1-Acetyl-3,5-diaryl-4,5-dihydro(1H)pyrazoles: Exhibiting anticancer activity through intracellular ROS scavenging and the mitochondria-dependent death pathway

Article abstract of DOI:10.1002/ardp.201400199

A series of 17 analogs of 1-acetyl-4,5-dihydro(1H)pyrazoles (JP-1 to JP-17) bearing two aromatic rings at positions 3 and 5, either of which ought to be heterocyclic, were synthesized and evaluated for their anti-proliferative potential against breast cancer (MCF-7 and T-47D) and lung cancer (H-460 and A-549) cell lines for the first time. JP-1-7, -10, -11, -14, and -15 were observed to exhibit significant anti-proliferative activity against MCF-7 cells. Some notions about structure-activity relationships are reported. The investigated compounds were found to lower the intracellular reactive oxygen species in the H₂DCFDA assay and also caused mitochondria-dependent cell death in the MCF-7 cell line, indicating a plausible mechanism of their anticancer effect. Analogs of 1-acetyl-4,5-dihydro(1H)pyrazoles (JP-1-17) were synthesized and evaluated for their anti-proliferative activity in four cancer cell lines and for their intracellular ROS scavenging properties. An attempt was made to determine the mitochondrial membrane potential of MCF-7 cells treated with JP-1 and -14, aiming to elucidate the mechanism by which proliferation was curbed.

Full text of DOI:10.1002/ardp.201400199

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
1
Full Paper
1-Acetyl-3,5-diaryl-4,5-dihydro(1H)pyrazoles: Exhibiting
Anticancer Activity through Intracellular ROS Scavenging
and the Mitochondria-Dependent Death Pathway
Jimi M. Alex1, Sandeep Singh2, and Raj Kumar1
1
Laboratory for Drug Design and Synthesis, Centre for Chemical and Pharmaceutical Sciences, School of Basic
and Applied Sciences, Central University of Punjab, Bathinda, India
Centre for Genetic Diseases and Molecular Medicine, Central University of Punjab, Bathinda, India
2
A series of 17 analogs of 1-acetyl-4,5-dihydro(1H)pyrazoles (JP-1 to JP-17) bearing two aromatic rings at
positions 3 and 5, either of which ought to be heterocyclic, were synthesized and evaluated for their
anti-proliferative potential against breast cancer (MCF-7 and T-47D) and lung cancer (H-460 and A-549)
cell lines for the first time. JP-1–7, -10, -11, -14, and -15 were observed to exhibit significant anti-
proliferative activity against MCF-7 cells. Some notions about structure–activity relationships are
reported. The investigated compounds were found to lower the intracellular reactive oxygen species
in the H2DCFDA assay and also caused mitochondria-dependent cell death in the MCF-7 cell line,
indicating a plausible mechanism of their anticancer effect.
Keywords: Antiproliferative / MCF cell lines / Mitochondria-dependent cell death / Pyrazoles / Synthesis
Received: May 17, 2014; Revised: June 19, 2014; Accepted: June 24, 2014
DOI 10.1002/ardp.201400199
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
to the membrane structure [15, 16]. Heterocycles also possess
the ability to alter drug properties such as potency, selectivity,
lipophilicity, polarity, and aqueous solubility, thus playing
an extremely crucial role in the design of therapeutic
molecules [13]. Recently chalcones have gained considerable
attention as they act not only as precursors for the synthesis of
various derivatives, including pyrazoles, but also owing to the
fact that they themselves exhibit broad spectrum of biological
activities including anticancer [17–20]. Further, it was
observed that pyrazoles when incorporated across the double
bonds of chalcones [21] and combretastatins [22], resulted in
better cytotoxic activity. In addition, acetylating the N1 of the
pyrazole was observed to further enhance anticancer
activity [23]. This ability could be attributed to the twisted
structure or the angular conformation that pyrazolines could
adopt, as opposed to pyrazoles, which possess a planar
conformation, which showed significant interaction with the
biological target [24, 25].
Cancer can be defined as the abnormal growth of cells
resulting from multiple changes in gene expression, conse-
quently disturbing the balance between cell proliferation and
cell death and culminating in significant morbidity and death
if untreated [1–3]. Over the past few decades numerous
compounds have been synthesized, many of which, having
subsequently qualified clinical trials and testing, are eventu-
ally accredited as efficacious chemotherapeutic agents [4–10].
Many of these chemotherapeutic agents, for their anticancer
activity, are indebted to the heterocyclic scaffold [11–13] such
as pyrazole [14], indole, quinazoline, imidazoline, pyridine,
etc. Heterocycles have been known to greatly increase
solubility in water or to bring about the reversible distortion
Correspondence: Dr. Raj Kumar, Laboratory for Drug Design and
Synthesis, Centre for Chemical and Pharmaceutical Sciences, School
of Basic and Applied Sciences, Central University of Punjab, Bathinda
151 001, India.
E-mail: raj.khunger@gmail.com, rajcps@cup.ac.in
Fax: þ91 164 2240555
CUPB Library Communication No. P75/14.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Keeping in view of the above revelations, we thought of
Antiproliferative activity
designing N1-acetyl pyrazolines linked with two aromatic
moieties of which at least one is heteroaromatic (JP-1–17;
Fig. 1).
In order to test the cytotoxic potential of the synthesized
compounds, in vitro cytotoxic assay (MTT assay) was performed
for the synthesized pyrazoline derivatives (JP-1–17) against
breast (MCF-7 and T-47D) and lung (A-549 and H-460) cancer
cell lines. Approximately 8000–10,000 cells were seeded per
well of the 96-well plate and treatments with indicated drug
concentrations were given in triplicate for 48 h followed by
MTT assay. Results were expressed as inhibitory potential
possessed by the synthesized compounds. Etoposide was
used as standard anticancer agent [27]. The synthesized target
compounds when tested on peripheral human blood
lymphocytes were found to be non-toxic indicating that
compounds had no effect on the normal cells. Although the
synthesized pyrazoline derivatives were found to be devoid of
activity against the lung cancer cell lines A-549 and H-460,
they exhibited potent activity against MCF-7 (Fig. 2) cell lines
and only to a small extent in T-47D (Fig. 3). The results of
the IC50 values, of some of the potential compounds, thus
obtained are summarized in Table 2. The potency against the
MCF-7 cell line indicates their effectiveness against non-
metastatic hormonal dependent breast cancer cells while the
higher grade cancer cell line seemed less sensitive toward
the treatments. The unresponsiveness of the lung cancer
Results and discussion
Synthesis
All the final compounds (JP-1–17) were synthesized via a two-
step process. Briefly, a heteroaryl/aryl ketone (1) and an
aldehyde (2) were allowed to undergo base catalyzed Claisen–
Schmidt condensation [26] to afford chalcones JA-1–17 (yield
75–88%). The subsequent step involved Michael condensation
of hydrazine hydrate with chalcones followed by cyclization
(via imine bond formation) and N-acetylation in the presence
of acetic acid under reflux to give 1-acetyl-3,5-diaryl-4,5-
dihydro-(1H)-pyrazoles (JP-1–17; Scheme 1). The target com-
pounds were purified through recrystallization from metha-
nol. All the target compounds (Table 1) were characterized
by m.p., IR, NMR, and HRMS. The H1 NMR of pyrazolines
obtained showed typical ABX system characterized by three
double doublets, out of which one double doublet was
sufficiently deshielded and appeared more downfield than the
other two.
