Design, Synthesis, and Biological Evaluation of Pyrazole Derivatives

Article abstract of DOI:10.1111/cbdd.12699

In this in vitro study, a series of novel pyrazole derivatives were designed, synthesized, and evaluated against five human cancer cell lines (PC3, A549, HL60, HCT116, and SW620) for their antiproliferative and p53-MDM2 binding inhibitory activities. Although biological evaluations showed that this series of compounds possessed weak p53-MDM2 inhibitory activities, most of them displayed moderate to potent antiproliferative activities against the tested cells lines. Compound 11c exhibited the best potency for MDM2 (FP-IC₅₀ = 29.22 μm) and demonstrated antiproliferative activities in response to the five tested cell lines (IC₅₀ = 4.09-16.82 μm). Compared with the positive control Nutlin-1, there was enhanced antiproliferative activity to p53-mutated or p53-deficient cell lines (SW620, HL60, and PC3).

Full text of DOI:10.1111/cbdd.12699

Chem Biol Drug Des 2016; 87: 673–679
Research Article
Design, Synthesis, and Biological Evaluation
of Pyrazole Derivatives
1
1
2,
Chunqi Hu , Yali Gao and Wenting Du *
protein interaction inhibitors. Although these amino-thio-
phene derivatives also possessed favorable antiproliferative
activities, in vivo treatment revealed that the amino group
on thiophene provided no clear benefits. In this study, the
scaffold of amino-thiophene was replaced with pyrazole
1
College of Chemistry and Chemical Engineering,
Shaoxing University, Shaoxing 312000, China
Department of Pharmacy, Zhejiang Medical College, 481
Binwen Road, Hangzhou 310053, China
2
(Figure 1) and the substituents on amino-thiophene were
*
Corresponding author: Wenting Du, ddwwtt@163.com
retained. Meanwhile, a methylene group was introduced to
one of the phenyl groups to test for any improvements in
potency. The resulting molecules were tested for their p53-
MDM2 binding inhibitory and antiproliferative activities.
In this in vitro study, a series of novel pyrazole deriva-
tives were designed, synthesized, and evaluated
against five human cancer cell lines (PC3, A549, HL60,
HCT116, and SW620) for their antiproliferative and
p53-MDM2 binding inhibitory activities. Although bio-
logical evaluations showed that this series of com-
pounds possessed weak p53-MDM2 inhibitory
activities, most of them displayed moderate to potent
antiproliferative activities against the tested cells lines.
Compound 11c exhibited the best potency for MDM2
Experimental
Molecular docking
Molecular docking was performed using CDOCKER, which
is
a
CHARMm-based molecular dynamics docking
(
FP-IC50 = 29.22 lM) and demonstrated antiproliferative
activities in response to the five tested cell lines
IC50 = 4.09–16.82 lM). Compared with the positive
algorithm on DISCOVERY STUDIO 2.1 (Accelrys, College of
Pharmaceutical Sciences, Zhejiang University, Hangzhou,
China). The MDM2 structure cocrystallized with an imida-
zoline compound was obtained from the PDB data bank
(
control Nutlin-1, there was enhanced antiproliferative
activity to p53-mutated or p53-deficient cell lines
(
(PDB code: 1RV1). A protein clean process and a
SW620, HL60, and PC3).
CHARMm-force field were applied sequentially. The area
around imidazoline compounds was chosen as the active
Key words: anticancer, molecular docking, p53-MDM2, pyra-
zole derivatives, synthesis
ꢀ
site, with the radius set as 9 A. After removing the ligand
from the structure of the complex, a binding sphere was
constructed around the active site in the three axis direc-
tions. All default parameters were used in the docking pro-
cess. CHARMm-based molecular dynamics (1000 steps)
were used to generate random conformations. Final
energy minimization was set to full potential mode. The
final binding conformation of compound 11b was deter-
mined on the basis of CDOCKING ENERAGE. For each
compound, the most stable binding mode among the top
ten docking poses is presented in Figure 2.
