α-Ketoamides as Broad-Spectrum Inhibitors of Coronavirus and Enterovirus Replication: Structure-Based Design, Synthesis, and Activity Assessment

Article abstract of DOI:10.1021/acs.jmedchem.9b01828

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral po

Full text of DOI:10.1021/acs.jmedchem.9b01828

Subscriber access provided by University of Newcastle, Australia
Article
Alpha-ketoamides as broad-spectrum inhibitors of
coronavirus and enterovirus replication Structure-
based design, synthesis, and activity assessment
Linlin Zhang, Daizong Lin, Yuri Kusov, Yong Nian, Qingjun Ma, Jiang Wang, Albrecht von Brunn, Pieter
LEYSSEN, Kristina Lanko, Johan Neyts, Adriaan H. de Wilde, Eric J. Snijder, Hong Liu, and Rolf Hilgenfeld
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01828 • Publication Date (Web): 11 Feb 2020
Downloaded from pubs.acs.org on February 11, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 20
Journal of Medicinal Chemistry
Alpha-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus
replication
1
2
3
4
5
6
7
8
9
Structure-based design, synthesis, and activity assessment
†
Linlin Zhang1,2,‡, Daizong Lin1,2,3,‡, , Yuri Kusov1, Yong Nian3, Qingjun Ma1, Jiang Wang3, Albrecht von
Brunn4, Pieter Leyssen5, Kristina Lanko5, Johan Neyts5, Adriaan de Wilde6, Eric J. Snijder6, Hong
Liu3*, Rolf Hilgenfeld1,2,3
*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, 23562 Lübeck, Germa-
ny.
2German Center for Infection Research (DZIF), Hamburg - Lübeck - Borstel - Riems Site, University of Lübeck, Lübeck,
Germany.
3Shanghai Institute of Materia Medica, 201203 Shanghai, China
4Max von Pettenkofer Institute, Ludwig-Maximilians-University Munich, 80336 Munich, Germany
5Rega Institute for Medical Research, University of Leuven, 3000 Leuven, Belgium
6Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
‡These authors contributed equally.
KEYWORDS: coronavirus main protease, enterovirus 3C protease, antiviral drug design, X-ray crystallography, SARS,
MERS, human coronavirus 229E, human coronavirus NL63, Wuhan pneumonia coronavirus, 2019 novel coronavirus, enter-
ovirus A71, Coxsackievirus B3, enterovirus D68, human rhinovirus
ABSTRACT: The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and
a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral
polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-
equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued structure-based
design of peptidomimetic a-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease:inhibitor com-
plexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in
viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the a-
ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u
(P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoro-
naviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against Middle East
Respiratory Syndrome coronavirus.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 20
INTRODUCTION
the protease is designated 3Cpro). In coronaviruses, non-structural
protein 5 (Nsp5) is the main protease (Mpro). Similar to the enteroviral
3Cpro, it is a cysteine protease in the vast majority of cases and has
therefore also been called "3C-like protease" (3CLpro). The first crys-
tal structure of a CoV Mpro or 3CLpro (ref. 22) revealed that two of the
three domains of the enzyme together resemble the chymotrypsin-like
fold of the enteroviral 3Cpro, but there is an additional a-helical do-
main that is involved in the dimerization of the protease (Fig. 1A).
This dimerization is essential for the catalytic activity of the CoV
Mpro, whereas the enteroviral 3Cpro (Fig. 1B) functions as a monomer.
Further, the enteroviral 3Cpro features a classical Cys...His...Glu/Asp
catalytic triad, whereas the CoV Mpro only has a Cys...His dyad.22 Yet,
there are a number of common features shared between the two types
of proteases, in particular their almost absolute requirement for Gln in
the P1 position of the substrate and space for only small amino-acid
residues such as Gly, Ala, or Ser in the P1' position, encouraging us to
explore the coronaviral Mpro and the enteroviral 3Cpro as a common
target for the design of broad-spectrum antiviral compounds. The fact
that there is no known human protease with a specificity for Gln at the
cleavage site of the substrate increases the attractivity of this viral
target, as there is hope that the inhibitors to be developed will not
show toxicity versus the host cell. Indeed, neither the enterovirus
3Cpro inhibitor rupintrivir, which was developed as a treatment of the
common cold caused by HRV, nor the peptide aldehyde inhibitor of
the coronavirus Mpro that was recently demonstrated to lead to com-
plete recovery of cats from the normally fatal infection with Feline
Infectious Peritonitis Virus (FIPV), showed any toxic effects on
humans or cats, respectively.23,24
1
2
3
4
5
6
7
8
Seventeen years have passed since the outbreak of severe acute
respiratory syndrome (SARS) in 2003, but there is yet no approved
treatment for infections with the SARS coronavirus (SARS-CoV).1
One of the reasons is that despite the devastating consequences of
SARS for the affected patients, the development of an antiviral drug
against this virus would not be commercially viable in view of the
fact that the virus has been rapidly contained and did not reappear
since 2004. As a result, we were empty-handed when the Middle-East
respiratory syndrome coronavirus (MERS-CoV), a close relative of
SARS-CoV, emerged in 2012.2 MERS is characterized by severe
respiratory disease, quite similar to SARS, but in addition frequently
causes renal failure3. Although the number of registered MERS cases
is low (2494 as of November 30, 2019; www.who.int), the threat
MERS-CoV poses to global public health may be even more serious
than that presented by SARS-CoV. This is related to the high case-
fatality rate (about 35%, compared to 10% for SARS), and to the fact
that MERS cases are still accumulating seven years after the discov-
ery of the virus, whereas the SARS outbreak was essentially con-
tained within 6 months. The potential for human-to-human transmis-
sion of MERS-CoV has been impressively demonstrated by the 2015
outbreak in South Korea, where 186 cases could be traced back to a
single infected traveller returning from the Middle East.4 SARS-like
coronaviruses are still circulating in bats in China,5-8 from where they
may spill over into the human population; this is probably what
caused the current outbreak of atypical pneumonia in Wuhan, which
is linked to a seafood and animal market. The RNA genome (Gen-
Bank accession code: MN908947.2; http://virological.org/t/initial-
genome-release-of-novel-coronavirus/319, last accessed on January
11, 2020) of the new betacoronavirus features around 82% identity to
that of SARS-CoV.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In spite of the considerable threat posed by SARS-CoV and related
viruses, as well as by MERS-CoV, it is obvious that the number of
cases so far does not warrant the commercial development of an
antiviral drug targeting MERS- and SARS-CoV even if a projected
steady growth of the number of MERS cases is taken into account. A
possible solution to the problem could be the development of broad-
spectrum antiviral drugs that are directed against the major viral
protease, a target that is shared by all coronavirus genera as well as, in
a related form, by members of the large genus Enterovirus in the
picornavirus family. Among the members of the genus Alphacorona-
virus are the human coronaviruses (HCoV) NL63 (ref. 9) and 229E10
that usually cause only mild respiratory symptoms in otherwise
healthy individuals, but are much more widespread than SARS-CoV
or MERS-CoV. Therapeutic intervention against alphacoronaviruses
is indicated in cases of accompanying disease such as cystic fibrosis11
or leukemia,12 or certain other underlying medical conditions.13 The
enteroviruses include pathogens such as EV-D68, the causative agent
of the 2014 outbreak of the "summer flu" in the US,14 EV-A71 and
Coxsackievirus A16 (CVA16), the etiological agents of Hand, Foot,
and Mouth Disease (HFMD),15 Coxsackievirus B3 (CVB3), which
can cause myocardic inflammation,16 and human rhinoviruses (HRV),
notoriously known to lead to the common cold but also capable of
causing exacerbations of asthma and COPD.17 Infection with some of
these viruses can lead to serious outcome; thus, EV-D68 can cause
polio-like disease,18 and EV-A71 infection can proceed to asepticꢀ
meningitis, encephalitis, pulmonary edema, viral myocarditis, and
acute flaccid paralysis.15,19-20 Enteroviruses cause clinical disease
much more frequently than coronaviruses, so that an antiviral drug
targeting both virus families should be commercially viable.
Figure 1: Crystal structures of SARS-CoV main protease (Mpro, ref.
26; PDB entry 2BX4) and Coxsackivirus B3 3C protease (3Cpro; Tan
et al., unpublished; PDB entry 3ZYD). Catalytic residues are indicat-
ed by spheres (yellow, Cys; blue, His; red: Glu). A. The coronavirus
Mpro is a homodimer, with each monomer comprising three domains.
B. The structure of the monomeric CVB3 3Cpro resembles the N-
terminal two domains of the SARS-CoV Mpro. Structure is on the
same scale as image A. C. Superimpostion of residues from the two
structures involved in ligand binding. Superimposition was carried out
by aligning the catalytic Cys-His pair of each protease. Residues of
the SARS-CoV Mpro are shown with carbon atoms in cyan, CVB3
3Cpro residues have orange carbons and are labeled with an asterisk
(*).
However, enteroviruses are very different from coronaviruses.
While both of them have a single-stranded RNA genome of positive
polarity, that of enteroviruses is very small (just 7 - 9 kb) whereas
coronaviruses feature the largest RNA genome known to date (27 - 34
kb). Enteroviruses are small, naked particles, whereas coronaviruses
are much larger and enveloped. Nevertheless, a related feature shared
by these two groups of viruses is their type of major protease,21 which
in the enteroviruses is encoded by the 3C region of the genome (hence
2
ACS Paragon Plus Environment

Page 3 of 20
Journal of Medicinal Chemistry
removing the acetyl group of compounds 9. In the final step, the
oxidation of the exposed alcohol group in compounds 10 generated
our target a-ketoamides 11.
We chose the chemical class of peptidomimetic a-ketoamides to
assess the feasibility of achieving antiviral drugs targeting corona-
viruses and enteroviruses with near-equipotency. Here we describe
the structure-based design, synthesis, and evaluation of inhibitory
activity of a series of compounds with broad-spectrum activities
afforded by studying the structure-activity relationships mainly with
respect to the P2 position of the peptidomimetics. One of the com-
pounds designed and synthesized exhibits excellent activity against
MERS-CoV.
1
2
3
4
5
6
7
The inhibitory potencies of candidate a-ketoamides were evaluated
against purified recombinant SARS-CoV Mpro, HCoV-NL63 Mpro
,
CVB3 3Cpro, and EV-A71 3Cpro. The most potent compounds were
further tested against viral replicons and against SARS-CoV, MERS-
CoV, or a whole range of enteroviruses in cell culture-based assays
(Tables 1 - 3 and Supplementary Table 1).
8
9
RESULTS
a
Structure-based design of -ketoamides
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Our efforts to design novel a-ketoamides as broad-spectrum inhibi-
tors of coronavirus Mpros and enterovirus 3Cpros started with a detailed
analysis of the following crystal structures of unliganded target en-
zymes: SARS-CoV Mpro (ref. 25-27; PDB entries 1UJ1, 2BX3,
2BX4); bat coronavirus HKU4 Mpro as a surrogate for the closely
related MERS-CoV protease (our unpublished work (Ma, Xiao et al.;
PDB entry 2YNA; see also ref. 27); HCoV-229E Mpro (ref. 27,28;
PDB entry: 1P9S); Coxsackievirus B3 3Cpro (our unpublished work;
Tan et al., PDB entry 3ZYD); enterovirus D68 3Cpro (ref. 29; PDB
entry: 3ZV8); and enterovirus A71 3Cpro (ref. 30; PDB entry: 3SJK).
During the course of the present study, we determined crystal struc-
tures of a number of lead a-ketoamide compounds in complex with
SARS-CoV Mpro, HCoV-NL63 Mpro, and CVB3 3Cpro, in support of
the design of improvements in the next round of lead optimization.
Notably, unexpected differences between alpha- and betacoronavirus
Mpro were found in this study. The structural foundation of these was
elucidated in detail in a subproject involving the Mpro of HCoV NL63;
because of its volume, this work will be published separately (Zhang
et al., in preparation) and only some selected findings are referred to
here. The main protease of the newly discovered coronavirus linked to
the Wuhan outbreak of respiratory illness is 96% identical (98%
similar) in amino-acid sequence to that of SARS-CoV Mpro (derived
from the RNA genome of BetaCoV/Wuhan/IVDC-HB-01/2019,
Genbank accession code: MN908947.2; http://virological.org/t/initial-
genome-release-of-novel-coronavirus/319, last accessed on January
11, 2020), so all results reported here for inhibition of SARS-CoV
will most likely also apply to the new virus.
Scheme 1: Synthesis of
a
-ketoamides. Reaction conditions: (a)
BrCH2CN, LiHMDS, THF; (b) PtO2, H2, CHCl3, MeOH; (c) NaOAc,
MeOH; (d) TFA, CH2Cl2; (e) TEA, CH2Cl2; (f) 1 M NaOH or LiOH,
MeOH; (g) HATU, DMF; (h) NaBH4, MeOH; (i) DMP, NaHCO3,
CH2Cl2; (j) isocyanide, AcOH, CH2Cl2; (k) 1 M NaOH or LiOH,
MeOH; (l) DMP, NaHCO3, CH2Cl
Viral replicons
To enable the rapid and biosafe screening of antivirals against co-
rona- and enteroviruses, a non-infectious, but replication-competent
SARS-CoV replicon was used33 along with subgenomic replicons of
CVB334 and EV-A71 (kind gift of B. Zhang, Wuhan, China). The
easily detectable reporter activity (firefly or Renilla luciferase) of
these replicons has previously been shown to reflect viral RNA syn-
thesis.33-35 In-vitro RNA transcripts of the enteroviral replicons were
also used for transfection. For the SARS-CoV replicon containing the
CMV promoter, only the plasmid DNA was used for transfection.
As the proteases targeted in our study all specifically cleave the
peptide bond following a P1-glutamine residue (HCoV-NL63 Mpro
uniquely also accepts P1 = His at the Nsp13/Nsp14 cleavage site31),
we decided to use a 5-membered ring (g-lactam) derivative of gluta-
mine (henceforth called GlnLactam) as the P1 residue in all our a-
ketoamides (see Scheme 1). This moiety has been found to be a good
mimic of glutamine and enhance the power of the inhibitors by up to
10-fold, most probably because compared to the flexible glutamine
side-chain, the more rigid lactam leads to a reduction of the loss of
entropy upon binding to the target protease.29,32 Our synthetic efforts
therefore aimed at optimizing the substituents at the P1', P2, and P3
positions of the a-ketoamides.
Initial inhibitor design steps
The initial compound to be designed and synthesized was 11a,
which carries a cinnamoyl N-cap in the P3 position, a benzyl group in
P2, the glutamine lactam (GlnLactam) in P1, and benzyl in P1' (Table
1). This compound showed good to mediocre activities against re-
combinant SARS-CoV Mpro (IC50 = 1.95 µM; for all compounds, see
Tables 1 - 3 for standard deviations), CVB3 3Cpro (IC50 = 6.6 µM),
and EV-A71 3Cpro (IC50 = 1.2 µM), but was surprisingly completely
inactive (IC50 > 50 µM) against HCoV-NL63 Mpro. These values were
mirrored in the SARS-CoV and in the enterovirus replicons (Table 2).
