Discovery of potent anilide inhibitors against the severe acute respiratory syndrome 3CL protease

Article abstract of DOI:10.1021/jm050184y

A diversified library of peptide anilides was prepared, and their inhibition activities against the SARS-CoV 3CL protease were examined by a fluorogenic tetradecapeptide substrate. The most potent inhibitor is an anilide derived from 2-chloro-4-nitroanili

Full text of DOI:10.1021/jm050184y

J. Med. Chem. 2005, 48, 4469-4473
4469
Discovery of Potent Anilide Inhibitors against the Severe Acute Respiratory
Syndrome 3CL Protease
Jiun-Jie Shie,† Jim-Min Fang,*,†,‡ Chih-Jung Kuo,§ Tun-Hsun Kuo,§ Po-Huang Liang,*,§ Hung-Jyun Huang,†
Wen-Bin Yang,‡ Chun-Hung Lin,§ Jiun-Ling Chen,† Yin-Ta Wu,‡ and Chi-Huey Wong‡,|
Department of Chemistry, National Taiwan University, Taipei, 106, Taiwan, Genomics Research Center, Academia Sinica,
Taipei, 115, Taiwan, Institute of Biological Chemistry, Academia Sinica, Taipei, 115, Taiwan, and Department of Chemistry
and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037
Received February 28, 2005
A diversified library of peptide anilides was prepared, and their inhibition activities against
the SARS-CoV 3CL protease were examined by a fluorogenic tetradecapeptide substrate. The
most potent inhibitor is an anilide derived from 2-chloro-4-nitroaniline, L-phenylalanine and
4-(dimethylamino)benzoic acid. This anilide is a competitive inhibitor of the SARS-CoV 3CL
protease with Ki ) 0.03 µM. The molecular docking experiment indicates that the P1 residue
of this anilide inhibitor is distant from the nucleophilic SH of Cys145 in the active site.
Introduction
boxylate,11 and phthalhydrazide-substituted keto-
glutamine analogues.12
Severe Acute Respiratory Syndrome (SARS) first
occurred in Guandong (China) in November of 2002 and
spread through many countries in 2003. This illness is
caused by infection with a novel human coronavirus
(SARS-CoV).1 The fatality rate of SARS-CoV infection
is rather high, estimated to be 14-15%. In only a few
months, nearly 1000 lives were claimed.2 The natural
source of SARS-CoV is unclear, though studies on the
molecular evolution of SARS-CoV indicate that the virus
may have emerged from wild animals.3 At present, no
efficacious therapy for SARS is available. Therefore, a
search for effective antivirals for the SARS-CoV is of
current interest.
As a part of our efforts directed toward the develop-
ment of anti-SARS agents, we prepared several chro-
mogenic peptides (AA)x-Gln-pNA, e.g. QTSITSAVLQ-
pNA containing a p-nitroaniline moiety at the C-terminal
glutamine, to test their activities as substrates for the
SARS-CoV 3CL protease. Unlike typical nitroanilide-
based peptides which are readily hydrolyzed by serine
and cysteine proteases,13 these (AA)x-Gln-pNA peptides
were not efficiently cleaved by the SARS-CoV 3CL
protease but displayed weak inhibition against the
enzyme. This observation, along with an earlier cell-
based assay showing the inhibitory activity of N-(2-
chloro-4-nitrophenyl)-5-chloro-2-hydroxybenzamide
(Niclosamide) against the replication of SARS-CoV,14 led
us to explore peptide nitroanilides as inhibitors of the
SARS-CoV 3CL protease.
SARS coronavirus is a positive-strand RNA virus,4
that encodes two replicase polyproteins pp1a and pp1b.