Figure 1. Design of the target compounds (JP-1–17)
Scheme 1. Synthesis of the target compounds.
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Synthesis and Anticancer Activity of N-Acetyl Pyrazolines
3
Table 1. Chemical structures of target compounds.
Entry
Ring A
Ring B
Synthesized compounds
Yield (%)a)
1
65
2
55
3
56
4
67
5
85
6
7
61
72
(Continued)
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Table 1. (Continued)
Entry
Ring A
Ring B
Synthesized compounds
Yield (%)a)
8
64
9
70
10
59
11
12
68
74
13
14
63
77
(Continued)
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Table 1. (Continued)
Synthesis and Anticancer Activity of N-Acetyl Pyrazolines
5
Entry
Ring A
Ring B
Synthesized compounds
Yield (%)a)
15
56
16
60
17
55
a)
Isolated yield.
cell line highlights the enhanced tumorigenicity and self-
renewal capacity of these cell lines [28].
-5, -7, -14, and -15 exhibited promising inhibitory potential as
compared to JP-2, -3, -10, and -11, which showed comparatively
little inhibitory potential. At higher concentration of 50 mM,
JP-1, -5, -7, and -14 portrayed significant inhibitory potential
with JP-2, -3, -10, and -11 showing mild activity whereas some
of the other synthesized compounds either showed negligible
activity or were completely devoid of activity. An attempt to
assess the anti-proliferative potential of the target compounds
MCF-7 cells exhibited significant sensitivity toward the
synthesized compounds especially JP-1, -5, -7, and -14, which
displayed potent inhibitory potential at low concentration of
5 mM. At the same concentration, moderate inhibitory
potential was observed for JP-5 and -7 whereas JP-3, -4, -10,
and -11 showed very low inhibitory potential. At 25 mM, JP-1,
Figure 2. Percent inhibition of MCF-7 cells in response to treatment
with synthesized compounds at concentrations of 5, 25, and 50 mM
for a time duration of 48 h. Data are expressed as mean values ꢀ SD
of three independent experiments.
Figure 3. Percent inhibition of T-47D cells in response to treatment
with synthesized compounds at concentrations of 5, 25, and 50 mM
for a time duration of 48 h. Data are expressed as mean values ꢀ SD
of three independent experiments.
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Table 2. IC50 of synthesized compounds.
bearing compounds exhibited excellent inhibitory potential
against the MCF-7 cells as compared to furan containing
compounds. However, analogs containing phenyl ring (JP-14
and -15) exhibited better activity than furan containing
compounds. Among the thiophene containing derivatives the
activity was better for bromo substitution (JP-1) than for
chloro (JP-4 and -5). Furan containing derivative with chloro
substitution (JP-7) showed appreciable activity although only
at higher concentration of 50 mM. Among the derivatives
bearing electron releasing group, –CH3 bearing thiophene
derivative (JP-2) showed almost twice the activity seen with
–OCH3 (JP-3), but in case of furan containing compounds,
–OCH3 (JP-10) exhibited potent activity whereas –CH3 (JP-9)
exhibited no inhibitory potential. On the contrary, the MTT
results obtained for the T-47D cell line suggested that the
compounds bearing electron releasing group especially in
–CH3 bearing furan derivative (JP-9) exhibited better inhibito-
ry potential than the electron withdrawing group (JP-7 and
-11). JP-12, a pyridine analogue, also showed comparable
activity. These observations helped us to formulate a basic
pharmacophore (Fig. 4) as shown below with the structural
features required for potent anticancer activity.
IC50 (mM)
Compound
MCF-7
T-47D
JP-1
JP-2
JP-3
JP-4
7
>50
>50
48
NT
>100
>100
>100
NT
JP-5
30
JP-7
28
NT
JP-10
JP-11
JP-14
JP-15
Etoposide
>50
>50
24
49
20.9 [27]
>100
>100
NT
>100
ND
NT, not tested due to solubility problem; ND, not determined.
against T-47D, another breast cancer cell line, exhibited
remarkably weak activity at all the three concentrations.
Nevertheless, JP-2 displayed potent inhibitory activity at
25 mM and JP-10 and -15 showed inhibitory potential to a very
small extent. This loss of activity in case of T-47D cell line as
compared to MCF-7 cell line can be attributed to the highly
metastatic and aggressive nature of T-47D cell line in addition
to the fact that it possesses proteins such as G1/S-specific
cyclin-D3 and prohibitin, which stimulate cell growth,
antiapoptosis, and carcinogenesis [29].
Compounds are scavengers of intracellular reactive
oxygen species (ROS)
Reactive oxygen species (ROS) and reactive nitrogen species
are classified under unstable moieties, produced in vivo in
low-to-moderate concentrations in response to normal cell
metabolism [30–32]. Oxidative stress results as a consequence
of the imbalance created when these substances are produced
in higher concentrations [33]. Oxidative stress following a
cascade of reactions eventually culminates in events having
dire consequences such as damage to pyrimidine and purine
bases, which may initiate the process of carcinogenesis [31].
In order to evaluate the antioxidant potential of the
synthesized compounds, the representative derivatives JP-1,
-5, -8, -12, and -14 were evaluated using the H2DCFDA dye,
which gave an estimation of the intracellular ROS concentra-
tion (Fig. 5). In this assay, free radicals formation was first
induced in primary human cells and then their scavenging by
Structure–activity relationships
Structural–activity relationships emerged were based on the
role played by ring A and ring B and substituents on them.
Significant inhibitory potential was observed against the
MCF-7 cells with phenyl on ring A (JP-1–11), which was also
comparable to compounds bearing thiophene ring (JP-14 and
-15). The compounds with pyridyl as ring A were observed to
be completely devoid of activity in both MCF-7 as T-47D cells
(JP-12, -13, -16, and -17). Among the active compounds,
comparing ring B of JP-1, -2, -3, -4, -5 with JP-11, -9, -10, -7, and
-8, respectively, it can be concluded that the thiophene
Figure 4. Features decisive for the structure–activity relationships of the synthesized compounds.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Synthesis and Anticancer Activity of N-Acetyl Pyrazolines
7
Figure 5. Graphical representation of the
absorbance of the blood cells in response to
the compounds JP-5, -8, -12, and -14.
