Synthesis and evaluation of pyrazolone compounds as SARS-coronavirus 3C-like protease inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.09.050

A series of pyrazolone compounds as possible SARS-CoV 3CL protease inhibitors were designed, synthesized, and evaluated by in vitro protease assay using fluorogenic substrate peptide in which several showed potent inhibition against the 3CL protease. Interestingly, one of the inhibitors was also active against 3C protease from coxsackievirus B3. These inhibitors could be potentially developed into anti-coronaviral and anti-picornaviral agents.

Full text of DOI:10.1016/j.bmc.2010.09.050

Bioorganic & Medicinal Chemistry 18 (2010) 7849–7854
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of pyrazolone compounds as SARS-coronavirus
3C-like protease inhibitors
R. Ramajayam a, Kian-Pin Tan b, Hun-Ge Liu b, Po-Huang Liang a,b,
⇑
a Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Taipei 11529, Taiwan
b Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrazolone compounds as possible SARS-CoV 3CL protease inhibitors were designed, synthe-
sized, and evaluated by in vitro protease assay using fluorogenic substrate peptide in which several
showed potent inhibition against the 3CL protease. Interestingly, one of the inhibitors was also active
against 3C protease from coxsackievirus B3. These inhibitors could be potentially developed into anti-
coronaviral and anti-picornaviral agents.
Received 10 August 2010
Revised 20 September 2010
Accepted 21 September 2010
Available online 25 September 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Pyrazolone
3CL protease
SARS-CoV
Coxsackievirus
Computer modeling
1. Introduction
which cause life-threatening infectious diseases. The 3Cpro essen-
tial for viral replication had served as a drug target.7 Both 3Cpro
Severe acute respiratory syndrome (SARS) is a newly emerging
infectious disease caused by a novel coronavirus, SARS-coronavirus
(SARS-CoV). SARS has been recognized as a global threat since the
initial outbreak of SARS first identified in Guangdong Province,
China in November 2002. This outbreak spread to several countries
and has had significant health and economic impact. SARS is a life-
threatening form of atypical pneumonia characterized by high
fever, malaise rigor, headache, chills, cough, and progressive radio-
graphic changes of the chest and lymphophenia.1–3 The mortality
rate is nearly 10%.4 The SARS-CoV is a positive-strand RNA virus
that uses a complex set of enzymes to replicate the largest RNA
genomes currently known for RNA viruses and synthesize an
extensive set of 50 leader-containing subgenomic mRNAs that
encode the viral structural proteins and several species-specific
proteins with unknown functions. These processes are mediated
primarily by the 3C-like protease (3CLpro) with chymotrypsin fold.
The active site of SARS-CoV 3CLpro contains Cys145 and His41 to
constitute a catalytic dyad, in which cysteine functions as the
common nucleophile in the proteolytic process.5,6 Because of the
essential role in viral processing, the 3CLpro is considered as an
attractive target for anti-SARS and other coronavirus infections.
3CLpro is named after the 3C proteases (3Cpro) from picornaviruses
such as enterovirus (EV), coxsackievirus (CV), and rhinovirus (RV)
and 3CLpro have similar 3-D structures, but unlike the dimeric
3CLpro, 3Cpro is monomeric and utilizes Glu-His-Cys triad for
catalysis.8
To date various SARS-CoV protease inhibitors have been reported
from both screened compound libraries and designed compounds
based on the substrate structure or active site properties. Their scaf-
folds are diverse, including C2-symmetric diols,9 3-quinolinecarb-
oxylic acid derivatives,10 thiophene-2-carboxylate derivatives,11
cinanserin,12 calmodulin,13 keto-glutamine analogs,14 anilide,15
bifunctional boronic acid compounds,16 isatin derivatives,17 pyr-
midinone,18 benzotriazole19 as well as glutamic acid and glutamine
peptides possessing a trifluoromethyl ketone group,20
a,b-unsatu-
rated esters,21 and etacrynic acid derivatives.22 With metal-coordi-
nated structures, some molecules make a covalent bond with Cys-
145 at the active site of SARS-CoV 3CLpro 23
However, no effective
.
therapy has been developed so far and recent isolation of strains of
SARS-CoV emphasizes the possibility of a reemergence. Therefore,
it is still a great challenge to explore new chemical classes of SARS-
CoV 3CLpro inhibitors that can be used in anti-SARS therapy in case
the disease re-emerges.
Compounds containing a pyrazole and its related analogs have
received significant attention in chemical, medicinal, and pharma-
ceutical research as this structural scaffold is found in a variety of
drugs. As shown in Figure 1, a new pyrazolone compound,
edaravone (A), also known as MCI-186, has been developed as a
medical drug for brain ischemia24 and has also been reported to
be effective for myocardial ischemia.25 Compound (B) is claimed
⇑
Corresponding author. Tel.: +886 2 2362 0261x3091; fax: +886 2 2363 5038.
E-mail address: phliang@gate.sinica.edu.tw (P.-H. Liang).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.050

7850
R. Ramajayam et al. / Bioorg. Med. Chem. 18 (2010) 7849–7854
Table 1
Structure and IC50
CH3
(l
M) of compounds 2a–u
O
N
N
NH
N
N
H3C
N
O
HO
O
R2
O
N
O
O
N
A
B
R1
OH
COOH
O
2a-u
Br
Br
Compd
R1
R2
SARS (lM)
CVB3 (lM)
O
2a
2b
2c
2d
2e
2f
H
H
H
H
H
H
H
N.I.
N.I.
N.I.
18.0
N.I.
N.I.
N.I.
N.I.
13.9
N.I.
N.I.
N.I.
12.0
N.I.
N.I.
5.5
N.I.
N.I.
N.I.
51.1
N.I.
N.I.
N.I.
N.I.
25.0
N.I.
N.I.
N.I.
16.6
N.I.
N.I.
