Synthesis and biological evaluation of 3-alkyl-1,5-diaryl-1H-pyrazoles as rigid analogues of combretastatin A-4 with potent antiproliferative activity

Article abstract of DOI:10.1371/journal.pone.0128710

A series of novel 3-alkyl-1,5-diaryl-1H-pyrazoles were synthesized as combretastatin A-4 (CA-4) analogues and evaluated for antiproliferative activity against three human cancer cell lines (SGC-7901, A549 and HT-1080). Most of the target compounds displayed moderate to potent antiproliferative activity, and 7k was found to be the most potent compound. Structure-activity relationships indicated that compounds with a trimethoxyphenyl A-ring at the N-1 position of the pyrazole skeleton were more potent than those with the A-ring at the C-5 position. Tubulin polymerization and immunostaining experiments revealed that 7k potently inhibited tubulin polymerization and disrupted tubulin microtubule dynamics in a manner similar to CA-4. Computational modelling demonstrated that the binding of 7k to the colchicine binding site on microtubules may involve a similar mode as CA-4.

Full text of DOI:10.1371/journal.pone.0128710

RESEARCH ARTICLE
Synthesis and Biological Evaluation of 3-
Alkyl-1,5-Diaryl-1H-Pyrazoles as Rigid
Analogues of Combretastatin A-4 with Potent
Antiproliferative Activity
Qile Xu1, Huan Qi2, Maolin Sun1, Daiying Zuo2, Xuewei Jiang2, Zhiyong Wen1,
Zhiwei Wang1, Yingliang Wu2*, Weige Zhang1*
1 Department Key Laboratory of Structure-Based Drug Design and Discovery Ministry of Education,
Shenyang Pharmaceutical University, Shenyang, China, 2 Department of Pharmacology, Shenyang
Pharmaceutical University, Shenyang, China
a11111
* yingliang_1016@163.com (YW); zhangweige2000@sina.com (WZ)
Abstract
OPEN ACCESS
Citation: Xu Q, Qi H, Sun M, Zuo D, Jiang X, Wen Z,
et al. (2015) Synthesis and Biological Evaluation of 3-
Alkyl-1,5-Diaryl-1H-Pyrazoles as Rigid Analogues of
Combretastatin A-4 with Potent Antiproliferative
Activity. PLoS ONE 10(6): e0128710. doi:10.1371/
journal.pone.0128710
A series of novel 3-alkyl-1,5-diaryl-1H-pyrazoles were synthesized as combretastatin A-4
(CA-4) analogues and evaluated for antiproliferative activity against three human cancer
cell lines (SGC-7901, A549 and HT-1080). Most of the target compounds displayed moder-
ate to potent antiproliferative activity, and 7k was found to be the most potent compound.
Structure-activity relationships indicated that compounds with a trimethoxyphenyl A-ring at
the N-1 position of the pyrazole skeleton were more potent than those with the A-ring at the
C-5 position. Tubulin polymerization and immunostaining experiments revealed that 7k po-
tently inhibited tubulin polymerization and disrupted tubulin microtubule dynamics in a man-
ner similar to CA-4. Computational modelling demonstrated that the binding of 7k to the
colchicine binding site on microtubules may involve a similar mode as CA-4.
Academic Editor: Mohammad Shahid, Aligarh
Muslim University, INDIA
Received: December 17, 2014
Accepted: April 29, 2015
Published: June 10, 2015
Copyright: © 2015 Xu et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Introduction
Microtubules are an essential component of the cytoskeleton and have many cellular functions,
including regulation of motility, cell division, organelle transport, maintenance of cell mor-
phology and signal transduction [1]. Their importance in mitosis and cell division make micro-
tubules an obvious and important target for the design and development of antitumour drugs
[2]. Microtubule-targeting agents can be grouped into two classes: those that inhibit the poly-
merization process (microtubule destabilising agents) and those that promote the polymeriza-
tion of tubulin (microtubule stabilising agents) [3]. Both classes disrupt microtubule/tubulin
dynamics by binding to the protein tubulin, an α,β-heterodimer that forms the core of the mi-
crotubule and arrests cells during mitosis, leading to cell death. There are at least four charac-
terised binding sites on tubulin: the taxane site, the laulimalide site, the vinca site and the
colchicine site [4]. Many compounds that bind tubulin, such as paclitaxel, vinblastine and eri-
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by the
Programme for Innovative Research Team of the
Ministry of Education and the Programme for
Liaoning Innovative Research Team in University, the
National Science and Technology Major Project of the
Ministry of Science and Technology of China
(1.2012ZX09103101-060, http://www.nmp.gov.cn/
zxjs/ WZ, YW), and the National Natural Science
Foundation of China (2.30973614,http://www.nsfc.
gov.cn/ WZ). The funders had no role in study design, bulin, are in clinical use for chemotherapy, and these drugs act by binding to the taxane site or
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
data collection and analysis, decision to publish, or
preparation of the manuscript.
the vinca site. While there are no clinically approved anticancer agents that bind to the colchi-
cine site on tubulin, a number of effective inhibitors of tubulin are currently being investigated
in clinical studies [5].
Competing Interests: The authors have declared
that no competing interests exist.
Combretastatin A-4 (CA-4, 1) is one of the most effective inhibitors of tubulin found to
bind to the colchicine site and whose impressive vascular disrupting activity leads to its clinical
development [6]. CA-4 (1) was first isolated by Pettit et al. from the bark of the South African
willow tree Combretum caffrum in 1989 [7], and it exhibits potent antiproliferative activity
against a broad spectrum of human cancer lines, including those that show multidrug resis-
tance [8]. Unfortunately, the cis double bond of 1 readily isomerizes to the more stable trans
isomer under external stimuli, such as light or heat, resulting in complete loss of cytotoxicity
[9]. Therefore, to retain the appropriate configuration of the two adjacent aryl groups required
for bioactivity, researchers have synthesized a wide variety of cis-restricted analogues of 1
[4,10,11]. Structure-activity relationship (SAR) studies indicated that the common key struc-
tural feature of these analogues was two cis-orientated aromatic rings, the A-ring and B-ring,
with the A-ring preferred as a 3,4,5-trimethoxy-substituted phenyl group and the B-ring pre-
ferred as a p-methoxy-substituted phenyl group [12].
To date, a variety of five-membered heterocyclic analogues of 1, such as oxazoles [13], isoxa-
zoles [14], imidazoles and triazoles [15,16], have been reported as cis-restricted, biologically ac-
tive analogues of CA-4. Because pyrazoles play an important role among biologically active
heterocyclics [17–19], several pyrazole-based CA-4 analogues (Fig 1) such as 3-substituted-
4,5-diaryl-pyrazoles (2, 3, 4 and 5) and 1,5-diaryl-pyrazole (6) were synthesized and evaluated
[15,20,21]. Herein, we present the synthesis and antiproliferative activity of 4-(un)substituted-
3-alkyl-1,5-diaryl-pyrazoles (7a-s) with the A-ring on the 1-position and 3-methyl-1,5-diaryl-
pyrazoles (8a-i) with the A-ring on the 5-position, as analogues of combretastatin A-4 (Fig 2).
The representative compounds 7i and 7k were investigated for their inhibition of tubulin poly-
merization. In addition, computational modelling studies have been performed to understand
the interaction between tubulin and the most active inhibitor 7k.
Results and Discussion
Chemistry
The synthesis of target pyrazole derivatives 7a-s were performed according to Fig 3. 3,4,5-Tri-
methoxy-benzoic acid (9) was treated with concentrated nitric acid in acetic acid at 40°C to yield
1,2,3-trimethoxy-5-nitrobenzene (10), which was then reduced to amine 11 with hydrazine, fer-
ric chloride hexahydrate and activated carbon in methanol at 65°C [22,23]. 3,4,5-Trimethoxy-
phenylhydrazine hydrochloride (12) was prepared from 11 via diazotization with sodium nitrite,
followed by reduction with stannous chloride dihydrate [24]. Compounds 14a-g were prepared
via a Claisen condensation between 13a-g and ethyl acetate, ethyl propionate or ethyl butanoate
in the presence of sodium [25]. After that compounds 14b, 14h, 14i were nitrated to give com-
pounds 14j-l. Subsequently, target compounds 7a-h and 7p-q were prepared in excellent yield by
treating 1-benzoylacetone 14 with compound 12 and sodium acetate trihydrate in ethanol under
reflux [26]. Dealkylation of compounds 7f and 7g with titanium tetrachloride afforded, respec-
tively the phenol derivatives, 7j and 7i [27]. Reduction of the nitro group of 7h, 7p and 7q in a
mixture of hydrazine hydrate, ferric chloride hexahydrate and activated carbon in methanol pro-
vided the corresponding 7k, 7r and 7s. Finally, 7i was formylated according to the Vilsmeier-
Haack protocol with phosphorus oxychloride and dimethylformamide, and the resulting alde-
hyde 7l was reduced to the alcohol 7m using sodium borohydride in methanol at room tempera-
ture [28]. A mixture of 7h and NBS in CCl4 was stirred at room temperature to give 7n followed
by reduction of the nitro group to form the desired compound 7o [29].
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
Fig 1. Structures of CA-4 and some pyrazole derivatives.
doi:10.1371/journal.pone.0128710.g001
The target compounds 8a-i were prepared as shown in Fig 4. 3,4,5-Trimethoxyphenyl-etha-
none (15) [30]. The synthesis of 16, 18 and 8a-i was accomplished according to the chemistry
described above with the corresponding starting materials.