Received 21 August 2015, revised 14 October 2015 and
accepted for publication 24 November 2015
Heterocyclic compounds have been connected with a
broad spectrum of biological activities. On account of their
interesting biological and pharmaceutical activities, the
important pharmacophores known as pyrazole derivatives
have been researched extensively for more than a century
to elucidate anticancer, antibacterial, antispasmodic, plant
growth regulation, and platelet inhibition functions (1,2). The
tumor suppressor p53 plays a prominent role in the regula-
tion of the cell cycle, apoptosis, and DNA repair via a com-
plex network of proteins known as the p53 pathway (3),
which is regulated by its principal negative regulator MDM2
Chemistry
Melting points were determined on a B u€ chi B-540 appara-
tus (College of Pharmaceutical Sciences, Zhejiang Univer-
1
sity, Hangzhou, China) and are uncorrected. HNMR
(4,5). Nowadays, a promising strategy for tumor treatment
spectra were recorded on a Bruker 400 M spectrometer
is the use of small molecule inhibitors to disrupt p53-MDM2
interaction and, thus, restore p53 function (6,7).
with CDCl as solvent. Chemical shifts were reported in d
values (ppm), relative to internal TMS, and J values were
3
reported in Hertz.). Mass spectra (MS), ESI (positive), were
recorded on an Esquire-LC-00075 spectrometer (College of
Pharmaceutical Sciences, Zhejiang University, Hangzhou,
In our previous research (8), a series of amino-thiophene
derivatives were identified as potent p53-MDM2 protein–
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12699
673

Hu et al.
Figure 1: Structure of 3-carboxylate
pyrazole derivatives, designed as p53-
MDM2 binding inhibitors.
Figure 2: Compound 11b occupying three hydrophobic binding pockets of MDM2 (1RV1).
China). Reagents and solvents were purchased from com-
mercial suppliers and were used without further purification.
diazoacetate as a light yellow liquid which was used
directly to the next step.
Ethyl 2-diazoacetate (2)
1, 2-Bis (4-chlorophenyl) ethanone (5)
To a 50-mL reactor was added water (15 mL), glycine
ethyl ester hydrochloride (14 g, 0.1 mol), sodium acetate
Oxalyl chloride (4.82 mL) was added to 4-chlorophenyla-
cetic acid (10.0 g, 0.05 mol) in anhydrous methylene
chloride and then stirred at 0 °C for 30 min. The
excess oxalyl chloride was removed under vacuum to give
an oily stuff 4, which was used without further purification.
(
0.33 g, 3 mmol), and sodium nitrite (8.0 g, 0.12 mol).
The reaction mixture was stirred and under 2 °C. Ether
8 mL) and cold 10% H SO (0.3 mL) were added drop-
(
2
4
wise until the reaction temperature started to rise. The
addition was stopped, and ice was added to chill down
the reaction temperature below 15 °C. The organic
phase (ether) was separated and washed with saturated
The obtained 4 from above was slowly added to cold mix-
ture of AlCl3 (10.0 g, 0.075 mol) in chlorobenzene (5.0 g,
0.05 mol), stirred at room temperature for 45 min, cold
diluted hydrochloric acid was added to stop the reaction,
extracted with methylene chloride (3 9 20 mL), the organic
2 3 2 4
aqueous K CO and dried over Na SO . The solvent
was removed under reduced pressure to afford ethyl
6
74
Chem Biol Drug Des 2016; 87: 673–679

Pyrazole Derivatives as Anticancer Agents
layer was kept, washed with brine (3 9 15 mL), and dried
with anhydrous sodium sulfate. The solvent was removed,
the obtained solid was recrystallized by methanol to give a
white solid 7. Yield: 90%; M.P.: 112 ~ 114 °C [Ref (9): 113–
4-(4-Chlorobenzyl)-5-(4-chlorophenyl)-1H-pyrazole-
3-carboxylic acid (10)
Ethyl 4-(4-chlorobenzyl)-5-(4-chlorophenyl)-1H-pyrazole-3-
carboxylate 8 (200 mg, 0.53 mmol) was dissolved in a
mixture of ethanol-THF (1: 1, 10 mL), added with 10%
NaOH (5 mL). The reaction mixture was stirred at 60 °C
overnight and then the THF was removed under vacuum.