20.8
98.8
17.3
125.5
22.4
9.6
N
N
3-OCH3
4-NHCOCH3
4-COOH
4-N(CH3)2
3-NO2
NH
O
N
N
N
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
2t
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-OCH3
4-CH(CH3)2
4-C(CH3)3
4-CN
4-OCF3
3-Cl
3,4-Cl2
4-F
3-NO2
H
4-Cl
Cl
Cl
Cl
Cl
C
4-COOH
4-NHCOCH3
4-OCH3
4-OH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
4-COOH
D
Cl
Figure 1. Pyrazole compounds as drugs or enzyme inhibitors.
to have potent anti-orthopoxvirus activity.26 Compound (C) 3,5-
dioxopyrazolidine has been reported as SARS-CoV 3CLpro inhibi-
tor.27 Recently, we identified from high throughput screening cer-
tain pyrazolines, particularly those displaying
substitution pattern (D), were active against SARS-CoV 3CLpro
CoV-229E 3CLpro, CVB3 3Cpro, EV71 3Cpro, and RV14 3Cpro 28
In
42.0
10.8
24.3
6.8
a 1,3,5-triaryl
,
2u
8.4
.
the present study, we synthesized the pyrazolone compounds as
SARS 3CL protease inhibitors and explored their structure–activity
N.I.: no inhibition at 50 lM.
relationship (SAR) in inhibiting 3CLpro and 3Cpro
.
In view of the facts mentioned above, 21 compounds containing
the pyrazolone template were synthesized and screened for their
3C and 3CL protease inhibitory activities. Some of the synthesized
compounds displayed potent inhibition against CVB3 3Cpro and
SARS-CoV 3CLpro. The synthesis of the target compounds 2a–u was
envisioned following the synthetic route illustrated in Scheme 1.
(see Table 1). Compound 2p is the most potent inhibitor showing
an IC50 of 5.5 M and 2t is the second with IC50 of 6.8 M against
SARS-3CoV 3CLpro. Interestingly, 2u showed inhibitory activity sig-
nificantly against both SARS-3CoV 3CLpro (IC50 = 8.4
M) and CVB3
3Cpro (IC50 = 9.6
M). The cytotoxicity of the test compounds was
tested by performing the MTT assay and found that all compounds
are devoid of cytotoxicity at 200 M.
l
l
l
l
l
In search of a computer model of the associated complex be-
tween the compound 2u and the proteases to rationalize its inhib-
itory activities, the orientation of the ligand has the N1-phenyl
group situated in the S10 pocket of the 3CLpro. One of the oxygen
of the nitro group is in close proximity 2.7 Å and forms H-bond
to the Gly-143 (Fig. 2A). The C@O in the central pyrazolone ring
is close to Glu-166 with the distance of 3.0 Å to form a H-bond.
C-3 phenyl ring fits into the S2 pocket, having hydrophobic inter-
actions with Met-49, Arg-188, and Gln-189 (hiding behind these
resides in Fig. 2A). The carboxyl benzylidene group is situated in
the S3 pocket of the 3CLpro. The oxygen of the carboxyl group forms
a hydrogen bond with the side chain of Gln-192 at a distance of
2. Results and discussion
Compounds 1a–k were synthesized by refluxing the corre-
sponding b-ketoester and the substituted phenylhydrazine
hydrochloride in acetic acid.29 The pyrazolones were treated with
the appropriate aromatic aldehyde in presence of piperidine in
ethanol to obtain target compounds 2a–u in 70–87% yields.30
From the preliminary investigation, as summarized in Table 1,
we noted that compounds with substituent R2, carboxyl group at
4th position in benzylidene aryl ring shows significant inhibition
against SARS-CoV 3CLpro. Compounds having R1 substitution like
halogens, cyano, and nitro group increase the inhibitory action
CHO
R2
N
NHNH2 HCl
R1
O
O
NEt3
R2
N
N
O
O
CH3COOH
N
Piperidine
EtOH
O
R1
R1
1a-k
2a-u
Scheme 1. General synthesis of compounds 2a–u.

R. Ramajayam et al. / Bioorg. Med. Chem. 18 (2010) 7849–7854
7851
Figure 2. Docking studies of 2u binding in the active site of SARS 3CLpro (A) and CVB3 3Cpro (B).
3.2 Å. It is important for inhibition activity since the compounds
lacking carboxy functionality in the benzylidene lost the activity.
Electron withdrawing R1 substituents like cyano (2p), fluoro (2t),
and nitro (2u), accompanied with R2 carboxyl group favors the
inhibitory activity.
The mixture was stirred at reflux temperature for 20 h. The solvent
was removed by evaporation, and the residue was extracted with
AcOEt. The organic phase was dried over anhydrous Na2SO4 and
evaporated under reduced pressure to get crude solid. The crude
product was recrystallised from methanol to yield pure pyrazolone
1a–k. The spectroscopic data of the 1a–k are described below in
details.
In further evaluating the inhibitors against CVB3 3Cpro, we
found 2p and 2t were moderate inhibitors against CVB3 3Cpro
(IC50 = 20.8 and 22.4
l
M, respectively), but 2u was more active
against CVB3 3Cpro (IC50 = 9.6
lM). According to the modeling
4.1.2.1. 1,3-Diphenyl-5-pyrazol-5(4H)-one (1a). 89% yield, mp:
139–141 °C. 1H NMR (CDCl3, 500 MHz) d 3.86 (s, 2H), 7.23–7.26
(m, 1H), 7.44–7.49 (m, 5H), 7.80 (d, J = 8.0 Hz, 2H), 8.01 (d,
J = 7.9 Hz, 2H); ESI-TOF-MS: 237.10 (C15H13N2O, [M+H]+).
shown in Figure 2B, the R1 nitro group of 2u forms H-bond with
Gly-145 (2p and 2t without nitro group fail to form such a H-bond)
and benzylidene carboxylate of 2u is H-bonded to Glu-71 in the ac-
tive site of 3Cpro. It was predicted that the C-3 phenyl ring of 2u is
pointed to S1 site and the carboxyl benzylidene group is relocated
to S2 in order to form the H-bond in 3Cpro due to the subtle differ-
4.1.2.2. 3-Phenyl-1-(4-chlorophenyl)pyrazol-5(4H)-one (1b).