Antioxidant action of the compounds in case
of pre- as well as post-treatment with either the
compounds or H2O2.
the compounds was evaluated. H2O2 served as the free radical
inducer while free radical production was studied using
fluorescent H2DCFDA stain by spectrophotometry. Among the
tested compounds, JP-5 was observed to exhibit significant
antioxidant potential especially after 24 h treatment.
cell death. Although various techniques are available for
estimating the membrane potential, most commonly used
method is employing cationic dyes such as JC-1 (5,50,6,60-
tetrachloro-1,10,3,30-tetraethylbenzimidazolyl carbocyanine
iodide). These dyes redistribute between various compart-
ments depending on the membrane potential forming
multimeric units called J-aggregates. These J-aggregates are
associated with the changes in the mitochondrial membrane
potential. The dual fluorescence characteristics of the JC-1
can be used in the assessment by comparing the relative
fluorescence units (RFU) ratios of 590–600 nm (red)/527 nm
(green). In the healthy cells, characterized by high potential,
JC-1 forms complexes exhibiting red fluorescence as opposed
to the green in apoptotic cells [37]. When mitochondrial
depolarization occurs, the red fluorescence decreases and the
green fluorescence remains constant or increases, indicating
cell death.
Two representative compounds with good IC50 values, JP-1
and -14, were selected in order determine whether the cell
death was mitochondria dependent or independent. For this
purpose, the MCF-7 cell line was used and approximately 8000
cells were seeded and treated with 5 mM of the compounds.
Consequently, JC-1 dye was added to each of the wells and
fluorescence read at 590 nm. A green fluorescence was
observed, which confirmed that the treatments administered
resulted in decreased mitochondrial membrane potential.
At lower mitochondrial potential as in these cells, the JC-1
dye fails to form the multimeric form, remaining in the
monomeric form, thus resulting in green color. However, in
the control cells, in the absence of synthesized compounds,
the cells maintained their integrity without altering the
mitochondrial membrane potential thus displaying red
fluorescence. Figure 6 depicts that the RFU is significantly
lower than that of control cells indicating that the
compounds showed mitochondria-dependent cell death of
the MCF-7 cells.
In Fig. 5, bar 1 represents the treatment of blood cells with
the representative compounds only (JP-1, -5, -8, -12, and -14)
whereas bar 2 represents the treatment of cells with the
representative compounds followed by treatment with
hydrogen peroxide for 24 h and bar 3 represents the
incubation of cells with the representative compounds for
24 h followed by treatment with hydrogen peroxide after
30 min. In case of JP-5, addition of H2O2 to the cells
immediately after treatment with the compounds resulted
in generation of free radicals as indicated by the bar 2 but
when the pre-treated cells were allowed to incubate for 24 h
and then followed by the addition of H2O2, there was observed
a marked decrease in the intracellular free radical concentra-
tion as depicted in bar 3. On the contrary, in case of JP-8, -12,
and -14, even after incubating the cells with compound for
24 h, there was no decrease in the free radical concentration.
Compounds cause mitochondria-dependent cell death
Literature has evidences that cellular stresses, such as
DNA damage and oxidative stress, trigger the release of
caspase activating factors especially cytochrome-c [34]. The
cytochrome-c released from the mitochondria result in
the inception of a cascade of biochemical reactions culminat-
ing in cell death [35]. Numerous surveys have suggested
that this release from the mitochondria and the mitochon-
drial depolarization occur simultaneously, thus making
mitochondrial membrane potential a crucial factor not
only for mitochondrial function but also for onset of
apoptosis [36]. The membrane potential may range from
high to low in healthy and apoptotic cells, respectively,
thereby indicating mitochondria-dependent or -independent
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
400 and 100 MHz frequencies, respectively, using TMS (d ¼ 0) as
internal standard and HRMS experiment at SAIF, Panjab
University, Chandigarh.
General procedure for the preparation of chalcones JA-1–17
To a stirred mixture of an aryl or heteroaryl ketone (1; 1 equiv.)
and an aryl or heteroaryl aldehyde (2; 1 equiv.) in methanol,
sodium hydroxide (5%, 4 mL) was added at 0°C. The mixture was
stirred at room temperature for about 15 min to 24 h (TLC). Solid
was obtained after filtration and was recrystallized from
methanol to afford the pure chalcone [26].
General procedure for the preparation of 1-acetyl-3,5-
diaryl-4,5-dihydro(1H)pyrazoles (JP-1–17)
To a solution of an appropriate chalcone (JA-1–17; 1 mmol,
1 equiv.) in 5 mL acetic acid, hydrazine hydrate 80% (1.5 mmol)
was added. The mixture was refluxed for about 4–6 h (TLC). Then
the mixture was poured into ice water mixture to get crude
pyrazole derivatives, which were then purified by recrystalliza-
tion from methanol to afford the pure compounds (JP-1–17).
Figure 6. Graphical representation of the RFU of JC-1 in control
cells as compared to JP-1 and -14. Data is expressed as mean
values ꢀ SD of three independent experiments.
1-(3-(4-Bromophenyl)-5-(thiophen-2-yl)-4,5-dihydro(1H)-
pyrazol-1-yl)ethanone (JP-1) [38]
Conclusions
Light yellow solid; yield: 65%; m.p.: 164–165°C; lmax (MeOH):
325 nm. IR (KBr cmꢁ1): 3014 (C–H), 1660 (C O), 1591 (C N
–
–
–
–
stretch), 1246 (C–N stretch), 1136 (C–S stretch). 1H NMR (400 MHz,
CDCl3, TMS ¼ 0) d: 7.61 (2H, d, J ¼ 8.64 Hz), 7.57 (2H, d, J ¼ 8.62 Hz),
7.18 (1H, dd, J ¼ 1.04 and 5.08 Hz), 7.01 (1H, d, J ¼ 3.36 Hz), 6.92
(1H, dd, J ¼ 3.56 and 5.08 Hz), 5.91 (1H, dd, J ¼ 4.04 and 11.48 Hz),
3.70 (1H, dd, J ¼ 11.48 and 17.6 Hz), 3.31 (1H, dd, J ¼ 4.08 and
17.6 Hz), 2.38 (3H, s).
We have designed and synthesized some 1-acetyl-4,5-dihydro-
(1H)pyrazoles that have shown encouraging antiproliferative
activity in vitro with low micromolar IC50 values in MCF-7
breast cancer cell line. The compounds were found to be less
cytotoxic to the normal cells. Our preliminary mechanistic
interventions of biological activity suggested that these
compounds possess the ability to scavenge the intracellular
ROS, which are usually up-regulated in the carcinogenesis.
