Discovery of novel small molecule inhibitors of dengue viral NS2B-NS3 protease using virtual screening and scaffold hopping

Article abstract of DOI:10.1021/jm300146f

By virtual screening, compound 1 was found to be active against NS2B-NS3 protease (IC₅₀ = 13.12 ± 1.03 μM). Fourteen derivatives (22) of compound 1 were synthesized, leading to the discovery of four new inhibitors with biological activity. In order to expand the chemical diversity of the inhibitors, small-molecule-based scaffold hopping was performed on the basis of the common scaffold of compounds 1 and 22. Twenty-one new compounds (23, 24) containing quinoline (new scaffold) were designed and synthesized. Protease inhibition assays revealed that 12 compounds with the new scaffold are inhibitors of NS2B-NS3 protease. Taken together, 17 new compounds were discovered as NS2B-NS3 protease inhibitors with IC₅₀ values of 7.46 ± 1.15 to 48.59 ± 3.46 μM, and 8 compounds belonging to two different scaffolds are active to some extent against DENV based on luciferase reporter replicon-based assays. These novel chemical entities could serve as lead structures for discovering therapies against DENV.

Full text of DOI:10.1021/jm300146f

Article
pubs.acs.org/jmc
Discovery of Novel Small Molecule Inhibitors of Dengue Viral NS2B-
NS3 Protease Using Virtual Screening and Scaffold Hopping
Jing Deng,†,⊥ Ning Li,‡,⊥ Hongchuan Liu,‡,⊥ Zhili Zuo,§,⊥ Oi Wah Liew,∥ Weijun Xu,§ Gang Chen,§
Xiankun Tong,‡ Wei Tang,*,‡ Jin Zhu,† Jianping Zuo,‡ Hualiang Jiang,†,‡ Cai-Guang Yang,*,‡ Jian Li,*,†
and Weiliang Zhu*,‡
†Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei
Long Road, Shanghai 200237, China
‡Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
§Centre for Biomedical and Life Sciences, Singapore Polytechnic, 500 Dover Road, Singapore 139651
∥Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore
117599
S
* Supporting Information
ABSTRACT: By virtual screening, compound 1 was found to be active against NS2B-NS3 protease (IC50 = 13.12 1.03 μM).
Fourteen derivatives (22) of compound 1 were synthesized, leading to the discovery of four new inhibitors with biological
activity. In order to expand the chemical diversity of the inhibitors, small-molecule-based scaffold hopping was performed on the
basis of the common scaffold of compounds 1 and 22. Twenty-one new compounds (23, 24) containing quinoline (new
scaffold) were designed and synthesized. Protease inhibition assays revealed that 12 compounds with the new scaffold are
inhibitors of NS2B-NS3 protease. Taken together, 17 new compounds were discovered as NS2B-NS3 protease inhibitors with
IC50 values of 7.46 1.15 to 48.59 3.46 μM, and 8 compounds belonging to two different scaffolds are active to some extent
against DENV based on luciferase reporter replicon-based assays. These novel chemical entities could serve as lead structures for
discovering therapies against DENV.
globally in recent years.6 Currently there is no approved vaccine
1. INTRODUCTION
or a targeted drug therapy available for treatment of dengue
Dengue viruses (DENVs), transmitted by Aedes aegypti
mosquitoes, belong to the Flavivirus genus that includes other
serious pathogens such as tick-borne encephalitis viruses, West
Nile viruses (WNVs), yellow fever viruses (YFVs), and
infection.7 Accordingly, development of a safe, effective
therapeutic agent for dengue viruses is urgently needed.
DENV is an enveloped RNA virus with ∼11 kb positive-
Japanese encephalitis viruses (JEVs).1 There are four serotypes
strand RNA genome, which is transcribed and translated into a
single polypeptide upon entering the host cell.8 Subsequently,
of dengue (DENV 1−4), each of which can cause dengue
disease. The infection severity ranges from the mild dengue
the single polypeptide is processed by host cell proteases and
the virus-encoded NS2B-NS3 protease to generate three
fever (DF) to the more serious dengue hemorrhagic fever
(DHF) and dengue shock syndrome (DSS).2 The World
structural proteins (capsid protein, precursor-membrane
protein, and envelope protein) and seven nonstructural
Health Organization has estimated that the dengue viruses
proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).9
infect 50−100 million people each year and threaten up to 2.5
The 184-residue NS3pro, with multiple enzyme activities that
billion people in more than 100 countries, especially in the
tropical and subtropical regions.3−5 Approximately 500 000
infected people develop DHF and DSS, leading to 22 000
deaths, and the infection numbers have been on the rise
Received: November 28, 2011
Published: June 28, 2012
© 2012 American Chemical Society
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are required for viral replication, is a trypsin-like protease with a
serine protease catalytic triad (His51, Asp75, and Ser135).10,11
The proteolytic activity of NS3pro can be markedly enhanced
when it is coupled to its cofactor, the central hydrophilic
portion (residues 49−95) of NS2B.12,13 Optimal catalytic
activity of NS3 depends on the presence of NS2B.14 The
NS2B-NS3 protease mediated post-translational proteolytic
processing of the viral polyprotein is essential for the
maturation and pathogenesis of dengue virus.15,16 Conse-
quently, NS2B-NS3 protease is considered as a promising
target for antidengue virus drug development.
A number of different strategies have been employed to
search for DENV NS2B-NS3 protease inhibitors,17,18 including
synthesizing rationally designed substrate-based peptidomimet-
ics19−21 or cyclopeptide22 and structure-based virtual screen-
ing,23−25 as well as screening natural products26−28 and small
compound libraries.29 However, only a few peptide or small
molecule inhibitors of the DENV NS2B-NS3 protease with
moderate activity have so far been reported.17,18 Although
several peptide or cyclopeptide inhibitors against DENV NS2B-
NS3 protease have been reported with IC50 values lower than
submicromolar range,19−21 the undesirable physicochemical
features and high toxicity may limit their further development.
Lam and co-workers22 have prepared some kalata B1 analogues
and revealed that two oxidized forms of cyclopeptide could be
substrate-competitive inhibitors of the dengue viral NS2B-NS3
docking-based virtual screening on ∼600 000 compounds in the
ACD database, 27 predicted hits were bought and tested for
NS2B-NS3 protease inhibitory activity, leading to the discovery
of three active small molecule compounds (1−3) with different
scaffolds (Figure 2). Compound 1 was selected as the starting
point for further structural optimization in considering
inhibitory activity, structural variability, and synthetic accessi-
bility. Then functional group modification and scaffold hopping
approaches were used in turn for structural optimization of
compound 1. In total, 35 new analogues (22a−n, 23a−m, and
24a−h, Figure 3) have been designed, synthesized, and tested.
Taken together, in this study 17 compounds (1, 22a, 22j,
22l,m, 23a−e, 23g−j, 23l, and 24g,h) were discovered to show
moderate dengue viral NS2B-NS3 protease inhibition in vitro,
and eight compounds (1, 22j, 23b, 23i,j, 23l, and 24g,h) are
active to some extent against the replication of the dengue viral
replicon in vitro. These compounds could be used as leads for
further search for small molecule dengue viral NS2B-NS3
protease inhibitors with better potency.