Extensive proteolytic processing of these nonstructural
polyproteins is required to provide the functional pro-
teins for viral propagation. These processes are medi-
ated primarily by the main protease (Mpro), which is also
known as dimeric chymotrypsin-like protease (3CLpro).5
The active site of SARS-CoV 3CL protease contains
Cys145 and His41 to constitute a catalytic dyad, in
which cysteine functions as the common nucleophile in
the proteolytic process.5 The 3CL protease cleaves pp1a
at no less than 11 conserved sites with a sequence of
(Leu,Met,Phe)-GlnV(Ser,Ala,Gly), in which a P1 glutamine
residue invariably occupies the S1 site.5,6 The 3CL
protease is essential for the propagation of the virus and
thus serves as a key target for the discovery of anti-
SARS drugs.
So far, only a few inhibitors have been validated by
in vitro protease assays. These protease inhibitors
include C2 symmetric peptidomimetic compounds,7 zinc-
conjugated compounds,8 bifunctional aryl boronic acids,9
a quinolinecarboxylate derivative,10 a thiophenecar-
* To whom correspondence should be addressed. Tel: 8862-
23637812. Fax: 8862-23636359. E-mail: jmfang@ntu.edu.tw.
† National Taiwan University.
Results and Discussion
‡ Genomics Research Center, Academia Sinica.
§ Institute of Biological Chemistry, Academia Sinica.
| The Scripps Research Institute.
We chose to prepare a series of peptide anilides
having L-phenylalanine as the P1 residue, on the basis
10.1021/jm050184y CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/04/2005

4470 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Shie et al.
Scheme 1. Synthesis of Ketomethylene Isosteres of Tripeptidesa
a Reagents and conditions: (i) NaH, THF, 0 °C to room temperature, 24 h. (ii) CF3CO2H, CH2Cl2, rt, 24 h. (iii) H2, Pd/C, Boc2O, MeOH,
rt, 10 h. (iv) allyl iodide, Cs2CO3, DMF, 45 °C, 5 h. (v) HCl, 1,4-dioxane, rt, 2 h. (vi) N-methylmorpholine, CH2Cl2, 0-25 °C, 2 h. (vii)
Pd(PPh3)4, morpholine, THF, 25 °C, 3 h. (viii) HOBt, EDCI, (i-Pr)2NEt, CH2Cl2, 0 °C to room temperature, 20 h.
of another study on AG7088 analogues that the inhibi-
tory activity can be improved by using L-phenylalanine
to replace L-glutamine or its γ-lactam isostere (see Table
3 in Supporting Information). Anilide 1 was prepared
by condensation of 2-chloro-4-nitroaniline with the acyl
chloride derivative of Boc-Phe-OH. Using the previously
reported amide formation in a microtiter plate,15 the
coupling reactions of a 60-member library of carboxylic
acids with the amine generated by removal of the Boc
group from anilide 1 afforded a 60-member library of
anilide 2. Tripeptide anilides 3a-x (24 members) and
tetrapeptide anilides 4a-x (24 members) were also
created by coupling of 1 with appropriate peptides. The
dimeric peptide anilides 5a-c and 6a-c were prepared
by using a diacid, e.g. succinic acid, S-malic acid, and
(2R,3R)-tartaric acid, as the core structure to link with
appropriate amino acids or peptides.
Figure 1. Lineweaver-Burk plot for inhibition of the SARS-
CoV 3CL protease by anilide 2a (Ki ) 0.03 µM).
inhibitory activities against the SARS-CoV 3CL pro-
tease, whereas the ketomethylene isosteres 7 were less
potent than the tripeptide counterpart 3. Surprisingly,
Niclosamide showed no inhibitory activity at a concen-
tration of 50 µM.8 The IC50 data for some anilide
inhibitors, either prepared in microtiter plate without
isolation or in pure form, are listed in Table 1. Anilide
2a (JMF1507) derived from 2-chloro-4-nitroaniline, L-
phenylalanine, and 4-(dimethylamino)benzoic acid is the
most potent inhibitor, showing an IC50 of 0.06 µM and
Ki ) 0.030 µM.
Lineweaver-Burk plots of kinetic data were fitted
with the computer program KinetAsyst II (IntelliKinet-
ics, PA) by nonlinear regression to obtain the Ki values.