82% yield, mp: 162–164 °C. 1H NMR (CDCl3, 500 MHz) d 3.88 (s,
2H), 7.41 (d, J = 8.8 Hz, 2H), 7.49 (m, 3H), 7.78–7.80 (m, 2H), 7.99
(d, J = 8.8 Hz, 2H); ESI-TOF-MS: 271.06 (C15H12ClN2O, [M+H]+).
ences between the structures of 3CLpro and 3Cpro 7
. However, it
should be noted that computer modeling is speculation based on
energy minimization to fit the SAR data. In conclusion, 2p and 2t
are selective against 3CLpro, but 2u is a common inhibitor of 3CLpro
and 3Cpro, which may be potentially developed into anti-coronav-
iral and anti-picornaviral drugs.
4.1.2.3. 3-Phenyl-1-(4-methoxyphenyl)pyrazol-5(4H)-one (1c).
77% yield, mp: 127–129 °C. 1H NMR (DMSO-d6, 500 MHz) d 3.75 (s,
3H), 3.86 (s, 2H), 6.88 (d, J = 8.15 Hz, 2H), 7.19–7.22 (m, 1H), 7.29–
7.31 (m, 2H), 7.64 (d, J = 8.3 Hz, 2H), 7.70–7.74 (m, 2H); ESI-TOF-
MS: 267.11 (C16H15N2O2, [M+H]+).
3. Conclusion
4.1.2.4. 3-Phenyl-1-(4-isopropylphenyl)pyrazol-5(4H)-one (1d).
81% yield, mp: 132–133 °C. 1H NMR (CDCl3, 500 MHz) d 1.29 (s,
6H), 2.93–2.99 (m, 1H), 3.85 (s, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.47–
7.48 (m, 3H), 7.79 (d, 2H), 7.88 (d, J = 8.4 Hz, 2H); ESI-TOF-MS:
279.15 (C18H19N2O, [M+H]+).
As reported here, pyrazolone compounds (2p, 2t, and 2u) with a
4-carboxylbenzylidene aryl ring attaching to C4 of pyrazolone
showed potent 3CLpro inhibition, while 3-nitro-phenyl group at-
tached to N1 atom (2u) gave the simultaneously inhibitory activity
against 3Cpro from CVB3.
4.1.2.5. 3-Phenyl-1-(4-tert-butylphenyl)pyrazol-5(4H)-one (1e).
78% yield, mp: 120–122 °C. 1H NMR (CDCl3, 500 MHz) d 1.35 (s,
9H), 3.86 (s, 2H), 7.46–7.48 (m, 5H), 7.80 (d, J = 8.6 Hz, 2H), 7.88
(d, J = 8.6 Hz, 2H); ESI-TOF-MS: 293.16 (C19H21N2O, [M+H]+).
4. Experimental
4.1. Chemistry
4.1.1. General
4.1.2.6.
3-Phenyl-1-(4-cyanophenyl)pyrazol-5(4H)-one (1f).
All chemicals (reagent grade) used were purchased from Sig-
ma–Aldrich (USA) and Acros organics Co., Ltd (USA). ESITOF-MS
spectra were recorded on a Bruker BioTOF II mass spectrometer
and 1H NMR spectra were recorded on a AV-400 or AV-500 spec-
trometer at 25 °C with TMS as an internal standard. Chemical shifts
(d) are reported in ppm and were adjusted relative to the residual
solvent peak.
73% yield, mp: 211–212 °C. 1H NMR (CDCl3, 500 MHz) d 3.93 (s,
2H), 7.51–7.52 (m, 3H), 7.74 (d, J = 8.75 Hz, 2H), 7.80–7.82 (m,
2H), 8.22 (d, J = 8.75 Hz, 2H); ESI-TOF-MS: 262.10 (C16H12N3O,
[M+H]+).
4.1.2.7. 3-Phenyl-1-(4-trifluoromethoxyphenyl)pyrazol-5(4H)-
one (1g). 65% yield, mp: 114–116 °C. 1H NMR (CDCl3, 500 MHz)
d 3.90 (s, 2H), 7.30 (d, J = 8.5 Hz, 2H), 7.49–7.50 (m, 3H), 7.79–
7.80 (m, 2H), 8.07 (d, J = 8.95 Hz, 2H); ESI-TOF-MS: 321.08
(C16H12F3N2O2, [M+H]+).
4.1.2. General procedure for the preparation of compounds 1a–k
An equimolar solution of ethyl benzoylacetate and substituted
phenylhydrazine hydrochloride was treated with triethylamine.

7852
R. Ramajayam et al. / Bioorg. Med. Chem. 18 (2010) 7849–7854
4.1.2.8. 3-Phenyl-1-(3-chlorophenyl)pyrazol-5(4H)-one (1h).
61% yield, mp: 101–103 °C. 1H NMR (CDCl3, 500 MHz) d 3.89 (s,
2H), 7.41–7.43 (m, 3H), 7.47–7.50 (m, 1H), 7.56–7.58 (m, 2H),
7.61 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.91 (s, 1H); ESI-
TOF-MS: 271.06 (C15H12ClN2O, [M+H]+).
4.1.3.7. 3-Phenyl-1-(4-chlorophenyl)-4-benzylidenepyrazol-5(4H)-
one (2g). 73% yield, mp: 245–247 °C. 1H NMR (DMSO-d6,
500 MHz) d 7.19–7.21 (m, 4H), 7.23–7.32 (m, 5H), 7.40–7.41 (m,
2H), 7.55 (d, J = 8.85 Hz, 2H), 7.88 (d, J = 8.9 Hz, 2H); ESI-TOF-MS:
359.09 (C22H16ClN2O, [M+H]+).
4.1.2.9. 3-Phenyl-1-(3,4-dichlorophenyl)pyrazol-5(4H)-one (1i).
71% yield, mp: 139–141 °C. 1H NMR (CDCl3, 500 MHz) d 3.89 (s,
2H), 7.49–7.50 (m, 4H), 7.79–7.81 (m, 2H), 7.95–7.98 (m, 1H),
8.20 (d, 1H); ESI-TOF-MS: 305.02 (C15H11Cl2N2O, [M+H]+).
