Structure-activity relationships of heteroaromatic esters as human rhinovirus 3C protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.04.114

Human rhinovirus 3C protease (HRV 3C^pro) is known to be a promising target for development of therapeutic agents against the common cold because of the importance of the protease in viral replication as well as its expression in a large number of serotypes. To explore non-peptidic inhibitors of HRV 3C^pro, a series of novel heteroaromatic esters was synthesized and evaluated for inhibitory activity against HRV 3C^pro, to determine the structure-activity relationships. The most potent inhibitor, 7, with a 5-bromopyridinyl group, had an IC₅₀ value of 80 nM. In addition, the binding mode of a novel analog, 19, with the 4-hydroxyquinolinone moiety, was explored by molecular docking, suggesting a new interaction in the S1 pocket.

Full text of DOI:10.1016/j.bmcl.2009.04.114

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3632–3636
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationships of heteroaromatic esters as human
rhinovirus 3C protease inhibitors
Isak Im a, Eui Seung Lee b, Soo Jeong Choi a, Ju-Yeon Lee a, Yong-Chul Kim a,
*
a Research Center for Biomolecular Nanotechnology, Department of Life Science, Gwangju Institute of Science and Technology, Republic of Korea
b BK21 Center for Biomedical Human Resources, Chunnam Medical School, Chunnam National University, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Human rhinovirus 3C protease (HRV 3Cpro) is known to be a promising target for development of thera-
peutic agents against the common cold because of the importance of the protease in viral replication as
well as its expression in a large number of serotypes. To explore non-peptidic inhibitors of HRV 3Cpro, a
series of novel heteroaromatic esters was synthesized and evaluated for inhibitory activity against HRV
3Cpro, to determine the structure–activity relationships. The most potent inhibitor, 7, with a 5-bromop-
yridinyl group, had an IC50 value of 80 nM. In addition, the binding mode of a novel analog, 19, with the 4-
hydroxyquinolinone moiety, was explored by molecular docking, suggesting a new interaction in the S1
pocket.
Article history:
Received 26 February 2009
Revised 21 April 2009
Accepted 24 April 2009
Available online 3 May 2009
Keywords:
Rhinovirus
3C Protease
Heteroaromatic esters
Inhibitors
Ó 2009 Elsevier Ltd. All rights reserved.
Picornaviruses include important human pathogens such as
human rhinovirus (HRV), enterovirus (EV), coxsackievirus (CV),
poliovirus (PV), and hepatitis A virus (HAV). HRVs are the single
major cause of the common cold in populations of all ages.1,2
Although rhinovirus infection is self-limiting, complications still
occur in patients with asthma, congestive heart failure, bronchiec-
tasis, and cystic fibrosis.1 To date, no agent has been approved by
the FDA for treatment of rhinovirus infection. In addition, the large
number (>100) of known HRV serotypes makes vaccine develop-
ment difficult.3 Clinical treatments are directed toward relief of
the most prominent symptoms of each clinical syndrome. Previous
work on anti-rhinoviral therapeutic agents focused principally on
capsid-binding anti-picornaviral compounds4 and HRV 3C protease
(3Cpro) inhibitors.5,6
structure and cleavage specificity. To date, one peptidomimetic
analog, rupintrivir (AG7088) (1), shown in Figure 1, is the only
HRV 3CPro inhibitor that has advanced to the stage of human
clinical trials. Phase II clinical trials of rupintrivir showed that
intranasal delivery reduced the mean total daily symptom score
by 33%.12 Recently, several efforts to develop orally bioavailable
anti-rhinoviral agents have been reported; these include a rupintri-