The double reciprocal plot of the initial rate vs substrate
concentration indicates that all these compounds are
competitive inhibitors. A representative example for
inhibition of the SARS-CoV 3CL protease by anilide 2a
is shown in Figure 1. It is worth to note that anilide 2a
functions as a potent inhibitor with Ki ) 0.03 µM, rather
than a substrate for the SARS-CoV 3CL protease. The
HPLC and absorption spectral analyses indicated that
no decomposition of anilide 2a occurred under the
enzymatic conditions for a period over 16 h (see the
Supporting Information). In hydrolysis of N-nitrophen-
ylamide, alignment of n,π-orbitals is required for the
facile leaving of nitroaniline. Due to the steric effect of
the chlorine atom at the ortho-position, the 2-chloro-4-
nitrophenyl ring and amido group cannot be in a
coplanar conformation, thus making hydrolysis unfavor-
able. This speculation is in agreement with the com-
puter modeling shown in Figure 2.
On the basis of the previously reported synthesis of
AG7088,16 we also devised an expedient method for the
synthesis of anilides 7a-x, the ketomethylene isosteres
of tripeptides 3a-x (Scheme 1).
These anilide products 2-7 were characterized by
mass analyses and directly subjected, without isolation,
to the inhibition assay against the SARS-CoV 3CL
protease according to the previously described fluoro-
metric method.7,17 The initial velocities of the inhibited
reactions using 50 nM of SARS-CoV 3CL protease and
6 µM of the fluorogenic substrate were plotted against
the different inhibitor concentrations to obtain the IC50
values. On the basis of the preliminary results of assays,
some of the most promising inhibitor candidates were
selected for the scale-up synthesis. The IC50 values and
inhibition constants (Ki) of the pure samples were then
measured to validate their activities.
To know the structure-activity relationship, a series
of the 2a analogues was synthesized and the inhibitory
activity was examined. None of the analogues 8a-f
showed adequate activity (IC50 > 10 µM). Deletion of
The results of preliminary assays indicated that the
2-chloro-4-nitroanilides 2-6 generally possessed good

Inhibitors of SARS-CoV 3CL Protease
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4471
Table 1. IC50 Values for Some 2-Chloro-4-nitroanilide Inhibitors against the SARS-CoV 3CL Protease
structure type
niclosamide
anilide
compd
R
R′
IC50 (µM)
Ki (µM)a
>50a
>50a
0.06a
3b
1
t-BuO
Me2NC6H4
2a
2b
2c
2d
2e
3a
3c
3d
3f
0.03 ( 0.001
C14H29CH(Br)
3,4-(NH2)2C6H3
2b
(indol-3-yl)-CHdCH
(2-NH2-1,3-thiazol-4-yl)-C(dNOCH3)
3b
7b
tripeptide anilide
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
Et
Ph
7b
4b
t-BuO
morpholino
thien-2-yl
>10a
19b
5a
3h
3o
3p
4a
4f
4g
4j
4k
4q
4s
5a
5b
5c
6a
6b
6c
7a
7c
7d
7f
2.29 ( 1.01
2.90 ( 1.27
4.3 ( 1.9
PhCH2
PhCH2
i-Bu
5-Me-isoxazol-3-yl
7a
Thien-2-yl
Et
morpholino
t-Bu
5a
tetrapeptide anilide
7b
i-Bu
16b
2b
PhCH2
PhCH2
PhCH2
4-FC6H4CH2
4-FC6H4CH2
H
(S)-OH
(R)-OH
H
5-Me-isoxazol-3-yl
5a
1.61 ( 1.03
1.51 ( 0.95
PhCH2O
Et
Ph
H
6a
5b
2b
dimeric anilide
>50b
4a
H
3.1 ( 0.4
(R)-OH
H
H
(R)-OH
Et
Ph
5b
2b
(S)-OH
(R)-OH
i-Bu
2b
2b
ketomethylene anilide
27b
21b
19b
29b
22b
6b
i-Bu
i-Bu
i-Bu
i-Bu
t-BuO
morpholino
thien-2-yl
5-Me-isoxazol-3-yl
Thien-2-yl
7h
7o
7p
PhCH2
PhCH2
16b
a The sample is in pure form. b The sample is synthesized in a microtiter plate and assayed in situ without isolation.
replacing the dimethylamino group in 2a with a nitro
group.
