Design, synthesis, and bioevaluation of viral 3C and 3C-like protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.09.070

A class of tripeptidyl transition state inhibitors containing a P1 glutamine surrogate, a P2 leucine, and a P3 arylalanines, was found to potently inhibit Norwalk virus replication in enzyme and cell based assays. An array of warheads, including aldehyde, α-ketoamide, bisulfite adduct, and α-hydroxyphosphonate transition state mimic, was also investigated. Tripeptidyls 2 and 6 possess antiviral activities against noroviruses, human rhinovirus, severe acute respiratory syndrome coronavirus, and coronavirus 229E, suggesting a broad range of antiviral activities.

Full text of DOI:10.1016/j.bmcl.2013.09.070

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6317–6320
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and bioevaluation of viral 3C and 3C-like protease
inhibitors
Allan M. Prior a, Yunjeong Kim b, Sahani Weerasekara a, Meghan Moroze a, Kevin R. Alliston c,
Roxanne Adeline Z. Uy c, William C. Groutas c, Kyeong-Ok Chang b, Duy H. Hua a,
⇑
a Department of Chemistry, Kansas State University, 213 CBC Building, Manhattan, KS 66506, United States
b Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, United States
c Department of Chemistry, Wichita State University, Wichita, KS 67260, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A class of tripeptidyl transition state inhibitors containing a P1 glutamine surrogate, a P2 leucine, and a
P3 arylalanines, was found to potently inhibit Norwalk virus replication in enzyme and cell based assays.
Received 20 August 2013
Revised 17 September 2013
Accepted 23 September 2013
Available online 30 September 2013
An array of warheads, including aldehyde,
a-ketoamide, bisulfite adduct, and a-hydroxyphosphonate
transition state mimic, was also investigated. Tripeptidyls 2 and 6 possess antiviral activities against
noroviruses, human rhinovirus, severe acute respiratory syndrome coronavirus, and coronavirus 229E,
suggesting a broad range of antiviral activities.
Keywords:
Viral 3C and 3C-like protease inhibitors
Norovirus
Ó 2013 Elsevier Ltd. All rights reserved.
Human rhinovirus
Severe acute respiratory syndrome
coronavirus
Coronavirus 229E
Viruses that belong to the Picornaviridae, Caliciviridae, and Coro-
naviridae families encode a 3C or 3C-like protease (3Cpro or
3CLpro, respectively), which cleaves the viral polyproteins into ma-
ture or intermediate proteins and is essential for viral replication.1–3
Viruses in the Picornaviridae family include enteroviruses (EV),4
coxsakieviruses (CV), human rhinoviruses (HRV),5 and poliovirus
(PV); viruses in the Caliciviridae family include human noroviruses
such as Norwalk virus (NV) and MD145,2 and murine norovirus
(MNV); and viruses in the Coronaviridae family include severe
acute respiratory syndrome coronavirus (SARS-CoV)6 and human
coronavirus 229E. These viruses can cause outbreaks of acute gas-
troenteritis (NV),7 severe systemic disease with high mortality
(SARS-CoV),8,9 or a severe form of hand, foot and mouth disease
(HFMD)10 associated with fatal encephalitis, paralysis, and myo-
carditis (EV71). Despite great efforts made in the discovery of pre-
ventive and therapeutic measures for these viruses,11 no antiviral
drug or vaccine (except for poliovirus) is currently available.
Viral 3Cpro and 3CLpro are cysteine proteases and share a typ-
ical chymotrypsin-like folding, a nucleophilic cysteine residue in
the active site, and a preference for a glutamine or glutamic acid
residue in the primary binding residue (P1 site) of the substrate
proteins.12 The 3Cpro or 3CLpro of these viruses are appealing
targets for the discovery of antiviral therapeutics, and the conser-
vation of the proteases may provide an excellent platform for
broad-spectrum antivirals for those important viruses.12 We report
herein our design, synthesis, and bio-evaluation of a class of broad-
spectrum anti-viral tripeptidyl inhibitors as depicted in Figure 1.
Our antiviral design focused on transition state inhibitors of
3Cpro and 3CLpro by incorporating a recognition element such as
a peptidyl fragment that is compatible with the known substrate
specificity of the targeted enzymes and a warhead such as an alde-
hyde, a a-hydroxy phospho-
-ketoamide, bisulfite adduct,13,14 or an
nate transition state mimic. Since the primary substrate specificity
residue of 3Cpro and 3CLpro is a glutamine residue, a glutamine
surrogate15,16 was therefore utilized in the P1 site of the inhibitors.
Like others,17,18 we found that a hydrophobic amino acid such as
leucine residue at the P2 site enhances the recognition.
Results of our cell-based assays have shown that a hydrophobic
residue at P3 in the tripeptidyl compounds is important for mani-
festing antiviral activity, and those with arylalanine at P3 are
highly active in cell-based assays as well as enzyme assays, sug-
gesting that arylalanine at P3 is important for cell penetration.
Hence, a series of tripeptidyl molecules containing an aldehyde
warhead of glutamine surrogate at P1, leucine at P2, and arylala-
nine at P3, such as 1–5, was first synthesized and evaluated.
Tripeptidyl aldehydes 1–5 were readily synthesized using a
five-step reaction sequence starting from glutamine surrogate
⇑
Corresponding author. Tel.: +1 (785) 532 6699; fax: +1 (785) 532 6666.
E-mail address: duy@ksu.edu (D.H. Hua).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.070

