Synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent SARS coronavirus 3CL protease inhibitor

Article abstract of DOI:10.1021/jm0603926

A potent SARS coronavirus (CoV) 3CL protease inhibitor (TG-0205221, K _i = 53 nM) has been developed. TG-0205221 showed remarkable activity against SARS CoV and human coronavirus (HCoV) 229E replications by reducing the viral titer by 4.7 log (at 5 μM) for SARS CoV and 5.2 log (at 1.25 μM) for HCoV 229E. The crystal structure of TG-0205221 (resolution = 1.93 A?) has revealed a unique binding mode comprising a covalent bond, hydrogen bonds, and numerous hydrophobic interactions. Structural comparisons between TG-0205221 and a natural peptide substrate were also discussed. This information may be applied toward the design of other 3CL protease inhibitors.

Full text of DOI:10.1021/jm0603926

J. Med. Chem. 2006, 49, 4971-4980
4971
Synthesis, Crystal Structure, Structure-Activity Relationships, and Antiviral Activity of a
Potent SARS Coronavirus 3CL Protease Inhibitor
Syaulan Yang,† Shu-Jen Chen,†,# Min-Feng Hsu,‡,§,|,# Jen-Dar Wu,†,# Chien-Te K. Tseng, Yu-Fan Liu,† Hua-Chien Chen,†
Chun-Wei Kuo,† Chi-Shen Wu,† Li-Wen Chang,† Wen-Chang Chen,† Shao-Ying Liao,† Teng-Yuan Chang,† Hsin-Hui Hung,†
Hui-Lin Shr,§,| Cheng-Yuan Liu,† Yu-An Huang,† Ling-Yin Chang,† Jen-Chi Hsu,† Clarence J. Peters,*,
Andrew H.-J. Wang,*,‡,§,| and Ming-Chu Hsu*,†
TaiGen Biotechnology Co., Taipei 114, Taiwan, ROC, Institute of Biochemical Sciences, National Taiwan UniVersity, Taipei 106, Taiwan,
ROC, Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, ROC, Core Facility for Protein X-ray Crystallography,
Academia Sinica, Taipei 115, Taiwan, ROC, and Department of Microbiology and Immunology, UniVersity of Texas Medical Branch,
GalVeston, Texas 77555
ReceiVed April 4, 2006
A potent SARS coronavirus (CoV) 3CL protease inhibitor (TG-0205221, Ki ) 53 nM) has been developed.
TG-0205221 showed remarkable activity against SARS CoV and human coronavirus (HCoV) 229E
replications by reducing the viral titer by 4.7 log (at 5 µM) for SARS CoV and 5.2 log (at 1.25 µM) for
HCoV 229E. The crystal structure of TG-0205221 (resolution ) 1.93 Å) has revealed a unique binding
mode comprising a covalent bond, hydrogen bonds, and numerous hydrophobic interactions. Structural
comparisons between TG-0205221 and a natural peptide substrate were also discussed. This information
may be applied toward the design of other 3CL protease inhibitors.
Introduction
inhibitor exhibited potent activities in suppressing the replication
of SARS CoV (4.7 log viral titer reduction at 5 µM concentra-
tion, IC50 ) 0.6 µM, and cytotoxicity IC50 > 20 µM) and human
coronavirus HCoV 229E (5.2 log viral titer reduction at 1.25
µM concentration). The tight binding between TG-0205221 and
the 3CLpro enzyme is through a covalent bond, hydrogen bonds,
and unprecedented hydrophobic interactions as evidenced by
the crystal structure of the corresponding complex.
The emergence of Severe Acute Respiratory Syndrome
(SARS) and its causative agent, a new coronavirus (SARS
CoV),1-5 in 2002 and 2003 has caused about 900 deaths among
more than 8400 infected people (a fatality rate of 10-15%) in
5 continents and 32 countries.6 The social impact and economic
loss in the affected regions, particularly in East and Southeast
Asia, have been enormous. Although regional preventive
measures are being implemented, vaccine and therapeutic drugs
are being sought.
SARS CoV is a positive-sense, single-stranded RNA virus
featuring the largest viral RNA genomes known to date.7-9 It
consists of about 29 700 nucleotides with its replicase gene alone
encompassing more than 21 000 nucleotides and encoding two
overlapping polyproteins, pp1a (486 kDa) and pplab (790
kDa).10 The functional polypeptides are released from each
polyprotein through extensive proteolytic processing, primarily
by the 34.6 kDa protease, which is also called the 3C-like
protease (3CLpro). Designing small molecule inhibitors to the
3CL protease is an attractive strategy for the development of
anti-SARS drugs. Such an antiviral strategy has been success-
fully applied to the aspartyl protease of the human immunode-
ficiency virus (HIV) to treat AIDS.11
Synthesis. The preparation of 15 (TG-0205221) was carried
out as described in Scheme 1 (the synthesis from compound 6
to 9 is similar to the ones described in ref 12):12 L-glutamic
acid (5) was silylated with trimethylsilyl chloride and esterified
in methanol at 0 °C, followed by Boc protection to afford
compound 6 in 95% yield. Sequential treatment of 6 with lithium
hexamethyl disilazide in THF under -78 °C and then with
bromoacetonitrile at -70 °C yielded alkylated product 7 in 83%
yield. Compound 7 was then dissolved in acetic acid and shaken
with 10% palladium on carbon under hydrogen gas in a Parr
shaker at 70 psi to give crude residue 8. This residue was used
without purification and treated with triethylamine in THF and
then stirred overnight at 60 °C to afford cyclized product 9 in
61% overall yield. After removal of the Boc protecting group,
intermediate 10 was coupled with 2-tert-butoxycarbonylamino-
3-(S)-cyclohexyl-propionic acid in the presence of EDC, HOBt,
and N-methylmorpholine at 0-5 °C in CH2Cl2 to give a high
yield (93%) of compound 11. The removal of Boc from 11 was
carried out by using ethereal HCl solution to afford intermediate
12, followed by peptide coupling of 12 with 2-benzyloxycar-
bonylamino-3-(S)-tert-butoxy-butyric acid in the presence of
EDC, HOBt, and N-methylmorpholine at 0-5 °C in CH2Cl2 to
give compound 13 in 64% yield. Reduction of 13 with lithium
borohydride in THF provided alcohol 14 in a 74% yield, which
was then oxidized with sulfur trioxide-pyridine complex in
DMSO to afford target product 15 (TG-0205221) in 30% yield.
Herein, we report our findings on a potent 3CLpro inhibitor
TG-0205221 (Ki ) 53 nM, Table 1) of SARS coronavirus with
distinct functional groups at the P1 to P4 sites compared to those
of reference compound 1, a 3C protease inhibitor to rhinovirus
possessing low activity toward 3CLpro. The newly designed
* To whom correspondence should be addressed. Tel: +886-2-
2790-1861 ext. 1702. Fax: +886-2-2796-3606. e-mail: mchsu@
taigenbiotech.com.tw (M.C.H.). Tel: +886-2-2788-1981. Fax: +886-2-
2788-2043. E-mail: ahjwang@gate.sinica.edu.tw (A.H.-J.W.). Tel: +1-
409-772-0090. Fax: +1-409-747-0762. E-mail: cjpeters@utmb.edu (C.J.P.).
