Design, synthesis and crystallographic analysis of nitrile-based broad-spectrum peptidomimetic inhibitors for coronavirus 3C-like proteases

Article abstract of DOI:10.1016/j.ejmech.2012.10.053

Coronaviral infection is associated with up to 5% of respiratory tract diseases. The 3C-like protease (3CL^pro) of coronaviruses is required for proteolytic processing of polyproteins and viral replication, and is a promising target for the deve

Full text of DOI:10.1016/j.ejmech.2012.10.053

European Journal of Medicinal Chemistry 59 (2013) 1e6
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design, synthesis and crystallographic analysis of nitrile-based broad-spectrum
peptidomimetic inhibitors for coronavirus 3C-like proteases
,
Chi-Pang Chuck a, Chao Chen b, Zhihai Ke b, David Chi-Cheong Wan c, Hak-Fun Chow b, Kam-Bo Wong a
*
a School of Life Sciences and Center for Protein Science and Crystallography, The Chinese University of Hong Kong, Shatin, Hong Kong
b Department of Chemistry, State Key Laboratory of Synthetic Chemistry, and Center of Novel Functional Molecules, The Chinese University of Hong Kong, Shatin, Hong Kong
c School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong
a r t i c l e i n f o
a b s t r a c t
Article history:
Coronaviral infection is associated with up to 5% of respiratory tract diseases. The 3C-like protease
(3CLpro) of coronaviruses is required for proteolytic processing of polyproteins and viral replication, and
is a promising target for the development of drugs against coronaviral infection. We designed and
synthesized four nitrile-based peptidomimetic inhibitors with different N-terminal protective groups
and different peptide length, and examined their inhibitory effect on the in-vitro enzymatic activity of
3CLpro of severe-acute-respiratory-syndrome-coronavirus. The IC50 values of the inhibitors were in the
Received 19 September 2012
Received in revised form
29 October 2012
Accepted 31 October 2012
Available online 7 November 2012
range of 4.6e49 m
M, demonstrating that the nitrile warhead can effectively inactivate the 3CLpro auto-
Keywords:
cleavage process. The best inhibitor, Cbz-AVLQ-CN with an N-terminal carbobenzyloxy group, was w10x
more potent than the other inhibitors tested. Crystal structures of the enzymeeinhibitor complexes
showed that the nitrile warhead inhibits 3CLpro by forming a covalent bond with the catalytic Cys145
residue, while the AVLQ peptide forms a number of favourable interactions with the S1eS4 substrate-
binding pockets. We have further showed that the peptidomimetic inhibitor, Cbz-AVLQ-CN, has broad-
spectrum inhibition against 3CLpro from human coronavirus strains 229E, NL63, OC43, HKU1, and
Peptidomimetics
Coronavirus
X-ray crystallography
Protease
infectious bronchitis virus, with IC50 values ranging from 1.3 to 3.7 mM, but no detectable inhibition
against caspase-3. In summary, we have shown that the nitrile-based peptidomimetic inhibitors are
effective against 3CLpro, and they inhibit 3CLpro from a broad range of coronaviruses. Our results provide
further insights into the future design of drugs that could serve as a first line defence against coronaviral
infection.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
(3CLpro) [5,6]. Inhibition of the proteolytic processing can reduce
viral replication and viral-induced cytotoxic effect [5,6]. The ability
Coronaviruses (CoVs) cause a number of respiratory tract and
gastroenteritis diseases in human and animals [1]. Coronaviruses
can be classified into three main groups: alphacoronavirus (group
1), betacoronavirus (group 2), and gammacoronavirus (group 3),
while the group 2 is further sub-divided into group 2a and 2b [2]. In
addition to the SARS-CoV (group 2b) that caused the outbreak of
pneumonia in 2003, the four human group 1 (229E and NL63) and 2
(OC43 and HKU1) strains are associated with up to 5% of respiratory
tract disease cases reported [3,4]. The single-stranded RNA genome
of CoVs encodes two polyproteins, which are processed into at least
15 mature proteins by papain-like protease and 3C-like protease
of coronaviruses to generate novel strains with high virulence
through high frequency of mutations and recombination remains
a potential threat to human health. Therefore, it is desirable to
develop a broad-spectrum drug that can combat against all kinds of
coronavirus infection. Because 3CLpro is essential to viral replication
and its active-site structure is conserved, the protease is an
attractive drug target for the development of such a broad-
spectrum inhibitor [7]. The SARS-CoV 3CLpro can be inactivated by
peptidomimetic inhibitors that are composed of a substrate-like
peptide and a reactive warhead such as an aldehyde [8,9],
a Michael acceptor [7,10,11], a halo-methyl ketone [10,12], or an
epoxide [13]. The peptide interacts with the substrate-binding cleft
of the protease to form a non-covalent complex, and then the
warhead covalently links with the catalytic cysteine residue and
inhibits the enzyme.
