Synthesis, biological evaluation and molecular docking studies of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacrylamide derivatives as inhibitors of HDAC activity

Article abstract of DOI:10.1016/j.bmc.2012.05.031

In present study, a series of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N- phenylacrylamide derivatives (5a-8d) were designed, synthesized, and evaluated for HDAC inhibition and tumor cell antiproliferation. All of these compounds are reported for the first time,

Full text of DOI:10.1016/j.bmc.2012.05.031

Bioorganic & Medicinal Chemistry 20 (2012) 4430–4436
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular docking studies
of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacrylamide derivatives
as inhibitors of HDAC activity
Xi Li a, Jia-Lin Liu a, Xian-Hui Yang a, Xiang Lu a, Ting-Ting Zhao a, Hai-Bin Gong b, , Hai-Liang Zhu a,
⇑
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b Xuzhou Central Hospital, Xuzhou 221009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In present study, a series of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacrylamide derivatives (5a–8d)
were designed, synthesized, and evaluated for HDAC inhibition and tumor cell antiproliferation. All of
these compounds are reported for the first time, the chemical structures of these compounds were con-
firmed by means of 1H NMR, ESI-MS and elemental analyzes. Among the compounds, compound 8c
Received 27 March 2012
Revised 14 May 2012
Accepted 14 May 2012
Available online 26 May 2012
showed the most potent biological activity against HCT116 cancer cell line (IC50 of 0.42 0.02 lM for
HDAC-1 and IC50 = 0.62 0.02 for HCT116). Docking simulation was performed to position compound
8c into the HDAC active site to determine the probable binding model. The results of antiproliferative
assay and western-blot demonstrated that compound 8c with potent inhibitory activity in tumor growth
inhibition may be a potential anticancer agent against HCT116 cancer cell.
Keywords:
3-(1,3-Diphenyl-1H-pyrazol-4-yl)-N-
phenylacrylamide derivatives
Histone deacetlylase (HDAC)
Structure–activity relationship
Molecular docking
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
ently in clinical trial for the treatment of a variety of haematolog-
ical and solid tumors, including colon cancer.10–12
The role of gene regulation by physical alterations of either DNA
or the structural components of chromatin has recently been high-
lighted as a major process in neoplastic transformation and main-
tenance of the malignant phenotype.1 The reversible acetylation of
-N-acetyl groups of lysine residues, in the N-terminal tails of core
histones, plays a critical role in the regulation of gene expression
by altering the accessibility of transcriptional factors to DNA via
conformational changes in the structure of the nucleosome.2–4 This
restructuring of chromatin is regulated by the balance of histone
acetyl transferase (HAT) and histone deacetylase (HDAC) activ-
ity.5,6 Perturbations of this balance have been linked to gene
expression, the inappropriate recruitment of HDACs provides at
least one mechanism by which oncogenes can alter gene expres-
sion in favor of excessive proliferation.7 Moreover, evidence dem-
onstrates that inhibition of HDAC triggers growth arrest,
differentiation, and/or apoptosis in many types of tumor cells by
reactivating the transcription of a small number of genes.8,9 These
findings suggest that modulation of HDAC’s function might be tar-
geted for the prevention and/or therapeutic intervention of cancer.
Based on pre-clinical findings, several HDAC inhibitors are pres-
Till date, several structurally distinct compounds targeting
HDAC enzyme have been reported, and with few exceptions, these
can be divided into a few structural classes, including small-molec-
ular hydroxmates, carboxylates, benzamides, electrophilic ketones
and cyclic peptides.13 Recent publication of the X-ray crystallogra-
phy revealed that the structure of the HDAC active site is com-
prised of a tubular hydrophobic pocket and active site zinc.14
A
typical HDAC inhibitor includes three molecular fragments,
namely, a metal-binding domain, which interacts with the active
site, a linker domain, which occupies the channel, and a surface
recognition domain, which interacts with residues on the rim of
the active site.15,16 Capitalizing on published SAR, we designed
and synthesized a variety of compounds building off of an acyla-
mide template, with the presumption that the carbonyl-oxygen
group coordinate to the active site zinc and the double bond acts
as a hydrocarbon linker to fill out the narrow tunnel.17 On the
other hand, acylamide is an important pharmacophore of natural
products and the synthetic precursors to various drugs.18,19 On that
basis, a double bond was introduced into the amide moiety to
construct an
a,b-unsaturated acylamide, with the reasoning that
a
,b-unsaturated enones could also react with the nuclephilic
groups of residues in HDAC domain.20 Furthermore, structure-
guided designs suggested that a substituted five-membered ring
projecting from the 3-position of N-cinnamamide would best
facilitate productive interaction between a binding moiety and
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail addresses: ghbxzh@yahoo.com.cn (H.-B. Gong), zhuhl@nju.edu.cn (H.-L.
