Design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors

Article abstract of DOI:10.1021/jm050548m

Design, synthesis, and biological evaluation of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease (SARS-3CLpro) inhibitors for severe acute respiratory syndrome coronavirus (SARS-CoV) are described. These inhibitors exhibited antiviral activity against SARS-CoV in infected cells in the micromolar range. An X-ray crystal structure of our lead inhibitor (4) bound to SARS-3CLpro provided important drug-design templates for the design of small-molecule inhibitors.

Full text of DOI:10.1021/jm050548m

© Copyright 2005 by the American Chemical Society
Volume 48, Number 22
November 3, 2005
Letters
This epidemic affected more than 8000 reported indi-
viduals by July 2003 and resulted in 774 deaths. Thus
far, no effective therapy exists for this virus. During
viral replication, the RNA-dependent RNA polymerase
is extensively processed by two viral proteases, chymo-
trypsin-like protease (3CLpro) and papain-like protease
(PLpro).3 As a consequence, these proteases are recog-
nized as attractive targets for drug development for
SARS and related infections.4
Design and Synthesis of Peptidomimetic
Severe Acute Respiratory Syndrome
Chymotrypsin-like Protease Inhibitors
Arun K. Ghosh,*,§,† Kai Xi,§,† Kiira Ratia,
Bernard D. Santarsiero, Wentao Fu,
Brian H. Harcourt,¶ Paul A. Rota,¶ Susan C. Baker,#
Michael E. Johnson, and Andrew D. Mesecar
Departments of Chemistry and Medicinal Chemistry,
Purdue University, West Lafayette, Indiana 47907,
Department of Chemistry, University of Illinois at Chicago,
Chicago, Illinois 60607, Center for Pharmaceutical
Biotechnology and Department of Medicinal Chemistry and
Pharmacognosy, University of Illinois at Chicago, 900 S.
Ashland, Illinois 60607, Department of Microbiology and
Immunology, Loyola University of Chicago, Stritch School of
Medicine, Maywood, Illinois, and Center for Disease Control
and Prevention, Atlanta, Georgia
The structure and activity of the coronavirus 3CLpro
has already been eludicated.5 It contains a catalytic
dyad in the active site where a cysteine residue acts as
a nucleophile and a histidine residue acts as the general
acid base. The design of inhibitors of 3CLpro as thera-
peutics was particularly interesting because 3CLpro is
functionally analogous to the main picornaviral protease
3Cpro and significant drug-design efforts for human
rhinoviral 3Cpro are already well precedented.6 These
studies have provided important molecular insights that
may aid the design of novel inhibitors of SARS-3CLpro.
Herein, we report our preliminary investigation that led
to the design and synthesis of peptidomimetic inhibitors
of SARS-3CLpro inhibitors. The inhibitors have been
shown to block the replication of SARS-CoV in cell
culture assay. Furthermore, we have determined the
X-ray structure of an inhibitor complexed with SARS-
3Clpro at a resolution of 1.9 Å.
Our initial design of SARS-3CLpro inhibitors was
based on examination of the superimposed X-ray crystal
structure of the related enzyme of porcine transmissible
gastroenteritis (corona)virus (TGEV MPro) and a sub-
strate-analogue hexapeptidyl chloromethyl ketone (CMK)
inhibitor (1) and from structural information derived
from modeled lead inhibitor AG-7088 (2) in the 3CL-
pro active site (Figure 1).5,7 Inhibitor 2 is ineffective
against SARS-CoV in cell culture assay. Antiviral
activity for 2 was reported to be >100 µg/mL.8 It
appeared that the vinylogous group of the ethyl ester
of 2 may interact with residues similar to the chlorom-
ethyl ketone functionality of inhibitor 1. Both inhibitors
bind to their respective targets through covalent bond-
ing with the active site cysteine residue. The S2-subsites
Received June 10, 2005
Abstract: Design, synthesis, and biological evaluation of
peptidomimetic severe acute respiratory syndrome chymo-
trypsin-like protease (SARS-3CLpro) inhibitors for severe
acute respiratory syndrome coronavirus (SARS-CoV) are de-
scribed. These inhibitors exhibited antiviral activity against
SARS-CoV in infected cells in the micromolar range. An X-ray
crystal structure of our lead inhibitor (4) bound to SARS-
3CLpro provided important drug-design templates for the
design of small-molecule inhibitors.
Severe acute respiratory syndrome (SARS) is an acute
respiratory illness caused by a novel human corona-
virus, SARS-CoV.1 Since its first report in Guangdong
Province, China, in November 2002, SARS has spread
to other Asian countries, North America, and Europe.1,2
* To whom correspondence should be addressed. Phone: 765-494-
5323. Fax: 765-496-1612. E-mail: akghosh@purdue.edu.
§ Purdue University.
† Department of Chemistry, University of Illinois at Chicago.
Center for Pharmaceutical Biotechnology and Department of
Medicinal Chemistry and Pharmacognosy, University of Illinois at
Chicago.
¶ Center for Disease Control and Prevention.
# Loyola University of Chicago.
10.1021/jm050548m CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/04/2005