Experimental evidences suggested that the investigated
compounds were able to disrupt the potential of mitochon-
drial membrane, as evidenced by JC-1 staining, resulting in
mitochondrial depolarization indicating pro-apoptotic mech-
anism of breast cancer cell death. Further, detailed inves-
tigations of structure–activity relationships as well as
explorations of antitumor activity are currently in progress.
Compound JP-1 has been identified for the further studies.
1-(5-(Thiophen-2-yl)-3-(p-tolyl)-4,5-dihydro(1H)pyrazol-1-
yl)ethanone (JP-2) [39]
Yellow solid; yield: 55%; m.p.: 180–182°C; lmax (MeOH): 304 nm.
IR (KBr cmꢁ1): 3007 (C–H stretch), 1660 (C O), 1609 (C N stretch),
–
–
–
–
1587 (C C aromatic), 1176 (C–N stretch), 1014 (C–S stretch). 1H
–
–
NMR (400 MHz, CDCl3, TMS ¼ 0) d: 7.64 (2H, d, J ¼ 8.32 Hz), 7.25
(2H, d, J ¼ 8.32 Hz), 7.17 (1H, dd, J ¼ 1.0 and 5.04 Hz), 7.01 (1H, d,
J ¼ 3.36 Hz), 6.91 (1H, m), 5.89 (1H, dd, J ¼ 3.9 and 11.4 Hz), 3.70
(1H, dd, J ¼ 11.4 and 17.56 Hz), 3.33 (1H, dd, J ¼ 3.9 and 17.56 Hz),
2.40 (3H, s), 2.39 (3H, s).
1-(3-(4-Chlorophenyl)-5-(thiophen-2-yl)-4,5-dihydro(1H)-
pyrazol-1-yl)ethanone (JP-4) [38]
Experimental
Yellow solid; yield: 67%; m.p.: 125–127°C; lmax (MeOH): 307 nm.
IR (KBr cmꢁ1): 3010 (C–H stretch), 1660 (C O), 1593 (C N stretch),
–
–
–
–
Chemistry
General
1215 (C–N stretch), 1142 (C–S), 702 (C–Cl stretch). 1H NMR
(400 MHz, CDCl3, TMS ¼ 0) d: 7.69 (2H, d, J ¼ 8.42 Hz), 7.41 (2H, d,
J ¼ 8.42 Hz), 7.39 (1H, s), 7.18 (1H, dd, J ¼ 1.0 and 5.08 Hz), 7.01 (1H,
d, J ¼ 3.36 Hz), 6.94 (1H, m), 5.92 (1H, dd, J ¼ 4.0 and 11.44 Hz), 3.70
(1H, dd, J ¼ 11.48 and 17.6 Hz), 3.31 (1H, dd, J ¼ 4.0 and 17.56 Hz),
2.39 (3H, s).
All the reagents used in the synthetic process were of analytical
grade, purchased from Sigma–Aldrich, Loba-Chemie Pvt. Ltd., S.D.
Fine Chemicals, Sisco Research Laboratory. The completion of the
reaction was ascertained by thin layer chromatography and
the prepared plates were visualized by MAC Ultraviolet Fluores-
cence Analysis Cabinet. Melting points were recorded on Stuart
SMP-30 melting point apparatus with open glass capillary tube
and are uncorrected. Infrared spectra were recorded on Bruker IR
spectrophotometer. Nuclear magnetic resonance (1H and 13C)
measurements were recorded on Bruker Advance II instrument at
1-(3-Phenyl-5-(thiophen-2-yl)-4,5-dihydro(1H)pyrazol-1-yl)-
ethanone (JP-6) [39]
Dark brown solid; yield: 61%; m.p.: 110–112°C; lmax (MeOH):
249 nm. IR (KBr cmꢁ1): 2925 (C–H stretch), 1658 (C O), 1620 (C N
–
–
–
–
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Synthesis and Anticancer Activity of N-Acetyl Pyrazolines
9
–
stretch), 1523 (C C aromatic), 1232 (C–N stretch), 1090 (C–S
J ¼ 4.76 and 12 Hz), 3.86 (1H, dd, J ¼ 11.96 and 18.56 Hz), 3.38
(1H, dd, J ¼ 4.76 and 18.6 Hz), 2.44 (3H, s).
–
stretch). 1H NMR (400 MHz, CDCl3, TMS ¼ 0) d: 7.70 (2H, d,
J ¼ 2.32 Hz), 7.46 (3H, m), 7.19 (1H, dd, J ¼ 1.12 and 5.08 Hz), 7.03
(1H, d, J ¼ 3.52 Hz), 6.93 (1H, dd, J ¼ 3.52 and 5.04 Hz), 5.9 (1H, dd,
J ¼ 3.76 and 11.44 Hz), 3.9 (1H, dd, J ¼ 11.48 and 18.08 Hz), 3.5 (1H,
dd, J ¼ 3.8 and 18.04 Hz), 2.3 (3H, s).
1-(3-(Pyridin-2-yl)-5-(thiophen-2-yl)-4,5-dihydro(1H)-
pyrazol-1-yl)ethanone (JP-17) [44]
Yellow solid; yield: 55%; m.p.: 113–114°C (120–121°C); lmax
(MeOH): 304 nm. IR (KBr cmꢁ1): 3013 (C–H stretch), 1657 (C O),
–
–
1-(5-(Furan-2-yl)-3-(p-tolyl)-4,5-dihydro(1H)pyrazol-1-yl)-
–
–
1585 (C N stretch), 1522 (C C aromatic), 1148 (C–S stretch), 1215
–
–
(C–N stretch). 1H NMR (400 MHz, CDCl3, TMS ¼ 0) d: 8.63–8.61 (1H,
m), 8.09 (1H, d, J ¼ 8.0 Hz), 7.70 (1H, d, J ¼ 7.72 Hz), 7.34–7.30 (1H,
m), 7.17 (1H, d, J ¼ 6.2 Hz), 7.03 (1H, s), 6.91 (1H, d, J ¼ 5.04 Hz),
5.92 (1H, dd, J ¼ 4.20 and 11.52 Hz), 3.82 (1H, dd, J ¼ 11.56 and
18.52 Hz), 3.59 (1H, dd, J ¼ 4.20 and 18.52 Hz), 2.41 (3H, s). 13C
NMR (100 MHz, CDCl3, TMS ¼ 0) d ¼ 169.1, 155.4, 150.6, 149.4,
144.1, 136.3, 126.7, 124.8, 124.6, 124.4, 121.2, 55.5, 41.7, 22.0.