† TaiGen Biotechnology Co.
‡ National Taiwan University.
§ Institute of Biological Chemistry, Academia Sinica.
| Core Facility for Protein X-ray Crystallography, Academia Sinica.
University of Texas Medical Branch.
Results and Discussion
Crystal Structure and Structure-Activity Relationships.
# These authors contributed equally to this work.
The structure of SARS 3CLpro bound with TG-0205221 (resolu-
10.1021/jm0603926 CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/14/2006

4972 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Yang et al.
Table 1. 3CL Protease Inhibitor on SARS Coronavirus, TG-0205221, and Reference Compound 1, Originally for 3C Protease Inhibitor on Rhinovirus
a Ki in µM for the in vitro inhibition of SARS coronavirus 3CL protease. b Ki in µM for the in vitro inhibition of HCoV 229E 3CL protease. c Drug
concentration in µM showing 50% inhibition on the HCoV 229E infected in vitro in the MRC-5 cells. d Drug concentration in µM showing 50% inhibition
on the SARS coronavirus infected in vitro in the Vero-E6 cells. e ND ) not determined.
Scheme 1a
a Reagents and conditions: (a) TMSCl, MeOH, 0 °C f rt, 15 h; (b) (Boc)2O, Et3N, rt, 0.5-1h; (c) LiHMDS, THF, N2,-78 °C, 1.5h, then BrCH2CN,
-78 °C, 3h; (d) H2(70 psi), 10% Pd/C, CH3COOH,rt, 2h; (e) Et3N, THF, 60 °C, overnight; (f) 4M HCl in dioxane, rt, 0.5h; (g) Boc-â-cyclohexyl-Ala-OH,
EDC, HOBt, NMM, DCM, rt, 2h; (h) 4M HCl in dioxane, rt, 0.5h; (i) Cbz-Thr(tBu)-OH, EDC, HOBt, NMM, DCM, rt, 2h; (j) LiBH4, THF, 0 °C f rt,
2h; (k) Et3N, DMSO, 15 °C, then SO3-Pyridine, 15 °Cfrt, 1h.
tion ) 1.93 Å) is shown in Figure 1A. Related parameters for
X-ray data collection and refinements are listed in Table 2. The
active site consists of a Cys145-His41 catalytic dyad at the
S1′ pocket, an oxyanion hole formed by the aldehyde oxygen
and N-H of Cys145 and Gly143 at the S1 pocket, and a large
hydrophobic cavity at the S2 to S4 pockets. The inhibitor
exhibits a unique and unsymmetrical binding mode (Figure 1B)
with the covalent bond, and most of the hydrogen bond inter-
actions (7 out of 10 in total) gathered near the P1 site and the
aldehyde acceptor, whereas numerous hydrophobic interactions
are grouped in the P2, P3, and P4 sites. At the acceptor site,
Cys145 attacks the carbonyl group of the aldehyde to form a
covalent C-S bond (bond length ) 1.24 Å) and induces the
formation of two hydrogen bonds between the aldehyde oxygen

Potent SARS 3CL Protease Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4973
Figure 1. (A) Stereoview of the SARS 3CL protease bound with the TG-0205221 (15) inhibitor shown by electrostatic potential. TG-0205221
(cyan stick) is fitted into the active site pocket of 3CLpro, and the surrounding residues of 3CLpro are shown as green lines. (B) Interactions between
SARS 3CLpro and TG-0205221. Schematic representation of key contacts between TG-0205221 and 3CLpro. The covalent bond between the S atom
of C145 and the carbon atom of the P1′ aldehyde group of TG-0205221 is shown by a purple line. The diagram was generated with LIGPLOT.23
(C) Superposition of 3CLpro-TG-0205221 and 3CLpro-product structures. The 3CLpro of 3CLpro-TG-0205221 and 3CLpro-product (C-terminus)
(SGVTFQ) complex structures are shown in ribbon format. In the 3CLpro-TG-0205221 complex structure, TG-0205221 is shown as cyan sticks,
and the residues with conformational change are shown by blue lines. In the 3CLpro-product structure, the product is shown by yellow sticks, and
the residues with conformational change are shown by orange lines.
and the N-H of Gly143 and Cys145 itself (Figure 1B). The five-
member lactam ring on P1 forms three hydrogen bonds with
His163, Phe140, and Glu166 as well as hydrophobic contacts
with the S1 environment.
Unlike P1, interacting mainly with hydrogen bonds, the P2
site forms extensive hydrophobic contacts with its environment.
As shown in Figure 1B, most of the cyclohexyl carbon atoms
(5 out of 6 in total) are involved in hydrophobic interactions.
The peptide backbone of P2-P4 residues also forms three
hydrogen bonds by the N-H on P2 with the oxygen on Gln189
and the oxygen on P3 with the N-H on Glu166 as well as the
N-H on P3 with the oxygen on Glu166. These hydrogen bonds
also contribute to the strong interactions of TG-0205221 with
the enzyme.
Because of the much easier synthetic procedures and very
similar properties of structure, enzyme, and cell activity against
3C protease in comparison with those of AG7088,12a,13 we
selected compound 1 as the reference compound for our project.

4974 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Yang et al.
Table 2. X-ray Data Collection and Refinement Statistics of SARS
3CLpro-TG-0205221 Complex
induced hydrophobic interactions with the phenyl ring of the
benzoxy group, which is folded and faces P3 by sticking to the
methylene group in a small corner pocket near A191 as pre-
viously described. Note that the loop region of a.a. 190-194
(Figure 1A), like a pen holder, is holding the end of the P6 site
like a pen tip in the product-bound structure (Figure 1C, yellow
object) and keeping the backbone away from the bottom of the
active site, whereas in the inhibitor-bound structure (Figure 1C,
cyan object), the tert-butyl threonine on the P3 forms induced
hydrophobic interactions with the P4 benzene ring by a folded
conformation that snugly fits the backbone into the hydrophobic
S3 and S4 pockets and stabilizes the three hydrogen bond
interactions (two with Glu166 and one with Gln189) (Figure
1B). The inhibitor-bound structure (TG-0205221) is found to
fit more tightly to the active site pockets by a larger calculated
contact area of 1119.79 Å2 than the product-bound structure
(SGVTFQ) (1050.95 Å2).
Data Collection
space group
cell dimensions
a, b, c (Å)
C2
109.0, 81.2, 53.3
104.7
50.0-1.93 (2.00-1.93)
0.052 (0.549)
24.87 (2.18)
99.8 (99.3)
â (°)
resolution (Å)a
b
Rmerge
I/σ(I)
completeness (%)
redundancy
4.1 (3.6)
Refinement Statistics
no. reflections
28,192
20.45/24.93
c
Rwork/Rfree
average B-values (Å2)
(no. of atoms)
protein
38.5 (2371)
47.1 (257)
33.9 (43)
0.005
water
inhibitor
rmsd from ideal
bond length (å)
rmsd from ideal
bond angles (°)
In addition to enzyme activity, we further focused on the
issues of cell activity, stability, and other drug-like properties.
