Identification of fused bicyclic derivatives of pyrrolidine and imidazolidinone as dengue virus-2 NS2B-NS3 protease inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.09.063

A series of fused ring derivatives of pyrrolidine and imidazolidinone were designed, synthesized, characterized and assayed against the DENV-2 NS2B-NS3 protease and wild-type DENV-2 virus. The linear dipeptide compound 1 and the non-peptidic fused ring compound 2 show comparable activities against DENV-2 NS2B-NS3 protease and wild-type DENV-2 virus in a viral replication assay. The preliminary SAR reveals that a substituent and its stereochemistry at C-3 position, substitution (X) at N-2 arene and a linker (Y) between C-3 position and its attached arene are important for the fused-ring scaffold of pyrrolidino [1,2-c]imidazolidinone to block the active site of NS2B-NS3 protease. This promising structural core will facilitate the discovery of non-peptidic, potent NS2B-NS3 protease inhibitors to stop dengue virus infections.

Full text of DOI:10.1016/j.ejmech.2016.09.063

Accepted Manuscript
Identification of the fused bicyclic derivatives of pyrrolidine and imidazolidinone as
dengue virus-2 NS2B-NS3 protease inhibitors
Zhibing Weng, Xiaoxia Shao, Dominik Graf, Chunguang Wang, Christian Klein, Jing
Wang, Guo-Chun Zhou
PII:
S0223-5234(16)30800-5
10.1016/j.ejmech.2016.09.063
EJMECH 8929
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 July 2016
Revised Date: 19 September 2016
Accepted Date: 20 September 2016
Please cite this article as: Z. Weng, X. Shao, D. Graf, C. Wang, C. Klein, J. Wang, G.-C. Zhou,
Identification of the fused bicyclic derivatives of pyrrolidine and imidazolidinone as dengue virus-2
NS2B-NS3 protease inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.09.063.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Table of contents graphic
O
N
O
N
O
HN
3
3
N
N
N
H N
NO2
2
O
NO2
NO2
1
2
3
S
O N
O N
2
2
IC50 = 1.2 ± 0.4 µM to NS2B-NS3
EC50 = 38.7 ± 5.4 µM to virus plaque
IC50 = 1.2 ± 0.4 µM to NS2B-NS3
EC50 = 39.4 ± 6.2 µM to virus plaque
inactive
previous work
this work

ACCEPTED MANUSCRIPT
Identification of the fused bicyclic derivatives of pyrrolidine and
imidazolidinone as dengue virus-2 NS2B-NS3 protease inhibitors
†
a
†b
c
b
c
Zhibing Weng, Xiaoxia Shao, Dominik Graf, Chunguang Wang, Christian Klein,
d
a
Jing Wang* and Guo-Chun Zhou*
a
School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 211816, Jiangsu, PR China.
Institute of Protein Research, Tongji University, Shanghai 200092, PR China.
b
c
Medicinal Chemistry, Institute of Pharmacy and Molecular Biotechnology IPMB, Heidelberg University, Im
Neuenheimer Feld 364, D-69120 Heidelberg, Germany.
d
College of Sciences, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China.
Correspondingauthors:
wangjing@njau.edu.cn (jw)
These authors contributed equally to this paper.
Tel:
86-25-58139415,
E-mail:
gczhou@njtech.edu.cn
(gcz)
andE-mail:
†
1
/ 28

ACCEPTED MANUSCRIPT
Abstract
A series of fused ring derivatives of pyrrolidine and imidazolidinone were designed, synthesized,
characterized and assayed against the DENV-2 NS2B-NS3 protease and wild-type DENV-2 virus.
The linear dipeptide compound 1 and the non-peptidic fused ring compound 2 show comparable
activities against DENV-2 NS2B-NS3 protease and wild-type DENV-2 virus in the plaque assay.
The preliminary SAR reveals that a substituent and its stereochemistry at C-3 position,
substitution (X) at N-2 arene and a linker (Y) between C-3 position and its attached arene are
important for the fused-ring scaffold of pyrrolidino[1,2-c]imidazolidinone to block the active site
of NS2B-NS3 protease. This promising structural core will facilitate the discovery of non-peptidic,
potent NS2B-NS3 protease inhibitors to stop dengue virus infections.
Keywords: Dengue virus NS2B-NS3 protease inhibitor; pyrrolidino[1,2-c]imidazolidinone;
stereochemistry; substitution and substituent; linker; structure-activity relationship (SAR)
2
/ 28

ACCEPTED MANUSCRIPT
Table of contents graphic
O
N
O
N
O
3
3
N
N
N
O
H N
2
HN
NO
2
NO2
NO2
1
2
3
S
O N
O N
2
2
IC50 = 1.2 ± 0.4 µM to NS2B-NS3
EC50 = 38.7 ± 5.4 µM to virus plaque
IC50 = 1.2 ± 0.4 µM to NS2B-NS3
EC50 = 39.4 ± 6.2 µM to virus plaque
inactive
previous work
this work
3
/ 28

ACCEPTED MANUSCRIPT
1
. Introduction
Dengue virus (DENV), a member of the genus Flavivirus (family Flaviviridae), is one of
the most rapidly spreading mosquito-borne human pathogens in the tropical and
subtropical regions [1]. The dengue virus is transmitted by the Aedes aegypti and
Aedesalbopictus mosquitos, and causes a wide spectrum of clinical manifestations in
humans ranging from an epidemic disease, known as dengue fever (DF), to the more severe
dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [1,2]. In recent
decades, the number of occurring infections, severe cases, and regions reporting
autochthonous infections for the first time has dramatically increased [3] and WHO
estimates 50−100 million infections worldwide per year.
There are four distinctive, but closely related, serotypes of DENVs named as DENV-1,
DENV-2, DENV-3 and DENV-4. It was observed that cross-immunity to the other
serotypes after recovery is only partial and temporary, and more dangerously, subsequent
infection by any of the other three serotypes may enhance the risk of developing severe
dengue, which was called “antigen-dependent enhancement” effect (ADE) [1,4]. Due to
ADE, the vaccine development against DENVs is extremely difficult and the first
tetravalent vaccine against dengue virus was approved only recently in December, 2015 in
Mexico.
The genome of DENV is comprised of a 10.7 kb, single, positive-stranded RNA
encoding a single polypeptide, which is subsequently processed by the virus-encoded
trypsin-like NS2B-NS3 protease and by host proteases to generate three structural proteins
(
capsid protein, precursor membrane protein, and envelope protein) and seven
nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [5]. The
N-terminal part of NS3 contains a protease domain (termed NS3pro) with a classic serine
proteinase catalytic triad (His51, Asp75, and Ser135) and optimal catalytic activity of NS3
depends on the presence of NS2B [6]. Since the NS2B-NS3 protease is essential for the
maturation and pathogenesis of dengue virus, it is considered to be a promising target for
anti-dengue virus drug development [7]. Therefore, the inhibition of NS2B-NS3 protease
4
/ 28

ACCEPTED MANUSCRIPT
activity is an attractive antiviral therapeutic treatment against DENV infections and many
efforts have been made to develop anti-dengue virus NS2B-NS3 agents [8,9].
We have recently reported a series of linear chain dipeptides as inhibitors of DENV-2
NS2B-NS3 protease, and compound
1 (Fig. 1) was demonstrated as the most potent
NS2B-NS3 protease inhibitor among them [10]. As part of the ongoing research to develop
small molecular entities targeting DENV NS2B-NS3 protease, structural modifications
based on compound
1 were explored. In this work, we describe the design, synthesis,
absolute configuration determination and bioassay of the fused bicyclic derivatives of
pyrrolidine and imidazolidinone as DENV-2 NS2B-NS3 protease inhibitors. It was
discovered that non-peptidic compound
2 (3α-isomer) (Fig. 1), with an IC value of 1.2 ±
50
0
.4 µM, showed comparable inhibitory activity to DENV-2 NS2B-NS3 protease as
compound
1 (IC =1.2 ± 0.4 µM), which possesses Ki value of 4.9 µM as reported [10];
50
however, its C-3 epimeric counterpart
protease. Furthermore, both and are active against wild-type DENV-2 virus plaque
formation with EC values of around 39 µM.
3 (3β-isomer) was inactive against NS2B-NS3
1
2
5
0
<Fig. 1>
2
. Results and Discussion
2
.1. Design of cyclic modifications
The crystal structures of NS2B-NS3 without any ligand [11] and complexed with bovine
pancreatic trypsin inhibitor (BPTI) [12] exhibit an “open state” (inactive form), in which
the C-terminal of NS2B was mostly dissociated from NS3pro; however, NMR studies in
solution suggested that inhibitors like BPTI and a p-guanidino-benzoyl group (esterified to
the catalytic serine) induce a conformational change in NS2B-NS3pro from the “open
state” (inactive form) to the “closed state” (active form). In the latter, one or more groups
of the inhibitors can promote the association of NS2B with NS3 [13]. Experimental studies
and molecular docking showed that some S-S disulphide bridged cyclopeptides improved
the binding affinity to NS2B-NS3 [14]. Furthermore, it was recently uncovered that the
head-to-tail connection of a cyclopeptide also enhanced the inhibitory activity
5
/ 28