Zhu).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.031

X. Li et al. / Bioorg. Med. Chem. 20 (2012) 4430–4436
4431
Figure 1. Three main types of scaffold found in reported HDAC inhibitors. Pharmacophoric features of HDAC inhibitors in the title compounds.
the protein surface.21 As is known, pyrazole derivatives are a very
interesting class of heterocyclic compounds that have remarkable
pharmacological activities as antibacterial–antifungal, tumor
necrosis inhibitor and antiangiogenic agent.22–26 Given the phar-
macological function of pyrazole derivatives, N-arylpyrazole is
chosen as a surface recognition domain to interact with the amino
acids near the entrance of the active site. In an effort to enhance
the affinity and selective of HDAC, we synthesized a series of com-
pounds building off of an acylamide template combined with pyr-
azole considering that they might exhibit synergistic effect in
anticancer activities by the inhibition against HDAC (Fig. 1).
In this paper, we synthesized a series of 3-(1,3-diphenyl-1H-
pyrazol-4-yl)-N-phenylacrylamid, in an attempt to extend previ-
ous research on the benzamide class of HDAC inhibitors. Herein,
in continuation to further our research on antitumor compounds
with HDAC structure inhibitory activity, advances have been made
in designing new phenylacrylamid derivatives, which share the
core template with the benzamide class can be as developments
to enrich and optimize HDAC inhibition activity. The anticancer
activities of the synthesized compounds were evaluated in
HCT116 and the preliminary mechanism of the active compound
was further examined by docking study.
2. Results and discussion
2.1. Chemistry
The synthetic route of the 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-
phenylacrylami derivatives is outlined in Scheme 1. As reported,27
the synthesis of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacryl-
amid begins with the interaction of substituted 1,3-diphenyl-1H-
pyrazole-4-acrylic acid and substituted anilines with the help of
DMAP and K2CO3 in anhydrous methylene dichloride to give the
desired compounds. These compounds are all reported for the first
Scheme 1. General synthesis of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacrylamid derivates derivatives (5a–8d). Reagents and conditions: (i) ethanol, 50–60 °C, 3 h; (ii)
DMF, POCl3, 50–60 °C, 5 h; (iii) piperidine/CH2(COOH)2,40–50 °C, 5 h;(iv)DMAP, K2CO3, dichloromethane, 50–60 °C, 12 h.

4432
X. Li et al. / Bioorg. Med. Chem. 20 (2012) 4430–4436
Table 1
structure variation and modifications. By investigating the varia-
Structure of compounds 5a–8d
tion in selectivity of the tested compounds over the two cell lines,
it was obviously revealed that different substitutes on the A-ring
determine the primary order of potency. A comparison of the
para-substituents on the A-ring demonstrated that NO2 group have
dramatically improved antiproliferative activity, with the pre-
sumption that NO2 group at the para-position of ring A may bind
potently into HDAC through
p-cation interaction. In addition,
para-OMe group substituent is also well tolerated in this position,
suggesting that para-substituents of A-ring play a key role in react-
ing with the function groups of residues in HDAC domain. How-
ever, Me and Br group showed minimal effects for the reason
that they may lack chemical bonds with HDAC. In the case of con-
stant A-ring substituents, change of substituents on the B-ring
could also affect the activities of these compounds. Among the
compounds, the compounds with Br and Cl group as electron-with-
drawing substituents on ring B are of better antitumor activity
comparing to those with electron-donating, the potency of para-
substituents on the B-ring is ordered as: Br > Cl > H > MeO, fol-
lowed that 5d,7d are not tolerated. Among all the compounds, 8c
with para-NO2 group on the A-ring and para-Br on the B-ring,
respectively, led to a noteworthy best activity.