In virus-infected cell cultures, the results obtained were also good to
mediocre (Table 3): SARS-CoV (EC50 = 5.8 µM in Vero E6 cells),
Synthesis of -ketoamides
a
Synthesis (Scheme 1) started with the dianionic alkylation of N-
Boc glutamic acid dimethyl ester with bromoacetonitrile. As ex-
pected, this alkylation occurred in a highly stereoselective manner,
giving 1 as the exclusive product. In the following step, the cyano
group of 1 was subjected to hydrogenation. The in-situ cyclization of
the resulting intermediate afforded the lactam 2. The lactam deriva-
tive 3 was generated by removal of the protecting group of 2. On the
other hand, the amidation of acyl chloride and a-amino acid methyl
ester afforded the intermediates 4, which gave rise to the acids 5 via
alkaline hydrolysis. The key intermediates 6 were obtained via the
condensation of the lactam derivative 3 and the N-capped amino acids
5. The ester group of compounds 6 was then reduced to the corre-
sponding alcohol. Oxidation of the alcohol products 7 by Dess-Martin
periodinane generated the aldehydes 8, which followed by nucleo-
philic addition with isocyanides gave rise to compounds 9 under
acidic conditions. Then, the a-hydroxyamides 10 were prepared by
MERS-CoV (EC50 = 0.0047 µM in Huh7 cells), HCoV 229E (EC50
=
11.8 µM in Huh7 cells), or a host of enteroviruses (EC50 = 9.8 µM
against EV-A71 in RD cells; EC50 = 0.48 µM against EV-D68 in
HeLa Rh cells; EC50 = 5.6 µM against HRV2 in HeLa Rh cells). In all
cell types tested, the compound generally proved to be non-toxic, with
selectivity indices (CC50/EC50) usually >10 (Table 3).
Crystal structures of compound 11a in complex with SARS-CoV
Mpro, HCoV-NL63 Mpro, and CVB3 3Cpro demonstrated that the a-
keto-carbon is covalently linked to the active-site Cys (no. 145, 144,
and 147, resp.) of the protease (Fig. 2, 3a-c). The resulting thiohe-
miketal is in the R configuration in the SARS-CoV and HCoV-NL63
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 20
Mpro but in the S configuration in the CVB3 3Cpro complex. The
some activity against HRV2 in HeLa Rh cells (EC50 = 9.0 µM). A
crystal structure of 11f bound to HCoV-NL63 Mpro demonstrated that
the P2-Boc group entered the S2 pocket (Fig. 3d). In conclusion,
although there is probably room for further improvement, we decided
to maintain the original design with P1' = benzyl and P3 = cinnamoyl,
and focussed on improving the P2 substituen.
reason for this difference is that the oxygen atom of the thiohemiketal
accepts a hydrogen bond from the catalytic His40 in the CVB3 prote-
ase, rather than from the main-chain amides of the oxyanion hole as in
the SARS-CoV and HCoV-NL63 enzymes (Fig. 3a,b,c insets). It is
remarkable that we succeeded in obtaining a crystal structure of
compound 11a in complex with the HCoV-NL63 Mpro, even though it
has no inhibitory effect on the activity of the enzyme (IC50 > 50 µM),
(Fig. 2c). Apparently, the compound is able to bind to this Mpro in the
absence of peptide substrate, but cannot compete with substrate for
the binding site due to low affinity. A similar observation has been
made in one of our previous studies, where we were able to determine
the crystal structure of a complex between the inactive Michael-
acceptor compound SG74 and the EV-D68 3Cpro (ref. 29; PDB entry
3ZV9).
1
2
3
4
5
6
7
8
Properties of the S2 pockets of the target enzymes
The crystal structures of SARS-CoV Mpro, HCoV-NL63 Mpro, and
CVB3 3Cpro in complex with 11a revealed a fundamental difference
between the S2 pockets of the coronavirus proteases and the enterovi-
rus proteases: The cavities are covered by a "lid" in the former but are
open to one side in the latter (Fig. 2,b-d). In SARS-CoV Mpro, the lid
is formed by the 310 helix 46 - 51 and in HCoV-NL63 Mpro by the
loop 43-48. Residues from the lid, in particular Met49 in the case of
SARS-CoV Mpro, can thus make hydrophobic interactions with the P2
substitutent of the inhibitor, whereas such interaction is missing in the
enterovirus 3Cpros. In addition to the lid, the S2 pocket is lined by the
"back-wall" (main-chain atoms of residues 186 and 188 and Cb atom
of Asp187), the side-walls (Gln189, His41), as well as the "floor"
(Met165) in SARS-CoV Mpro. In HCoV-NL63 Mpro, the correspond-
ing structural elements are main-chain atoms of residues 187 and 188
as well as the Cb atom of Asp187 (back-wall), Pro189 and His41
(side-walls), and Ile165 (floor). Finally, in CVB3 3Cpro, Arg39,
Asn69, and Glu71 form the back-wall, residues 127-132 and His40
form the side-walls, and Val162 constitutes the floor.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In addition, the S2 pocket is of different size in the various proteas-
es. The SARS-CoV enzyme features the largest S2 pocket, with a
volume of 252 Å3 embraced by the residues (Gln189, His41) defining
the side-walls of the pocket in the ligand-free enzyme, as calculated
by using Chimera,36 followed by the CVB3 3Cpro S2 pocket with
about 180 Å3 (space between Thr130 and His40). The HCoV-NL63
Mpro has by far the smallest S2 pocket of the three enzymes, with a
free space of only 45 Å3 between Pro189 and His41, according to
Chimera.
In agreement with these observations, a good fit is observed be-
tween the P2 benzyl group of 11a and the S2 subsite of the SARS-
CoV Mpro as well as that of the CVB3 3Cpro (Fig. 3a,c). In contrast,
the crystal structure of the complex between 11a and HCoV-NL63
Mpro, against which the compound is inactive, demonstrates that the
P2-benzyl group cannot fully enter the S2 pocket of the enzyme
because of the restricted size of this site (Fig. 3b).
Thus, the properties of our target proteases with respect to the S2
pocket were defined at this point as "small" and "covered by a lid" for
HCoV-NL63 Mpro, "large" and "covered" for SARS-CoV Mpro, and
"large" and "open" for CVB3 3Cpro. Through comparison with crystal
structures of other proteases of the same virus genus (HCoV-229E
Mpro for alphacoronaviruses28 (PDB entry 1P9S); HKU4 Mpro for
betacoronaviruses (Ma, Xiao et al., unpublished; PDB entry 2YNA);
and EV-A71 3Cpro for enteroviruses30 (PDB entry 3SGK), we ensured
that our conclusions drawn from the template structures were valid for
other family members as well.
Figure 2: Fit of compound 11a (pink carbon atoms) to the target
proteases (wheat surfaces) as revealed by X-ray crystallography of the
complexes. A. Fo - Fc difference density (contoured at 3s) for 11a in
the substrate-binding site of the SARS-CoV Mpro (transparent sur-
face). Selected side-chains of the protease are shown with green
carbon atoms. B. Another view of 11a in the substrate binding site of
the SARS-CoV Mpro. Note the "lid" formed by residue Met49 and its
neighbors above the S2 pocket. C. 11a in the substrate-binding site of
HCoV-NL63 Mpro. Because of the restricted size of the S2 pocket, the
P2 benzyl group of the compound cannot enter deeply into this site.
Note that the S2 pocket is also covered by a "lid" centred around
Thr47. D. 11a in the substrate-binding site of the CVB3 3Cpro. The S2
site is large and not covered by a "lid".
To explore the sensitivity of the S2 pocket towards a polar substit-
uent in the para position of the benzyl group, we synthesized com-
pound 11m carrying a 4-fluorobenzyl group in P2. This substitution
abolished almost all activity against the SARS-CoV Mpro (IC50 > 50
µM), and the compound proved inactive against HCoV-NL63 Mpro as
well, whereas IC50 values were 2.3 µM against the EV-A71 3Cpro and
8.7 µM against CVB3 3Cpro. From this, we concluded that the intro-
duction of the polar fluorine atom is not compatible with the geometry
of the S2 pocket of SARS-CoV Mpro, whereas the fluorine can accept
a hydrogen bond from Arg39 in EV-A71 3Cpro (ref. 30) and probably
also CVB3 3Cpro. In SARS-CoV Mpro, however, the carbonyl groups
of residues 186 and 188 might lead to a repulsion of the fluorinated
benzyl group.
P1' and P3 substituents
The crystal structures indicated that the fits of the P1' benzyl group
of 11a in the S1' pocket and of the P3 cinnamoyl cap in the S3 subsite
might be improved (see Fig. 3a-c). Compounds 11b - 11e and 11g -
11l were synthesized in an attempt to do so; however, none of them
showed better inhibitory activity against the majority of the recombi-
nant proteases, compared to the parent compound, 11a (see Supple-
mentary Results). To investigate whether the P3 residue of the inhibi-
tor is dispensible, we synthesized compound 11f, which only com-
prises P2 = Boc, P1 = GlnLactam, and P1' = benzyl. 11f was inactive
against all purified proteases and in all replicons tested, but showed
4
ACS Paragon Plus Environment

Page 5 of 20
Journal of Medicinal Chemistry
Table 1: Inhibition of viral proteases by α-ketoamides (IC50, μM)
1
2
3
4
5
6
Compound No.
11a
Formula
EV-A71 3Cpro
CVB3 3Cpro
SARS-CoV Mpro
HCoV-NL63 Mpro
1.22 ± 0.12
6.56 ± 3.10
1.95 ± 0.24
>50
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11f
11m
11n
11o
>50
>50
>50
>50
>50
2.32 ± 1.21
13.80 ± 4.17
3.24 ± 1.69
8.69 ± 1.28
3.82 ± 1.21
5.20 ± 0.28
>50
0.33 ± 0.04
8.50 ± 3.71
1.08 ± 0.09
>50
11p
11q
8.50 ± 1.81
8.93 ± 2.39
10.68 ± 7.34
6.27 ± 2.87
17.37 ± 3.97
11.50 ± 5.05
>30
15.38 ± 4.12
11r
1.69 ± 0.47
0.95 ± 0.15
0.71 ± 0.36
12.27 ± 3.56
11s
11t
18.47 ± 4.25
10.81 ± 2.62
4.73 ± 0.94
4.25 ± 1.40
4.78 ± 1.13
1.93 ± 0.43
0.24 ± 0.08
1.44 ± 0.40
1.27 ± 0.34
1.37 ± 0.35
3.43 ± 2.45
5.41 ± 2.31
11u
nd – not done
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 20
Table 2:
a
-ketoamide-induced inhibition of subgenomic RNA synthesis using replicons in a cell-based assay (EC50, μM)
1
2
3
Compound No.
EV-A71
CVB3
SARS-CoV
4
5
6
7
8
11a
11f
4.5 ± 0.5
>>10
>20
4.5 ± 0.5
>>10
nd
2.0 ± 0.2
>40
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11m
11n
11o
nd
>>10
>20
>>10
nd
7.2 ± 0.2
nd
11p
11r
nd
nd
>20
6.95 ± 0.05
(0.85 ± 0.05)*
2.35 ± 0.05
(0.45 ± 0.05)*
1.4 ± 0.1
1.9 ± 0.1
6.7 ± 0.2
3.6 ± 0.1
11s
11t
11u
>>20
18 ± 2
>20
(10.9 ± 0.1)*
4.3 ± 0.2
(2.2 ± 0.2)*
8.9 ± 0.1
(3.65 ± 0.15)*
5.1 ± 0.1
(4.9 ± 2.6)*
nd - not done; * - values in brackets obtained by RNA-launched transfection.
6
ACS Paragon Plus Environment

Page 7 of 20
Journal of Medicinal Chemistry
1
2
3
Table 3: Cytotoxicity and antiviral activity of α-ketoamides against selected entero- and coronaviruses in a live-virus cell-based assay
4
5
6
7
8
9
Cells
Huh-T7
--
Huh-T7
Huh7
HCoV-229E
EC50 (μM)
11.8 ± 2.1
0.6 ± 0.1
1.8 ± 0.6
1.3 ± 0.4
nd
Huh7
Vero
MERS-CoV
EC50 (μM)
13.4
Vero E6
SARS-CoV
EC50 (μM)
5.8
RD
--
RD
Hela Rh
--
Hela Rh
EV-D68
EC50 (μM)
0.48 ± 0.09
4.44 ± 0.02
0.48 ± 0.09
nd
Hela Rh
HRV2
Hela Rh
HRV14
Virus
CVB3
MERS-CoV
EC50 (μM)
EV-A71
EC50 (μM)
9.8 ± 1.1
72 ± 14
(Strain)
(Nancy)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Compound
11a
CC50 (μM) EC50 (μM)
CC50 (μM)
97 ± 7
CC50 (μM)
35.5 ± 2.7
103 ± 5
10.9 ± 1.8
103 ± 2
65 ± 7
EC50 (μM)
5.6 ± 0.7
8.9 ± 2.5
0.64 ± 0.05
3.8 ± 0.2
3.7 ± 0.4
0.51 ± 0.05
EC50 (μM)
4.3 ± 0.1
>265
>188
14 ± 1
24 ± 5
0.0048 ± 0.001
0.0046 ± 0.001
11n
9.2
14.2
115 ± 14
17.3 ± 2.9
106 ± 8
100 ± 2
30.8 ± 0.1
13.40 ± 0.01
0.70 ± 0.09
5.5 ± 1.2
0.0004 ±
0.0003
11r
43 ± 2
2.77 ± 0.03
10.4 ± 0.1
10.4 ± 0.1
8.1 ± 0.8
5.0 ± 0.4
10.9 ± 1.7
9.8 ± 1.5
11.1 ± 0.2
2.1 ± 1.2
18.4 ± 6.7
7.0 ± 2.3
4.9 ± 1.2
3.7 ± 0.2
46 ± 7
11s
>189
0.08 ± 0.01
0.02 ± 0.01
11t
124 ± 1
47.5 ± 2.3
41 ± 3
nd
4.2 ± 0.1
11u
2.5 ± 0.6
0.007 ± 0.006
4.20 ± 0.02
33.2 ± 0.9
nd
0.42 ± 0.02
nd – not done
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 20
thiohemiketal is S. The side-chains of Ser144 and Arg188 have been
omitted for clarity.
1
2
3
P2-alkyl substituents of varying sizes
As the P2-benzyl group of 11a was apparently too large to fit into
the S2 pocket of the HCoV-NL63 Mpro, we replaced it by isobutyl in
11n. This resulted in improved activities against SARS-CoV Mpro
(IC50 = 0.33 µM) and in a very good activity against HCoV-NL63
Mpro (IC50 = 1.08 µM, compare with the inactive 11a). For EV-A71
3Cpro, however, the activity decreased to IC50 = 13.8 µM, different
from CVB3 3Cpro, where IC50 was 3.8 µM. Our interpretation of this
result is that the smaller P2-isobutyl substituent of 11n can still inter-
act with the "lid" (in particular, Met49) of the SARS-CoV Mpro S2
site, but is unable to reach the "back-wall" of the EV-A71 3Cpro pock-
et and thus, in the absence of a "lid", cannot generate sufficient en-
thalpy of binding. We will see from examples to follow that this trend
persists among all inhibitors with a smaller P2 substituent: Even
though the SARS-CoV Mpro S2 pocket has a larger volume than that
of the enterovirus 3Cpro, the enzyme can be efficiently inhibited by
compounds carrying a small P2 residue that makes hydrophobic
interactions with the lid (Met49) and floor (Met165) residues.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The EC50 of 11n was >10 µM against the EV-A71 and CVB3 re-
plicons, and even in the SARS-CoV replicon, the activity of 11n was
relatively weak (EC50 = 7.0 µM; Table 2). In agreement with the
replicon data, 11n proved inactive against EV-A71 in RD cells and
showed limited activity against HRV2 or HRV14 in HeLa Rh cells
(Table 3). Only the comparatively good activity (EC50 = 4.4 µM)
against EV-D68 in HeLa Rh cells was unexpected. The activity of
11n against HCoV 229E in Huh7 cells was good (EC50 = 0.6 µM),
and against MERS-CoV in Huh7 cells, it was excellent, with EC50
=
0.0048 µM, while in Vero cells, the EC50 against MERS-CoV was as
high as 9.2 µM. Similarly, the EC50 against SARS-CoV in Vero cells
was 14.2 µM (Table 3).