ACCEPTED MANUSCRIPT
againstNS2B-NS3 [15]. These results suggest that an appropriate cyclic inhibitor may fit
well into the binding cleft and effectively block the active site of NS2B-NS3 protease. In
addition, cyclization avoids the entropic penalty that is associated with the binding of
highly flexible (e.g., peptidic) inhibitors. Based on compound
1, a linear dipeptide from
our previous study [10], a series of fused bicyclic compounds of pyrrolidino[1,2-
c
]
imidazolidinone derivatives (Fig. 1) were designed, synthesized and tested against
DENV-2 NS2B-NS3 protease. It is expected that these non-peptidic compounds are
resistant to peptide enzymes. As noted, the structural skeleton of pyrrolidino[1,2-c]
imidazolidinone [16] is very stable and appears in natural products [16a], pharmaceutical
entities [16b-16d], chiral organo-catalysts [16e] or chiral catalytic ligands [16f].
2
.2. Chemical Synthesis
The general synthetic route is outlined in Scheme 1 and the reaction results are listed in Table 1.
Scheme 1>
were synthesized according to the procedure
reported previously [10]. Briefly, the condensation of Boc- -proline with -nitroaniline
provided , which was followed by the deprotection of Boc group in the presence of
trifluoroacetic acid (TFA) to provide the salt of . Then, various aldehydes were refluxed
in ethanol or toluene with compound in the presence of triethylamine (TEA) to afford
compounds 6a series and 6b series, respectively. Specifically, the reaction of linear
aliphatic aldehydes (not including phenylacetaldehydes) (Table 1, entries 1-4) with in
refluxing ethanol or refluxing toluene gave the thermodynamic products of
α-diastereoisomers (trans); meanwhile, the reaction of phenylacetaldehydes (Table 1,
entries 5 and 6) with happened only in refluxing toluene and yielded 3α-diastereoisomers
trans), which is a similar result as with TFA catalysis [16a]. It is interesting that the
<
Firstly, the intermediates of
4 and 5
L
p
4
5
5
2, 3,
5
3
5
(
reaction of
5
with aromatic aldehydes without substitution (Table 1, entry 7) or with
-position (Table 1, entries 8 and 9) in refluxing
toluene led to only 3α-diastereoisomers (trans); whereas, starting from aromatic aldehydes
bearing electron-withdrawing group (EWG) at the -position (Table 1, entries 10-15) in
electron-donating group (EDG) at the
p
p
refluxing toluene afforded two diastereoisomers in the ratio of around 4 to 1 of
/ 28
6

ACCEPTED MANUSCRIPT
thermodynamic 3α-diastereoisomer (trans) [16e] to 3β-diastereoisomer (cis), which could
be separated by column chromatography. Even though chlorine is a weak
electron-withdrawing substituent, the corresponding products obeyed the above rule and
these results suggest that the electron-deficiency of aromatic ring and aldehyde group
dominates the reaction mechanism. However, due to the hindrance at the
-nitro phenyl aldehyde only gave trans-isomer 6a12 (Table 1, entry 16). It is valued that
free bases and salts of these compounds are stable as expected in our study.
Table 1>
m-position,
m
<
2
.3. Determination of stereochemistry
Some absolute configurations of pyrrolidino[1,2-
c
]imidazolidinone compounds were
1
analyzed in relevant references [16] but the determination of C-3 stereochemistry by H
NMR assignment is still very difficult. Therefore, X-ray crystallography analysis and
2
D-NOESY NMR study were used here to determine the relationship between the absolute
1
configurations of C-3 and their H NMR data (Table 1). The absolute configuration of the
chloro analog 6a11
Fig. 2a), indicating that the reaction condition used in this work did not affect the
stereochemistry of position C-7 , which originates from L-proline; accordingly, the
absolute configuration of compound in crystals (Fig. 2b) is trans isomer as 6a11 and the
aromatic ring of -NO -Ph in position 3 is α-oriented. Therefore, -NO -Ph and -Cl-Ph at
C-3 in and 6b11, respectively, are
and 3β-Hs of and 6a11 (4.01 and 5.90, 3.98 and 5.76, respectively) with those of 7
and 3α-Hs of
(p-Cl-Ph is α-oriented) was determined by X-ray diffraction analysis
(
a
2
p
p
p
2
2
3
β
-oriented. Comparing the chemical shifts of 7
a
α-Hs
α-Hs
2
a
3
and 6b11 (4.05-4.28 and 6.55, 3.98-4.20 and 6.40, respectively), we
conclude that 3β-Hs (trans-isomers) are less than 6.0 ppm but 3α-Hs (cis-isomers) are
higher than 6.0 ppm. Based on these results, we deduce that the 3-aromatic compounds of
2,
6a7, 6a8, 6a9, 6a10 and 6a11 are trans-isomers and those of 3, 6b10 and 6b11 are
cis-isomers.
<
Fig. 2>
was also analyzed by a NOESY experiment (Fig. s1,
Supporting Materials). The NOESY data of showed that no obvious NOE correlation
/ 28
The detailed assignment of
2
2
7

ACCEPTED MANUSCRIPT
between 3β-H (5.90 ppm) and 7
NOE correlations between 7 α-H with 2’-H (7.71 ppm) and 2”-H (7.50 ppm) of two
aromatic rings (Fig. s1, Supporting Materials), supporting that 3β-H and 7 α-H are in trans
configuration. The observations that 3β-H (5.90 ppm) exhibited strong NOE correlations
with both 5-Hs (3.55 and 2.94 ppm) but there was lack of NOE correlation between 7 α-H
aα-H (4.01 ppm) was observed while there were strong
a
a
a
and any one of 5-Hs propose the α-orientation of the lone-pair electrons of N-4.
Meanwhile, the presence of strong NOE correlation between 3β-H with 2’-H and the
absence of NOE correlation between 7aα-H and 2’-H indicated that the p-nitro-phenyl
group at N-2 is β-oriented. These results lead to the deduction that 3β-H is surrounded by
two adjacent nitrogen atoms and shielded by an aromatic ring at N-2, which are in
agreement to the crystal structures of
2 and 6a11.
In order to deduce the stereochemistry of 6a5, which was derived from
phenylacetaldehyde, NOESY experiment (Fig. s2, Supporting Materials) was conducted.
The strong NOE correlation between 7
”-H (7.18 ppm) and the absence of a NOE between 7
that benzylic group is trans-configuration to 1-carbonyl. Similar to
ppm) and 5β-H (2.61 ppm) have NOE correlations with 3β-H and there is no NOE
correlation between 7 α-H and 5α-Hs; meanwhile, no NOE correlation was observed
between 7 α-H with 2’-H. It was found that benzylic Hs (α-oriented) and 3β-H have NOE
correlations with 2’-H (7.84 ppm) but stronger NOE correlation of 3β-H with 2’-H was
observed than that of benzylic H with 2’-H, indicating that the -nitro-phenyl group at N-2
is more β-oriented. Based on these results, it is deduced that the aliphatic Rs in 6a2 6a3
a4 and the -nitro-benzylic group in 6a6 are in α-orientation as 6a5
.4. Inhibitory activity against DENV-2 NS2B-NS3 protease
a
α-H (3.61 ppm) with benzylic H (3.00 ppm) and
α-H with 3β-H (5.15 ppm) support
, both 5α-H (3.26
2
a
2
a
a
p
,
,
6
p
.
2
The inhibitory activity of these compounds to DENV-2 NS2B-NS3 protease was assayed
according to our previous procedure [10] and the activity results are listed in Table 1.
<
Fig. 3>
6a3 and 6a4, derived from aliphatic aldehydes, were
assayed and it was discovered that they showed moderate inhibitory activities and
/ 28
Initially, compounds 6a1, 6a2,
8

ACCEPTED MANUSCRIPT
established good structure-activity relationship (SAR) against DENV-2 NS2B-NS3
protease (Table 1, entries 1-4 and Fig. 3). Along with the increase of R group from H (6a1
to Me (6a2) and Et (6a3), the inhibitory activity was stepwise evaluated; however,
branched alkyl R of bulky -Pr (6a4) decreased the inhibitory activity, as seen by
)
i
comparison of 6a4 with 6a3. These findings created a new and non-peptidic scaffold as
DENV-2 NS2B-NS3 protease inhibitors. Based upon these results, diverse compounds
with this scaffold were explored as DENV-2 NS2B-NS3 protease inhibitors.
In order to enhance the inhibitory activity, the replacement of the linear aliphatic group
with a benzylic group was conducted and compound 6a5 (Table 1, entry 5) exhibited a
moderate inhibition as 6a4 with
i
-Pr group; however, compound 6a6 bearing a
p
-nitro-benzylic group sharply lost the inhibitory activity (Table 1, entry 6). It seems that
the p-nitro group seriously diverts the conformation of whole molecule, which negatively
affects the binding between NS2B-NS3 with compound 6a6
.
<Table 2>
Further studies by the replacement of linear aliphatic group with aromatic groups were
conducted and it was discovered that compound 6a7 with phenyl group (Table 1, entry 7)
exhibited very weak inhibitory activity to NS2B-NS3 protease and compounds 6a8 (Table
1
, entry 8) and 6a9 (Table 1, entry 9) bearing
p-EDG phenyl groups did not show any
activity against NS2B-NS3 protease. Then, -EWG phenyl groups were introduced and the
p
results showed that 4-chloro-phenyl and 4-trifluoromethyl phenyl derivatives (Table 1,
entries 10-13) were also not active against NS2B-NS3 protease. Interestingly, it was
disclosed that compound
exhibited good inhibitory activity (IC50 value of 1.2 ± 0.4 µM) and almost the same
inhibition to NS2B-NS3 protease as parent compound [10] (Table 1, entry 17).
However, compound , the corresponding cis-isomer (Table 1, entry 15) did not exhibit
inhibitory activity at NS2B-NS3 protease, and compound 6a12 trans-isomer) with -nitro
phenyl group (Table 1, entry 16) showed no activity. These activity differences suggest
that the stereochemistry of the 3α-( -nitro) phenyl group provides an appropriate binding
demand of to NS2B-NS3.
2, with R = p-nitro-phenyl, of trans-isomer (Table 1, entry 14)
1
3
(
m
p
2
9
/ 28