2. METHODS
2.1. Computation. 2.1.1. Structure Based Virtual Screen-
ing. Here we used the highest quality structure of unliganded
DEN NS3pro-NS2B (PDB code 2FOM)31 with resolution of
1.5 Å for virtual screening. The structure represents the
virtually active protease, as NS2B has a pronounced effect on
both stability and the catalytic activity of NS3pro (3300- to
6600-fold).13 This structure was actually used in our previous
study for molecular docking.14 About 600 000 compounds in
the chemical database ACD (available chemical database,
http://www.mdli.com/products/experiment/available_chem_
dir/index.jsp) were prepared for virtual screening. Two
molecular docking programs DOCK 4.032,33 and GOLD 3.034
are quite suitable for the system according to our previous VS
protocol and result.14 All water molecules were removed from
the NS3pro-Ns2B structures. Hydrogen atoms were added, and
Amber95 atomic charges were assigned to all protein atoms
using standard Sybyl, version 6.8,35 protocols. To estimate the
binding strength between ligands and target protein, a three-
dimensional grid of 0.3 Å resolution centered on a proper
protein pocket, which is near the catalytic site,14 was generated
with CHEMGRID using an all atom model, where a 1/(4r)
distance-dependent dielectric factor and a 10 Å distance cutoff
were set for nonbonded interactions. Residues around Leu149
at a radius of 10 Å were isolated for the construction of the grid
for docking simulation. This radius was large enough to include
all residues that were involved in putative inhibitor binding site.
On average, 100 orientations were sampled for each small
molecule. The bump filter was used with bump maximum of 3.
In addition, all ligand configurations were subjected to
maximum 50 steps of simplex rigid body minimization
(convergence of 0.1 Å). The distance filter was used in the
matching calculation. Neither chemical labeling nor critical
points were used in the docking calculation. No decoy database
was used for validating the VS protocol, as few that are potent
could be found during the early stage of this study.
protease with Ki of 1.39
0.35 and 3.03
0.75 μM,
respectively. Watowich et al.23,24 reported two compounds with
inhibitory activity against the DENV protease in vitro and
antiviral activity against DENV-2 in cell culture (EC50 of 4.2
1.9 and 35 8 μM). After further studies, they improved the
inhibitory activities of anthracene-based compounds ranging
between ∼2- and 60-fold (with Ki decreased). Just recently, a
small molecule inhibitor BP2109 with moderate enzymatic
activity against DENV NS2B-NS3 protease (IC50 = 15.43
2.12 μM) was reported to show more potency in the dengue
virus replicon-based assay (EC50 = 0.17 0.01 μM).29 Other
known compounds23−25,30 also need further optimization to be
therapeutically useful, as they either have limited potency or are
not druglike compounds.
Toward this end, we initiated a program aimed at seeking
novel small molecule inhibitors of dengue viral NS2B-NS3
protease. A schematic representation of the discovery process
of the inhibitors is shown in Figure 1. Briefly, by use of a
2.1.2. Small Molecule Based Scaffold Hopping. Functional
group modifications on R1−R4 substituents in compound 1
(Figure 3) resulted in four new inhibitors with moderate
inhibition activity in vitro. We switched to perform structural
optimization that mainly focused on the core structure
replacement (scaffold hopping)36 in compound 1 (red part,
Figure 3). Given the docked binding pose of compound 1 in
Figure 1. Schematic representation of the inhibitors' discovery strategy
adopted.
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Figure 2. Structures of DENV NS2B-NS3 protease inhibitors 1−3 discovered by virtual screening and their inhibitory activities.
Figure 3. Evolution process from lead compound 1 to analogues 22, 23, and 24 is shown. The structures of R1−R8 are presented in Tables 1−3.
the binding pocket of NS2B-NS3 protease, the phenyl moiety
(old scaffold) of compound 1 (middle, Figure 3) could be
replaced by different small molecules (new scaffold). To
quickly find a new scaffold, we prepared a small molecule data
set containing approximately 60 000 small molecules from the
ZINC library (xLogP ≤ 2.5; MW ≤ 250; the number of
rotatable bonds of ≤5). The 3D structures of all the small
molecules were generated by Concord Standalone with the
ionization state at pH 7, which were subsequently optimized
using the Powell method with Tripos force field and
Gasteiger−Marsilli charges in Sybyl.35 The same parameter
settings were used in the docking step as those for virtual
screening. The small molecule data set was screened, and the
top 6000 small molecules generated from the energy scoring
function of DOCK 4.032,33 were then reevaluated by GOLD
3.034 (genetic optimization for ligand docking). The top 500
molecules from GOLD 3.0 were selected for similarity and
synthetic feasibility analysis. The default parameters in GOLD
3.0 were used except that a maximum number of 300 000
operations and a population of 200 individuals were imposed.
The most reasonable binding poses of different small molecules
in the pocket of NS3-NS2B were identified by the sequential
evaluation of GoldScore and ChemScore fitness function,
which then were used for overlay with the binding pose of
compound 1. After visual inspection of the docked compounds
in the binding site, the fragments fitting nicely with the phenyl
moiety of compound 1 were selected as potential new moieties
for scaffold-hopping (red part, Figure 3). The whole process
was called scaffold hopping using 3D overlay in the binding site.
All the virtual screening and small-molecule-based scaffold
hopping were performed on SGI Origin3800 computer at the
Shanghai Institute of Materia Medica.
2.2. Chemistry. 2.2.1. Design of Analogues of Compound
1. Compound 1 (Figure 2) was obtained from the ACD
database by virtual screening. To provide expedient and
significant structure−activity relationship (SAR) information
and improve the inhibitory activity of lead compound 1,
chemical modifications were performed in two cycles (Figure
3). In the first round, we incorporated various steric, electronic,
and hydrophobic groups at substituted positions 3 and 5 on the
benzene ring (R3 and R4, Figure 3) in compound 1; thus, 11
analogues (22a−k) were designed and prepared (Table 1). To
test if the double hydrazone substituents phenolic hydroxy (R2,
Figure 3) and carbethoxy (R1, Figure 3) on the benzene ring
are necessary for ligand−enzyme interaction, we designed and
synthesized three more analogues (compounds 22l−n, Table
1). In the second round, to further improve inhibitory activity
and expand chemical diversity, we discovered two new series of
compounds 23 and 24 (Figure 3) through small-molecule-
based scaffold hopping. In total, 21 compounds (23a−m and
24a−h), containing a quinoline moiety (new scaffold) and
different side chain substituents, were synthesized to explore
their inhibitory activity on NS2B-NS3 protease (Tables 2 and
3).
2.2.2. Synthetic Procedures. Scheme 1 depicts the synthetic
route for the preparation of compound 1 and its derivatives
22a−l. Esterification of 4-hydroxy-3,5-dimethylbenzoic acid 4
in reflux ethanol easily afforded ethyl 4-hydroxy-3,5-dimethyl-
benzoate 5, followed by acetylation with acetic anhydride to
give ethyl 4-acetoxy-3,5-dimethylbenzoate 6 in good yield.
Oxidation of this intermediate with chromium trioxide/sulfuric
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Table 1. Chemical Structures of Compounds 1 and 22a−n and Their Activities
a
Data are the mean values of three independent experiments.
acid in a mix solvent of acetic anhydride and acetic acid at 0 °C
afforded compounds 7a and 7b, respectively. Target
hydrazones 1 and 22a−l were synthesized by reacting 7a and
7b with hydrazines, hydrazides, aminoguanidines, or semi-
carbazides. Compounds 22m−n were prepared from 8 and 11
similarly (Schemes 2 and 3).