vir analog, 2,13 with an improved pharmacokinetic profile, and the
non-peptidic inhibitors 3 and 4.14–16
The crystal structure of severe acute respiratory syndrome
(SARS) coronavirus 3C-like protease (3CL )
pro 17 revealed a high sim-
ilarity of active site architecture to that of HRV 3Cpro, suggesting
that picornavirus 3Cpro and SARS 3CLpro inhibitors should cross-re-
act. In fact, SARS 3CLpro inhibitors were developed by modifying
rupintrivir18 and common inhibitors with IC50 values in the micro-
molar range were recently identified for both picornavirus 3Cpro
and coronavirus 3CLpro using high-throughput screening.19,20 The
pyridinyl thiophene ester 5 was discovered by screening a small
molecule library (50,000 molecules) and has significant inhibitory
HRV 3Cpro is essential for virus replication, specifically catalyz-
ing the cleavage of glutamine-glycine peptide bonds in a 230 kDa
viral polyprotein produced by cellular translation of the viral
RNA genome. HRV 3Cpro belongs to the cysteine protease family,
showing structural similarity to trypsin proteases, but sharing
minimal homology with common mammalian enzymes.7,8 Because
of the importance of the enzyme in viral replication, and the high
conservation of enzyme active site components in all known HRV
serotypes, potent and selective HRV 3Cpro inhibitors could be
potential broad-spectrum anti-rhinoviral therapeutic agents.9–11
As part of an effort to develop HRV 3Cpro inhibitors, peptidom-
imetic approaches have been widely employed based on active site
activity against both HAV 3Cpro and SARS 3CLpro (IC50 = 0.5 M).19,21
l
Further work on a series of heteroaromatic esters resulted in the
discovery of the most potent ester 6, inhibiting SARS 3CLpro with
an IC50 of 30 nM and an antiviral activity (EC50 value) of
6.9 l
M.22 Another analog, 7, also showed potent inhibition of
HAV 3Cpro with an IC50 of 53 nM.23 Studies on the inhibitory and
molecular docking mechanisms of heteroaromatic esters have
shown that the 5-halopyridine moiety is crucial in binding to the
S1 pocket, because both 3Cpro and 3CLpro show high specificity
for Gln as the P1 residue.23
* Corresponding author. Tel.: +82 62 970 2502; fax: +82 62 970 2484.
E-mail address: yongchul@gist.ac.kr (Y.-C. Kim).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.114

I. Im et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3632–3636
3633
Herein, we report on the structure–activity relationships of
heteroaromatic esters with respect to inhibitory activity against
HRV 3Cpro. Thirty-one heteroaromatic esters, including 15 newly
designed derivatives, were synthesized and screened against HRV
3Cpro using a fluorescent peptide substrate. We also performed
molecular docking studies to understand the potential inhibitor
binding modes to the active site of HRV 3Cpro
general synthetic method for heteroaromatic esters is
.
A
described in Scheme 1. A series of compounds was synthesized
Table 1
% Inhibition and IC50 values of compounds 7–19 tested against HRV16 3Cpro
Compd
R1
% Inhibition at 1
l
M
IC50 (lM)
7
90
8
2
0.08 0.03
8
9
3
NIa
NI
87
NI
NI
24
NI
—
—
—
—
—
—
—
10
11
12
13
14
15
Figure 1. Structure of HRV 3Cpro inhibitors (1–4) and SARS 3CLpro inhibitors (5–7).
3
3
Scheme 1. Reagents and conditions: (a) Pyridine (1.2 equiv), CH2Cl2, rt, 30 min–2 h,
30–90% yield; (b) EDC (1.2 equiv), HOBt (1.2 equiv), DIPEA (1.2 equiv), DMF, rt, 2–
8 h, 50–80% yield; (c) two steps, (i) SOCl2 (1.3 equiv), DMF (two drops), CH2Cl2, rt,
24 h; (ii) pyridine (1.2 equiv), CH2Cl2, rt, 30 min–2 h, 30–60% yield.
16
17
14
NI
6
—
—
18
5
2
—
19
98
1
0.20 0.05
Scheme 2. Synthesis of 4-hydroxyquinolinone. Reagents and conditions: (a)
CH2Cl2/THF, 60 °C, 9 h, 78%; (b) Eaton’s reagent, 70 °C, 4 h, 72%.
a
NI = no inhibition.