Figure 2. Computer modeling of compound 2a binding to
SARS-CoV 3CL protease (1uk4). Compound 2a is colored in
yellow. The van der Waals filling space is generated by 1.0
scale and colored according to atom type. Clusters of possible
docking modes were sorted by computed binding free energy
and the docking mode with lowest docking energy (-9.1 kcal/
mol in this case) is generated by MGLTOOLS.
The crystal structure of SARS-CoV 3CL protease in
complex with a specific inhibitor of hexapeptidyl chlo-
romethyl ketone, Cbz-Val-Asn-Ser-Thr-Leu-Gln-CH2Cl,
has been reported (coded 1uk4 in the Protein Data Bank
deposition).5b On this basis, the docking experiment
(Autodock version 3.0.5)18 using p-nitroaniline as the
core structure formed three main clusters (RMSD ) 2
Å). The clusters with lowest binding free energy occupies
the chloro, nitro, or dimethylamino substituents from
anilide 2a significantly deteriorated potency as did

4472 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13
Shie et al.
was purified by flash column chromatography with elution of
EtOAc/hexane (1:9) to provide N-[4-(dimethylamino)phenyl]-
phenylalanine 2-chloro-4-nitroanilide (2a, 0.29 g, 61%) as light
yellow solids.
the pocket formed by Cys145, Ser144, His163, and
Phe140 or the pocket formed by Thr25, His41, Cys44,
Thr45, and Ala46. The 2-chloro-4-nitroanilide moiety of
2a was found to occupy the second favorite pocket
described above (Figure 2). The nitro group in 2a is
predicted to be hydrogen bonded with the HN of Ala46,
while the chlorine atom is within 3 Å from γ-S atom of
Cys145 and ꢀ-N2 atom of His41, providing a possible
key interaction with the catalytic dyad. The (dimethyl-
amino)phenyl group is fitted to the cleft formed by
Gln189-Gln192 and Met165-Pro68. The P1 phenyl
residue in 2a is positioned in the S1 pocket, which may
be modified to increase the interactions with Phe140,
His163, and Glu166.
The docking study showed that anilide 2a has the
lowest binding free energy of -9.1 kcal/mol in compari-
son with the anilides 8a-e (∆G* ) -7.5 to -8.7 kcal/
mol, see the Supporting Information). This docking
experiment supports the observation in the enzymatic
assay, which reveals the important roles of the 2-chloro-
4-nitroanilide and (dimethylamino)phenyl moieties in
inhibition of the SARS-CoV 3CL protease. The docking
model also shows that the P1 site of anilide 2a is distant
(∼4.95 Å) from the nucleophilic SH of Cys145. This
model is in agreement with the observation that anilide
2a is stable in the SARS 3CL protease.
4-(Dimethylamino)benzoyl-L-Phe-(2-chloro-4-nitro-
anilide) (2a). Light yellow solid; mp 205-207 °C; λmax) 320
nm; TLC (EtOAc/hexane, 1:1) Rf ) 0.5; IR (KBr) 3304, 2926,
1710, 1607, 1512, 1344, 1279, 1185 cm-1; 1H NMR (CDCl3, 400
MHz) δ 9.17 (1 H, s), 8.67 (1 H, d, J ) 9.2 Hz), 8.24 (1 H, d, J
) 2.4 Hz), 8.14 (1 H, dd, J ) 9.2, 2.4 Hz), 7.60 (2 H, d, J ) 8.9
Hz), 7.35-7.31 (3 H, m), 7.30-7.27 (2 H, m), 6.65 (2 H, d, J )
8.9 Hz), 6.41 (1 H, d, J ) 7.1 Hz), 5.08-5.06 (1 H, m), 3.34 (2
H, d, J ) 7.1 Hz), 3.04 (6 H, s); 13C NMR (CDCl3, 100 MHz) δ
170.5 (C), 168.1 (C), 152.9 (C), 143.0 (C), 140.4 (C), 136.2 (CH),
129.3 (C), 129.0 (CH, 2 × ), 128.7 (CH, 2 × ), 127.4 (CH, 2 ×
), 124.7 (CH), 123.3 (CH), 122.9 (C), 120.4 (C), 119.2 (CH),
111.0 (CH, 2 × ), 55.6 (CH), 40.0 (CH3, 2 × ), 36.7 (CH2); FAB
MS m/z 467.1 (M+ + H); HRMS calcd for C24H24ClN4O4:
467.1486 (M+ + H); found: 467.1488; Anal. Calcd for C24H23-
ClN4O4: C 61.74, H 4.97, N 12.00; found: C 61.71, H 5.01, N
11.96.