* Corresponding author. School of Life Sciences and Center for Protein Science
and Crystallography, The Chinese University of Hong Kong, Rm 289 Science Centre,
Shatin, Hong Kong. Tel.: þ852 3943 8024; fax: þ852 2603 7246.
E-mail address: kbwong@cuhk.edu.hk (K.-B. Wong).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.053

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C.-P. Chuck et al. / European Journal of Medicinal Chemistry 59 (2013) 1e6
In this study, we have shown that the nitrile group is an
effective warhead against 3CLpro. Here, based on the auto-cleavage
sequence of SARS-CoV 3CLpro (TSAVLQY), four nitrile-based
inhibitors with different length of peptide sequences and
protective groups were designed and synthesized. We have shown
that these inhibitors were effective against 3CLpro from six
different strains of CoVs with IC50 values in the
mM range. The
crystal structures of the four enzymeeinhibitor complexes were
determined to reveal the mechanism of inhibition and the
enzymeeinhibitor interactions.
2. Results and discussion
2.1. SARS-CoV 3CLpro is inhibited by nitrile-based peptidomimetic
inhibitors
Based on the autocleavage tetrapeptide sequence (AVLQ) of
SARS-CoV 3CLpro, three nitrile-based inhibitors with different
protective groups, 5-methylisoxazole-3-carboxyl (Mic), tert-buty-
loxycarbonyl (Boc) and carboxybenzyl (Cbz), were synthesized
(Scheme 1). The protease activity in the presence of inhibitors was
measured by using a fluorescent-protein substrate as described
previously [14,15]. The IC50 values of ‘Mic-AVLQ-CN’, ‘Boc-AVLQ-
CN’, and ‘Cbz-AVLQ-CN’ were 49 ꢀ 2, 49 ꢀ 2, and 4.6 ꢀ 0.2
mM,
respectively (Fig. 1). Interestingly, the inhibitor with the Cbz
protective group was w10x more potent than the other inhibitors
tested. To test the effect of increasing the length of the peptido-
mimetic inhibitor, we synthesized a hexapeptide inhibitor ‘Cbz-
TSAVLQ-CN’. The IC50 value for this inhibitor was 39 ꢀ 1
mM (Fig. 1),
suggesting that increasing the length of the inhibitor did not
improve inhibition and the Cbz group at the P4 position may be
responsible for the higher potency of ‘Cbz-AVLQ-CN’.
Fig. 1. IC50 values of the nitrile-based inhibitors against SARS-CoV 3CLpro
. (A) A
schematic diagram shows chemical structures and IC50 values of the inhibitors. (B)
SARS-CoV 3CLpro was pre-mixed with ‘Miu-AVLQ-CN’ (circle), ‘Boc-AVLQ-CN’ (triangle),
‘Cbz-AVLQ-CN’ (square) and ‘Cbz-TSAVLQ-CN’ (diamond) at 0e256
mM, followed by
measuring the protease activity to determine the inhibitory effect. The IC50 values were
determined by fitting the curve to a four-parameter logistics equation.