On the basis of the structure of potent compound 4 (Ki ) 58
nM), we replaced the leucine residue at P2 with the more
lipophilic cyclohexyl alanine to improve the cell activity. This
strategy resulted in an enhancement of cell activity for our
overall pharmacophore, as is evidenced by the prominent
antiviral activity of TG-0205221 described in Table 1 and the
next section. Although the P1′ group of the 1,4-Michael acceptor
(e.g., R, â-vinyl ethyl ester, -CHdCH-C(O)-OEt) in com-
pounds 1-4 functioned as an efficient center to interact with
Cys145 for irreversible binding, such an ethyl ester group is
easily hydrolyzed to carboxylic acid by carboxyl esterases
present in plasma; for example, AG7088 lost its activity in the
plasma of rodents and rabbits.16,17 As a result, we replaced the
whole vinyl ethyl ester group with another efficient cystine
acceptor, aldehyde, for our pharmacophore. As shown in Table
4, TG-0205221 displays quite stable profile in mouse, rat, and
human plasma.
1.45
b
a Values in parentheses refer to the highest resolution bin. Rmerge)Σ|(Ihkl
)
- <I>|/Σ(Ihkl), where Ihkl is the integrated intensity of a given reflection.
c Rwork ) (Σ|Fo - Fc|)/(ΣFo), where Fo and Fc are observed and calculated
structure factors.
On the basis of the backbone structure of compound 1 (Table
3), each site of P1′-P4 was modified in a systematic fashion
in order to study structure-activity relationships with the
enzyme. We have learned that the P1 site favors glutamine type
of residues, including glutamine and the five-member lactam
ring.13,14 A five-member lactam ring was found to provide much
better enzyme activity (>15-fold) than the ones with glutamine
residue (data not shown). The strong binding feature of the five-
member lactam ring is evidenced by the multi-hydrogen-bond
formation in the crystal structure (Figure 1B). We then fixed
all of the groups from P1′ to P3 and replaced the methyl
isoxazole at P4 with a series of small alkyls or aryls and
heteroaryls extended with small alkyls for activity screenings.
The benzoxy group was found to give a more than 4-fold
increase in enzyme activity (Table 3, compound 1 and 2) and
became the best group for this site. As is demonstrated in the
crystal structure (Figure 1A), the benzoxy group locks in a
unique folding conformation with the methylene group residing
in the small corner pocket, whereas the phenyl ring aligns
parallel to the wall-shaped area near A191. Strong lipophilic
interactions between the benzene ring and its environment are
observed by the measured hydrophobic contacts from most of
the carbon atoms (4 out of 6) on the ring (Figure 1B).
For the P2 site, substituting the phenylalanine or 4-fluoro-
phenylalanine with a leucine group increased enzyme activity
by about 4-fold in our study (Table 3, compound 2 and 3). This
is probably due to the rigid and planar properties of the phenyl
ring, which are not favorable for binding in the S2 hydrophobic
pocket. As is shown in Figure 1C, the cyclohexyl alanine ring
of the 3CLpro-TG-0205221 complex (inhibitor-bound) structure
(cyan object) fits deeper into the S2 pocket in a stable chair
form than the rigid phenylalanine of the 3CLpro-product
complex (product-bound) structure (yellow object, SGVTFQ).15
The P3 site residue was predicted to have no specificity for
binding and may orient toward the bulk solvent12 or shift to the
P2 site.13 However, we found that inserting a lipophilic tert-
butyl group at this site further enhances the binding affinity
more than 10-fold (Table 3, compound 3 and 4). This enhance-
ment may be explained by the crystal structure in Figure 1A
showing that the tert-butyl group shifts to the P4 site and forms
Antiviral Activity. To conveniently monitor antiviral activity,
we employed a screening system of human coronavirus HCoV
229E and MRC-5 cells as a surrogate assay. TG-0205221 was
found to reduce HCoV 229E viral load from the original value
of 6.5 log TCID50/mL to 3.0 log at 0.625 µM and further down
to 1.3 log at 1.25 µM (Figure 2A) with an estimated IC50
)
0.14 µM. These results were further confirmed by a viral plaque
reduction assay (Figure 2B) in which MRC-5 cells were infected
with a 10-fold dilution of HCoV 229E viral stock (1.1 × 106
pfu/mL). The cytopathic effect (CPE) resulting from the viral
replication produced visible plaques in the MRC-5 cell layer.
TG-0205221 at 1 µM (Figure 2C) protected cells from all six
viral dilutions, whereas the control plate without drug treat-
ment gave visible plaques even at a 10-5 dilution of the viral
stock.
The experiments of the antiviral activity of TG-0205221
against SARS coronavirus replication in Vero E6 cells were
performed in a biosafety level-3 (BSL-3) laboratory at the
University of Texas Medical Branch at Galveston (UTMB). TG-
0205221 reduced the SARS CoV viral titer from 6.7 to 6.4,
6.0, 5.0, and 2.0 log TCID50/mL at 0.625, 1.25, 2.5, 5.0 µM,
respectively (Figure 3A), with a 4.7 log viral titer reduction at
5 µM drug concentration. CPE measurements were also
performed with Vero E6 cells infected by SARS CoV (original
stock: 1 × 107 TCID50/mL). Partial and complete protections
on the cells against SARS CoV by TG-0205221 were observed
at 2.5 and 5 µM, respectively (Figure 3B-E).

Potent SARS 3CL Protease Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4975
Table 3. Enzyme and Cell Activity of TG-0205221 Analogues
Table 4. Plasma Stability of TG-0205221a
Experimental Section
% of initial
incubation
General Methods. Melting points were obtained on a Buchi
B-545 apparatus and are uncorrected. 1H and 13C NMR spectra were
recorded on a Varian Mercury Plus 300 spectrometer. Chemical
time (min)
rat
mouse
human
0
30
120
100 ( 19
70 ( 1
73 ( 7
100 ( 6
84 ( 1
71 ( 12
100 ( 7
81 ( 3
83 ( 7
1
shifts are reported downfield from tetramethylsilane () 0) for H
NMR. Mass spectra (MS) were determined on an Agilent 1100
series mass spectrometer (ESI-MS). High-Resolution Mass spectra
(HR-MS) were determined on a Finnigan MAT 95S. Reagents and
solvents were used as obtained from commercial suppliers without
further purification. Chromatographic purification of the compounds
was performed on silica gel 60 (63-200 µm) purchased from Merck
Co. and a Pre-Packed Column Merck KGaA RT 250-25. HPLC
was measured by a Waters 2795 system, with a Waters 2996
PDA detector, and an Agilent/ZORBAX Eclipse/XDB-C18/4.6 ×
150 mm column as well as a Gilson 215 system, with a Gilson
UV/Vis-156 detector, and a LiChrospher 100 RP-18e column.