Water was added, and the residue was acidified with 1N
hydrochloric acid. The resulted solid was filtered, washed
by water, and dried to give a white solid 10. Yield: 92%;
115 °C].
1, 3-Bis (4-chlorophenyl) propan-2-one (6)
A
solution of 4-chlorophenylacetic acid (2.0 g,
14.7 mmol) in dry methylene chloride (80 mL) was
+
added slowly to a solution of DCC (2.8 g, 14.7 mmol)
and DMAP (0.45 g, 3.6 mmol) in dry methylene chloride.
After stirring at room temperature for 4 h, the solvent
was removed under vacuum; the residue was extracted
by ethyl estate and water; organic layer was taken,
washed with brine (3 9 15 mL), and dried with anhy-
drous sodium sulfate; the organic solvent was removed
to give a yellow solid 6. Yield: 75%; M.P.: 93 ~ 94 °C
M.P. 240 ~ 242 °C; MS (ESI): 346 [M] .
General procedure for synthesis of 11a-11e, 13a-
13e
9 or 10 (0.15 mM), DCC (62 mg, 0.30 mM), and HOBt
(21 mg, 0.15 mM) were dissolved in anhydrous methylene
chloride (10 mL). The reaction was protected under N2
and stirred at 0 °C for 30 min while added a solution of
corresponding secondary amine (22 mg, 0.30 mM) in
methylene chloride (10 mL). After 3 h, the solid was fil-
tered and the filtrate was washed with brine (3 9 10 mL)
and dried with anhydrous sodium sulfate. Purification of
the residue by flash chromatography (silica, petroleum
ether/ethyl estate/triethylamine = 3: 1: 0.01 ~ 1: 1: 0.01)
resulted in 11a-11e, 12a-12e.
[Re (10): 93 °C].
Ethyl 4, 5-bis (4-chlorophenyl)-1H-pyrazole-3-
carboxylate (7)
Compound 5 (260 mg, 2.28 mmol) and DBU (489 mg,
3.21 mmol) were slowly added to the solution of 2
(500 mg, 1.87 mmol) in acetonitrile (5 mL), protected by
N . The reaction was stirred at room temperature for 3 h.
2
The reaction solvent was removed under vacuum and
extracted by saturated sodium carbonate/methylene chlo-
ride (1: 1, 100 mL). The organic layer was kept, washed
with brine, and dried with anhydrous sodium sulfate. Purifi-
cation of the residue by flash chromatography (silica, ethyl
(4, 5-Bis (4-chlorophenyl)-1H-pyrazol-3-yl)
(pyrrolidin-1-yl) methanone (11a)
Yield: 83%; M.P.: 215 ~ 219 °C; H NMR (500 MHz,
1
CDCl ) d 7.34 (m, Ar-H, 6H), 7.23 (m, Ar-H, 2H), 3.57 (t,
3
estate/petroleum ether = 1:9 ~ 1: 1) resulted in a white oily
J = 7.0 Hz, CH
2H), 1.75 (m, CH
2
, 2H), 3.08 (m, CH
2 2
, 2H), 1.87 (m, CH ,
1
+
stuff 7. Yield: 50%; H NMR (500 MHz, CDCl
Ar-H, 4H), 7.25 (m, 4H), 4.22 (d, J = 7.1 Hz, CH
3
) d 7.35 (m,
, 2H),
2
, 2H); MS (ESI): 386 [M+H] .