Figure 3: Detailed interactions of peptidomimetic a-ketoamides
(pink carbon atoms) with target proteases (green carbon atoms).
Hydrogen bonds are depicted as blue dashed lines. The inset at the top
of the images shows the stereochemistry of the thiohemiketal formed
by the nucleophilic attack of the catalytic Cys residue onto the a-keto
group. A. Binding of 11a to SARS-CoV Mpro. The thiohemiketal is in
the R configuration, with its oxygen accepting two hydrogen bonds
from the oxyanion-hole amides of Gly143 and Cys145. The amide
oxygen accepts an H-bond from His41. The side-chains of Ser144 and
Arg188 have been omitted for clarity. B. The P2-benzyl substituent of
11a cannot fully enter the S2 pocket of the HCoV-NL63 Mpro, which
is much smaller and has less plasticity than the corresponding pocket
of SARS-CoV Mpro (cf. A). The benzyl therefore binds above the
pocket in the view shown here; this is probably the reason for the total
inactivity (IC50 > 50 µM) of compound 11a against HCoV-NL63 Mpro
The small size of the pocket is due to the replacement of the flexible
Gln189 of the SARS-CoV Mpro by the more rigid Pro189 in this
enzyme. The stereochemistry of the thiohemiketal is R. The side-
chains of Ala143 and Gln188 have been omitted for clarity. C. Bind-
ing of 11a to the CVB3 3Cpro. The stereochemistry of the thiohe-
miketal is S, as the group accepts a hydrogen bond from His41,
whereas the amide keto group accepts three H-bonds from the oxyan-
ion hole (residues 145 - 147). The side-chain of Gln146 has been
omitted for clarity. D. The crystal structure of 11f in complex with
HCoV-NL63 Mpro shows that this short (inactive) compound lacking a
P3 residue has its P2-Boc group inserted into the S2 pocket of the
protease. The stereochemistry of the thiohemiketal is S. The side-
chains of Ala143 and Gln188 have been omitted for clarity. E. In
contrast to P2 = benzyl in 11a, the isobutyl group of 11n is small and
flexible enough to enter into the narrow S2 pocket of the HCoV-NL63
Mpro. The thiohemiketal is in the R configuration. The side-chains of
Ala143 and Gln188 have been omitted for clarity. F. In spite of its
small size, the cyclopropylmethyl side-chain in the P2 position of 11s
can tightly bind to the S2 subsite of the SARS-CoV Mpro, as this
pocket exhibits pronounced plasticity due to the conformational
flexibility of Gln189 (see also Fig. 4). The stereochemistry of the
We managed to obtain crystals of 11n in complex with the Mpro of
HCoV NL63 and found the P2 isobutyl group to be well embedded in
the S2 pocket (Fig. 3e). This is not only a consequence of the smaller
size of the isobutyl group compared to the benzyl group, but also of
its larger conformational flexibility, which allows a better fit to the
binding site.
When we replaced the P2-isobutyl residue of 11n by n-butyl in
11o, the activities were as follows: IC50 = 8.5 µM for SARS-CoV
Mpro, totally inactive (IC50 > 50 µM) against HCoV-NL63 Mpro, IC50
=
3.2 µM for EV-A71 3Cpro, and 5.2 µM for CVB3 3Cpro. The de-
creased activity in case of SARS-CoV Mpro and the total inactivity
against HCoV-NL63 Mpro indicate that the n-butyl chain is too long
for the S2 pocket of these proteases, whereas the slight improvement
against EV-A71 3Cpro and CVB3 3Cpro is probably a consequence of
the extra space that is available to long and flexible substituents
because of the lack of a lid covering the enterovirus 3Cpro pocket.
.
As the n-butyl substituent in P2 of 11o was obviously too long, we
next synthesized a derivative with the shorter propargyl (ethinylme-
thyl) as the P2 residue (compound 11p). This led to very mediocre
activities against all tested proteases. Using cyclopropyl as the P2
residue (compound 11q), the IC50 values were even higher against
most of the proteases tested. Obviously, the P2 side-chain requires a
methylene group in the b-position in order to provide the necessary
flexibility for the substituent to be embedded in the S2 pocket.
Modifying ring size and flexibility of P2-cycloalkylmethyl sub-
stituents
Having realized that in addition to size, flexibility of the P2 sub-
stituent may be an important factor influencing inhibitory activity, we
introduced flexibility into the phenyl ring of 11a by reducing it. The
cyclohexylmethyl derivative 11r exhibited IC50 = 0.7 µM against
SARS-CoV Mpro, 12.3 µM against HCoV-NL63 Mpro, 1.7 µM against
EV-A71 3Cpro, and 0.9 µM against CVB3 3Cpro. Thus, the replace-
8
ACS Paragon Plus Environment

Page 9 of 20
Journal of Medicinal Chemistry
ment of the phenyl group by the cyclohexyl group led to a significant
improvement of the inhibitory activity against the recombinant
SARS-CoV Mpro and to a dramatic improvement in case of CVB3
3Cpro. Even for the HCoV-NL63 Mpro, against which 11a was com-
pletely inactive, greatly improved albeit still weak activity was ob-
served (Table 1). In the viral replicons, 11r performed very well, with
EC50 = 0.8 - 0.9 µM for the EV-A71 replicon, 0.45 µM for CVB3,
and 1.4 µM for SARS-CoV (Table 2). In the virus-infected cell cul-
ture assays (Table 3), 11r exhibited EC50 = 3.7 µM against EV-A71 in
RD cells and EC50 = 0.48 - 0.7 µM against EV-D68, HRV2, and
HRV14 in HeLa cells. Against HCoV 229E in Huh7 cells, the EC50
was surprisingly low (1.8 µM). Interestingly, the compound proved
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
extremely potent against MERS-CoV in Huh7 cells, with EC50
=
0.0004 µM (400 picomolar!). Even in Vero cells, EC50 against
MERS-CoV was 5 µM, and the EC50 against SARS-CoV in Vero E6
cells was 1.8 - 2.1 µM, i.e. the best activity we have seen for an Mpro
inhibitor against SARS-CoV in this type of cells. The therapeutic
index (CC50/EC50) of 11r against EV-D68, HRV2, and HRV14 was
>15 in HeLa Rh cells as well as against CVB3 in Huh-T7 cells, but
only ~5 for EV-A71 in RD cells.
Figure 4: A pronounced plasticity of the S2 pocket of SARS-CoV
Mpro is revealed by a comparison of the geometry of the subsite in the
complexes with 11a (P2 = benzyl; inhibitor cyan, protein green) and
11s (P2 = cyclopropylmethyl; inhibitor orange, protein pink). The
main differences here concern the main chain around Gln189 (note
the flip of the 189 - 190 peptide bond) as well as the side-chain of this
flexible residue, conformational change of which allows the S2 pock-
et to "shrink" and adapt to the small size of the P2-substituent in 11s.
This change also enables the formation of a hydrogen bond between
the main-chain amide of the P2 residue and the side-chain oxygen of
Gln189. The side-chains of Arg188 and Thr190 as well as the P1'
substituent of the inhibitors have been omitted for clarity.
At this point, we decided to systematically vary the size of the ring
system in P2. The next substituent to be tried was cyclopropylmethyl
(compound 11s, which showed good activities against SARS-CoV
Mpro (IC50 = 0.24 µM) and HCoV-NL63 Mpro (1.4 µM), but poor
values against EV-A71 3Cpro (IC50 = 18.5 µM) and CVB3 3Cpro (IC50
= 4.3 µM) (Table 1). 11s was shown to inhibit the SARS-CoV repli-
con with an EC50 of about 2 µM, whereas activity against the EV-A71
and CVB3 replicons was poor (EC50 values > 20 µM) (Table 2). The
replicon results were mirrored by the antiviral activity of 11s in enter-
ovirus-infected cells (Table 3), which was weak or very weak. By
contrast, the compound inhibited HCoV 229E and MERS-CoV in
Huh7 cells with EC50 of 1.3 and 0.08 µM, respectively. The activity
against the latter virus in Vero cells was poor (EC50 ~11 µM), and so
was the anti-SARS-CoV activity in Vero E6 cells (Table 3).
We next introduced cyclobutylmethyl in the P2 position (com-
pound 11t) and obtained the following results: IC50 = 1.4 µM for
SARS-CoV Mpro, 3.4 µM for HCoV-NL63 Mpro, 10.8 µM for EV-A71
3Cpro, and 4.8 µM for CVB3 3Cpro (Table 1). Experiments with the
viral replicons confirmed this trend, although the EC50 value for
SARS-CoV (6.8 µM) was surprisingly high (Table 2). In Huh7 cells
infected with MERS-CoV, this compound exhibited EC50 = 0.1 µM
(but 9.8 µM in Vero cells), whereas EC50 was 7.0 µM against SARS-
CoV in Vero E6 cells. The compound was largely inactive against
EV-A71 in RD cells and inhibited the replication of the two HRV
subtypes tested (in HeLa Rh cells) with EC50 values of ~4 µM. The
CC50 of 11t in HeLa cells was 65 µM, i.e. the therapeutic index was
well above 15 (Table 3).
We next analyzed the crystal structure of the complex between
SARS-CoV Mpro and compound 11s (Fig. 3f). The cyclopropylmethyl
substituent was found to be incorporated deeply into the S2 pocket,
making hydrophobic interactions with Met49 (the lid), Met165 (the
floor), and the Cb of Asp187 (the back-wall). In spite of the small size
of the P2 substituent, this is possible because the S2 pocket of SARS-
CoV Mpro is flexible enough to contract and enclose the P2 moiety
tightly. This plasticity is expressed in a conformational change of
residue Gln189, both in the main chain and in the side-chain. The
main-chain conformational change is connected with a flip of the
peptide between Gln189 and Thr190. The c1 torsion angle of the
Gln189 side-chain changes from roughly antiperiplanar (ap) to (-)-
synclinal (-sc) (Fig. 4). The conformational variability of Gln189 has
been noted before, both in Molecular Dynamics simulations26 and in
other crystal structures.37 As a consequence of these changes, the side-
chain oxygen of Gln189 can accept a 2.54-Å hydrogen bond from the
main-chain NH of the P2 residue in the 11s complex (see Fig. 4). The
affinity of 11s for the S2 pocket of HCoV-NL63 Mpro is good because
of an almost ideal match of size and not requiring conformational
changes, which this enzyme would not be able to undergo because of
the replacement of the flexible Gln189 by the more rigid Pro. On the
other hand, docking of the same compound into the crystal structure
of the CVB3 3Cpro revealed that the cyclopropylmethyl moiety was
probably unable to generate sufficient Free Energy of binding because
of the missing lid and the large size of the S2 pocket in the enterovi-
rus 3Cpro, thereby explaining the poor inhibitory activity of 11s
against these targets.
Obviously, this substituent was still a bit too small for the enterovi-
rus proteases, so as the next step, we tested P2 = cyclopentylmethyl
(compound 11u). This turned out to be the one compound with ac-
ceptable IC50 values against all tested enzymes: 1.3 µM against
SARS-CoV Mpro, 5.4 µM against HCoV-NL63 Mpro, 4.7 µM against
EV-A71 3Cpro, and 1.9 µM against CVB3 3Cpro (Table 1). The activi-
ty against the replicons was between 3.6 and 4.9 µM (Table 2). In
Huh7 cells infected with HCoV 229E or MERS-CoV, 11u showed
EC50 = 2.5 or 0.03 µM (11.1 µM for MERS-CoV in Vero cells), while
EC50 was 4.9 µM against SARS-CoV in Vero E6 cells (Table 3).
11u appeared so far the best compromise compound, yet for each
of the individual viral enzymes, the following compounds proved
superior: P2 = cyclopropylmethyl (compound 11s) for SARS-CoV
Mpro, P2 = isobutyl (compound 11n) and P2 = cyclopropylmethyl
(11s) for HCoV-NL63 Mpro, P2 = benzyl (11a) or cyclohexylmethyl
(11r) for EV-A71 3Cpro, and 11r for CVB3 3Cpro. In other words, the
nearly equipotent 11u is indeed a compromise. Therefore, in view of
the surprisingly good antiviral activity of 11r against HCoV 229E in
Huh7 cells, we relaxed the condition that the universal inhibitor
should show good activity against the recombinant HCoV-NL63 Mpro
,
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 20
and selected 11r (P2 = cyclohexylmethyl) as the lead compound for
further development. This compound exhibited submicromolar IC50
values against CVB3 3Cpro and SARS-CoV Mpro, and IC50 = 1.7 µM
against EV-A71 3Cpro (Table 1), as well as similarly low EC50 values
in the replicons of these viruses (Table 2). In Huh7 cells infected with
MERS-CoV, the performance of this compound was excellent, with
EC50 = 0.0004 µM, and even against HCoV 229E in Huh7 cells and
SARS-CoV in Vero E6 cells, EC50 values of 1.8 and 2.1 µM, respec-
tively, were observed (Table 3). Also in enterovirus-infected cell
culture, the compound performed well, with EC50 values of 0.7 µM or
below against HRV2, HRV14, and EV-D68 in HeLa (Rh) cells and
selectivity values >15. The only concern is the activity of the com-
pound against EV-A71 in RD cells, for which the EC50 value was 3.7
µM, resulting in too low a therapeutic index. On the other hand, only
weak toxicity was detected for 11r in Vero or Huh-T7 cells. Prelimi-
nary pharmacokinetics tests with the compound in mice did not indi-
cate a toxicity problem (to be published elsewhere).
While our study was underway, Zeng et al.42 published a series of
1
2
3
4
5
6
7
8
a-ketoamides as inhibitors of the EV-A71 3Cpro. These authors main-
ly studied the structure-activity relationships of the P1' residue and
found small alkyl substituents to be superior to larger ones. Interest-
ingly, they also reported that a six-membered d-lactam in the P1
position led to 2 - 3 times higher activities, compared to the five-
membered g-lactam. At the same time, Kim et al.43 described a series
of five a-ketoamides with P1' = cyclopropyl that showed submi-
cromolar activity against EV-D68 and two HRV strains.