2.2. Crystal structures of enzymeeinhibitor complexes
To better understand the enzymeeinhibitor interaction, we
determined the crystal structures of SARS-CoV 3CLpro in complex
ꢀ
with the four inhibitors at 1.95e2.5 A resolution (Table S1,
Supplementary data). The 3CLpro formed a dimer in the crystal
structure, and the inhibitors occupied the substrate binding site in
both protomers. Electron density was observed for the tetrapeptide
(AlaeValeLeueGln) at P1 to P4 positions for all inhibitor complexes
(Fig. S1, Supplementary material). Overlaying these inhibitor
structures demonstrated that the tetrapeptides adopted the same
conformation when bound to the 3CLpro. On the other hand, the
protective group of ‘Mic-AVLQ-CN’, the P5-Ser and P6-Thr of ‘Cbz-
TSAVLQ-CN’ were disordered and not observable in the electron
density.
Scheme 1. Reaction scheme of nitrile-based peptidomimetic inhibitor synthesis. Step
(a) was the coupling of peptides by using N,N0-dicyclohexylcarbodiimide and N-
hydroxysuccinimide. Step (b) was the coupling of peptides by the mixed anhydride
method. Step (c) was the deprotection of benzyl carbamates by using palladium on
charcoal. Step (d) was the deprotection of Boc, Trt, Pbf groups and tertiary butyl esters by
using trifluoroacetic acid. Step (e) was the transformation of primary amide to nitrile by
using trifluoroacetic anhydride. Final products were highlighted with dashed box.

C.-P. Chuck et al. / European Journal of Medicinal Chemistry 59 (2013) 1e6
3
In SARS-CoV, the catalytic dyad consists of Cys145 and His41
2.3. Nitrile-based peptidomimetic inhibitor exhibits broad-
spectrum inhibition against 3CLpro from different groups of
coronaviruses
[16]. The inhibitor was covalently bonded with the thiol group of
Cys145 via the carbon atom of the nitrile warhead (Fig. 2A). This
mechanism is consistent with that observed in other cysteine
proteases [17]. The side chains of P1-Gln, P2-Leu and P4-Ala
occupied the S1, S2 and S4 subsites, respectively, of SARS-CoV
Next, we tested if the ‘Cbz-AVLQ-CN’ inhibitor has broad-
spectrum inhibition against 3CLpro from different groups of coro-
naviruses. We measured and compared the IC50 values of ‘Cbz-
AVLQ-CN’ against 3CLpro from human coronavirus (HCoV) strains
229E (group 1), NL63 (group 1), OC43 (group 2a), HKU1 (group 2a),
SARS-CoV (group 2b), and infectious bronchitis virus (IBV) (group
3). Our results showed that ‘Cbz-AVLQ-CN’ can inhibit all 3CLpro
3CLpro
, while the side-chain of P3-Val pointed towards the
solvent (Fig. 2A). A number of hydrogen bonds are involved in
enzymeeinhibitor interactions. The P1-Gln side chain was
hydrogen-bonded with the backbone O atom of Phe140 and the
side-chain of His163 and Glu166. The backbone amide groups of P2
to P4 formed hydrogen bonds with Glu166, Gln189 and Thr190, and
tested, with values of IC50 ranging from 1.3 to 4.6
contrast, the ‘Cbz-AVLQ-CN’ had no observable inhibition effect on
caspase-3 at 0.5e256 M (Fig. S2, Supplementary material), sug-
mM (Fig. 3). In
these hydrogen bonds help the P3 and P4 residues to adopt a
strand like conformation (Fig. 2B).
b-
m
Noteworthy, the aromatic ring of the Cbz group is docked into
a pocket where the ring can form favourable hydrophobic inter-
actions with Pro168 and P3-Val (Fig. 2C). These extra interactions,
which are absent in other enzymeeinhibitor complexes, may
enhance the binding affinity between 3CLpro and increase the
potency of the ‘Cbz-AVLQ-CN’ inhibitor.
gesting that the inhibitor is specific for 3CLpro from coronaviruses.