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I. Im et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3632–3636
by esterification of various carboxylic acids and hydroxy-heteroar-
omatic groups. Initial replacement of the pyridine moiety with
various heteroaromatic rings was performed with retention of
the 2-furoyl group. Commercially available hydroxy-heteroaro-
matic rings were esterified with 2-furoyl chloride in pyridine and
CH2Cl2 (7–18). Compound 19 was prepared from 4-hydroxyquin-
olinone (39) in two steps (Scheme 2). The structure and inhibitory
activities of furan-2-carboxylate analogs (7–19) are shown in Table
1. As a second series of analogs, with 5-halopyridyl esters, we used
5-chloro (or -bromo)-3-pyridinol for esterification with diverse
carboxylic acid moieties. Compounds 20–29 were prepared with
commercially available carboxylic acids. Compound 30 was syn-
thesized by arylation of the -N–H group of the imidazole ring of
28, in a reaction with 4-trifluoromethylphenyl boronic acid fol-
lowed by pyridine, with use of cupric acetate and 4-Å molecular
sieves in CH2Cl2.24 The synthesis of the 2-substituted oxazole-car-
boxylic acids of compounds 31–35 is outlined in Scheme 3. Briefly,
acylation of 2-amino diethyl malonate (40a–e) using various acid
chlorides followed by dehydrative cyclization employing trifluoro-
acetic anhydride or trichloroacetyl chloride under microwave con-
ditions at 160 °C for 5–10 min afforded the oxazoles 41a–e,25
which were subjected to basic hydrolysis to yield the correspond-
ing oxazole-carboxylic acids 42a–e. The structures and activities of
the 5-chloro/bromopyridyl ester analogs are shown in Table 2.
A FRET-based assay system was used to measure the enzyme
activity of HRV16 3Cpro, expressed from cDNA26, using the fluoro-
genic substrate NMN-ALFQGPPVK-DNP.27
esters acting in the active site of SARS 3CLpro,20,22 Such esters
initially bind competitively and strongly to the active site, being
Table 2
% Inhibition and IC50 values of compounds 20–37 tested against HRV16 3Cpro
Compd
R2
X
% Inhibition at 1
lM
IC50
—
(lM)
20
Cl
64
59
80
79
89
60
4
2
4
3
2
4
21
22
23
24
25
26
Cl
Cl
Cl
Cl
Cl
Cl
—
—
—
—
—
80 10
0.71 0.15
As shown in Table 1, the 5-bromopyridine moiety of compound
7, a representative analog of the heteroaromatic inhibitors, was
replaced by various heteroaromatic groups. The 3-pyridyl furonate,
27
Cl
75
6
1
—
8, showed weak inhibition of HRV 3Cpro (8% inhibition at 1
lM),
whereas the 2- and 4-pyridinyl furonates, 9 and 10 showed no
inhibitory effects. Chloro- and bromo-substituents at the 5 position
of the pyridinyl ring significantly increased inhibitory effects (com-
pounds 7, 11), with compound 7 showing the most potent activity
with an IC50 value of 80 nM. 5-Halopyridine esters functioned well
as HRV 3Cpro inhibitors as a result of S1 pocket similarities with
28
29
30
Cl
Cl
Cl
82
NIa
40
0.29 0.11
—
—
both HAV 3Cpro and SARS 3CLpro
.
3
Molecular docking of compound 7 into the active site of X-ray
crystal structure of HRV 3Cpro (PDB: 1CQQ) was performed by a
CHARMm force field based docking tool, CDOCKER of Discovery Studio
2.0 (Accelrys Inc., San Diego, CA). In a rigid receptor structure, ran-
dom orientations of ligand conformations were subjected to simu-
lated annealing molecular dynamics. Final minimized poses of
docked ligand were sorted by CHARMm energy (interaction energy
plus ligand strain). Mass spectrometry and HPLC studies have
already revealed the inhibition mechanism of heteroaromatic
31
32
Cl
Cl
NI
5
—
—
2
33
34
Cl
Cl
34
87
7
2
—
0.69 0.22
35
36
Cl
5
1
—
Br
Br
34
63
2
1
Scheme 3. Synthesis of oxazole-4-carboxylic acids. Reagents and conditions: (a)
TEA, CH2Cl2, R = benzoyl, phenylacetyl, 2-naphthoyl, 2,2-diphenylacetyl, or trans-
cinnamoyl; 75–91%; (b) TFAA or CF3COCl, trifluorotoluene, microwave heating,
160 °C, 5–10 min, 35–82%; (c) 10% (w/v) KOH (aq), EtOH, rt, 1 h, 65–88%.
37
a
NI = no inhibition.