Compd
R1
R2
Compd
R1
R2
5a
5b
5c
5d
6a
6b
6c
6d
Me
Me
Me
OMe
OMe
OMe
OMe
OMe
H
Cl
Br
OMe
H
Cl
Br
OMe
7a
7b
7c
7d
8a
8b
8c
8d
Br
Br
Br
Br
NO2
NO2
NO2
NO2
H
Cl
Br
OMe
H
Cl
Br
OMe
On the basis of previous research, it is revealed that the class of
benzamide HDAC inhibitors bore exclusively HDAC-1 and HDAC-2
inhibition.17 To further examine whether the compounds inhibit
HDAC, we screen compounds 5a–8d against the HDAC-1 and
HDAC-2. The results were summarized in Table 2. Most of the
tested compounds displayed potent HDAC-1 and HDAC-2 inhibi-
tory. Among them, compounds 8c showed the most potent inhibi-
time. All of the synthesized compounds 5a–8d (Table 1) gave sat-
isfactory elementary analytical and spectroscopic data. 1H NMR
and ESI-MS spectra were consistent with the assigned structures.
2.2. Bioactivity
tory with IC50 of 0.42 0.02
lM against HDAC-1 and IC50 of
0.55 0.03 M against HDAC-2. The results of HDAC inhibitory
l
To test the anticancer activities of the synthesized compounds,
we evaluated antiproliferative activities of compounds 5a–8d
against HCT116 and A549 cells. The results were summarized in
Table 2. As illustrated in Table 2, with few exception, the active
activity of the tested compounds were corresponded to the struc-
ture relationships (SAR) of their antitumor activities. This demon-
strated that the potent antitumor activities of the synthetic
compounds were probably correlated to their HDAC inhibitory
activities.
In an effort to study the preliminary mechanism of the com-
pound with potent inhibitory activity, the Western-blot experi-
ment was performed to assay the effect of compound 8c. The
Western-blot results were summarized in Figure 2. The result indi-
cated that compound 8c showed excellent HDAC inhibitory activity
against HCT116.
analogs showed
a remarkable potential antitumor activity,
suggesting that 1H-pyrazole-4-phenylacrylamid pharmacophore
significantly enhanced anticancer potency. For the given com-
pounds, it was observed that compound 8c showed the most
potent biological activity (IC50 = 0.62 0.02 for HCT116 and
IC50 = 3.71 0.12 for A549).
From the results listed in Table 2 we can arrive at the conclusion
that the activity of the tested compounds may be correlated to
In order to gain more insight into the potency of the studied
compounds and guide further structure–activity relationship stud-
ies, we proceeded to examine the interaction of compound 8c with
human HDAC-2 crystal structure (3MAX.pdb). The molecular dock-
ing was performed by inserting compound 8c into the active site of
HDAC-2. All docking runs were applied LigandFit Dock protocol of
Discovery Studio 3.1. The binding mode of compound 8c and
HDAC-2 were depicted in Figure 3a. All the amino acid residues
which had interactions with HDAC-2 were exhibited in Figure 4.
In the binding mode, compound 8c is potently bound to the active
site of HDAC-2 via hydrophobic interactions and binding is stabi-
Table 2
Inhibition (IC50) of HCT116, A549 cells proliferation and inhibition of HDAC-1, HDAC-
2 by compounds 5a–8d
Compd
IC50 SD (lM)
HCT116a
A549a
HDAC-1b
HDAC-2b
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
10.36 0.56
7.65 0.63
7.16 0.91
9.81 0.14
2.09 0.12
1.56 0.08
1.53 0.06
2.12 0.10
10.71 1.03
8.37 0.98
8.16 0.79
9.69 1.09
0.85 0.03
0.64 0.04
0.62 0.02
1.08 0.03
<0.10
16.13 0.72
15.41 0.89
11.79 0.83
>20
9.85 0.65
8.04 0.79
7.47 0.74
8.59 1.07
>20
15.75 0.91
12.18 1.13
>20
5.18 0.47
3.72 0.16
3.71 0.12
5.17 0.64
<0.10
4.06 0.10
3.65 0.13
3.33 0.21
4.05 0.14
1.76 0.12
1.09 0.08
1.05 0.06
1.73 0.10
3.43 0.13
2.98 0.18
2.83 0.09
3.31 0.09
0.92 0.03
0.46 0.04
0.42 0.02
1.02 0.02
<0.10
8.75 0.67
5.86 0.46
5.02 0.38
6.45 0.33
2.67 0.14
2.36 0.12
2.17 0.10
3.02 0.45
6.52 0.43
4.99 0.28
4.87 0.26
5.35 0.27
1.15 0.09
0.57 0.04
0.55 0.03
1.15 0.08
<0.10
lized by a hydrogen bond and two
p-cation interactions. The
hydrogen atom of the amide group involved in a hydrogen bond
8d
MS-275
a
Inhibition of the growth of tumor cell lines.
Inhibition of HDAC.