As structures of 3CLpro from group 1, 2a, 2b, and 3 are similar, our
modelling study suggests that the ‘Cbz-AVLQ-CN’ inhibitor can fit
nicely to the active site of 3CLpro from all groups of coronaviruses
and inhibit the protease via the same mechanism (Fig. S3,
Supplementary material).
Fig. 2. Crystal structure of 3CLpro-inhibitor complex. (A) The 3CLpro was shown in surface representation, while the inhibitor was shown in stick representation. The side-chains of
P1-Gln, P2-Leu and P4-Ala occupy the S1, S2, S4 subsites of 3CLpro, while the side-chain of P3-Val points towards the solvent. (B) The hydrogen bonds (dotted lines) between the
inhibitor (orange) and the 3CLpro (grey) were shown. (C) The Cbz protective group of the ‘Cbz-AVLQ-CN’ docks into a pocket formed by Glu166, Leu167, and Pro168 of SARS-CoV
3CLpro. Surface was shown for atoms of 3CLpro and P1-P4 residues of the inhibitors. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

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C.-P. Chuck et al. / European Journal of Medicinal Chemistry 59 (2013) 1e6
Fig. 3. Broad-spectrum inhibition of ‘Cbz-AVLQ-CN’ against 3CLpro from (A) HCoV-NL63, (B) HCoV-299E, (C), HCoV-OC43, (D) HCoV-HKU1, (E) SARS-CoV, and (F) IBV. The proteases
were pre-mixed with ‘Cbz-AVLQ-CN’ at 0e32
m
M. The IC50 values were determined by fitting the curve to a four-parameter logistics equation.
3. Conclusion
4. Experimental section
In this study, we have shown that nitrile-based peptidomi-
metic inhibitors display broad-spectrum inhibition against 3CLpro
from all groups of coronaviruses. Crystal structures of enzymee
inhibitor complexes provide interesting insights in the design of
peptidomimetic inhibitors. First, the enzymeeinhibitor binding is
mainly determined by the tetrapeptide sequence, AVLQ. This
suggestion is consistent with the observation that extending the
length of the inhibitor to hexapeptide (‘Cbz-TSAVLQ-CN’) did not
improve inhibition (Fig. 1). Second, we found that the Cbz
protective group at the N-terminus of the tetrapeptide can result
in w10 fold increases in potency in the ‘Cbz-AVLQ-CN’ inhibitor
than the other inhibitors tested. This observation suggests that
modification of the peptidomimetics, e.g. cross-linking the P3-Val
with the Cbz groups, may further improve the efficacy of inhibi-
tion. Because of the high recombination frequency among the RNA
genome of CoVs, development of a broad-spectrum inhibitor is an
attractive strategy to combat against infection of all kinds of CoVs
[7]. Here, we have shown that the nitrile-based inhibitor ‘Cbz-
AVLQ-CN’ can inhibit 3CLpro from all groups of CoVs tested with
4.1. Inhibitor synthesis
The reaction scheme for inhibitor synthesis is summarized in
Scheme 1. 5-Methylisoxazole-3-carboxylic acid (Mic-OH), N,N0-
dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (HOSu)
were purchased from SigmaeAldrich. Identities of the final prod-
ucts were verified by 1H nuclear magnetic resonance and mass
spectrometry analysis.