ACCEPTED MANUSCRIPT
It is interesting and valuable that the SARs of NS2B-NS3 protease inhibitors determined
by X and Y (Table 2) in fused ring derivatives of
our previous report [10] of linear dipeptides (Table 2), in which a
affects the activity when Y is methylene whereas -nitro positively exerts the activity when
2
and 6a7
,
6a5 and 6a6 also existed in
p-nitro group negatively
p
Y is empty, indicating an elementary molecular recognition requirement for these series of
NS2B-NS3 protease inhibitors. These results imply that the suitable combination of X and
Y can determine optimal orientation of a specific group, which is crucial for the binding
and interaction of an inhibitor with the enzyme.
2
.5.Antiviral activity of 1 and 2 against wild-type DENV-2 virus
As and possess the most potent inhibitory activity to DENV-2 NS2B-NS3 protease
among these compounds, antiviral testing of and was conducted in wild-type DENV-2
virus plaque assays and the results are listed in Table 3. Both of and are moderately
active against wild-type DENV-2 virus plaque formation with similar EC50 values of 38.7
5.4 µM and 39.4 ± 6.2 µM, respectively; meanwhile, the CC50 values of and are
1
2
1
2
1
2
±
1
2
higher than 100 ꢀM at Huh-7 cells, indicating that they have no cytotoxic effect in the
tested concentration range. Importantly, these observations illustrate that these series
compounds could be as lead compounds to discover more potent inhibitors to treat and
prevent dengue virus infections.
<
Table 3>
.6. Docking analysis of inhibitory activity to DENV-2 NS2B-NS3 protease
To understand the relationship of the structural feature with the binding mode, the putative
binding poses of compounds [10], and to DENV-2 NS2B-NS3 protease were
2
1
2
3
compared by means of molecular docking. The docking model was generated from the
chain A of the crystal structure of NS2B-NS3 (PDB: 2FOM [11]) by using Discovery
Studio 3.5 and the results are shown in Fig. 4. In the fitted low energy conformation,
are binding close to the catalytic pocket (His51, Asp75 and Ser135) of NS2B-NS3
protease (Fig. 4a and Fig. 4b) even though there are slight binding variations in detail
between compound and dipeptide -Nitro phenyl in to substitute pyrrole ring in
occupies the cleft of Tyr 150, Ser135 and Pro132, and the oxygen of the -nitro phenyl
0 / 28
1 and
2
2
1.
p
2
1
p
1

ACCEPTED MANUSCRIPT
group in
2
interacts with Ser135 from the catalytic triad via a hydrogen bond. Importantly,
the fused ring of
2 is close to Asp75 and His51 of the catalytic triad, and its N-4 nitrogen
atom donates its lone-pair electrons to imidazole of His51 to form a hydrogen bond. Both
p
-nitro-anilide moieties in
Val52, but the -nitro-anilide in
-nitro-anilide in
network could be an important factor to stabilize the binding and block the catalytic
activity that both of two hydrogen bonds by the binding of are related with His51 and
Ser135 from the catalytic triad; meanwhile, one of three hydrogen bonds formed by
toward Ser135 from the catalytic pocket. The comparable inhibitory activity of and
1
and
2
are oriented toward the channel of Gly133, Ile36 and
p
1 is more towards the cleft of Ile36 and Val52 while the
p
2 is more towards the cleft of Ile36 and Gly133. The hydrogen bond
2
1
2
is
at
1
NS3 suggests that their predicted binding poses are complementary and the binding
discrepancy between and is compensated by hydrogen bonds with the catalytic pocket.
1
2
These results will be helpful for us to design more potent NS3 inhibitors.
Fig. 4>
<
It is noted that the putative binding conformation of 2 (Fig. 4d) is different from the free
conformation of 2 in crystal structure (Fig. 2b and Fig. 4e), especially the relative orientations of
two phenyl groups and the fused ring, illustrating that the conformational change of 2 may be
necessary to generate more favorable interactions between 2 and NS3.
In comparison of the docking results of 2 and 3 to NS3, it is proposed that their different
configurations at 3-position lead to a modified orientation of p-nitro-phenyl at C-3 and p-nitro
aniline at N-2 in 2 and 3, in which p-nitro-phenyl and p-nitro aniline in 3 (Fig. 4c) are oriented
towards the channel of Gly133, Ile36 and Val52, and the cleft of Tyr 150, Ser135 and Pro132,
respectively. There are two hydrogen bonds between 3 and NS3 and the fused ring is located very
close to His51; however, the twisted conformation of the fused ring in 3 renders the N-4 nitrogen
unable to form a hydrogen bond with the imidazole ring of His51 from the catalytic pocket due to
unfavorable chirality of the C-3 position in 3, which might explain at least in part the loss of its
binding and inhibitory activity to NS3 observed in 3.
3
. Conclusions
1
1 / 28

ACCEPTED MANUSCRIPT
In summary, a series of the fused ring derivatives of hexahydropyrrolo[1,2-c]-
imidazol-1-one were designed, synthesized, determined and assayed against DENV-2
NS2B-NS3 protease and wild-type DENV-2 virus. The data in Table 1 and Table 2 show
that non-peptidic fused-ring compound
similar potency against DENV-2 NS2B-NS3 protease as linear dipeptide
.4050, calculated from Chem3D). It is noteworthy that and possess moderate activity
against wild-type DENV-2 virus plaque formation (Table 3). From the point of molecular
docking, and have a similar putative binding mode even though their bindings to NS3
2
(CLogP = 3.7286, calculated from Chem3D) has
1
(CLogP =
1
1
2
1
2
slightly differ in some aspects. The preliminary SAR reveals that R and its stereochemistry
at C-3 position, X of a substituent at arene and Y of a linker between C-3 position and
arene are important for the fused-ring scaffold to block the active site of NS2B-NS3
protease. Although these inhibitors are not very potent at the current stage, it appears likely
that the further discovery of highly active non-peptidic NS2B-NS3 protease inhibitors will
be facilitated, based on the promising structural core element of the compounds. Further
studies will focus on designing more potent inhibitors for the treatment and prevention of
dengue virus infections and to decipher the underlying structural features and binding
mechanism.
4
. Experimental
4
.1. Chemistry
Unless stated, materials were obtained from commercial suppliers and used without further
1
13
purification. H and C NMR spectra were recorded on a Bruker AV-400 spectrometer at
00 and 101 MHz, respectively. Coupling constants ( ) are expressed in hertz (Hz).
Chemical shifts ( ) of NMR are reported in parts per million (ppm) units relative to the
4
J
δ
solvent. The high resolution of MS (HRMS) was recorded on an Applied Biosystems
Q-STAR Elite ESI-LC-MS/MS mass spectrometer. Melting points were measured using a
YRT-3 melting point apparatus (Shanghai, China) and were uncorrected.
4
.1.1. Preparation of intermediates 4 and 5
To the solution of Boc- -Proline (2.0 g, 9.3 mmol) and
0 ml anhydrous dichloromethane (DCM), dicyclohexylcarbodiimide (DCC) (1.9 g, 9.3
L
p-nitroamine (1.2 g, 9.0 mmol) in
6
mmol) was added and the reaction mixture was stirred for 5h. After resulting precipitate
was filtered and the solvent was evaporated, the residue was recrystallized from ethanol to
1
2 / 28