9
Occasionally, individual a-ketoamides have been reported in the
literature as inhibitors of both the enterovirus 3C protease and the
coronavirus main protease. A single capped dipeptidyl a-ketoamide,
Cbz-Leu-GlnLactam-CO-CO-NH-iPr, was described that inhibited
the recombinant transmissible gastroenteritis virus (TGEV) and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
SARS-CoV Mpros as well as human rhinovirus and poliovirus 3Cpro
in the one-digit micromolar range.44 Coded GC-375, this compound
showed poor activity in cell culture against EV-A71 though (EC50
s
DISCUSSION
=
We describe here the structure-based design, the synthesis, and the
assessment of capped dipeptide a-ketoamides that target the main
protease of alpha- or betacoronaviruses as well as the 3C protease of
enteroviruses. Through crystallographic analyses of a total of six
inhibitor complexes of three different proteases in this study, we
found the a-ketoamide warhead (-CO-CO-NH-) to be sterically more
versatile than other warheads such as Michael acceptors (-CH=CH-
CO-) and aldehydes (-CH=O), because it features two acceptors for
hydrogen bonds from the protein, namely the a-keto oxygen and the
amide oxygen, whereas the other warheads have only one such accep-
tor. In the various complexes, the hydroxy group (or oxyanion) of the
thiohemiketal that is formed by the nucleophilic attack of the active-
site cysteine residue onto the a-keto carbon, can accept one or two
hydrogen bonds from the main-chain amides of the oxyanion hole. In
addition, the amide oxygen of the inhibitor accepts a hydrogen bond
from the catalytic His side-chain. Alternatively, the thiohemiketal can
interact with the catalytic His residue and the amide oxygen with the
main-chain amides of the oxyanion hole. Depending on the exact
interaction, the stereochemistry at the thiohemiketal C atom would be
different. We have previously observed a similar difference in case of
aldehyde inhibitors, where the single interaction point — the oxyan-
ion of the thiohemiacetal — can accept a hydrogen bond either from
the oxyanion hole or from the catalytic His side-chain,37 resulting in
different stereochemistry of the thiohemiacetal carbon. Both a-
ketoamides and aldehydes react reversibly with the catalytic nucleo-
phile of proteases, whereas Michael acceptors form irreversible ad-
ducts.
15.2 µM), probably because P2 was isobutyl. As we have shown here,
an isobutyl side-chain in the P2 position of the inhibitors is too small
to completely fill the S2 pocket of the EV-A71 3Cpro and the CVB3
3Cpro
.
Among a series of aldehydes, Prior et al.45 described the capped
tripeptidyl a-ketoamide Cbz-1-naphthylalanine-Leu-GlnLactam-CO-
CO-NH-iPr, which showed IC50 values in the 3-digit nanomolar range
against HRV 3Cpro and SARS-CoV Mpro, as well as EC50 values of
0.03 µM against HRV18 and 0.5 µM against HCoV 229E in cell
culture. No optimization of this compound was performed and no
toxicity data have been reported.
For compounds with warheads other than a-ketoamides, in-vitro
activity against both corona- and enteroviruses has also occasionally
been reported. Lee et al.46 described three peptidyl Michael acceptors
that displayed inhibitory activity against the Mpros of SARS-CoV and
HCoV 229E as well as against the 3Cpro of CVB3. These inhibitors
had an IC50 10 - 20 times higher for the CVB3 enzyme, compared to
SARS-CoV Mpro. P2 was invariably isobutyl (leucine) in these com-
pounds, suggesting that further improvement might be possible.
In addition to Michael acceptors, peptide aldehydes have also been
used to explore the inhibition of coronavirus Mpros as well as enterovi-
rus 3Cpros. Kim et al.44 reported a dipeptidyl aldehyde and its bisulfite
adduct, both of which exhibited good inhibitory activities against the
isolated 3C proteases of human rhinovirus and poliovirus as well as
against the 3C-like proteases of a number of coronaviruses, but antivi-
ral activities in cell culture against EV-A71 were poor (EC50 >10
µM), again most probably due to P2 being isobutyl (leucine).
In addition to better matching the H-bonding donor/acceptor prop-
erties of the catalytic center through offering two hydrogen-bond
acceptors instead of one, a-ketoamides have another big advantage
over aldehydes and a,b-unsaturated esters (Michael acceptors) in that
they allow easy extension of the inhibitors to probe the primed speci-
ficity subsites beyond S1', although this has so far rarely been ex-
plored (e.g., ref. 38 in case of calpain).
In our series of compounds, we used P1 = GlnLactam (g-lactam)
throughout, because this substituent has proven to be an excellent
surrogate for glutamine.29,32 While we made some efforts to optimize
the P1' residue of the compounds as well as the N-cap (P3), we main-
ly focussed on optimization of the P2 substituent. In nearly all studies
aiming at discovering peptidomimetic inhibitors of coronavirus Mpros,
P2 is invariably isobutyl (leucine), and this residue has also been used
in the efforts to design compounds that would inhibit enterovirus
3Cpros as well (see above). From crystal structures of our early lead
compound, 11a (cinnamoyl-Phe-GlnLactam-CO-CO-NH-Bz), in
complex with the Mpros of HCoV NL63 (as representative of the
alphacoronavirus proteases) and SARS-CoV (beta-CoV) as well as
the 3Cpro of Coxsackievirus B3 (enterovirus proteases), we found that
the S2 pocket has fundamentally different shapes in these enzymes. In
the SARS-CoV Mpro, the S2 subsite is a deep hydrophobic pocket that
is truly three-dimensional in shape: the "walls" of the groove are
formed by the polypeptide main chain around residues 186 - 188 as
well as by the side-chains of His41 (of the catalytic dyad) and
Gln189, whereas the "floor" is formed by Met165 and the "lid" by
residues 45 - 51, in particular Met49. The two methionines provide
The most prominent a-ketoamide drugs are probably telaprivir and
boceprivir, peptidomimetic inhibitors of the HCV NS3/4A prote-
ase,39,40 which have helped revolutionize the treatment of chronic
HCV infections. For viral cysteine proteases, a-ketoamides have only
occasionally been described as inhibitors and few systematic studies
have been carried out.
A number of capped dipeptidyl a-ketoamides have been described
as inhibitors of the norovirus 3C-like protease.41 These were opti-
mized with respect to their P1' substituent, whereas P2 was isobutyl in
most cases and occasionally benzyl. The former displayed IC50 values
one order of magnitude lower than the latter, indicating that the S2
pocket of the norovirus 3CL protease is fairly small. Although we did
not include the norovirus 3CLpro in our study, expanding the target
range of our inhibitors to norovirus is probably a realistic undertaking.
10
ACS Paragon Plus Environment

Page 11 of 20
Journal of Medicinal Chemistry
important interaction points for the P2 substituents of inhibitors; while
of 11r against MERS-CoV and SARS-CoV. Whereas the inhibitor
exhibits excellent activity against MERS-CoV when Huh7 cells are
the host cells (400 pM), the inhibitory activity is weaker by a factor of
up to 12,500 when Vero cells are used (EC50 = 5 µM). On the other
hand, 11r exhibits excellent anti-MERS-CoV activity in human Calu3
lung cells, i.e. in the primary target cells where the compound will
have to act in a therapeutic setting (A. Kupke, personal communica-
tion). As we tested antiviral activity against SARS-CoV exclusively
in Vero cells, the EC50 values determined for our compounds against
this virus are in the one-digit micromolar range or higher; the best is
again compound 11r with EC50 = 2.1 µM. Interestingly, the relatively
weaker activity (or even inactivity) of our inhibitors against RNA
viruses in Vero cells was observed independently in the virology
laboratories in Leuven and in Leiden. It is thus unlikely that the lack
of activity in Vero cells is related to problems with the experimental
set-up. In preliminary experiments, we replaced the P3 cinnamoyl
group of 11r by the fluorophor coumaryl and found by fluorescence
microscopy that much more inhibitor appeared to accumulate in Huh7
cells compared to Vero cells (D.L., R.H. & Irina Majoul, un-
published).
these interactions are mostly hydrophobic in character, we have
previously described the surprising observation of the carboxylate of
an aspartic residue in P2 that made polar interactions with the sulfur
atoms of these methionines.37 Because the pocket offers so many
opportunities for interaction and features a pronounced plasticity, P2
substituents such as isobutyl (from Leu), which are too small to fill
the pocket entirely, can still generate sufficient binding enthalpy.
Accordingly, the S2 pocket of SARS-CoV Mpro is the most tolerant
among the three enzymes investigated here, in terms of versatility of
the P2-substituents accepted.
1
2
3
4
5
6
7
8
9
In the S2 pocket of the HCoV-NL63 Mpro, Gln189 is replaced by
proline and this change is accompanied by a significant loss of flexi-
bility; whereas the side-chain of Gln189 of SARS-CoV Mpro is found
to accommodate its conformation according to the steric requirements
of the P2 substituent, the proline is less flexible, leading to a much
smaller space at the entrance to the pocket. As a consequence, a P2-
benzyl substituent is hindered from penetrating deeply into the pock-
et, whereas the smaller and more flexible isobutyl group of P2-Leu is
not.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Regardless of which cell system is the most suitable one for the
testing of peptidomimetic antiviral compounds, we next plan to test
11r in small-animal models for MERS and for Coxsackievirus-
induced pancreatitis. In parallel, we aim to refine the experiments to
quantify the accumulation of peptidomimetic protease inhibitors in
different host-cell types, in the hope to find an explanation for the
observed cell-type dependencies.
Finally, in the 3Cpros of EV-A71 and CVB3, the S2 pocket lacks a
lid, i.e. it is open to one side. As a consequence, it offers less interac-
tion points for P2 substituents of inhibitors, so that such substituents
must reach the "back-wall" of the pocket (formed by Arg39, Asn69,
Glu71) in order to create sufficient binding energy. Hence, large
aromatic substituents such as benzyl are favored by the enterovirus
3Cpros.
CONCLUSIONS
When we introduced a fluoro substituent in the para position of the
P2-benzyl group of our lead compound, 11a, we observed good
activity against the enterovirus 3Cpros but complete inactivity against
the coronavirus Mpros (see Table 1, compound 11m). This is easily
explained on the basis of the crystal structures: In the enterovirus
3Cpros, the fluorine can accept a hydrogen bond from Arg39 (ref. 30),
whereas in the coronavirus Mpros, there would be electrostatic repul-
sion from the main-chain carbonyls of residues 186 and 188. In
agreement with this, rupintrivir (which has P2 = p-fluorobenzyl) is a
good inhibitor of the enteroviral 3Cpros,46 but not of the coronaviral
main proteases, as we predicted earlier.28
This work demonstrates the power of structure-based approaches in
the design of broad-spectrum antiviral compounds with roughly
equipotent activity against coronaviruses and enteroviruses. We
observed a good correlation between the inhibitory activity of the
designed compounds against the isolated proteases, in viral replicons,
and in virus-infected Huh7 cells. One of the compounds (11r) exhibits
excellent anti-MERS-CoV activity in virus-infected Huh7 cells.
Because of the high similarity between the main proteases of SARS-
CoV and the novel BetaCoV/Wuhan/2019, we expect 11r to exhibit
good antiviral activity against the new coronavirus as well.
EXPERIMENTAL SECTION
Crystallization and X-ray structure determination of complex-
a
es between viral proteases and -ketoamides
In this structure-based inhibitor optimization study, we achieved
major improvements over our original lead compound, 11a, by sys-
tematically varying the size and the flexibility of the P2-substituent.
The compound presenting so far the best compromise between the
different requirements of the S2 pockets (SARS-CoV Mpro: large and
Crystallization. The recombinant production and purification of
SARS-CoV Mpro with authentic N and C termini was described in
detail previously.48,49 Using an Amicon YM10 membrane (EMD
Millipore), the purified SARS-CoV Mpro was concentrated to 21
mg·mL-1 in buffer A (20 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 1
mM EDTA, pH 7.5). Crystallization was performed by equilibrating 1
µL protein (mixed with 1 µL precipitant solution) against 500 µl
reservoir containing 6 – 8% polyethylene glycol (PEG) 6,000, 0.1 M
MES (pH 6.0), at 20℃ using the vapor diffusion sitting-drop method.
Compounds 11a and 11s were dissolved in 100% DMSO at 50 mM
and 200 mM stock concentrations, respectively. A crystal of the free
enzyme was soaked in cryo-protectant buffer containing 20% MPD,
6% PEG 6,000, 0.1 M MES, 7.5 mM 11a, pH 6.0, for 2 h at 20℃.
Another set of free enzyme crystals was soaked in another cryo-
protectant buffer with 6% PEG 6,000, 5% MPD, 0.1 M MES, 15%
glycerol, 10 mM 11s, pH 6.0, for 2 h. Subsequently, crystals were
fished and flash-cooled in liquid nitrogen prior to data collection.
covered, HCoV-NL63 Mpro: small and covered, and CVB3 3Cpro
:
large and open) is 11u (P2 = cyclopentylmethyl), which has satisfac-
tory broad-spectrum activity against all proteases tested. However,
with regard to its antiviral activities in cell cultures, it is inferior to
11r (P2 = cyclohexylmethyl). The latter compound exhibits very good
inhibitory activity against the SARS-CoV Mpro as well as the enterovi-
rus 3Cpro, and its performance in the SARS-CoV and enterovirus
replicons is convincing. Being in the low micromolar range (EV-A71,
CVB3), the data for the antiviral activity in cell culture for 11r corre-
late well with the inhibitory power of the compound against the re-
combinant proteases as well as in the replicon-based assays. This is
not true, though, for the surprisingly good in-cellulo activity of 11r
against HCoV 229E in Huh7 cells. Also, the correlation does not
seem to hold for LLC-MK2 and CaCo2 cells. We tested the antiviral
activity of many of our compounds against HCoV NL63 in these two
cell types and found that all of them had low- or submicromolar EC50
values against this virus in LLC-MK2 cells but were largely inactive
in CaCo2 cells (not shown). Furthermore, 11r and all other com-
pounds that we synthesized are inactive (EC50 > 87 µM) against
CVB3 in Vero cells (not shown), but exhibit good to excellent activi-
ties against the same virus in Huh-T7 cells. We have previously
observed similar poor antiviral activities in Vero cells not only for a-
ketoamides, but also for Michael acceptors (Zhu et al., unpublished
work). A similar cell-type dependence is seen for the antiviral activity
Crystals of HCoV-NL63 Mpro with 11a were obtained using cocrys-
tallization. The concentrated HCoV-NL63 Mpro (45 mg·mL-1) was
incubated with 5 mM 11a for 4 h at 20℃, followed by setting up
crystallization using the vapor diffusion sitting-drop method at 20℃
with equilibration of 1 µL protein (mixed with 1 µL mother liquor)
against 500 µL reservoir composed of 0.1 M lithium sulfate monohy-
drate, 0.1 M sodium citrate tribasic dihydrate, 25% PEG 1,000, pH
6.0. The crystals were protected by a cryo-buffer containing 0.1 M
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 20
lithium sulfate monohydrate, 0.1 M sodium citrate tribasic dihydrate,
360 nm, using a Flx800 fluorescence spectrophotometer (BioTek).
25% PEG 1,000, 15% glycerol, 2 mM 11a, pH 6.0 and flash-cooled in
liquid nitrogen.
Curves of relative fluorescence units (RFU) against substrate concen-
tration were linear for all substrates up to beyond 50 µM, indicating a
minimal influence of the inner-filter effect. Stock solutions of the
compounds were prepared by dissolving them in 100% DMSO. The
UV absorption of 11a was found to be negligible at l = 360 nm, so
that no interference with the FRET signal through the inner-filter
effect was to be expected. For the determination of the IC50, different
proteases at a specified final concentration (0.5 µM SARS-CoV or
HCoV-NL63 Mpro, 2 µM CVB3 3Cpro, 3 µM EV-A71 3Cpro) were
separately incubated with the inhibitor at various concentrations (0 to
100 μM) in reaction buffer at 37℃ for 10 min. Afterwards, the reac-
tion was initiated by adding FRET peptide substrate at 20 μM final
concentration (final volume: 50 μl). The IC50 value was determined by
using the GraphPad Prism 6.0 software (GraphPad). Measurements of
enzymatic activity were performed in triplicate and are presented as
the mean ± standard deviations (SD).