Compounds 23a−m were synthesized through the approach
outlined in Scheme 4. Treatment of isatin 14 with acetone in
potassium hydroxide solution under reflux afforded 2-
methylquinoline-4-carboxylic acid 15, followed by esterification
with ethanol to yield ethyl 2-methylquinoline-4-carboxylate 16.
Oxidation of this intermediate with selenium dioxide in dioxane
solution under reflux afforded compound 17 in high yield.
Then compound 17 was substituted by different phenyl-
hydrazines, giving 18, which was converted to the correspond-
ing hydrazide 19 by refluxing with hydrazine hydrate. Reaction
of 19 with different aldehydes in ethanol afforded compounds
23a−m.
Compounds 24a−h were synthesized through the route
shown in Scheme 5. 2-Formylquinoline-4-carboxylic acid 20
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Table 2. Chemical Structures of Compounds 23a−m and
Their Activities
Table 3. Chemical Structures of Compounds 24a−h and
Their Activities
a
Data are the mean values of three independent experiments.
for 15 min. Inhibitor solutions were prepared from stock in
DMSO (final DMSO concentration in the assay was less than
1%). Then catalysis was initiated by adding substrate (Dabcyl-
Lys-Gln-Arg-Arg-Gly-Arg-Ile-Glu-Edans, purchased from
Shanghai GL Biochem Ltd. and directly used without further
purification) to a final concentration of 10 μM. The
fluorescence increase was continuously monitored every 2
min for 30 min at room temperature using Flex Station 3
(Molecular Devices), and the excitation wavelength of 340 nm
and the emission wavelength of 450 nm were used. IC50 values
were determined from plots of percent activity over compound
concentration using sigmoidal dose−response by GraphPad
Prism software (San Diego, CA, U.S.). Triplicate measurements
were taken for each data point. All data are reported as the
mean SE.
2.3.2. In Vitro Antiviral Assays. 2.3.2.1. Dengue Virus
Replicon. A DENV-2 replicon carrying a firefly luciferase gene
as reporter was constructed using an infectious full length
DENV2 plasmid pD2FT, which was derived from the DENV2
(New Guinea C strain) cDNA. Initially, the structure protein
coding sequence of CprME was deleted by PCR method. The
Luc-IRES-APH gene cassette was constructed in a pMD18
vector. Then the PCR amplified gene cassette was ligated with
pD2FT which, lacking most of the structure gene coding
sequence, resulted in a DENV2 replicon plasmid driven by a T7
protomer named pD2RepT. Replicon genome diagram is
shown in Figure 4. The plasmid was linearized by XbaI and
replicon RNA was transcribed using the Ribomax T7 RNA
synthesis kit (Promega) in the presence of cap analogue
m7GpppA (NEB). Replicon RNA was then transfected into
BHK cell using Lipofectamin 2000 reagent. Cells were cultured
a
Data are the mean values of three independent experiments.
was prepared by the oxidation of compound 15 with selenium
dioxide in dioxane solution. Amidation of compound 20 with 3-
phenylpropan-1-amine or 4-phenylbutan-1-amine mediated by
1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDCI) read-
ily provided the desired compounds 21. Target hydrazones
24a−h were synthesized by reacting compounds 21 with
different phenylhydrazines.
2.3. Biological Assays. 2.3.1. In Vitro Protease Inhibition
Assays. The hydrophilic core sequence of the NS2B cofactor
(residues 49−95) linked to the NS3 protease domain (residues
1−184) via a flexible glycine linker (GGGGSGGGG) was used
for expression of NS2B-NS3 protease.37 The purification of
NS2B-NS3 protease was performed as described in a known
procedure.38 For protease inhibition assays, the small molecule
inhibitors in 10 concentrations (ranging from 0.8 to 400 μM)
were mixed with DENV-2 NS2B-NS3pro (final concentration
at 100 nM) in the assay buffer (50 mM Tris-HCL, 1 mM
CHAPS, 20% glycerol, pH 9.0) and then preincubated at 25 °C
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a
Scheme 1
a
Reagents and conditions: (a) conc H2SO4, EtOH, reflux, 15 h; (b) Ac2O, reflux, overnight; (c) (i) CrO3, Ac2O, conc H2SO4, AcOH, 0 °C, 20 h; (ii)
conc H2SO4, EtOH, reflux, 1 h; (d) AcOH, EtOH, 80 °C, 2 h.
a
Scheme 2
a
Reagents and conditions: (a) conc H2SO4, EtOH, reflux, 15 h; (b) (i) CrO3, Ac2O, conc H2SO4, AcOH, 0 °C, 20 h; (ii) conc H2SO4, EtOH, reflux,
1 h; (c) AcOH, EtOH, 80 °C, 2 h.
a
Scheme 3
a
Reagents and conditions: (a) Ac2O, reflux, overnight; (b) (i) CrO3, Ac2O, conc H2SO4, AcOH, 0 °C, 20 h; (ii) conc H2SO4, EtOH, reflux, 1 h; (c)
AcOH, EtOH, 80 °C, 2 h.
a
Scheme 4
a
Reagents and conditions: (a) (i) acetone, aq KOH, reflux, 15 h; (ii) conc HCl; (b) conc H2SO4, EtOH, reflux, 18 h; (c) SeO2, dioxane, reflux, 4 h;
(d) AcOH, EtOH, 80 °C, 2 h; (e) N2H4·H2O, 120 °C, 20 h; (f) AcOH, EtOH, 80 °C, 2 h.
in DMEM medium containing 10% FBS and 400 μg/mL G418
(Invitrogen). Resistant clones expressing the highest amount of
luciferase and viral nonstructural proteins were selected and
used as a DENV2 replicon cell line named BHK-D2RepT
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a
Scheme 5
a
Reagents and conditions: (a) SeO2, dioxane, reflux, 4 h; (b) EDCI, HOBt, TEA, CH2Cl2, rt, overnight; (c) AcOH, EtOH, 80 °C, 2 h.
Figure 4. Schematic diagram of the DENV-2 reporter replicon (DV2Rep).
Figure 5. Compound 1 directed scaffold hopping based on the six small molecules (new scaffold) selected from the ZINC library.
(Figure 4). Cells were propagated in DMEM medium
containing 10% FBS and 200 μg/mL G418.
concentration that led to 50% of the control luciferase signals)
and CC50 (the concentration that led to 50% of the control cell
number) of selected compounds.
2.3.2.2. Inhibition of the Replication of the Dengue Virus
Replicon. BHK-D2RepT cells were seeded in white 96-well
plates (Costar) at densities of 5× 104 cells per well for 24 h.
Compounds were dissolved in DMSO as stocking solution,
then diluted to appropriate concentration using medium and
added into cell culture medium. Final concentration of DMSO
in the culture medium was less than 0.2% (v/v). Cells were
then cultured for another 48 h in the presence of compounds.
Before luciferase readout, culture medium was replaced with
DMEM medium and luciferase signal was assayed by Bright-
Glo firefly luciferase assay kit (Promega). In each 96-well plate,
incubations were done in triplicate and six control wells were
also included. The inhibitory rate was calculated as the
percentage of luciferase signals compared with control wells
without compound. Experiments carried out in the 96-well
plate format, with BHK cells or BHK-D2RepT-replicon
containing cells, allowed us to calculate IC50 (the inhibitory
2.3.3. Cytotoxicity Assays. Besides the above-mentioned
antiviral assays, an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] assay was performed to estimate
compound cytotoxicity. Approximately 5 × 104 BHK cells
(nontransfected cells) per well were seeded to a 96-well plate.