I. Im et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3632–3636
3635
Figure 2. Stereo view of initial binding mode of compound 7 with HRV 3Cpro. The nitrogen, oxygen, and sulfur atoms are colored blue, red, and orange, respectively. Hydrogen
bonds are displayed as green dashed lines.
Figure 3. Stereo view of initial binding mode of compound with 19 HRV 3Cpro. The nitrogen, oxygen, and sulfur atoms are colored blue, red, and orange, respectively.
Hydrogen bonds are displayed as green dashed lines.
recognized by S1 pocket. The carbonyl carbon is then attacked by
the nucleophilic Cys residue, and become covalently connected
with the protease prior to eventual release. Thus in our study, pos-
sible prehydrolysis-binding complex models of 7 was generated
(Fig. 2). The lone pair electron of nitrogen moiety of 5-bromopyri-
dine in 7 occupies the S1 binding pocket, forming a hydrogen bond
effect (5% inhibition at 1 lM, 21% inhibition at 10 lM), it was nota-
ble that compound 19 showed a potent inhibitory effect at 1 lM
(98% inhibition), with an IC50 value of 200 nM.
To gain molecular insight into the binding mode of the 4-quin-
olinone moiety, compound 19 was also docked in the HRV 3Cpro
active site (Fig. 3). The oxygen of quinolinone forms a hydrogen
e2
e2
with N of His161 (3.1 Å). The bromo group occupies S1 pocket
near the protein surface and, notably, a hydrogen bond with the
–NH of Gly166 (3.0 Å) was observed, explaining the importance
of the presence of a halogen group on the pyridine ring. The ester
carbonyl oxygen forms a hydrogen bond with the –NH of Gly164
(2.5 Å). This predicted binding model is consistent with the binding
bond with N of His161 (2.8 Å) and the benzene ring of quinoli-
none occupies the outer space of the S1 pocket as does the bromo
group of 5-bromopyridine, but there is no hydrogen bond with the
–NH of Gly166. The hydrogen bond of the ester carbonyl oxygen
with the –NH of Gly164 (2.0 Å) was also conserved as well.
Although there is different spatial position of the 4-quinolinone
carbonyl oxygen of 19 from the pyridyl nitrogen of 7, the require-
ment of hydrogen bond angle between the carbonyl oxygen and
mode of halopyridinyl esters to SARS 3CLpro and HAV 3Cpro
.
To explore the possibility of new binding interaction, new
motives for the substitution of 2-position of the pyridine ring were
e
2
N
of His161 was satisfied with the ideal hydrogen bond at 30–
e
designed for pointing toward N 2 of His161 in S1 pocket. Therefore,
60° to the O@C axis within 30° of the carbonyl plane, while the
e
2
2-substituted pyridinyl compounds (12–16) were investigated.
Amine (14) and amide (16) substituted compounds showed mod-
erate activity at 1 lM in the absence of chlorine at the 5 position.
The new S1 pocket binding moiety was also evaluated using
para, meta benzamide (17, 18) and 4-quinolinone (19), instead of
5-halopyridine. Compounds 17–19 may possibly mimic Gln, the
substrate cleavage moiety. While 17 showed weak inhibitiory
sp2 lone pair of pyridyl nitrogen of 7 is lying toward N of
His161 forming optimal hydrogen bond angle (180°). This precise
hydrogen bond with His161 seems to be the key initial binding
unit which mimics the Gln moiety (the P1 residue), which resulted
in the dramatic inhibitory activity changes depending on the sub-
stituents R1. Interaction energy of compound 7ꢀ19 with 3Cpro was
calculated by CDOCKER program. The most potent compounds 7 and

3636
I. Im et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3632–3636
19 showed À24.5 and À26.3 kcal/mol, respectively. Comparison of
the interaction energy of the ortho (9, À21.9 kcal/mol), meta (8,
À22.2 kcal/mol), and para (10, À20.0 kcal/mol) position of the pyr-
idine nitrogen showed a parallel results with the biological activi-
ties, suggesting that the meta position could prefer the orientation
for the hydrogen bond. Compound 12–16 with additional hydro-
gen bond acceptor at 2-position of the pyridine ring might disturb
the optimal hydrogen bond of the pyridyl nitrogen showing the
less interaction energies (À19.9 kcal/mol to À23.5 kcal/mol).