Inhibition Assay against the SARS-CoV 3CL Protease.
A fluorometric assay17 was utilized to determine the inhibition
constants of the prepared samples. Briefly, a fluorogenic
peptide Dabcyl-KTSAVLQSGFRKME-Edans is used as the
substrate, and the enhanced fluorescence due to cleavage of
this substrate catalyzed by the protease was monitored at 538
nm with excitation at 355 nm. The IC50 value of individual
sample was measured in a reaction mixture containing 50 nM
of the SARS 3CL protease and 6 µM of the fluorogenic
substrate in 20 mM Bis-Tris (pH 7.0). The enzyme stock
solution was kept in 12 mM Tris-HCl (pH 7.5) containing 120
mM NaCl, 0.1 mM EDTA, and 1 mM DTT plus 7.5 mM â-ME
before adding to the assay solution. The Ki measurements were
performed at two fixed inhibitor concentrations and various
substrate concentrations.
Under the assay conditions similar to that for the
SARS-CoV 3CL protease, the IC50 values of anilide 2a
against trypsin, chymotrypsin, and papain were mea-
sured to be 110, 200, and 220 µM, respectively. In
comparison, anilide 2a is a potent inhibitor for the
SARS-CoV 3CL protease with an IC50 value of 0.06 µM.
Acknowledgment. We thank National Science Coun-
cil (Taiwan) for financial support, and Shih-Jia Shiao
and Su-Lung Tang for preparation of some anilide
inhibitors.
Experimental Section
Materials and Methods. SARS-CoV 3CL protease was
prepared according to the previously described procedure.17
Reactions requiring dry conditions were carried out under an
inert atmosphere using standard techniques. All the reagents
and solvents were reagent grade and were used without
further purification unless otherwise specified. THF was
distilled from sodium benzophenone ketyl under N2.
Supporting Information Available: Physical and spec-
troscopic properties of new compounds, inhibition assay, HPLC
and UV-vis analyses, molecular modeling, and NMR spectra.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Representative Procedure of Coupling Reactions. A
solution of N-Boc phenylalanine (2.65 g, 10 mmol) and 2-chloro-
4-nitroaniline (1.73 g, 10 mmol) in pyridine (30 mL) was cooled
to -15 °C, and phosphorus oxychloride (1 mL, 11 mmol) was
added dropwise with vigorous stirring. After the mixture was
stirred for 1.5 h at -15 °C, the reaction was quenched by
pouring into ice-water (100 mL). The mixture was extracted
with EtOAc (1 × 50 mL and 3 × 30 mL). The combined organic
phase was washed with saturated NaHCO3 (2 × 50 mL) and
brine (30 mL). The organic phase was dried over MgSO4 and
filtered, and the filtrate was concentrated under reduced
pressure. The residue was purified by flash silica gel column
chromatography with elution of EtOAc/hexane (5:95) to give
N-Boc phenylalanine 2-chloro-4-nitroanilide (1, 3.25 g, 78%)
as white solids.