Figure 2. Compound 8c was examined by western blotting. Data are representative
of three independent experiments.
b

X. Li et al. / Bioorg. Med. Chem. 20 (2012) 4430–4436
4433
zinc, in an attempt to access the pockt as deeply. On the other
hand, the benzene ring with nitro moiety formed two -cation
p
interactions, respectively, with Phe C:155 (bond length: 6.36 Å)
and Phe C:210 (bond length: 3.78 Å), Phe 155 and Phe 210 are part
of the lipophilic tube, which leads from the surface to this machin-
ery. The enzyme surface model was showed in Figure 3b, which re-
vealed that the molecule was well embedded in the active pocket.
This molecular docking result, along with the biological assay data,
suggested that compound 8c was a potential inhibitor of HDAC.
3. Conclusion
In this study, a series of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-
phenylacrylamid derivatives have been synthesized and evaluated
for their antitumor activities. These compounds exhibited potent
antiproliferative activities against HTC116 and HDAC inhibitory
activities. Among all of the synthesized compounds, compound
8c demonstrated the most potent inhibitory activity (IC50 of
0.42 0.02 lM for HDAC-1 and IC50 of 0.55 0.03 lM for HDAC-
2). Antiproliferative assay results also showed that compound 8c
(IC50 = 0.62 0.02 for HCT116) had the potential to be developed
for antiproliferative agents against HCT116. Docking simulation
was performed to position compound 8c into the human HDAC-2
active site to determine the probable binding model. Analysis of
the compound 8c binding conformation in active site displayed
the compound 8c was stabilized by a hydrogen bond with
His146 and two
p-cation interactions, respectively, with Phe155
and Phe210. Western-blot results also showed the compound 8c
was a potential antitumor agent. The information of this work
might be helpful for the design and synthesis of a leading com-
pound 8c toward the development of new therapeutic agent to
fight against cancer.
4. Experiments
Figure 3. (a) Compound 8c (colored by atom: carbons: gray; nitrogen: blue;
oxygen: red) is bond into human HDAC-2(entry 3MAX in the Protein Data Bank).
The dotted lines show the hydrogen bond and the yellow line show the
interaction. (b) The surface model structure of compound 8c binding model with
p-cation
4.1. Materials and measurements
HDAC-2 complex.
All chemicals and reagents used in the current study were of
analytical grade. All the 1H NMR spectra were recorded on a Bruker
DPX300 model Spectrometer in CDCl3 and chemical shifts were re-
ported in ppm (d). ESI-MS spectra were recorded on a Mariner Sys-
tem 5304 mass spectrometer. Elemental analyzes were performed
on a CHN-O-Rapid instrument. TLC was performed on the glass
backed silica gel sheets (Silica Gel 60 GF254) and visualized in
UV light (254 nm). Column chromatography was performed using
silica gel (200–300 mesh) eluting with ethyl acetate and petroleum
ether.
4.2. General procedure for 1,3-diphenyl-1H-pyrazole-4-acrylic
acid (5–8)
The starting material substituted 1,3-diphenyl-1H-pyrazole-4-
acrylic acid (5–8) was synthesized as following: para-substituted
acetophenone(1–4) (20 mmol) interact with phenylhydrazine
hydrochloride (25 mmol) couple with sodium acetate (40 mmol)
in anhydrous ethanol to form 1-phenyl-2-(1-phenylethyli-
dene)hydrazine, which was then dissolved in a cold mixed solution
of DMF (10 mL) and POCl3 (8 mL), stirred at 50–60 °C for 6 h. The
resulting mixture was poured into ice-cold water, a saturated solu-
tion of sodium hydroxide was added to neutralize the mixture, the
obtained solid precipitate was filtered, washed with water, dried
and recrystallized from ethanol. The outcome drawn from the
above processes was dissolved in pyridine solution with the help
of piperidine and malonic acid to oxidize to give the desired start-
ing material para-substituted acetophenone.28
Figure 4. 2D Ligand interaction diagram of compound 8c with HDAC-2 using
Discovery Studio program with the essential amino acid residues at the binding site
are tagged in circles. The purple circles show the amino acids which participate in
hydrogen bonding, electrostatic or polar interactions and the green circles show the
amino acids which participate in the Van der Waals interactions.
with the sidechain of His C:146 (bond length: 2.44 Å; bond angle:
107.5°), moreover, the nitrogen of the amide group chelates the

4434
X. Li et al. / Bioorg. Med. Chem. 20 (2012) 4430–4436
4.3. General procedure for 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-
phenylacrylamid derivates (5a–8d)
MS: 429.12 (C25H21ClN3O2, [M+H]+). Anal. Calcd for C25H20ClN3O2:
C, 69.85; H, 4.69; Cl, 8.25; N, 9.77; O, 7.44. Found: C, 69.63; H, 4.32;
Cl, 8.12; N, 9.61; O, 7.28.