ACCEPTED MANUSCRIPT
afford intermediate tert-butyl (
4) (2.4 g, 72% yield).
S)-2-((4-nitrophenyl)carbamoyl)pyrrolidine-1-carboxylate
(
In a round-bottomed flask, compound
4 (2.9 mmol) was dissolved in 30 ml dry DCM.
o
The solution was cooled to 0 C and treated with trifluoroacetic acid (TFA) (TFA/DCM
(
V/V) = 1/4) and stirred for 2h. Upon completion, the solvent was evaporated and the
residue was compound ( )-2-((4-nitrophenyl)carbamoyl)pyrrolidin-1-ium trifluoroacetate
) and used for the next reaction without further purification.
.1.2. General preparation of compounds 6a1-6a4
(349 mg, 1.0 mmol) was dissolved in 8 ml ethanol. The solution was treated
S
(
5
4
Intermediate
5
with 0.15 ml triethylamine and distinct aldehyde (1.0 mmol). The reaction mixture was
stirred at 85℃ for 30 min. Upon completion, the solution was evaporated under the reduced
pressure and the residue was dissolved in ethyl acetate. The organic layer was washed with
water (3 × 50 mL) and brine (50 ml), dried over anhydrous Na SO and filtered and
2
4
concentrated under reduced pressure. The residue was purified by column chromatography
on silica gel (petroleum ether/ethyl acetate 10/1 to 6/1) to give 6a1-6a4
.1.2.1. ( )-2-(4-Nitrophenyl)hexahydro-1 -pyrrolo[1,2- ]imida-zol-1-one (6a1). 60%
yield, white solid, m.p. 197.5-203.3 C. H NMR (400 MHz, CDCl ) 8.35-8.16 (m, 2H,
), 4.67 (d, = 8.0 Hz, 1H,
), 3.34-3.29 (m, 1H, 5- ), 2.69 (dd, = 16.0, 8.0
), 1.99-1.79 (m, 2H, 6- ); C NMR (101 MHz,
), 143.4 (Ar- ), 125.0 (Ar-
), 27.9 (7- ), 25.2 (6-
.
4
S
H
c
o
1
δ
3
Ar-
H
), 7.87-7.71 (m, 2H, Ar-
), 3.96 (dd, = 4.0, 8.8 Hz, 1H, 7
Hz, 1H, 5- ), 2.36-2.11 (m, 2H, 7-
CDCl3) 175.1 (
), 66.5 (3-
H
), 5.03 (d,
J
= 8.0 Hz, 1H, 3-
H
J
3
-
H
J
a
-
H
H
J
1
3
H
H
H
δ
C
=O), 143.6 (Ar-
C
C
C
), 118.0 (Ar-
C
), 70.0
+
(
7
a-
C
C
), 55.9 (5-
C
C
C
); HRMS (ESI) m/z 248.1033 [M+H] ,
calculated for C H N O , 248.1035.
1
2
14
3
3
4
.1.2.2. (3R,7αS)-3-Methyl-2-(4-nitrophenyl)hexahydro-1H-pyrrolo[1,2-c]imidazol-1-
o
1
one (6a2). 74% yield, white solid, m.p. 154.2-157.3 C. H NMR (400 MHz, CDCl )
δ
3
8
J
2
.31-8.22 (m, 2H, Ar-
H
), 7.87-7.76 (m, 2H, Ar-
), 3.34-3.29 (m, 1H, 5-
), 1.94-1.80 (m, 2H, 6- ), 1.49 (d,
174.7 ( =O), 143.7 (Ar- ), 142.3 (Ar-
), 55.3 (5- ), 27.2 (7- ), 24.7 (6-
H
), 5.06 (q,
), 2.63 (dd,
= 6.0 Hz, 3H, CH3); C NMR
), 125.0 (Ar- ), 119.7
), 20.5 ( H ); HRMS
J
= 8.0 Hz, 1H, 3-
H
), 4.06 (dd,
= 12.0, 4.0 Hz, 1H, 7
a
-
H
H
J
= 16.0, 8.4 Hz, 1H, 5-
H
),
1
3
.33-2.08 (m, 2H, 7-
H
H
J
(
101 MHz, CDCl3)
δ
C
C
C
C
(
Ar-
C
), 77.2 (3-
C
), 64.5 (7
a
-
C
C
C
C
C
3
+
(
ESI) m/z 262.1188 [M+H] , calculated for C H N O , 262.1192.
1
3
16
3
3
4
.1.2.3.
(3R,7αS)-3-Ethyl-2-(4-nitrophenyl)hexahydro-1H-pyrrolo[1,2-c]imidazol-1-
o
1
one (6a3). 70% yield, white solid, m.p. 151.1-157.3 C. H NMR (400 MHz, CDCl )
3
δ
4
2
1
8.30-8.21 (m, 2H, Ar-
.01 (dd, = 8.0, 4.0 Hz, 1
.32-2.18 (m, 1H, 7- ), 2.10 (m, 1H, 7-
.61 (m, 1H, one of CH2CH ), 1.03 (t,
H
), 7.83-7.74 (m, 2H, Ar-
, 7 ), 3.34-3.29 (m, 1H, 5-
), 1.96-1.72 (m, 3H, 6-
= 7.4 Hz, 3H, CH CH3); C NMR (101 MHz,
H
), 4.80 (dd,
J
= 8.0, 4.0 Hz, 1H, 3-
H
),
J
H
a
-
H
H), 2.71-2.65 (m, 1H, 5-
H
),
H
H
H and one of CH2CH3),
1
3
J
3
2
CDCl3)
δ
175.3 (
C
=O), 143.7 (Ar-
C
), 143.1 (Ar-
3 / 28
C), 124.94 (Ar-C), 120.1 (Ar-C), 82.9
1

ACCEPTED MANUSCRIPT
(
3-
C
), 65.0 (7
a-
C
), 56.5 (5-
C
), 27.8 (7-C), 27.2 (CH CH ), 24.9 (6-C), 9.2 (CH2CH );
2 3 3
+
HRMS (ESI) m/z 276.1346 [M+H] , calculated for C H N O , 276.1348.
1
4
18
3
3
4
.1.2.4. (3R,7αS)-3-Isopropyl-2-(4-nitrophenyl)hexahydro-1H-pyrrolo[1,2-c]imidazol
o
1
-
1-one (6a4). 66% yield, white solid, m.p. 145.0-148.8 C. H NMR (400 MHz, CDCl )
3
δ
8.30-8.22 (m, 2H , Ar-
H
), 7.78-7.66 (m, 2H, Ar-
= 5.0, 4.8 Hz, 1H, 7 ), 3.36-3.31 (m, 5- ), 2.80-2.69 (m, 1H, 5-
(CH ) ), 2.08-1.95 (m, 2H, 7- ), 1.89-1.75 (m, 2H, 6- ), 1.04 (d,
= 6.8 Hz, 3H, one CH3 of CH(CH ) ); C NMR (101
3 2
H
), 4.77 (d,
J
= 4.0 Hz, 1H, 3-
), 2.29-2.17 (m,
= 6.8 Hz, 3H,
H), 3.98
(
dd,
J
a
-
H
H
H
1
H, C
H
H
H
J
3
2
1
3
one CH3 of CH(C
MHz, CDCl3) 174.8 (
7.0 (3- ), 66.4 (7a- ), 58.6 (5-
H of CH( H ) ), 14.8 (one H of CH(
H
3)2), 0.80 (d,
=O), 144.0 (Ar-
), 31.4( 7-
J
δ
C
C
), 142.8 (Ar-
), 29.0 (
C
), 124.9 (Ar-
C), 121.6 (Ar-C),
8
C
C
C
C
C
H(CH ) ), 25.2 (6-
C
), 18.4 (one
3
2
+
C
C
C
C
H ) ); HRMS (ESI) m/z 290.1498 [M+H] ,
3
3 2
3
3 2
calculated for C H N O , 290.1505.
1
5
20
3
3
4
.1.3. General preparation of compounds 6a5-12, 6b10, 6b11, 2 and 3
To a stirred solution of intermediate (349 mg, 1.0 mmol) and triethylamine (0.15 ml) in
5
toluene, phenylacetaldehyde or aromatic aldehyde (1.0 mmol) was added. The reaction
mixture was refluxed for 2h and then evaporated under reduced pressure to remove the
volatile solvents. The obtained crude was dispersed by water (50 mL) and extracted with
ethyl acetate (3 × 50 mL). The resulting organic phase was washed with water (3 × 50 mL)
and brine (50 mL), dried over Na SO , filtered, and concentrated under vacuum to afford a
2
4
yellow powder. The crude solid was purified by flash silica gel column chromatography
dichloromethane/ethyl acetate 10/0 to 10/1) to afford 6a5-12, 6b10, 6b11, 2 and
.1.3.1. (3 ,7αS)-3-Benzyl-2-(4-nitrophenyl)hexahydro-1 -pyrrolo[1,2- ]imidazol-1-
one (6a5). 68% yield, white solid, m.p. 145.1-150.4 C. H NMR (400 MHz, CDCl )
(
3.
4
R
H
c
o
1
3
δ
4
4
7
1
8.36-8.27 (m, 2H, Ar-
.8 Hz, 1H, 3- ), 3.61 (dd,
.9 Hz, 2H, Ar-CH2), 2.64-2.57 (m, 1H, 5-
H
), 7.91-7.83 (m, 2H, Ar-
= 9.2, 4.4 Hz, 1H, 7
), 2.20-2.11 (m, 1H, 7-
); C NMR (101 MHz, CDCl ) 174.9 (
), 127.2 (Ar- ), 125.0 (Ar-
H ), 27.8 (7- ), 24.8 (6-
H
), 7.35-7.17 (m, 5H, Ar-
H
), 5.15 (t,
J
J
=
=
H
J
a
-
H
), 3.32-3.25 (m, 1H, 5-
H), 3.00 (d,
H
H
), 2.10-1.97 (m, 1H,
=O), 143.8 (Ar- ),
), 120.1
); HRMS
1
3
-
H
), 1.92-1.75 (m, 2H, 6-
43.1 (Ar- ), 135.5 (Ar- ), 129.8 (Ar-
Ar- ), 82.1 (3- ), 64.7 (7
H
δ
C
C
3
C
C
C
), 128.5 (Ar-
C
C
C
(
C
C
a
-
C
), 56.3 (5- ), 39.9 (Ar-
C
C
C
C
2
+
(
ESI) m/z 338.1499 [M+H] , calculated for C H N O , 338.1505.
1
9
20
3
3
4
.1.3.2.
(3R
,7αS)-3-(4-Nitrobenzyl)-2-(4-nitrophenyl)hexahydro-1
H
-pyrrolo[1,2-c]-
o
1
imidaz-ol-1-one (6a6). 64% yield, red brown solid, m.p. 159.7-166.4 C. H NMR (400
MHz, DMSO-d6
Ar- ), 7.69 (d,
3.2 Hz, 1H, one of Ar-CH2), 4.40 (dd,
.39-3.34 (m, 1H, 5- ), 2.38-2.24 (m, 1H, 5-
171.9 ( =O), 148.5 (Ar-
), 124.2 (Ar- ), 122.3 (Ar- ), 119.1 (Ar-
)
δ
8.29-8.18 (m, 2H, Ar-
= 13.8 Hz, 1H, one of Ar-CH2), 7.34-7.24 (m, 2H, Ar-
= 8.4, 3.2 Hz, 1H, 3- ), 3.54-3.43 (m, 1H, 7
), 2.08-1.92 (m, 4H, 7- and 6- );
), 145.0 (Ar- ), 142.3 (Ar-
), 96.4 (3- ), 63.8
H), 8.00-7.93 (m, 2H, Ar-H), 7.92-7.84 (m, 2H,
H
J
H
), 5.21 (d,
J
=
),
C
1
3
J
H
a-H
1
3
H
H
H
H
NMR (101 MHz, DMSO- 6
d
)
δ
C
C
C
C),
1
41.2 (Ar- ), 124.9 (Ar-
C
C
C
C
C
C
1
4 / 28