A solution of N-Boc phenylalanine 2-chloro-4-nitroanilide
(0.42 g, 1 mmol) in 1,4-dioxane (3 mL) was treated with a
solution of HCl in 1,4-dioxane (4 M, 2 mL) at 25 °C. The
mixture was stirred for 1.5 h and concentrated under reduced
pressure to give a crude aminium chloride salt. The material
was dissolved in CH2Cl2 (5 mL), cooled to 0 °C, and treated
with 4-methylmorpholine (0.3 mL, 2.5 mmol) and 4-(dimethyl-
amino)benzoyl chloride (0.22 g, 1.2 mmol) sequentially. The
ice bath was removed, and the mixture was stirred for 2 h at
25 °C. The reaction was quenched by addition of brine (15 mL).
The mixture was extracted with CH2Cl2 (2 × 20 mL). The
organic phase was dried over Na2SO4 and filtered, and the
filtrate was concentrated under reduced pressure. The residue
References
(1) (a) 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. A Novel Coronavirus Associated
with Severe Acute Respiratory Syndrome N. Engl. J. Med. 2003,
348, 1953-1966. (b) Drosten, C.; Gu¨nther, S.; Preiser, W.; van
der Werf, S.; Brodt, H.-R.; Becker, S.; Rabenau, H.; Panning,
M.; Kolesnikova, L.; Fouchier, R. A. M.; Berger, A.; Burguiere,
A.-M.; Cinatl, J.; Eickmann, M.; Escriou, N.; Grywna, K.;
Kramme, S.; Manuguerra, J.-C.; Muller, S.; Rickerts, V.; Stur-
mer, M.; Vieth, S.; Klenk, H.-D.; Osterhaus, A. D. M. E.; Schmitz,
H.; Doerr, H. W. Identification of a Novel Coronavirus in
Patients with Severe Acute Respiratory Syndrome. N. Engl. J.
Med. 2003, 348, 1967-1976. (c) 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.
Coronavirus as a Possible Cause of Severe Acute Respiratory
Syndrome Lancet 2003, 361, 1319-1325.
(2) World Helth Organization, Communicable Disease Surveillance
&
Response, website: http://www.who.int/csr/sars/archive/
2003_05_07a/en and http://www.who.int/csr/sars/country/en/
country2003_08_15.pdf. Summary table of SARS cases by coun-
try (1 November 2002 to 7 August 2003).
(3) He, J.-F.; Peng, G.-W.; Min, J.; Yu, D.-W.; Liang, W.-J.; 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.-

Inhibitors of SARS-CoV 3CL Protease
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4473
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. Molecular Evolution
of the SARS Coronavirus During the Course of the SARS
Epidemic in China. Science 2004, 303, 1666-1669.
(8) Hsu, J. T.-A.; Kuo, C.-J.; Hsieh, H.-P.; Wang, Y.-C.; Huang, K.-
K.; Lina, C. P.-C.; Huang, P.-F.; Chen, X.; Liang, P.-H. Evalu-
ation of Metal-Conjugated Compounds as Inhibitors of 3CL
Protease of SARS-CoV. FEBS Lett. 2004, 574, 116-120.
(9) Bacha, U.; Barrila, J.; Velazquez-Campoy, A.; Leavitt, S. A.;
Freire, E. Identification of Novel Inhibitors of the SARS Coro-
navirus Main Protease 3CLpro. Biochemistry 2004, 43, 4906-
4912.
(4) (a) 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.; Gu¨nther, S.; Osterhaus, A. D. M. E.; Drosten,
C.; Pallansch, M. A.; Anderson, L. J.; Bellini, W. J. Characteriza-
tion of a Novel Coronavirus Associated with Severe Acute
Respiratory Syndrome. Science 2003, 300, 1394-1399. (b) Marra,
M. A.; Jones, S. J. M.; Astell, C. R.; Holt, R. A.; Brooks-Wilson,
A.; Butterfield, Y. S. N.; Khattra J.; Asano, J. K.; Barber, S. A.;
Chan, S. Y.; Cloutier, A.; Coughlin, S. M.; Freeman, D.; Girn,
N.; Griffith, O. L.; Leach, S. R.; Mayo, M.; McDonald, H.;
Montgomery, S. B.; Pandoh, P. K.; Petrescu, A. S.; Robertson,
A. G.; Schein, J. E.; Siddiqui, A.; Smailus, D. E.; Stott, J. M.;
Yang, G. S.; Plummer, F.; Andonov, A.; Artsob, H.; Bastien, N.;
Bernard, K.; Booth, T. F.; Bowness, D.; Czub, M.; Drebot, M.;
Fernando, L.; Flick, R.; Garbutt, M.; Gray, M.; Grolla, A.; Jones,
S.; Feldmann, H.; Meyers, A.; Kabani, A.; Li, Y.; Normand, S.;
Stroher, U.; Tipples, G. A.; Tyler, S.; Vogrig, R.; Ward, D.;
Watson, B.; Brunham, R. C.; Krajden, M.; Petric, M.; Skowron-
ski, D. M.; Upton, C.; Roper, R. L. The Genome Sequence of the
SARS-Associated Coronavirus. Science 2003, 300, 1399-1404.
