Identification of novel thiadiazoloacrylamide analogues as inhibitors of dengue-2 virus NS2B/NS3 protease

Article abstract of DOI:10.1016/j.bmc.2014.09.057

Dengue virus is endemic throughout tropical and subtropical regions, and cause severe epidemic diseases. The NS2B/NS3 protease is a promising drug target for dengue virus. Herein, we report the discovery and modification of a novel class of thiadiazoloacrylamide derivatives with potent inhibitory activity against the NS2B/NS3 protease. Thiadiazolopyrimidinone 1 was firstly determined as a new chemical structure against NS2B/NS3 from a commercial compound library. Then, we sought to identify similar compounds with the thiadiazoloacrylamide core that would exhibit better activity. A series of analogues were synthesized and fourteen of them were identified with strong inhibitory activities, in which the nitrile group in the linker part was discovered as an essential group for the inhibitory activity. The best of these (8b) demonstrated an IC₅₀ at 2.24 μM based on in vitro DENV2 NS2B-NS3pro assays.

Full text of DOI:10.1016/j.bmc.2014.09.057

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of novel thiadiazoloacrylamide analogues as inhibitors
of dengue-2 virus NS2B/NS3 protease
a
a
Hailong Liu a,b, , Ruoming Wu c, , Yanyan Sun c, Yan Ye a, Jing Chen a, , Xiaomin Luo , Xu Shen ,
⇑
Hong Liu a,
⇑
a Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
b School of Pharmacy, China Pharmaceutical University, Jiangsu, Nanjing 210009, China
c School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Dengue virus is endemic throughout tropical and subtropical regions, and cause severe epidemic dis-
eases. The NS2B/NS3 protease is a promising drug target for dengue virus. Herein, we report the discovery
and modification of a novel class of thiadiazoloacrylamide derivatives with potent inhibitory activity
against the NS2B/NS3 protease. Thiadiazolopyrimidinone 1 was firstly determined as a new chemical
structure against NS2B/NS3 from a commercial compound library. Then, we sought to identify similar
compounds with the thiadiazoloacrylamide core that would exhibit better activity. A series of analogues
were synthesized and fourteen of them were identified with strong inhibitory activities, in which the
nitrile group in the linker part was discovered as an essential group for the inhibitory activity. The best
Received 12 August 2014
Revised 24 September 2014
Accepted 26 September 2014
Available online xxxx
Keywords:
Thiadiazo
Dengue virus
NS2B/NS3
of these (8b) demonstrated an IC50 at 2.24
lM based on in vitro DENV2 NS2B-NS3pro assays.
Ó 2014 Published by Elsevier Ltd.
1. Introduction
Dengue virus contain a ꢀ11 kb positive-strand RNA genome
that is transcribed as a single polyprotein arranged in order NH2-
Dengue virus (DENV), together with other serious pathogens
such as West Nile viruses (WNVs), Yellow Feverviruses (YFVs) and
Japanese encephalitis viruses (JEVs), belong to the Flaviviridae
family. DENV infection causes severe epidemic disease ranging
from the mild dengue fever (DF) to the life-threatening dengue
hemorrhagic fever (DHF) and dengue shock syndrome (DSS).1 The
U.S. Center for Disease Control and Prevention estimates that 2.5
billion people worldwide are at risk of DENV infection, especially
in tropical and subtropical regions. Approximately 500,000 indi-
viduals cases develop DHF and DSS, with ꢀ25,000 fatalities every
year.2 There are four distinct serotypes of dengue (DENV 1–4), each
of which can cause dengue disease, but DEN2 being the most pre-
valent. It has been suggested that subsequent infection by different
serotypes enhances the likelihood of developing more serious
forms of Dengue such as DHF and DSS,3,4 making vaccine studies
more difficult to perform. To date, there is no approved vaccine
or targeted drug therapy to combat this disease. Therefore, there
is an urgent need for efforts to develop effective anti-dengue drugs.
C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH.5
Subse-
quently, the polyprotein precursor is cleaved by both host prote-
ases and the viral protease complex NS2B/NS3 to generate three
structural proteins and seven nonstructural proteins, which are
required for viral replication.6 The N-terminal region of the non-
structural protein 3 (NS3) is a trypsin-like protease with a serine
protease catalytic triad (His51, Asp75 and Ser135).7,8 To exert the
full protease activity of NS3pro, the NS2B must be presence acting
as a cofactor.9 There is a precedent for the therapeutic exploitation
of protease inhibition in the treatment of HIV and hepatitis C infec-
tion, and given the indispensable role of the NS2B/NS3 protease
complex in DENY replication, it serves as an ideal target for anti-
dengue virus drug development.
Recently, intensive efforts have been made to find DENV NS2B/
NS3 protease inhibitors. Candidates identified to date have
included peptidomimetics with electrophilic warheads such as
aldehydes,10–12 ketones,13 cyclopetide,14,15 retro peptide-hybrids,16
and non-petidyl agents such as anthracene-based,17 aminobenza-
mides,18 arylcyanoacrylamides,19 benz(d)isothiazol-3(2H)-one
derivatives,20 1,2-benzisothiazol-3(2H)-one derivatives.21 Compu-
tational approaches have also been made to drug discovery in this
field.22,23 However, most of the antiviral agents reported to date
⇑
Corresponding authors. Tel./fax: +86 21 50807042.
E-mail addresses: jingchen@simm.ac.cn (J. Chen), hliu@mail.shcnc.ac.cn (H. Liu).