Biological activities
In vitro antiproliferative activities. All of the target compounds, 7a-s and 8a-i, along
with the positive control, CA-4 (1), were evaluated for their antiproliferative activity against
three human cancer cell lines: fibrosarcoma cell line HT-1080, gastric adenocarcinoma cell line
SGC-7901 and pulmonary adenocarcinoma cell line A549. The majority of synthesized com-
pounds displayed antiproliferative activity with IC50 values in the sub-micromolar to micro-
molar range. The IC50 values of the compounds are shown in Table 1.
Fig 2. Structures of the synthesized pyrazole derivatives.
doi:10.1371/journal.pone.0128710.g002
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
Fig 3. Reagents and conditions. (a) HNO3, AcOH, 40°C, 2 h; (b) NH2NH2ÁH2O, FeCl3, activated carbon, MeOH, reflux, 1 h; (c) i) NaNO2, HCl, H2O, 0–4°C;
ii) SnCl2Á2H2O, HCl; (d) I) ethyl acetate, Na, reflux, 1 h; II) ethyl propionate, Na, reflux, 1h; III) ethyl butanoate, Na, reflux, 1 h; (e) KNO3, H2SO4, 0°C, 3 h; (f)
12, NaOAc, EtOH, reflux, 2 h; (g) POCl3, DMF, 0–60°C, 3 h; (h) NaBH4, MeOH, r.t., 2 h; (i) NBS, CCl4, 1.5 h.
doi:10.1371/journal.pone.0128710.g003
As given in Table 1, compound 7k exhibited the most potent antiproliferative activity, with
IC50 values between 0.076 and 0.12 μM against the three cancer cell lines. CA-4 (1), the positive
control, gave IC50 values of 0.016–0.035 μM. Compounds 7b, 7c and 7i also significantly inhib-
ited the growth of two or three cell lines at sub-micromolar concentrations. A comparison of
the IC50 values for 7a-k and 8a-i revealed that compounds with a trimethoxyphenyl A-ring at
Fig 4. Reagents and conditions. (a) MeLi, THF, -20°C, 3 h; (b) ethyl acetate, Na, reflux, 1 h; (c) i) NaNO2,
HCl, H2O, 0–4°C; ii) SnCl2Á2H2O, HCl; (d) 16, NaOAc, EtOH, reflux, 2 h.
doi:10.1371/journal.pone.0128710.g004
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
Table 1. Antiproliferative activity of compounds 7a-s, 8a-i and CA-4 (1).
Antiproliferative activity (IC50 SD, μM) a
Compd
HT-1080
SGC-7901
A549
7a
7b
7c
7d
7e
7f
>100
>100
>100
1.01 0.12
1.23 0.15
>100
0.73 0.09
0.53 0.20
20.7 1.2
3.05 0.33
23.6 2.3
6.45 0.22
5.22 0.19
0.275 0.020
25.9 2.3
0.076 0.009
16.4 1.5
8.42 0.32
>100
15.2 2.2
2.35 0.33
>100
5.52 0.26
>100
2.84 0.52
>100
7g
7h
7i
>100
>100
>100
>100
0.93 0.13
>100
1.12 0.12
>100
7j
7k
7l
0.122 0.013
23.8 1.8
7.11 0.23
>100
0.096 0.011
45.7 3.3
9.04 0.25
>100
7m
7n
7o
7p
7q
7r
15.9 3.2
>100
14.3 3.1
>100
79.1 2.0
>100
>100
>100
>100
8.16 0.12
23.2 0.11
>100
4.62 0.09
35.7 0.08
>100
3.55 0.09
56.6 1.0
>100
7s
8a
8b
8c
8d
8e
8f
28.2 1.9
15.6 1.5
>100
19.0 2.0
11.8 1.2
31.1 2.5
2.43 0.09
>100
9.39 0.21
16.2 1.0
48.6 1.1
28.2 1.7
>100
11.9 1.9
>100
8g
8h
8i
24.7 2.3
>100
21.2 1.6
>100
21.4 1.2
>100
15.0 0.9
0.023 0.012
13.7 1.2
0.016 0.010
21.8 2.1
0.035 0.009
1b
a IC50: Concentration of the compound (μM) producing 50% cell growth inhibition after 72 h of drug exposure, as determined by the MTT assay. Each
experiment was carried out in triplicate. b Used as a positive control.
doi:10.1371/journal.pone.0128710.t001
the N-1 position of the pyrazole skeleton were more potent than those with the A-ring at the
C-5 position. With the A-ring tethered to the N-1 position of the pyrazole ring, analogues
monosubstituted at the para position of the B-ring (7b, 7c and 7d) were found to have slightly
increased activity compared to the unsubstituted compound (7a). To study the effect of disub-
stitution on the B-ring, alkoxy, amino and hydroxy groups were introduced in the para and
meta positions (7e-k). Interestingly, the 3-amino-4-methoxy-substituted compound 7k exhib-
ited the most potent antiproliferative activity; however, the 3-hydroxy-4-methoxy-substituted
compound 7i, which most closely resembles CA-4 (1), displayed only moderate activity. Fur-
thermore, introduction of a formyl (7l), hydroxymethyl (7m) or bromo (7o) group at the C-4
position of the pyrazole skeleton resulted in reduced activity compared to the corresponding
compounds (7i and 7k). Additionally, replacement of the methyl group (7k) with an ethyl (7r)
or a propyl group (7s) at C-3 position of the pyrazole ring tended to great reduce the activity.
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
These data indicate that a large group (such as an ethyl or a propyl group) at C-3 position of
the pyrazole skeleton was not tolerated in this series.
Inhibition of tubulin polymerization activity. To investigate whether the antiprolifera-
tive activity of the target compounds was related to their interaction with tubulin, the most ac-
tive compound 7k and the moderately active 7i were evaluated for their inhibition of tubulin
polymerization in a cell-free in vitro assay. The assay used the polymerization suppressor 1 as a
positive control. As shown in Fig 5, compounds 7i and 7k, as well as 1, inhibited tubulin poly-
merization in a dose-dependent manner. Compound 7k (IC50 = 1.7 μM) was slightly less active
than the reference compound 1 (IC50 = 0.9 μM). Compound 7i, which exhibited less antiproli-
ferative activity than 7k, also showed lower potency as an inhibitor of tubulin polymerization
(IC50 = 2.5 μM). An excellent correlation was observed between the antiproliferative activity
and the inhibition of tubulin polymerization for 7i and 7k, indicating that the molecular target
of this series of combretastatin A-4 analogues was most likely tubulin.
Immunofluorenscence studies. To demonstrate the effect of inhibition of tubulin poly-
merization in cells, we investigated microtubule structure and distribution in cultured HT-
1080 and SGC-7901 cells. Microtubules were labelled using the indirect immuno-fluorescence
method and were analysed by fluorescence microscopy. Cells treated with DMSO (control
cells) stained with a tubulin monoclonal antibody demonstrated a well-organised microtubule
network throughout the cells (Figs 6 and 7). In contrast, cells treated with 1 and 7k (at their re-
spective 2-fold IC50 concentrations, respectively) showed a disruption of the tubulin network
and the appearance of a bundle at a pole of the cell (Figs 6 and 7). Taken together, these results
confirm that tubulin is the molecular target of compound 7k. Comparing the tubulin inhibition
with the corresponding antiproliferative activity revealed a correlation, and it also suggests that
tubulin is the target for this series of combretastatin A-4 analogues.
Molecular modelling
To further expand the understanding of our experimental findings, molecular modelling stud-
ies on 7k and 1 were conducted using CDOCKER within the Discovery Studio 3.0 programme
package. Compounds 7k and 1 were docked into the colchicine binding site of tubulin (PDB
ID: 1SA0). In the CDOCKER protocol run results, we report the energy as a score, so the higher
(more positive) value indicates a more favorable binding. The “-CDOCKER Interaction Ener-
gy” are 48.63 and 46.83 kcal/mol for CA-4 and 7k respectively. The binding energies correlate
with the experimental IC50 values of CA-4 and 7k. These studies also showed that 7k, the most
active compound, can occupy the colchicine binding site of tubulin, as can 1 (Fig 8A). Indeed,
the trimethoxyphenyl moiety in the A-rings of 1 and 7k was buried in the β subunit binding
cavity. The thiol group of Cys β241 formed a hydrogen bond with the oxygen atom of the para
methoxy group, and formed another hydrogen bond to the oxygen atom of the meta methoxy
group (Fig 8B). Several amino acids of β-tubulin formed hydrophobic interactions with the tri-
methoxyphenyl moiety of 7k. The methoxy oxygen atom in ring B of 7k formed a hydrogen
bond with the main chain nitrogen atom of Val α181. Additionally, there is direct bond be-
tween compound 7k and the Ala β250 residue. Thus, the results of this docking study are in
good agreement with the potent antiproliferative activity of 7k and its ability to inhibit
tubulin polymerization.
Conclusions
In summary, a series of 3-alkyl-1,5-diaryl-1H-pyrazoles were synthesized and evaluated for
antiproliferative activity and tubulin polymerization inhibition. The majority of synthesized
compounds displayed moderate to potent antiproliferative activity. Structure-activity
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
Fig 5. Dose-response effects of CA-4 (1), 7i and 7k on the inhibition of tubulin polymerization. Purified
bovine tubulin and GTP were mixed in a 96-well plate. The reaction was initiated by warming the solution from
4°C to 37°C. DMSO was used as a vehicle control. The effect on the assembly of tubulin was monitored using
a plate reader at 1 min intervals for 90 min at 37°C.
doi:10.1371/journal.pone.0128710.g005
relationships indicated that compounds with a 3,4,5-trimethoxyphenyl A-ring at the N-1 posi-
tion of the pyrazole skeleton were more potent than those with the A-ring at the C-5 position.