Although weak inhibitors of benzamide analogs, 17–18 could form
hydrogen bonds with His161 (À24.2 kcal/mol for 17, À22.6 kcal/
mol for 18), the distance between ester carbonyl carbon and the
nucleophilic –SH group of Cys might be changed unfavorably
resulting in weak or no inhibitory activity.
Medicine and Public Health, University of Wisconsin, Madison,
WI) for supplying us with cDNA encoding HRV16.
References and notes
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synthesized and tested. This R2 carboxylic acids were expected to
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covalently connected binding mode at the active site. Most com-
pounds showed moderate-to-good inhibitory effects at 1 lM ex-
cept for 29 and 31 (Table 2). Compounds with thiophen-2-
carbonyl (20), benzoyl (21), phenylpropanoyl groups (36), and cin-
namoyl (37) showed lower activities than did the 2-furoyl analogs
(7 and 11). Substitution of the 5 position of the furan ring with aro-
matic groups allowed retention of good activity (22–25). The steric
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reaction state by
p
-stacking interaction with His4022 rather than
tight binding to S2 pocket. The 2-naphthoyl (26), 1-naphthoyl
(27), and imidazole (28) groups were useful building blocks, show-
ing potent inhibitory activities (IC50 of 290 nM for 28). However,
arylation of the imidazole ring of 28 showed twofold decrease in
activity (30), which could be caused by unfavorable constraint
compared to furan ring.
In further efforts to replace the furoyl ring with other heterocy-
clic carboxylate moieties, isoxazole and oxazole groups were inves-
tigated. In the case of 3-methylisooxazole derivative, 29, the
replaced position of furan oxygen by carbon atom resulted in the
loss of activity significantly. However, oxazole derivatives (31–
35) demonstrated a broad range of inhibitory activities depending
on substitutions at the 2 position of the oxazole group. A cinnam-
yloxazole analog, 34, showed the highest activity among these
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compounds with 87% inhibition at 1 lM and an IC50 value of
690 nM. Lower electron density of oxazol ring may result in weak-
er binding affinity than furan or imidazol moiety, but additional
hydrophobic phenyl group in a proper position connected to 2-
oxazolic position with two carbon chain (34) significantly en-
hanced the inhibitory activity compared to the compounds with
shorter chains or bulky aromatic groups (31–33, 35).
In conclusion, 31 heteroaromatic esters were synthesized and
screened as non-peptidic inhibitors against HRV 3Cpro. Compound
7, which was one of the most potent inhibitors in an earlier series,
with activity against both SARS 3CLpro and HAV 3Cpro, was also
found to be the most potent in the current work (IC50 of 80 nM).
Substitution of 5-halopyridine with various heteroaromatic rings
resulted in the discovery of the 4-quinolinone group as an alterna-
tive key structure. Further optimization of 4-quinolinone ester
analogs as Gln skeleton mimics is underway, with the aim of
enhancing inhibitory activities.
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26. The HRV16 3Cpro coding region was amplified by PCR, subcloned into the
pTYB12 expression vector, and transformed into E. coli strain BL21(DE3).
HRV16 3Cpro was purified using the IMPACT-CN system (New England Biolabs,
Beverly, MA). A protease stock solution was maintained in 20 mM HEPES/
NaOH, 100 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol (pH 7.9).
27. Assays were performed at 30 °C in 96-well microplates in reaction volumes of
100 lL with 50 mM Tris (pH 7.6), 1 mM EDTA, 50 lM substrate, 200 nM HRV16
3Cpro, and various concentrations of test compounds. Inhibitors and enzymes
were incubated for 10 min in reaction buffer and reactions were initiated by
addition of FRET-substrate. Fluorescence values were monitored at 340 nm
(excitation) and 440 nm (emission). Heteroaromatic esters were initially tested
at 1 lM. Mean % inhibition was calculated from 3 to 4 repeated experiments.
IC50 values of six potent inhibitors were determined using various
concentrations with three independent experiments.
Acknowledgments
This study was supported by a grant of the National R&D
Program for Cancer Control, Ministry of Health & Welfare, Republic
of Korea (0720430). We thank Dr. Wai-Ming Lee (School of