Synthesis, docking studies, and evaluation of pyrimidines as inhibitors of SARS-CoV 3CL protease

Article abstract of DOI:10.1016/j.bmcl.2010.04.118

A series of 2-(benzylthio)-6-oxo-4-phenyl-1,6-dihydropyrimidine as SARS-CoV 3CL protease inhibitors were developed and their potency was evaluated by in vitro protease inhibitory assays. Two candidates had encouraging results for the development of new anti-SARS compounds.

Full text of DOI:10.1016/j.bmcl.2010.04.118

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3569–3572
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, docking studies, and evaluation of pyrimidines as inhibitors
of SARS-CoV 3CL protease
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 2-(benzylthio)-6-oxo-4-phenyl-1,6-dihydropyrimidine as SARS-CoV 3CL protease inhibitors
were developed and their potency was evaluated by in vitro protease inhibitory assays. Two candidates
had encouraging results for the development of new anti-SARS compounds.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 6 March 2010
Revised 20 April 2010
Accepted 27 April 2010
Available online 20 May 2010
Keywords:
SARS-CoV
Pyrimidines
Severe acute respiratory syndrome (SARS) has been recognized
as a global threat. SARS is characterized by high fever, malaise rig-
or, headache, chills, cough, and progressive radiographic changes
of the chest and lymphophenia.1–3 The initial outbreak of SARS
was first identified in Guangdong Province, China in November
2002. This outbreak spread to several countries and has had signif-
icant health and economic impact. The mortality rate is nearly
10%.4 With rigorous effort by the world health organization
(WHO), researchers found that SARS is caused by a novel coronavi-
rus, SARS-CoV.1,2,5 The SARS-CoV is a positive-strand RNA virus and
the genome is ꢀ30 kb (Tor2 strain). The genome is constituted of
five major open reading frames namely replicase polyproteins,
nucleocapsid proteins, spike (S), envelope (E), and membrane (M)
glycoproteins.
Resulting of structural and functional studies of coronaviral life-
cycle has provided a number of significant targets for ceasing the
viral replication. During the viral replication, the replicase polypro-
tein undergoes extensive processing by two viral proteases
namely, chymotrypsin-like protease (3CLpro) and papain-like pro-
tease (PLpro), reside within the polyprotein. They catalyze their
own release from the polyprotein and other non-structural pro-
teins (nsps) from the polyproteins and initiate virus mediated
RNA replication.6 Because of their essential roles in viral replica-
tion, both proteases are recognized as attractive targets for devel-
opment of anti-SARS agents.
pounds based on the substrate structure or active site properties.
Their scaffolds are diverse, including C2-symmetric diols,7 3-quin-
olinecarboxylic acid derivatives,8 thiophene-2-carboxylate deriva-
tives,9 cinanserin,10 calmodulin,11 keto-glutamine analogues,12
anilide,13 bifunctional boronic acid compounds,14 isatin deriva-
tives,15 benzotriazole16 as well as glutamic acid and glutamine
peptides possessing a trifluoromethyl ketone group,17
a,b-unsatu-
rated esters,18 and etacrynic acid derivatives.19 With metal-conju-
gated structures, some molecules make a coordinate bond with
Cys-145 at the active site of SARS-CoV 3CL .
pro 20 However, no effec-
tive 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 clas-
ses of SARS-CoV 3CLpro inhibitors that can be used in anti-SARS
therapy in case the disease re-emerges.
In our previous study, from high throughput screening we have
identified various heterocycles as novel anti-SARS agents with
selective inhibition ranging from IC50 2–10 lM against SARS–CoV
3CL protease (compounds 1–4, Fig. 1).21,22 In this paper, as a part
of our ongoing efforts to delineate a complete pharmacophore
model, we designed several 2-(benzylthio)-6-oxo-4-phenyl-1,
6-dihydropyrimidine derivatives as anti-SARS agents (compounds
6a–n, Fig. 1).
From a synthetic point view, the preparation of the target com-
pounds was envisioned following the synthetic routes illustrated
in Scheme 1. The synthesis of 6-aryl-5-cyano-2-thiouracils 5a–n
was prepared by reaction between substituted benzaldehyde, ethyl
cyanoacetate, and thiourea using the literature procedure.23 The
regioselective S-alkylation of 2-thiouracil 5a–n achieved by slow
addition of the respective halides to a solution of 5a–n in DMF
To date various SARS-CoV protease inhibitors have been re-
ported from both screened compound libraries and designed com-
* Corresponding author. Tel.: +886 2 2362 0261x3091; fax: +886 2 2363 5038.
E-mail address: phliang@gate.sinica.edu.tw (P.-H. Liang).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.118