Among the synthesized compounds, 7k showed the most potent antiproliferative activity against
SGC-7901 cells with an IC50 value of 0.076 μM. Consistent with its antiproliferative activity, 7k
also exhibited potent antitubulin activity (IC50 = 1.7 μM), similar to that of CA-4 (1). In addition,
7k also strongly affected cell morphology and microtubule networking comparable to CA-4 (1).
Molecular docking analysis of the binding conformation of 7k into the tubulin colchicine binding
site showed hydrogen bonds and hydrophobic interactions with protein residues, which may be
responsible for the antitubulin polymerization and antiproliferative activities. These results may
be helpful for the future design of structurally related tubulin inhibitors.
Experimental Section
Synthesis
Unless otherwise noted, all of the materials were obtained from commercially available sources
and were used without purification. The progress of reactions was monitored by TLC using
Fig 6. Immunostaining of tubulin assembly in HT-1080 cells. HT-1080 cells were treated with 7k
(0.18 μM) for 48 h (scale bar = 10 μm). The left, middle and right panels represent tubulin assembly stained
with FITC, DAPI and a merge of the corresponding left and middle panels, respectively. Images were taken
using a confocal microscope.
doi:10.1371/journal.pone.0128710.g006
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
Fig 7. Immunostaining of tubulin assembly in SGC-7901 cells. SGC-7901 cells were treated with 7k
(0.14 μM) for 48 h (scale bar = 10 μm). The left, middle and right panels represent the tubulin assembly
stained with FITC, DAPI and a merge of the corresponding left and middle panels, respectively. Images were
taken using a confocal microscope.
doi:10.1371/journal.pone.0128710.g007
silica gel plates (250 μm, F-254) under UV light. The purification of products was performed
using column chromatography (60 Å, 200–300 mesh, Qingdao Ocean Chemicals) or thin layer
chromatography on silica gel plates (0.25 mm layer, Qingdao Ocean Chemicals) with the desig-
nated solvents. Melting points were measured on a hot stage microscope (X-4, Beijing Taike
Ltd.) and are uncorrected. Mass spectra (MS) were measured on an Agilent 1100-sl mass spec-
trometer with an electrospray ionisation source. NMR spectra were recorded on a Bruker
AVANCE 400 (1H, 400 MHz; 13C, 100 MHz) in CDCl3 (S1 File). Chemical shifts are expressed
Fig 8. (A) tubulin is displayed as a flat ribbon with α-tubulin coloured lavender and β-tubulin coloured cyan.
Superimposition of the presumptive conformation of active 7k (pink model) with that of 1 (green model). The
structures were docked into the colchicine binding site of tubulin (PDB ID: 1SA0). (B) the amino acids of
tubulin within 5.0 Å of colchicine are shown as lines models. Hydrogen bonds (distance: <3.5 Å) are shown as
dotted line.
doi:10.1371/journal.pone.0128710.g008
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
as parts per million downfield from tetramethylsilane. Splitting patterns have been designated
as follows: s (singlet), d (doublet), dd (doublet of doublets), t (triplet) and m (multiplet).
1,2,3-Trimethoxy-5-nitrobenzene (10). A flask immersed in a room temperature oil bath
was charged with 9 (50 g, 0.235 mol) and acetic acid (150 mL) and heated to 40°C. Over a 10
min period, HNO3 (70%, 33 mL) was added dropwise with stirring. The deep orange solution
was stirred for an additional 120 min. The reaction was quenched upon addition of 200 g of
ice. A yellow precipitate formed, which was filtered and washed with H2O. The crude product
was recrystallized from methanol to give pure 10. Yield: 66%.
3,4,5-Trimethoxyaniline (11). A mixture of 1,2,3-trimethoxy-5-nitrobenzene 10 (32.7 g,
0.153 mol), activated carbon (3.27 g, 50–200 mesh, Fisher Scientific Co.), ferric chloride hexa-
hydrate (3.27 g) and methanol (250 mL) was refluxed for 10 min with stirring. Hydrazine hy-
drate (80%, 65 mL) was added over 30 min to the boiling solution. The mixture was stirred
under reflux for an additional 1 h, cooled and evaporated. The resulting slurry was dissolved in
dichloromethane (250 mL), washed with water (2 × 100 mL) and dried (Na2SO4). Evaporation
of the solvent gave amine 11, which was used without further purification. Yield: 95%.
1-(3,4,5-Trimethoxyphenyl)butane-1,3-dione (16). Methyl-lithium (1.3M in diethyl
ether, 39.8 mL, 51.8 mmol) was added dropwise to a solution of 3,4,5-trimethoxybenzoic acid 9
(5 g, 23.6 mmol) in anhydrous THF (50 mL) at -20°C under a nitrogen atmosphere. The mix-
ture was stirred at -20°C for 1 h, warmed to room temperature and stirred for an additional 3
h. The solvent was removed, and the residue was dissolved in excess CH2Cl2. The solution was
washed with H2SO4 (0.1N, 80 mL) and deionised water (3 × 30 mL), dried over anhydrous
Na2SO4, filtered and dried under vacuum. The product 15 was used without further purifica-
tion. Yield: 91%. A mixture of 3,4,5-trimethoxyacetophenone 15 (4.5 g, 21.4 mmol) in freshly
purified anhydrous ethyl acetate (50 mL) was added to sodium sand (0.99 g, 42.8 mmol) under
a nitrogen atmosphere. The mixture was stirred, and upon the initiation of an exothermic reac-
tion, the remainder of the ketone solution was added dropwise at such a rate as to maintain re-
fluxing. Stirring and refluxing were continued for another 120 min. The reaction mixture was
diluted with water (100 mL) and extracted with ethyl acetate (3 × 100 mL). The combined or-
ganic phases were washed with water, dried over Na2SO4, filtered and concentrated under vac-
uum. The crude product was purified by chromatography on silica gel to afford the desired 16.
Yield: 90%; white solid; m.p. 86–87°C; 1H-NMR (400 MHz, CDCl3): δ 2.20 (3H, s), 3.93 (9H,
s), 6.12 (1H, s), 7.13 (2H, s), minor tautomer inter alia 2.29 (3H, s), 3.93 (9H, s), 4.07 (2H, s),
7.22 (2H, s).
1-Phenylbutane-1,3-dione (14a). This compound was obtained from acetophenone 13a
by following the procedure described above for 16. Yield: 87%; yellow solid; m.p. 53–54°C;
1H-NMR (400 MHz, CDCl3): δ 2.20 (3H, s), 6.18 (1H, s), 7.44–7.52 (3H, m), 7.87–7.88 (2H, d,
J = 7.26 Hz), 16.15 (1H, s), minor tautomer inter alia 2.30 (3H, s), 4.10 (2H, s), 7.44–7.52 (3H,
m), 7.93–7.95 (2H, d, J = 7.26 Hz).
1-(4-Methoxyphenyl)butane-1,3-dione (14b). This compound was obtained from 1-
(4-methoxyphenyl)ethanone 13b by following the procedure described above for 16. Yield:
88%; orange-red solid; m.p. 49–50°C; 1H-NMR (400 MHz, CDCl3): δ 2.17 (3H, s), 3.86 (3H, s),
6.11 (1H, s), 6.94 (2H, d, J = 8.37 Hz), 7.86 (2H, d, J = 8.37Hz), minor tautomer inter alia 2.28
(3H, s), 3.86 (3H, s) 4.04 (2H, s), 6.94 (2H, d, J = 8.37 Hz).
1-P-tolylbutane-1,3-dione (14c). This compound was obtained from 1-p-tolylethanone
13c by following the procedure described above for 16. Yield: 83%; yellow solid; m.p. 54–55°C;
1H-NMR (400 MHz, CDCl3): δ 2.16 (3H, s), 2.38 (3H, s), 6.14 (1H, s), 7.22 (2H, d, J = 7.24 Hz),
7.76 (2H, d, J = 7.24 Hz), minor tautomer inter alia 2.26 (3H, s), 2.38 (3H, s), 4.04 (2H, s), 7.22
(2H, d, J = 7.24 Hz), 7.82 (2H, d, J = 7.24 Hz).
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
1-(4-Fluorophenyl)butane-1,3-dione (14d). This compound was obtained from 1-
(4-fluorophenyl)ethanone 13d by following the procedure described above for 16. Yield: 79%;
orange-red solid; m.p. 47–48°C; 1H-NMR (400 MHz, CDCl3): δ 2.19 (3H, s), 6.13 (1H, s),
7.10–7.15 (2H, m), 7.86–7.92 (2H, m), 16.15 (1H, s), minor tautomer inter alia 2.30 (3H, s),
4.08 (2H, s), 7.10–7.15 (2H, m), 7.94–8.02 (2H, m).
1-(3-Fluoro-4-methoxyphenyl)butane-1,3-dione (14e). This compound was obtained
from 1-(3-fluoro-4-methoxyphenyl) ethanone 13e by following the procedure described above
for 16. Yield: 78%; pale yellow solid; m.p. 107–108°C; 1H-NMR (400 MHz, CDCl3): δ 2.18 (3H,
s), 3.95 (3H, s), 6.08 (1H, s), 6.97–7.02 (1H, m), 7.61–7.68 (2H, m), minor tautomer inter alia
2.29 (3H, s), 3.95 (3H, s), 4.04 (2H, s), 6.97–7.02 (1H, m), 7.61–7.68 (2H, m).