These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.09.057
0968-0896/Ó 2014 Published by Elsevier Ltd.
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2
H. Liu et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
have manifest only moderate activity, and novel inhibitors with
good activity are still needed.
In this study, we present a detailed report of the synthesis of
novel thiadiazoloacrylamide derivatives as potential inhibitors of
the DENV2 NS2B/NS3 protease, along with biochemical and struc-
ture–activity relationship (SAR) studies.
Tables 1 and 2. Binding affinity assay was conducted to confirm
the results of the molecular modeling and the protease inhibition
assay. The results are listed in Figure 5.
4. Results and discussion
A series of structurally-diverse thiadiazoloacetamides were
readily obtained as shown in Schemes 1 and 2, and their activities
against DENV2 NS2B/NS3 protease were investigated. The struc-
tural modification of lead compound 1 was based on two main
considerations: (a) it was envisaged that the thiadiazoloacetamide
core would interact with the protease through multiple hydrogen
bonds, essential for its inhibitory activity. Thus the thiadiazoloace-
tamide core was retained. (b) Based on previously reported molec-
ular modelling studies,15,18–20 it was hypothesized that the two
aromatic rings linked by the thiadiazoloacetamide core would pro-
vide hydrophobic interaction with the protease. Therefore, a wide
range of functional testing was performed on both aromatic rings
in various conformations with the aim of enhancing the pharmaco-
logical activity of the compound.
The inhibitory effects of compounds 7a–i and 8a–e against
DENV2 NS2B/NS3pro were evaluated (Fig. 2) and the results are
shown in Table 1. It can be generally inferred from the SAR that
the alteration of R1 contributed to improve the inhibitory activity
(e.g. 7c and 7e). The substituent in the benzylthio ring influence
the inhibitory activity followed the order at F > OCH3 > Br > Cl.
The derivatives with p- or o-chloro groups displayed similar activ-
ity, corresponding to the results for compounds 7f, and 7i. An
increase the size of the ring in the R1 part was associated with
decreased inhibitory activity, based on the results of compounds
7b and 7g. Of note, compound 7a, which utilized trifluoromethyl
to replace benzylthio, retained inhibitory activity against NS2B/
NS3pro. Variation of the R2 group impacted on the inhibitory
potency significantly. Both fluorine and nitrile groups in the
p- and o-benzyl group could enhance the inhibitory activity and
compounds carrying these groups (8a, 8b and 8e) displayed strong
inhibition of over 85%.
2. Chemistry
Our work was initiated with a high-throughput screening of a
commercial compound library containing ꢀ7000 compounds and
identified the thiadiazolopyrimidinone 1 as a potential NS2B/NS3
protease inhibitor. This compound (1, Fig. 1) was commercially
obtained and exhibited inhibitory activity against DENV2 NS2B/
NS3 protease with an IC50 value of 6.09 lM.
We aimed to undertake a study of structure–activity relation-
ships for compound 1. However, in the synthesis of this compound
(Scheme 1), we found that we can only obtain the transamidation
product 7b rather than the desired cyclization product 1 using the
previously reported methods.24,25 This result was confirmed from
the IR spectrum of the reaction product, in which a characteristic
nitrile stretch was found in the region 2260–2220 cmÀ1. We then
tested the activity of thiadiazoloacrylamide compound 7b using
in vitro DENV2 NS2B/NS3 pro assays. To our delight, this product
exhibited a better inhibitory activity than compound 1, with IC50
value of 4.44 lM. As the thiadiazoloacrylamide 7b has a better
molecular flexibility than the screening compound 1, making it clo-
ser to a drug-like structure, we decided to use 7b as the benchmark
compound for further study.
The synthesis of thiadiazoloacrylamide analogues 7a–i and 8a–
e reported in this study are summarized in Scheme 2. The amino
thiadiazoles 4a–i were readily prepared from cyclodehydration
between thiosemicarbazide 2 and trifluoroacetic anhydride26 (for
4a) or alkylation of amino thiadiazoles 3 under base conditions27
(for 4b–i). Another fragments 6a–f were synthesized via condensa-
tion between different N-protected indolaldehydes 5a–f with ethyl
cyanacetate. Treatment with the two building blocks (4a–i and
5a–f) in a refluxing MeONa/MeOH mixture then yielded the
corresponding thiadiazoloacetamides 7a–i and 8a–e.
To study the role of the linker component of in the thiadiazolo-
acrylamides in determining the biological activity, another chemi-
cal modification was performed; synthetic routes leading to
compounds 12a–b are illustrated in the synthetic Scheme 3. The
starting intermediate 4a was coupled with acryl chlorides 9, fol-
lowed by dehydration with N-protected indolaldehyde 5 to form
thiadiazoloacrylamide 11. The target product 12a was obtained
after decarboxylation of compound 11. Subsequently, it was hydro-
genated to yield compound 12b.
Considering that the strong electro-affinity of the linker part
may incur risk of toxicity, another chemical optimization was per-
formed to modify the enamide fragment. This provided the impe-
tus for the synthesis of the denitrile product 12a and the olefins
hydrogenation product 12b. Surprisingly, both of the compounds
were found to be inactive (data not shown), which may suggest
an essential role of the enamide part in determining inhibitory
activity against NS2B/NS3pro. We speculate that the nitrile group
provides a hydrogen bond acceptor that interacts with the prote-
ase, and which is therefore crucial to its activity.