4.1.1. Peptide coupling using N,N0-dicyclohexylcarbodiimide (DCC)
and N-hydroxysuccinimide (HOSu)
In part 1, to a stirred suspension of N-protected-amino acid-OH
or N-protected-peptide-OH (1.0 equiv) and HOSu (1.1 equiv) in
anhydrous tetrahydrofuran (THF) (20 mL/mmol), DCC (1.1 equiv) in
THF was added to the solution at 0 ꢁC. The reaction mixture was
stirred for 30 min at the same temperature and 6 h at 25 ꢁC. N,N0-
dicyclohexylurea was then removed by filtration and the filtrate
was collected. In part 2, the amino acid was dissolved in THF/H2O
(v/v ¼ 1:1), then the pH of the solution was adjusted to 9 with 2 M
K2CO3. The filtrate from part 1 was added to the solution at 0 ꢁC.
This mixture was stirred for 30 min at 0 ꢁC and overnight at 25 ꢁC.
Solvents were then evaporated in vacuo and the pH of the residue
was adjusted to acidic with citric acid at 0 ꢁC and extracted with
ethyl acetate (EtOAc). The combined extracts were washed with
brine, dried (MgSO4), filtered and evaporated in vacuo. The residue
IC50 values in the mM range. As nitrile-based protease inhibitors
(e.g. odanacatib [18], vildagliptin [19]) have been successful in
clinical applications [20], our work demonstrated that the inhib-
itors described in this study may serve as a lead for the
developmentof antiviral drugs that target a broad-spectrum of
coronaviruses.

C.-P. Chuck et al. / European Journal of Medicinal Chemistry 59 (2013) 1e6
5
was purified by flash chromatography on silica gel to give the
desired compound.
was purified by flash chromatography on silica gel to afford the
target compound (yield ¼ 67%). Rf: 0.1 (EtOAc).
1H NMR (300 MHz, d6-DMSO)
d (ppm): 0.68e0.96 (12H, m,
CH(CH3)2), 1.15 (3H, d, J ¼ 6.9 Hz, CH3), 1.37 (9H, s, C(CH3)3), 1.41e
1.52 (2H, m, CH2), 1.52e1.66 (1H, m, CH), 1.67e1.87 (1H, m, CH),
1.87e2.09 (2H, m, CH2), 2.33e2.49 (2H, m, CH2CO), 3.86e4.04 (1H,
m, NHCH), 4.06e4.33 (3H, m, NHCH), 7.09 (1H, d, J ¼ 7.5 Hz, NH),
7.17 (1H, s, NH), 7.32 (1H, s, NH), 7.60 (1H, d, J ¼ 8.7 Hz, NH), 7.93
(1H, d, J ¼ 8.1 Hz, NH). 8.06 (1H, d, J ¼ 7.8 Hz, NH). ESI-HRMS (m/z):
calculated for C24H42N6O6 þ Naþ: 533.3058, found: 533.2850.
4.1.2. Peptide coupling using mixed anhydride method
N-protected-amino acid-OH or N-protected-peptide-OH
(1 equiv) was dissolved in THF and cooled to ꢂ15 ꢁC. To the stirred
solution, N-methylmorpholine (NMM) (1 equiv) and isobutyl-
chloroformate (1 equiv) were added consecutively. After precipi-
tation of N-methylmorpholine hydrochloride, the amine (1 equiv)
was added and the mixture was allowed to warm to 25 ꢁC within
30 min. It was stirred for additional 90 min, and the solvent was
removed under reduced pressure. The residue was dissolved in
EtOAc and the solution was washed with H2O, saturated NaHCO3,
5% citric acid and brine. The solvent was dried (MgSO4) and evap-
orated. The crude residue was purified by flash column chroma-
tography to give the product.
4.1.6.3. Cbz-AVLQ-CN. After this compound was synthesized by
following the procedure as described in Section 4.1.2, the product
was purified by flash chromatography on silica gel to afford the
target compound (yield ¼ 73%). Rf: 0.65 (CH2Cl2/MeOH ¼ 1/4).