1-(4-(Allyloxy)-3-methoxyphenyl)butane-1,3-dione (14f). To a solution of 1-(4-hy-
droxy-3-methoxyphenyl)ethanone (0.20 g, 1.22 mmol) in dry acetone (20 mL) was added an-
hydrous K2CO3 (0.67 g, 4.88 mmol) over 10 min, followed by addition of allyl bromide (0.17 g,
1.34 mmol), and the mixture was stirred under reflux for an additional 3 h. The mixture was
cooled and evaporated, and the resulting slurry was dissolved in dichloromethane (30 mL),
washed with water (2 × 15 mL), and dried over Na2SO4. Evaporation of the solvent gave 13f as
colourless oil. The intermediate 14f was obtained from 1-(4-(allyloxy)-3-methoxyphenyl)-
ethanone 13f by following the procedure described above for 16. Yield: 73%; pale yellow solid;
m.p. 51–52°C; 1H-NMR (400 MHz, CDCl3): δ 2.16 (3H, s), 3.92 (3H, s), 4.67 (2H, d, J = 5.19
Hz), 5.32 (1H, d, J = 10.52 Hz), 5.43 (1H, d, J = 16.50 Hz), 6.03–6.12 (2H, m), 6.88 (1H, d,
J = 8.34 Hz), 7.45–7.52 (2H, m), 16.25 (1H, s), minor tautomer inter alia 2.28 (3H, s), 3.93 (3H,
s), 4.05 (2H, s), 4.67 (2H, d, J = 5.19 Hz), 5.32 (1H, d, J = 10.52 Hz), 5.43 (1H, d, J = 16.50 Hz),
6.03–6.12 (1H, m), 6.88 (1H, d, J = 8.34 Hz), 7.45–7.52 (2H, m).
1-(3-(Allyloxy)-4-methoxyphenyl)butane-1,3-dione (14g). This compound was obtained
from 1-(3-hydroxy-4-methoxyphenyl) ethanone by following the procedure described above
for 14f. Yield: 75%; orange-red solid; m.p. 59–60°C; 1H-NMR (400 MHz, CDCl3): δ 2.17 (3H,
s), 3.93 (3H, s), 4.67 (2H, d, J = 5.02 Hz), 5.32 (1H, d, J = 10.32 Hz), 5.43 (1H, d, J = 16.56 Hz),
6.06–6.14 (2H, m), 6.90 (1H, d, J = 8.27 Hz), 7.47–7.59 (2H, m), minor tautomer inter alia 2.29
(3H, s), 3.93 (3H, s), 4.04 (2H, s) 4.67 (2H, d, J = 5.02 Hz), 5.32 (1H, d, J = 10.32 Hz), 5.43 (1H,
d, J = 16.56 Hz), 6.06–6.14 (1H, m), 6.90 (1H, d, J = 8.27 Hz), 7.47–7.59 (2H, m).
1-(4-Methoxy-3-nitrophenyl)butane-1,3-dione (14j). 1-(4-Methoxyphenyl)butane-
1,3-dione (5.56 g, 0.192 mol) was dissolved in 100 mL of coned. sulfuric acid at laboratory tem-
perature and the solution cooled to 0°C. A solid of KNO3 (3.27 g, 0.101 mol) was sollowly
added with 20 min. The deep orange solution was stirred for an additional 160 min. The reac-
tion was quenched upon addition of 200 g of ice. A yellow precipitate formed, which was fil-
tered and washed with H2O. The crude product was recrystallized from methanol to give pure
14j. Yield: 85%; brown solid; m.p. 109–110°C; 1H-NMR (400 MHz, CDCl3): δ 2.21 (3H, s),
4.04 (3H, s), 6.14 (1H, s), 7.16 (1H, d, J = 8.76 Hz), 8.10 (1H, d, J = 8.76 Hz), 8.36 (1H, s), 16.06
(1H, s).
3-Methyl-5-phenyl-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole (7a). (3,4,5-Trimethoxy-
phenyl)hydrazine hydrochloride (0.14 g, 0.617 mmol), 1-benzoylacetone (0.10 g, 0.617 mmol)
and NaOAc (0.05 g, 0.074 mmol) were combined in EtOH (50 mL). The reaction was refluxed
for 1 h, cooled to room temperature concentrated in vacuo. The resulting slurry was dissolved
in dichloromethane (50 mL), washed with water (2 × 30 mL), dried over Na2SO4, filtered and
concentrated under vacuum. The crude product was purified by chromatography on silica gel
to afford the desired 5-phenyl-1-(3,4,5-trimethoxyphenyl)- 3-methyl-1H-pyrazole 7a. Yield:
74%; yellow solid; m.p. 77–78°C; 1H-NMR (400 MHz, CDCl3): δ 2.41 (3H, s), 3.66 (6H, s), 3.83
(3H, s), 6.32 (1H, s), 6.50 (2H, s), 7.24–7.28 (2H, m), 7.31–7.32 (3H, m); 13C-NMR (100 MHz,
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
CDCl3): δ 13.5, 56.0 (2C), 60.9, 102.7 (2C), 107.6, 128.2, 128.4 (2C), 128.7 (2C), 130.8, 135.6,
137.0, 143.8, 149.2, 153.0 (2C); ESI-MS: m/z = 325.1[M+H]+, 649.3[2M+H]+, 671.3 [2M+Na]+.
3-Methyl-5-(4-methoxyphenyl)-1-(3,4,5-trimethoxy-phenyl)-1H-pyrazole (7b). The
title compound was obtained according to the procedure described above for 7a. Yield: 76%;
pale yellow solid; m.p. 118–119°C; 1H-NMR (400 MHz, CDCl3): δ 2.42 (3H, s), 3.70 (6H, s),
3.80 (3H, s), 3.83 (3H, s), 6.27 (1H, s), 6.52 (2H, s), 6.85 (2H, d, J = 8.60 Hz), 7.18 (2H, d,
J = 8.60 Hz); 13C-NMR (100 MHz, CDCl3): δ 13.6, 55.3, 56.0 (2C), 61.0, 102.9 (2C), 107.0,
113.8 (2C), 123.2, 129.9 (2C), 135.9, 136.9, 143.6, 149.2, 153.0 (2C), 159.5; ESI-MS: m/z = 355.1
[M+H]+, 731.3 [2M+Na]+.
3-Methyl-5-(4-methylphenyl)-1-(3,4,5-trimethoxy-phenyl)-1H-pyrazole (7c). The title
compound was obtained according to the procedure described above for 7a. Yield: 79%; brown
liquid; 1H-NMR (400 MHz, CDCl3): δ 2.33 (3H, s), 2.38 (3H, s), 3.67 (6H, s), 3.83 (3H, s), 6.27
(1H, s), 6.50 (2H, s), 7.11 (2H, d, J = 8.28 Hz), 7.14 (2H, d, J = 8.28 Hz); 13C-NMR (100 MHz,
CDCl3): δ 13.5, 21.1, 56.0 (2C), 60.9, 102.8 (2C), 107.3, 127.9, 128.5 (2C), 129.0 (2C), 135.9,
137.0, 138.0, 143.8, 149.1, 153.0 (2C); ESI-MS: m/z = 339.2 [M+H]+, 677.3 [2M+H]+, 699.3
[2M+Na]+.
3-Methyl-5-(4-fluorophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole (7d). The title
compound was obtained according to the procedure described above for 7a. Yield: 83%; yellow
solid; m.p. 104–105°C; 1H-NMR (400 MHz, CDCl3): δ 2.38 (3H, s), 3.69 (6H, s), 3.83 (3H, s),
6.28 (1H, s), 6.47 (2H, s), 6.98–7.04 (2H, m), 7.21–7.25 (2H, m); 13C-NMR (100 MHz, CDCl3):
δ 13.4, 56.1 (2C), 61.0, 102.9 (2C), 107.6, 115.5 (2C, d, J = 21.9 Hz), 126.7, 130.5 (2C, d, J = 8.3
Hz), 135.3, 137.3, 142.8, 149.2, 153.2 (2C), 161.6 (d, J = 249.0 Hz); ESI-MS: m/z = 343.1 [M
+H]+, 707.3 [2M+Na]+.
3-Methyl-5-(3-fluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole
(7e). The title compound was obtained according to the procedure described above for 7a.
Yield: 78%; brown solid; m.p. 86–87°C; 1H-NMR (400 MHz, CDCl3): δ 2.38 (3H, s), 3.71 (6H,
s), 3.84 (3H, s), 3.88 (3H, s), 6.27 (1H, s), 6.50 (2H, s), 6.86–6.90 (1H, m), 6.93–6.95 (1H, m),
7.00–7.03 (1H, m); 13C-NMR (100 MHz, CDCl3): δ 13.5, 56.1 (2C), 56.2, 60.9, 103.0 (2C),
107.3, 113.1, 116.3 (d, J = 19.54 Hz), 123.5 (d, J = 6.73 Hz), 124.7 (d, J = 3.56 Hz), 135.7, 137.3,
142.3, 147.5 (d, J = 10.23 Hz), 149.2, 151.6 (d, J = 246.92 Hz), 153.2 (2C); ESI-MS: m/z = 373.2
[M+H]+, 745.3 [2M+H]+.
3-Methyl-5-(4-allyloxy-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole
(7f). The title compound was obtained according to the procedure described above for 7a.