4.1.3.8. 3-Phenyl-1-(4-chlorophenyl)-4-(4-chlorobenzylidene)pyr-
azol-5(4H)-one (2h). 70% yield, mp: 174–176 °C. 1H NMR (DMSO-
d6, 500 MHz) d 7.20 (d, J = 8.35 Hz, 2H), 7.25–7.28 (m, 4H), 7.36 (d,
J = 8.5 Hz, 2H), 7.40 (m, 2H), 7.55 (d, J = 8.8 Hz, 2H), 7.86 (d,
J = 8.85 Hz, 2H); ESI-TOF-MS: 393.05 (C22H15Cl2N2O, [M+H]+).
4.1.2.10. 3-Phenyl-1-(4-fluorophenyl)pyrazol-5(4H)-one (1j).
65% yield, mp: 158–160 °C. 1H NMR (CDCl3, 500 MHz) d 3.88 (s,
2H), 7.13–7.16 (m, 2H), 7.49–7.51 (m, 3H), 7.78–7.80 (m, 2H),
7.96–7.99 (m, 2H); ESI-TOF-MS: 255.09 (C15H12FN2O, [M+H]+).
4.1.3.9. 3-Phenyl-1-(4-chlorophenyl)-4-(4-carboxybenzylidene)-
pyrazol-5(4H)-one (2i). 85% yield, mp: 249–250 °C. 1H NMR
(DMSO-d6, 500 MHz) d 7.23–7.33 (m, 5H), 7.40–7.44 (m, 4H), 7.54
(d, J = 8.85 Hz, 2H), 7.88 (d, J = 8.9 Hz, 2H), 7.89 (s, 1H), 12.52 (br,
1H); ESI-TOF-MS: 403.08 (C23H16ClN2O3, [M+H]+).
4.1.2.11.
3-Phenyl-1-(3-nitrophenyl)pyrazol-5(4H)-one (1k).
80% yield, mp: 172–174 °C. 1H NMR (CDCl3, 500 MHz) d 3.94 (s,
2H), 7.50–7.52 (m, 3H), 7.62 (t, J = 8.2 Hz, 1H), 7.82–7.84 (m, 2H),
8.08 (d, J = 7.85 Hz, 1H), 8.48 (d, J = 8.05 Hz, 1H), 8.89 (s, 1H);
ESI-TOF-MS: 282.08 (C15H12N3O3, [M+H]+).
4.1.3.10. 3-Phenyl-1-(4-chlorophenyl)-4-(4-acetamidobenzylid-
ene)pyrazol-5(4H)-one (2j). 69% yield, mp: 182–184 °C. 1H NMR
(DMSO-d6, 500 MHz) d 1.99 (s, 3H), 7.10 (d, J = 8.55 Hz, 2H),
7.18–7.21 (m, 3H), 7.26 (d, J = 6.95 Hz, 2H), 7.32–7.35 (m, 2H),
7.42–7.43 (m, 1H), 7.48 (d, J = 9.0 Hz, 2H), 7.98 (d, J = 7.1 Hz, 2H),
9.83 (s, 1H); ESI-TOF-MS: 416.11 C24H19ClN3O2, [M+H]+).
4.1.3. General procedure for the preparation of compounds 2a–u
An equimolar of pyrazolone 1a–k, substituted benzaldehyde
and piperidine in ethanol (50 ml) were refluxed for 3–5 h. The ex-
cess of ethanol was evaporated and the residue was poured into
water. The solid product was filtered, dried, and recrystallized from
methanol. The spectroscopic data of the synthesized compounds
are described below in details.
4.1.3.11. 3-Phenyl-1-(4-chlorophenyl)-4-(4-methoxybenzyli-
dene)pyrazol-5(4H)-one (2k). 80% yield, mp: 190–191 °C. 1H
NMR (DMSO-d6, 500 MHz) d 3.65 (s, 3H), 6.74 (d, J = 7.25 Hz,
2H), 7.14–7.18 (m, 4H), 7.24 (d, J = 7.05 Hz, 2H), 7.31–7.34 (m,
2H), 7.46 (d, J = 9.0 Hz, 2H), 8.0 (d, J = 9.0 Hz, 2H); ESI-TOF-MS:
389.10 (C23H18ClN2O2, [M+H]+).
4.1.3.1. 1,3-Diphenyl-4-benzylidenepyrazol-5(4H)-one (2a).
74% yield, mp: 232–233 °C. 1H NMR (DMSO-d6, 500 MHz) d 7.08–
7.14 (m, 4H), 7.16–7.27 (m, 6H), 7.34–7.37 (m, 4H), 8.02–8.03
(m, 2H); ESI-TOF-MS: 325.13 (C22H17N2O, [M+H]+).
4.1.3.12.
3-Phenyl-1-(4-chlorophenyl)-4-(4-hydroxybenzylid-
ene)pyrazol-5(4H)-one (2l). 83% yield, mp: 218–220 °C. 1H NMR
(DMSO-d6, 500 MHz) d 6.97 (d, J = 8.5 Hz, 2H), 7.14–7.17 (m, 2H),
7.24 (d, J = 6.95 Hz, 2H), 7.28–7.31 (m, 1H), 7.45 (d, J = 7.15 Hz,
2H), 7.52–7.54 (m, 1H), 7.70–7.72 (m, 1H), 8.03 (d, J = 6.9 Hz,
2H), 8.58 (d, J = 8.85 Hz, 1H), 9.03 (s, 1H); ESI-TOF-MS: 375.09
(C22H16ClN2O2, [M+H]+).
4.1.3.2. 1,3-Diphenyl-4-(4-methoxybenzylidene)pyrazol-5(4H)-
one (2b). 81% yield, mp: 222–224 °C. 1H NMR (DMSO-d6,
500 MHz) d 3.64 (s, 3H), 7.10–7.15 (m, 5H), 7.22–7.27 (m, 5H),
7.34–7.37 (m, 3H), 8.01–8.03 (m, 2H); ESI-TOF-MS: 355.14
(C23H19N2O2, [M+H]+).