2
+
3
1.17 (t, J = 7.1 Hz, CH , 3H); GC/MS (ESI): 360 [M] .
(
(
4, 5-Bis (4-chlorophenyl)-1H-pyrazol-3-yl)
piperidin-1-yl) methanone (11b)
1
Ethyl 4-(4-chlorobenzyl)-5-(4-chlorophenyl)-1H-
pyrazole-3-carboxylate (8)
Yield: 73%; M.P.: 191 ~ 195 °C; H NMR (500 MHz, CDCl )
3
d 7.28 (dt, J = 7.8, 6.0 Hz, Ar-H, 3H), 7.16 (d, J = 8.4 Hz,
Referring to above reaction, using 6 in replace of 5
Ar-H, 2H), 3.60 (m, CH
2
, 2H), 3.06 (m, CH
2
, 2H), 1.48 (m,
+
resulted to the white solid 8. Yield 80%; M.P.: 146 ~
CH 9 2, 4H), 1.02 (m, CH
2
2
, 2H); MS (ESI): 400 [M+H] .
1
1
7
1
47 °C; H NMR (500 MHz, CDCl
3
) d 7.81 (s, Ar-H, 2H),
.31 (m, Ar-H, 6H), 4.65 (s, CH , 2H), 4.15 (m, CH , 2H),
.28 (m, CH
2
2
+
3
, 3H); MS (ESI): 375 [M+H] .
(4, 5-Bis (4-chlorophenyl)-1H-pyrazol-3-yl)(4-
methylpiperazin-1-yl) methanone (11c)
Yield: 93%; M.P.: 225 ~ 230 °C; H NMR (500 MHz,
1
4
, 5-Bis (4-chlorophenyl)-1H-pyrazole-3-carboxylic
CDCl
CH 9 2, 4H), 1.87 (m, CH
MS (ESI): 415 [M+H] .
3
) d 7.34 (m, Ar-H, 6H), 7.23 (m, Ar-H, 2H), 3.57 (m,
acid (9)
2
2
+ CH , 5H), 1.75 (m, CH , 2H);
3
2
+
Ethyl 4, 5-bis (4-chlorophenyl)-1H-pyrazole-3-carboxylate
7
(200 mg, 0.56 mmol) was dissolved in a mixture of etha-
nol-THF (1: 1, 10 mL), added with 10% NaOH (5 mL). The
reaction mixture was stirred at 60 °C overnight, and then
the THF was removed under vacuum. Water was added,
and the residue was acidified with 1N hydrochloric acid.
The resulted solid was filtered, washed by water, and
4, 5-Bis (4-chlorophenyl)-N,N-diethyl-1H-pyrazole-
3-carboxamide (11d)
Yield: 82%; M.P.: 195 ~ 197 °C; H NMR (500 MHz,
1
CDCl ) d 7.43 (m Ar-H, 2H), 7.27 (m, Ar-H, 4H), 7.25 (m,
3
dried to give a white solid 9. Yield: 90%; M.P. 236 ~
Ar-H, 2H), 2.43 (q, J = 7.1 Hz, CH
2
9 2, 4H), 0.94 (t,
+
+
238 °C; MS (ESI): 332 [M] .
J = 7.1 Hz, CH 9 2, 6H); MS (ESI): 388 [M+H] .
3
Chem Biol Drug Des 2016; 87: 673–679
675

Hu et al.