Yield: 80%; white solid; m.p. 78–79°C; 1H-NMR (400 MHz, CDCl3): δ 2.42 (3H, s), 3.70 (9H, d,
J = 2.26 Hz), 3.83 (3H, s), 4.60–4.62 (2H, m), 5.28–5.31 (1H, m), 5.37–5.42 (1H, m), 6.01–6.12
(1H, m), 6.31 (1H, s), 6.54 (2H, s), 6.73 (1H, s), 6.82 (2H, s); 13C-NMR (100 MHz, CDCl3): δ
12.9, 55.9, 56.3 (2C), 61.0, 69.8, 103.3 (2C), 107.1, 112.1, 113.1, 118.3, 120.9, 121.5, 122.1, 132.7,
133.9, 137.8, 144.5, 148.6, 149.2, 153.3 (2C); ESI-MS: m/z = 411.2 [M+H]+, 821.4 [2M+H]+,
843.4 [2M+Na]+.
3-Methyl-5-(3-allyloxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole
(7g). The title compound was obtained according to the procedure described above for 7a.
Yield: 85%; brown solid; m.p. 79–80°C; 1H-NMR (400 MHz, CDCl3): δ 2.42 (3H, s), 3.72 (6H,
s), 3.85 (3H, s), 3.90 (3H, s), 4.45 (2H, d, J = 5.37 Hz), 5.20–5.23 (1H, m), 5.26–5.30 (1H, m),
5.88–5.97 (1H, m), 6.29 (1H, s), 6.53 (2H, s), 6.74 (1H, d, J = 1.80 Hz), 6.84 (1H, d, J = 8.18 Hz),
6.89 (1H, dd, J = 8.18 Hz, J = 1.80 Hz); 13C-NMR (100 MHz, CDCl3): δ 13.4, 56.0, 56.1 (2C),
60.9, 69.8, 103.0 (2C), 107.0, 111.4, 114.0, 118.0, 121.7, 122.9, 132.8, 135.6, 137.2, 143.7, 147.6,
149.1, 149.5, 153.1 (2C); ESI-MS: m/z = 411.2 [M+H]+, 821.4 [2M+H]+, 843.4 [2M+Na]+.
3-Methyl-5-(4-methoxy-3-nitrophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole (7h).
The title compound was obtained according to the procedure described above for 7a. Yield:
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
75%; yellow solid; m.p. 145–146°C; 1H-NMR (400 MHz, CDCl3): δ 2.41 (3H, s), 3.75 (6H, s),
3.86 (3H, s), 3.97 (3H, s), 6.37 (1H, s), 6.52 (2H, s), 7.01 (1H, d, J = 8.82 Hz), 7.33 (1H, dd,
J = 8.82 Hz, J = 2.26 Hz), 7.86(1H, d, J = 2.26Hz); 13C-NMR (100 MHz, CDCl3): δ 13.3, 56.3
(2C), 56.7, 61.0, 103.3 (2C), 107.6, 113.5, 122.8, 125.5, 133.9, 134.8, 137.9, 139.4, 141.2, 149.3,
152.6, 153.4 (2C); ESI-MS: m/z = 400.1 [M+H]+, 422.1 [M+Na]+, 799.3 [2M+H]+, 821.3
[2M+Na]+.
3-Methyl-5-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole
(7i). TiCl4 (0.5 mL) was added to a solution of 7g (0.2 g, 0.49 mmol) in CH2Cl2 (20 mL) and
the reaction mixture was stirred for 0.5 h at -20°C. The reaction mixture was warmed to room
temperature and concentrated in vacuo. The resulting slurry was dissolved in dichloromethane
(30 mL), washed with water (2 × 15 mL), dried over Na2SO4, filtered and concentrated under
vacuum. The crude product was purified by chromatography on silica gel to give 7i. Yield:
93%; pale yellow solid; m.p. 130–131°C; 1H-NMR (400 MHz, CDCl3): δ 2.37 (3H, s), 3.69 (6H,
s), 3.82 (3H, s), 3.86 (3H, s), 6.23 (1H, s), 6.52 (2H, s), 6.68 (1H, dd, J = 8.32 Hz, J = 2.00 Hz),
6.76 (1H, d, J = 8.32 Hz), 6.87 (1H, d, J = 2.00 Hz); 13C-NMR (100 MHz, CDCl3): δ 13.5, 56.0,
56.1 (2C), 60.9, 103.0 (2C), 107.2, 110.5, 115.0, 120.7, 123.8, 135.7, 137.1, 143.6, 145.5, 146.7,
149.1, 153.0 (2C); ESI-MS: m/z = 371.2 [M+H]+, 741.3 [2M+H]+, 763.3 [2M+Na]+.
3-Methyl-5-(4-hydroxy-3-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole
(7j). The title compound was obtained according to the procedure described above for 7i.
Yield: 91%; white solid; m.p. 197–198°C; 1H-NMR (400 MHz, CDCl3): δ 2.40 (3H, s), 3.71 (9H,
s), 3.82 (3H, s), 6.28 (1H, s), 6.53 (2H, s), 6.68 (1H, d, J = 1.61 Hz), 6.81 (1H, dd, J = 8.28 Hz,
J = 1.61 Hz), 6.87 (1H, d, J = 8.28 Hz); 13C-NMR (100 MHz, CDCl3): δ 13.5, 55.9, 56.2 (2C),
61.0, 103.0 (2C), 107.0, 111.3, 114.5, 122.1, 122.4, 135.6, 137.2, 143.9, 145.9, 146.3, 149.1, 153.1
(2C); ESI-MS: m/z = 371.2 [M+H]+, 741.3 [2M+H]+, 763.3 [2M+Na]+.
3-Methyl-5-(3-amino-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole
(7k). The title compound was obtained according to the procedure described above for 11.
Yield: 95%; pale yellow solid; m.p. 129–130°C; 1H-NMR (400 MHz, CDCl3): δ 2.36 (3H, s),
3.70 (6H, s), 3.82 (3H, s), 3.83 (3H, s), 3.91 (2H, s), 6.21 (1H, s), 6.54 (2H, s), 6.58 (1H, dd,
J = 8.28 Hz, J = 1.94 Hz), 6.65 (1H, d, J = 1.94 Hz), 6.69 (1H, d, J = 8.28 Hz); 13C-NMR (100
MHz, CDCl3): δ 13.5, 55.5, 56.0 (2C), 60.9, 102.7 (2C), 107.1, 110.0, 114.9, 119.0, 123.5, 136.0,
136.1, 136.8, 144.0, 147.2, 149.0, 152.9 (2C); ESI-MS: m/z = 370.1 [M+H]+, 739.3 [2M+H]+,
761.3 [2M+Na]+.
4-Formyl-3-methyl-5-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-
pyrazole (7l). DMF (8 mL) was cooled in an ice bath, and POCl3 (1 mL, 10.8 mmol) was
added dropwise over 10 min. The mixture was stirred for 30 min at 0°C, followed by dropwise
addition of a solution of 7i (0.06 g, 0.17 mmol) in DMF (2 mL) over 15 min. The ice bath was
removed, and the mixture was stirred at 60°C for 3 h. The reaction was quenched upon addi-
tion of 30 g of ice. The resulting mixture was extracted with EtOAc (3 × 30 mL), and the ex-
tracts were dried over Na2SO4, concentrated in vacuo and purified by chromatography on
silica gel to afford the desired 7l. Yield: 81%; pale yellow solid; m.p. 97–98°C; 1H-NMR (400
MHz, CDCl3): δ 2.61 (3H, s), 3.68 (6H, s), 3.82 (3H, s), 3.92 (3H, s), 6.49 (2H, s), 6.80 (1H, dd,
J = 8.39 Hz, J = 1.94 Hz), 6.87–6.89 (2H, m), 9.75 (1H, s); 13C-NMR (100 MHz, CDCl3): δ 12.7,
55.1 (3C), 60.0, 101.8 (2C), 109.7, 115.4, 118.7, 119.5, 121.6, 133.3, 136.6, 144.9, 146.9, 147.8,
150.0, 152.1 (2C), 185.4; ESI-MS: m/z = 399.1 [M+H]+, 421.1 [M+Na]+, 699.3 [2M+H]+, 761.3
[2M+Na]+.
4-Hydroxymethyl-3-methyl-5-(3-hydroxy-4-methoxy-phenyl)-1-(3,4,5-trimethoxyphe-
nyl)-1H-pyrazole (7m). NaBH4 (0.02 g, 0.63 mmol) was added with stirring to a suspension
of the aldehyde 7l (0.05 g, 0.13 mmol) in MeOH (20 mL) and the reaction mixture was stirred
for 2 h at room temperature. The reaction mixture was concentrated in vacuo. The resulting
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
slurry was dissolved in dichloromethane (40 mL), washed with water (2 × 20 mL), dried over
Na2SO4, filtered and concentrated under vacuum. The crude product was purified by chroma-
tography on silica gel to afford the desired 7m. Yield: 96%; white solid; m.p. 166–167°C;
1H-NMR (400 MHz, CDCl3): δ 2.44 (3H, s), 3.67 (6H, s), 3.80 (3H, s), 3.91 (3H, s), 4.52 (2H, s),
6.46 (2H, s), 6.75 (1H, d, J = 7.70 Hz), 6.83 (1H, d, J = 7.70Hz), 6.88 (1H, s); 13C-NMR (100
MHz, CDCl3): δ 11.8, 55.0, 56.0 (2C), 58.4, 60.9, 102.5 (2C), 110.6, 116.1, 118.5, 122.1, 123.1,
135.6, 136.9, 142.0, 145.6, 146.9, 148.8, 153.0 (2C); ESI-MS: m/z = 401.1 [M+H]+, 801.2 [2M
+H]+, 823.2 [2M+Na]+.