6318
A. M. Prior et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6317–6320
H
N
O
H
N
H
O
O
N
4
3
1
H
N
O
H
N
H
N
H
O
N
O
H
O
H
N
N
H
H
H
H
N
O
O
N
H
N
H
O
O
O
O
O
O
O
O
O
Cap
P2
P3
warhead
P1
3
2
1
H
N
H
O
O
N
H
N
O
H
N
O
H
N
H
N
H
H
O
O
N
H
N
H
O
O
O
O
O
O
5
4
H
N
H
H
N
O
O
N
O
H
N
O
H
N
O
H
N
OEt
O
O
O
S
O
*
HN
P
OEt
N
HN
O
O
N
H
*
HN
O
N
H
N
O
O
H
O
O
ONa
OH
H
O
O
O
OH
O
8
6
7
Figure 1. Synthesized and bioevaluated viral cysteine protease inhibitors 1–8.
H
N
O
H
N
EDCI, DMAP
DMF, CH2Cl2
25oC
O
O
H
N
O
O
H
N
+
OH
OMe
O
N
H
OMe
O
(72% yield)
H2N
O
O
O
9
11
10
H
N
1) 10% TFA
O
CH2Cl2, 25oC
1) NaBH4, CH2Cl2
MeOH, 0oC
1 (80% yield)
2 (90% yield)
3 (93% yield)
4 (93% yield)
5 (79% yield)
2) EDCI, DMAP
O
R
O
H
N
OMe
O
R
O
N
H
2) Dess-Martin
periodinane,
CH2Cl2
N
H
OH
O
N
O
O
H
O
12
13 : R =
14 : R =
CH2Ph
CH2
(87% yield)
(72% yield)
12a : (S)-N-Cbz-phenylalanine
12b : (S)-N-Cbz-1-naphthylalanine
12c : (R)-N-Cbz-1-naphthylalanine
12d : (S)-N-Cbz-2-naphthylalanine
12e : (S)-N-Cbz-(p-phenyl)-
phenylalanine
15 : R =
CH2
(83% yield)
CH2
CH2
16 : R =
17 : R =
(54% yield)
(73% yield)
Scheme 1. Synthesis of viral cysteine protease inhibitors 1–5.