Compounds 5a–8d were synthesized from a stirring mixture of
the starting material substituted 1,3-diphenyl-1H-pyrazole-4-ac-
rylic acid (5–8) (1 ml) and substituted anilines (0.8 ml) with the
help of DMAP and K2CO3 in anhydrous methylene dichloride at
the temperate of 40–50 °C for 12 h.29 The products were extracted
with ethyl acetate. The extract was washed successively with 10%
HCl, saturated K2CO3 and water, respectively, then dried over
anhydrous NaCl, filtered and evaporated. The residue was recrys-
tallized from ethanol to gain the desired compounds.
4.3.7. N-(4-Bromophenyl)-3-(3-(4-methoxyphenyl)-1-phenyl-
1H-pyrazol-4-yl)acrylamide
Yellow solid, yield 69%, mp: 246–247 °C; 1H NMR (300 MHz,
CDCl3, d ppm): 7.95(s, 1H), 7.82(s, 2H), 7.68(t, J = 3.93 Hz, 4H),
7.39–7.48 (m, 6H), 7.34(d, J = 4.53 Hz, 2H), 7.13(d, J = 5.49 Hz,
2H), 4.13 (s, 3H). ESI-MS: 473.1 (C25H21BrN3O2, [M+H]+). Anal.
Calcd for C25H20BrN3O2: C, 63.30; H, 4.25; Br, 16.84; N, 8.86; O,
6.75. Found: C, 62.19; H, 4.39; Br, 16.71; N, 8.48; O, 6.61.
4.3.1. N-Phenyl-3-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-
acrylamide
4.3.8. N-(4-Methoxyphenyl)-3-(3-(4-methoxyphenyl)-1-phenyl-
1H-pyrazol-4-yl)acrylamide
Yellow solid, yield 81%, mp: 226–227 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 8.77(s, 1H), 8.35 (s, 1H), 8.17(s, 1H), 7.79 (d,
J = 4.64 Hz, 2H), 7.46–7.52 (m, 6H), 7.36 (d, J = 2.72 Hz 1H), 7.25–
7.33(m, 4H), 7.29(d, J = 4.57 Hz, 2H), 2.39 (s, 3H). ESI-MS: 379.2
(C25H22N3O, [M+H]+). Anal. Calcd for C25H21N3O: C, 79.13; H,
5.58; N, 11.07; O, 4.22. Found: C, 78.26; H, 5.43; N, 12.12; O, 4.08.
Yellow powders, yield 70%, mp: 255–256 °C; 1H NMR (300 MHz,
CDCl3, d ppm): 8.54 (s, 1H), 7.60(d, J = 5.41 Hz, 2H), 7.48–7.56 (m,
5H), 7.41–7.45(m, 4H), 7.27–7.31 (m, 3H), 6.84 (d, J = 8.79 Hz,
2H), 4.01(s, 3H), 3.88(s, 3H). ESI-MS: 425.2 (C26H24N3O3 [M+H]+).
Anal. Calcd for C26H23N3O3: C, 73.39; H, 5.45; N, 9.88; O, 11.28.
Found: C, 73.21; H, 5.57; N, 9.71; O, 11.45.
4.3.2. N-(4-Chlorophenyl)-3-(1-phenyl-3-(p-tolyl)-1H-pyrazol-
4-yl)acrylamide
4.3.9. 3-(3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-N-
phenylacrylamide
Yellow solid, yield 79%, mp: 229–231 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 8.78(s, 1H), 8.40 (s, 1H), 8.17(s, 1H), 7.81 (d,
J = 4.82 Hz, 2H), 7.48–7.59 (m, 4H), 7.37 (d, J = 3.13 Hz, 2H), 7.25–
7.33(m, 6H), 2.39 (s, 3H). ESI-MS: 413.1 (C25H21ClN3O, [M+H]+).
Anal. Calcd for C25H20ClN3O: C, 72.55; H, 4.87; Cl, 8.57; N, 10.15;
O, 3.87. Found: C, 71.45; H, 5.08; Cl, 8.13; N, 9.85; O, 3.74.