4.1.3.13. 3-Phenyl-1-(4-methoxyphenyl)-4-(4-carboxybenzylid-
ene)pyrazol-5(4H)-one (2m). 72% yield, mp: 230–232 °C. 1H
NMR (DMSO-d6, 500 MHz) d 3.74 (s, 3H), 7.0 (d, J = 8.15 Hz, 2H),
7.23 (s, 1H), 7.27 (d, J = 8.25 Hz, 2H), 7.32–7.35 (m, 5H), 7.65 (d,
J = 8.2 Hz, 2H), 7.84 (d, J = 8.35 Hz, 2H), 14.28 (br, 1H); ESI-TOF-
MS: 399.13 (C24H19N2O4, [M+H]+).
4.1.3.3. 1,3-Diphenyl-4-(4-acetamidobenzylidene)pyrazol-5(4H)-
one (2c). 75% yield, mp: 184–186 °C. 1H NMR (DMSO-d6,
500 MHz) d 2.0 (s, 3H), 7.09–7.15 (m, 6H), 7.23–7.26 (m, 5H),
7.36 (d, J = 8.2 Hz, 2H), 8.02 (d, J = 8.15 Hz, 2H), 9.76 (s, 1H); ESI-
TOF-MS: 382.15 (C24H20N3O2, [M+H]+).
4.1.3.14. 3-Phenyl-1-(4-isopropylphenyl)-4-(4-carboxybenzylid-
ene)pyrazol-5(4H)-one (2n). 69% yield, mp: 206–208 °C. 1H NMR
(DMSO-d6, 500 MHz) d 1.21 (s, 6H), 2.86–2.89 (m, 1H), 7.16 (d,
J = 7.55 Hz, 2H), 7.22–7.24 (m, 2H), 7.25–7.28 (m, 4H), 7.31 (d,
J = 8.25 Hz, 2H), 7.80 (d, J = 8.35 Hz, 2H), 7.86 (d, J = 8.5 Hz, 2H),
12.59 (br, 1H); ESI-TOF-MS: 411.17 (C26H23N2O3, [M+H]+).
4.1.3.4. 1,3-Diphenyl-4-(4-carboxybenzylidene)pyrazol-5(4H)-
one (2d). 83% yield, mp: 194–196 °C. 1H NMR (DMSO-d6,
500 MHz) d 7.09–7.16 (m, 4H), 7.22–7.28 (m, 4H), 7.31–7.38 (m,
3H), 7.80 (d, J = 8.95 Hz, 2H), 8.01 (d, J = 8.85 Hz, 2H), 12.65 (br,
1H); ESI-TOF-MS: 369.12 (C23H17N2O3, [M+H]+).
4.1.3.5. 1,3-Diphenyl-4-(4-dimethylaminobenzylidene)pyrazol-
4.1.3.15. 3-Phenyl-1-(4-tert-butylphenyl)-4-(4-carboxybenzylid-
ene)pyrazol-5(4H)-one (2o). 71% yield, mp: 198–200 °C. 1H NMR
(DMSO-d6, 500 MHz) d 1.29 (s, 9H), 7.21–7.24 (m, 4H), 7.27–7.28
(m, 2H), 7.31 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.75 Hz, 2H), 7.74 (d,
J = 8.75 Hz, 2H), 7.87 (d, J = 8.35 Hz, 2H), 12.47 (br, 1H); ESI-TOF-
MS: 425.18 (C27H25N2O3, [M+H]+).
5(4H)-one (2e). 76% yield, mp: 194–196 °C. 1H NMR (DMSO-d6,
500 MHz)
d 2.80 (s, 6H), 6.60 (d, J = 8.85 Hz, 2H), 7.03 (d,
J = 8.65 Hz, 2H), 7.09–7.15 (m, 4H), 7.24–7.26 (m, 4H), 7.34–7.37
(m, 3H); ESI-TOF-MS: 368.17 (C24H22N3O, [M+H]+).
4.1.3.6. 1,3-Diphenyl-4-(3-nitrobenzylidene)pyrazol-5(4H)-one
(2f). 79% yield, mp: 230–232 °C. 1H NMR (DMSO-d6, 500 MHz) d
7.11–7.16 (m, 5H), 7.23–7.29 (m, 5H), 7.37 (t, 2H), 7.52–7.62 (m,
1H), 8.01 (d, 1H), 8.09 (s, 1H); ESI-TOF-MS: 370.12 (C22H16N3O3,
[M+H]+).
4.1.3.16. 3-Phenyl-1-(4-cyanophenyl)-4-(4-carboxybenzylidene)-
pyrazol-5(4H)-one (2p). 87% yield, mp: 179–181 °C. 1H NMR
(DMSO-d6, 500 MHz) d 7.21–7.25 (m, 2H), 7.28 (s, 1H), 7.29–7.32
(m, 1H), 7.38–7.41 (m, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.92 (d,

R. Ramajayam et al. / Bioorg. Med. Chem. 18 (2010) 7849–7854
7853
J = 8.75 Hz, 2H), 8.01–8.03 (m, 1H), 8.13 (d, J = 8.2 Hz, 2H), 8.15 (d,
J = 8.85 Hz, 2H), 12.58 (br, 1H); ESI-TOF-MS: 394.11 (C24H16N3O3,
[M+H]+).
in 5% CO2 at 37 °C. Cells with 70% confluence at density were trea-
ted with each compound at designated concentrations for 24 h.
After the incubation, 10 lL of MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) stock solution was added into
4.1.3.17. 3-Phenyl-1-(4-trifluoromethoxyphenyl)-4-(4-carboxy-
benzylidene)pyrazol-5(4H)-one (2q). 81% yield, mp: 164–166 °C.
1H NMR (DMSO-d6, 500 MHz) d 7.13–7.15 (m, 3H), 7.19–7.32 (m,
5H), 7.36 (d, J = 8.75 Hz, 2H), 7.75 (d, J = 8.2 Hz, 2H), 8.14 (d,
J = 9.05 Hz, 2H), 12.45 (br, 1H); ESI-TOF-MS: 453.11 (C24H16F3N2O4,
[M+H]+).
each well. The conversion of MTT to formazan by viable cells was
performed at 37 °C for another 4 h. After the reaction, 100 lL of
DMSO solution were added into each well following the removal
of culture media in order to solubilize the formazan precipitates.
The levels of formazan were determined by optical density at
540 nm using an ELISA reader and represented as cell viability.