The following compounds were found to be unreported.
ethanone (JP-9) [39]
Light brown solid; yield: 70%; m.p.: 120–121°C; lmax (MeOH):
297 nm. IR (KBr cmꢁ1): 2923 (C–H stretch), 1661 (C O), 1640 (C N
–
–
–
–
–
stretch), 1591 (C C aromatic), 1250 (C–O stretch), 1210 (C–N
–
stretch). 1H NMR (400 MHz, CDCl3, TMS ¼ 0) d: 7.66 (1H, s), 7.63
(1H, s), 7.29 (1H, s), 7.24 (1H, s), 7.25 (1H, s), 6.31 (1H, s), 6.30 (1H, d,
J ¼ 1.4 Hz), 5.68 (1H, dd, J ¼ 4.72 and 11.64 Hz), 3.56 (1H, dd,
J ¼ 11.64 and 17.48 Hz), 3.42 (1H, dd, J ¼ 4.76 and 17.48 Hz), 2.40
(3H, s), 2.38 (3H, s).
1-(3-(4-Methoxyphenyl)-5-(thiophen-2-yl)-4,5-dihydro(1H)-
1-(3-(4-Bromophenyl)-5-(furan-2-yl)-4,5-dihydro(1H)-
pyrazol-1-yl)ethanone (JP-3)
pyrazol-1-yl)ethanone (JP-11) [40]
Light brown; yield: 56%; m.p.: 89–90°C; lmax (MeOH): 315 nm. IR
–
–
Light brown solid; yield: 68%; m.p.: 141–143°C; lmax (MeOH):
(KBr cmꢁ1): 3007 (C–H stretch), 1657 (C O), 1608 (C N stretch),
–
–
393 nm. IR (KBr cmꢁ1): 3011 (C–H stretch), 1659 (C O), 1594 (C N
1517 (C C aromatic), 1113 (C–S stretch), 1253 (C–O stretch), 1216
–
–
–
–
–
–
–
stretch), 1532 (C C aromatic), 1252 (C–O stretch), 1215 (C–N
(C–N stretch). 1H NMR (400 MHz, CDCl3, TMS ¼ 0) d: 7.7 (1H, d,
J ¼ 9.64 Hz), 7.69 (1H, d, J ¼ 2.0 Hz), 7.22 (1H, d, J ¼ 5.08 Hz), 7.16
(1H, dd, J ¼ 1.2 Hz), 6.90–6.94 (2H, m), 6.95 (1H, s), 5.89 (1H, dd,
J ¼ 3.88 and 11.36 Hz), 3.69 (1H, dd, J ¼ 11.36 and 17.44 Hz), 3.31
(1H, dd, J ¼ 3.96 and 17.48 Hz), 3.85 (3H, s) 2.38 (3H, s). HRMS
–
stretch), 754 (C–Br stretch). 1H NMR (400 MHz, CDCl3, TMS ¼ 0) d:
7.62 (1H, s), 7.61 (1H, s), 7.56 (1H, s), 7.55 (1H, s), 7.29 (1H, m), 6.32
(1H, s), 6.31 (1H, s), 5.69 (1H, dd, J ¼ 4.84 and 11.76 Hz), 3.56 (1H,
dd, J ¼ 11.72 and 17.44 Hz), 3.41 (1H, dd, J ¼ 4.88 and 17.48 Hz),
2.38 (3H, s).
(TOF-ESI) calcd. for
C
16H16N2O2S, 300.0932 (M)þ; observed:
301.1133 (MþH)þ.
1-(5-(4-Chlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro(1H)-
pyrazol-1-yl)ethanone (JP-14) [41]
Yellow solid; yield: 77%; m.p.: 107–109°C; lmax (MeOH): 317 nm.
1-(3-(2,4-Dichlorophenyl)-5-(thiophen-2-yl)-4,5-
dihydro(1H)pyrazol-1-yl)ethanone (JP-5)
IR (KBr cmꢁ1): 3007 (C–H stretch), 1657 (C O), 1632 (C N stretch),
Yellow solid; yield: 85%; m.p.: 115–116°C; lmax (MeOH): 293 nm.
–
–
–
–
–
–
–
1523 (C C aromatic), 1232 (C–N stretch), 1089 (C–S stretch), 709
IR (KBr cmꢁ1): 3012 (C–H stretch), 1658 (C O), 1609 (C N stretch),
–
–
–
(C–Cl stretch). 1H NMR (400 MHz, CDCl3, TMS ¼ 0) d: 7.44 (1H, dd,
J ¼ 1.08 and 5.08 Hz), 7.28 (1H, s), 7.21 (1H, dd, J ¼ 1.04 and
3.68 Hz), 7.18 (1H, s), 7.16 (1H, s), 7.16 (1H, s), 7.07 (1H, dd, J ¼ 3.68
and 5.0), 5.55 (1H, dd, J ¼ 4.68 and 11.84 Hz), 3.76 (1H, dd, J ¼ 11.84
and 17.48 Hz), 3.18 (1H, dd, J ¼ 4.72 and 17.52 Hz), 2.38 (3H, s).
1589 (C C aromatic), 1176 (C–S stretch), 1215 (C–N), 702 (C–Cl
–
–
stretch). 1H NMR (400 MHz, CDCl3, TMS ¼ 0) d: 7.7 (1H, d,
J ¼ 8.48 Hz), 7.46 (1H, s), 7.3 (1H, s), 7.19 (1H, dd, J ¼ 1.08 and
5.04 Hz), 7.03 (1H, d, J ¼ 3.2 Hz), 6.93 (1H, m), 5.91 (1H, dd, J ¼ 3.76
and 11.44 Hz), 3.91 (1H, dd, J ¼ 11.48 and 18.08 Hz), 3.49 (1H, dd,
J ¼ 3.80 and 18.04 Hz), 2.37 (3H, s). HRMS (TOF-ESI) calcd. for
C
15H12Cl2N2OS, 338.0047 (M)þ; observed: 339.1031 (MþH)þ.
1-(5-Phenyl-3-(thiophen-2-yl)-4,5-dihydro(1H)pyrazol-1-yl)-
ethanone (JP-15) [42]
1-(3-(4-Chlorophenyl)-5-(furan-2-yl)-4,5-dihydro(1H)-
Yellow solid; yield: 56%; m.p.: 118–120°C; lmax (MeOH): 311 nm.