The IC50 against the DENV NS2B/NS3 protease was determined
for those compounds with inhibitory rates over 75%. The results
are summarized in Table 2. Six of the synthetic derivatives tested
demonstrated significant inhibitory activity, with IC50 values of
3. Biochemistry
<5 l
M. It can be observed that variation of R2 was a more effective
The in vitro protease inhibition assay was used to screen the
lead compound and evaluate the analogues as previously
described.28 In this assay, aprotinin acted as a positive control
and exhibited the significant inhibitory effect against DENV2
NS2B/NS3pro. The results are summarized in Figures 2 and 3 and
way to improve inhibitory activity than variation of the R1 groups.
Furthermore, small substitutes that can act as a hydrogen bond
receptor in the R2 group (e.g., –F and –CN) produce better results.
The most effective compound 8b (IC50 = 2.24
in this way, which increased the activity by 3 times compared with
the lead compound 1 (IC50 = 6.09 M).
lM) was obtained
l
To understand the structural basis for the binding affinities of
the inhibitors, we scrutinized the 3D binding poses of designed
compounds 7a, 7b, 8b, 12a by molecular docking (Fig. 4A–G). In
a preliminary sense, the docking results corroborate the activity
trends observed in these four compounds. The key feature in the
docked conformation of active compounds (7a, 7b, 8b) is that the
nitrile group is involved in hydrogen-bonding interactions with
N
O
S
N
N
S
N
NH
1
Figure 1. The structure of hit compound 1.
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H. Liu et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
N
O
S
N
N
S
N
NH
1
S
NH2
S
Ph
N
N
NaOMe
MeOH reflux
O
O
CN
N
H
N
CN
S
S
N
R
N
N
O
7b
Scheme 1. Results of the cyclization reaction.
S
S
a
H2N
CF3
H2N
NH
NH2
N
N
R1
H
N
S
CN
6a
e
Ph
2
N
4a
N
N
O
S
S
R1
H2N
H2N
SH
R-Cl
7a-i
N
N
4b-i
N
N
3
b
O
d
CN
O
O
O
CN
R2-Br
c
O
O
H
N
R2
H
H
N
HN
R2
6a-f
5
5a-f
R2
H
N
CN
S
S
Ph
4b
e
N
N
N
O
8a-e
Cl
F
Br
R1
=
S
CF3
S
S
S
4c, 7c
S
4a, 7a
4b, 7b
4d, 7d
4f, 7f
O
Cl
S
Me
S
S
4g, 7g
4e, 7e
4h, 7h
F3C
4i, 7i
F
CF3
F
F3C
CN
R2
=
6a
6b, 8a
6e, 8d
6c, 8b
6f, 8e
6d, 8c
Scheme 2. Reagents and conditions: (a) (CF3O)2O, 40 °C; (b) MeOH/MeONa, rt; (c) NaH/DMF; (d) Piperidine/EtOH reflux; (e) MeOH/MeONa, reflux.
Linker
H
N
CN
S
S
N
N
N
O
O
7b
O
O
H
N
N
N
O
O
S
4a
a
5
b
O
F3C
N
P
h
NH
S
F3C
O
Cl
N
N
O
10
O
9
11
H
S
c
F3C
Ph
H
N
d
S
N
N
F3C
Ph
N
N
N
N
N
O
O
12a
12b
Scheme 3. Reagents and conditions: (a) Et3N/CH2Cl2, 0 °C; (b) Piperidine/EtOH reflux; (c) (1), NaOH/MeOH/H2O (2), HCl, reflux; (d) H2/Pd/C, MeOH.
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4
H. Liu et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5. Conclusion
In summary, we have demonstrated that the thiadiazoloaceta-
mide core constitutes a novel scaffold upon which DENV NS2B/
NS3 protease inhibitors can be developed. Basis on the structure
of the initial screening hit, compound 1, and structural modifica-
tion was performed and a series of derivatives were synthesized.
Most of the products displayed strong inhibitory rates and six were
found with IC50 <5
compound 8b presented better inhibitory activity than the lead
compound 1 (IC50 = 2.24 vs 6.09 M). Furthermore, the perfor-
lM against the target enzyme. The most potent
l
mances of docking and binding affinity studies give an insight into
the binding modes of these novel compounds. Their combination
of potency and synthetic tractability means that the new chemical
structures described in this study pave the way for the discovery of
small molecule DENV NS2B-NS3 inhibitors.
Figure 2. The inhibitory activities of compounds 7a–i and 8a–e at 20 lM against
DENV2 NS2B/NS3pro. The compounds (20
NS3pro (200 nM) at 37 °C for 30 min, then addition of the substrate (Bz-Nle-Lys-
Arg-Arg-AMC, 200 M) started the reaction. The inhibitory rate was calculated from
lM) were incubated with DENV2 NS2B/
l
the relative maximum reaction rate in the presence and absence of tested
compound. Every assay was performed in triplicate.
6. Experimental section
6.1. General
the side chain of Ser83 in pocket S2 and the main chain of Met84.