White powders, yield 75%, mp: 231–232 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 8.56(s, 1H), 8.39(s, 1H), 8.10(s, 1H), 7.69–7.74 (m,
4H), 7.51–7.56(m, 4H), 7.29–7.43(m, 7H). ESI-MS: 443.1
(C24H19BrN3O, [M+H]+). Anal. Calcd for C24H18BrN3O: C, 64.88; H,
4.08; Br, 17.98; N, 9.46; O, 3.60. Found: C, 65.27; H, 3.91; Br,
17.48; N, 9.02; O, 3.78.
4.3.3. N-(4-Bromophenyl)-3-(1-phenyl-3-(p-tolyl)-1H-pyrazol-
4-yl)acrylamide
4.3.10. 3-(3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-N-(4-
chlorophenyl)acrylamide
Yellow solid, yield 81%, mp: 230–231 °C; 1H NMR (300 MHz,
CDCl3, d ppm): 8.77(s, 1H), 8.31 (s, 1H), 8.16(s, 1H), 7.82 (d,
J = 4.85 Hz, 2H), 7.56 (d, J = 5.16 Hz, 2H), 7.48–7.52 (m, 5H), 7.35
(d, J = 2.75 Hz, 1H), 7.29(d, J = 4.53 Hz, 2H), 6.89 (d, J = 5.49 Hz,
2H), 2.40 (s, 3H). ESI-MS: 457.1 (C25H21BrN3O, [M+H]+). Anal. Calcd
for C25H20BrN3O: C, 65.51; H, 4.40; Br, 17.43; N, 9.17; O, 3.49.
Found: C, 64.31; H, 5.01; Br, 16.89; N, 9.08; O, 3.30.
White powders, yield 67%, mp: 237–238 °C. 1H NMR (300 MHz,
CDCl3, d ppm): 8.77(s, 1H), 8.40(s, 1H), 8.17(s, 1H), 7.81(d,
J = 5.02 Hz, 2H), 7.57(dd, J = 4.98 Hz, 4H), 7.50(t, J = 4.86 Hz, 2H),
7.29–7.38(m, 6H). ESI-MS: 477.0 (C24H18BrClN3O, [M+H]+). Anal.
Calcd for C24H17BrClN3O: C, 60.21; H, 3.58; Br, 16.69; Cl, 7.41; N,
8.78; O, 3.34. Found: C, 59.09; H, 3.43; Br, 16.77; Cl, 7.21; N,
8.92; O, 3.56.
4.3.4. (4-Methoxyphenyl)-3-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-
yl)acrylamide
4.3.11. N-(4-Bromophenyl)-3-(3-(4-bromophenyl)-1-phenyl-
1H-pyrazol-4-yl)acrylamide
Yellow solid, yield 72%, mp: 242–243 °C; 1H NMR (300 MHz,
CDCl3, d ppm): 8.78(s, 1H), 8.31 (s, 1H), 8.16(s, 1H), 7.82 (d,
J = 3.04 Hz, 2H), 7.46–7.54 (m, 7H), 7.36 (s, 1H), 7.30(d,
J = 3.67 Hz, 2H), 6.90 (d, J = 5.18 Hz, 2H), 3.81 (s, 3H), 2.41 (s, 3H).
ESI-MS: 409.2 (C26H24N3O2, [M+H]+). Anal. Calcd for C26H23N3O2:
C, 76.26; H, 5.66; N, 10.26; O, 7.81. Found: C, 76.34; H, 5.78; N,
10.13; O, 7.79.
White powders, yield 68%, mp: 238–240 °C; 1H NMR (300 MHz,
CDCl3, d ppm): 8.77(s, 1H), 8.39(s, 1H), 8.11(s, 1H), 7.68(d,
J = 4.87 Hz, 2H), 7.63(d, J = 5.16 Hz, 2H), 7.47–7.53(m, 8H),
7.38(dd, J = 4.51 Hz, 2H). ESI-MS: 521.0 (C24H18Br2N3O, [M+H]+).
Anal. Calcd for C24H17Br2N3O: C, 55.09; H, 3.27; Br, 30.54; N,
8.03; O, 3.06. Found: C, 54.78; H, 3.06; Br, 30.27; N, 7.87; O, 3.01.