On the following day, the cells were incubated with various
concentrations of the compound for 48 h. Cell viability was
then quantified using an MTT assay.
3. RESULTS AND DISCUSSION
3.1. Identification of Compound 1 by Virtual Screen-
ing. Targeting the crystal structure of DENV NS2B-NS3
complex, we screened ∼600 000 compounds in the ACD
database by molecular docking. On the basis of the docking
score and binding modes, we purchased 27 samples for activity
test. Finally, compounds 1−3 were found to be active against
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Figure 6. Compound 23g dose-dependently inhibited DENV-2 NS2B-NS3 protease in the cleavage of substrate peptide NH2-FAAGRKSLTL-
COOH by reversed-phase HPLC. HPLC traces showed the substrate peptide (red) and the inhibition of cleavage of substrate peptide by 1 μM
protease in the presence of inhibitor 23g at 0 μM (black), 12.5 μM (green), 25 μM (magenta), and 50 μM (cyan).
DENV-2 NS2B-NS3 protease with IC50 of 13.1−242.4 μM.
Compound 1 was then identified as lead structure for further
optimization after considering the biological activity, structural
variability, and synthetic accessibility.
μM. For the primary assay, the percent inhibition against
DENV NS2B-NS3 protease of the tested compounds at 100
μM was measured. All compounds with hydrazone fluorescence
would appear as false positives in the protease assay, so the
testing condition we used was carefully optimized for
minimizing the potential interference using the same
compound and peptide substrate without protease as reference.
Next, to rule out the false-positive inhibitory activities of our
compounds, we performed a reversed-phase HPLC-based
assay39 that is orthogonal to the fluorescence-based assay. We
used the most potent compound 23g as an example to test its
inhibitory activity against NS2B-NS3 protease (for the
materials and methods of this assay, see Supporting
Information). As shown in Figure 6, compound 23g was
inhibitory active in a dose-dependent manner with 63.3%,
37.1%, and 21.4% of inhibition rate at 50, 25, and 12.5 μM,
respectively, based on the decrease of peak area of peptide
substrate in the HPLC trace. These inhibition data are
consistent with the results observed in the fluorescence-based
inhibition assay, which proves that the fluorescence-based
protease inhibition assays employed in this study are reliable.
Then the compounds with inhibitory rates of more than 50%
were used for further study to determine their half-maximal
inhibitory concentration (IC50) against DENV NS2B-NS3
protease. A typical inhibitory progress curve of compound 1 in
different concentrations is shown in Figure 7A. Figure 7B
shows that compound 1 dose-dependently inhibited DENV
NS2B-NS3 protease in the cleavage of substrate by IC50 of
13.12 1.03 μM. These results indicated that compound 1 was
a good candidate inhibitor worthy of structure modification and
optimization.
3.2. Discovery of New Scaffold by Scaffold Hopping.
We initially used the DOCK 4.0 program to screen a small
molecule database of the ZINC library containing about 60 000
small molecules (xLogP ≤ 2.5; MW ≤ 250; the number of
rotatable bonds of ≤5). The top 6000 hits were then evaluated
by GoldScore fitness function and ChemScore fitness function
sequentially, and the best-fitting binding modes of ranked 500
small molecules were selected for conformational comparison
with the phenyl moiety of compound 1 (red part, Figure 3). By
overlay with the binding pose of compound 1 in the pocket of
NS2B-NS3, 6 small molecules out of the top 500 compounds
with higher 3D similarity to the phenyl moiety of compound 1
were chosen after visual inspection. Among them,
ZINC00036641 has been proved to possess reliable synthetic
feasibility by querying the Beilstein database (CrossFire
Beilstein) and its quinoline moiety could be used for
replacement of the phenyl moiety of compound 1 (Figure 5).
On this basis, we continued to design a variety of derivatives by
combining the core moiety of ZINC00036641 with various
substitutions of compound 1, leading to compounds 23a−m
and 24a−h.
3.3. Analogue Design and Synthesis. In total, 36
compounds (1, 22a−n, 23a−m, and 24a−h) were designed
and synthesized, and their chemical structures are shown in
Tables 1−3. These compounds were synthesized through the
routes outlined in Schemes 1−5, and the details for synthetic
procedures and structural characterizations are described in the
Experimental Section. All of the compounds are dissolvable in
dimethylsulfoxide (DMSO) and methanol (MeOH) with high
lipophilicity. All compounds (1, 22a−n, 23a−m, and 24a−h)
were confirmed to have 95% purity (Supporting Information
Table 1S).
In the first round of structure modification, we tested the
DENV NS2N-NS3 protease activities of compound 1 and its
derivatives 22a−n. The results are summarized in Table 1. Of
the synthetic derivatives tested, only four derivatives (22a, 22j,
and 22l,m) displayed moderate inhibitory activity (IC50 ranging
3.4. Biological Activities. 3.4.1. In Vitro Protease Activity
Inhibition. For DENV-2 NS2B-NS3 protease activity assays, an
internally quenched fluorescent peptide substrate Dabcyl-
KQRRGRIE-Edans was designed and commercially synthe-
sized. The Km of our purified DENV NS2B-NS3 protease on
Dabcyl-KQRRGRIE-Edans was determined to be 16.31 4.15
from 14.58
2.06 to 48.59
3.46 μM), less potent than
compound 1 (IC50 = 13.12 1.03 μM). The results of the first
cycle modification of compound 1 turned out to be
unsatisfactory but still acceptable.
After we obtained compounds 23a−m and 24a−h through
scaffold hopping, 12 new compounds (23a−e, 23g−j, 23l, and
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Table 4. Antiviral Activities of the Compounds Tested
a
b
c
entry
compd
CC50 (μM) IC50 (μM)
SI
0.4
1
2
1
12.6
29.7
19.7
35.9
8.0
22j
3.7
0.3
3
23b
23i
61.3
24.7
74.9
73.4
30.9
11.0
4.2
4
>100
>4.1
0.3
5
23j
25
6
23l
16.4
64.3
49.7
14.8
1.9
0.2
7
24g
24h
curcumin
2.1
8
4.5
9
3.5
10
mycophenolic acid (MPA)
0.7
2.7
a
b
CC50 values were calculated with nontransfected cells. IC50 values
c
were determined with the 96-well plate assay. SI: selectivity index. SI
= CC50/IC50.
compound 1 and its derivatives 22. Of 12 compounds tested,
six compounds showed antiviral activity with IC50 ranging from
11.0 to 74.9 μM and their CC50 ranging from 19.7 to >100 μM.
We were delighted to observe a loss of cytotoxicity over
DENV-2 luciferase reporter replicon with some compounds
from this series (e.g., 23i and 24h), which demonstrated the
efficiency of the strategy of scaffold hopping. The reason for
loss of cytotoxicity might be attributed to the removal of one
hydrazone substituent in the chemical structures of 23 and 24.