By contrast, the inactive compound 12a lose these two hydrogen-
bonding interactions, supporting an essential role for the nitrile
group in determining to the activity of the compound. The benzyl
group in the pocket S3 may have hydrophobic interactions with
residues, including Val154, Val155, Gly159 and Ile86, which lead
to the different binding pattern of 7b (Fig. 4A) from its non-benzyl
counterpart 7a (Fig. 4C). In addition, the benzyl group in the indole
fragment of compound 7b appears to occupy the pocket S2 better
compared with compound 7a (Fig. 4B and D). These interactions
may explain the increase of inhibitory activity observed in benzyl
thiol derivatives. The p-fluoro in the phenyl group forms H-bonds
with the side chain of Arg54 in the S2 region (Fig. 4E), resulting
in a better binding affinity with the S2 pocket. This may permit
the molecule to bend to the protein more tightly, leading to the
enhanced activity of compound 8b. Next, the binding affinities of
7b and its analogues, including 7a, 8b and 12a, were also examined
by Surface Plasmon Resonance (SPR) technology based Biacore
3000 instrument (GE, Healthcare.). As shown in Fig. 5, 7b bound
Unless otherwise stated, all reagents used were commercially
purchased without further purification. NMR spectra were
recorded on Varian-MERCURY Plus-400 in CDCl3 and DMSO-d6 on
a standard spectrometer operating at 400, and 500 MHz (1H
400 MHz, 13C 125 MHz). Infrared spectra were recorded on
NICOLET FTIR 6700 spectrophotometer using KBr pellets. Column
chromatography was performed with on silica gel (200À300
mesh). TLC was performed on Silica Gel GF254 for TLC and spots
were visualized by irradiation with UV light (254 nm). Melting
points were measured uncorrected. High-resolution mass spectra
were recorded using the EI method on Thermo-DFS. Melting points
were measured uncorrected.
6.2. Chemistry
6.2.1. Synthesis of 5-(trifluoromethyl)-1,3,4-thiadiazol-2-amine
4a
A mixture of thiosemicarbazide (2.0 g, 21 mmol), trifluoroacetic
acid anhydride (4.6 g, 21 mmol) was stirred at À5 °C for 1 h under
nitrogen atmosphere, then reaction mixture was heated at 40 °C
overnight. The reaction mixture was poured into ice water
(20 mL) and made alkaline with NaHCO3. Then the mixture was fil-
tered, washed with ice water and dried to afford white solid in a
yield of 72%. 1H NMR (400 MHz, DMSO-d6): 7.44 (s, 2H).
to NS2B/NS3 by KD at 3.59 lM, 7a at 9.27 lM, and 8b at 2.07 lM,
while 12a failed to bind to NS2B/NS3. These results were consis-
tent with their enzymatic inhibitory activities against NS2B/NS3
(Fig. 3), further confirming the conclusions from the molecular
modeling.
Figure 3. The dose-dependent effects of compounds 7a, 7b, 7c, 7e (A) and 8a, 8b, 8e (B) against DENV2 NS2B/NS3pro.
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H. Liu et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Table 1
Structures and percent inhibition against DENV-2 NS2B/NS3 protease
H
N
R1
CN
S
N
N
R2
N
O
Inhibitor
Apro
R1
R2
Inhibition ratea
97.59 1.4
Inhibitor
R1
R2
H
Inhibition ratea
50.05 1.6
S
7g
S
1
72.01 2.2
76.05 1.1
78.95 1.9
83.38 0.9
7h
7i
H
69.38 2.1
63.48 2.2
88.89 0.3
86.48 1.6
S
7a
7b
7c
–CF3
H
H
H
H
Cl
S
S
S
8a
8b
2-F
4-F
S
F
S
S
S
S
S
7d
7e
7f
H
H
H
65.78 1.5
76.34 1.4
63.40 1.8
8c
8d
8e
4-CF3
2,4-CF3
2-CN
45.87 0.51
54.06 5.5
85.83 0.7
Br
OMe
Cl
S
a
The inhibition rate was determined as at 20 lM of the tested compounds.
Table 2
d 5.77 (2H, s), 7.15 (1H, s), 7.10 (1H, s), 7.12–7.23 (4H, m), 7.30
(1H, s), 7.36–7.38 (2H, m), 7.76 (1H, d, J = 8.2 Hz), 9.95 (1H, s).
IC50values against DENV-2 NS2B/NS3 protease
Inhibitor
IC50 (lM)
6.2.4. General synthesis of (2E)-ethyl-3-(1-benzyl-1H-indol-2-
yl)-2-cyanoprop-2-enoates 6a–f
7a
7b
7c
7e
8a
8b
8e
10.07 0.49
4.44 0.31
3.72 0.22
3.44 0.36
3.58 0.36
2.24 0.32
2.86 0.26
To a solution of 5a (500 mg, 2.13 mmol) in ethanol was add eth-
ylcyanoacetate (240 mg, 2.13 mmol) and three drops of piperidine.
The mixture was stirred at 80 °C for 6 h. Then cooled to room tem-
perature and precipitate formed was collected by suction filtration
to give 6a as a yellow solid (632 mg, 90%). 1H NMR (400 MHz,
CDCl3) d 8.64 (d, J = 3.7 Hz, 2H), 7.91–7.83 (m, 1H), 7.39–7.30 (m,
6H), 7.22–7.16 (m, 2H), 5.45 (s, 2H), 4.40 (q, J = 7.1 Hz, 2H), 1.42
(t, J = 7.1 Hz, 3H).
6.2.2. General synthesis of 5-(benzylsulfanyl)-1,3,4-thiadiazol-2-
amines 4b–i
To solution of 5-amino-1,3,4-thiadiazole-2-thiol 3 (500 mg,
3.75 mmol) in MeOH (20 mL) was added MeONa (405.6 mg,
7.5 mmol) and stirred. Then benzyl chloro (475 mg, 3.75 mmol)
was added to the reaction mixture at room temperature and stirred
until a white solid was precipitated. The solid was filtered, washed
with methanol to afford 4b (710 mg, 85%) and it was available for
the next reaction. 1H NMR (400 MHz, CDCl3) d 7.41–7.29 (m, 5H),
5.31 (br, 1H), 4.38 (s, 2H).