ACCEPTED MANUSCRIPT
(
7
a-
C
), 48.7 (5-C), 40.1 (Ar-CH ), 30.7 (7-C), 23.3 (6-C); HRMS (ESI) m/z 383.1354
2
+
[
M+H] , calculated for C H N O , 383.1355.
1
9
19
4
5
4
.1.3.3. (3R,7αS)-2-(4-Nitrophenyl)-3-phenylhexahydro-1H-pyrrolo[1,2-c]imidazol-1-
o
1
one (6a7). 50% yield, white solid, m.p. 126.7-131.9 C. H NMR (400 MHz, CDCl )
δ
3
8
2
.19-8.13 (m, 2H, Ar-
H
), 7.77-7.70 (m, 2H, Ar-
H
), 7.44-7.33 (m, 3H, Ar-
= 6.4 Hz, 1H, 7 ), 3.61-3.49 (m, 1H, 5-
), 2.01-1.88 (m, 2H, 6-
H
), 7.32-7.27 (m,
), 2.91
); C NMR (101 MHz,
), 137.5 (Ar- ), 129.4 (Ar- ), 129.1
), 64.1 (7a- ), 56.0 (5- ), 27.4
H, Ar- ), 5.80 (s, 1H, 3-
H
H
), 4.07 (t,
), 2.30-2.20 (m, 2H, 7-
J
a
-
H
H
1
3
(
m, 1H, 5-
H
H
H
CDCl3)
δ
175.6 (
C
=O), 143.6 (Ar-
C
), 143.4 (Ar-
C
C
C
(
Ar-
C
), 125.8 (Ar-
), 24.7 (6-
24.1348.
.1.3.4.
]imidazol-1-one (6a8). 75% yield, white solid, m.p. 100.7-105.3 C. H NMR (400
MHz, CDCl3) 8.18-8.15 (m, 2H, Ar- ), 7.75-7.73 (m, 2H, Ar- ), 7.24-7.21 (m, 4H,
Ar- ), 5.81 (s, 1H, 3- ), 4.20-4.05 (m, 1H, 7 ), 3.80-3,55 (m, 1H, C (CH ) ),
.92-2.86 (m, 2H, 5-H), 2.28-2.25 (m, 2H,7-H), 1.96 (s, 2H, 6-H), 1.21 ( dd, = 7.2, 2 Hz,
C
), 124.8 (Ar- ), 119.5 (Ar-
C
C
), 82.9 (3-
C
C
C
+
(
7-
C
C
); HRMS (ESI) m/z 324.1344 [M+H] , calculated for C H N O ,
1
8
18
3
3
3
4
(3R,7αS)-3-(4-Isopropylphenyl)-2-(4-nitrophenyl)-hexa-hydro-1H-pyrrolo
o
1
[1,2-c
δ
H
H
H
H
a
-
H
H
3
2
2
6
J
1
3
H, CH(CH3) ); C NMR (101 MHz, CD OD)
δ
177.1 (
), 127.5 (Ar-
H(CH ) ), 28.5 (7-
C
=O), 151.1 (Ar-
C
), 145.2
), 121.7
2
3
(
(
(
Ar-
Ar-
C
C
), 144.5 (Ar-
C
), 137.4 (Ar-
C
), 128.2 (Ar-
C
C
), 125.5 (Ar-
C
), 83.8 (3-
C
), 65.41 (7a-
C
), 56.7 (5-
C
), 35.1 (
C
C
), 25.6 (6-
C
), 24.3
3
2
+
CH(
.1.3.5.
,7αS)-3-(4-Methoxyphenyl)-2-(4-nitrophenyl)hexa-hydro-1
C
H ) ); HRMS (ESI) m/z 366.1811 [M+H] , calculated for C H N O , 366.1818.
3
2
21 24
3
3
4
(
3
R
H
-pyrrolo[1,2-c]
o
1
imidazol-1-one (6a9). 67% yield, white solid, m.p. 142.7-145.3 C. H NMR (400 MHz,
CDCl3)
δ
8.26-8.14 (m, 2H, Ar-
.97-6.88 (m, 2H, Ar- ), 5.78 (s, 1H, 3-
.56-3.46 (m, 1H, 5- ), 2.91-2.85 (m, 1H, 5-
); C NMR (101 MHz, CDCl3) 176.0 (
), 130.1 (Ar- ), 127.1 (Ar- ), 124.7 (Ar-
4.1 (7 ), 55.8 (5- ), 55.4 ( H ), 27.4 (7-
M+H] , calculated for C H N O , 354.1454.
H
), 7.80-7.70 (m, 2H, Ar-
), 4.14-4.03 (m, 1H, 7
), 2.32-2.22 (m, 2H, 7-
=O), 160.0 (Ar- ), 143.5 (Ar-
), 119.5 (Ar- ), 114.7 (Ar- ), 82.6 (3-
), 24.8 (6- ); HRMS (ESI) m/z 354.1450
H
), 7.28-7.20 (m, 2H, Ar-
), 3.81 (s, 3H, CH3),
), 1.96-1.91 (m, 2H,
), 132.0
),
H),
6
3
6
H
H
a-H
H
H
H
1
3
-
H
δ
C
C
C
(
Ar-
C
C
C
C
C
C
C
6
a
-
C
C
C
C
C
3
+
[
1
9
20
3
4
4
.1.3.6.
(3R
,7αS)-2-(4-Nitrophenyl)-3-(4-(trifluoromethyl)-phenyl)-hexahydro-1
H
-
o
1
pyrrolo[1,2-
NMR (400 MHz, CDCl3)
.2 Hz, 2H, Ar- ), 7.42 (d,
Hz, 1H, 7 ), 3.55-3.50 (m, 5-
.99-1.87 (m, 2H, 6-
Ar- ), 142.2 (Ar-
c
]-imidazol-1-one (6a10). 57% yield, white solid, m.p. 139.1-143.5 C. H
δ
8.23-8.14 (m, 2H, Ar-
= 8.4 Hz, 2H, Ar- ), 5.85 (s, 1H, 3-
), 2.92-2.86 (m, 1H, 5- ), 2.28-2.19 (m, 2H, 7-H
H
), 7.75-7.69 (m, 2H, Ar-
H
), 7.65 (d,
= 6.0, 7.6
),
J =
8
H
J
H
H
), 3.99 (dd,
J
a
-
H
H
H
1
3
1
H
); C NMR (101 MHz, CDCl )
δ
4
175.8 (
C
=O), 143.8 (Ar-
= 33 Hz, 4”-
C
), 143.1
3
9
6
1
9
(
(
C
C
), 131.3 (q, 131.7 , 131.4 , 131.1 , 130.8 ,
J
C
), 126.3
= 3 Hz,
4
1
7
126.4 , 126.4 , 126.3 , the last peak overlapped with the peak at 126.3 ppm,
J
1
5 / 28