N-tert-butyl-4, 5-bis (4-chlorophenyl)-1H-pyrazole-
(4-(4-Chlorobenzyl)-5-(4-chlorophenyl)-1H-pyrazol-
3-yl)(Morpholino)methanone (13c)
Yield: 82%; M.P.: 244 ~ 246 °C; H NMR (500 MHz,
3
-carboxamide (11e)
1
1
Yield: 72%; M.P.: 201 ~ 204 °C; H NMR (500 MHz,
CDCl ) d 7.43 (d, J = 8.4 Hz, Ar-H, 2H), 7.28 (dd, J = 8.2,
CDCl ) d 7.90 (m, Ar-H, 2H), 7.38 (dd, J = 25.6, 12.3 Hz,
3
3
6
1
.2 Hz, Ar-H, 4H), 7.25 (m, Ar-H, 2H), 5.79 (s, NH, 1H),
Ar-H, 6H), 4.72 (s, CH
2
, 2H), 3.45 (dt, J = 13.8, 6.9 Hz,
+
.29 (s, CH 9 3, 9H); MS (ESI): 388 [M+H] .
CH 9 2, 4H), 2.01 (m, CH , 2H), 1.89 (m, CH , 2H); MS
3
2
2
2
+
(
ESI): 416 [M+H] .
(4-(4-Chlorobenzyl)-5-(4-chlorophenyl)-1H-pyrazol-
3
-yl)(pyrrolidin-1-yl)methanone (13a)
(4-(4-Chlorobenzyl)-5-(4-chlorophenyl)-1H-pyrazol-
3-yl)(Morpholino)methanone (13d)
Yield: 78%; M.P.: 207 ~ 210 °C; H NMR (500 MHz,
1
Yield: 88%; M.P.: 235 ~ 239 °C; H NMR (500 MHz,
CDCl ) d 7.89 (m, Ar-H, 2H), 7.42 (dt, J = 90.2, 20.2 Hz,
Ar-H, 6H), 4.79 (s, CH , 2H), 3.68 (d, J = 4.4 Hz, CH 9
1
3
CDCl ) d 7.83 (d, J = 8.4 Hz, Ar-H, 2H), 7.28 (m, 6H),
2
2
3
2
, 4H), 3.60 (s, CH
2
, 2H), 3.45 (dd, J = 20.7, 15.6 Hz,
4.70 (s, CH
(t, J = 7.1 Hz, CH
2
, 2H), 2.43 (q, J = 7.1 Hz, CH
2
9 2, 4H), 0.94
+
+
CH , 2H); MS (ESI): 400 [M+H] .
2
3
9 2, 6H); MS (ESI): 402 [M+H] .
(
3
4-(4-Chlorobenzyl)-5-(4-chlorophenyl)-1H-pyrazol-
N-tert-butyl-4-(4-chlorobenzyl)-5-(4-chlorophenyl)-
1H-pyrazole-3-carboxamide (13e)
Yield: 78%; M.P.: 207 ~ 209 °C; H NMR (500 MHz,
-yl)(piperidin-1-yl)methanone (13b)
1
1
Yield: 86%; M.P.: 237 ~ 241 °C; H NMR (500 MHz,
CDCl ) d 7.86 (m, Ar-H, 2H), 7.31 (d, J = 9.3 Hz, Ar-H,
H), 5.51 (s, CH, 1H), 4.74 (s, CH, 1H), 3.54 (m, CH
H), 3.39 (m, CH , 2H), 1.68 (m, CH , 2H), 1.54 (d,
3
3
CDCl ) d 7.83 (d, J = 8.4 Hz, Ar-H, 2H), 7.28 (m, 6H),
6
2
2
,
5.79 (s, NH, 1H), 4.72 (s, CH
MS (ESI): 402 [M+H] .
2
, 2H), 1.29 (s, CH
3
9 3, 9H);
+
2
2
+
2
J = 3.5 Hz, CH 9 2, 4H); MS (ESI): 414 [M+H] .
2 2 3 3
Scheme 1: a. NaNO , AcONa, <2 °C; b. (COCl) , 0 °C, 30 min; c.AlCl , chlorobenzene, R.T.; d. DBU, CH CN; e. NaOH, EtOH; f. DCC,
HOBt, CH Cl , r. t.; g. Chloroethanol, K
2
2
2
CO3, DMF, 60 °C 1 h.