4.3.12. 3-(3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-N-(4-
methoxyphenyl)acrylamide
4.3.5. 3-(3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-N-
phenylacrylamide
White powders, yield 69%, mp: 242–243 °C; 1H NMR (300 MHz,
CDCl3, d ppm): 8.54(s, 1H), 8.21(s, 1H), 7.97(s, 1H), 7.73 (d,
J = 5.13 Hz, 4H), 7.40–7.48(m, 5H), 7.39(d, J = 5.49 Hz, 3H), 7.21(t,
J = 2.42 Hz, 2H), 3.78(s, 3H). ESI-MS: 473.1 (C25H21BrN3O2,
[M+H]+). Anal. Calcd for C25H20BrN3O2: C, 63.30; H, 4.25; Br,
16.84; N, 8.86; O, 6.75. Found: C, 63.11; H, 4.36; Br, 16.56; N,
8.48; O, 6.78.
Yellow solid, yield 77%, mp: 243–244 °C; 1H NMR (300 MHz,
CDCl3, d ppm): 9.19(s, 1H), 7.77–7.86 (m, 3H), 7.43–7.60 (m, 7H),
7.26–7.39 (m, 5H), 7.07 (d, J = 2.94 Hz, 2H), 3.77 (s, 3H). ESI-MS:
395.2 (C25H22N3O2, [M+H]+). Anal. Calcd for C25H21N3O2: C, 75.93;
H, 5.35; N, 10.63; O, 8.09. Found: C, 76.02; H, 5.29; N, 10.56; O,
8.17.
4.3.6. (4-Chlorophenyl)-3-(3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazol-4-yl)acrylamide
4.3.13. 3-(3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-N-
phenylacrylamide
Yellow solid, yield 73%, mp: 248–249 °C; 1H NMR (300 MHz,
CDCl3, d ppm): 8.36(s, 1H), 7.94(s, 1H), 7.64(d, J = 8.76 Hz, 2H),
7.61–7.69 (m, 4H), 7.53(d, J = 5.71 Hz, 2H), 7.44(d, J = 7.84 Hz,
2H), 7.27–7.33 (m, 3H), 7.23 (d, J = 7.25 Hz, 2H), 4.13 (s, 3H). ESI-
Yellow powders, yield 81%, mp: 240–241 °C; 1H NMR (300 MHz,
CDCl3, d ppm): 8.37(s, 1H), 7.91(s, 1H), 7.87(d, J = 8.44 Hz, 2H),
7.55–7.59(m, 5H), 7.31–7.47(m, 8H), 7.13(t, J = 4.46 Hz, 1H). ESI-
MS: 410.1 (C24H19N4O3, [M+H]+). Anal. Calcd for C24H18N4O3: C,

X. Li et al. / Bioorg. Med. Chem. 20 (2012) 4430–4436
4435
70.23; H, 4.42; N, 13.65; O, 11.69. Found: C, 71.01; H, 4.35; N,
13.47; O, 11.36.
and immune complexes allowed to form for 1 h at 4 °C. Protein A-
sepharose (10 mL of settled beads) were added and the compo-
nents mixed on a rotor overnight at 4 °C. Immune complexes were
collected by centrifugation and washed with cold TBST. The activ-
ities of HDAC-1 and HDAC-2 were assayed with a pan-HDAC sub-
strate (KI-104; Biomol Research Laboratories Inc., PA, USA) and
the reaction was carried out in half-volume white 96-well plates.
The assay components were incubated at 37 °C for 40 min. The
4.3.14. N-(4-Chlorophenyl)-3-(3-(4-nitrophenyl)-1-phenyl-1H-
pyrazol-4-yl)acrylamide
Yellow powders, yield 81%, mp: 242–243 °C; 1H NMR (300 MHz,
CDCl3, d ppm): 8.26(s, 1H), 7.92(d, J = 4.81 Hz, 2H), 7.77–7.91(m,
4H), 7.40–7.53(m, 5H), 7.32–7.40(m, 5H). ESI-MS: 444.1
(C24H18ClN4O3, [M+H]+). Anal. Calcd for C24H17ClN4O3: C, 64.80;
H, 3.85; Cl, 7.97; N, 12.59; O, 10.79. Found: C, 64.09; H, 3.48; Cl,
7.90; N, 12.63; O, 10.90.
reaction was quenched with the addition of 50
Developer (KI-105, Biomol) 20Â stock diluted in KI-143 buffer with
M TSA. The plates were incubated 30 min at room temperature
lL HDAC-FDL
2
l
to allow the fluorescent signal to develop. The fluorescence gener-
ated was monitored at 355/460 nm (excitation/emission)
wavelength.