ACCEPTED MANUSCRIPT
7
5
5
3
3
”-
F3), 119.4 (Ar-
ESI) m/z 392.1217 [M+H] , calculated for C H F N O , 392.1222.
C
), 126.3 (Ar-
C
), 126.4 (Ar-
C
), 123.7 (q, 127.7 , 125.0 , 122.3 , 119.6 , = 272 Hz,
J
C
C
), 82.2 (3- ), 64.1 (7
C
a
-
C
), 56.1 (5- ), 27.5 (7- ), 24.8 (6-C); HRMS
C
C
+
(
1
9
17
3
3
3
4
.1.3.7.
(3S
,7αS)-2-(4-Nitrophenyl)-3-(4-(trifluoromethyl)-phenyl)hexahydro-1
H
-
o
1
pyrrolo[1,2-
c
]-imidazol-1-one (6b10). 12% yield, white solid, m.p. 82.1-83.5 C. H
NMR (400 MHz, CDCl3)
δ
8.18-8.12 (m, 2H, Ar-
), 6.48 (s, 1H, 3- ), 4.20-4.05 (m, 1H, 7
); C NMR (101 MHz, CDCl )
H
), 7.62 (d,
J
= 8.4 Hz, 2H, Ar-
H),
7
5
.46-7.35 (m, 4H, Ar-
H
H
a-H), 2.53-2.20 (m, 4H,
1
3
-H and 7- ), 1.85-1.70 (m, 2H, 6-
H
H
δ
175.9 (C=O), 143.9
3
2
1
7
6
(
Ar-
C
), 143.2 (Ar-
C
), 142.2 (Ar-
C
), 131.3 (q, 131.8 , 131.5 , 131.1 , 130.8 ,
J
= 31 Hz,
4
9
6
1
4
J
2
”-
C
), 126.4 (126.4 , 126.4 , 126.4 , the last peak overlapped with the peak at 126.4 ppm,
8
9
9
8
= 3 Hz, 3”-
C
), 126.5 (Ar-
C
), 126.4 (Ar-
C
), 123.7 (q, 127.7 , 125.0 , 122.3 , 119.6 ,
J
=
71 Hz, F3),119.7 (Ar-
C
C
), 82.2 (3-
C
), 64.1 (7 ), 56.1 (5- ), 27.5 (7- ), 24.9 (6-
a
-
C
C
C
C
);
+
HRMS (ESI) m/z 392.1218 [M+H] , calculated for C H F N O , 392.1222.
1
9
17
3
3
3
4
.1.3.8. (3R,7αS)-3-(4-Chlorophenyl)-2-(4-nitrophenyl)hexahydro-1H-pyrrolo[1,2-c]-
o
1
imidazol-1-one (6a11). 63% yield, white solid, m.p. 146.0-151.8 C. H NMR (400 MHz,
CDCl3)
δ
8.24-8.15 (m, 2H, Ar-
.30-7.20 (m, 2H, Ar- ), 5.76 (s, 1H, 3-
.55-3.45 (m, 1H, 5- ), 2.84 (m, 1H, 5- ), 2.34-2.18 (m, 2H, 7-
); C NMR (101 MHz, CDCl3) 175.9 ( =O), 143.7 (Ar- ), 143.2 (Ar-
), 134.9 (Ar- ), 129.5 (Ar- ), 127.3 (Ar- ), 124.8 (Ar- ), 119.5 (Ar- ), 82.2 (3-
4.1 (7 ), 55.9 (5- ), 27.4 (7- ), 24.7 (6- ); HRMS (ESI) m/z 358.0953 [M+H] ,
calculated for C H ClN O , 358.0958.
H
), 7.78-7.69 (m, 2H, Ar-
H
), 7.42-7.34 (m, 2H, Ar-
H
),
7
3
6
H
H
), 3.98 (dd,
J
= 13.2, 5.4 Hz, 1H, 7
a-
H
),
H
H
H
), 2.01-1.87 (m, 2H,
), 136.7
),
1
3
-
H
δ
C
C
C
(
Ar-
C
C
C
C
C
C
C
+
6
a
-
C
C
C
C
1
8
17
3
3
4
.1.3.9. (3S,7αS)-3-(4-Chlorophenyl)-2-(4-nitrophenyl)hexa-hydro-1H-pyrrolo[1,2-c]
o
1
imidazol-1-one (6b11). 15% yield, white solid, m.p. 80.3-91.5 C. H NMR (400 MHz,
CDCl3)
δ
8.18-8.11 (m, 2H, Ar-
= 7.9 Hz, 2H, Ar- ), 6.40 (s, 1H, 3-
), 1.85-1.70 (m, 2H, 6-H); C NMR (101 MHz, CDCl )
H
), 7.46-7.39 (m, 2H, Ar-
H
), 7.36-7.29 (m, 2H, Ar-
), 2.54-2.18 (m,
176.0 ( =O),
43.6 (Ar-C), 143.2 (Ar-C), 136.7 (Ar-C), 134.9 (Ar-C), 129.5 (Ar-C), 127.3 (Ar-C), 124.8
-C), 55.9 (5-C), 27.4 (7-C), 24.8 (6-C); HRMS
ESI) m/z 358.0954 [M+H] , calculated for C H ClN O , 358.0958.
H),
7
4
1
.19 (d,
H, 5-
J
H
H
), 4.20-3.98 (m, 1H, 7a-H
1
3
H
and 7-
H
δ
C
3
(
Ar-C), 119.4 (Ar-C), 82.2 (3-C), 64.1 (7a
+
(
1
8
17
3
3
4
.1.3.10. (3R,7αS)-2,3-bis(4-Nitrophenyl)hexahydro-1H-pyrrolo-[1,2-c]imidazol-1-one
o
1
(
2). 60% yield, white solid, m.p. 125.3-142.1 C. H NMR (400 MHz, CDCl )
δ
8.25 (d,
J
3
=
8.8 Hz, 2H, Ar-
Hz, 2H, Ar- ), 5.90 (s, 1H, 3-
.93 (m, 1H, 5- ), 2.33-2.19 (m, 2H, 7-
175.3 ( =O), 148.3 (Ar- ), 144.9 (Ar-
), 124.6 (Ar- ), 119.5 (Ar-
H
), 8.22-8.16 (m, 2H, Ar-
), 4.01 (dd,
), 2.02-1.89 (m, 2H, 6-H
H
), 7.76-7.65 (m, 2H, Ar-
H
), 7.49 (d,
J
= 8.8
H
H
J
= 7.8, 5.2 Hz, 1H, 7
a
-
H
), 3.56 (m, 1H, 5-
H
),
); C NMR (101 MHz,
), 142.8 (Ar- ), 127.1
), 64.1 (7 ), 56.2 (5- ), 27.5
1
3
2
H
H
CDCl3)
δ
C
C
C
), 144.0 (Ar-
C
C
(
Ar-
C
), 125.0 (Ar-
C
C
C
), 81.8 (3-
C
a
-
C
C
1
6 / 28

ACCEPTED MANUSCRIPT
+
(
7-
C
), 24.9 (6-
69.1199.
.1.3.11. (3
C
); HRMS (ESI) m/z 369.1194 [M+H] , calculated for C H N O ,
1
8
17
4
5
3
4
S
,7αS)-2,3-bis(4-Nitrophenyl)hexahydro-1
H
-pyrrolo-[1,2-
c
]imidazol-1-one
8.25-8.20
), 7.45-7.39 (m, 2H,
and 7- ), 1.86 (s,
), 144.9 (Ar- ), 144.0
), 119.4 (Ar- ), 81.8 (3- ),
C); HRMS (ESI) m/z 369.1198 [M+H] ,
o
1
(
3). 15% yield, white solid, m.p. 65.0-70.1 C. H NMR (400 MHz, CDCl ) δ
3
(
m, 2H, Ar-
H
), 8.19-8.13 (m, 2H, Ar-
), 4.28-4.05 (m, 1H, 7
); C NMR (101 MHz, CDCl ) 175.4 (
), 125.0 (Ar- ), 124.6 (Ar-
), 27.5 (7- ), 24.9 (6-
H
), 7.48 (d,
), 2.54-2.19 (m, 4H 5-
=O), 148.2 (Ar-
J = 7.7 Hz, 2H, Ar-H
Ar-
H, 6-
Ar- ), 142.8 (Ar-
4.1 (7 ), 56.2 (5-
H
), 6.55 (s, 1H, 3-
H
a
-
H
H
H
1
3
2
H
δ
C
C
C
3
(
C
C
), 127.1 (Ar-
C
C
C
C
C
+
6
a
-
C
C
C
calculated for C H N O , 369.1199.
1
8
17
4
5
4
.1.3.12.
(3R,7αS)-3-(3-Nitrophenyl)-2-(4-nitrophenyl)hexahydro-1H-pyrrolo[1,2-c]
o
1
imidazol-1-one (6a12). 55% yield, white solid, m.p. 109.9-126.1 C. H NMR (400 MHz,
CDCl3)
δ
8.35-8.30 (m, 1H, Ar-
.79-7.70 (m, 2H, Ar- ), 7.66-7.57 (m, 2H, Ar-
.71 (m, 1H, 5- ), 3.05 (m, 1H, 5- ), 2.41-2.29 (m, 2H, 7-
175.5 ( =O), 148.8 (Ar- ), 144.0 (Ar-
), 130.4 (Ar- ), 125.0 (Ar- ), 124.0 (Ar-
), 64.1 (7 ), 56.1 (5- ), 27.5 (7- ), 24.9 (6-
H
), 8.26-8.24 (m, 1H, Ar-
), 6.01 (s, 1H, 3-
), 2.12-1.98 (m, 2H, 6-
), 142.9 (Ar- ), 140.7
), 121.6 (Ar- ), 119.7
); HRMS (ESI) m/z
H
), 8.22-8.18 (m, 2H, Ar-
H
),
7
3
H
H
H
), 4.15 (m, 1H, 7
a-
H
),
1
3
H
H
H
H); C
NMR (101 MHz, CDCl3)
δ
C
C
C
C
(
(
Ar-
Ar-
C
C
), 131.5 (Ar-
C
C
C
C
C
), 81.8 (3-
C
a-
C
C
C
C
+
3
69.1195 [M+H] , calculated for C H N O , 365.1199.
1
8
17
4
5
4
.2. General information for bioassay
All compounds were dissolved in DMSO and diluted into gradient stock solution before use, and
the final concentration of DMSO in every well including control well was 0.5%.
4
.2.1. Inhibitory activity assay against DENV-2 NS2B-NS3 protease
DENV-2 NS2B-NS3 protease was expressed with the plasmid kindly provided by Dr. Siew
Phengsuozeg Lim, Novartis Institute for Tropical Diseases, Singapore [17]. The assay was
conducted as our previous report [10,15]. Briefly, the inhibitory activity of a compound
o
against DENV-2 NS2B-NS3 protease were measured at 37 C in the reaction buffer (50
mM Tris–HCl, pH 9.0, 10 mM NaCl, 20% glycerol, 1 mM CHAPS, and 0.04% NaN ) and
3
Benzoyl-Nle-Lys-Arg-Arg-AMC (33 ꢀM) was used as a substrate. The protease was
o
incubated with different concentrations of an inhibitor at 37 C for 3 min before the
substrate (33 ꢀM) was added. The remaining enzyme activity was measured with a
fluorescence spectrophotometer using 356 and 438 nm as the excitation and emission
wavelengths, respectively. Vehicle control was normalized as 100% remaining NS3
1
0
enzymatic activity and active compound
1 reported in our previous work was used as a
reference (Table 1, entry 17). IC50 value of an inhibitor was calculated based on the
relationship of its remaining NS3 enzymatic activity vs its corresponding concentration.
The assay data were averaged from triplicate measurements and the results are shown in
Table 1, Table 2 and Fig 3.
1
7 / 28