1H NMR (300 MHz, d6-DMSO)
d (ppm): 0.62e1.00 (12H, m,
CH(CH3)2), 1.18 (3H, d, J ¼ 6.9 Hz, CHCH3), 1.20e1.30 (1H, m, CH2),
1.32e1.52 (2H, m, CH2), 1.52e1.66 (1H, m, CH), 1.66e1.86 (1H, m,
CH(CH3)2), 1.86e2.14 (2H, m, CH2CONH2), 2.30e2.46 (1H, m,
CH(CH3)2), 4.00e4.18 (2H, m, NHCH), 4.18e4.36 (2H, m, NHCH),
5.01 (2H, s, ArCH2), 7.18 (1H, s, NH), 7.20e7.44 (6H, ArH, NH) 7.53
(1H, d, J ¼ 7.5 Hz, NH), 7.77 (1H, d, J ¼ 8.7 Hz, NH), 7.94 (1H, d,
J ¼ 8.1 Hz, NH). 8.05 (1H, d, J ¼ 7.5 Hz, NH). ESI-HRMS (m/z):
calculated for C27H40N6O6 þ Naþ: 567.2907, found: 567.2902.
4.1.3. Deprotection of benzyl carbamates using palladium on
charcoal (Pd/C)
To a stirred solution of the benzyl carbamate (1.0 equiv) in
MeOH, palladium on charcoal (benzyl carbamate/palladium on
charcoal ¼ 1 g/100 mg) was added. The solution was evacuated,
placed under H2 (1 atm), and stirred for 1 h. The reaction was
monitored by thin layer chromatography (TLC). The suspension was
then filtered through Celite and washed with MeOH. The filtrate
was concentrated under reduced pressure to afford the desired
amine.
4.1.6.4. Cbz-TSAVLQ-CN. After this compound was synthesized by
following the procedure as described in Section 4.1.2., the product
was purified by flash chromatography on silica gel to afford the
target compound (yield ¼ 48%). Rf: 0.75 (MeOH/EtOAc ¼ 3/1).
1H NMR (300 MHz, d6-DMSO)
d (ppm): 0.68e0.96 (12H, m,
4.1.4. Deprotection of Boc, Trt groups and tertiary butyl esters using
trifluoroacetic acid (TFA)
CH(CH3)2), 1.06 (3H, d, J ¼ 6.3 Hz, CHCH3), 1.21e1.32 (4H, m, CH3,
CH), 1.36e1.53 (2H, m, CH2), 1.53e1.68 (1H, m, CHH), 1.68e1.88 (1H,
m, CHH), 1.88e2.12 (2H, m, CH2CONH2), 2.33e2.46 (1H, m, CH),
3.44e3.70 (2H, m, CH2OH), 3.84e4.00 (1H, m, CH3CHOH), 4.00e
4.14 (2H, m, NHCH), 4.14e4.40 (4H, m, NHCH), 4.92e4.94 (1H, m,
OH), 4.96e5.13 (3H, m, ArCH2, OH), 7.02 (1H, d, J ¼ 8.1 Hz, NH), 7.16
(1H, s, NH), 7.26 (1H, s, NH), 7.28e7.44 (5H, m, ArH), 7.73 (1H, d,
J ¼ 8.1 Hz, NH), 7.85 (1H, d, J ¼ 8.1 Hz, NH), 7.89e8.02 (2H, m, NH),
8.13 (1H, d, J ¼ 6.9 Hz, NH). ESI-HRMS (m/z): calculated for
C34H52N8O10 þ Naþ: 755.3698, found: 755.3669.
To a stirred solution of the starting materials (1.0 equiv) in
CH2Cl2, TFA (16.0 equiv) was added. The solution was stirred at
25 ꢁC and monitored by TLC. The CH2Cl2/TFA solvent was removed
under reduced pressure. The residue was diluted with toluene and
the solvent was removed once again under reduced pressure.
4.1.5. Transformation from primary amide to nitrile using
trifluoroacetic anhydride
To a stirred cold solution (0 ꢁC) of the primary amide (1.0 equiv)
in dry THF and Et3N (2.2 equiv), trifluoroacetic anhydride (1.1 equiv)
was added over 5 min. The solution was allowed to warm to 25 ꢁC.