4-Bromo-3-methyl-5-(4-methoxy-3-nitrophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyra-
zole (7n). A mixture of 7h (0.08 g, 0.20 mmol) and NBS (0.04 g, 0.02 mmol) in CCl4 (4 mL)
was stirred at room temperature for 1.5 h. The progress of the reaction was monitored by thin
layer chromatography (TLC). The by-product succinimide was filtered and washed with CCl4
(5 mL). The filtrate was washed with water (2 × 10 mL) and dried over MgSO4. The solvent
was removed under reduced pressure. The crude product was purified by chromatography on
silica gel to afford the desired 7n. Yield: 88%; pale yellow solid; m.p. 182–183°C; 1H-NMR (400
MHz, CDCl3): δ 2.39 (3H, s), 3.71 (6H, s), 3.83 (3H, s), 3.99 (3H, s), 6.44 (2H, s), 7.08 (1H, d,
J = 8.62 Hz), 7.41 (1H, dd, J = 8.62 Hz, J = 2.36 Hz), 7.92 (1H, d, J = 2.36 Hz); 13C-NMR (100
MHz, CDCl3): δ 11.3, 55.3 (2C), 55.7, 60.0, 96.2, 102.0 (2C), 112.6, 120.1, 126.0, 133.5, 134.3,
137.0, 137.4, 138.4, 147.4, 152.0, 152.4 (2C); ESI-MS: m/z = 478.1 [M+H]+.
4-Bromo-3-methyl-5-(3-amino-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyr-
azole (7o). The title compound was obtained according to the procedure described above for
11. Yield: 92%; yellow solid; m.p. 128–129°C; 1H-NMR (400 MHz, CDCl3): δ 2.36 (3H, s), 3.67
(6H, s), 3.81 (3H, s), 3.85 (3H, s), 6.47 (2H, s), 6.66 (1H, d, J = 8.60 Hz), 6.71 (1H, s), 6.77 (1H,
d, J = 8.60 Hz); 13C-NMR (100 MHz, CDCl3): δ 12.5, 55.5, 56.0 (2C), 60.9, 96.6, 102.1 (2C),
110.1, 115.9, 120.4, 121.9, 135.8, 136.2, 136.9, 141.1, 147.6, 148.0, 152.9 (2C); ESI-MS: m/
z = 448.1 [M+H]+, 470.1 [M+Na]+.
3-Ethyl-5-(4-methoxy-3-nitrophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole (7p).
The title compound was obtained according to the procedure described above for 7a. Yield:
85%; yellow solid; m.p. 151–152°C; 1H-NMR (400 MHz, CDCl3): δ 1.34 (3H, t), 2.75 (2H, q),
3.73 (6H, s), 3.84 (3H, s), 3.96 (3H, s), 6.38 (1H, s), 6.50 (2H, s), 7.00 (1H, d, J = 8.85 Hz), 7.31
(1H, dd, J = 8.85 Hz, J = 2.21 Hz), 7.86(1H, d, J = 2.21Hz); 13C-NMR (100 MHz, CDCl3): δ
12.8, 20.4, 55.2 (2C), 55.7, 60.0, 102.4 (2C), 105.1, 112.4, 122.2, 124.5, 132.9, 134.3, 136.7, 138.4,
139.9, 151.4, 152.4 (2C), 154.5; ESI-MS: m/z = 414.1 [M+H]+, 827.3 [2M+H]+.
3-Propyl-5-(4-methoxy-3-nitrophenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole (7q).
The title compound was obtained according to the procedure described above for 7a. Yield:
75%; yellow solid; m.p. 149–150°C; 1H-NMR (400 MHz, CDCl3): δ 1.03 (3H, t), 1.69–1.78 (2H,
m), 2.69 (2H, t), 3.72 (6H, s), 3.84 (3H, s), 3.95 (3H, s), 6.36 (1H, s), 6.50 (2H, s), 7.00 (1H, d,
J = 11.05 Hz), 7.31 (1H, dd, J = 11.05 Hz, J = 2.37 Hz), 7.84(1H, d, J = 2.37Hz); 13C-NMR (100
MHz, CDCl3): δ 13.0, 21.8, 29.2, 55.2 (2C), 55.7, 60.0, 102.4 (2C), 105.6, 112.4, 122.2, 124.5,
132.9, 134.3, 136.7, 138.4, 139.8, 151.4, 152.4 (2C), 153.2; ESI-MS: m/z = 428.1 [M+H]+.
3-Ethyl-5-(3-amino-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole (7r).
The title compound was obtained according to the procedure described above for 11. Yield:
95%; pale yellow solid; m.p. 114–115°C; 1H-NMR (400 MHz, CDCl3): δ 1.32 (3H, t), 2.74 (2H,
q), 3.70 (6H, s), 3.82 (3H, s), 3.84 (3H, s), 6.24 (1H, s), 6.54 (2H, s), 6.58 (1H, dd, J = 8.37 Hz,
J = 2.05 Hz), 6.64 (1H, d, J = 2.05 Hz), 6.69 (1H, d, J = 8.37 Hz); 13C-NMR (100 MHz, CDCl3):
δ 13.9, 21.5, 55.6, 56.1 (2C), 61.0, 102.8 (2C), 105.5, 110.1, 115.3, 119.4, 123.6, 135.5, 136.1,
136.8, 143.9, 147.4, 153.0 (2C), 15/5.1; ESI-MS: m/z = 384.2 [M+H]+, 767.4 [2M+H]+, 789.3
[2M+Na]+.
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
3-Propyl-5-(3-amino-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrazole
(7s). The title compound was obtained according to the procedure described above for 11.
Yield: 90%; pale yellow solid; m.p. 125–126°C; 1H-NMR (400 MHz, CDCl3): δ 1.03 (3H, t),
1.64–1.80(2H, m), 2.68 (2H, t), 3.69 (6H, s), 3.82 (3H, s), 3.83 (3H, s), 4.35 (2H, s), 6.23 (1H, s),
6.54 (2H, s), 6.58 (1H, dd, J = 8.30 Hz, J = 2.10 Hz), 6.64 (1H, d, J = 2.10 Hz), 6.69 (1H, d,
J = 8.30 Hz); 13C-NMR (100 MHz, CDCl3): δ 14.1, 22.9, 30.4, 55.5, 56.1 (2C), 61.0, 102.8 (2C),
106.0, 110.1, 115.0, 119.1, 123.7, 136.0, 136.1, 136.8, 143.8, 147.2, 152.9 (2C), 153.8; ESI-MS:
m/z = 398.1 [M+H]+.
3-Methyl-1-phenyl-5-(3,4,5-trimethoxyphenyl)-1H-pyrazole (8a). The title compound
was obtained according to the procedure described above for 7a. Yield: 74%; yellow solid; m.
p. 88–89°C; 1H-NMR (400 MHz, CDCl3): δ 2.38 (3H, s), 3.63 (6H, s), 3.84 (3H, s), 6.32 (1H, s),
6.40 (2H, s), 7.27–7.52 (5H, m); 13C-NMR (100 MHz, CDCl3): δ 13.4, 55.9 (2C), 60.8, 105.8
(2C), 106.0, 107.2, 125.5 (2C), 127.5, 128.9 (2C), 138.0, 139.8, 143.8, 149.1, 153.0 (2C); ESI-MS:
m/z = 325. 1 [M+H]+.
3-Methyl-1-(4-methoxyphenyl)-5-(3,4,5-trimethoxy-phenyl)-1H-pyrazole (8b). The
title compound was obtained according to the procedure described above for 7a. Yield: 78%;
brown solid; m.p. 67–68°C; 1H-NMR (400 MHz, CDCl3): δ 2.41 (3H, s), 3.67 (6H, s), 3.80 (3H,
s), 3.85 (3H, s), 6.31 (1H, s), 6.42 (2H, s), 6.88 (2H, d, J = 8.60 Hz), 7.24 (2H, d, J = 8.60 Hz);
13C-NMR (100 MHz, CDCl3): δ 13.5, 55.5, 55.9 (2C), 60.9, 105.9 (2C), 106.6, 114.0 (2C), 125.8,
126.9 (2C), 133.2, 137.9, 143.7, 148.8, 153.0 (2C), 158.8; ESI-MS: m/z = 355.1 [M+H]+, 731.3
[2M+Na]+.
3-Methyl-1-(4-methylphenyl)-5-(3,4,5-trimethoxy-phenyl)-1H-pyrazole (8c). The title
compound was obtained according to the procedure described above for 7a. Yield: 73%; yellow
solid; m.p. 70–71°C; 1H-NMR (400 MHz, CDCl3): δ 2.33 (3H, s), 2.37 (3H, s), 3.64 (6H, s), 3.84
(3H, s), 6.29 (1H, s), 6.40 (2H, s), 7.14 (4H, m); 13C-NMR (100 MHz, CDCl3): δ 12.5, 20.0, 54.9
(2C), 59.9, 105.0 (2C), 105.9, 124.3 (2C), 124.9, 128.4 (2C), 136.2, 136.7, 136.9, 142.6, 148.0,
151.9 (2C); ESI-MS: m/z = 339.2 [M+H]+.
3-Methyl-1-(2-methyl-5-nitrophenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazole (8d).