6.2.5. General synthesis of (2E)-3-(1-benzyl-1H-indol-3-yl)-N-(5-
(benzylsulfanyl)-1,3,4-thiadiazol-2-yl)-2-cyanoprop-2-
enamides 7a–i and 8a–e
To a solution of sodium metal (46 mg, 2 mmol) in dry methanol
(15 mL), 4a (112 mg, 0.5 mmol) and 6a (165 mg, 0.5 mmol) were
added and resulting mixture was refluxed for 2 h. Then cooling
the mixture and acidified with acetic acid, the separated yellow
solid was collected by filtration and dried to afford the desired
products.
6.2.3. General synthesis of 1-benzyl-1H-indole-2-carbaldehydes
5a–f
6.2.5.1. (E)-3-(1-benzyl-1H-indol-3-yl)-2-cyano-N-(5-(trifluoro-
A mixture of 1H-indole-2-carbaldehyde 5 (1 g, 8.54 mmol) and
NaH (410 mg, 17 mmol) in dry DMF was stirred at 0 °C under nitro-
gen atmosphere. To this solution was add benzyl bromo (1.46 g,
8.54 mmol). Then the mixture was stirred at room temperature
until the reaction was deemed complete by TLC. The reaction mix-
ture was extracted with ethyl acetate (60 mL) and saturated NH4Cl
solution (50 mL Â 2), washed with brine (50 mL). After filtration
and concentration, the crude mixture obtained was purified by
chromatography on silica gel with 4:1 PE/EA mixture as eluent,
to give 5a as a white solid (1.4 g 79%). 1HNMR (400 MHz, CDCl3)
methyl)-1,3,4-thiadiazol-2-yl)enamide (7a).
Yellow solid
(155 mg, 68%), mp >250 °C 1H NMR (400 MHz, CDCl3) d 9.78 (br,
1H), 8.85 (s, 1H), 8.65 (s, 1H), 7.91 (d, J = 8 Hz, 1H), 7.40–7.34 (m,
6H), 7.22–7.20 (m, 2H), 5.47 (s, 2H). HRMS (EI): Calcd for C22H14F3
N5OS 453.0871; Found 453.0877.
6.2.5.2.
(E)-3-(1-benzyl-1H-indol-3-yl)-N-(5-(benzylsulfanyl)-
1,3,4-thiadiazol-2-yl)-2-cyanoprop-2-enamide (7b).
Yellow
solid (146 mg, 73%), mp 207–208 °C. 1H NMR (400 MHz, DMSO) d
8.84 (s, 1H), 8.69 (s, 1H), 8.09–8.02 (m, 1H), 7.73–7.66 (m, 1H),
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H. Liu et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
(A)
(B)
(C)
(D)
(E)
(G)
(F)
(H)
Figure 4. Putative binding mode of selected compounds 7a (A, B), 7b (C, D), 8b (E, F) and 12a (G, H) interacting with catalytic site of DENV2 NS2B/NS3 protease. The poses
were prepared using PyMol (http://pymol.sourceforge.net/). The ligands are shown as sticks, and the non-carbonatoms are colored by atom types. Hydrogen bonds are shown
as dotted lines. The NS2B-NS3 surface is colored according to electrostatic potential.
7.44–7.39 (m, 3H), 7.37–7.31 (m, 5H), 7.29–7.25 (m, 2H), 7.20 (t,
J = 7.5 Hz, 1H), 5.73 (s, 2H), 4.50 (s, 2H). 13C NMR (125 MHz, DMSO)
d 163.14, 161.20, 144.16, 137.12, 136.47, 134.53, 133.22, 133.20,
130.15, 130.08, 129.50, 129.07, 128.72, 128.11, 124.37, 122.95,
119.57, 116.22, 116.05, 112.30, 110.03. HRMS (EI): Calcd for
6.2.5.3. (E)-3-(1-benzyl-1H-indol-3-yl)-2-cyano-N-(5-(((4-fluo-
rophenyl)methyl)sulfanyl)-1,3,4-thiadiazol-2-yl)prop-2-ena-
mide (7c).
Yellow solid (133 mg, 54%), mp 246–247 °C. 1H
NMR (400 MHz, DMSO) d 8.58 (s, 1H), 8.45 (s, 1H), 7.90–7.88 (m,
1H), 7.73 (d, J = 8.2 Hz, 2H), 7.56–7.54 (m, 1H), 7.44 (d, J = 8.1 Hz,
2H), 7.39–7.37 (m, 2H), 7.34–7.30 (m, 2H), 7.27–7.26 (m, 3H),
C28H21N5OS2 507.1188; Found 507.1188.
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H. Liu et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
Figure 5. The binding affinities of compounds 7a (A), 7b (B), 8b (C) and 12a (D) to DENV2 NS2B/NS3pro.
5.76 (s, 2H), 4.36 (s, 2H). HRMS(EI): Calcd for C28H20FN5OS2
525.1093; Found 525.1082.