All animal procedures were approved by the Animal Care
Committee of TaiGen Biotechnology Co., Ltd.
a The drug was added to 90% rat, mouse, or human plasma and incubated
for 0, 30, and 120 min in respective wells. The solution was then added to
10 times the volume of acetonitrile for denaturization and analyzed by liquid
chromatography/mass spectrometry (LC/MS).
Conclusions
In summary, we have discovered a potent SARS 3CLpro
inhibitor that shows promising antiviral activities against
SARS coronavirus and human coronavirus HCoV 229E. The
resolved crystal structure showed that this inhibitor binds to
the enzyme with extensive hydrophobic contacts by the interac-
tion of its 16 carbons with 10 residues on the enzyme. These
hydrophobic contacts combine with 10 hydrogen bonds and 1
covalent bond to form a strong binding conformation for this
3CLpro inhibitor. The crystal structure has revealed binding
characteristics of a SARS inhibitor that are different from
early reports13,14 and shall provide new understanding for
designing inhibitors of other viral proteases with the sequence
and/or structure homologous to that of the SARS CoV protease.
TG-0205221 and its analogues showed favorable pharmacoki-
netic profiles in rodents (data not shown). Further testing of
the inhibitors in animal disease models is underway. Through
the enormous effort of the southeastern Asian health authorities,
the spread of SARS CoV in 2002 was effectively brought under
control in a short period of time. However, we believe that an
antiviral drug, such as TG-0205221, should be developed as a
defensive measure against a potential future outbreak of the
infection.
2-tert-Butoxycarbonylamino-pentanedioic Acid Dimethyl
Ester (6). To a stirred suspension of commercially available L-glu-
tamic acid (50.0 g, 340 mmol) in dry MeOH (1100 mL, 0.3 M)
was added dropwise TMSCl (162 g, 1490 mmol, 190 mL) at
0 °C. After the addition was completed, the reaction was allowed
to warm-up to room temperature and stirred until TLC analysis
showed no starting material (about 15 h). Then, Et3N (222 g,
2190 mmol, 306 mL) and (Boc)2O (82 g, 376 mmol) were
sequentially added at the same temperature. The reaction mixture
was stirred until TLC analysis showed completed protection. The
solvent was removed under reduced pressure, and the residue was
triturated and washed with Et2O using a pad of Celite. The
combined organic layers were evaporated, and the residue was
purified by silica gel column chromatography (15-20% EtOAc in
n-hexane as the eluent) to afford N-Boc-L-(+)-glutamic acid
1
dimethyl ester (6, 88.0 g, 320 mmol) in 94% yield as an oil. H
NMR (CDCl3) δ 1.40 (9H, s), 1.91 (1H, m), 2.14 (2H, m), 2.37
(2H, m), 3.64 (3H, s), 3.70 (3H, s), 4.29 (1H, br s); ESI-MS (m/z):
276 (M + H)+.

4976 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Yang et al.
Figure 3. (A) Reduction of infectious titers of SARS-CoV by TG-
0205221. Vero E6 cells were infected with SARS-CoV in the presence
of the drug at the indicated concentrations for 24 h. The culture medium
was harvested and TCID50 determined by re-infecting a fresh batch of
Vero E6 cells. (B)-(E) Prevention of the cytopathic effect (CPE) of
SARS-CoV in Vero E6 cells by TG-0205221. Vero E6 cells were
cultured in DMEM and 10% FCS medium (D-10) (B). Then 1 × 107
TCID50/mL of stock virus was diluted with D-10. The cells were
infected with 100 TCID50 per well, or multiplicity of infection (MOI)
of 0.001-0.002, without the presence of the drug (C), with 2.5 µM
(D), or 5.0 µM (E) drug. SARS-CoV usually induced CPE in Vero E6
cells within 2 days. The cells were protected and displayed slight to
no CPE at the drug concentrations. The CPE was observed and scored
daily until the experiments were terminated 6 days after infection. The
Urbani strain of SARS coronavirus, kindly provided by Dr. T. G.
Ksiazek (Centers for Disease Control, Atlanta, Georgia), was used
throughout this study. The experiments were conducted in a BSL-3
laboratory at the University of Texas Medical Branch at Galveston
(UTMB).
Figure 2. Inhibition of HCoV 229E replication by TG-0205221. (A)
Reduction of infectious titers: MRC-5 cells were infected with human
coronavirus HCoV 229E, kindly provided by Dr. Michael Lai (Aca-
demia Sinica, Taiwan), in the presence or absence of the drug for 72
h. The culture medium was harvested and TCID50 determined by re-
infecting a fresh batch of MRC-5 cells. (TCID50 ) 50% tissue culture
infective dose. This is the reciprocal of the highest dilution of virus
that causes 50% CPE of the cultured well.) (B) and (C) Plaque reduction
assay: A stock virus of 1.1 × 106 pfu/mL was serially diluted 10-fold
with D-10, as labeled in each plate, and infected with MRC-5 without
the drug (B), or with 1 µM drug (C) (pfu ) plaque forming unit). The
plates were overlayed with 0.3% agarose on day 2 and fixed with 10%
formalin and stained with 0.5% crystal violet on day 6.
mL). After stirred for 10 min, the cooling bath was removed and
replaced with a water bath. The reaction mixture was allowed to
warm up to 0 ( 5 °C and then poured into brine solution (10 g of
NaCl in 100 mL water) in a 1 L extractor. The aqueous layer was
separated, and the organic layer was concentrated to afford a dark
brown oil. Silica gel (25 g) and methylene chloride (60 mL) were
added to the Rotorvap flask and spun on a Rotorvap for 1 h without
heat and in a vacuum. The slurry was then filtered and washed
with more methylene chloride (100 mL). The light brown filtrate
was concentrated and purified by silica gel column chromatography
(50% EtOAc in n-hexane as the eluent) to afford compound 7 (19.0
2-tert-Butoxycarbonylamino-4-cyanomethyl-pentanedioic Acid
Dimethyl Ester (7). To a solution of N-Boc-L-(+)-glutamic acid
dimethyl ester (6, 20 g, 72.6 mmol) in THF (50 mL) was added
dropwise a solution of LiHMDS (26.3 g, 157 mmol) in THF (250
mL) at -78 °C under nitrogen atmosphere. The resulting mixture
was stirred at -78 °C for 1.5 h. Bromoacetonitrile (13 g, 108 mmol)
was added dropwise to the dianion solution over a period of 1 h
while maintaining the temperature below -70 °C. The reaction
mixture was further stirred at -78 °C until the disappearance of
the starting material was confirmed by TLC analysis (1-2 h).
The reaction was quenched with precooled methanol (10 mL)
in one portion and stirred for 10 min. The resulting methoxide was
then quenched with a precooled acetic acid (9 mL) in THF (60
1
g, 60.4 mmol) in 83% yield. H NMR (CDCl3) δ 1.42 (9H, s),
2.10-2.17 (2H, m), 2.77-2.90 (3H, m), 3.73 (3H, s), 3.74 (3H,
s), 4.32-4.49 (1H, m), 5.12 (1H, d, J ) 6.0 Hz); ESI-MS (m/z)
315 (M + H)+.