3.5. SAR. The SAR analysis of a set of 36 compounds
provided important insights into the essential structural
requirement for effective dengue NS2B-NS3 protease inhib-
ition. An analysis of the data shown in Tables 1−3 reveals some
noteworthy observations of the SAR for compounds 1, 22a−l,
23a−m, and 24a−h: (1) It can be seen from compound 1 and
its derivatives 22l−n, obtained from structure-based virtual
screening and subsequent chemical modification, respectively,
that the absence of the double hydrazone substituents, phenolic
hydroxyl, or carbethoxy on benzene ring causes these analogues
to decrease antiviral activity (1 vs 22l−n) (see section 3.6). The
presence of electron withdrawing groups on R3 or R4 improves
the biological activity (22a vs 22j,k), which urged us to use
electron withdrawing groups on R5 or R7. (2) It can be
observed from scaffold hopping that the high hit rate for
inhibitors might be due to both the van der Waals and
hydrogen bond interactions influenced by the quinoline ring
and the hydrogen bond interactions on the carbonyl bond of
the amide−hydrazine linker moiety of 23 and 24, respectively
(see section 3.6). (3) For compound 23g, it was observed from
modeling (see section 3.6) that the binding interaction between
sulfonamide in R5 and DENV NS2B-NS3 protease (such as
Thr120 and Lys73) contribute to the inhibitory activity.
Therefore, suggesting polar decoration on the R5 or R7 of the
molecules may increase the biological activity (23d, 23e, 23g,
23j, 23l, 24g, and 24h). (4) The analogues with two
unsaturated bonds on the R6 have better inhibitory activity
(23a vs 23c and 23e vs 23g). The effect of the linker length
between hydrazide and phenyl is limited. (5) Between
molecular series 23 and 24, the former is better for the
inhibitory activity of the derivatives. For derivates 24, the
significant loss of inhibitory activity may be attributable to a
flexible acid amide chain (24a vs 23f, 24b vs 23e, 24c vs 23i,
24d vs 23h, 24e vs 23b, 24f vs 23a). It seems that more rigid
substituents on R6 or R8 may contribute to the biological
activity.
Figure 7. Compound 1 inhibited DENV-2 NS2B-NS3 protease
activity in vitro. (A) Shown is the inhibitory progress curve of
compound 1 in different concentrations ranging from 0.8 to 100 μM.
(B) Compound 1 dose-dependently inhibited DENV-2 NS2B-NS3
protease in the cleavage of substrate Dabcyl-KQRRGRIE-Edans with
IC50 of 13.12 1.03 μM.
24g,h) containing new scaffold (quinoline moiety) kept the
moderate inhibitory activity against DENV NS2B-NS3 protease
with IC50 ranging from 7.46 1.15 to 48.59 3.46 μM (Tables
2 and 3). Values of two compounds 23d (IC50 = 7.83 0.94
μM, Table 2) and 23g (IC50 = 7.46
increased almost 2 times that of lead compound 1 (IC50
1.15 μM, Table 2)
=
13.12 1.03 μM) in vitro. To our surprise, analogues 24a−h
with the same scaffold as 23a−m but different side chain
substitutions decreased the inhibitory activity (Table 3).
Notably, the hit rate for inhibitors is 57% (12/21, defined as
the number of active compounds divided by the number of
compounds experimentally tested), higher than that of the first
round (29%, 4/14), demonstrating that the scaffold hopping
strategy employed in this study is efficient and valuable for
discovering new DENV NS2B-NS3 protease inhibitors.
3.4.2. Antiviral Activity of Compounds 1, 22a,j,l,m, 23a−
e,g−j,l, and 24g,h. To further validate the in vitro antiviral
effects of these inhibitors, we constructed a subgenomic BHK-
D2RepT-replicon (Figure 4) and compounds with in vitro
inhibitory rate of more than 50% (1, 22a, 22j, 22l,m, 23a−e,
23g−j, 23l, and 24g,h) have been evaluated on this DENV-2
luciferase reporter replicon-based assays (Table 4).
After virtual screening and chemical optimization, we tested
in vitro antiviral activity of compound 1 and its derivatives 22a,
22j, and 22l,m. Only compound 22j showed moderate activity
against DENV (IC50 = 8.0 μM, CC50 = 29.7 μM, Table 4)
among the derivatives tested. There is no big improvement on
the antiviral activity compared to lead compound 1 (IC50
=
35.9 μM, CC50 = 12.6 μM, Table 4). This unsatisfactory result
urged us to design new series of compounds 23 and 24,
obtained by scaffold hopping based on the common moiety of
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Figure 8. Three-dimensional (3D) interaction schemes of docked poses of 1 (A, B), 23i (C, D) and 23g (E, F) in the binding site of DENV-2
NS2B-NS3 protease. The poses were prepared using PyMol (http://pymol.sourceforge.net/). The ligands are shown as sticks, and the non-carbon
atoms are colored by atom types (receptor carbon in white and ligand carbon in green). Hydrogen bonds are shown as dotted lines. The NS2B-NS3
surface is colored according to electrostatic potential. The subsites are labeled as P1, P2, P3, and P4.
3.6. Binding Models. To understand the structural basis
for the binding affinities of the inhibitors for DENV NS2B-NS3
protease, we scrutinized the binding poses of compounds 1 and
23i, obtained from virtual screening and scaffold hopping,
respectively, by molecular docking (Figure 8A−D). For hit
compound 1, on the left side, the benzimidazole nitrogen form
hydrogen-bonding interaction with side chain of Asn167 in the
positive region P1 (Figure 8B), while phenol oxygen and the
first nitrogen on hydrazone are involved in the hydrogen
bonding network with the side chains of Asn167 and Lys74
(Figure 8A). On the right side, the benzimidazole moiety forms
hydrophobic interactions with Trp69 and Trp83 in the
hydrophobic region P3 (Figure 8B). And the ester group has
both hydrophobic and electrostatic interactions with the
hydrophobic region P2 including residues of Leu85 and
Leu149 (Figure 8B), which might explain the decrease of
inhibitory activity observed in derivatives 22l−n. For
compound 23i, the quinoline moiety has hydrophobic
interactions with Leu76 and Ile165 (Figure 8C). On the left
side, the first nitrogen on hydrazone and quinoline nitrogen
form hydrogen-bonding interactions with Asn152 side chain
(Figure 8C). The second nitrogen on the hydrazone also forms
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high-resolution mass spectrometry (LRMS and HRMS) conducted
with electric, electrospray, and matrix-assisted laser desorption
ionization (EI, ESI, and MALDI) produced by a Finnigan MAT-95,
LCQ-DECA spectrometer and IonSpec 4.7 T. Compounds 1, 22a−n,
23a−m, and 24a−h were confirmed to have ≥95% purity (Supporting
Information Table 1S). The details for purity analyses of these
compounds are described in the Supporting Information.
Ethyl 4-Hydroxy-3,5-dimethylbenzoate (5). To a solution of 4-
hydroxy-3,5-dimethylbenzoic acid (0.5 g) in 20 mL of EtOH was
added 1 mL of H2SO4 (conc). The resulting reaction mixture was
refluxed for 15 h. Solvent was removed under reduced pressure, and
the residue was partitioned between EtOAc and saturated NaHCO3
solution. The organic layer was washed with water and brine, dried
over anhydrous Na2SO4, filtered, and condensed to give compound 5
as a white solid. Yield: 93%. Mp 116 °C. 1H NMR (500 MHz, CDCl3)
δ: 1.36 (t, J = 7.1 Hz, 3H), 2.26 (s, 6H), 4.34 (q, J1 = 7.1 Hz, J2 = 14.3
Hz, 2H), 7.72 (s, 2H).