6
76
Chem Biol Drug Des 2016; 87: 673–679

Pyrazole Derivatives as Anticancer Agents
General procedure for synthesis of 12 and 14
1b or 13b (0.20 mmol) was dissolved in DMF (5 mL), and
potassium carbonate (0.21 mmol) was added. After stirring
for a while, chloroethanol was added to the system
J = 8.4 Hz, Ar-H, 2H), 7.46 (d, J = 8.4 Hz, Ar-H, 2H),
1
7.37 (d, J = 8.5 Hz, Ar-H, 2H), 4.82 (s, CH
CH , 2H), 3.65 (m, CH 9 2, 4H), 3.44 (m, CH
(d, J = 4.7 Hz, CH , 2H), 1.57 (d, J = 4.2 Hz, CH 9 2,
2
, 2H), 3.77 (m,
2
2
2
, 2H), 1.66
2
2
+
(0.24 mmol). The reaction was stirred under 60 °C for about
4H); MS (ESI): 458 [M+H] .
1
h till there is no starting material left. Then, cool the reac-
tion mixture to room temperature, extract with water/ethyl
estate, keep the organic layer, wash with brine, and dry with
anhydrous sodium sulfate. Purification of the residue by
flash chromatography (silica, petroleum ether/ethyl estate/
triethylamine = 3:1 ~ 1:1) resulted in white solid 12 and 14.
Biological evaluation
Fluorescence polarization competitive binding
assay
Measurements were performed using an EnVision Multil-
abel Plate Reader with a 480 nM excitation filter and a
535 nM emission filter. Assays were conducted in Corning
384-well black plates. Nutlin-1 and DMSO were employed
as the positive and negative controls, respectively. Assays
were performed in duplicate and repeated at least twice
on separate days. Competition experiments were carried
out in a total volume of 20 lL 40 mM Tris–HCl (pH 7.5),
150 mM NaCl, and 1 mM DTT, 4% DMSO. Probe peptide
and MDM2 were present at final concentrations of 1 nM
and 10 lM, respectively. Prior to measurement, plates
were incubated at room temperature for one hour. Inhibi-
tion was calculated via the following formula: Inhibition
(%) = 1–(FP–FPmin)/(FPmax–FPmin). The concentration of
drug required to inhibit 50% of the protein (FP-IC50) was
calculated using a microcomputer with DOSE-EFFECT ANALYSIS
software.
(
4,5-bis(4-chlorophenyl)-1-(2-hydroxyethyl)-1H-
pyrazol-3-yl)(piperidin-1-yl)methanone (12)
Yield: 42%; M.P.: 177 ~ 181 °C; H NMR (500 MHz, CDCl )
d 7.36 (d, J = 8.5 Hz, Ar-H, 2H), 7.29 (dd, J = 15.0, 8.5 Hz,
Ar-H, 4H), 7.13 (d, J = 8.5 Hz, Ar-H, 2H), 4.37 (d,
J = 12.2 Hz, CH
1
3
2 2 2
, 2H), 4.12 (m, CH , 2H), 3.66 (m, CH , 2H),
3
4
.09 (s, CH, 1H), 2.83 (s, CH, 1H), 1.50 (d, J = 50.9 Hz, CH ,
H), 1.18 (s, CH, 1H), 0.62 (s, CH, 1H); MS (ESI): 444 [M+H] .
2
+
(4-(4-chlorobenzyl)-5-(4-chlorophenyl)-1-(2-
hydroxyethyl)-1H-pyrazol-3-yl)(piperidin-1-yl)
methanone (14)
Yield: 40%; M.P.: 186 ~ unstable; H NMR (500 MHz,
1
3
CDCl ) d 7.90 (d, J = 8.5 Hz, Ar-H, 2H), 7.52 (d,
Scheme 2: a. NaNO
2
, AcONa, <2 °C; b. DCC, DMAP, CH
2
Cl
2
; c. DBU, CH
3
CN; d. NaOH, EtOH; e. DCC, HOBt, CH
2
Cl
2
, r. t.;
f. Chloroethanol, K CO3, DMF, 60 °C 1 h.