(c) Ruan, Y.; Wei, C. L.; Lin, A. E.; Vega, V. B.; Thoreau, H.; Se
Thoe, S. Y.; Chia, J. M.; Ng, P.; Chiu, K. P.; Lim, L.; Zhang, T.;
Chan, K. P.; Lin, E. L. O.; Ng, M. L.; Leo, S. Y.; Ng, L. F.; Ren,
E. C.; Stanton, L. W.; Long, P. M.; Liu, E. T. Comparative Full-
Length Genome Sequence Analysis of 14 SARS Coronavirus
Isolates and Common Mutations Associated with Putative
Origins of Infection. Lancet 2003, 361, 1779-1785.
(5) (a) Anand, K.; Ziebuhr, J.; Wadhwani, P.; Mesters, J. R.;
Hilgenfeld, R. Coronavirus Main Proteinase (3CLpro) Structure:
Basis for Design of Anti-SARS Drugs. Science 2003, 300, 1763-
1767. (b) 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. The Crystal Structures of
Severe Acute Respiratory Syndrome Virus Main Protease and
Its Complex with an Inhibitor. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 13190-13195. (c) Chou, K.; Wei, D.; Zhong, W.
Binding Mechanism of Coronavirus Main Proteinase with
Ligands and Its Implication to Drug Design Against SARS.
Biochem. Biophys. Res. Commun. 2003, 308, 148-151.
(6) (a) Fan, K.; Wei, P.; Feng, Q.; Chen, S.; Huang, C.; Ma, L.; Lai,
B.; Pei, J.; Liu, Y.; Chen, J.; Lai, L. Biosynthesis, Purification,
and Substrate Specificity of Severe Acute Respiratory Syndrome
Coronavirus 3C-Like Proteinase. J. Biol. Chem. 2004, 279,
1637-1642. (b) Huang, C.; Wei, P.; Fan, K.; Liu, Y.; Lai, L. 3C-
Like Proteinase from SARS Coronavirus Catalyzes Substrate
Hydrolysis by a General Base Mechanism. Biochemistry 2004,
43, 4568-4574. (c) Du, Q.-S.; Wang, S.-Q.; Zhu, Y.; Wei, D.-Q.;
Guo, H.; Sirois, S.; Chou, K.-C. Polyprotein Cleavage Mechanism
of SARS CoV Mpro and Chemical Modification of the Octapeptide.
Peptides 2004, 25, 1857-1864.
(10) Kao, R. Y.; Tsui, W. H. W.; Lee, T. S. W.; Tanner, J. A.; Watt, R.
M.; Huang, J.-D.; Hu, L.; Chen, G.; Chen, Z.; Zhang, L.; 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. Identification of
Novel Small-Molecule Inhibitors of Severe Acute Respiratory
Syndrome-Associated Coronavirus by Chemical Genetics. Chem.
Biol. 2004, 11, 1293-1299.
(11) Blanchard, J. E.; Elowe, N. H.; Huitema, C.; Fortin, P. D.;
Cechetto, J. D.; Eltis, L. D.; Brown1, E. D. High-Throughput
Screening Identifies Inhibitors of the SARS Coronavirus Main
Proteinase. Chem. Biol. 2004, 11, 1445-1453.