2-tert-Butoxycarbonylamino-3-(2-oxo-pyrrolidin-3-yl)-propi-
onic Acid Methyl Ester (9). Compound 7 (10.0 g, 31.8 mmol)
was dissolved in AcOH (240 mL) and shaken with 10% Pd/C (20

Potent SARS 3CL Protease Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4977
g) under H2 gas (70 psi) for 2 h. The mixture was filtered over
Celite. The filtrate was evaporated under reduced pressure, and the
residue was repeatedly evaporated from methyl tert-butyl ether to
yield a light pink solid. Then, the crude compound was dissolved
in THF, and Et3N (20 mL) was added to the solution. The resulting
mixture was stirred at 60 °C overnight. The reaction was quenched
with H2O (50 mL). The layers were separated, and the aqueous
layer was further extracted with methylene chloride. The organic
layers were combined, dried (MgSO4), and filtered. The light brown
filtrate was concentrated and purified by silica gel column chro-
matography (50-100% EtOAc in n-hexane as eluent) to afford
compound 9 (5.55 g, 19.4 mmol) in 61% yield. 1H NMR (CDCl3)
δ 1.41 (9H, s), 1.77-1.88 (2H, m), 2.06-2.15 (1H, m), 2.39-
2.49 (2H, m), 3.30-3.35 (2H, m), 3.71 (3H, s), 4.25-4.33 (1H,
m), 5.49 (1H, d, J ) 7.8 Hz), 6.00 (1H, s); 13C NMR (75 MHz,
CDCl3) δ 28.31, 28.48, 34.30, 38.32, 40.55, 52.47, 52.57, 80.08,
155.96, 173.13, 179.92; ESI-MS (m/z): 287 (M + H)+.
2-(2-tert-Butoxycarbonylamino-3-cyclohexyl-propionylamino)-
3-(2-oxo-pyrrolidin-3-yl)-propionic Acid Methyl Ester (11). (a)
A commercially available solution of HCl in 1,4-dioxane (4.0 M,
3.5 mL) was added to compound 9 (0.404 g, 1.41 mmol) and stirred
at room temperature for 30 min. The resulting solution was
concentrated to remove 1,4-dioxane under vacuum. CH2Cl2 (10 mL)
was then added to the residue and cooled to 0-5 °C. N-
Methylmorpholine (0.62 mL, 5.64 mmol) was added and stirred
for 10 min. (b) In the meantime, 2-tert-butoxycarbonylamino-(S)-
3-cyclohexyl-propionic acid (0.383 g, 1.41 mmol) was mixed with
EDC (0.324 g, 1.69 mmol), and HOBt (0.229 g, 1.69 mmol) in
CH2Cl2 (4 mL) and stirred for 20 min.
Solution (a) was then added to solution (b) and stirred at room
temperature for 2 h. The reaction mixture was mixed with brine
(10 mL) and extracted with CH2Cl2 (3 × 10 mL). The organic layers
were combined and dried with MgSO4 and concentrated. Purifica-
tion of the residue by flash column chromatography (3% MeOH
in CH2Cl2 as eluent) afforded a white solid of 11 (0.576 g, 1.31
mmol) in 93% yield. 1H NMR (CDCl3) δ 0.85-0.99 (2H, m), 1.12-
1.24 (4H, m), 1.41 (9H, s), 1.60-1.69 (5H, m), 1.76-1.90 (3H,
m), 2.12-2.21 (1H, m), 2.37-2.46 (2H, m), 3.29-3.37 (3H, m),
3.70 (3H, s), 4.17-4.25 (1H, m), 4.48-4.55 (1H, m), 5.00 (1H, d,
J ) 7.8 Hz), 6.44 (1H, s), 7.50 (1H, d, J ) 6.9 Hz); 13C NMR (75
MHz, CDCl3) δ 26.28, 26.43, 26.60, 28.30, 28.46, 32.84, 33.42,
33.78, 34.14, 38.42, 40.60, 40.84, 51.21, 52.50, 79.92, 155.78,
172.40, 173.51, 179.97; ESI-MS (m/z): 462 (M + Na)+.
128.22, 128.36, 128.71, 136.36, 156.35, 169.68, 172.29, 172.41,
179.89; ESI-MS (m/z): 631 (M + H)+.
(2-tert-Butoxy-1-{2-cyclohexyl-1-[2-hydroxy-1-(2-oxo-pyrro-
lidin-3-ylmethyl)-ethylcarbamoyl]-ethylcarbamoyl}-propyl)-car-
bamic Acid Benzyl Ester (14). To a stirring solution of compound
13 (0.450 g, 0.713 mmol) in THF (4.7 mL) was added LiBH4 (2.0
M in THF, 1.8 mL, 3.6 mmol) in several portions at 0 °C under a
nitrogen atmosphere. The reaction mixture was stirred at 0 °C for
1 h, then allowed to warm up to room temperature, and stirred for
an additional 2 h. The reaction was quenched by the dropwise
addition of 1.0 M HCl(aq) (3.6 mL) with cooling in an ice bath.
The solution was diluted with ethyl acetate (11.7 mL) and H2O
(5.8 mL). The phases were separated, and the aqueous layer was
extracted with ethyl acetate (3 × 35 mL). The organic phases were
combined together, dried over MgSO4, filtered, and concentrated
on a rotorvap to give a yellow oily residue. Column chromato-
graphic purification of the residue (6% MeOH in CH2Cl2 as the
eluent) afforded a white solid (14, 0.318 g, 74%). 1H NMR (CDCl3)
δ 0.85-0.98 (2H, m), 1.07 (3H, d, J ) 6.6 Hz), 1.11-1.38 (4H,
m), 1.24 (9H, s), 1.45-1.84 (9H, m), 2.00 (1H, m), 2.36-2.42
(2H, m), 3.26-3.29 (2H, m), 3.49-3.59 (3H, m), 3.98 (1H, m),
4.13-4.18 (2H, m), 4.35 (1H, m), 5.06 (1H, AB quartet, J ) 12.3
Hz), 5.13 (1H, AB quartet, J ) 12.3 Hz), 5.77 (1H, br s), 6.02
(1H, d, J ) 5.1 Hz), 7.29-7.36 (6H, m), 7.52 (1H, d, J ) 7.5 Hz);
13C NMR (75 MHz, CDCl3) δ 17.66, 26.21, 26.40, 26.54, 28.43,
28.57, 32.75, 32.83, 33.79, 34.42, 38.39, 39.97, 40.72, 50.32, 52.05,
59.34, 65.86, 67.02, 67.20, 75.60, 128.29, 128.41, 128.76, 136.39,
156.54, 170.03, 172.84, 181.28; ESI-MS (m/z): 603 (M + H)+
(2-tert-Butoxy-1-{2-cyclohexyl-1-[1-formyl-2-(2-oxo-pyrroli-
din-3-yl)-ethylcarbamoyl]-ethylcarbamoyl}-propyl)-carbamic Acid
Benzyl Ester (15) (TG-0205221). To a solution of compound 14
(0.125 g, 0.207 mmol, 1 equiv.) in methylsulfoxide (1.0 mL) was
added triethylamine (0.1 mL). The resulting solution was cooled
to 15 °C with an ice bath followed by the addition of the sulfur
trioxide-pyridine complex (0.23 g, 3 equiv). The reaction was
removed from the ice bath and stirred at room temperature for 1 h.