ACCEPTED MANUSCRIPT
4
.2.2. Cytotoxicity assay for compounds 1 and 2 to Huh-7 cells
All cells were cultivated in Dulbecco's Modified Eagle Medium(DMEM) supplemented
with 10 % fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin at 37
°
C, 5 % CO and 95 % relative humidity. For assessing cytotoxicity, Huh-7 cells were
2
4
seeded into 96-well plates with a density of 10 cells per well in presence of increasing
concentrations of the tested compounds. After overnight incubation, medium was removed
and 50 µl of fresh DMEM was added to the cells. Cell viability was determined using
Cell-Titer Glo Luminescent Viability Assay (Promega) by recording luminescence for 2 s
with a FluoStar Omega microplate luminometer (BMG Labtech). All concentrations were
measured in triplicates and compared to an untreated control.
4
.2.3. Wild-type DENV-2 virus plaque assay for compounds 1 and 2
Antiviral activity of compounds
1
and
2
were determined by plaque assay. First, Huh-7
4
cells were seeded into 96-well plates with a density of 10 cells per well and infected with
wild type DENV serotype 2 with a multiplicity of infection (MOI) of 1. After one hour, a
gradient of non-toxic concentrations of the compounds were added to the infected cells and
plates were incubated for 48 h at 37 °C. Each concentration was assayed in triplicates.
Following this, the supernatant was harvested and used for determination of the virus yield
reduction by plaque assay using Vero E6 cells. Therefore Vero E6 cells were seeded in
5
2
4-well plates with a density of 2.5 x 10 cells per well and incubated overnight at 37 °C.
-
1
-6
The virus containing medium was diluted with DMEM ranging from 10 to 10 .
Afterwards 100 µl of these dilutions were used to infect the Vero E6 cells with agitation
for 1 h. Then, the medium was removed and 1 ml of plaque medium was added. After
further incubation for 7 days at 37 °C the cells were fixed with 5 % (v/v) formaldehyde for
2
h, stained with crystal violet and plaques were counted. Reduction of the viral titer was
monitored by comparison to an untreated control.
Acknowledgements
Authors thank Dr. Qian Li for his kind help of the molecular docking. This work was
supported by Natural Science Foundation of China (U0632001) and Six Major Talents of
Jiangsu Province of China (2014). CK appreciates financial support by the Deutsche
Forschungsgemeinschaft, Grant KL-1356/3-1.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version, at
1
http://dx.doi.org/. These data include 2D NOESY spectra of compounds 2 and 6a5, H NMR and
1
3
C NMR spectra of new compounds, MOL file of reported compound of 1 and new compound 2
described in this article.
1
8 / 28

ACCEPTED MANUSCRIPT
Notes and references
[
1] M. G. Guzman, S. B. Halstead, H. Artsob, P. Buchy, J. Farrar, D. J. Gubler, E. Hunsperger, A. Kroeger,
H. S. Margolis, E. Martinez, M. B. Nathan, J. L. Pelegrino, C. Simmons, S. Yoksan, R. W. Peeling, Nat.
Rev. Microbiol. 8 (2010) 7-16.
[
[
2] J. G. R. Perez, G. G. Clark, D. J. Gubler, P. Reiter, E. J. Sanders, A. V. Vorndam, Lancet 352 (1998)
9
71-976.
3] (a) D. Normile, Science 342 (2013) 415; (b) S. Bhatt, P. W. Gething, O. J. Brady, J. P. Messina, A. W.
Farlow, C. L. Moyes, J. M. Drake, J. S. Brownstein, A. G. Hoen, O. Sankoh, M. F. Myers, D. B. George,
T. Jaenisch, G. R. W. Wint, C. P. Simmons, T. W. Scott, J. J. Farrar and S. I. Hay, Nature 496 (2013)
5
04–507.
4] A. P. Goncalvez, R. E. Engle, M. S. Claire, R. H. Purcell, C. Lai, Proc. Natl. Acad. Sci. U.S.A. 104 (2007)
422-9427.
5] (a) C. M. Rice, E. M. Lenches, S. R. Eddy, S. J. Shin, R. L. Sheets, J. H. Strauss, Science 229 (1985) 726;
b) T. J. Chambers, C. S. Hahn, R. Galler, C. M. Rice, Annu. Rev. Microbiol. 44 (1990) 649-688. (c) S. J.
[
[
9
(
Kaptein, J. Neyts, Curr. Opin. Pharmacol. 30 (2016) 1-7. (d) M. Saudi, J. Zmurko, S. Kaptein, J.
Rozenski, B. Gadakh, P. Chaltin, A. Marchand, J. Neyts, A. V. Aerschot, Euro. J. Med. Chem. 121
(
2016) 158-168. (e) Y.-L. Chen, F. Yokokawa, P.-Y. Shi, Antiviral Res. 122 (2015) 12–19. (f) C. G.
Noble, S. P. Lim, R. Arora, F. Yokokawa, S. Nilar, C. C. Seh, S. K. Wright, T. E. Benson, P. W. Smith,
P. Y. Shi. J. Biol. Chem. 291 (2016) 8541-8548.
[
[
6] (a) S. Melino, S. Fucito, A. Campagna, F. Wrubl, A. Gamarnik, D. O. Cicero and M. Paci, FEBS J. 273
(
(
2006) 3650-3662; (b) R.Yusof, S.Clum, M.Wetzel, H. M.Murthy, R.Padmanabhan,J. Biol. Chem. 275
2000) 9963-9969.
7] (a) J. Lescar, D. Luo, T. Xu, A. Sampath, S. P. Lim, B. Canard, S. G. Vasudevan, Antiviral Res. 80 (2008)
4; (b) B. Falgout, M. Pethel, Y. M. Zhang, C. J. Lai, J. Virol. 65 (1991) 2467-2475; (c) C. G. Noble, Y.
L. Chen, H. P. Dong, F. Gu, S. P. Lim, W. Schul, Q. Y. Wang, P. Y. Shi, Antiviral Res. 85 (2010)
50-462.
9
4
[
[
8] (a) C. Nitsche, S. Holloway, T. Schirmeister, C. D. Klein, Chem. Rev. 114 (2014) 11348-11381. (b) S.
Bhakat, W. Karubiu, V. Jayaprakash, M. E.S. Soliman, Euro. J. Med. Chem. 87 (2014) 677-702. (c) S. P.
Lim, Q.-Y. Wang, C. G. Noble, Y.-L. Chen, H. Dong, B. Zou,F. Yokokawa, S. Nilar, P. Smith, D. Beer,
J. Lescar, P.-Y. Shi, Antiviral Res. 100 (2013) 500–519.
9] (a) H. L. Liu, R. M. Wu, Y. Y. Sun, Y. Ye, J. Chen, X. M. Luo, X. Shen, H. Liu, Bioorg. Med. Chem. 22
(
2014) 6344-6352; (b) S. Pambudi, N. Kawashita, S. Phanthanawiboon, M. D. Omokoko, P. Masrinoul,
A. Yamashita, K.Limkittikul, T. Yasunaga, T. Takagi, K. Ikuta, T. Kurosu, Biochem. Biophys. Res.
Commun. 440 (2013) 393-398; (c) S. K. V. Vernekar,L. Qiu,J. Zhang, J. Kankanala, H. M. Li, R. J.
Geraghty and Z. Q. Wang, J. Med. Chem. 58 (2015) 4016-4028; (d) X. Y. K. Stenta, J. Joy, S. F. Wang,
P. Z. Kwek, J. L. K. Wee, K. F. Wan, S. Gayen, A. S. Y. Chen, C. B. Kang, M. A. Lee, A. Poulsen, S. G.
Vasudevan, J. Hill, K. Nacro, Drug Des. Dev. Ther. 9 (2015) 6389-6399; (e) L.Ramos, F. D. Sousa, H.
M. Wu, L. Nebo, W. Kiefer, M. Kanitz, J. Bodem, W. E. Diederich, T. Schirmeister, P. C. Vieira, Bioorg.
Med. Chem. 23 (2015) 466-470; (f) T. T. H. Nguyen, S. Lee, H. K. Wang, H. Y. Chen, Y. T. Wu, S. C.
Lin, D. W. Kim and D. Kim, Molecules 18 (2013) 15600-15612; (g) U. Viswanathan, S. M. Tomlinson,
J. M. Fonner, S. A. Mock and S. J. Watowich, J. Chem. Inf. Model. 54 (2014) 2816-2825; (h) A. K.
Timiri, S. Subasri, M. Kesherwani, V. Vishwanathan, B. N. Sinha, D. Velmurugan, V. Jayaprakash,
Bioorg. Chem. 62 (2015) 74-82; (i) L. F. Li, C. Basavannacharya, K. W. K. Chan, L. Q. Shang, S. G.
Vasudevan and Z. Yin, Chem. Biol. Drug Des. 86 (2015) 255-264. (j) A. K. Timiri, B. N. Sinha, V.
1
9 / 28