3570
R. Ramajayam et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3569–3572
OCH3
H3CO
N
N
S
O
N
O
S
O
O
N
O
2
CH3
O
7.2 µM
1
O
N
NC
3.3 µM
NH
S
n
R2
6a-n
R1
N
N
OCH3
Br
N
S
N
N
N
N
O
Cl
H
N
N
O
Cl
4
3
Cl
2.0 µM
2.5 µM
Figure 1. Hits from this study (6) and previous studies21,22 in our laboratory.
O
Br
n
NC
O
S
CHO
NH
NC
NH
O
H2N
NH2
n = 1-2
N
S
NC
H
N
S
O
n
DMF, K2CO3
0 ºC-RT
R1
R1
5a-n
R2
6a-n
R1
R2 = H, 4-NO2
Scheme 1. Synthesis of Inhibitors 6a–n.
Table 1
using K2CO3 as a base at 0–5 °C to yield the corresponding com-
pounds 6a–n.24 Both analytical and spectral data of all target com-
pounds are accordant with the structures.
Structure and activity of compounds 6a–n.
O
The target compounds were tested for anti-SARS activity
NC
NH
against SARS-CoV 3CLpro, using previously developed assay meth-
od25 containing 0.05
l
M SARS 3CLpro, 6
lM fluorogenic substrate
N
S
n
Dabcyl-KTSAVLQSGFRKME-Edans, and 50
l
M of test compounds.
R2
Enhanced fluorescence of the reactions in the buffer of 20 mM
Bis–Tris at pH 7.0 was monitored at 538 nm with excitation at
355 nm using a fluorescence plate reader. The compounds which
6a-n
R1
Compound
R1
R2
n
IC50 (lM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
H
H
H
H
H
4-NO2
H
H
4-NO2
H
H
4-NO2
H
4-NO2
H
4-NO2
H
1
2
1
1
2
1
1
2
1
2
1
2
1
1
>50
>50
35.2
>50
20.3
26.3
>50
>50
>50
>50
inhibited more than 50% of the protease activity at 50 lM were se-
lected for the next assay run at 10 lM for IC50 calculation. Com-
pound 6m with R2 group of nitro functionality at C-4 position is
4-OCH3
4-OCH3
4-OCH3
4-CH3
4-CH3
4-CH3
3-NO2
3-NO2
4-Cl
the most potent inhibitor with an enzyme inhibitory activity
against SARS-CoV 3CLpro with an IC50 of 6.1
lM. The structure
and IC50 values are given in Table 1. The cytotoxicity of the test
compounds was tested by performing the MTT assay and found
that all compounds are devoid of cytotoxicity.24
To obtain molecular insight into the binding properties of these
active compounds, 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.26
Docking process was performed using an automated ligand-docking
subprogram of the Discovery Studio Modeling 1.2 SBD (Accelrys Inc.,
San Diego, CA), with a set of parameters chosen to control the precise
operation of the genetic algorithm. Docking runs were carried out
10.6 1.2
16.9 1.3
6.1 1.1
>50
4-Cl
3-Cl
using standard default settings ‘grid resolution’ of 5 Å, ‘site opening’
of 12 Å, and ‘binding site’ selected for defining the active site
cavity.

R. Ramajayam et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3569–3572
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Figure 2. Computer modeling of 6m binding in the active site of SARS 3CLpro
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In conclusion, we disclosed the inhibitory potency of compound
6m, containing pyrimidine unit, as a SARS-CoV 3CLpro inhibitor.
The measured inhibitory activity coupled with possible structure
modifications revealed by 3D docking give us new directions for
a fast development of much more potent inhibitors. Further inves-
tigations on this new family of compounds are currently in pro-
gress in our laboratory.
24. Ramajayam, R.; Mahera, N. B.; Neamati, N.; Yadav, M. R.; Giridhar, R.; Arch.
Pharm. 2009, 342, 710. General procedure for the synthesis of compound 6a–n: To
a mixture of 6-aryl-5-cyano-2-thiouracil (1 mmol) and K2CO3 (1.5 mmol) in
DMF (10 mL), alkyl iodide (1.2 mmol) was added dropwise with stirring while
maintaining the temperature of the reaction mixture at 0–5 °C. Stirring was
continued for 3 h at this temperature and continued for additional 2 h at room
temperature. Water was added to the mixture and filtered. The aqueous filtrate
was neutralized with acetic acid and the precipitate was filtered and purified.
The selected data of representative compounds 6k and 6m were as follows:
Compound 6k: Yield: 51%; mp 236–237 °C; IR (KBr): 3078, 2221, 1666, 1521,
References and notes
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1473, 1346, 1249, 1112, 1010, 916, 891, 785 cmÀ1 1H NMR (400 MHz, DMSO-
;
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J.; Yee, W. K.-S.; Yan, W. W.; Cheung, M.-T.; Cheng, V. C.-C.; Chan, K.-H.; Tsang,
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d6) d: 4.59 (s, 2H, CH2), 7.63 –7.65 (d, J = 8.72 Hz, 2H, ArH), 7.79 (t, J = 8.04 Hz,
1H, ArH), 8.17 –8.20 (d, J = 8.72 Hz, 2H, ArH), 8.31–8.33 (d, J = 8.0 Hz, 1H, ArH),
8.41–8.44 (m, 1H, ArH), 8.69 –8.70 (m, 1H, ArH), 12.9 (br, 1H, NH); MS (CI) m/z:
410 [M+H] +. Anal. Calcd for C18H11N5O5S: C, 52.81; H, 2.71; N, 17.11. Found: C,
52.68; H, 2.85; N, 17.04;

3572
R. Ramajayam et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3569–3572
Compound 6m: Yield: 66%; mp 254–257 °C; IR (KBr): 3000, 2218, 1651, 1517,
1467, 1346, 1244, 1009, 1004, 997, 856, 779 cmÀ1 1H NMR (400 MHz, DMSO-
25. Kuo, C. J.; Chi, Y. H.; Hsu, J. T. A.; Liang, P. H. Biochem. Biophys. Res. Commun.
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;
d6) d: 4.60 (s, 2H, CH2), 7.51–7.53 (d, J = 8.64 Hz, 2H, ArH), 7.60–7.62 (d,
J = 8.76 Hz, 2H, ArH), 7.89–7.91 (d, J = 8.64 Hz, 2H, ArH), 8.13–8.15 (d,
J = 8.72 Hz, 2H, ArH), 12.88 (br, 1H, NH); MS (CI) m/z: 399 [M+H]+. Anal.
Calcd for C18H11ClN4O3S: C, 54.21; H, 2.78; N, 14.05. Found: C, 54.18; H, 2.85;
N, 14.01.
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