After 30 min, the reaction was quenched with H2O. The THF was
removed under reduced pressure, and the product was extracted
with EtOAc. The combined extracts were washed with saturated
NaHCO3, 5% citric acid and brine, dried (MgSO4), filtered and
evaporated in vacuo. The residue was purified by flash chroma-
tography on silica gel to give the desired compound.
4.2. Protease assay and determination of IC50 values
The detailed procedures for the production of 3CLpro, fluorescent
substrate, and protease assay for 3CLpro were described previously
[14,15]. 3CLpro at 0.5e1
mM were pre-mixed with 0.5e256
mM of
inhibitors for 5 min, followed by addition of 35 M substrate. The
m
decay of fluorescence at 530 nm was fitted to a single-exponential
decay to yield the observed rate constant (kobs) and kobs/[3CLpro].
The values of kobs/[3CLpro] at different concentration of inhibitors
were normalized by those without inhibitors to determine the
relative protease activities, which were fitted to a four-parameter
logistics equation to obtain the IC50 values.
4.1.6. Identification of products by NMR and mass spectrometry
4.1.6.1. Mic-AVLQ-CN. After this compound was synthesized by
following the procedure as described in Section 4.1.2, the product
was purified by flash chromatography on silica gel to afford the
target compound (yield ¼ 64%). Rf: 0.7 (EtOAc/MeOH ¼ 4/1).
1H NMR (300 MHz, d6-DMSO)
d
(ppm): 0.50e1.08 (12H, m,
4.3. Structure determination of protease-inhibitor complexes
CH(CH3)2), 1.30 (3H, d, J ¼ 6.8 Hz, CH3), 1.46e1.49 (2H, m, CH2),
1.52e1.67 (1H, m, CH), 1.74e1.86 (1H, m, CH), 1.88e2.15 (2H, m,
CH2), 2.23e2.42 (2H, m, CH2CONH2), 2.46 (3H, s, CH3C ¼ CH), 3.96e
4.40 (3H, m, NHCH), 4.42e4.65 (1H, m, NHCH), 6.56 (1H, s, C]CH),
7.03 (1H, s, NH), 7.26 (1H, s, NH) 7.94 (2H, d, J ¼ 8.4 Hz, NH), 8.08
(1H, d, J ¼ 7.2 Hz, NH), 8.62 (1H, d, J ¼ 7.2 Hz, NH); ESI-HRMS (m/z):
calculated for C24H37N7O6 þ Naþ: 542.2703, found: 542.2706.
SARS-CoV 3CLpro was crystallized at 16 ꢁC using the hanging-
drop-vapour-diffusion method by mixing 5 mg/mL 3CLpro in 1:1
ratio with 50 mM 2-(N-morpholino)ethanesulfonic acid, pH 5.5,
8.5% (w/v) polyethylene glycol 6000, 10% (v/v) glycerol, 3% (v/v)
DMSO, 1 mM ethylenediaminetetraacetic acid and 1 mM dithio-
threitol. Single crystals were transferred to 5
mL of the mother
liquor with 600
m
M inhibitors. After incubation at 16 ꢁC overnight,
4.1.6.2. Boc-AVLQ-CN. After this compound was synthesized by
following the procedure as described in Section 4.1.2, the product
the crystals were cryoprotected by 20% (v/v) glycerol, and diffrac-
tion data were collected at 110 K in an in-house Rigaku FREþ X-ray

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C.-P. Chuck et al. / European Journal of Medicinal Chemistry 59 (2013) 1e6
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source. The phase was solved by molecular replacement, the
models were built interactively by the program COOT [21] and
refined by PHENIX [22].
Acknowledgements
This work was supported by the Research Fund for the Control of
Infectious Diseases, Hong Kong SAR Government (project number:
06060432 and 09080282).
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
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