(400 MHz, DMSO) d 8.72 (s, 1H), 8.67 (s, 1H), 8.22 (d, J = 8.5 Hz,
1H), 8.03–7.96 (m, 2H), 7.89 (d, J = 8.2 Hz, 1H), 7.65–7.54
(m, 4H), 7.46–7.42 (m, 1H), 7.39–7.27 (m, 7H), 5.66 (s, 2H), 4.95
(s, 2H). HRMS (EI): Calcd for C32H23N5OS2 547.1344; Found
557.1326.
6.2.5.4. (E)-3-(1-benzyl-1H-indol-3-yl)-N-(5-(((4-bromophenyl)
methyl)sulfanyl)-1,3,4-thiadiazol-2-yl)-2-cyanoprop-2-enamide
(7d).
Yellow solid (125 mg, 58%), mp 225–226 °C. 1H NMR
(400 MHz, DMSO) d 8.86 (s, 1H), 8.73 (s, 1H), 8.10–8.03 (m, 1H),
7.68–7.62 (m, 1H), 7.57–7.52 (m, 2H), 7.40–7.29 (m, 9H), 5.68 (s,
2H), 4.49 (s, 2H). HRMS (EI): Calcd for C28H20BrN5OS2 585.0293;
Found 585.0300.
6.2.5.8. (E)-3-(1-benzyl-1H-indol-3-yl)-2-cyano-N-(5-(((2-meth-
ylphen-yl)methyl)sulfanyl)-1,3,4-thiadiazol-2-yl)prop-2-ena-
mide (7h).
Yellow solid (107 mg, 65%), mp 216–218 °C. 1H
NMR (400 MHz, DMSO) d 8.82 (s, 1H), 8.71 (s, 1H), 8.08–8.01 (m,
1H), 7.67–7.62 (m, 1H), 7.39–7.28 (m, 8H), 7.24–7.19 (m, 2H),
7.18–7.13 (m, 1H), 5.68 (s, 2H), 4.50 (s, 2H), 2.39 (s, 3H). HRMS
(EI): Calcd for C29H23N5OS2 521.1344; Found 521.1342.
6.2.5.5. (E)-3-(1-benzyl-1H-indol-3-yl)-2-cyano-N-(5-(((4-meth-
oxyphenyl)methyl)sulfanyl)-1,3,4-thiadiazol-2-yl)prop-2-ena-
mide (7e).
Yellow solid (152 mg, 50%), mp 213–215 °C. 1H
NMR (400 MHz, DMSO) d 8.86 (s, 1H), 8.73 (s, 1H), 8.11–8.04 (m,
1H), 7.66–7.63 (m, 1H), 7.38–7.29 (m, 9H), 6.92–6.88 (m, 2H),
5.68 (s, 2H), 4.46 (s, 2H), 3.74 (s, 3H). HRMS (EI): Calcd for
6.2.5.9. (E)-3-(1-benzyl-1H-indol-3-yl)-N-(5-(((2-chlorophenyl)-
methyl)sulfanyl)-1,3,4-thiadiazol-2-yl)-2-cyanoprop-2-ena-
mide (7i).
Yellow solid (107 mg, 53%), mp 210–211 °C. 1H
C
22H14N5O2S2 537.1293; Found 537.1303.
NMR (400 MHz, DMSO) d 8.88 (s, 1H), 8.74 (s, 1H), 8.12–8.04 (m,
1H), 7.70–7.62 (m, 1H), 7.52 (td, J = 7.2, 1.8 Hz, 2H), 7.39–7.29
(m, 9H), 5.69 (s, 2H), 4.58 (s, 2H). HRMS (EI): Calcd for C28H20ClN5
OS2 541.0798; Found 541.0803.
6.2.5.6. (E)-3-(1-benzyl-1H-indol-3-yl)-N-(5-(((4-chlorophenyl)-
methyl)sulfanyl)-1,3,4-thiadiazol-2-yl)-2-cyanoprop-2-ena-
mide (7f).
Yellow solid (102 mg, 70%), mp 221–222 °C. 1H
NMR (400 MHz, DMSO) d 8.87 (s, 1H), 8.73 (s, 1H), 8.12–8.03 (m,
1H), 7.69–7.63 (m, 1H), 7.48–7.29 (m, 11H), 5.68 (s, 2H), 4.51 (s,
6.2.5.10.
cyano-3-(1-((2-fluorophenyl)methyl)-1H-indol-3-yl)prop-2-
enamide (8a).
Yellow solid (121 mg, 65%), mp 201–202 °C. 1H
NMR (400 MHz, DMSO) d 8.84 (s, 1H), 8.69 (s, 1H), 8.09–8.01 (m,
1H), 7.73–7.66 (m, 1H), 7.44–7.38 (m, 3H), 7.37–7.32 (m, 5H),
7.30–7.24 (m, 2H), 7.20 (t, J = 7.5 Hz, 1H), 5.73 (s, 2H), 4.50 (s,
(E)-N-(5-(benzylsulfanyl)-1,3,4-thiadiazol-2-yl)-2-
2H). HRMS (EI): Calcd for
541.0807.
C28H20ClN5OS2 541.0798; Found
6.2.5.7. (E)-3-(1-benzyl-1H-indol-3-yl)-2-cyano-N-(5-((naphtha-
len-1-ylmethyl)sulfanyl)-1,3,4-thiadiazol-2-yl)prop-2-enamide
(7g).
2H). HRMS (EI): Calcd for
525.1084.
C28H20FN5OS2 525.1093; Found
Yellow solid (115 mg, 63%), mp 198–200 °C.1H NMR
Please cite this article in press as: Liu, H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.057

8
H. Liu et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
6.2.5.11.