4.1.3.18. 3-Phenyl-1-(3-chlorophenyl)-4-(4-carboxybenzylid-
ene)pyrazol-5(4H)-one (2r). 85% yield, mp: 161–163 °C. 1H
NMR (DMSO-d6, 500 MHz) d 7.14–7.18 (m, 3H), 7.23–7.30 (m,
5H), 7.38–7.42 (m, 1H), 7.81 (d, J = 8.35 Hz, 2H), 8.03 (d,
J = 9.45 Hz, 2H), 8.13 (t, J = 9.0 Hz, 1H), 12.74 (br, 1H); ESI-TOF-
MS: 403.08 (C23H16ClN2O3, [M+H]+).
4.4. Docking studies
To gain further molecular insight into the mode of inhibition of
active compound, we conducted docking studies in the 3CLpro ac-
tive site. For modeling analysis, the crystal structure of SARS 3CLpro
in complex with a peptide inhibitor (PDB code 1UK4) was used.33
Docking process was performed using an automated ligand-dock-
ing subprogram of the Discovery Studio Modeling 1.2 SBD (Accel-
rys Inc., San Diego, CA), with a set of parameters chosen to
control the precise operation of the genetic algorithm. Docking
runs were carried out using standard default settings ‘grid resolu-
tion’ of 5 Å, ‘site opening’ of 12 Å, and ‘binding site’ selected for
defining the active site cavity.
4.1.3.19. 3-Phenyl-1-(3,4-dichlorophenyl)-4-(4-carboxybenzy-
lidene)pyrazol-5(4H)-one (2s). 83% yield, mp: 185–187 °C. 1H
NMR (DMSO-d6, 500 MHz)
d
7.14–7.30 (m, 5H), 7.62 (d,
J = 9.0 Hz, 2H), 7.81 (d, J = 8.35 Hz, 2H), 8.08 (d, J = 8.9 Hz, 2H),
8.31 (m, 2H), 12.62 (br, 1H); ESI-TOF-MS: 437.04 (C23H15Cl2N2O3,
[M+H]+).
4.1.3.20. 3-Phenyl-1-(4-fluorophenyl)-4-(4-carboxybenzylid-
References and notes
ene)pyrazol-5(4H)-one (2t). 78% yield, mp: 190–192 °C. 1H
1. Ksiazek, T. G.; Erdman, D.; Goldsmith, C. S.; Zaki, S. R.; Peret, T.; Emery, S.; Tong, S.;
Urbani, C.; Comer, J. A.; Lim, W.; Rollin, P. E.; Dowell, S. F.; Ling, A.-E.; Humphrey,
C. D.; Shieh, W.-J.; Guarner, J.; Paddock, C. D.; Rota, P.; Fields, B.; DeRisi, J.; Yang, J.-
Y.; Cox, N.; Hughes, J. M.; LeDuc, J. W.; Bellini, W. J.; Anderson, L. J. N. Engl. J. Med.
2003, 348, 1953.
2. Peiris, J. S. M.; Lai, S.-T.; Poon, L. L.-M.; Guan, Y.; Yam, L. Y.-C.; Lim, W.; Nicholls,
J.; Yee, W. K.-S.; Yan, W. W.; Cheung, M.-T.; Cheng, V. C.-C.; Chan, K.-H.; Tsang,
D. N.-C.; Yung, R. W.-H.; Ng, T. K.; Yuen, K.-Y. Lancet 2003, 361, 1319.
3. Rota, P. A.; Oberste, M. S.; Monroe, S. S.; Nix, W. A.; Campagnoli, R.; Icenogle, J. P.;
Penaranda, S.; Bankamp, B.; Maher, K.; Chen, M.-H.; Tong, S.; Tamin, A.; Lowe, L.;
Frace, M.; DeRisi, J. L.; Chen, Q.; Wang, D.; Erdman, D. D.; Peret, T. C. T.; Burns, C.;
Ksiazek, T. G.; Rollin, P. E.; Sanchez, A.; Liffick, S.; Holloway, B.; Limor, J.;
McCaustland, K.; Olsen-Rasmussen, M.; Fouchier, R.; Gunther, S.; Osterhaus, A. D.
M. E.; Drosten, C.; Pallansch, M. A.; Anderson, L. J.; Bellini, W. J. Science 2003, 300,
1394.
NMR (DMSO-d6, 400 MHz)
d
7.12–7.28 (m, 5H), 7.31 (d,
J = 8.24 Hz, 2H), 7.80 (d, J = 8.28 Hz, 2H), 8.0 (d, J = 8.52 Hz, 2H),
8.03 (d, J = 8.4 Hz, 2H), 8.1 (s, 1H), 12.62 (br, 1H); ESI-TOF-MS:
387.11 (C23H16FN2O3, [M+H]+).
4.1.3.21. 3-Phenyl-1-(3-nitrophenyl)-4-(4-carboxybenzylidene)-
pyrazol-5(4H)-one (2u). 72% yield, mp: 175–177 °C. 1H NMR
(DMSO-d6, 500 MHz) d 7.13–7.36 (m, 7H), 7.69–7.30 (m, 1H),
7.85 (d, J = 8.1 Hz, 2H), 8.03 (d, J = 7.3 Hz, 1H), 8.48 (d, J = 8.1 Hz,
2H), 8.88 (s, 1H), 12.71 (br, 1H); ESI-TOF-MS: 414.10
(C23H16N3O5, [M+H]+).
4. He, J.-F.; Peng, G.-W.; Min, J.; Yu, D.-W.; Liang, W.-L.; Zhang, S.-Y.; Xu, R.-H.;
Zheng, H.-Y.; Wu, X.-W.; Xu, J.; Wang, Z.-H.; Fang, L.; Zhang, X.; Li, H.; Yan, X.-G.;
Lu, J.-H.; Hu, Z.-H.; Huang, J.-C.; Wan, Z.-Y.; Hou, J.-L.; Lin, J.-Y.; Song, H.-D.; Wang,
S.-Y.; Zhou, X.-J.; Zhang, G.-W.; Gu, B.-W.; Zheng, H.-J.; Zhang, X.-L.; He, M.;
Zheng, K.; Wang, B.-F.; Fu, G.; Wang, X.-N.; Chen, S.-J.; Chen, Z.; Hao, P.; Tang, H.;
Ren, S.-X.; Zhong, Y.; Guo, Z.-M.; Liu, Q.; Miao, Y.-G.; Kong, X.-Y.; He, W.-Z.; Li, Y.-
X.; Wu, C.-I.; Zhao, G.-P.; Chiu, R. W. K.; Chim, S. S. C.; Tong, Y.-K.; Chan, P. K. S.;
Tam, J. S.; Lo, Y. M. D. Science 2004, 303, 1666.