6768 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Letters
Scheme 1
Figure 1. Structures of inhibitors 1 and 2.
Scheme 2
Figure 2. Key fragments for inhibitors 3 and 4.
diethyl ether provided the aldehyde, which was reacted
with lithium propiolate derived from the treatment of
ethyl propiolate and lithium diisopropylamide to furnish
the acetylenic alcohol 10 as an inseparable mixture
(3.4:1) of diastereomers. Catalytic hydrogenation of 10
followed by acid-catalyzed lactonization of the resulting
γ-hydroxy ester with a catalytic amount of acetic acid
in toluene at reflux afforded γ-lactone 11 in 56% yield
after silica gel chromatography. For introduction of the
desired alkyl group at C-2, lactone 11 was treated with
lithium diisopropylamide (2.2 equiv) in THF at -78 °C
(30 min). The resulting enolate was reacted with benzyl
bromide (1.1 equiv) or 4-bromo-2-methyl-2-butene (1.1
equiv) for 30 min at -78 °C to provide the respective
alkylated lactone 12 or 13.9 Lithium hydroxide promoted
hydrolysis of lactones 12 and 13 followed by protection
of the resulting γ-hydroxyl group with tert-butyldi-
methylsilyl chloride in the presence of imidazole in DMF
provided the carboxylic acids 6 and 7 in 64% and 71%
yields, respectively.
The synthesis of P2-lactam ligand 8 is outlined in
Scheme 2. Optically pure glutamic acid was converted
into N-Boc-L-(+)-glutamic acid dimethyl ester 14 as
described previously.10 Diester 14 was converted into
alkylated product 15 by utilizing 1,3-asymmetric induc-
tion by the dianionic alkylation protocol developed by
Hanessian.11 Thus, treatment of 14 with LHMDS (2.2
equiv) at -78 °C provided the enolate, which was
alkylated with bromoacetonitrile (1.1 equiv) to give
alkylated product 15 as a single diastereomer. The
rationale for similar diastereoselectivity was previously
of both enzymes are quite different. The P2-residue of
inhibitor 1 is Leu, and the corresponding P2 side chain
for inhibitor 2 is a p-fluorophenylmethyl group. There-
fore, it appears that the P2-p-fluorophenylmethyl group
in 2 is too large for the S2-pocket.5a Our preliminary
modeling studies indicate that a P2-phenylmethyl or
other designed side chains may result in potent inhibi-
tors for SARS-3CLpro. Since the autocleavage site of
SARS-CoV contains a P2-Phe, incorporation of the P2-
benzyl side chain may be accommodated as well.6 On
the basis of this rationale, we have prepared and
evaluated inhibitors containing phenylmethyl and pre-
nyl side chains as the P2-ligand (Figure 2). These
inhibitors possess a P1/P1′-Michael acceptor R,â-unsat-
urated ester functionality, which is expected to link to
the Cys-145 covalently. In addition to varying the P2-
side chains, we have also investigated a hydroxyethyl-
ene isostere in place of the ketoethylene isostere. Since
covalent bound inhibitors often exhibit toxicity, our
ultimate goal is to design noncovalent and reversible
SARS-3CLpro inhibitors.
The 3CLpro inhibitors depicted in Figure 2 were
synthesized by assembly of key fragments isoxazole
carboxylic acid 5, dipeptide isostere (6 and 7), and
γ-lactam derivative 8. The synthesis of dipeptide iso-
steres for inhibitors 3 and 4 is outlined in Scheme 1.
Commercial Boc-L-valine was converted to Weinreb
amide 9 by treatment with isobutyl chloroformate and
1-methylpiperidine followed by treatment of the result-
ing mixed anhydride with N,O-dimethylhydroxylamine.
Reduction of 9 with lithium aluminum hydride in