1
2
3
4
5
6
7
8
Crystals of HCoV-NL63 Mpro with 11n or 11f were generated by
using the soaking method. Several free-enzyme crystals were soaked
in cryo-protectant buffer containing 0.1 M lithium sulfate monohy-
drate, 0.1 M sodium citrate tribasic dihydrate, 25% PEG 1,000, 15%
glycerol, 5 mM 11n (or 11f), pH 6.0. Subsequently, the soaked crys-
tals were flash-cooled in liquid nitrogen.
Freshly prepared CVB3 3Cpro at a concentration of 21.8 mg·mL-1
was incubated with 5 mM 11a pre-dissolved in 100% DMSO at room
tempature for 1 h. Some white precipitate appeared in the mixture.
Afterwards, the sample was centrifuged at 13,000 x g for 20 min at 4
℃. The supernatant was subjected to crystallization trials using the
following, commercially available kits: SigmaTM (Sigma-Aldrich),
IndexTM, and PEG RxTM (Hampton Research). Single rod-like crystals
were detected both from the IndexTM screen, under the condition of
0.1 M MgCl2 hexahydrate, 0.1 M Bis-Tris, 25% PEG 3,350, pH 5.5,
and from the SigmaTM screen at 0.2 M Li2SO4, 0.1 M Tris-HCl, 30%
PEG 4,000, pH 8.5. Crystal optimization was performed by using the
vapor-diffusion sitting-drop method, with 1 μL CVB3 3Cpro-inhibitor
complex mixed with 1 μL precipitant solution, and equilibration
against 500 μL reservoir containing 0.1 M Tris-HCl, 0.2 M MgCl2, pH
8.5, and PEG 3,350 varied from 22% to 27%. Another optimization
screen was also performed against a different reservoir, 0.1 M Tris-
HCl, 0.2 M MgCl2, pH range from 7.5 to 8.5, and PEG 4,000 varied
from 24% to 34%. Crystals were fished from different drops and
protected by cryo-protectant solution consisting of the mother liquor
and 10% glycerol. Subsequently, the crystals were flash-cooled with
liquid nitrogen.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Assessment of inhibitory activity of a-ketoamides using viral re-
plicons and virus-infected cells
Cells and viruses. Hepatocellular carcinoma cells (Huh7; ref. 58)
and their derivative constitutively expressing T7 RNA polymerase
(Huh-T7; ref. 59) were grown in Dulbecco's modified minimal essen-
tial medium (DMEM) supplemented with 2 mM glutamine, 100
U·mL-1 penicillin, 100 µg·mL-1 streptomycin sulfate, and fetal calf
serum (10% in growth medium and 2% in maintenance medium).
Huh-T7 cells were additionally supplemented with geneticin (G-418
sulfate, 400 µg·mL-1). Huh-T7 cells were used for the enteroviral
replicons as well as for infection experiments with CVB3 strain
Nancy.
For enterovirus (except CVB3) infection experiments, human
rhabdomyosarcoma cells (RD; for EV-A71; BRCR strain) and HeLa
Rh cells (for EV-D68 and human rhinoviruses) were grown in MEM
Rega 3 medium supplemented with 1% sodium bicarbonate, 1% L-
glutamine, and fetal calf serum (10% in growth medium and 2% in
maintenance medium). For HCoV-229E (a kind gift from Volker
Thiel (Bern, Switzerland)), culture and infection experiments were
carried out as described.60 For MERS-CoV or SARS-CoV infection
experiments, Vero, Vero E6, and Huh7 cells were cultured as de-
scribed previously.61,62 Infection of Vero and Huh7 cells with MERS-
CoV (strain EMC/2012) and SARS-CoV infection of Vero E6 cells
(strain Frankfurt-1) at low multiplicity of infection (MOI) were done
as described before.61,63 All work with live MERS-CoV and SARS-
CoV was performed inside biosafety cabinets in biosafety level-3
facilities at Leiden University Medical Center, The Netherlands.
Diffraction data collection, structure elucidation and refine-
ment. Diffraction data from the crystal of the SARS-CoV Mpro in
complex with 11a were collected at 100 K at synchrotron beamline
PXI-X06SA (PSI, Villigen, Switzerland) using a Pilatus 6M detector
(DECTRIS). A diffraction data set from the SARS-CoV Mpro crystal
with compound 11s was collected at 100 K at beamline P11 of
PETRA III (DESY, Hamburg, Germany), using the same type of
detector. All diffraction data sets of HCoV-NL63 Mpro complex struc-
tures and of the complex of CVB3 3Cpro with 11a were collected at
synchrotron beamline BL14.2 of BESSY (Berlin, Germany), using an
MX225 CCD detector (Rayonics). All data sets were processed by the
program XDSAPP and scaled by SCALA from the CCP4 suite.50-52
The structure of SARS-CoV Mpro with 11a was determined by molec-
ular replacement with the structure of the complex between SARS-
CoV Mpro and SG85 (PDB entry 3TNT; Zhu et al., unpublished) as
search model, employing the MOLREP program (also from the CCP4
suite).52,53 The complex structures of HCoV-NL63 Mpro with 11a, 11f,
and 11n were also determined with MOLREP, using as a search
model the structure of the free enzyme determined by us (LZ et al.,
unpublished). The complex structure between CVB3 3Cpro and 11a
was determined based on the search model of the free-enzyme struc-
ture (PDB entry 3ZYD; Tan et al., unpublished). Geometric restraints
for the compounds 11a, 11f, 11n, and 11s were generated by using
JLIGAND52,54 and built into Fo-Fc difference density using the COOT
software.55 Refinement of the structures was performed with
REFMAC version 5.8.0131 (ref. 52,56,57).
Viral replicons. The DNA-launched SARS-CoV replicon harbour-
ing Renilla luciferase as reporter directly downstream of the SARS-
CoV replicase polyprotein-coding sequence (pp1a, pp1ab, Urbani
strain, acc. AY278741), in the context of a bacterial artificial chromo-
some (BAC) under the control of the CMV promoter, has been de-
scribed previously (pBAC-REP-RLuc).33 Apart from the replicase
polyprotein, the replicon encodes the following features: the 5'- and
3'-non-translated regions (NTR), a ribozyme (Rz), the bovine growth
hormone sequence, and structural protein N.
Subgenomic replicons of CVB3 (pT7-CVB3-FLuc34) and EV-A71
(pT7-EV71-RLuc) harbouring T7-controlled complete viral genomes,
in which the P1 capsid-coding sequence was replaced by the Firefly
(Photinus pyralis) or Renilla (Renilla renifor) luciferase gene, were
generously provided by F. van Kuppeveld and B. Zhang, respectively.
To prepare CVB3 and EV-A71 replicon RNA transcripts, plasmid
DNAs were linearized by digestion with SalI or HindIII (New Eng-
land Biolabs), respectively. Copy RNA transcripts were synthesized
in vitro using linearized DNA templates, T7 RNA polymerase, and
the T7 RiboMax™ Large-Scale RNA Production System (Promega)
according to the manufacturer’s recommendations.
Inhibitory activity assay of alpha-ketoamides. A buffer contain-
ing 20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, pH
7.3, was used for all the enzymatic assays. Two substrates with the
cleavage sites of Mpro and 3Cpro, respectively (indicated by the arrow,
↓), Dabcyl-KTSAVLQ¯SGFRKM-E(Edans)-NH2 and Dabcyl-
KEALFQ¯GPPQF-E(Edans)-NH2 (95% purity; Biosyntan), were
employed in the fluorescence resonance energy transfer (FRET)-
based cleavage assay, using a 96-well microtiter plate. The dequench-
ing of the Edans fluorescence due to the cleavage of the substrate as
catalyzed by the proteases was monitored at 460 nm with excitation at
12
ACS Paragon Plus Environment

Page 13 of 20
Journal of Medicinal Chemistry
Transfection. Huh-T7 cells grown in 12-well plates to a confluen-
cy of 80% - 90% (2 - 3 x 105 cells/well) were washed with 1 mL
OptiMEM (Invitrogen) and transfected with 0.25 µg of the replica-
tion-competent replicon and Lipofectamin2000 or X-tremeGENE9 in
300 µl OptiMEM (final volume) as recommended by the manufactur-
er (Invitrogen or Roche, respectively). The transfection mixtures were
incubated at 37°C for 4 to 5 h (Lipofectamin2000) or overnight (X-
tremeGENE9), prior to being replaced with growth medium contain-
ing the compound under investigation. For RNA-launched transfec-
tion of enteroviral replicons, DMRIE-C was used as transfection
reagent according to the manufacturer's recommendations (Invitro-
gen). All experiments were done in triplicate or quadruplicate and the
results are presented as mean values ± SD.
Antiviral assay with Human coronavirus 229E. For HCoV-229E
infection experiments, 5x104 Huh7 cells were infected in triplicate in
24-well plates in 100 µl DMEM at 0.1 pfu/mL. After 1.5 h incubation
at 37oC, virus inocula were removed. Cells were washed with DMEM
and complete DMEM (10% FCS, 1% Pen./Strep.) containing the
desired concentration of inhibitors (0, 1, 2.5, 5, 10, 20, and 40 μM)
were added. After 48 h, the supernatant was collected. Viral RNA was
isolated using the Bioline ISOLATE II RNA Mini Kit (#BIO-52072)
according to the manufacturer’s instructions and eluted in 30 μl
RNase-free water. qPCR was performed using the Bioline SensiFAST
Probe Hi-ROX One-Step Kit (# BIO-77001) in a Roche Light Cy-
cler96. cDNA was synthesized at 48°C for 1800 sec and 95°C for 600
sec, followed by 45 cycles at 95°C for 15 sec and 60°C for 60 sec at a
temperature ramp of 4.4°C/sec. qPCR primer sequences (adapted
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Testing for inhibitory activity of candidate compounds. Initial-
ly, we performed a quick assessment of the inhibitory activity of the
candidate compounds towards the enteroviral and coronaviral repli-
cons at a concentration of 40 µM in Huh-T7 cells. Compounds that
were relatively powerful and non-toxic at this concentration, were
assayed in a dose-dependent manner to estimate their half-maximal
effective concentration (EC50) as well as their cytotoxicity (CC50), as
described.29 In brief, different concentrations of a-ketoamides (40 µM
in screening experiments or increasing concentration (0, 1.25, 2.5, 5,
10, 20, 40 µM) when determining the EC50) were added to growth
medium of replicon-transfected Huh-T7 cells. Twenty-four hours
later, the cells were washed with 1 mL phosphate-buffered saline
(PBS or OPTIMEM, Invitrogen) and lysed in 0.15 mL Passive lysis
buffer (Promega) at room temperature (RT) for 10 min. After freezing
(-80oC) and thawing (RT), the cell debris was removed by centrifuga-
tion (16,000 x g, 1 min) and the supernatant (10 or 20 µl) was assayed
for Firefly or Renilla luciferase activity (Promega or Biotrend
Chemikalien) using an Anthos Lucy-3 luminescence plate reader
(Anthos Microsystem).
from
ref.
64)
were
229E-For:
HCoV-229E-Rev:
5´-
5′-
CTACAGATAGAAAAGTTGCTTT-3´,
ggTCGTTTAGTTGAGAAAAGT -3′, and 229E-ZNA probe: 5'- 6-
Fam-AGA (pdC)TT(pdU)G(pdU)GT(pdC)TA(pdC)T-ZNA-3-BHQ-1
-3' (Metabion). Standard curves were prepared using serial dilutions
of RNA isolated from virus stock. Data were analysed using
GraphPad Prism 5.0; EC50 values were calculated based on a 4-
parameter logistic statistics equation. In parallel to the qPCR assays
with inhibitors, cell viability assays were performed using Alamar-
Blue™ Cell Viability Reagent (ThermoFisher) according to the manu-
facturer’s instruction. CC50 values were calculated using inhibitor
versus normalized response statistics equation by including proper
controls (no inhibitor and 1% Triton-X-100- treated cells).
Determination of the cell toxicity of candidate compounds. The
CellTiter 96Aqueous One Solution Cell Proliferation Assay (MTS
test, Promega), the CellTiter Glo assay kit (Promega), the Non-
Destructive Cytotoxicity Bio-Assay (ToxiLight (measuring the re-
lease of adenylate kinase from damaged cells), Lonza Rockland), or
the AlamarBlue™ Cell Viability Reagent (ThermoFisher) were used
to determine the cytotoxic effect of compounds towards host cells
according to the manufacturers' recommendations.29,65
Antiviral assay with infectious enteroviruses. The antiviral activ-
ity of the compounds was evaluated in a cytopathic effect (CPE) read-
out
assay
using
MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-
inner-
carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium,
Chemical synthesis of -ketoamides
a
salt]-based assay. Briefly, 24 h prior to infection, cells were seeded in
96-well plates at a density of 2.5 x 104 (RD cells) or of 1.7 x 104
(HeLa Rh) per well in medium supplemented with 2% FCS. For
HRV2 and HRV14 infection, the medium contained 30 mM MgCl2.
The next day, serial dilutions of the compounds and virus inoculum
were added. The read-out was performed 3 days post infection as
follows: The medium was removed and 100 μl of 5% MTS in Phenol
Red-free MEM was added to each well. Plates were incubated for 1 h
at 37°C, then the optical density at 498 nm (OD498) of each well was
measured by microtiter plate reader (Saffire2, Tecan). The OD values
were converted to percentage of controls and the EC50 was calculated
by logarithmic interpolation as the concentration of compound that
results in a 50% protective effect against virus-induced CPE. For each
condition, cell morphology was also evaluated microscopically.
General procedure. Reagents were purchased from commercial
sources and used without purification. HSGF 254 (0.15 - 0.2 mm
thickness) was used for analytical thin-layer chromatography (TLC).
All products were characterized by their NMR and MS spectra. 1H
NMR spectra were recorded on 300-MHz, 400-MHz, or 500-MHz
instruments. Chemical shifts were reported in parts per million (ppm,
δ) down-field from tetramethylsilane. Proton coupling patterns were
described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), and broad (br). Mass spectra were recorded using a Bruker ESI
ion-trap HCT Ultra. HPLC spectra were recorded by LC20A or
LC10A (Shimadzu Corporation) with Shim-pack GIST C18 (5 µm,
4.6x150mm) with three solvent systems (methanol/water, methanol/
0.1% HCOOH in water or methanol/0.1% ammonia in water). Purity
was determined by reversed-phase HPLC and was ≥95% for all com-
pounds tested biologically.
Antiviral assays with SARS and MERS coronaviruses. Assays
with MERS-CoV and SARS-CoV were performed as previously
described.61,63 In brief, Huh7, Vero, or Vero E6 cells were seeded in
96-well plates at a density of 1 × 104 (Huh7 and Vero E6) or 2 × 104
cells (Vero) per well. After overnight growth, cells were treated with
the indicated compound concentrations or DMSO (solvent control)
and infected with an MOI of 0.005 (final volume 150 µl/well in
Eagle's minimal essential medium (EMEM) containing 2% FCS, 2
mM L-glutamine, and antibiotics). Huh7 cells were incubated for two
days and Vero/VeroE6 cells for three days, and differences in cell
viability caused by virus-induced CPE or by compound-specific side
effects were analyzed using the CellTiter 96 AQueous Non-Radioactive
Cell Proliferation Assay (Promega), according to the manufacturer’s
instructions. Absorbance at 490 nm (A490) was measured using a
Berthold Mithras LB 940 96-well plate reader (Berthold). Cytotoxic
effects caused by compound treatment alone were monitored in paral-
lel plates containing mock-infected cells.