IR (KBr cmꢁ1): 3013 (C–H stretch), 1655 (C O), 1590 (C N stretch),
–
–
pyrazol-1-yl)ethanone (JP-7)
–
–
1523 (C C aromatic), 1141 (C–S stretch), 1215 (C–N stretch). 1H
Whitish yellow solid; yield: 72%; m.p.: 156–157°C; lmax (MeOH):
–
–
297 nm. IR (KBr cmꢁ1): 3010 (C–H stretch), 1663 (C O), 1596 (C N
–
–
–
NMR (400 MHz, CDCl3, TMS ¼ 0) d: 7.43 (1H, dd, J ¼ 1.04 and
5.04 Hz), 7.32 (1H, d, J ¼ 1.16), 7.30 (1H, d, J ¼ 1.28 Hz), 7.26 (1H, s),
7.24–7.20 (3H, m), 7.06 (1H, dd, J ¼ 3.68 and 5.08 Hz), 5.59 (1H, dd,
J ¼ 4.56 and 11.76 Hz), 3.76 (1H, dd, J ¼ 11.84 and 17.48 Hz), 3.15
(1H, dd, J ¼ 4.60 and 11.44 Hz), 2.39 (3H, s).
–
–
stretch), 1502 (C C aromatic), 1252 (C–O stretch), 1215 (C–N
–
stretch). 1H NMR (400 MHz, CDCl3, TMS ¼ 0) d: 7.70 (1H, d,
J ¼ 2.36 Hz), 7.68 (1H, d, J ¼ 1.96 Hz), 7.41 (1H, s), 7.39 (1H, d,
J ¼ 2.4 Hz), 7.30 (1H, m), 6.33 (1H, s), 6.31 (1H, t), 5.69 (1H, dd,
J ¼ 4.84 and 11.72 Hz), 3.56 (1H, dd, J ¼ 11.72 and 17.52 Hz), 3.41
(1H, dd, J ¼ 4.84 and 17.52 Hz), 2.32 (3H, s). HRMS (TOF-ESI) calcd.
for C15H13ClN2O2, 288.0666 (M)þ; observed: 289.0046 (MþH)þ.
1-(5-Phenyl-3-(pyridin-2-yl)-4,5-dihydro(1H)pyrazol-1-yl)-
ethanone (JP-16) [43]
Yellow solid; yield: 60%; m.p.: 135–137°C; lmax (MeOH): 304 nm.
IR (KBr cmꢁ1): 3007 (C–H stretch), 1664 (C O), 1581 (C N stretch),
1-(3-(2,4-Dichlorophenyl)-5-(furan-2-yl)-4,5-dihydro(1H)-
–
–
–
–
1561 (C C aromatic), 1253 (C–N stretch). 1H NMR (400 MHz,
–
–
pyrazol-1-yl)ethanone (JP-8)
CDCl3, TMS ¼ 0) d: 8.61 (1H, m), 8.10 (1H, d, J ¼ 7.92), 7.78–7.4 (1H,
m), 7.33 (1H, d, J ¼ 2.16 Hz), 7.31 (1H, d, J ¼ 1.56 Hz), 7.29 (1H, s),
7.25 (1H, s), 7.23 (1H, d, J ¼ 6.28 Hz), 7.22 (1H, s), 5.61 (1H, dd,
Yellow solid; yield: 64%; m.p.: 115–116°C; lmax (MeOH): 295 nm.
IR (KBr cmꢁ1): 3013 (C–H stretch), 1711 (C O), 1665 (C N
–
–
–
–
–
aromatic), 1586 (C C aromatic), 1257 (C–O stretch), 1215 (C–N
–
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

10
J. M. Alex et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
stretch), 751 (C–Cl stretch). 1H NMR (400 MHz, CDCl3, TMS ¼ 0) d:
7.75 (1H, d, J ¼ 8.48 Hz), 7.46 (1H, s), 7.32–7.29 (2H, m), 6.33 (1H, s),
6.32 (1H, m), 5.67 (1H, dd, J ¼ 4.56 and 11.8 Hz), 3.79 (1H, dd,
J ¼ 11.8 and 17.96 Hz), 3.57 (1H, dd, J ¼ 4.96 and 17.92 Hz), 2.36
(3H, s). HRMS (TOF-ESI) calcd. for C15H12Cl2N2O2, 322.0276 (M)þ;
observed: 345.990 (MþNa)þ.
(200 mL per well of 96-well plate) was kept constant and all the
treatment volumes were accommodated within these ranges
only.
3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay
MTT assay was carried out in 96-well plates where total volume of
media was 200 mL/well [27]. Briefly cells after the treatments were
washed with 1% PBS and were mixed with 100 mL/mL well of
MTT (5 mg in 10 mL of 1% PBS) and incubated at room
temperature in dark for 4 h to allow formation of formazan
crystals. Each well was then mixed with 100 mL of DMSO to
dissolve the crystals followed by ELISA readings at 570 nm. The
results were then represented as mean ꢀ SD obtained from three
independent experiments.
1-(5-(Furan-2-yl)-3-(4-methoxyphenyl)-4,5-dihydro(1H)-
pyrazol-1-yl)ethanone (JP-10)
Dark brown solid; yield: 59%; m.p.: 122–124°C; lmax (MeOH):
301 nm. IR (KBr cmꢁ1): 3005 (C–H stretch), 1659 (C O), 1606 (C N
–
–
–
–
–
stretch), 1596 (C C aromatic), 1250 (C–O stretch), 1223 (C–N
–
stretch). 1H NMR (400 MHz, CDCl3, TMS ¼ 0) d: 7.71 (1H, d,
J ¼ 2.0 Hz), 7.69 (1H, d, J ¼ 2.04 Hz), 7.29 (1H, m), 6.95 (1H, d,
J ¼ 2.0 Hz), 6.93 (1H, d, J ¼ 2.0 Hz), 6.31 (1H, d, J ¼ 1.48 Hz), 6.30
(1H, d, J ¼ 1.44 Hz), 5.67 (1H, dd, J ¼ 4.68 and 11.6 Hz), 3.85 (3H, s),
3.55 (1H, dd, J ¼ 11.56 and 17.36 Hz), 3.41 (1H, dd, J ¼ 4.68 and
17.36 Hz), 2.38 (3H, s). HRMS (TOF-ESI) calcd. for C16H16N2O3,
284.1161 (M)þ; observed: 285.1680 (MþH)þ.
Antioxidant assay
Five milliliters of human blood was taken and processed with RBC
lysis buffer to get the lymphocytes and counting was done using
Trypan blue assay in the automated cell counter, 10,000 cells were
seeded into 12 wells of the microtiter plate [27]. The compounds to
be evaluated were added in triplicate to the respective cells in
concentration such that the final concentration of each well
would be 5 mM. Briefly cells were incubated for 24 h followed by
treatment either with compounds alone, compounds þ H2O2 or
H2O2 alone for 24 h followed by other treatments. After the
treatments, 4 mL of H2DCFDA dye was added in the cultured cells
and kept in dark for 30 min followed by absorbance at emission
wavelength of 530 nm. All the treatments were done in triplicates.