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6769
Scheme 3
Figure 3. Time course of inactivation of SARS 3CLpro by 3
(9) and 4 (2) versus a DMSO control (b).
reported by Hanessian.11 The nitrile derivative 15 was
subjected to hydrogenation in the presence of PtO2 and
chloroform.12 The resulting amine salt was treated with
Na2CO3 at reflux for 6 h to afford lactam ester 16.13
Selective reduction of the ester group in the presence
of the lactam was carried out with LiBH4 (1 N solution
in THF) in CH2Cl2 to provide alcohol 17 in 91% yield.
Alcohol 17 was converted into desired lactam fragment
18 by a one-pot oxidation followed by Wittig reaction of
the resulting aldehyde with carbethoxyphosphorane in
DMSO in 90% yield.
The synthesis of inhibitors 3 and 4 is outlined in
Scheme 3. Exposure of 18 to trifluoroacetic acid (20%
in CH2Cl2) effected the removal of the Boc group. To
the resulting TFA salt, Hunig’s base was added to
liberate the free amine. It was coupled with acids 6 and
7 to provide amide derivatives 19 and 20, respectively.
Treatment of these amides with trifluoroacetic acid in
dichloromethane afforded the corresponding amine,
which was coupled with known 5-methylisoxazole-3-
carboxylic acid 514 to furnish silyl derivatives 21 and
22. Removal of silyl group with 48% aqueous hydro-
fluoride acid in tetrahydrofuran and oxidation of the
resulting alcohol with Dess-Martin periodinane fur-
nished inhibitors 3 and 4. Inhibitor 23 with a hydroxy-
ethylene isostere was prepared by deprotection of the
silyl group from 21.
Table 1. Inactivation Rate Constants for SARS-CoV 3CLpro
by Compounds
inhibitor
kinact (min-1
)
3CLpro T1/2 (min)
E64
3
0.0022 ( 0.0013
0.014 ( 0.0017
0.045 ( 0.0095
0.0025 ( 0.00038
315 ( 302
49 ( 6
4
15 ( 3
23
277 ( 43
CoV 3CLpro is shown in Figure 3 for each of these
compounds, and the kinact and T1/2 values resulting from
a fit of the data to a single-exponential equation are
summarized in Table 1. The data indicate that 4 is the
best inhibitor with a T1/2 of ∼15 min. Compound 23,
with a hydroxyethylene isostere, and E64 did not inhibit
the enzyme under the conditions assayed.
The antiviral activity of synthetic inhibitors was also
evaluated in a neutral red assay developed on the basis
of the work of Huggins and co-workers.17 The antiviral
IC50 of 3 and 4 were shown to be approximately 45 and
70 µM, respectively.
The above synthetic inhibitors were evaluated in a
FRET-based microplate assay developed to measure the
activity of SARS-3CLpro.15 The inactivation rate con-
stant kinact and half-life T1/2 of SARS-3CLpro in the
presence of these compounds and in the presence of
E64,16 a standard cysteine protease inhibitor, have been
quantitated. The time course for inactivation of SARS-
No toxicity is observed up to the maximum concentra-
tion of compound tested, which was 100 µM. Significant
antiviral activity of E64d was not observed up to 100
Figure 4. X-ray crystal structure of 4 (thick stick with magenta carbon) with SARS 3CLpro. Hydrogen bonds between inhibitor
and 3Clpro are shown as green dotted lines.

6770 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Letters
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National Academies Press: Washington, DC, 2004; Chapter 4,
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anionic allylation reactions of amino acid derivatives. Synthesis
of functionally useful enantiopure glutamates, pipecolates, and
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µM, but observable toxicity was noted. Since inhibitor
4 with a P2-prenyl group is significantly more potent
than the P2-phenylmethyl containing inhibitor 3 in
vitro, it indicates that other side chain residues could
be accommodated by the S2-subsite of 3CLpro.
To gain molecular insight, we attempted to resolve
the inhibitor-bound crystal structure of SARS-3CLpro.
We successfully crystallized the nontagged SARS-
3CLpro apoenzyme in complex, i.e., covalently modified,
with inhibitor 4. The X-ray structure of SARS-3CLpro
inhibitor complex of 4 was determined to a resolution
of 1.89 Å.18 A stereoview of the inhibitor-bound structure
is shown in Figure 4. Analysis of this structure reveals
why 23 shows very little inhibitory activity of the
enzyme. It indicates that substitution of the carbonyl
oxygen atom with a hydroxyl group disrupts an impor-
tant hydrogen bond between the backbone amide nitro-
gen of Glu166 and the carbonyl oxygen of the inhibitor.
This SARS-3CLpro-bound inhibitor structure will pro-
vide molecular insight and facilitate design of more
effective inhibitors.
In conclusion, our preliminary investigation led to
identification of two peptidomimetic lead inhibitors for
SARS-3CLpro. These inhibitors are not only potent
against SARS-3CLpro but effective in SARS-CoV cell
culture assay. An inhibitor containing a hydroxyethyl-
ene isostere scaffold was not very effective. An X-ray
structure of 4-bound SARS-3CLpro revealed important
molecular insights into the molecular recognition of this
class of compounds by SARS-3CLpro. Further investi-
gations including detailed structure-activity relation-
ships and nonpeptidal inhibitor design from these
preliminary studies are currently underway.
Acknowledgment. This work was supported by the
National Institutes of Health (NIAID), Grant P01
AI060915. The work was also partially supported by
NIGMS (Grant GM 53386).
Supporting Information Available: Experimental meth-
ods for 3-23 and HPLC data for 3, 4, and 23. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
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The reactions were monitored at 25 °C in 96-well microplates
containing 50 mM HEPES (pH 7.5) using a Varian Eclipse
spectrofluorimeter. For time course of inactivation studies, 50
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percent remaining activity as a function of time was plotted, and
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and half-life T. Details of this assay will be published in due
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Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 6771
unit cell parameters of a ) 107.1 Å, b ) 83.2 Å, c ) 53.7 Å, and
Rcryst and Rfree values were 25.9% and 28.4%, respectively. The
â ) 104.4°. The structure was determined by molecular replace-
ment using PDB code 1M4H as a search model. The structure
was built and refined using the programs O and CNS. The final
final model includes the inhibitor covalently linked to Cys145.
JM050548M