Synthesis of (2S,4R)-dimethyl 2-(tert-butoxycarbonylamino)-4-
(cyanomethyl) pentanedioate (1).
To a solution of N-Boc-L-glutamic acid dimethyl ester (6.0 g, 21.8
mmol) in THF (60 mL) was added dropwise a solution of lithium
bis(trimethylsilyl)amide (LHMDS) in THF (47 mL, 1 M) at −78°C
under nitrogen. The resulting dark mixture was stirred at −78°C.
Meanwhile, bromoacetonitrile (1.62 mL, 23.3 mmol) was added
dropwise to the dianion solution over a period of 1 h while keeping
the temperature below −70°C. The reaction mixture was stirred at
−78°C for additional 2 h. After the consumption of the reactant was
confirmed by TLC analysis, the reaction was quenched by methanol
(3 mL), and acetic acid (3 mL) in precooled THF (20 mL) was added.
After stirring for 30 min, the cooling bath was removed. The reaction
mixture was allowed to warm up to room temperature and then
poured into brine (40 mL). The organic layer was concentrated and
purified by flash column chromatography (petroleum ether/ethyl
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 20
acetate = 4/1) to give product 1 (4.92 g, 72%) as colorless oil. 1H
was evaporated and the crude material purified on silica, eluted with a
NMR (CDCl3, 400 MHz) δ 5.23 (1H, d, J = 9.0 Hz), 4.43-4.36 (1H,
m), 3.77(1H, s), 3.76 (1H, s), 2.89-2.69 (3H, m), 2.20-2.14 (2H, m),
1.45 (9H, s). ESI-MS (m/z): 315 (M + H)+.
mixture of CH2Cl2/MeOH (40/1) to give the product 6 (62-84%
yield).
1
2
3
4
5
6
7
8
Synthesis of alcohols 7 (general procedure). Compound 6 (1.1
mmol) was dissolved in methanol (40 mL), then NaBH4 (0.34 g, 8.8
mmol) was added under ambient conditions. The reaction mixture
was stirred at 20oC for 2 h. Then the reaction was quenched with
water (30 mL). The suspension was extracted with ethyl acetate. The
organic layers were combined, dried, and filtered. The filtrate was
evaporated to dryness and could be used for the next step without
further purification (46-85% yield).
Synthesis of (S)-methyl 2-(tert-butoxycarbonylamino)-3-((S)-2-
oxopyrrolidin-3-yl)propanoate (2).
In a hydrogenation flask was placed compound 1 (4.0 g, 12.7
mmol), 5 mL of chloroform and 60 mL of methanol before the addi-
tion of PtO2. The resulting mixture was stirred under hydrogen at
20oC for 12 h. Then the mixture was filtered over Celite to remove the
catalyst. NaOAc (6.77 g, 25.5 mmol) was added to the filtrate before
the resulting mixture was stirred at 60oC for 12 h. The reaction was
quenched with water (30 mL). The suspension was extracted with
ethyl acetate. The organic layers were combined, dried (MgSO4), and
filtered. The light-brown filtrate was concentrated and purified by
silica gel column chromatography (petroleum ether/ethyl acetate =
4/1) to give the product 2 (2.20 g, 61%) as white solid. 1H NMR
(CDCl3) δ 6.02 (1H, br), 5.49 (1H, d, J = 7.8 Hz), 4.27-4.33 (1H, m),
3.72 (3H, s), 3.31-3.36 (2H, m), 2.40-2.49 (2H, m), 2.06-2.16 (1H,
m), 1.77-1.89 (2H, m), 1.41 (9H, s). ESI-MS (m/z): 287 (M + H)+.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of aldehydes 8 (general procedure). Compound 7 (0.75
mmol) was dissolved in CH2Cl2, then Dess-Martin periodinane (337
mg, 0.79 mmol) and NaHCO3 (66 mg, 0.79 mmol) were added. The
resulting mixture was stirred at 20oC for 1 h. The mixture was con-
centrated and purified by column chromatography on silica gel
(CH2Cl2/MeOH = 20/1) to give the product 8 as white solid (88-95%
yield).
Synthesis of compounds 9 (general procedure). Compound 8
(0.40 mmol) was dissolved in CH2Cl2, and then acetic acid (0.028 g,
0.47 mmol) and isocyanide (0.43 mmol) were added successively to
the solution. The reaction was stirred at 20oC for 24 h. Then the
solvent was evaporated and the crude material purified on silica,
eluted with a mixture of CH2Cl2/MeOH (20/1) to give the product 9
(46-84%).
Synthesis of (S)-methyl 2-amino-3-((S)-2-oxopyrrolidin-3-
yl)propanoate (3).
Compound 2 (1.0 g, 3.5 mmol) was dissolved in 10 mL dichloro-
methane (DCM), then 10 mL trifluoroacetic acid (TFA) was added.
The reaction mixture was stirred at 20oC for 0.5 h, and concentrated in
vacuo to get a colorless oil, which could be used for the following
step without purification.
ESI-MS (m/z): 187 (M + H)+.
Synthesis of α-hydroxyamides 10 (general procedure). 1 M
NaOH (0.5 mL) was added to a solution of compound 9 (0.164 mmol)
in methanol (5 mL). The reaction was stirred at 20oC for 0.5 h until
the consumption of compound 9 was confirmed by TLC analysis.
Then, 1 M HCl was added to the reaction solution until pH = 7. Fol-
lowing this, the solvent was evaporated to generate the product 10 as
white solid, which could be used directly in the next step.
Synthesis of methyl N-substituted amino-acid esters 4
General procedure. The methyl amino-acid ester hydrochloride
(6.0 mmol) was dissolved in 20 mL CH2Cl2, and then acyl chloride
(6.0 mmol), triethylamine (1.69 mL, 12.0 mmol) were added, before
the reaction was stirred for 2 h at 20oC. The reaction mixture was
diluted with 20 mL CH2Cl2, washed with 50 mL of saturated brine (2
× 25 mL), and dried over Na2SO4. The solvent was evaporated and the
product 4 was obtained as white solid (70-95% yield), which could be
used for the next step without further purification.
Synthesis of α-ketoamides 11 (general procedure). Compound
10 was dissolved in CH2Cl2, then Dess-Martin periodinane (74 mg,
0.176 mmol) and NaHCO3 (30 mg, 0.176 mmol) were added. The
resulting mixture was stirred at 20oC for 1 h. The mixture was con-
centrated and purified by column chromatography on silica gel
(CH2Cl2/MeOH = 20/1) to give the a-ketoamides 11 as light yellow
solid (52-79% in two steps).
(S)-methyl 2-cinnamamido-3-phenylpropanoate (4a).
The methyl L-phenylalaninate hydrochloride (1.30 g, 6.0 mmol)
was dissolved in 20 mL CH2Cl2, and then cinnamoyl chloride (1.00 g,
6.0 mmol), triethylamine (1.69 mL, 12.0 mmol) were added, before
the reaction was stirred for 2 h at room temperature. The reaction
mixture was diluted with 20 mL CH2Cl2, washed with 50 mL of
saturated brine (2×25 mL), and dried over Na2SO4. The solvent was
evaporated and the product 4a was obtained as white solid (1.75 g,
95%), which could be used for the next step without further purifica-
tion.
(S)-N-benzyl-3-((S)-2-cinnamamido-3-phenylpropanamido)-2-oxo-
4-((S)-2-oxopyrrolidin-3-yl)butanamide (11a).
75% yield. 1H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 7.6 Hz, 1H),
7.56 (d, J = 15.2 Hz, 1H), 7.45-7.43 (m, 2H), 7.35-7.19 (m, 12H),
7.02-6.98 (m, 1H), 6.48 (d, J = 15.2 Hz, 1H), 6.44-6.42 (m, 1H), 5.01-
4.92 (m, 2H), 4.46 (d, J = 8.4 Hz, 2H), 3.25-3.03 (m, 4H), 2.24-2.21
(m, 2H), 1.95-1.86 (m, 1H), 1.74-1.69 (m, 1H), 1.55-1.49 (m, 1H)
ppm. ESI-MS (m/z): 567 [M + H]+.
Synthesis of N-substituted amino acids 5 (general procedure). 1
M NaOH (5 mL) was added to a solution of compound 4 (3.0 mmol)
in methanol (5 mL). The reaction was stirred for 20 min at 20oC. Then
1 M HCl was added to the reaction solution until pH = 1. Then the
reaction mixture was extracted with 100 mL of CH2Cl2 (2 × 50 mL)
and the organic layer was washed with 50 mL of brine and dried over
Na2SO4. The solvent was evaporated and the crude material purified
on silica, eluted with mixtures of CH2Cl2/MeOH (20/1) to afford the
product 5 (90-96% yield) as a white solid.
For compounds 11b - 11e, see Supplementary Information.
tert-Butyl ((S)-4-(benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-
3-yl)butan-2-yl)carbamate (11f)
1H NMR (300 MHz, CDCl3) δ 7.32-7.22 (m, 5H), 6.47 (br, 1H),
5.10 (d, J = 8.4 Hz, 1H), 4.37-4.26 (m, 3H), 3.37-3.32 (m, 2H), 2.53-
2.47 (m, 2H), 2.05-1.98 (m, 1H), 1.85-1.79 (m, 1H), 1.62-1.56 (m,
1H), 1.44 (s, 9H) ppm. ESI-MS (m/z): 390 [M + H]+.
Synthesis of compounds 6 (General procedure). Compound 5
(2.7 mmol) was dissolved in 10 mL of dry CH2Cl2. To this solution,
1.5 equiv (1.54 g) of 1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) was
added, and the reaction was stirred for 0.5 h at 20oC. Then compound
3 (500 mg, 2.7 mmol) and TEA (0.70 mL, 5.42 mmol) was added to
the reaction. The reaction was stirred for another 6 h. The reaction
mixture was poured into 10 mL water. The aqueous solution was
extracted with 50 mL of CH2Cl2 (2 × 25 mL) and washed with 50 mL
of saturated brine (2 × 25 mL) and dried over Na2SO4. The solvent
For compounds 11g - 11l, see Supplementary Information.
(S)-N-benzyl-3-((S)-2-cinnamamido-3-(4-
fluorophenyl)propanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-
yl)butanamide (11m)
78% yield, 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 7.6 Hz, 1H),
7.55 (d, J = 15.2 Hz, 1H), 7.43-7.40 (m, 2H), 7.35-7.11 (m, 11H),
7.01-6.98 (m, 1H), 6.46 (d, J = 15.2 Hz, 1H), 6.35-6.31 (m, 1H), 4.99-
14
ACS Paragon Plus Environment

Page 15 of 20
Journal of Medicinal Chemistry
4.91 (m, 2H), 4.43 (d, J = 8.8 Hz, 2H), 3.27-3.12 (m, 3H), 3.05-2.99
(m, 1H), 2.24-2.21 (m, 2H), 2.03-1.96 (m, 1H), 1.72-1.54 (m, 2H)
ppm. ESI-MS (m/z): 585 [M + H]+.
6.48 (m, 1H), 5.35-5.27 (m, 1H), 4.95-4.65 (m, 1H), 4.45 (d, J = 6.5
Hz, 2H), 3.38-3.29 (m, 2H), 2.65-2.35 (m, 1H), 2.00-1.38 (m, 13H),
1.20-1.00 (m, 2H) ppm. ESI-MS (m/z): 559 [M + H]+.
1
2
3
4
(S)-N-((S)-4-(benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-
yl)butan-2-yl)-2-cinnamamido-4-methylpentanamide (11n)
57% yield, 1H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 7.6 Hz, 1H),
7.58 (d, J = 15.6 Hz, 1H), 7.43-7.35 (m, 5H), 7.33-7.14 (m, 4H), 7.02-
6.98 (m, 1H), 6.48 (d, J = 15.6 Hz, 1H), 6.37-6.32 (m, 1H), 4.94-4.86
(m, 1H), 4.68-4.62 (m, 1H), 4.46 (d, J = 8.4 Hz, 2H), 3.25-3.11(m,
1H), 3.09-3.06 (m, 1H), 2.25-2.21 (m, 2H), 1.99-1.92 (m, 1H), 1.73-
1.64 (m, 3H), 1.58-1.48 (m, 2H), 0.92 (d, J = 8.4 Hz, 3H), 0.88 (d, J =
8.4 Hz, 3H) ppm. ESI-MS (m/z): 533 [M + H]+.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(S)-N-((S)-4-(benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-
yl)butan-2-yl)-2-cinnamamidohexanamide (11o)
76% yield, 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 7.6 Hz, 1H),
7.55 (d, J = 15.2 Hz, 1H), 7.43-7.36 (m, 5H), 7.28-7.14 (m, 4H), 7.01-
6.98 (m, 1H), 6.45 (d, J = 15.2 Hz, 1H), 6.37-6.32 (m, 1H), 4.98-4.91
(m, 1H), 4.73-4.67 (m, 1H), 4.48 (d, J = 8.0 Hz, 2H), 3.25-3.11(m,
1H), 3.09-3.03 (m, 1H), 2.25-2.21 (m, 2H), 1.92-1.86 (m, 1H), 1.73-
1.64 (m, 3H), 1.56-1.52 (m, 1H), 1.36-1.25 (m, 4H), 0.93 (t, J = 8.4
Hz, 3H) ppm. ESI-MS (m/z): 533 [M + H]+.
(S)-N-((S)-4-(benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-
yl)butan-2-yl)-2-cinnamamidopent-4-ynamide (11p)
65% yield, 1H NMR (500 MHz, CDCl3) δ 8.56 (d, J = 7.5 Hz, 1H),
7.60 (d, J = 15.0 Hz, 1H), 7.53-7.46 (m, 2H), 7.38-7.17 (m, 7H), 6.53-
6.42 (m, 2H), 5.32-5.25 (m, 1H), 4.85-4.65 (m, 1H), 4.47 (d, J = 8.5
Hz, 2H), 3.43-3.29 (m, 3H), 2.59-2.45 (m, 1H), 2.20-1.60 (m, 7H)
ppm. ESI-MS (m/z): 515 [M + H]+.
(S)-N-benzyl-3-((S)-2-cinnamamido-2-cyclopropylacetamido)-2-
oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11q).
66% yield, 1H NMR (500 MHz, CDCl3) δ 8.62 (d, J = 7.5 Hz, 1H),
7.60 (d, J = 15.0 Hz, 1H), 7.53-7.43 (m, 2H), 7.35-7.17 (m, 7H), 6.76-
6.69 (m, 1H), 6.59-6.48 (m, 1H), 5.35-5.25 (m, 1H), 4.85-4.72 (m,
1H), 4.48 (d, J = 8.5 Hz, 2H), 3.38-3.22 (m, 2H), 2.62-2.45 (m, 1H),
2.12-1.63 (m, 4H), 1.20-0.92 (m, 1H), 0.46 (t, J = 7.0 Hz, 2H), 0.16-
0.07 (m, 2H) ppm. ESI-MS (m/z): 517 [M + H]+.