1-(5-(2,5-Dimethoxy)-3-phenyl-4,5-dihydro(1H)pyrazol-1-
yl)ethanone (JP-12)
Yellow solid; yield: 74%; m.p.: 112–114°C; lmax (MeOH): 328 nm.
IR (KBr cmꢁ1): 3015 (C–H stretch), 1660 (C O), 1583(C N stretch),
–
–
–
–
1215 (C–N stretch). 1H NMR (400 MHz, CDCl3, TMS ¼ 0) d: 8.86 (1H,
d, J ¼ 1.68 Hz), 8.64 (1H, dd, J ¼ 1.56 and 4.80 Hz), 8.09–8.06 (m,
1H), 7.36–7.33 (1H, m), 6.82 (1H, d, J ¼ 8 Hz), 6.75–6.73 (m, 1H),
6.58 (1H, d, J ¼ 2 Hz), 5.83 (1H, dd, J ¼ 4.68 and 11.84 Hz), 3.04 (1H,
dd, J ¼ 4.72 and 17.72 Hz), 3.41 (1H, dd, J ¼ 11.92 and 17.32 Hz),
3.81 (3H, s), 3.72 (3H, s), 2.46 (3H, s). HRMS (TOF-ESI) calcd. for
C18H19N3O3, 325.1430 (M)þ; observed: 348.0940 (MþH)þ.
Determination of mitochondrial membrane potential
Experimental and control cell populations were counted to be
5 ꢂ 105 cells/mL using the Trypan blue counting method, from
which a small aliquot was taken and the cell density measured.
The cells were concentrated to less than 3 ꢂ 106 cells/mL. In order
to obtain this target concentration, the cells were centrifuged at
<300g for 5–8 min. After discarding the supernatant enough
culture media was added and the cells were resuspended by
gentle vortexing. One milliliter per sample is placed into 15 mL
polypropylene centrifuge tubes and the sample are stained with
0.5 mL 1ꢂ working solution of MitoPT1 after centrifuging and
gentle mixing. This is then incubated 15–20 min at 37°C in the
dark. Cells were washed to remove excess MitoPT. Then the
cells are centrifuged and pelleted. Discarding the supernatant
2 mL 1ꢂ assay buffer or culture media is added. For the second
resuspension, the cells are centrifuged and pelleted. Discarding
the supernatant 0.5–1 mL 1ꢂ assay buffer or culture media is
added. Pipetted out 100–200 mL of each sample per well
(in triplicate) into a black round or flat-bottom 96-well microtiter
plate.
Jurkat and HL-60 cells were exposed to DMSO as the negative
control (left, dark orange bars) or 50 mM CCCP depolarizing agent
(right, light orange bars) for 15 min at 37°C, subsequently
incubated with MitoPT1 JC-1 for 20 min at 37°C, and washed.
100 mL aliquots were analyzed in triplicate in a black 96-well plate
using a Molecular Devices Gemini XS fluorescence plate reader set
at 488 nm excitation and 590 nm emission filter settings. The
amount of orange fluorescence was measured by the plate reader.
Healthy cells in the DMSO control populations exhibited a high
level of orange fluorescence; metabolically stressed cells in the
1-(5-(Furan-2-yl)-3-(pyridin-3-yl)-4,5-dihydro(1H)pyrazol-
1-yl)ethanone (JP-13)
Yellow solid; yield: 63%; m.p.: 95–97°C; lmax (MeOH): 301 nm. IR
(KBr cmꢁ1): 3011 (C–H stretch), 1662 (C O), 1595 (C N stretch),
–
–
–
–
–
1553 (C C aromatic), 1256 (C–O stretch), 1215 (C–N stretch).
–
1H NMR (400 MHz, CDCl3, TMS ¼ 0) d: 8.92 (1H, d, J ¼ 1.64), 8.67
(1H, dd, J ¼ 1.48 and 4.76 Hz), 8.10 (1H, t, J ¼ 1.84 Hz), 7.40–7.36
(1H, m), 7.30 (1H, m), 6.35 (1H, m), 6.32 (1H, m), 5.72 (1H, dd,
J ¼ 4.92 and 11.76 Hz), 3.61 (1H, dd, J ¼ 11.76 and 17.56 Hz), 3.47
(1H, dd, J ¼ 4.88 and 11.76 Hz), 2.39 (3H, s). HRMS (TOF-ESI) calcd.
for C14H13N3O2, 255.1008 (M)þ; observed: 256.0901 (MþH)þ.
Biology
Cell culture and treatment
All the cell lines were procured from National Cell Repository,
NCCS, Pune. A-549 and H-460 (liver) and MCF-7 and T-47D (breast)
cell lines representing different human cancers were grown in
DMEM media supplemented with 10% fetal bovine serum (FBS)
and antibiotic solution (1% Penstrip, all the reagents from
Invitrogen). Cells were cultured in DMEM media with 10% FBS,
50 U/mL penicillin G and 50 mg/mL streptomycin sulfate. The cells
were incubated at 37°C with 5% CO2 and 95% humidity
conditions. For experiments, cells were seeded in equal numbers
after Trypan blue cell counting (8000 cells per well of 96-well
plate). The compounds were dissolved in cell culture grade DMSO
up to concentration of 100 mM and further dilutions were done
in serum free DMEM media. The total amount of media per well
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Synthesis and Anticancer Activity of N-Acetyl Pyrazolines
11
CCCP-stimulated samples exhibited a reduced level of orange
fluorescence after the mitochondria became depolarized. As the
membrane potential gradient collapses, JC-1 will equilibrate out
of the mitochondria and into the cytosol, causing cells to lose
their red fluorescence.
[20] A. Repanas, A. Katsori, D. Hadjipavlou-Litina, Mini Rev. Med.
Chem. 2013, 13, 952–970.
[21] B. Bhat, K. Dhar, S. Puri, A. Saxena, M. Shanmugavel, G. Qazi,
Bioorg. Med. Chem. Lett. 2005, 15, 3177–3180.
[22] T. Liu, R. Cui, J. Chen, J. Zhang, Q. He, B. Yang, Y. Hu, Arch.
Pharm. 2011, 344, 279–286.
The authors thank Acting Vice Chancellor and Dean, Prof. P. Ramarao,
Central University of Punjab, Bathinda for providing the facilities to
carry out the present work.
[23] C. Congiu, V. Onnis, L. Vesci, M. Castorina, and P. C. Bioorg,
Med. Chem. 2010, 18, 6238–6248.
[24] R. LeBlanc, J. Dickson, T. Brown, M. Stewart, H. N. Pati,
D. VanDerveer, H. Arman, J. Harris, W. Pennington, H. L.
Holt, Jr., Bioorg. Med. Chem. 2005, 13, 6025–6034.