(12) Jain, R. P.; Pettersson, 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. Synthesis and Evaluation of Keto-
Glutamine Analogues as Potent Inhibitors of Severe Acute
Respiratory Syndrome 3CLpro. J. Med. Chem. (Web release
date: Nov. 5, 2004).
(13) (a) Zhang, R.; Malcolm, B. A.; Beyer, B. M.; Njoroge, F. G.;
Durkin, J. P.; Windsor, W. T. Peptide Substrates for Hepatitis
C Virus NS3 Protease Assays. U.S. Patent, 2001, 21 pp. (b)
Voelter, W.; Echner, H.; Philapitsch, A. Determining the C1-
Esterase Inhibitor Activity in a Sample. Ger. Offen. 1982, 22
pp. (c) Echner, H.; Voelter, W. New Chromogenic Substrates for
Determination of Serine Proteases. Chemiker-Zeitung 1988, 112,
117-124.
(14) Wu, C. J.; Jan, J. T.; Chen, C. M.; Hsieh, H. P.; Hwang, D. R.;
Liu, H. W.; Liu, C. Y.; Huang, H. W.; Chen, S. C.; Hong, C. F.;
Lin, R. K.; Chao, Y. S.; Hsu, J. T. A. Inhibition of Severe Acute
Respiratory Syndrome Coronavirus Replication by Niclosamide.
Antimicrob. Agents Chemother. 2004, 48, 2693-2696.
(15) (a) Wu, C.-Y.; Chang, C.-F.; Chen, J. S.-Y.; Wong, C.-H.; Lin,
C.-H. Rapid Diversity-Oriented Synthesis in Microtiter Plates
for In Situ Screening: Discovery of Potent and Selective R-Fu-
cosidase Inhibitors. Angew. Chem., Int. Ed. 2003, 42, 4661-4664.
(b) Chang, C.-F.; Ho, C.-W.; Wu, C.-Y.; Chao, T.-A.; Wong, C.-
H.; Lin, C.-H. Discovery of Picomolar Slow Tight-Binding Inhibi-
tors of R-Fucosidase Chem. Biol. 2004, 11, 1301-1306.
(16) (a) Tian, Q.; Nayyar, N. K.; Babu, S.; Chen, L.; Tao, J.; Lee, S.;
Tibbetts, A.; Moran, T.; Liou, J.; Guo, M.; Kennedy, T. P. An
Efficient Synthesis of a Key Intermediate for the Preparation
of the Rhinovirus Protease Inhibitor AG7088 via Asymmetric
Dianionic Cyanomethylation of N-Boc-L-(+)-Glutamic Acid Di-
methyl Ester. Tetrahedron Lett. 2001, 42, 6807-6809. (b) Tian,
Q.; Nayyar, N. K.; Babu, S.; Tao, J.; Moran, T. J.; Dagnino, R.;
Mitchell, L. J.; Remarchuk, T. P.; Melnick, M. J.; Bender, S. L.
Efficient Synthetic Routes for the Preparation of Rhinovirus
Protease Inhibitors and Key Intermediates. U.S. Pat. Appl.
6,355,807, 2002, 25 pp.
(17) Kuo, C.-J.; Chi, Y.-H.; Hsu, T.-A.; Liang, P.-H. Characterization
of SARS Main Protease and Inhibitor Assay Using a Fluorogenic
Substrate. Biochem. Biophys. Res. Commun. 2004, 318, 862-
867.
(18) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart,
W. E.; Belew, R. K.; Olson, A. J. Automated Docking Using a
Lamarckian Genetic Algorithm and an Empirical Binding Free
Energy Function. J. Comput. Chem. 1998, 19, 1639-1662.
(7) Wu, C.-Y.; Jan, J.-T.; Ma, H.-H.; Kuo, C.-J.; Juan, H.-F.; Cheng,
Y.-S. E.; 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. Small Molecules Targeting Severe Acute Respiratory Syn-
drome Human Coronavirus. Proc. Natl. Acad. Sci. U. S. A. 2004,
101, 10012-10017.
JM050184Y