Ethyl 4-Acetoxy-3,5-dimethylbenzoate (6). A solution of 5
(160 mg) in acetic anhydride (Ac2O, 5 mL) was heated at 140 °C for
18 h. Excess acetic anhydride and generated acetic acid were then
evaporated under reduced pressure, and the residue was extracted with
EtOAc/H2O three times. The combined organic layer was washed,
dried, filtered, and condensed to give 6 as an orange solid. Yield: 82%.
1H NMR (500 MHz, CDCl3) δ: 1.36 (t, J = 7.1 Hz, 3H), 2.18 (s, 6H),
hydrogen-bonding interaction with the carbonyl group of
Lys73. The bromobenzene part is projected toward the
hydrophobic region P4 consisting of hydrophobic residues,
such as Ile123 and Val154 (Figure 8D). On the right side of the
positive region P1, Lys74 is involved in tight hydrogen bonding
with the carbonyl of the amide−hydrazine linker moiety of
compound 23i, while the amide−hydrazine nitrogen is
observed to have hydrogen bonding with Ile165 main chain
(Figure 8C). The phenyl part is sandwiched between the alkyl
part of the Glu88 side chain and Ala166 side chain (Figure 8D).
These interactions may explain that compound 23i shows
better inhibitory activity against DENV NS2B-NS3 protease
than compound 1. To further understand the electronic effects
of the inhibitors, we scrutinized the binding hypothesis for the
sulfonamide in R5 of compound 23g. It was observed that the
sulfonamide in R5 of 23g could form more hydrogen bonds
with the P4 region such as Thr120 and Lys73 (Figure 8E and
Figure 8F), which might explain its higher inhibitory activity
compared with others. These binding interactions gave insight
into the structure optimization in a further study.
4. CONCLUSION
2.34 (s, 3H), 4.34 (q, J1 = 7.1 Hz, J2 = 14.3 Hz, 2H), 7.75 (s, 2H).
Ethyl 3,5-Bis(formyl)-4-hydroxybenzoate (7a) and Ethyl 3-
Formyl-4-hydroxy-5-methylbenzoate (7b). A solution of the 6
(350 mg) in acetic anhydride (Ac2O, 8 mL), acetic acid (8 mL,
AcOH), and concentrated H2SO4 (1 mL) was cooled to 0 °C, and
chromium trioxide (CrO3, 0.69 g) was added in small portions during
2 h. After another 20 h, the reaction mixture was poured onto ice.
Excess sodium metabisulfite solution was added, and it was extracted
with EtOAc (3 × 20 mL). The organic layer was washed with water
and brine, dried over anhydrous Na2SO4, filtered, and condensed. The
residue was dissolved in EtOH (10 mL), and 100 μL of concentrated
H2SO4 was added. The resulting reaction mixture was heated under
reflux for 1 h. Solvent was removed under reduced pressure, and the
residue was partitioned between EtOAc and saturated NaHCO3. The
organic layer was washed with water and brine, dried over anhydrous
Na2SO4, filtered, and condensed. The residue was purified by
chromatography on silica gel, eluting with EtOAc/petroleum ether
(1:6, v/v) to give 7b as a white solid. Yield: 20%. 1H NMR (500 MHz,
CDCl3) δ: 1.40 (t, J = 7.2 Hz, 3H), 2.25 (s, 3H), 4.40 (q, J1 = 7.2 Hz,
J2 = 14.4 Hz, 2H), 8.04 (s, 1H)), 8.15 (s, 1H), 9.95 (s, 1H). Further
elution afforded 7a as a yellow solid. Yield: 11%. 1H NMR (500 MHz,
CDCl3) δ: 1.36 (t, J = 7.1 Hz, 3H), 4.40 (q, J1 = 7.1 Hz, J2 = 14.2 Hz,
2H), 8.52 (s, 2H), 10.30 (s, 2H). EI-MS m/z 222.1 (M+).
In summary, we have discovered novel small molecule
inhibitors of DENV NS2B-NS3 protease using virtual screening
and scaffold hopping techniques. Compound 1, derived from
the ACD database by virtual screening, was found against
NS2B-NS3 protease activity. On the basis of the structure of
lead compound 1, 14 derivatives (22a−n) were designed,
synthesized, and tested for inhibition activity. On the basis of
the common scaffold of compound 1 and derivatives 22a−n,
we carried out scaffold hopping and identified a small
compound (ZINC00036641) bearing quinoline as its core
structure from the ZINC data set containing 60 000 small
compounds. Twenty-one new compounds (23a−m and 24a−
h) containing new scaffold (quinoline moiety) were designed,
synthesized, and evaluated for biological activity. In total, of 36
new compounds synthesized and tested, 17 compounds (1,
22a, 22j, 22l,m, 23a−e, 23g−j, 23l, and 24g,h) were found to
show DENV NS2B-NS3 protease inhibition activity and eight
compounds (1, 22j, 23b, 23i,j, 23l, and 24g,h) showed antiviral
activity against DENV in vitro, which may be good leads for
discovering new therapeutic agents for dengue viruses.
Molecular binding models give rational explanations about
SARs, which are in good agreement with pharmacological
results. Discovering small molecule DENV NS2B-NS3
inhibitors is an urgent need, so the new chemical structures
discovered in this study are of significance in terms of the
chemical scaffold diversity. These findings have paved the way
for the advancement of more potent compounds through
rational drug design and optimization.
Ethyl 4-Hydroxy-3,5-bis((2-(1H-benzo[d]imidazol-2-yl)-
hydrazinylidene)methyl)benzoate (1). To a solution of 7a (75
mg, 0.34 mmol) in EtOH (5 mL) were added 1-(1H-benzimidazol-2-
yl)hydrazine (110 mg, 0.74 mmol) (see Scheme 1 in Supporting
Information) and AcOH (50 μL). The mixture was refluxed for 2 h.
The reaction mixture was allowed to cool to room temperature and
filtered. Then the precipitate was collected, washed with H2O, and
1
dried to afford 1 (107 mg, 65%) as a yellow solid. Mp >300 °C. H
NMR (500 MHz, DMSO-d6) δ: 1.37 (t, J = 7.1 Hz, 3H), 4.36 (q, J1 =
7.0 Hz, J2 = 14.1 Hz, 2H), 7.00 (br, 4H), 7.22 (br, 4H), 8.25 (s, 2H),
8.42 (s, 2H), 11.55 (br, 4H), 12.23 (br, 1H). ESI-MS m/z 505.2 [M +
Na]+. HRMS (ESI) m/z calcd for C25H22N8O3 [M + H]+ 483.1893,
found 483.1879.