5. Anand, K.; Ziebuhr, J.; Wadhwani, P.; Mesters, J. R.; Hilgenfeld, R. Science 2003,
300, 1763.
6. Du, Q. S.; Wang, S. Q.; Zhu, Y.; Wei, D. Q.; Guo, H.; Sirois, S.; Chou, K. C. Peptides
2004, 25, 1857.
7. (a) Binford, S. L.; Maldonado, F.; Brothers, M. A.; Weady, P. T.; Zalman, L. S.;
Meador, J. W., 3rd; Matthews, D. A.; Patick, A. K. Antimicrob. Agents Chemother.
2005, 49, 619; (b) Lee, E. S.; Lee, W. G.; Yun, S. H.; Rho, S. H.; Im, I.; Yang, S. T.;
Sellamuthu, S.; Lee, Y. J.; Kwon, S. J.; Park, O. K.; Jeon, E. S.; Park, W. J.; Kim, Y. C.
Biochem. Biophys. Res. Commun. 2007, 358, 7; (c) Kuo, C. J.; Shie, J. J.; Fang, J. M.;
Yen, G. R.; Hsu, J. T.; Liu, H. G.; Tseng, S. N.; Chang, S. C.; Lee, C. Y.; Shih, S. R.; Liang,
P. H. Bioorg. Med. Chem. 2008, 16, 7388.
8. Lee, C. C.; Kuo, C. J.; Ko, T. P.; Hsu, M. F.; Tsui, Y. C.; Chang, S. C.; Yang, S.; Chen,
S. J.; Chen, H. C.; Hsu, M. C.; Shih, S. R.; Liang, P. H.; Wang, A. H. J. Biol. Chem.
2008, 284, 7646.
4.2. 3CLpro and 3Cpro activity assays
A fluorogenic peptide substrate (Dabcyl-KTSAVL QSGFRKME-
Edans) was used for assays of 3CLpro and 3Cpro activities. SARS-
CoV 3CLpro and CVB3 3Cpro were prepared as previously re-
ported.8,31 The proteases were stored in the buffer containing
12 mM Tris–HCl (pH 7.5), 120 mM NaCl, 0.1 mM EDTA, 7.5 mM
b-ME, and 1 mM DTT at ꢀ70 °C before use. The anti-SARS-3CLpro
activity of the test compounds were performed in the solution con-
taining 0.05
50 M of test compounds at 25 °C and the anti-CVB3 3Cpro activity
was assayed using 0.05
M CVB3 3Cpro. Enhanced fluorescence of
l lM fluorogenic substrate, and
M SARS 3CLpro, 6
l
l
the reactions in the buffer of 20 mM Bis-Tris at pH 7.0 was moni-
tored at 538 nm with excitation at 355 nm using a fluorescence
plate reader (Fluoroskan Ascent; ThermoLabsystems, Helsinki, Fin-
land). The compounds which inhibited more than 50% of the prote-
9. (a) Wu, C.-Y.; Jan, J.-T.; Ma, S.-H.; Kuo, C.-J.; Juan, H.-F.; Cheng, E. Y.-S.;
Hsu, H.-H.; Huang, H.-C.; Wu, D.; Brik, A.; Liang, F.-S.; Liu, R.-S.; Fang, J.-M.;
Chen, S.-T.; Liang, P.-H.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
10012; (b) Shao, Y.-M.; Yang, W.-B.; Peng, H.-P.; Hsu, M.-F.; Tsai, K.-C.; Kuo, T.-
H.; Wang, A. H.-J.; Liang, P.-H.; Lin, C.-H.; Yang, A.-S.; Wong, C.-H. ChemBioChem
2007, 8, 1654.
10. Kao, R. Y.; Tsui, W. H. W.; Lee, T. S. W.; Tanner, J. A.; Watt, R. M.; Huang, J. D.;
Hu, L. H.; Chen, G. H.; Chen, Z. W.; Zhang, L. Q.; He, T.; Chan, K. H.; Tse, H.; To, A.
P. C.; Ng, L. W. Y.; Wong, B. C. W.; Tsoi, H. W.; Yang, D.; Ho, D. D.; Yuen, K. Y.
Chem. Biol. 2004, 11, 1293.
ase activity at 50 lM were selected for the next assay run.
4.3. Cytotoxicity assay
Cell viability was determined by MTT 3-(4,5-dimethyl thiazol-
2-yl)-2,5–diphenyl tetrazolium bromide,32 using VybrantÒ MTT
cell proliferation assay kit purchased from Molecular Probes,
USA. Human embryonic kidney (HEK) 293 cells (2 ꢁ 105/ml) were
seeded into a 96-well culture plate containing 0.1 ml of Minimum
Essential Medium (MEM) (Gibico, Invitrogen, CA, USA) supple-
mented with 10% fetal bovine serum (FBS) (Gibico) and cultured
11. Blanchard, J. E.; Elowe, N. H.; Huitema, C.; Fortin, P. D.; Cechetto, J. D.; Eltis, L.
D.; Brown, E. D. Chem. Biol. 2004, 11, 1445.
12. (a) Chen, L. L.; Gui, C. S.; Luo, X. M.; Yang, Q. G.; Gunther, S.; Scandella, E.;
Drosten, C.; Bai, D.; He, X. C.; Ludewig, B.; Chen, J.; Luo, H. B.; Yang, Y. M.; Yang,

7854
R. Ramajayam et al. / Bioorg. Med. Chem. 18 (2010) 7849–7854
Y. F.; Zou, J. P.; Thiel, V.; Chen, K.; Shen, J. H.; Xu, S.; Jiang, H. L. J. Virol. 2005, 79,
7095; (b) Yang, Q.; Chen, L.; He, X.; Gao, Z.; Shen, X.; Bai, D. Chem. Pharm. Bull.