The reaction was then quenched with saturated brine (1 mL) and
extracted with ethyl acetate (3 × 5 mL). The combined organic
phases were dried over MgSO4, filtered, and concentrated to afford
a pale yellow oil. The oil was purified through chromatography
(100% EtOAc) to provide 15 as a white solid (0.037 g, 30%); mp
1
93.6-95.3 °C. H NMR (CDCl3) δ 0.86-1.31 (16H, m), 1.56-
2.02 (13H, m), 2.41 (2H, m), 3.31 (2H, d, J ) 7.8 Hz), 4.16 (2H,
br s), 4.42 (2H, m), 5.09 (2H, dd, J ) 20, 12 Hz), 5.89 (1H, br s),
7.33 (5H, m), 7.40 (1H, d, J ) 7.24 Hz), 8.01 (1H, br s), 9.48 (1H,
s); 13C NMR (75 MHz, CDCl3) δ 17.57, 26.20, 26.40, 26.56, 28.45,
28.78, 30.00, 32.84, 33.85, 34.43, 38.02, 40.17, 40.66, 51.71, 57.74,
59.22, 66.96, 67.22, 75.61, 128.30, 128.45, 128.78, 136.39, 156.46,
169.91, 173.09, 180.03, 199.72; ESI-MS (m/z): 601 (M + H)+;
HRMS (EI) m/z: C32H48N4O7 (M), calcd., 600.3518; found 600.3542.
4-(3-(4-Fluoro-phenyl)-2-{3-methyl-2-[(5-methyl-isoxazole-3-
carbonyl)-amino]-butyrylamino}-propionylamino)-5-(2-oxo-pyr-
rolidin-3-yl)-pent-2-enoic Acid Ethyl Ester (1). Compound 1 was
prepared by using a procedure similar to that described in the
published literature;12(a),(c) mp 143-144 °C. 1H NMR(CDCl3) δ 0.92
(6H, t, J ) 6 Hz), 1.28 (3H, t, J ) 6.9 Hz), 1.53-1.59 (1H, m),
1.74-1.81 (1H, m), 1.84-1.96 (1H, m), 2.11-2.38 (3H, m), 2.48
(3H, s), 3.02-3.04 (5H, m), 3.30-3.36 (2H, m), 4.17 (2H, q, J )
6.9 Hz), 4.34 (1H, t, J ) 7.8 Hz), 4.53-4.54 (1H, m), 4.86 (1H,
AB quartet, J ) 12.3 Hz), 5.70 (1H, d, J ) 15.9 Hz), 6.41 (1H, s),
6.68-6.75 (2H, m), 6.88 (2H, m), 7.10-7.15 (2H, m), 7.29-7.32
(1H, m), 7.75 (1H, d, J ) 8.1 Hz); 13C NMR (75 MHz, CDCl3)
δ12.55, 14.41, 18.42, 19.41, 28.42, 31.06, 35.20, 37.94, 38.60,
41.12, 49.04, 54.52, 59.15, 60.76, 101.62, 115.33, 115.62, 121.42,
131.20, 131.30, 132.24, 146.96, 158.40, 159.62, 160.45, 163.68,
166.39, 170.63, 171.0, 171.67, 180.58; ESI-MS (m/z): 600 (M +
H)+; HRMS (EI) m/z: C30H38FN5O7 (M), calcd., 599.2750; found
599.2770.
2-[2-(2-Benzyloxycarbonylamino-3-tert-butoxy-butyrylamino)-
3-cyclohexyl-propionylamino]-3-(2-oxo-pyrrolidin-3-yl)-propi-
onic Acid Methyl Ester (13). (a) A commercially available solution
of HCl in 1,4-dioxane (4.0M, 2.5 mL) was added to compound 11
(0.443 g, 1.01 mmol) and stirred at room temperature for 30 min.
The resulting solution was concentrated to remove 1,4-dioxane
under vacuum. CH2Cl2 (10 mL) was then added to the residue and
cooled to 0-5 °C, followed by the addition of N-methylmorpholine
(0.409 g, 0.45 mL, 4.04 mmol) and stirred for 10 min. (b) In the
meantime, 2-benzyloxycarbonylamino-(S)-3-tert-butoxy-butyric acid
(0.325 g, 1.05 mmol) was mixed with EDC (0.232 g, 1.21 mmol)
and HOBt (0.164 g, 1.21 mmol) in CH2Cl2 (4 mL) and stirred for
20 min.
Solution (a) was then added to solution (b) and stirred at room
temperature for 2 h. The reaction residue was added to brine (10
mL) and extracted with CH2Cl2 (3 × 10 mL). The organic layers
were combined and dried with MgSO4 and concentrated. Purifica-
tion of the residue by flash column chromatography (3% MeOH
in CH2Cl2 as eluent) provided a white solid (13, 0.408 g, 64%). 1H
NMR (CDCl3) δ 0.83-1.00 (2H, m), 1.06 (3H, d, J ) 6.0 Hz),
1.13-1.20 (3H, m), 1.25 (9H, s), 1.34 (1H, m), 1.47-1.90 (9H,
m), 2.15 (1H, m), 2.37 (2H, m), 3.27-3.30 (2H, m), 3.70 (3H, s),
4.15-4.17 (2H, m), 4.40 (1H, dd, J ) 13.5 Hz, 8.1 Hz), 4.51 (1H,
m), 5.06 (1H, AB quartet, J ) 12.3 Hz), 5.12 (1H, AB quartet, J )
12.3 Hz), 5.87 (2H, m), 7.28-7.35 (5H, m), 7.41 (1H, d, J ) 7.8
Hz), 7.58 (1H, d, J ) 6.9 Hz); 13C NMR (75 MHz, CDCl3) δ 17.40,
26.21, 26.36, 26.54, 28.25, 28.39, 32.88, 33.23, 33.72, 34.20, 38.34,
40.28, 40.63, 51.18, 51.53, 52.49, 59.08, 66.94, 67.11, 75.52,
4-[2-(2-Benzyloxycarbonylamino-3-methyl-butyrylamino)-3-
phenyl-propionylamino]-5-(2-oxo-pyrrolidin-3-yl)-pent-2-eno-
ic Acid Ethyl Ester (2). Compound 2 was prepared by using a

4978 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16
Yang et al.
