support through various funding mechanisms.
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References
1. R. Seigel, D. Naishadham, A. Jemal, Cancer statistics, 2013, CA Cancer J. Clin. 63
(2013) 11-30.
2. G. Kibria, H. Hatakeyama, H. Harashima, Cancer multidrug resistance: mechanisms
involved and strategies for circumvention using a drug delivery system, Arch. Pharm.
Res. 37 (2014) 4-15.
3. S.H. Kaufmann, W.C. Earnshaw, Induction of apoptosis by cancer chemotherapy, Exp.
Cell Res. 256 (2000) 42–49.
4. R.J. Bold, P.M. Termuhlen, D.J. McConkey, Apoptosis, cancer and cancer therapy Surg.
Oncol. 6 (1997) 133-142.
5. S.W. Fesik, Promoting apoptosis as a strategy for cancer drug discovery, Nat. Rev.
Cancer 5 (2005) 876–885.
6. V. Kumar, K. Kaur, G.K. Gupta, A.K. Sharma, Pyrazole containing natural products:
synthetic preview and biological significance, Eur. J. Med. Chem. 69 (2013) 735-753.
7. V. Kumar, R. Aggarwal, P. Tyagi, Synthesis and antibacterial activity of some new 1-
heteroaryl-5-mino-4-phenyl-3-trifluoromethylpyrazoles, Eur. J. Med. Chem. 40 (2005)
922-927.
8. R. Gondru, J. Banothu, R.K. Thatipamula, S.K. Althaf Hussain, R. Bavantula, 3-(1-
Phenyl-4-((2-(4-arylthiazol-2-yl) hydrazono) methyl)-1H-pyrazol-3-yl)-2H-chromen-2-
ones: one-pot three component condensation, in vitro antimicrobial, antioxidant and
molecular docking studies, RSC Adv. 5 (2015) 33562-33569.
9. J.B. Shi, W.J. Tang, R. Li, X.H. Liu, Novel pyrazole-5-carboxamide and pyrazole–
pyrimidine derivatives: Synthesis and anticancer activity, Eur. J. Med. Chem. 90 (2015)
889-896.
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