ACCEPTED MANUSCRIPT
Jayaprakash, Euro. J. Med. Chem. 117 (2016) 125-143. (k) C.-H. Tseng, C.-K. Lin, Y.-L. Chen, C.-Y.
Hsu, H.-N. Wu, C.-K. Tseng, J.-C. Lee, Euro. J. Med. Chem. 79 (2014) 66-76. (l) A. L. Carreño Otero, L.
Y. Vargas Méndez, J. E. Duque L., V. V. Kouznetsov, Euro. J. Med. Chem. 78 (2014) 392-400.
10] G.-C. Zhou, Z. Weng, X. Shao, F. Liu, X. Nie, J. Liu, D. Wan , C. Wang, K. Guo, Bioorg. Med. Chem.
Lett. 23 (2013) 6549-6554.
[
[
11] P. Erbel, N. Schiering, A. D’Arcy, M. Renatus, M. Kroemer, S.P. Lim, Z. Yin, T. H. Keller, S. G.
Vasudevan, U. Hommel, Nat. Struct. Mol. Biol. 13 (2006) 372-373.
[
[
12] C. G. Noble, C. C. Seh, A. T. Chao, P. Y. Shi, J. Virol. 86 (2012) 438-446.
13] (a) L. Cruz, T. H. D. Nguyen, K. Ozawa, J. Shin, B. Graham, T. Huber, G. Otting, J. Am. Chem. Soc.
1
5
33 (2011) 19205-19215; (b) W. N. Chen, K. V. Loscha, C. Nitsche, B. Graham, G. Otting, FEBS Lett.
88 (2014) 2206–2211.
[
[
[
14] (a) S. Idrees, U. A. Ashfaq, Asian Pac. J. Trop. Med. 7 (2014) 513-516; (b) A. J. Hell, D. J. A.
Crommelin, W. E. Hennink, E. Mastrobattista, Pharm. Res. 26 (2009) 2186-2193.
15] S. Q. Xu, H. Li, X. X. Shao, C. Fan, B. Ericksen,J. S. Liu, C. W. Chi, C. G. Wang, J. Med. Chem. 55
(
2012) 6881-6887.
16] (a) F. A. Khan, S. Ahmad, Tetrahedron Lett. 54 (2013) 2996–2998. (b). S. Oshiro, T. Nakura, T.
Okamoto, K. Okumura, Jpn. TokkyoKoho (1971) JP 46016990. (c) H.-J. Federsel, E. Konberg, L.
Lilljequist, B.-M. Swahn,J. Org. Chem. 55 (1990)2254-2256. (d) J. E. Mills, C. A. Maryanoff, D. F.
McComsey, R. C. Stanzione, L. Scott, L. J. Org. Chem. 52 (1987) 1857-1859 and references cited
therein. (e) Y. Uozumi, K. Mizutani, S.-i. Nagai, Tetrahedron Lett. 42 (2001) 407–410. (f) Y. Uozumi, K.
Shibatomi, Jpn. KokaiTokkyoKoho (2001) JP 2001328993 A 20011127.
[
17] J. Li, S. P. Lim, D. Beer, V. Patel, D. Wen, C. Tumanut, D. C. Tully, J. A. Williams, J. Jiricek, J. P.
Priestle, J. L. Harris, S. G. Vasudevan, J. Biol. Chem. 280 (2005) 28766-28774.
2
0 / 28

ACCEPTED MANUSCRIPT
2
"
6
7
2
"
3
"
5
7a
3
"
1
N 2
2
O
O
4N
O N 3α, 2
2
3
2'
H N
N
R =
2
HN
NO2
3'
R
2"
'
3'
O
3"
2"
3"
NO
2
3
α-R, 2 and 6a series
S
1
3
β-R, 3 and 6b series
3β, 3
O N
2
previous work
this work
Fig. 1 The chart of NS2B-NS3 protease inhibitor design
2
1 / 28

ACCEPTED MANUSCRIPT
Fig. 2 Crystal structure diagrams of 6a11 (a) and 2 (b). The crystal structures of 6a11 and 2 were deposited at the
Cambridge Crystallographic Data Centre (CCDC) with deposition numbers1474483 and1474481, respectively.
2
2 / 28

ACCEPTED MANUSCRIPT
Fig. 3 Relationships between the substituents (R) in 3-position of compounds 6a1 to 6a4 and their inhibitory
activity at the dengue NS2B-NS3 protease.
2
3 / 28

ACCEPTED MANUSCRIPT
Fig. 4 Three-dimensional (3D) diagrams of putative binding poses of 1 (a), 2 (b) and 3 (c) in the NS2B-NS3
active site (His51, Asp75 and Ser135). The figures were generated from chain A of 2FOM [11] from the
same view. Hydrogen bonds are indicated by green dashed lines and critical amino acid residues of the
binding pocket are labelled. The docking conformation (d) of
conformation (e) of was produced from Fig. 2b.
2 was extracted from (b) and the crystal
2
2
4 / 28

ACCEPTED MANUSCRIPT
Scheme 1
NH2
NO2
O
O
a
b
+
N
N
OH
HN
NO2
Boc
Boc
4
O
O
c
N
NO2
N
H
TFA
N
HN
NO2
R
5
2 and 6a series: α-R, designated as trans
3
and 6b series: β-R, designated as cis
o
Reagents and conditions: (a) DCM, DCC, rt, 5h. (b) DCM, TFA, 0 C, 2h. (c) aliphatic aldehydes (including
phenylacetaldehydes) or aromatic aldehydes, TEA, reflux in EtOH or toluene, 30min.
2
5 / 28

ACCEPTED MANUSCRIPT
Table 1 Reaction results and the inhibitory activity to NS2B-NS3 protease
a
Chemical shift (ppm)
b
c,d
entry cmpd No.
R (α or β)
Yield (%)
Activity to NS3
3
-H
7a-H
1
2
3
4
5
6
7
8
9
6a1
6a2
6a3
6a4
6a5
6a6
6a7
6a8
6a9
6a10
6b10
6a11
6b11
2
H
NA
5.06 (q, J = 8.0 Hz)
4.80 (dd, J = 4.0, 8.0 Hz)
4.77 (d, J = 4.0 Hz)
5.15 (t, J = 4.8 Hz)
4.40 (dd, J = 3.2, 8.4 Hz)
5.80 (s)
3.96 (dd, J = 4.0, 8.8 Hz)
4.06 (dd, J = 4.0, 12.0 Hz)
4.01 (dd, J = 4.0, 8.0 Hz)
3.98 (dd, J = 4.8, 5.0 Hz)
3.61 (dd, J = 4.4, 9.2 Hz)
3.43-3.54 (m)
60
74
70
66
68
64
50
75
67
57
12
63
15
60
15
55
61% (16)
Me (α)
Et (α)
i-Pr (α)
Bn (α)
58% (16)
40% (16)
66% (16)
58% (20)
p-NO
2
-Bn (α)
/
Ph (α)
4.07 (t, J = 6.4 Hz)
4.05-4.20 (m)
33% (200)
p-(i-Pr)-Ph (α)
p-OMe-Ph (α)
5.81 (s)
/
/
/
/
/
/
5.78 (s)
4.03 -4.14 (m)
1
0
p-CF
3
-Ph (α)
-Ph (β)
5.85 (s)
3.99 (dd, J = 6.0, 7.6 Hz)
4.05-4.20 (m)
1
1
2
3
4
5
6
7
p-CF
3
6.48 (s)
1
1
1
1
1
1
p-Cl-Ph (α)
p-Cl-Ph (β)
5.76 (s)
3.98 (dd, J = 5.4, 13.2 Hz)
3.98-4.20 (m)
6.40 (s)
p-NO
2
-Ph (α)
-Ph (β)
5.90 (s)
4.01 (dd, J = 5.2, 7.8 Hz)
4.05-4.28 (m)
IC50 = 1.2 ±0.4
3
p-NO
2
6.55 (s)
/
6a12
1
m-NO
2
-Ph (α)
6.01 (s)
4.15 (t, J = 8.0 Hz)
/
f
NA
IC50 = 1.2 ± 0.4
a
NMR experiments were conducted in CDCl except entry 6 (6a6 in DMSO-d6) due to insolubility of 6a6
3
b
c
in chloroform. Isolated yield. Percent residual activity of DENV-2 NS2B-NS3 (inhibitor concentration,
ꢀ
to 200 ꢀM of inhibitor. Not applicable.
d
M) or IC (ꢀM). “/” means that there is higher than 50% remaining activity of DENV-2 NS2B-NS3 up
50
f
2
6 / 28

ACCEPTED MANUSCRIPT
Table 2 SARs determined by substituent (X) and linker (Y) for two series of NS2B-BS3 inhibitors
Linear dipeptides
Fused ring derivatives
O
O
parameters
HN
Y
NH
X
N
NO2
N
O
N
2
Y
X
S
a
b
c
b
X
NO
H
Y
cmpd
activity to NS3
31% (15)
cmpd
activity to NS3
2
empty
empty
1 (1)
2
32% (15)
33% (200)
inactive
(12d)
(12e)
(12f)
inactive
6a7
6a6
6a5
NO
2
CH
2
2
36% (200)
58% (15)
H
a
CH
58% (20)
Compounds and their inhibitory activity, compound identifiers in parentheses from our previous report
10]. Percent residual activity of DENV-2 NS2B-NS3 (inhibitor concentration, ꢀM), and “inactive”
b
[
means that there is higher than 50% remaining activity of DENV-2 NS2B-NS3 at inhibitor concentrations
c
up to 200 ꢀM. From Table 1.
2
7 / 28

ACCEPTED MANUSCRIPT
Table 3 Antiviral activity of 1 and 2 against wild-type DENV-2 virus
entry cmpd No. EC50 (µM) against wild-type DENV-2 virus CC50 (µM) in Huh-7 cells
1
2
1
2
38.7 ± 5.4
39.4 ± 6.2
> 100
> 100
2
8 / 28