5. EXPERIMENTAL SECTION
Chemistry. General. The reagents (chemicals) were purchased
from Lancaster, Alfa Aesar, J&K, Acros, and Shanghai Chemical
Reagent Co. and used without further purification. Analytical thin-
layer chromatography (TLC) was done with HSGF 254 (150−200 μm
thickness; Yantai Huiyou Co., China). Reaction yields were not
optimized. Melting points were measured in a capillary tube on a SGW
X-4 melting point apparatus without correction. Nuclear magnetic
resonance (NMR) spectroscopy was performed on a Bruker AMX-500
and AMX-400 NMR (IS as TMS) instrument. Chemical shifts were
reported in parts per million (ppm, δ) downfield from tetramethylsi-
lane. Proton coupling patterns were described as singlet (s), doublet
(d), triplet (t), quartet (q), multiplet (m), and broad (br). Low- and
Ethyl 3-((2-(1H-Benzo[d]imidazol-2-yl)hydrazinylidene)-
methyl)-4-hydroxy-5-methylbenzoate (22l). To a solution of 7b
(62 mg, 0.3 mmol) in EtOH (5 mL) were added 1-(1H-benzimidazol-
2-yl)hydrazine (49 mg, 0.33 mmol) and AcOH (50 μL). The mixture
was refluxed for 2 h. the reaction mixture was allowed to cool to room
temperature and filtered. Then the precipitate was collected, washed
with H2O, and dried to afford 22l (81 mg, 80%) as a yellow solid. Mp
1
279−282 °C. H NMR (500 MHz, DMSO-d6) δ: 1.31 (t, J = 7.1 Hz,
3H), 2.28 (s, 3H), 4.27 (q, J1 = 7.1 Hz, J2 = 14.2 Hz, 2H), 6.96 (br,
2H), 7.13 (br, 2H), 7.71 (s, 1H), 7.87 (s, 1H), 8.37 (s, 1H), 11.55 (br,
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1H), 11.93 (br, 1H). EI-MS m/z 338.1 M+. HRMS (ESI) m/z calcd
for C18H18N4O3 [M + H]+ 339.1457, found 339.1442.
m/z 391.3 [M + Na]+. HRMS (ESI) m/z calcd for C13H16N6O3S2 [M
+ Na]+ 391.0623, found 391.0624.
Ethyl 4-Hydroxy-3,5-bis((2-(4-nitrophenyl)hydrazinylidene)-
methyl)benzoate (22a). In the same manner as that described for
the preparation of 1, 22a was prepared from 7a and 4-nitro-
phenylhydrazine. Yield: 55%. Mp >300 °C. 1H NMR (500 MHz,
DMSO-d6) δ: 1.37 (t, J = 7.2 Hz, 3H), 4.37 (q, J1 = 7.2 Hz, J2 = 14.4
Hz, 2H), 7.14 (d, J = 9.2 Hz, 4H), 8.20 (d, J = 9.2 Hz, 4H), 8.21 (s,
2H), 8.43 (s, 2H), 11.55 (s, 2H), 11.93 (s, 1H). EI-MS m/z 492.1
(M+). HRMS (ESI) m/z calcd for C23H20N6O7 [M − H]− 491.1315,
found 491.1345.
Ethyl 4-Hydroxy-3,5-bis((2-(allylcarbamothioyl)hydrazinyl)-
methylene)benzoate (22i). In the same manner as that described
for the preparation of 1, 22i was prepared from 7a and 4-
1
allylthiosemicarbazide. Yield: 39%. Mp 250−253 °C. H NMR (500
MHz, DMSO-d6) δ: 1.37 (t, J = 7.2 Hz, 6H), 4.32 (t, J = 4.8 Hz, 4H),
4.34 (q, J1 = 7.2 Hz, J2 = 14.0 Hz, 2H), 5.12−5.16 (m, 4H), 5.82−5.98
(m, 2H), 8.26 (s, 2H), 8.43 (s, 2H), 8.84 (t, J = 4.8 Hz, 2H), 11.56 (s,
2H). ESI-MS m/z 449.2 [M + H]+. HRMS (ESI) m/z calcd for
C19H24N6O3S2 [M + H]+ 449.1430, found 449.1404.
Ethyl 4-Hydroxy-3,5-bis((2-(4-isopropylphenyl)-
hydrazinylidene)methyl)benzoate (22j). In the same manner as
that described for the preparation of 1, 22j was prepared from 7a and
4-isopropylphenylhydrazine hydrochloride. Yield: 51%. Mp 262−264
°C. 1H NMR (500 MHz, DMSO-d6) δ: 1.19 (d, J = 6.8 Hz, 12H), 1.35
(t, J = 7.2 Hz, 3H), 2.79−2.84 (m, 2H), 4.35 (q, J1 = 7.2 Hz, J2 = 14.0
Hz, 2H), 6.95 (d, J = 8.4 Hz, 4H), 7.16 (d, J = 8.4 Hz, 4H), 8.12 (s,
2H), 8.22 (s, 2H), 10.54 (s, 2H), 12.45 (s, 1H). EI-MS m/z 486.3
(M+). HRMS (ESI) m/z calcd for C29H34N4O3 [M + H]+ 487.2709,
found 487.2684.
Ethyl 4-Hydroxy-3,5-bis((2-(benzo[d][1,3]dioxol-5-carbonyl)-
hydrazinylidene)methyl)benzoate (22k). In the same manner as
that described for the preparation of 1, 22k was prepared from 7a and
benzo[d][1,3]dioxol-5-carbohydrazide. Yield: 81%. Mp 253−258 °C.
1H NMR (500 MHz, DMSO-d6) δ: 1.36 (t, J = 7.2 Hz, 3H), 4.38 (q, J1
Ethyl 4-Hydroxy-3,5-bis((isoindoline-1,3-dione-2-yl-amino)-
methylene)benzoate (22b). In the same manner as that described
for the preparation of 1, 22b was prepared from 7a and N-
aminophthalimide. Yield: 60%. Mp 250−255 °C. 1H NMR (500 MHz,
DMSO-d6) δ: 1.37 (t, J = 7.2 Hz, 3H), 4.32 (q, J1 = 7.2 Hz, J2 = 14.4
Hz, 2H), 7.82−7.84 (m, 2H), 7.90−7.92 (m, 3H), 7.96−7.98 (m, 3H),
8.58 (s, 2H), 9.72 (s, 2H), 12.79 (br, 1H). EI-MS m/z 510.2 (M+).
HRMS (ESI) m/z calcd for C27H18N4O7 [M + Na]+ 533.1073, found
533.1063.
Ethyl 4-Hydroxy-3,5-bis((2-(pyridin-3-yl-carbonyl)-
hydrazinylidene)methyl)benzoate (22c). In the same manner as
that described for the preparation of 1, 22c was prepared from 7a and
nicotinic hydrazide. Yield: 53%. Mp 169−170 °C. 1H NMR (500
MHz, DMSO-d6) δ: 1.36 (t, J = 7.2 Hz, 3H), 4.37 (q, J1 = 7.2 Hz, J2 =
14.0 Hz, 2H), 7.58−7.61 (m, 3H), 8.28−8.31 (m, 2H), 8.37 (s, 2H),
8.79 (br, 3H), 9.12 (br, 2H), 12.45 (s, 2H), 13.28 (s, 1H). EI-MS m/z
460.2 (M+). HRMS (ESI) m/z calcd for C23H20N6O5 [M + H]+
461.1573, found 461.1557.
= 7.2 Hz, J2 = 14.0 Hz, 2H), 6.15 (s, 4H), 7.19 (d, J = 8.0 Hz, 2H),
7.50 (s, 2H), 7.58 (d, J = 8.0 Hz, 2H), 8.33 (s, 2H), 8.77 (s, 2H), 12.14
(s, 2H), 13.40 (s, 1H). ESI-MS m/z 569.1 [M + Na]+. HRMS (ESI)
m/z calcd for C27H22N4O9 [M + Na]+ 569.1284, found 569.1262.