(S)-N-benzyl-3-((S)-2-cinnamamido-3-cyclohexylpropanamido)-2-
oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11r)
71% yield, 1H NMR (500 MHz, CDCl3) δ 8.56 (t, J = 6.0 Hz, 1H),
7.61 (d, J = 16.0 Hz, 1H), 7.52-7.44 (m, 3H), 7.35-7.20 (m, 6H), 6.66-
6.59 (m, 1H), 6.48 (d, J = 13.0 Hz, 1H), 5.32-5.27 (m, 1H), 4.95-4.75
(m, 1H), 4.48 (d, J = 6.5 Hz, 2H), 3.39-3.29 (m, 2H), 2.65-2.35 (m,
2H), 2.09-1.68 (m, 10H), 1.29-1.16 (m, 4H), 1.00-0.88 (m, 2H) ppm.
ESI-MS (m/z): 573 [M + H]+.
(S)-N-benzyl-3-((S)-2-cinnamamido-3-cyclopropylpropanamido)-
2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11s).
64% yield, 1H NMR (500 MHz, CDCl3) δ 8.64 (d, J = 7.5 Hz, 1H),
7.60 (d, J = 15.0 Hz, 1H), 7.52-7.46 (m, 2H), 7.36-7.17 (m, 7H), 6.54-
6.42 (m, 2H), 5.35-5.25 (m, 1H), 4.85-4.75 (m, 1H), 4.46 (d, J = 8.5
Hz, 2H), 3.38-3.29 (m, 2H), 2.65-2.35 (m, 1H), 2.15-1.90 (m, 2H),
1.85-1.60 (m, 4H), 0.90-0.72 (m, 1H), 0.47 (t, J = 7.0 Hz, 2H), 0.15-
0.07 (m, 2H) ppm. ESI-MS (m/z): 531 [M + H]+.
(S)-N-benzyl-3-((S)-2-cinnamamido-3-cyclobutylpropanamido)-2-
oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11t)
77% yield, 1H NMR (500 MHz, CDCl3) δ 8.62 (t, J = 6.5 Hz, 1H),
7.60 (d, J = 15.0 Hz, 1H), 7.52-7.46 (m, 2H), 7.35-7.19 (m, 7H), 6.72-
6.60 (m, 1H), 6.48 (d, J = 15.0 Hz, 1H), 5.32-5.26 (m, 1H), 4.77-4.69
(m, 1H), 4.49 (d, J = 6.5 Hz, 2H), 3.40-3.31 (m, 2H), 2.60-2.35 (m,
3H), 2.09-1.68 (m, 11H) ppm. ESI-MS (m/z): 545 [M + H]+.
(S)-N-benzyl-3-((S)-2-cinnamamido-3-cyclopentylpropanamido)-2-
oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11u)
1
79% yield, H NMR (500 MHz, CDCl3) δ 7.60 (d, J = 15.0 Hz,
1H), 7.50-7.44 (m, 2H), 7.36-7.20 (m, 7H), 6.76-6.69 (m, 1H), 6.59-
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 20
and Prof. Volker Thiel (Berne) for HCoV 229E. We are grateful
to Dr. Naoki Sakai and the staff at synchrotron beamlines for help
with diffraction data collection.
ASSOCIATED CONTENT
1
2
3
Supporting Information
Detailed results of the variation of the P1' and P3 substitu-
ents
4
5
6
7
8
9
Synthesis of a-ketoamides 11b - 11e and 11g - 11l
Supplemental Table 1: inhibitory activities (IC50 (μM)) of
α-ketoamides with P1' and P3 modifications against viral
proteases
Supplemental Table 2: crystallographic data for complexes
between viral proteases and α-ketoamides
ABBREVIATIONS
3CLpro, 3C-like protease; 3Cpro, 3C protease; A490, absorbance at 490
nm; ap, antiperiplanar; BAC, bacterial artificial chromosome; CPE,
cytopathic effect; CVA16, Coxsackievirus A16; CVB3, Cox-
sackievirus B3; DMEM, Dulbecco's modified minimal essential
medium; EMEM, Eagle's minimal essential medium; EV, enterovirus;
FIPV, Feline Infectious Peritonitis Virus; FRET, fluorescence reso-
nance energy transfer; GlnLactam, glutamine lactam; HCoV, human
coronavirus; HFMD, Hand, Foot, and Mouth Disease; HRV, human
rhinovirus; MERS-CoV, Middle-East respiratory syndrome corona-
virus; Mpro, main protease; Nsp5, non-structural protein 5; NTR, non-
translated region; OD498, optical density at 498 nm; PEG, polyeth-
ylene glycol; RFU, relative fluorescence units; Rz, ribozyme; SARS,
severe acute respiratory syndrome; SARS-CoV, SARS coronavirus; -
sc, (-)-synclinal; SD, standard deviation; TLC, thin-layer chromato-
graphy
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Molecular formula strings and biological data (CSV)
Accession Codes
Authors will release the atomic coordinates and experi-
mental data upon article publication. Atomic coordinates
include SARS-CoV Mpro in complex with compounds 11a
(5N19), 11s (5N5O), HCoV-NL63 Mpro in complex with
11a (6FV2), 11n (6FV1), 11f (5NH0), and CVB3 3Cpro in
complex with (5NFS).
AUTHOR INFORMATION
Corresponding Author
* Prof. Rolf Hilgenfeld
Institute of Biochemistry
University of Lübeck
Ratzeburger Allee 160
23562 Lübeck
Germany
Tel.: +49-451-3101-3100; +49-1520-1450335
Fax: +49-451-3101-3104
Email: hilgenfeld@biochem.uni-luebeck.de
* Prof. Hong Liu
Shanghai Institute of Materia Medica
Zu Chong Zhi Road 555
Shanghai, 201203
China
Email: hliu@simm.ac.cn
Present Addresses
†Changchun Discovery Sciences Ltd., Changchun, China
Author Contributions
The manuscript was written through contributions by all authors. /
All authors have given approval to the final version of the manu-
script. / ‡These authors contributed equally.
Funding Sources
Financial support by the European Commission through its
SILVER project (contract HEALTH-F3-2010-260644 with RH,
JN, and EJS) and the German Center for Infection Research
(DZIF; TTU 01.803 to RH and AvB) is gratefully acknowledged.
HL thanks the Natural Science Foundation of China (grant no.
81620108027) for support.
ACKNOWLEDGMENT
We thank Doris Mutschall, Javier Carbajo-Lozoya, Dev Raj
Bairad, and Sebastian Schwinghammer for expert technical assis-
tance, Dr. Bo Zhang and Prof. Frank van Kuppeveld (Wuhan and
Utrecht, resp.) for the EV-A71 and CVB3 replicons, Prof. Ron
Fouchier (Rotterdam) for the EMC/2012 strain of MERS-CoV,
16
ACS Paragon Plus Environment

Page 17 of 20
Journal of Medicinal Chemistry
review and clinical highlights from the 2014 U.S. outbreak. Ann. Am.
Thorac. Soc. 2015, 12, 775-781.
REFERENCES
1
2
3
4
1. Hilgenfeld, R.; Peiris, M. From SARS to MERS: 10 years of re-
search on highly pathogenic human coronaviruses. Antiviral Res.
2013, 100, 286-295.
15. Xing, W.; Liao, Q.; Viboud, C.; Zhang, J.; Sun, J.; Wu, J. T.;
Chang, Z.; Liu, F.; Fang, V. J.; Zheng, Y.; Cowling, B. J.; Varma, J.
K.; Farrar, J. J.; Leung, G. M.; Yu, H., Hand, foot, and mouth disease
in China, 2008-12: an epidemiological study. Lancet Infect. Dis. 2014,
14, 308-318.
5
6
7
8
2. Zaki, A. M.; van Boheemen, S.; Bestebroer, T. M.; Osterhaus, A.
D.; Fouchier, R. A. Isolation of a novel coronavirus from a man with
pneumonia in Saudi Arabia. N. Engl. J. Med. 2012, 367, 1814-1820.
16. Massilamany, C.; Gangaplara, A.; Reddy, J. Intricacies of car-
diac damage in coxsackievirus B3 infection: implications for therapy.
Int. J. Cardiol. 2014, 177, 330-339.
3. Eckerle, I.; Muller, M. A.; Kallies, S.; Gotthardt, D. N.; Drosten,
C. In-vitro renal epithelial cell infection reveals a viral kidney tropism
as a potential mechanism for acute renal failure during Middle East
Respiratory Syndrome (MERS) Coronavirus infection. Virol. J. 2013,
10, 359.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17. Coleman, L.; Laing, I. A.; Bosco, A. Rhinovirus-induced asth-
ma exacerbations and risk populations. Curr. Opin. Allergy Clin.
Immunol. 2016, 16, 179-185.
4. Butler, D. South Korean MERS outbreak spotlights lack of re-
search. Nature 2015, 522, 139-140.
18. Greninger, A. L.; Naccache, S. N.; Messacar, K.; Clayton, A.;
Yu, G.; Somasekar, S.; Federman, S.; Stryke, D.; Anderson, C.; Yagi,
S.; Messenger, S.; Wadford, D.; Xia, D.; Watt, J. P.; Van Haren, K.;
Dominguez, S. R.; Glaser, C.; Aldrovandi, G.; Chiu, C. Y. A novel
outbreak enterovirus D68 strain associated with acute flaccid myelitis
cases in the USA (2012-14): a retrospective cohort study. Lancet
Infect. Dis. 2015, 15, 671-682.
5. Ge, X. Y.; Li, J. L.; Yang, X. L.; Chmura, A. A.; Zhu, G.; Ep-
stein, J. H.; Mazet, J. K.; Hu, B.; Zhang, W.; Peng, C.; Zhang, Y. J.;
Luo, C. M.; Tan, B.; Wang, N.; Zhu, Y.; Crameri, G.; Zhang, S. Y.;
Wang, L. F.; Daszak, P.; Shi, Z. L. Isolation and characterization of a
bat SARS-like coronavirus that uses the ACE2 receptor. Nature 2013,
503, 535-538.
19. Chang, L. Y.; Lin, T. Y.; Hsu, K. H.; Huang, Y. C. ; Lin, K. L.;
Hsueh, C.; Shih, S. R.; Ning, H. C.; Hwang, M. S.; Wang, H. S.; Lee,
C. Y. Clinical features and risk factors of pulmonary oedema after
enterovirus-71-related hand, foot, and mouth disease. Lancet 1999,
354, 1682-1686.
6. Menachery, V. D.; Yount, B. L., Jr.; Debbink, K.; Agnihothram,
S.; Gralinski, L. E.; Plante, J. A.; Graham, R. L.; Scobey, T.; Ge, X.
Y.; Donaldson, E. F.; Randell, S. H.; Lanzavecchia, A.; Marasco, W.
A.; Shi, Z. L.; Baric, R. S. A SARS-like cluster of circulating bat
coronaviruses shows potential for human emergence. Nat. Med. 2015,
21, 1508-1513. Erratum in Nat. Med. 2016, 22, 446
20. Wong, K. T.; Munisamy, B.; Ong, K. C.; Kojima, H.; Noriyo,
N.; Chua, K. B.; Ong, B. B.; Nagashima, K. The distribution of in-
flammation and virus in human enterovirus 71 encephalomyelitis
suggests possible viral spread by neural pathways. J. Neuropathol.
Exp. Neurol. 2008, 67, 162-169.
7. Yang X. L.; Hu B.; Wang B.; Wang M. N.; Zhang Q.; Zhang W.;
Wu L. J.; Ge X. Y.; Zhang Y. Z.; Daszak P.; Wang L. F.; Shi Z. L.
Isolation and characterization of a novel bat coronavirus closely
related to the direct progenitor of severe acute respiratory syndrome
coronavirus. J. Virol. 2016, 90, 3253-3256.
21. Gorbalenya, A. E.; Donchenko, A. P.; Blinov, V. M.; Koonin,
E. V. Cysteine proteases of positive strand RNA viruses and chymo-
trypsin-like serine proteases. A distinct protein superfamily with a
common structural fold. FEBS Lett. 1989, 243, 103-114.
8. Hu, B.; Zeng, L. P.; Yang, X. L.; Ge, X. Y.; Zhang, W.; Li, B.;
Xie, J. Z.; Shen, X. R.; Zhang, Y. Z.; Wang, N.; Luo, D. S.; Zheng, X.
S.; Wang, M. N.; Daszak, P.; Wang, L. F.; Cui, J.; Shi, Z. L. Discov-
ery of a rich gene pool of bat SARS-related coronaviruses provides
new insights into the origin of SARS coronavirus. PLoS Pathog. 2017,
13, e1006698.
22. Anand, K.; Palm, G. J.; Mesters, J. R.; Siddell, S. G.; Ziebuhr,
J.; Hilgenfeld, R. Structure of coronavirus main proteinase reveals
combination of a chymotrypsin fold with an extra alpha-helical do-
main. EMBO J. 2002, 21, 3213-3224.
9. van der Hoek, L.; Pyrc, K.; Jebbink, M. F.; Vermeulen-Oost, W.;
Berkhout, R. J.; Wolthers, K. C.; Wertheim-van Dillen, P. M.; Kaan-
dorp, J.; Spaargaren, J.; Berkhout, B. Identification of a new human
coronavirus. Nat. Med. 2004, 10, 368-373.
23. Hayden F. G.; Turner R. B.; Gwaltney J. M.; Chi-Burris K.;
Gersten M.; Hsyu P.; Patick A. K.; Smith G. J.; Zalman L. S. Phase II,
randomized, double-blind, placebo-controlled studies of rupintrivir
nasal spray 2-percent suspension for prevention and treatment of
experimentally induced rhinovirus colds in healthy volunteers. Anti-
microb. Agents Chemother. 2003, 47, 3907-3916.
10. Hamre, D.; Procknow, J. J. A new virus isolated from the hu-
man respiratory tract. Proc. Soc. Exp. Biol. Med. 1966, 121, 190-193.
24. Kim, Y.; Liu, H., Galasiti Kankanamalage, A. C.; Weerasekara,
S.; Hua, D. H.; Groutas, W. C.; Chang, K. O.; Pedersen, N. C. Rever-
sal of the progression of fatal coronavirus infection in cats by a broad-
spectrum coronavirus protease inhibitor. PLoS Pathog. 2016, 12,
e1005531. Erratum: PLoS Pathog. 2016, 12, e1005650.
11. da Silva Filho, L. V.; Zerbinati, R. M.; Tateno, A. F.; Boas, L.
V.; de Almeida, M. B.; Levi, J. E.; Drexler, J. F.; Drosten, C.; Pannuti,
C. S. The differential clinical impact of human coronavirus species in
children with cystic fibrosis. J. Infect. Dis. 2012, 206, 384-388.
12. Mayer, K.; Nellessen, C.; Hahn-Ast, C.; Schumacher, M.;
Pietzonka, S.; Eis-Hubinger, A. M.; Drosten, C.; Brossart, P.; Wolf, D.
Fatal outcome of human coronavirus NL63 infection despite success-
ful viral elimination by IFN-alpha in a patient with newly diagnosed
ALL. Eur. J. Haematol. 2016, 97, 208-210.
25. 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 respira-
tory syndrome virus main protease and its complex with an inhibitor.
Proc. Natl. Acad. Sci. USA 2003, 100, 13190-13195.
13. Lee, J.; Storch, G. A. Characterization of human coronavirus
OC43 and human coronavirus NL63 infections among hospitalized
children <5 years of age. Pediatr. Infect. Dis. J. 2014, 33, 814-820.