4.3.15. N-(4-Bromophenyl)-3-(3-(4-nitrophenyl)-1-phenyl-1H-
pyrazol-4-yl)acrylamide
Yellow powders, yield 82%, mp: 242–243 °C; 1H NMR (300 MHz,
CDCl3, d ppm): 8.77(s, 1H), 8.39(s, 1H), 8.16(s, 1H), 7.82(d,
J = 4.58 Hz, 2H), 7.46–7.54(m, 9H), 7.37(s, 1H), 7.30(d, J = 4.26 Hz,
2H). ESI-MS: 488.1 (C24H18BrN4O3 [M+H]+). Anal. Calcd for
C24H17BrN4O3: C, 58.91; H, 3.50; Br, 16.33; N, 11.45; O, 9.81.
Found: C, 58.78; H, 3.41; Br, 16.19; N, 11.23; O, 10.11.
4.6. Western-blot analysis
Compounds were dissolved in DMSO prior to addition to cell
culture, compound 8c was added in HCT116 cells in order to in-
duce histone H3 acetylation. Cells were treated for 4 h with 2.5-
fold serial dilutions of compound. Control DMSO-treated cells are
indicated with hyphen. Histone acetylation in whole cell lysates
was analyzed by Western-blotting. PCNA antibodies were used as
a control for protein loading. Dose-response signals were quanti-
fied in order to calculate the inhibitor concentration giving the
half-maximal response.
4.3.16. N-(4-Methoxyphenyl)-3-(3-(4-nitrophenyl)-1-phenyl-
1H-pyrazol-4-yl)acrylamide
Yellow powders, yield 82%, mp: 256–258 °C; 1H NMR (300 MHz,
CDCl3,
d ppm): 8.50(s, 1H), 8.31(d, J = 5.47 Hz, 2H), 8.08(d,
J = 5.18 Hz, 2H), 7.78(d, J = 4.56 Hz, 2H) 7.52(t, J = 5.01 Hz, 2H),
7.40–7.46(m, 4H), 7.33–7.36(m, 3H), 7.15(t, J = 4.21 Hz, 1H), 3.73
(dd, J = 4.20 Hz, 3H). ESI-MS: 440.1 (C25H21N4O4 [M+H]+). Anal.
Calcd for C25H20N4O4 C, 68.17; H, 4.58; N, 12.72; O, 14.53. Found:
C, 67.03; H, 4.41; N, 12.36; O, 14.31.
4.7. Docking simulations
The three-dimensional X-ray structure of human HDAC-2 crys-
tal structure (PDB code: 3MAX) was chosen as the template for the
modeling study of compound 8c bound to HDAC. The crystal struc-
ture was obtained from the RCSB Protein Data Bank (http://
www.rcsb.org/pdb/home/home.do). The molecular docking proce-
dure was performed by using LigandFit protocol within Discovery
Studio 3.1. For ligand preparation, the 3D structures of 8c was gen-
erated and minimized using Discovery Studio 3.1. For protein prep-
aration, the hydrogen atoms were added, and the water and
impurities were removed. The molecular docking was performed
by inserting compound 8c into the binding pocket of HDAC-2
based on the binding mode. Types of interactions of the docked
protein with ligand-based pharmacophore model were analyzed
after the end of molecular docking.
4.4. Antiproliferation assay
The antiproliferative activities of the prepared compounds
against HCT116 and A549 cells were evaluated as described with
some modifications.17 Target tumor cell lines were grown to log
phase in RPMI 1640 medium supplemented with 10% fetal bovine
serum. After diluting to 2 Â 104 cells mLÀ1 with the complete med-
ium, 100 lL of the obtained cell suspension was added to each well
of 96-well culture plates. The subsequent incubation was con-
ducted at 37 °C, 5% CO2 condition for 24 h before the cytotoxicity
assessments. The samples at pre-set concentrations were added
to six wells and MS275 was co-assayed as positive references. After
48 h exposure period, 40
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide))
was added to each well. Four hours later, 100 L per well extrac-
l
L of PBS containing 2.5 mg mLÀ1 of MTT
Acknowledgments
l
tion solution (10% SDS–5% isobutyl alcohol–0.01 M HCl) was
added. After incubation overnight at 37 °C, the optical density
was measured at 570 nm on an ELISA microplate reader. In all
experiments three replicate wells were measured for each drug
concentration. Each assay was carried out for at least three times.
The results were summarized in Table 2.
This work was financed by Agriculture Research Project
(NY1002), by PCSIRT (IRT1020), by Natural Science Foundation of
Jiangsu Province (BK2009234), and by the Public Benefits Project
of Ministry of Environmental Protection of PRC (201009023).
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