2008, 56, 1400.
Ghosh, A. K.; Xi, K.; Grum-Tokars, V.; Xu, X.; Ratia, K.; Fu, W.; Houser, K. V.;
Baker, S. C.; Johnson, M. E.; Mesecar, A. D. Bioorg. Med. Chem. Lett. 2007, 17,
5876.
13. Liu, Z.; Huang, C.; Fan, K.; Wei, P.; Chen, H.; Liu, S.; Pei, J.; Shi, L.; Li, B.; Yang, K.;
Liu, Y.; Lai, L. J. Chem. Inf. Model. 2005, 45, 10.
14. Jain, R. P.; Petterson, H. I.; Zhang, J.; Aull, K. D.; Fortin, P. D.; Huitema, C.; Eltis, L.
D.; Parrish, J. C.; James, M. N. G.; Wishart, D. S.; Vederas, J. C. J. Med. Chem. 2004,
47, 6113.
22. Kaeppler, U.; Stiefl, N.; Schiller, M.; Vicik, R.; Breuning, A.; Schmitz, W.;
Rupprecht, D.; Schmuck, C.; Baumann, K.; Ziebuhr, J.; Schirmeister, T. A. J. Med.
Chem. 2005, 48, 6832.
23. Hsu, J. T. A.; Kuo, C. J.; Hsieh, H. P.; Wang, Y. C.; Huang, K. K.; Lin, C. P. C.; Huang,
P. F.; Chen, X.; Liang, P. H. FEBS Lett. 2004, 574, 116.
15. Shie, J.-J.; Fang, J.-M.; Kuo, T.-H.; Kuo, C.-J.; Liang, P.-H.; Huang, H.-J.;
Yang, W.-B.; Lin, C.-H.; Chen, J.-L.; Wu, Y.-T.; Wong, C.-H. J. Med. Chem. 2005,
48, 4469.
24. (a) Watanabe, T.; Yuki, S.; Egawa, M.; Nishi, H. J. Pharmacol. Exp. Ther. 1994, 268,
1597; (b) Kawai, H.; Nakai, H.; Suga, M.; Yuki, S.; Watanabe, T.; Saito, K. I. J.
Pharmacol. Exp. Ther. 1997, 281, 921.
16. Bacha, U.; Barrila, J.; Velasquez-Campoy, A.; Leavitt, S. A.; Freire, E. Biochemistry
2004, 43, 4906.
17. Chen, L.-R.; Wang, Y.-C.; Lin, Y.-W.; Chou, S.-Y.; Chen, S.-F.; Liu, L.-T.; Wu,
Y.-T.; Kuo, C.-J.; Chen, T. S.-S.; Juang, S.-H. Bioorg. Med. Chem. Lett. 2005, 15,
3058.
25. Wu, T. W.; Zeng, L. H.; Wu, J.; Fung, K. P. Life Sci. 2002, 71, 2249.
26. Fan, X.; Zhang, X.; Zhou, L.; Keith, K. A.; Kernb, E. R.; Torrencea, P. F. Bioorg. Med.
Chem. Lett. 2006, 16, 3224.
27. Chen, L.; Chen, S.; Gui, C.; Shen, J.; Shen, X.; Jiang, H. J. Biomol. Screen. 2006, 11,
915.
18. Ramajayam, R.; Tan, K.-P.; Liu, H.-G.; Liang, P.-H. Bioorg. Med. Chem. Lett. 2010,
20, 3569.
28. Kuo, C. J.; Liu, H. G.; Lo, Y. K.; Seong, C. M.; Lee, K. I.; Jung, Y. S.; Liang, P. H. FEBS
Lett. 2009, 583, 549.
19. Wu, C.-Y.; King, K.-Y.; Kuo, C.-J.; Fang, J.-M.; Wu, Y.-T.; Ho, M.-Y.; Liao, C.-L.;
Shie, J.-J.; Liang, P.-H.; Wong, C.-H. Chem. Biol. 2006, 13, 4469.
20. (a) Bacha, U.; Barrila, J.; Gabelli, B.; Kiso, Y.; Amzel, L. M.; Freire, E. Chem. Biol.
Drug Des. 2008, 72, 34; (b) Regnier, T.; Sarma, D.; Hidaka, K.; Bacha, U.;
Freire, E.; Hayashi, Y.; Kiso, Y. Bioorg. Med. Chem. Lett. 2009, 19, 2722.
21. (a) Ghosh, A. K.; Xi, K.; Ratia, K.; Santarsiero, B. D.; Fu, W.; Harcourt, B. H.; Rota,
P. A.; Baker, S. C.; Johnson, M. E.; Mesecar, A. D. J. Med. Chem. 2005, 48, 6767; (b)
Shie, J.-J.; Fang, J.-M.; Kuo, T.-H.; Kuo, C.-J.; Liang, P.-H.; Huang, H.-J.; Wu, Y.-T.;
Jan, J.-T.; Cheng, E. Y.-S.; Wong, C.-H. Bioorg. Med. Chem. 2005, 13, 5240; (c)
29. Wang, X.-J.; Tan, J.; Grozinger, K. Tetrahedron Lett. 2000, 41, 4713.
30. Dhal, P. N.; Achary, T. E.; Nayak, A. J. Indian Chem. Soc. 1975, 52, 1196.
31. Kuo, C. J.; Chi, Y. H.; Hsu, J. T. A.; Liang, P. H. Biochem. Biophys. Res. Commun.
2004, 318, 862.
32. Terachi, T.; Stanescu, G.; Pontes, J. E.; Medof, M. E.; Caulfield, M. J. Cancer Res.
1991, 51, 2515.
33. Yang, H.; Yang, M.; Ding, Y.; Liu, Y.; Lou, Z.; Zhou, Z.; Sun, L.; Mo, L.; Ye, S.;
Pang, H.; Gao, G. F.; Anand, K.; Bartlam, M.; Hilgenfeld, R.; Rao, Z. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 13190.