2
Chem Biol Drug Des 2016; 87: 673–679
677

Hu et al.
Cell viability assay
duced soluble residues to improve the solubility of the
A549, HCT116, SW620, HL60, and PC3 cells were plated
in 96-well plates (4 9 10 /well) for 24 h and,
compounds. This resulted in compounds 12 and 14.
3
subsequently, treated with different concentrations of
compounds for 72 h. After MTT solution (5.0 mg/mL in
RPIM-1640, Sigma, St. Louis, MO, USA) was added
Chemistry
The synthetic routes for the pyrazole derivatives are sum-
marized in Schemes 1 and 2. The starting material, glycine
ethyl ester hydrochloride (compound 1), was reacted with
sodium acetate and sodium nitrite to give the correspond-
ing diazonium salt (compound 2). Subsequently, 4-chloro-
phenylaceticacid (compound 3) was treated with oxalyl
chloride to yield compound 4. Following a Fuke acyl reac-
tion with AlCl3 in chlorobenzene, compound 4 formed
compound 5. The intermediate compounds 2 and 5 were
catalyzed by DBU (11) to form the pyrazole intermediate 7.
Compound 9 was obtained by hydrolyzing compound 7
under alkaline conditions. Then, compound 9 underwent
amidation with the corresponding amine to give diphenyl-
3-carboxcylate pyrazole derivatives 11a-11e. Finally, 11
reacted with chloroethanol to get compound 12.
(10.0 lL/well), plates were incubated for a further four
hours at 37 °C. The purple formazan crystals were dis-
solved in 100 lL DMSO. After the crystals dissolved, the
plates were read on an automated microplate spectropho-
tometer (Bio-Tek Instruments, Winooski, VT, USA) at
5
70 nm. DOSE-EFFECT ANALYSIS software was used to com-
pute the drug concentration required to inhibit 50% of cells
IC50).
(
Results
Molecular docking and compounds design
In order to accurately study the binding affinity of the
series of compounds, a molecular docking study was
conducted using CDOCKER in DISCOVERY STUDIO 2.1 (Accel-
rys). According to the results, compound 11b could
occupy three hydrophobic pockets of MDM2 with a
CDOCKER interaction energy of 33.07 Kcal. As the amino
group on pyrazole was exposed to the solvent, we intro-
On the other hand, compound 6 was obtained by treating
compound 3 with DCC and DMAP (12). The intermediate
compounds 2 and 6 were catalyzed by DBU to yield the
pyrazole intermediate 8. Compound 10 was synthesized
by hydrolyzing compound 7 under alkaline conditions.
Table 1: P53-MDM2 binding inhibitory and antiproliferative activities of pyrazole derivatives
O
Cl
Cl
O
N
HO
H
N
N
HN
N
R
O
R
N
N
R
O
R
N
HN
HO
NH2
O
Cl
Cl
Cl
Cl Cl
O
Cl
7, 9, 11a-11e
Cl
Cl
8, 10, 13a-13e
12
14
SF
a,b
MTT-IC50 (lM)
No.