A. M. Prior et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6317–6320
6319
1) AcOH, EtOAc
with DMP (Scheme 2).13 Bisulfite adduct 7 and
a
-hydroxy phos-
phonate 8 were readily prepared as pairs of alcohol diastereomers
at the carbon adjacent to S or P from the addition of sodium
bisulfite14 and diethylphosphite,21 respectively, in ꢀ1.3:1 ratio in
both compounds based on 13C NMR spectral data.
The inhibitory activities of compounds 1–8 against NV were
studied using a FRET-based enzyme22 and cell-based assays using
the NV replicon cell system,23 and results are summarized in Ta-
ble 1. The half maximum inhibitory concentration (IC50) or half
maximum effective concentration (EC50) values of these com-
pounds, except for compound 6, were found to be in the submi-
cromolar ranges. Among different P3 residues, that is,
compounds 1–5, compound 2 displayed the highest inhibitory
:
C
N
2
6
2) K2CO3, MeOH
3) Dess-Martin
periodinane
(79% yield)
NaSO3H
EtOAc, EtOH
H2O
2
7
(quantitative yield)
HPO(OEt)2
Hunig base
CH2Cl2
activities both in the enzyme and cell-based assays. The D-amino
acid analog 3 exhibits lower activities than that of 2, and 2-naph-
thyl 4 and biphenyl 5 showed slightly weaker activity than that
of 2. Among different warheads, compounds 2 and 7 possess the
strongest activities. Bisulfite adduct 7 is a precursor and pro-drug
of 2 through equilibrium in aqueous solution.14 It is possible that
2
8
(54% yield)
Scheme 2. Synthesis viral cysteine protease inhibitors 6–8.
the activity of a-hydroxy phosphonate (warhead) varies in differ-
ent conditions in enzyme and cell-based assays. Nevertheless,
compound 8 showed strong activity in NV replicon cells.
Table 1
IC50 and EC50 values of compounds 1–8 from NV 3CLpro FRET-based assay and NV
replicon cells, respectively24
We further examined the broad-spectrum inhibitory activities
of compounds 2 and 6 against 3Cpro or 3CLpro from NV, MD145,
HRV, and SARS-CoV in the FRET-based enzyme assays (Table 2),
and against NV, MNV-1, HRV18, and coronavirus 229E in cell cul-
ture systems (Table 3). Both compounds effectively inhibited
tested proteases and viruses. Moreover, tripeptidyl 2 showed
nanomolar inhibitory activity against all viruses, suggesting that
Compound
IC50
(
l
M)
EC50 (lM)
1
2
3
4
5
6
7
8
0.21 0.1
0.14 0.2
0.7 0.3
0.15 0.1
0.18 0.06
2.6 1.5
0.06 0.03
0.04 0.02
0.45 0.4
0.08 0.03
0.05 0.05
4.2 2.4
a
broad-spectrum inhibitor against multiple viruses can be
0.24 0.1
35.5 5.7
0.04 0.03
0.1 0.3
achieved based on the conservation of the substrate binding pocket
of 3Cpro and 3CLpro.
The enzyme selectivity of a representative inhibitor, compound
2, was assessed using a panel of proteases, including human neu-
Table 2
trophil elastase,
a-chymotrypsin, trypsin, and thrombin. Inhibitor
IC50 values of two active tripeptidyl compounds 2 and 6 against various 3Cpro and
3CLpro using FRET-based enzyme assays24
2, up to 10 M, was found to display minimal or no activity against
l
these enzymes.
Compound
NV (
lM)
MD145 (
lM)
HRV (
lM)
SARS-CoV (lM)
Highly potent inhibitors against noroviruses, picornaviruses
and coronaviruses were discovered through the design of tripept-
idyl transition state inhibitors and transition state mimics capable
of binding to the active site of viral 3Cpro or 3CLpro. The cell-based
results suggest that a naphthylalanine residue at P3 site is impor-
tant for antiviral activity of the tripeptidyl compounds. These
inhibitors could be further developed as broad-spectrum antivirals
against these important viruses.
2
6
0.14 0.2
2.6 1.5
0.51 0.2
3.1 1.6
0.15 0.05
0.12 0.2
0.23 0.1
0.61 0.2
methyl ester 1015 (Scheme 1). Hence, condensation of 10 and (S)-
N-Boc-leucine (9) in the presence of 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide (EDCI) afforded dipeptide 11, which upon
removal of the Boc group and coupling with various N-Cbz-amino
acids 12a–e gave tripeptides 13–17, respectively in good yields.
Reduction of the methyl ester function with sodium borohydride
followed by oxidation with Dess–Martin periodinane (DMP) fur-
nished aldehydes 1–5. Compounds 12a–d are commercially avail-
able, while 12e was made by following a reported procedure19 via
a Suzuki coupling of (S)-N-Boc-4-bromophenylalanine and phenyl-
boronic acid followed by removal of the Boc group and installation
of the Cbz group with benzyl chloroformate.
Acknowledgments
We thank Mr. Laxman Pokhrel and Ms. Thi D.T. Nguyen for tech-
nical assistance with the synthesis. This work was generously sup-
ported by the NIH Grant U01AI081891. Moroze was funded
through the ASSURE program of the Department of Defense in
partnership with the NSF REU Site program number 1004991.
To avoid oxidative degradation and improve absorption and
in vivo pharmacokinetics, various warheads were installed13,14 by
Supplementary data
modifying the aldehyde function of compound 2.20
a-Ketoamide
6 was synthesized from a nucleophilic addition reaction of alde-
hyde 2 and isopropylisonitrile followed by hydration and oxidation
Supplementary data (synthetic procedure, analysis data,
cell line and cell culture, and protocols for cell-viability assay)
Table 3
EC50 values of two active tripeptidyl compounds 2 and 6, against various viruses in the cell-based assays and CC50 (concentration that causes 50% cell death) values24
Compound
NV (
lM)
MNV-1 (lM)
HRV18 (
lM)
Corona-virus 229E (
lM)
CC50 (lM)
2
6
0.04 0.02
4.2 2.4
0.6 0.2
4.2 2.1
0.015 0.03
0.03 0.04
0.2 0.1
0.5 0.2
87 6.3
>100

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A. M. Prior et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6317–6320
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