(E)-N-(5-(benzylsulfanyl)-1,3,4-thiadiazol-2-yl)-2-
layers were washed brine and dried over anhydrous MgSO4. After
removal of solvent, the oily crude product was purified by chroma-
tography with 40:1 CHCl2/MeOH mixture as eluent to afford 12a as
a white solid (160 mg, 60%). mp 202–203 °C. 1H NMR (400 MHz,
DMSO) d 12.86 (br, 1H), 8.14 (s, 1H), 7.97–7.89 (m, 2H), 7.58–
7.57 (m, 1H), 7.33–7.31 (m, 2H), 7.28–7.20 (m, 5H), 6.88 (d,
cyano-3-(1-((4-fluorophenyl)methyl)-1H-indol-3-yl)prop-2-
enamide (8b).
Yellow solid (127 mg, 48%), mp 219–220 °C.
1H NMR (400 MHz, DMSO) d 8.86 (s, 1H), 8.73 (s, 1H), 8.06 (br,
1H), 7.71–7.63 (m, 1H), 7.45–7.42 (m, 2H), 7.41–7.32 (m, 6H),
7.31–7.27 (m, 1H), 7.23–7.17 (m, 2H), 5.66 (s, 2H), 4.50 (s, 2H).
HRMS (EI): Calcd for C28H20FN5OS2 525.1093; Found 525.1092.
J = 16.0 Hz, 1H), 5.49 (s, 2H). 13C NMR (125 MHz, DMSO)
d
178.06, 137.75, 135.66, 129.14, 128.10, 127.65, 126.06, 123.31,
121.83, 120.65, 112.34, 111.88. HRMS (EI): Calcd for C21H15F3N4OS
428.0919; Found 428.0952.
6.2.5.12.
(E)-N-(5-(benzylsulfanyl)-1,3,4-thiadiazol-2-yl)-2-
cyano-3-(1-((4-(trifluoromethyl)phenyl)methyl)-1H-indol-3-
yl)prop-2-enamide (8c).
Yellow solid (130 mg, 70%), mp
218–220 °C. 1H NMR (400 MHz, DMSO) d 8.58 (s, 1H), 8.45 (s,
1H), 7.90–7.88 (m, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.56–7.54 (m,
1H), 7.44 (d, J = 8.1 Hz, 2H), 7.39–7.37 (m, 2H), 7.34–7.30 (m,
2H), 7.27–7.24 (m, 3H), 5.76 (s, 2H), 4.37 (s, 2H). HRMS (EI): Calcd
for C29H20F3N5OS2 575.1061; Found 575.1058.
6.2.9. Synthesis of (E)-3-(1-benzyl-1H-indol-2-yl)-N-(5-
(trifluoromethy-l)-1,3,4-thiadiazol- 2-yl)prop-2-enamide 12b
To a solution of 12a (100 mg, 0.224 mmol) in MeOH was added
Pt/C. The mixture was stirred at room temperature hydrogen under
atmosphere for 24 h. Then the mixture was filtered and the organic
layer was concentrated. The oily crude product was purified by
chromatography with 20:1 CHCl2/MeOH mixture as eluent to
afford 12b as a white solid (48 mg, 47%), mp 202–203 °C. 1H
NMR (400 MHz, DMSO) d 13.26 (br, 1H), 7.59 (d, J = 7.8 Hz, 1H),
7.39 (d, J = 8.1 Hz, 1H), 7.25 (s, 1H), 7.20–7.15 (m, 3H), 7.14–7.04
(m, 3H), 7.01 (t, J = 7.0 Hz, 1H), 5.33 (s, 2H), 3.09 (t, J = 7.3 Hz,
2H), 2.93 (t, J = 7.3 Hz, 2H). HRMS (EI): Calcd for C21H17F3N4OS
430.1075; Found 430.1067.
6.2.5.13. (E)-N-(5-(benzylsulfanyl)-1,3,4-thiadiazol-2-yl)-3-(1-
((2,4-bis(tri-fluoromethyl)phenyl)methyl)-1H-indol-3-yl)-2-
cyanoprop-2-enamide (8d).
Yellow solid (107 mg, 69%), mp
226–227 °C. 1H NMR (400 MHz, DMSO) d 8.80–8.79 (m, 2H),
8.09–8.04 (m, 4H), 7.70–7.69 (m, 1H), 7.43–7.41 (m, 2H),
7.36–7.27 (m, 5H), 5.88 (s, 2H), 4.49 (s, 2H). HRMS (EI): Calcd for
C30H19F6N5OS2 643.1035; Found 643.0933.
6.3. Biochemistry
6.2.5.14.
cyano-3-(1-((2-cyanophenyl)methyl)-1H-indol-3-yl)prop-2-
enamide (8e).
Yellow solid (140 mg, 70%), mp 224–226 °C. 1H
NMR (400 MHz, DMSO) d 8.87 (s, 1H), 8.71 (s, 1H), 8.11 (d, J = 8 Hz,
1H), 7.95 (d, J = 7.3 Hz, 1H), 7.66 (t, J = 7.7 Hz, 1H), 7.60 (d,
J = 7.1 Hz, 1H), 7.54 (t, J = 7.4 Hz, 1H), 7.43 - 7.42(m, 2H), 7.36–
7.29 (m, 5H), 7.07 (d, J = 8.2 Hz, 1H), 5.93 (s, 2H), 4.51 (s, 2H).
HRMS (EI): Calcd for C29H20N6OS2 532.1140; Found 532.1120.