26. Tan, J.; Verschueren, K. H.; Anand, K.; Shen, J.; Yang, M.; Xu,
Y.; Rao, Z.; Bigalke, J.; Heisen, B.; Mesters, J. R.; Chen, K.; Shen, X.;
Jiang, H.; Hilgenfeld, R. pH-dependent conformational flexibility of
the SARS-CoV main proteinase Mpro dimer: molecular dynamics
14. Oermann, C. M.; Schuster, J. E.; Conners, G. P.; Newland, J.
G.; Selvarangan, R.; Jackson, M. A. Enterovirus D68. A focused
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 20
simulations and multiple X-ray structure analyses. J. Mol. Biol. 2005,
W.; Parekh, T.; Pinto, P.; Popov, V.; Pike, R.; Ruan, S.; Santhanam,
354, 25-40.
B.; Vibulbhan, B.; Wu, W.; Yang, W.; Kong, J.; Liang, X.; Wong, J.;
Liu, R.; Butkiewicz, N.; Chase, R.; Hart, A.; Agrawal, S.; Ingravallo,
P.; Pichardo, J.; Kong, R.; Baroudy, B.; Malcolm, B.; Guo, Z.;
Prongay, A.; Madison, V.; Broske, L.; Cui, X.; Cheng, K. C.; Hsieh,
Y.; Brisson, J. M.; Prelusky, D.; Korfmacher, W.; White, R.; Bog-
danowich-Knipp, S.; Pavlovsky, A.; Bradley, P.; Saksena, A. K.;
Ganguly, A.; Piwinski, J.; Girijavallabhan, V.; Njoroge, F. G. Discov-
ery of (1R,5S)-N-[3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl]- 3-
[2(S)-[[[(1,1-dimethylethyl)amino]carbonyl]amino]-3,3-dimethyl-1-
oxobutyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2(S)-carboxamide
(SCH 503034), a selective, potent, orally bioavailable hepatitis C
virus NS3 protease inhibitor: a potential therapeutic agent for the
treatment of hepatitis C infection. J. Med. Chem. 2006, 49, 6074-6086.
1
2
3
4
5
6
7
8
27. Hilgenfeld, R. From SARS to MERS: crystallographic studies
on coronaviral proteases enable antiviral drug design. FEBS J. 2014,
281, 4085-4096.
28. Anand, K.; Ziebuhr, J.; Wadhwani, P.; Mesters, J. R.; Hilgen-
feld, R. Coronavirus main proteinase (3CLpro) structure: basis for
design of anti-SARS drugs. Science 2003, 300, 1763-1767.
9
29. Tan, J.; George, S.; Kusov, Y.; Perbandt, M.; Anemuller, S.;
Mesters, J. R.; Norder, H.; Coutard, B.; Lacroix, C.; Leyssen, P.;
Neyts, J.; Hilgenfeld, R. 3C protease of enterovirus 68: structure-
based design of Michael acceptor inhibitors and their broad-spectrum
antiviral effects against picornaviruses. J. Virol. 2013, 87, 4339-4351.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
41. Mandadapu, S. R.; Weerawarna, P. M.; Gunnam, M. R.; Allis-
ton, K. R.; Lushington, G. H.; Kim, Y.; Chang, K. O.; Groutas, W. C.
Potent inhibition of norovirus 3CL protease by peptidyl alpha-
ketoamides and alpha-ketoheterocycles. Bioorg. Med. Chem. Lett.
2012, 22, 4820-4826.
30. Lu, G.; Qi, J.; Chen, Z.; Xu, X.; Gao, F.; Lin, D.; Qian, W.; Liu,
H.; Jiang, H.; Yan, J.; Gao, G. F. Enterovirus 71 and Coxsackievirus
A16 3C proteases: binding to rupintrivir and their substrates and anti-
hand, foot, and mouth disease virus drug design. J. Virol. 2011, 85,
10319-10331.
42. Zeng, D.; Ma, Y.; Zhang, R.; Nie, Q.; Cui, Z.; Wang, Y.; Shang,
L.; Yin, Z. Synthesis and structure-activity relationship of alpha-keto
amides as enterovirus 71 3C protease inhibitors. Bioorg. Med. Chem.
Lett. 2016, 26, 1762-1766.
31. Wang, F.; Chen, C.; Tan, W.; Yang, K.; Yang, H. Structure of
main protease from human coronavirus NL63: insights for wide
spectrum anti-coronavirus drug design. Sci. Rep. 2016, 6, 22677.
43. Kim, Y.; Kankanamalage, A. C.; Damalanka, V. C.; We-
erawarna, P. M.; Groutas, W. C.; Chang, K. O. Potent inhibition of
enterovirus D68 and human rhinoviruses by dipeptidyl aldehydes and
alpha-ketoamides. Antiviral Res. 2016, 125, 84-91.
32. Dragovich, P. S.; Zhou, R.; Skalitzky, D. J.; Fuhrman, S. A.;
Patick, A. K.; Ford, C. E.; Meador, J. W., 3rd; Worland, S. T. Solid-
phase synthesis of irreversible human rhinovirus 3C protease inhibi-
tors. Part 1: Optimization of tripeptides incorporating N-terminal
amides. Bioorg. Med. Chem. 1999, 7, 589-598.
44. Kim, Y.; Lovell, S.; Tiew, K. C.; Mandadapu, S. R.; Alliston,
K. R.; Battaile, K. P.; Groutas, W. C.; Chang, K. O. Broad-spectrum
antivirals against 3C or 3C-like proteases of picornaviruses, no-
roviruses, and coronaviruses. J. Virol. 2012, 86, 11754-11762.
33. Kusov, Y.; Tan, J.; Alvarez, E.; Enjuanes, L.; Hilgenfeld, R. A
G-quadruplex-binding macrodomain within the "SARS-unique do-
main" is essential for the activity of the SARS-coronavirus replica-
tion-transcription complex. Virology 2015, 484, 313-322.
45. Prior, A. M.; Kim, Y.; Weerasekara, S.; Moroze, M.; Alliston,
K. R.; Uy, R. A.; Groutas, W. C.; Chang, K. O.; Hua, D. H. Design,
synthesis, and bioevaluation of viral 3C and 3C-like protease inhibi-
tors. Bioorg. Med. Chem. Lett. 2013, 23, 6317-6320.
34. van Kuppeveld, F. J.; Galama, J. M.; Zoll, J.; Melchers, W. J.
Genetic analysis of a hydrophobic domain of coxsackie B3 virus
protein 2B: a moderate degree of hydrophobicity is required for a cis-
acting function in viral RNA synthesis. J. Virol. 1995, 69, 7782-7790.
46. Lee, C. C.; Kuo, C. J.; Ko, T. P.; Hsu, M. F.; Tsui, Y. C.;
Chang, S. C.; Yang, S.; Chen, S. J.; Chen, H. C.; Hsu, M. C.; Shih, S.
R.; Liang, P. H.; Wang, A. H. Structural basis of inhibition specifici-
ties of 3C and 3C-like proteases by zinc-coordinating and pep-
tidomimetic compounds. J. Biol. Chem. 2009, 284, 7646-7655.
35. Lanke, K. H.; van der Schaar, H. M.; Belov, G. A.; Feng, Q.;
Duijsings, D.; Jackson, C. L.; Ehrenfeld, E.; van Kuppeveld, F. J.
GBF1, a guanine nucleotide exchange factor for Arf, is crucial for
Coxsackievirus B3 RNA replication. J. Virol. 2009, 83, 11940-11949.
47. Binford, S. L.; Maldonado, F.; Brothers, M. A.; Weady, P. T.;
Zalman, L. S.; Meador, J. W., 3rd; Matthews, D. A.; Patick, A. K.
Conservation of amino acids in human rhinovirus 3C protease corre-
lates with broad-spectrum antiviral activity of rupintrivir, a novel
human rhinovirus 3C protease inhibitor. Antimicrob. Agents
Chemother. 2005, 49, 619-626.
36. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.;
Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera – a
visualization system for exploratory research and analysis. J. Comput.
Chem. 2004, 25, 1605-1612.
37. Zhu, L.; George, S.; Schmidt, M. F.; Al-Gharabli, S. I.; Rade-
mann, J.; Hilgenfeld, R. Peptide aldehyde inhibitors challenge the
substrate specificity of the SARS-coronavirus main protease. Antivi-
ral Res. 2011, 92, 204-212.
48. Xue, X.; Yang, H.; Shen, W.; Zhao, Q.; Li, J.; Yang, K.; Chen,
C.; Jin, Y.; Bartlam, M.; Rao, Z. Production of authentic SARS-CoV
M(pro) with enhanced activity: application as a novel tag-cleavage
endopeptidase for protein overproduction. J. Mol. Biol. 2007, 366,
965-975.
38. Qian, J.; Cuerrier, D.; Davies, P. L.; Li, Z.; Powers, J. C.;
Campbell, R. L. Cocrystal structures of primed side-extending alpha-
ketoamide inhibitors reveal novel calpain-inhibitor aromatic interac-
tions. J. Med. Chem. 2008, 51, 5264-5270.
49. Verschueren, K. H.; Pumpor, K.; Anemuller, S.; Chen, S.; Mes-
ters, J. R.; Hilgenfeld, R. A structural view of the inactivation of the
SARS coronavirus main proteinase by benzotriazole esters. Chem.
Biol. 2008, 15, 597-606.
39. Romano, K. P.; Ali, A.; Aydin, C.; Soumana, D.; Ozen, A.;
Deveau, L. M.; Silver, C.; Cao, H.; Newton, A.; Petropoulos, C. J.;
Huang, W.; Schiffer, C. A. The molecular basis of drug resistance
against hepatitis C virus NS3/4A protease inhibitors. PLoS Pathog.
2012, 8, e1002832.
50. Krug M.; Weiss M. S.; Heinemann U.; Mueller U. XDSAPP: a
graphical user interface for the convenient processing of diffraction
data using XDS. J. Appl. Crystal. 2012, 45, 568-572.
40. Venkatraman, S.; Bogen, S. L.; Arasappan, A.; Bennett, F.;
Chen, K.; Jao, E.; Liu, Y. T.; Lovey, R.; Hendrata, S.; Huang, Y.; Pan,
18
ACS Paragon Plus Environment

Page 19 of 20
Journal of Medicinal Chemistry
51. Evans, P. R. An introduction to data reduction: space-group de-
interactome: identification of cyclophilins as target for pan-
termination, scaling and intensity statistics. Acta Crystallogr. D Biol.
Crystallogr. 2011, 67, 282-292.
coronavirus inhibitors. PLoS Pathog. 2011, 7, e1002331
1
2
3
4
5
6
7
8
61. de Wilde, A. H.; Raj, V. S.; Oudshoorn, D.; Bestebroer, T. M.;
van Nieuwkoop, S.; Limpens, R. W.; Posthuma, C. C.; van der Meer,
Y.; Barcena, M.; Haagmans, B. L.; Snijder, E. J.; van den Hoogen, B.
G. MERS-coronavirus replication induces severe in vitro cytopathol-
ogy and is strongly inhibited by cyclosporin A or interferon-alpha
treatment. J. Gen. Virol. 2013, 94, 1749-1760.
52. Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.;
Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A.
G.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.;
Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S.
Overview of the CCP4 suite and current developments. Acta Crystal-
logr. D Biol. Crystallogr. 2011, 67, 235-242.
62. Snijder, E. J.; van der Meer, Y.; Zevenhoven-Dobbe, J.;
Onderwater, J. J.; van der Meulen, J.; Koerten, H. K.; Mommaas, A.
M. Ultrastructure and origin of membrane vesicles associated with the
severe acute respiratory syndrome coronavirus replication complex. J.
Virol. 2006, 80, 5927-5940.
9
53. Vagin, A.; Teplyakov, A. Molecular replacement with
MOLREP. Acta Crystallogr. D Biol. Crystallogr. 2010, 66, 22-25.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
54. Lebedev, A. A.; Young, P.; Isupov, M. N.; Moroz, O. V.;
Vagin, A. A.; Murshudov, G. N. JLigand: a graphical tool for the
CCP4 template-restraint library. Acta Crystallogr. D Biol. Crystallogr.
2012, 68, 431-440.
63. de Wilde, A. H.; Jochmans, D.; Posthuma, C. C.; Zevenhoven-
Dobbe, J. C.; van Nieuwkoop, S.; Bestebroer, T. M.; van den Hoogen,
B. G.; Neyts, J.; Snijder, E. J. Screening of an FDA-approved com-
pound library identifies four small-molecule inhibitors of Middle East
respiratory syndrome coronavirus replication in cell culture. Antimi-
crob. Agents Chemother. 2014, 58, 4875-4884.
55. Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features
and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 2010,
66, 486-501.
56. Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of
macromolecular structures by the maximum-likelihood method. Acta
Crystallogr. D Biol. Crystallogr. 1997, 53, 240-255.
64. Chan, J. F.; Choi, G. K.; Tsang, A. K.; Tee, K. M.; Lam, H. Y.;
Yip, C. C.; To, K. K.; Cheng, V. C.; Yeung, M. L.; Lau, S. K.; Woo,
P. C.; Chan, K. H.; Tang, B. S.; Yuen, K. Y. Development and evalu-
ation of novel real-time reverse transcription-PCR assays with locked
nucleic acid probes targeting leader sequences of human-pathogenic
coronaviruses. J. Clin. Microbiol. 2015, 53, 2722-2726.
57. Murshudov, G. N.; Skubak, P.; Lebedev, A. A.; Pannu, N. S.;
Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A.
REFMAC5 for the refinement of macromolecular crystal structures.
Acta Crystallogr. D Biol. Crystallogr. 2011, 67, 355-367.
65. Carbajo-Lozoya, J.; Ma-Lauer, Y.; Malesevic, M.; Theuerkorn,
M.; Kahlert, V.; Prell, E.; von Brunn, B.; Muth, D.; Baumert, T. F.;
Drosten, C.; Fischer, G.; von Brunn, A. Human coronavirus NL63
replication is cyclophilin A-dependent and inhibited by non-
immunosuppressive cyclosporine A-derivatives including Alisporivir.
Virus Res. 2014, 184, 44–53.
58. Nakabayashi, H.; Taketa, K.; Miyano, K.; Yamane, T.; Sato, J.
Growth of human hepatoma cells lines with differentiated functions in
chemically defined medium. Cancer Res. 1982, 42, 3858-3863.
59. Schultz, D. E.; Honda, M.; Whetter, L. E.; McKnight, K. L.;
Lemon, S. M. Mutations within the 5' nontranslated RNA of cell
culture-adapted hepatitis A virus which enhance cap-independent
translation in cultured African green monkey kidney cells. J. Virol.
1996, 70, 1041-1049.
66. Weiss M. S.; Hilgenfeld R. On the use of the merging R factor
as a quality indicator for X-ray data. J. Appl. Cryst. 1997, 30, 203-205.
60. Pfefferle, S.; Schöpf, J.; Kögl, M.; Friedel, C. C.; Müller, M. A.;
Carbajo-Lozoya, J.; Stellberger, T., von Dall'Armi, E.; Herzog, P.;
Kallies, S.; Niemeyer, D.; Ditt, V.; Kuri, T.; Züst, R.; Pumpor, K.;
Hilgenfeld, R.; Schwarz, F.; Zimmer, R.; Steffen, I.; Weber, F.; Thiel,
V.; Herrler, G.; Thiel, H. J.; Schwegmann-Wessels, C.; Pöhlmann, S.;
Haas, J.; Drosten, C.; von Brunn, A. The SARS-coronavirus-host
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 20
SYNOPSIS TOC
Table of Contents Graphic
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
20
ACS Paragon Plus Environment