The title compound was obtained according to the procedure described above for 7a. Yield:
71%; pale yellow solid; m.p. 102–103°C; 1H-NMR (400 MHz, CDCl3): δ 2.09 (3H, s), 2.39 (3H,
s), 3.63 (6H, s), 3.81 (3H, s), 6.33 (2H, s), 6.39 (1H, s), 7.42 (1H, d, J = 8.44 Hz), 8.17 (1H, dd,
J = 8.44 Hz, J = 2.36 Hz), 8.23 (1H, d, J = 2.36 Hz); 13C-NMR (100 MHz, CDCl3): δ 12.4, 17.1,
55.0 (2C), 59.9, 104.2 (2C), 105.3, 122.6, 122.7, 130.8, 137.5, 138.8, 138.9, 143.1, 144.5, 145.5,
149.0, 152.3 (2C); ESI-MS: m/z = 384.2 [M+H]+, 406.1 [M+Na]+, 422.1 [M+K]+, 789.3 [2M
+Na]+.
3-Methyl-1-(5-amino-2-methylphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazole (8e).
The title compound was obtained according to the procedure described above for 11. Yield:
90%; pale yellow solid; m.p. 69–70°C; 1H-NMR (400 MHz, CDCl3): δ 1.78 (3H, s), 2.37 (3H,s),
3.64 (6H, s), 3.82 (3H, s), 6.34 (1H, s), 6.43 (2H, s), 6.68 (2H, m), 6.98 (1H, d, J = 8.37 Hz);
13C-NMR (100 MHz, CDCl3): δ 13.5, 16.5, 55.9 (2C), 60.9, 104.8 (2C), 104.9, 115.3, 116.5,
125.6, 126.0, 131.5, 137.8, 140.3, 144.2, 148.9, 152.9 (2C); ESI-MS: m/z = 354.2 [M+H]+.
3-Methyl-1-(4-methoxy-3-nitrophenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazole (8f).
The title compound was obtained according to the procedure described above for 7a. Yield:
78%; pale yellow solid; m.p. 118–119°C; 1H-NMR (400 MHz, CDCl3): δ 2.37 (3H, s), 3.73 (6H,
s), 3.87 (3H, s), 3.97 (3H, s), 6.31 (1H, s), 6.43 (2H, s),7.02 (1H, d, J = 8.79 Hz), 7.41 (1H, dd,
J = 8.79 Hz, J = 2.61 Hz), 7.92 (1H, d, J = 2.61 Hz); 13C-NMR (100 MHz, CDCl3): δ 13.5, 56.2
(2C), 56.8, 60.9, 106.2 (2C), 108.0, 113.5, 122.0, 125.4, 130.1, 132.7, 138.5, 139.1, 143.8, 150.0,
151.4, 153.3 (2C).
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
3-Methyl-1-(3-amino-4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazole
(8g). The title compound was obtained according to the procedure described above for 11.
Yield: 89%; white solid; m.p. 56–57°C; 1H-NMR (400 MHz, CDCl3): δ 2.35 (3H, s), 3.68 (6H, s),
3.82 (3H, s), 3.84 (3H, s), 3.96 (2H, s), 6.27 (1H, s), 6.45 (2H, s), 6.54 (1H, dd, J = 8.52 Hz,
J = 2.16 Hz), 6.68 (1H, d, J = 8.52 Hz), 6.80 (1H, d, J = 2.16 Hz); 13C-NMR (100 MHz, CDCl3): δ
13.4, 55.7, 55.9 (2C), 60.8, 105.8 (2C), 106.4, 109.9, 112.4, 115.7, 126.0, 133.6, 136.3, 137.8, 143.5,
146.6, 148.6, 152.9 (2C); ESI-MS: m/z = 370.1 [M+H]+, 739.3 [2M+H]+, 761.3 [2M+Na]+.
3-Methyl-1-(3-allyloxy-4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazole
(8h). The title compound was obtained according to the procedure described above for 7a.
Yield: 77%; brown solid; m.p. 98–99°C; 1H-NMR (400 MHz, CDCl3): δ 2.40 (3H, s), 3.68 (6H,
s), 3.85 (3H, s), 3.87(3H, s), 4.50 (2H, d, J = 5.07 Hz), 5.18–5.21 (1H, m), 5.27–5.32 (1H, m),
5.90–6.00 (1H, m), 6.31 (1H, s), 6.42 (2H, s), 6.81–6.87 (3H, m); 13C-NMR (100 MHz, CDCl3):
δ 13.5, 56.0 (2C), 56.2, 60.9, 69.9, 106.0 (2C), 106.8, 111.2, 111.3, 118.1 (2C), 126.0, 132.6,
133.2, 137.9, 143.6, 147.9, 148.7, 148.9, 153.0 (2C).
3-Methyl-1-(3-hydroxy-4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazole
(8i). The title compound was obtained according to the procedure described above for 7i.
Yield: 90%; grey solid; m.p. 167–168°C; 1H-NMR (400 MHz, CDCl3): δ 2.36 (3H, s), 3.66 (6H,
s), 3.80 (3H, s), 3.84 (3H, s), 6.29 (1H, s), 6.44 (2H, s), 6.67 (1H, dd, J = 8.59 Hz, J = 2.26 Hz),
6.72 (1H, d, J = 8.59 Hz), 6.99 (1H, d, J = 2.26 Hz); 13C-NMR (100 MHz, CDCl3): δ 13.4, 56.0
(3C), 60.9, 105.9 (2C), 106.5, 110.5, 113.1, 117.4, 125.8, 133.4, 137.9, 143.8, 146.1, 146.6, 148.8,
152.9 (2C); ESI-MS: m/z = 371.2 [M+H]+, 741.3 [2M+H]+, 763.3 [2M+Na]+.
Biology
Cell line and culture conditions. Pulmonary adenocarcinoma cell lines (A549) and gas-
tric adenocarcinoma cell lines (SGC-7901) were provided as gifts (purchased from the Cell Re-
source Center of Shanghai Institutes for Biological Sciences) from the China-Japan Research
Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University. Fibro-
sarcoma cell lines (HT-1080) were purchased from the Cell Resource Center of Shanghai Insti-
tutes for Biological Sciences. All cells were maintained in DMEM containing 1% antibiotics
(100 units of penicillin and 10 mg streptomycin per mL in 0.9% normal saline) and 10% foetal
bovine serum in a humidified atmosphere containing 5% CO2 at 37°C.
MTT assay. Antiproliferative activities of CA-4 and the target compounds were measured
using a colourimetric assay using 3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyltetrazolium bro-
mide (MTT). Approximately 3 × 104 cells were seeded in a 96-well plate. After 24 h of incuba-
tion at 37°C, cells were exposed to compounds of differing concentrations (0.0008 μg/mL,
0.004 μg/mL, 0.02 μg/mL, 0.10 μg/mL, 0.50 μg/mL, 2.5 μg/mL, 12.5 μg/mL, 62.5 μg/mL) for 72
h. After treatment, cells were washed with 1X PBS followed by addition of 100 μL of 0.05%
MTT reagent to each well, followed by incubation for 4 h at 37°C. After incubation, the super-
natant from each well was carefully removed and the formazan crystals were dissolved in
100 μL of DMSO. The colour density was measured spectrophotometrically at 490 nm using a
microplate reader (SpectraMax Plus384, Molecular Devices Corp., USA). The percentage of
cell growth inhibition was calculated as follow: inhibition ratio % = (Acontrol—Atreated) / Acontrol
× 100%. The 50% inhibitory concentration (IC50) was defined as the concentration that re-
duced the absorbance of the untreated wells by 50% of the vehicle in the MTT assay.
Tubulin assembly assay. The effect of compounds 7i and 7k on the polymerization of pu-
rified brain tubulin was determined employing a fluorescence-based tubulin polymerization
assay kit (Cat. #BK011P, Cytoskeleton, Inc., USA) according to the manufacturer’s protocol.
Tubulin was re-suspended in ice-cold G-PEM buffer (80 mM PIPES, 2 mM MgCl2, 0.5 mM
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Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
EGTA, 1 mM GTP, 20% (v/v) glycerol) and added to wells on a 96-well plate containing the
designated concentration of drugs or vehicle. Samples were mixed well, and tubulin assembly
was monitored (emission wavelength: 450 nm; excitation wavelength: 360 nm) at 1 min inter-
vals for 90 min at 37°C using a plate reader (FASCalibur, BD Biosciences, USA). IC50 values
were calculated at 80 min using SPSS software in three experiments.
Immunostaining of tubulin assembly and DAPI nuclear staining. Immunostaining
was performed to detect microtubule-associated tubulin protein after exposure to CA-4 and
compound 7k. The SGC-7901 and HT-1080 cells were seeded at a density of 1 × 104 per well
on a 24-well plate and grown for 24 h. Cells were treated with vehicle or 2-fold IC50 concen-
trations of CA-4 or 7k for 48 h. The control and treated cells were fixed in acetone:methanol
(1:1) for 30 min at -20°C, washed with 1X PBS, incubated for 30 min in 1X PBS containing
2% BSA, and then 0.1% Triton X-100. The primary tubulin antibody (Cell Signaling Technol-
ogy, MA, USA) was diluted (1:100) with 2% BSA in 1X PBS and incubated overnight at 4°C.
The cells were washed with 1X PBS to remove unbound primary antibody, and then cells
were incubated for 3 h at 37°C with FITC-conjugated anti-mouse secondary antibody diluted
1:1000 with 2% BSA in PBS. The cells were washed with 1X PBS to remove unbound second-
ary antibody, the nucleus was stained with 4,6-diamino-2-phenolindole dihydrochloride
(DAPI), and immunofluorescence was detected using an Olympus inverted fluorescence
microscope.
Docking study
The initial coordinates for tubulin were taken from the crystal structure of tubulin in complex
with colchicine (PDB ID: 1SA0) obtained from the Protein Data Bank. Molecular docking was
performed using the Discovery Studio 3.0 software package’s CDOCKER protocol with the de-
fault settings. The protein was prepared by removing all of the ions and substructures present
and then adding hydrogen atoms. In the docking process, the active site was defined along with
the CA-4 (1) complex. Compound 7k was docked into the active site and the docking simula-
tions were performed using the Discovery studio 3.0 programme.