(E)-N-(5-(benzylsulfanyl)-1,3,4-thiadiazol-2-yl)-2-
6.3.1. DENV2 NS2B/NS3pro expression and purification
The plasmid of pET15b-DEN2 containing CF40-Gly-NS3pro185
was donated from Prof. Chunguang Wang (Institute of Protein
Research, Tongji University). The plasmid was transformed into
Escherichia coli BL21, and then DENV2 NS2B-NS3 expression and
purification was performed as previously described.28
6.3.2. In vitro DENV2 NS2B/NS3pro assays and inhibition studies
6.2.6. Synthesis of ethyl-2-((5-(trifluoromethyl)-1,3,4-
thiadiazol-2-yl)carbamoyl)acetate 10
Protease assay was carried out in Greiner Black 96 well plates.
Each assay consisted of the reaction mixture of 100
50 mM Tris-HCl, pH 8.5, 10 mM NaCl, 20% v/v glycerol, 1 mM
CHAPS, 200nM DENV2 NS2B-NS3 pro and 20 M tested compound.
The concentration of positive inhibitor (Aprotinin) was 1 M. The
lL containing
A solution of 4a (1 g, 4.14 mmol) in dry DCM was stirred at 0 °C
under nitrogen atmosphere, then ethyl malonyl chloride (686 mg,
4.56 mmol) was slowly dropped to the solution. Then 0.5 mL of tri-
ethylamine was added to the mixture and the mixture was stirred
at room temperature until the reaction was deemed complete by
TLC. The reaction mixture was extracted with DCM (60 mL) and
saturated NaHCO3 solution (50 mL Â 2), washed with brine
(50 mL). After filtration and concentration, the crude mixture
obtained was purified by chromatography on silica gel with 4:1
PE/EA mixture as eluent, to give 10 as white solid (860 mg, 59%).
1H NMR (400 MHz, CDCl3) d 12.63 (s, 1H), 4.30 (q, J = 7.1 Hz, 2H),
3.79 (s, 2H), 1.34 (t, J = 7.1 Hz, 3H).
l
l
compounds were dissolved in dimethylsulfoxide (DMSO) and the
final DMSO concentration in the assay was 1% v/v. The enzyme
and the compound were pre-incubated at 37 °C for 30 min, and
then the substrate (Bz-Nle-Lys-Arg-Arg-AMC, 200 lM) was added
to initiate the reaction. The fluorescence was continuously moni-
tored every 10s for 5 min with a spectrofluorometer (SpectraMax
M5) at 380 nm excitation/460 nm emission.
For determining IC50 values, the tested compound in eleven
concentrations (0.1, 1, 10, 100 nM, 1, 5, 10, 20, 50, 100, 200 lM)
were mixed with enzyme in the assay buffer. IC50 values were
calculated using the OriginPro 8. Every assay was performed in
triplicate.
6.2.7. Synthesis of ethyl (E)-3-(1-benzyl-1H-indol-2-yl)-2-((5-
(trifluoromethyl)-1,3,4-thiadiazol-2-yl)carbamoyl)prop-2-
enoate 11
The enoate 11 was synthesized using the way similar with 6a.
Yellow solid (370 mg, 87%).1H NMR (400 MHz, CDCl3) d 12.87 (s,
1H), 9.38 (s, 1H), 8.95 (s, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.42–7.30
(m, 6H), 7.23 (d, J = 6.8 Hz, 2H), 5.52 (s, 2H), 4.47 (q, J = 7.2 Hz,
2H), 1.51 (t, J = 7.1 Hz, 3H).
6.3.3. SPR-based ligand binding assay
The equilibrium dissociation constant (KD) of the compound
binding to DENV2 NS2B/NS3pro was detected by Biacore 3000
based on SPR technology. Briefly, purified DENV2 NS2B/NS3pro
was immobilized onto CM5 sensor chip by standard amine-
coupling procedure in 10 mM sodium acetate buffer (pH 3.95).
Compounds were then serially diluted by HBS buffer (0.01 M
HEPES, 0.15 M NaCl, 3 mM EDTA, pH 7.4, 0.005% surfactant P20)
6.2.8. Synthesis of (E)-3-(1-benzyl-1H-indol-2-yl)-2-formyl-N-
(5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)prop-2-enamide 12a
A mixture of 11 (300 mg, 0.594 mmol) in MeOH/H2O (1:2 v/v)
was added 5 M aqueous NaOH (1 mL) and stirred at room temper-
ature for 4 h. Then the mixture was acidified with concentrated
HCl and heated to 120 °C. After the conversion was complete, the
mixture was extracted with EA (50 mL Â 2). The combined organic
and injected into the channels at a flow rate of 30 lL/min for
180 s, followed by dissociation for 240 s. BIAevaluation software
(version 3.1; Biacore) was used to determine the equilibrium
dissociation constant (KD) of the compounds against the protein.
Please cite this article in press as: Liu, H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.057

H. Liu et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
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In this study, we use the crystal structure of DENV3 NS2B-
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Acknowledgments
We gratefully acknowledge financial support from the National
Natural Science Foundation of China (Grants 81220108025,
21021063, 91229204, and 81025017), National S&T Major Projects
(2012ZX09103101-072, 2012ZX09301001-005, and 2013ZX09507-
001), Program of Shanghai Subject Chief Scientist (Grant
12XD1407100) and Silver Project (260644).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.09.057.
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Please cite this article in press as: Liu, H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.09.057