procedure similar to that used for compound 1 by replacing
5-methyl-isoxazole-3-carbonyl chloride with benzyl chloroformate
and (S)-2-amino-3-(4-fluoro-phenyl)-propionic acid with (S)-2-
amino-3-phenyl-propionic acid; mp 126-132 °C. 1H NMR (CDCl3)
δ 0.83 (3H, d, J ) 6.6 Hz), 0.88 (3H, d, J ) 6.9 Hz), 1.27 (3H, t,
J ) 7.2 Hz), 1.46-1.54 (1H, m), 1.65-1.78 (1H, m), 1.83-1.94
(1H, m), 1.99-2.01 (1H, m), 2.12-2.22 (1H, m), 2.26-2.30 (1H,
m), 2.80 (1H, b), 3.05 (2H, d, J ) 6.3 Hz), 3.21-3.30 (2H, m),
3.95 (1H, t, J ) 7.2 Hz), 4.16 (2H, q, J ) 6.9 Hz), 4.53 (1H, m),
5.08 (2H, AB quartet, J ) 12.3 Hz), 5.45 (1H, d, J ) 8.4 Hz), 5.72
(1H, d, J ) 15.3 Hz), 6.54 (1H, b), 6.70 (1H, dd, J ) 16.5 Hz, 5.4
Hz), 7.15-7.34 (10H, m), 7.57 (1H, d, J ) 7.5 Hz); 13C NMR (75
MHz, CDCl3) δ14.45, 18.03, 19.38, 28.41, 31.04, 35.28, 38.53,
38.63, 41.25, 49.05, 54.61, 60.68, 61.00, 121.52, 127.121, 128.24,
128.43, 128.72, 128.78, 129.55, 129.72, 136.42, 136.55, 146.89,
156.81, 166.39, 171.09, 171.49, 180.76; ESI-MS (m/z): 607 (M +
H)+; HRMS (EI) m/z: C33H42N4O7 (M), calcd. 606.3048; found
606.3070.
4-[2-(2-Benzyloxycarbonylamino-3-methyl-butyrylamino)-4-
methyl-pentanoylamino]-5-(2-oxo-pyrrolidin-3-yl)-pent-2-eno-
ic Acid Ethyl Ester (3). A procedure similar to that used for
compound 2 was used to prepare this compound (3) by replacing
(S)-2-amino-3-phenyl-propionic acid with (S)-2-amino-4-methyl-
pentanoic acid; mp 156-163 °C. 1H NMR(CDCl3) δ 0.838 (12H,
m), 1.24 (3H, t, J ) 7.2 Hz), 1.46-1.59 (4H, m), 1.70-1.76 (1H,
m), 1.99-2.10 (2H, m), 2.31-2.37 (2H, m), 3.28-3.31 (2H, m),
3.97 (1H, t, J ) 6.9 Hz), 4.14 (2H, q, J ) 7.2 Hz), 4.59 (2H, m),
5.06 (2H, s), 5.54-5.57 (1H, m), 5.90 (1H, d, J ) 15.6 Hz), 6.81
(1H, dd, J ) 15.3 Hz, 4.8 Hz), 7.31 (5H, b), 7.80 (1H, d, J ) 7.8
Hz); 13C NMR (75 MHz, CDCl3) δ14.42, 18.10, 19.42, 22.09,
23.15, 25.09, 28.28, 31.16, 35.48, 38.70, 41.20, 41.92, 48.79, 52.26,
60.71, 60.94, 67.35, 121.55, 128.20, 128.42, 128.77, 136.40, 147.22,
156.84, 166.42, 171.64, 172.62, 180.79; ESI-MS (m/z): 573 (M +
H)+; HRMS (EI) m/z: C30H44N4O7 (M), calcd. 572.3205; found
572.3221.
Tris-HCl at pH 8.0, 1 mM EDTA, and 1 M NaCl). The flow-through
fractions containing 3CLpro were pooled and concentrated for
growing crystals.
Crystallization of SARS 3CLpro-Inhibitor Complexes. The
3CLpro protein was stored in a buffer containing 10 mM Tris-HCl
(pH 7.5), 1 mM dithiothreitol (DTT), and 1 mM EDTA and
concentrated to 10 mg/mL. The protein was mixed with the inhibitor
TG-0205221 (15) dissolved in dimethyl sulfoxide (DMSO) in a
1:10 molar ratio and preincubated for at least 15 min prior to
crystallization. The complex was cocrystallized by the sitting drop
diffusion method at 18 °C for 4 days. The best crystals were
obtained by mixing 2 µL of 3CLpro protein-inhibitor solution with
2 µL of the reservoir solution (0.1 M MES at pH 6.5 and 4∼8%
(v/v) DMSO or MPD, 1 mM DTT and 3∼8% (v/v) PEG6000) onto
a sitting drop post, equilibrated with 1 mL of the reservoir solution.
X-ray Crystallography Data Collection and Processing. The
crystals used for data collection were rinsed with the reservoir
solution and cryo-cooled in liquid nitrogen. The X-ray data was
collected by an RU300 X-ray generator with a Rigaku R-Axis IV++
image plate system. Data integration and scaling were performed
by using the programs HKL2000.18 Data were reduced in the C2
space group. The unit cell dimensions are a ) 109.0 Å, b ) 81.2
Å, c ) 53.3 Å, and â ) 104.7°. There is one monomer in an
asymmetric unit. The data collection statistics are summarized in
Table S2.
Structure Solution and Refinement. The structures of the
enzyme-inhibitor complex were determined by the molecular
replacement method using the native structure taken from the
Protein Data Bank (pdb code 1Z1I) as the starting model. Cross-
rotation function and translation function searches were performed
with the program CCP4.19 The geometric adjustments were made
with XtalView20 under the guidance of (2Fo-Fc) sum difference
maps. The crystallography and NMR System (CNS) program21 was
used for structure refinement, including simulated annealing
procedure, positional, and B-factor refinements.
4-[2-(2-Benzyloxycarbonylamino-3-tert-butoxy-butyrylamino)-
4-methyl-pentanoylamino]-5-(2-oxo-pyrrolidin-3-yl)-pent-2-eno-
ic Acid Ethyl Ester (4). Replacing (S)-2-amino-3-methyl-butyric
acid with (S)-2-amino-3-tert-butoxy-butyric acid and following the
Graphics. The figures were prepared with PyMOL (DeLano
Scientific; http://pymol. sourceforge.net/), ChemDraw, and
LIGPLOT.
Coordinates. Coordinates and structure factors for the structure
of the SARS 3CLpro-TG-0205221 have been deposited in the
Protein Data Bank under the pdb ID 2GX4.
1
procedure used for 3 afforded compound 4; mp 63-65 °C. H
NMR(CDCl3) δ 0.90-0.95 (6H, m), 1.02-1.04 (3H, m), 1.22-
1.26 (12H, m), 1.50-1.66 (4H, m), 1.70-2.07 (2H, m), 2.31-
2.44 (3H, m), 3.25-3.31 (2H, m), 5.03-5.13 (4H, m), 4.46-4.47
(1H, m), 4.57-4.58 (1H, m), 5.08 (2H, m), 5.90 (2H, d, J ) 15.6
Hz), 6.36 (1H, m), 6.81 (1H, dd, J ) 15.3 Hz, 5.1 Hz), 7.29-7.40
(6H, m), 7.66 (1H, d, J ) 6.6 Hz); 13C NMR (75 MHz, CDCl3)
δ14.43, 17.67, 22.19, 23.18, 25.15, 28.46, 35.45, 38.61, 41.42,
41.63, 48.90, 52.53, 59.29, 60.71, 67.01, 67.27, 75.67, 76.81, 77.44,
121.67, 128.34, 128.48, 128.80, 136.37, 147.21, 156.44, 166.41,
170.06, 172.25, 180.91; ESI-MS (m/z) 631 (M + H)+; HRMS (EI)
m/z: C33H50N4O8 (M), calcd. 630.3623; found 630.3596.