R
Inhibitory (10 lM)
FP-IC50 (lM)
HCT116
A549
SW620
PC3
HL60
7
8
9
Ethyoxyl
Ethyoxyl
OH
28.1%
3.55%
NA
80.02
NA
NA
>50
19.38
>50
NT
29.29
15.56
NT
>50
15.58
NT
4.43
13.33
NT
6.78
c
NT
1
1
1
1
1
1
1
1
1
1
1
1
1
0
OH
Pyrrolidyl
Piperidyl
N-methyl piperazinyl
Diethylamine
t-butylamine
Piperidyl
Pyrrolidyl
Piperidyl
Morpholinyl
Diethylamine
t-butylamine
Piperidyl
NA
NA
NT
>50
NT
>50
NT
>50
NT
>50
8.89
4.59
3.47
16.31
5.69
>50
14.37
>50
11.51
16.36
22.24
16.14
21.58
NT
>50
2.22
5.61
22.75
8.46
13.9
>50
>50
>50
>50
5.72
>50
1a
1b
1c
1d
1e
2
27.27%
4.00%
50.55%
NA
9.54%
6.38%
0.71%
4.96%
2.13%
NA
33.43
70.34
29.22
NA
NA
NA
NA
NA
NA
NA
13.64
16.82
8.98
6.09
20.02
>50
8.55
25.29
>50
6.95
11.12
15.12
21.95
13.51
>50
13.98
>50
33.19
14.84
>50
3.58
4.09
13.84
3.12
7.58
>50
11.94
37.63
>50
2.72
>50
17.82
19.18
3a
3b
3c
3d
3e
4
NA
NA
100%
88.36%
NA
NA
1.24
3.36
9.9
29.26
3.49
7.02
SF
N-1
–
–
2.53
11.26
NT
5.87
a
Values are means of three experiments.
NA, no activity.
NT, not tested.
b
c
6
78
Chem Biol Drug Des 2016; 87: 673–679

Pyrazole Derivatives as Anticancer Agents
Then, compound 10 underwent amidation with the corre-
sponding amine to yield 4-benzy-5-phenyl-3-carboxcylate
pyrazole derivatives 13a-13e. Finally, 13 reacted with
chloroethanol to get compound 14.
(No. 81502926). We are also grateful to the support pro-
vided by Zhejiang Provincial Program for the Cultivation of
High-level Innovative Health Talents (2014) and The 151
Talents Project in new century in Zhejiang Province (the
third level, 2011).
Pharmacology
All synthesized compounds were evaluated for their p53-
MDM2 binding inhibitory activities using a fluorescence
polarization (FP)-based binding assay. Nutlin-1 was used
as the positive control. The results of this evaluation are
summarized in Table 1.
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7, 9, 11a-11e, and 12) exhibited higher potencies than 4-
benzy-5-phenyl-3-carboxcylate pyrazole derivatives (corre-
sponding compounds 8, 10, 13a-13e, and 14) against the
p53-MDM2 binding. When R was hydroxyl group (com-
pounds 9 and 10), compounds exhibited no binding affini-
ties. The introduction of soluble residues did not lead to
promising potencies (compounds 12 and 14). In both series,
compounds containing the pyrrolidine group showed no
activity in response to five cell lines. Compounds containing
diethylamine and t-butylamine in the form of open-ring frag-
ments exhibited less potency for MDM2 protein than those
comprising closed-ring fragments. Compared with Nutlin-1,
most of these compounds exhibited moderate antiprolifera-
tive activities. In diphenyl-3-carboxcylate pyrazole deriva-
tives, the compound containing N-methyl piperazine
(compound 11c) showed the best potency for MDM2 (FP-
IC50 = 29.22 lM) and promising antiproliferative activities
against five tested cell lines (IC50 = 4.09–16.82 lM). In par-
ticular, compared with Nutlin-1, there was enhanced
antiproliferative activity to p53-mutated or p53-deficient cell
lines (SW620, HL60, and PC3), which denotes different
mechanisms for the treatment of tumor cell lines. However,
the best potency exhibited, 11c showed less potency than
the lead compound, judging from the docking result, the R
group may be too bulky to feed in the PHE pocket; thus, to
further improve the potencies, we may introduce less bulky
groups, which may provide a new strategy to enhance the
potency of this series of compounds.
Acknowledgments
This study was financially supported by Zhejiang Provincial
Natural Science Foundation (No. LY16H300004 and No.
LQ13H300001), the Natural Science Foundation of China
Chem Biol Drug Des 2016; 87: 673–679
679