Supporting Information
S1 File. Contents: 1H and 13C NMR spectra of all target compounds and representative in-
termediates. Supporting Information include the NMR (1H and 13C) spectra of the synthesized
pyrazole derivatives and representative intermediates.
(PDF)
Acknowledgments
This work was supported by the Programme for Innovative Research Team of the Ministry of
Education and the Programme for Liaoning Innovative Research Team in University.
Author Contributions
Conceived and designed the experiments: WZ YW. Performed the experiments: QX HQ XJ
MS. Analyzed the data: QX DZ Z. Wen. Contributed reagents/materials/analysis tools: Z.
Wang. Wrote the paper: QX. Helped analyze the data: HQ.
References
1. Jordan MA, Wilson L (2004) Microtubules as a target for anticancer drugs. Nat Rev Cancer 4: 253–
265. PMID: 15057285
PLOS ONE | DOI:10.1371/journal.pone.0128710 June 10, 2015
16 / 18

Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
2. Dumontet C, Jordan MA (2010) Microtubule-binding agents: a dynamic field of cancer therapeutics. Nat
Rev Drug Discov 9: 790–803. doi: 10.1038/nrd3253 PMID: 20885410
3. Amos LA (2004) Microtubule structure and its stabilization. Org Biomol Chem 2: 2153–2160. PMID:
15280946
4. Lu Y, Chen JJ, Xiao M, Li W, Miller DD (2012) An overview of tubulin inhibitors that interact with the col-
chicine binding site. Pharm Res 29: 2943–2971. doi: 10.1007/s11095-012-0828-z PMID: 22814904
5. Kanthou C, Tozer GM (2009) Microtubule depolymerizing vascular disrupting agents: Novel therapeutic
agents for oncology and other pathologies. Int J Exp Pathol 90: 284–294. doi: 10.1111/j.1365-2613.
2009.00651.x PMID: 19563611
6. Tron GC, Pirali T, Sorba G, Pagliai F, Busacca S, Genazzani AA (2006) Medicinal chemistry of combre-
tastatin A4: present and future directions. J Med Chem 49: 3033–3044. PMID: 16722619
7. Pettit GR, Singh SB, Hamel E, Lin CM, Alberts DS, Garcia-Kendall D (1989) Isolation and structure of
the strong cellgrowth and tubulin inhibitor combretastatin A-4. Experientia 45: 209–211. PMID: 2920809
8. McGown AT, Fox BW (1990) Differential cytotoxicity of Combretastatins A1 and A4 in two daunorubi-
cin-resistant P388 cell lines. Cancer Chem Pharm 26: 79–81.
9. Pettit GR, Toki BE, Herald DL, Boyd MR, Hamel E, Pettit RK, et al. (1999) Antineoplastic agents. 410.
Asymmetric hydroxylation of trans-combretastatin A-4. J Med Chem 42: 1459–1465. PMID: 10212132
10. Lee L, Davis R, Vanderham J, Hills P, Mackay H, Brown T, et al. (2008) 1,2,3,4-Tetrahydro-2-thioxopyr-
imidine analogs of combretastatin-A4. Eur J Med Chem 43: 2011–2015. doi: 10.1016/j.ejmech.2007.
11.030 PMID: 18226429
11. Blanch NM, Chabot GG, Quentin L, Scherman D, Bourg S, Dauzonne D (2012) In vitro and in vivo bio-
logical evaluation of new 4,5-disubstituted 1,2,3-triazoles as cis-constrained analogs of combretastatin
A4. Eur J Med Chem 54: 22–32. doi: 10.1016/j.ejmech.2012.04.017 PMID: 22647220
12. Tron GC, Pagliai F, Del GE, Genazzani AA, Sorba G (2005) Synthesis and cytotoxic evaluation of com-
bretafurazans. J Med Chem 48: 3260–3268. PMID: 15857132
13. Biersack B, Effenberger K, Knauer S, Ocker M, Schobert R (2010) Ru(h6-arene) complexes of combre-
tastatin-analogous oxazoles with enhanced anti-tumoral impact. Eur J Med Chem 45: 4890–4896. doi:
10.1016/j.ejmech.2010.07.061 PMID: 20727621
14. Kaffy J, Pontikis R, Carrez D, Croisy A, Monneret C, Florent JC (2006) Isoxazole-type derivatives relat-
ed to combretastatin A-4, synthesis and biological evaluation. Bioorg Med Chem 14: 4067–4077.
PMID: 16510288
15. Wang L, Woods KW, Li Q, Barr KJ, McCroskey RW, Hannick SM, et al. (2002) Potent, orally active het-
erocycle-based combretastatin A-4 analogues: synthesis, structure-activity relationship, pharmacoki-
netics, and in vivo antitumor activity evaluation. J Med Chem 45: 1697–1711. PMID: 11931625
16. Zhang Q, Peng Y, Wang XI, Keenan SM, Arora S, Welsh WJ (2007) Highly potent triazole-based tubu-
lin polymerization inhibitors. J Med Chem 50: 749–754. PMID: 17249649
17. Kumara V, Kaur K, Gupta GK, Sharmac AK (2013) Pyrazole containing natural products: Synthetic pre-
view and biological significance. Eur J Med Chem 69: 735–753. doi: 10.1016/j.ejmech.2013.08.053
PMID: 24099993
18. Liu JJ, Zhao M, Zhang X, Zhao X, Zhu HL (2013) Pyrazole Derivatives as Antitumor, Anti-Inflammatory
and Antibacterial Agents. Mini-Reviews in Med Chem 10: 1957–1966.
19. Li M; Zhao BX (2014) Progress of the synthesis of condensed pyrazole derivatives (from 2010 to mid-
2013). Eur J Med Chem 85: 311–340. doi: 10.1016/j.ejmech.2014.07.102 PMID: 25104650
20. Tsyganov DV, Konyushkin LD, Karmanova IB, Firgang SI, Strelenko YA, Semenova MN, et al. (2013)
cis-Restricted 3-aminopyrazole analogues of combretastatins: synthesis from plant polyalkoxyben-
zenes and biological evaluation in the cytotoxicity and phenotypic sea urchin embryo assays. J Nat
Prod 76: 1485–1491. doi: 10.1021/np400310m PMID: 23924236
21. Burja B, Cimbora-Zovko T, Tomic S, Jelušic T, Kocevar M, Polanc S, et al. (2010) Pyrazolone-fused
combretastatins and their precursors: synthesis, cytotoxicity, antitubulin activity and molecular model-
ing studies. Bioorg Med Chem 18: 2375–2387. doi: 10.1016/j.bmc.2010.03.006 PMID: 20338766
22. Gong JX, Huang K, Wang F, Yang L, Feng Y, Li H, et al. (2009) Preparation of two sets of 5,6,7-trioxy-
genated dihydroflavonol derivatives as free radical scavengers and neuronal cell protectors to oxidative
damage. Bioorg Med Chem 17: 3414–3425. doi: 10.1016/j.bmc.2009.03.032 PMID: 19362850
23. Clive DLJ, Angoh G, Bennett SM (1987) Radical spirocyclization: synthesis of an appropriately oxygen-
ated spiro compound related to the antitumor antibiotic Fredericamycin A. J Org Chem 52: 1339–1342.
24. Ferlin MG, Marzano C, Via LD, Chilin A, Zagotto G, Guiotto A, et al. (2005) New water soluble pyrrolo-
quinoline derivatives as new potential anticancer agents. Bioorg Med Chem Lett 13: 4733–4739.
PMID: 15936202
PLOS ONE | DOI:10.1371/journal.pone.0128710 June 10, 2015
17 / 18

Synthesis and Biological Evaluation of 3-Alkyl-1,5-Diaryl-1H-Pyrazoles
25. Fehnel EA (1958) Quinoline analogs of podophyllotoxin. I. preliminary experiments. syntheses of some
4-phenylquinoline derivatives. J Org Chem 23: 432–434.
26. Genin MJ, Biles C, Keiser BJ, Poppe SM, Swaney SM, Tarpley WG, et al. (2000) Novel 1,5-diphenyl-
pyrazole nonnucleoside HIV-1 reverse transcriptase inhibitors with enhanced activity versus the dela-
virdine-resistant P236L mutant: lead identification and SAR of 3- and 4-substituted derivatives. J Med
Chem 43: 1034–1040. PMID: 10715167
27. Kadam SM, Nayak SK, Banerji A (1992) Low-valent titanium: a new approach to deprotection of allyl
and benzyl groups. Tetrahedron Lett 33: 5129–5132.
28. Frey RR, Curtin ML, Albert DH, Glaser KB, Pease LJ, Soni NB, et al. (2008) 7-Aminopyrazolo[1,5- a] py-
rimidines as potent multitargeted receptor tyrosine kinase inhibitors. J Med Chem 51: 3777–3787. doi:
10.1021/jm701397k PMID: 18557606
29. Zhao ZG, Wang ZX (2007) Halogenation of pyrazoles using N-Halosuccinimides in CCl4 and in water.
Synth Commun 37: 137–147.
30. Serrette AG, Lai CK, Swager TM (1994) Complementary shapes in columnar liquid crystals: structural
control in homo- and heteronuclear bimetallic assemblies. Chem Mater 6: 2252–2268.
PLOS ONE | DOI:10.1371/journal.pone.0128710 June 10, 2015
18 / 18