3CL Protease Enzyme Assays. Fusion protein in which the
SARS 3C-like protease or 229E 3C-like protease has been fused
to the E. coli glutathione-S-transferase (GST) was expressed in E.
coli BL21(DE3)pLysS cells (Novagen). The fusion protein GST-
3CLpro was purified by Glutathione Sepharose 4 Fast Flow
(Pharmacia) affinity chromatography and cleaved with factor Xa
to release 3CLpro. After removing the cleaved GST with glutathione
sepharose, the recombinant protease was concentrated to 25 µM.
The enzymatic activity of SARS 3CLpro (75 nM) and 229E 3CLpro
(75 nM) was determined by incubation with 15 µM substrate peptide
(SITSAVLQSGFRKMA for SARS 3CLpro and VSYGSTLQAGL-
RKMA for 229E 3CLpro, respectively) at 25 °C in 20 mM Tris-
HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 1 mM dithiothretol,
and 1 mg/mL of bovine serum albumin for 30 min. The reaction
was terminated by adding equal volume of 0.2% trifluoroacetic acid,
and the reaction mixture was analyzed with reverse-phase HPLC
using a C18 column. Cleavage products were resolved using a
5-95% linear gradient of acetonitrile in 0.9% trifluoroacetic acid.
Quantification of peak areas was used to determine the extent of
substrate conversion. To determine the inhibitory effect of various
compounds, the compound and the enzyme were preincubated at
25 °C for 20 min prior to the addition of the substrate. Ki was
calculated using the equation of Cheng and Prusoff.22
Plasma Stability of TG-0205221. Mouse, rat, or human plasma/
well (90 µL) was transferred to a 96 deep well plate and
preincubated at 37 °C for 5 min., followed by the addition of 10
µL (100 µM in 1% DMSO) of test compound to make a 90%
plasma solution at different time points of 0, 30, and 120 min,
respectively. The plasma sample was shaken at 400 rpm at 37 °C
for the different reaction times. The sample was then added to 900
µL/well acetonitrile-containing internal standard and shaken at 400
rpm for 5 min. The 96-well plate sample was centrifuged at 3500
rpm at 10 °C for 10 min and 20 µL of clear supernatant was
transferred 180 µL of H2O for LC/MS analysis. The sample was
diluted to 0.05 µM, and 50 µL was injected into a Waters Alliance
2795 LC/MS system with a C-18 column by using 5-95% MeOH/
H2O + 0.1% formic acid as the mobile phase for analysis.
Cloning, Expression, and Purification of SARS 3CL Protease.
SARS-CoV 3CLpro was cloned into pGEX-6p-1 plasmid DNA
(Pharmacia) with a Factor Xa cutting site at the upstream of our
target gene. GST-tagged protein was purified using a GST column,
and after tag cleavage, the mixture was loaded onto a HiTrap 16/
10 QFF column (Pharmacia) and washed with the buffer (20 mM
Human Coronavirus HCoV 229E Viral Load Reduction
Assay. Human coronavirus 229E was obtained from American Type
Culture Collection (ATCC, VR-740) and cell-free viral stocks were
prepared by infecting the human lung fibroblast cell line, MRC-5
(ATCC CCL-171), with HCoV 229E for 4 days. The aliquots of
cell-free viral stock were stored at -80 °C. For viral load reduction
assay, the MRC-5 cells were preabsorbed with HCoV 229E (40

Potent SARS 3CL Protease Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4979
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J.-Y.; Cox, N.; Hughes, J. M.; LeDuc, J. W.; Bellini, W. J.; Anderson,
L. J.; and the SARS Working Group. A novel coronavirus associated
with severe acute respiratory syndrome. N. Engl. J. Med. 2003, 348,
1953-1966.
pfu/well) for 1 h. Serially diluted compounds were added to the
culture following the removal of the unabsorbed virus. Cell culture
supernatants were harvested after 96 h, and TCID50 for individual
samples were determined using MRC-5 cells.
Human Coronavirus HCoV 229E Plaque Reduction Assay.
MRC-5 cells were grown to 90% confluence in a 6-well plate and
then incubated with serially diluted HCoV 229E for 1 h. Cultures
were overlayed with 0.3% agarose after the removal of the
unabsorbed virus. Test compounds were added to the culture
medium for 4 days. At the end of incubation, the cells were fixed
with 10% formalin and stained with 0.5% crystal violet after the
removal of agarose.
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SARS Antiviral Activity Measurements. The initial screening
for the compounds was carried out in 96-well microtiter plates.
Two concentrations of individual compounds, for example, 20 and
10 mM, were tested in duplicated wells against SARS-CoV-induced
CPE. We also included duplicate wells for treatment with com-
pounds alone (w/o virus) to exclude any potential CPE of Vero E6
cells as a result of chemical toxicity. For each microtiter plate,
uninfected and SARS-CoV-infected cells in quadruplicate were
included as negative and positive controls, respectively. Thus, for
a 96-well plate, we could test 11 different compounds. The
maximum CPE (e.g., cellular round up and detachment of mono-
layers) and viral yields in the viral infected controls were
consistently detected within 2 days post infection. Day 1: (a)
Established permissive Vero E6 cells confluent monolayers with
DMEM/10% FCS medium (D-10) in 96-well flat-bottomed micro-
titer plates by seeding 2 × 104 cells per well in 100 mL of D-10
medium. It usually takes 16-18 h to reach greater than 90%
confluence. (b) Made dilutions of compounds in D-10 medium.
Two concentrations, 80 and 40 mM, were prepared for individual
compounds. Day 2: (Most of the manipulation of the procedures
were done in the BSL-3 laboratory.) (a) Added 50 mL of D-10
medium or diluted compounds into designated wells for individual
compounds, cell alone and virus-infected controls. This was done
in the BSL-2 laboratory. (b) Made infectious SARS-CoV stock at
2 × 103 TCID50/mL in D-10 medium. The original stock is 1 ×
107 TCID50/mL. (c) Added 50 mL of D-10 medium (for negative
or compound alone controls) or diluted viral stock (for positive
controls and test wells; 100 TCID50 particles/well or MOI is 0.001-
0.002). Incubated the sealed microtiter plates at 37 °C. The final
concentrations of each compound will be 20 and 10 mM. (d)
Observed and scored the CPE daily. According to our experience,
SARS-CoV-induced CPE in Vero E6 cells usually appears within
2 days even at an extremely low MOI. Thus, we terminated the
experiments between day 5 and 6 after infection.
Supporting Information Available: HPLC and 1H NMR data
of major compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
Acknowledgment. This work was partially supported by
grants SDRU01 and 93SDRU17 to TaiGen Biotechnology from
the National Science Council, Taiwan and by grants to A.H.-
J.W. from Academia Sinica and the National Science Council
(NSC-93-3112-B-001-011-Y).
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