The authors have declared no conflict of interest.
[25] M. Lee, O. Brockway, A. Dandavati, S. Tzou, R. Sjoholm,
V. Satam, C. Westbrook, S. L. Mooberry, M. Zeller, B. Babu,
Eur. J. Med. Chem. 2011, 46, 3099–3104.
References
[26] K. Nepali, G. Singh, A. Turan, A. Agarwal, S. Sapra, R. Kumar,
U. C. Banerjee, P. K. Verma, N. K. Satti, M. K. Gupta, Bioorg.
Med. Chem. 2011, 19, 1950–1958.
[1] R. W. Ruddon, in Cancer Biology, Oxford University Press,
New York 2007.
[27] A. T. Baviskar, U. C. Banerjee, M. Gupta, R. Singh, S. Kumar,
M. K. Gupta, S. Kumar, S. K. Raut, M. Khullar, S. Singh, Bioorg.
Med. Chem. 2013, 21, 5782–5793.
[2] R. Siegel, J. Ma, Z. Zou, A. Jemal, CA: Cancer J. Clin. 2014, 64,
9–29.
[3] D. Hanahan, R. A. Weinberg, Cell 2011, 144, 646–674.
[4] W. A. Denny, Anti-Cancer Drug Des. 1989, 4, 241–263.
[28] Y. Shi, X. Fu, Y. Hua, Y. Han, Y. Lu, J. Wang, PLoS ONE 2012, 7,
e33358.
[5] M. J. Moore, D. Goldstein, J. Hamm, A. Figer, J. R. Hecht,
S. Gallinger, H. J. Au, P. Murawa, D. Walde, R. A. Wolff, J. Clin.
Oncol. 2007, 25, 1960–1966.
[29] J. A. Aka, S.-X. Lin, PLoS ONE 2012, 7, e31532.
[30] S. Blein, S. Berndt, A. D. Joshi, D. Campa, R. G. Ziegler,
E. Riboli, D. G. Cox, Free Radic. Res. 2014, 48, 380–386.
[6] N. J. Robert, V. Diéras, J. Glaspy, A. M. Brufsky, I. Bondarenko,
O. N. Lipatov, E. A. Perez, D. A. Yardley, S. Y. Chan, X. Zhou, J.
Clin. Oncol. 2011, 29, 1252–1260.
[31] G. Borges Bubols, D. da Rocha Vianna, A. Medina-Remon,
G. von Poser, R. Maria Lamuela-Raventos, V. Lucia Eifler-
Lima, S. Cristina Garcia, Mini Rev. Med. Chem. 2013, 13, 318–
334.
[7] D. J. Kuhn, Q. Chen, P. M. Voorhees, J. S. Strader, K. D. Shenk,
C. M. Sun, S. D. Demo, M. K. Bennett, F. W. B. van Leeuwen,
A. A. Chanan-Khan, Blood 2007, 110, 3281–3290.
[32] M. Valko, D. Leibfritz, J. Moncol, M. T. Cronin, M. Mazur,
J. Telser, Int. J. Biochem. Cell Biol. 2007, 39, 44–84.
[8] P. G. Richardson, B. Barlogie, J. Berenson, S. Singhal, S. Jagannath,
D. Irwin, S. V. Rajkumar, G. Srkalovic, M. Alsina, R. Alexanian,
N. Engl. J. Med. 2003, 348, 2609–2617.
[33] V. Fuchs-Tarlovsky, Nutrition 2013, 29, 15–21.
[34] C. Adrain, S. J. Martin, Trends Biochem. Sci. 2001, 26, 390–397.
[35] X. Jiang, X. Wang, Annu. Rev. Biochem. 2004, 73, 87–106.
[9] C. B. Yoo, P. A. Jones, Nat. Rev. 2006, 5, 37–50.
[10] F. Zagouri, T. N. Sergentanis, D. Chrysikos, M. Filipits,
[36] K. M. Heiskanen, M. B. Bhat, H.-W. Wang, J. Ma, A.-L.
R. Bartsch, Gynecol. Oncol. 2012, 127, 662–672.
Nieminen, J. Biol. Chem. 1999, 274, 5654–5658.
[11] N. M. N. Hari, V. Pratheepa, M. Ramos, V. Vasconcelos,
[37] M. Reers, T. W. Smith, L. B. Chen, Biochemistry 1991, 30, 4480–
P. Fernandes, Curr. Drug Targets 2014, 15, 681–688.
4486.
[12] W. R. Pitt, D. M. Parry, B. G. Perry, C. R. Groom, J. Med. Chem.
2009, 52, 2952–2963.
[38] A. K. Tengli, S. Badami, B. R. P. Kumar, S. K. H. Dongre,
S. Ravi, A. K. T. Durai, Indian J. Heterocycl. Chem. 2007, 16,
333–336.
[13] A. Gomtsyan, Chem. Heterocycl. Compd. 2012, 48, 7–10.
[39] M. M. El-Kerdawy, M. N. Tolba, A. A. El-Agamey, Egypt. J. Chem.
1976, 17, 7–14.
[14] J. M. Alex, R. Kumar, J. Enzyme Inhib. Med. Chem. 2014, 29, 427–
442.
[40] H.-M. Guo, Acta Crystallogr. 2007, 63, 3165.
[15] R. Brown, R. Fischer, J. Blunk, K. D. Berlin, K. Ramalingam,
N. N. Durham, Proc. Okla. Acad. Sci. 1976, 56, 15–17.
[41] D. M. Ferguson, J. Goodell, Regents of the University of
Minnesota. Pyrazoline derivatives for treatment of viral
infections. USA. Patent WO 2007038425, 2007.
[16] N. N. Durham, R. W. Chesnut, D. F. Haslam, K. D. Berlin, Ann.
Okla. Acad. Sci. 1974, 4, 77–86.
[42] A. V. Ingle, A. G. Doshi, A. W. Raut, Orient. J. Chem. 2010, 26,
951–9957.
[17] V. Sharma, V. Kumar, P. Kumar, Anticancer Agents Med. Chem.
2013, 13, 422–432.
[43] A. D. B. Ciupa, A. Paul, M. F. Mahon, P. J. Wood, L. Caggiano,
[18] B. P. Bandgar, S. S. Gawande, R. G. Bodade, N. M. Gawande,
Med. Chem. Commun. 2013, 4, 956–996.
C. N. Khobragade, Bioorg. Med. Chem. 2009, 17, 8168–8173.
[44] A. M. Attia, M. Michael, Acta. Chim. Hung. 1983, 114, 337–
[19] U. K. Jain, R. K. Bhatia, A. R. Rao, R. Singh, A. K. Saxena,
348.
I. Sehar, Trop. J. Pharm. Res. 2014, 13, 73–80.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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