Ethyl 3,5-Dimethylbenzoate (9). In the same manner as that
described for the preparation of 5, 9 was prepared from 3,5-
Ethyl 4-Hydroxy-3,5-bis((2-(4-(aminosulfonyl)phenyl)-
hydrazinylidene)methyl)benzoate (22d). In the same manner as
that described for the preparation of 1, 22d was prepared from 7a and
4-sulfonamidophenylhydrazine hydrochloride. Yield: 82%. Mp 242−
1
dimethylbenzoic acid 8. Yield: 98%. H NMR (500 MHz, CDCl3) δ:
1
248 °C. H NMR (500 MHz, DMSO-d6) δ: 1.37 (t, J = 7.2 Hz, 3H),
1.36 (t, J = 7.2 Hz, 3H), 2.36 (s, 6H), 4.38 (q, J1 = 7.2 Hz, J2 = 14.0
Hz, 2H), 7.18 (s, 1H), 7.72 (s, 2H).
4.36 (q, J1 = 7.2 Hz, J2 = 14.0 Hz, 2H), 7.11 (d, J = 8.8 Hz, 8H), 7.73
(d, J = 8.8 Hz, 4H), 8.22 (s, 2H), 8.35 (s, 2H), 11.11 (s, 2H), 12.13 (s,
1H). EI-MS m/z 583.1 (M+). HRMS (ESI) m/z calcd for
C23H24N6O7S2 [M + H]+ 561.1226, found 561.1199.
Ethyl 3,5-Diformylbenzoate (12). In the same manner as that
described for the preparation of 9a, 12 was prepared from 11. Yield:
28%. 1H NMR (500 MHz, CDCl3) δ: 1.46 (t, J = 7.2 Hz, 3H), 2.36 (s,
6H), 4.50 (q, J1 = 7.2 Hz, J2 = 14.0 Hz, 2H), 8.56 (t, J = 7.8 Hz, 1H),
8.79 (d, J = 7.8 Hz, 2H), 10.19 (s, 2H). EI-MS m/z 206.1 (M+).
Ethyl 3,5-Bis((2-(1H-benzo[d]imidazol-2-yl)hydrazinylidene)-
methyl)benzoate (22m). In the same manner as that described for
the preparation of 1, 22m was prepared from 10 and 1-(1H-
Ethyl 4-Hydroxy-3,5-bis((2-(phenylcarbamoyl)-
hydrazinylidene)methyl)benzoate (22e). In the same manner as
that described for the preparation of 1, 22e was prepared from 7a and
4-phenylsemicarbazide. Yield: 75%. Mp >300 °C. 1H NMR (500 MHz,
DMSO-d6) δ: 1.36 (t, J = 6.8 Hz, 3H), 4.35 (q, J1 = 7.2 Hz, J2 = 14.4
Hz, 2H), 7.03 (t, J = 7.6 Hz, 2H), 7.31 (t, J = 7.6 Hz, 4H), 7.58 (d, J =
8.0 Hz, 4H), 8.33 (s, 2H), 8.38 (s, 2H), 9.07 (s, 2H), 10.79 (s, 2H),
12.32 (br, 1H). ESI-MS m/z 511.2 [M + Na]+. HRMS (ESI) m/z
calcd for C25H24N6O5 [M + Na]+ 511.1706, found 511.1715.
Ethyl 4-Hydroxy-3,5-bis((2-carbamoylhydrazinyl)-
methylene)benzoate (22f). In the same manner as that described
for the preparation of 1, 22f was prepared from 7a and semicarbazide.
Yield: 54%. Mp 194−198 °C. 1H NMR (500 MHz, DMSO-d6) δ: 1.37
(t, J = 6.8 Hz, 3H), 4.32 (q, J1 = 7.2 Hz, J2 = 14.4 Hz, 2H), 7.24 (s,
2H), 7.72 (s, 2H), 8.31 (s, 6H), 8.42 (s, 2H). ESI-MS m/z 357.2 [M +
Na]+. HRMS (ESI) m/z calcd for C13H18N8O3 [M + H]+ 335.1580,
found 335.1566.
Ethyl 4-Hydroxy-3,5-bis((2-(ethylcarbamoyl)hydrazinyl)-
methylene)benzoate (22g). In the same manner as that described
for the preparation of 1, 22g was prepared from 7a and 4-ethyl-3-
thiosemicarbazide. Yield: 43%. Mp 263−265 °C. 1H NMR (500 MHz,
DMSO-d6) δ: 1.17 (t, J = 7.2 Hz, 6H), 1.34 (t, J = 7.2 Hz, 3H), 3.56−
3.63 (m, 4H), 4.34 (q, J1 = 7.2 Hz, J2 = 14.0 Hz, 2H), 8.27 (s, 2H),
8.42 (s, 2H), 8.64 (t, J = 4.8 Hz, 2H), 11.53 (s, 2H). EI-MS m/z 424.2
(M+). HRMS (ESI) m/z calcd for C17H24N6O3S2 [M + H]+ 425.1430,
found 425.1435.
1
benzimidazol-2-yl)hydrazine. Yield: 37%. Mp 312−315 °C. H NMR
(500 MHz, DMSO-d6) δ: 1.37 (t, J = 7.2 Hz, 3H), 4.32 (q, J1 = 7.2 Hz,
J2 = 14.0 Hz, 2H), 6.90−7.00 (m, 4H), 7.24−7.27 (m, 4H), 8.15 (s,
2H), 8.27 (s, 2H), 8.38 (s, 1H), 8.40 (s, 1H), 8.47 (s, 1H), 8.56 (s,
1H). EI-MS m/z 466.2 (M+). HRMS (ESI) m/z calcd for C25H22N8O2
[M + H]+ 467.1944, found 467.1923.
2,6-Dimethylphenyl Acetate (12). In the same manner as that
described for the preparation of 6, 12 was prepared from 11. Yield:
1
88%. H NMR (500 MHz, CDCl3) δ: 2.19 (s, 6H), 2.36 (s, 3H),
7.09−7.15 (m, 3H).
2-Hydroxybenzene-1,3-dialdehyde (13). In the same manner
as that described for the preparation of 7a, 13 was prepared from 12.
Yield: 30%. 1H NMR (500 MHz, CDCl3) δ: 6.94 (t, J = 7.6 Hz, 1H),
7.41 (d, J = 7.6 Hz, 2H), 9.89 (s, 2H), 11.27 (s, 1H).
2-Hydroxyisophthalaldehyde-1,3-bis(2-(1H-benzo[d]-
imidazol)hydrazone) (22n). In the same manner as that described
for the preparation of 1, 22n was prepared from 13 and 1-(1H-
benzimidazol-2-yl)hydrazine. Yield: 66%. Mp >300 °C. 1H NMR (500
MHz, DMSO-d6) δ: 6.96−7.00 (m, 4H), 7.12 (t, J = 8.0 Hz, 1H),
7.20−7.22 (m, 4H), 7.74 (d, J = 8.0 Hz, 2H), 8.40 (s, 2H), 10.26 (s,
1H), 10.47 (s, 2H), 11.44 (br, 2H). EI-MS m/z 410.2 (M+). HRMS
(ESI) m/z calcd for C22H18N8O [M + H]+ 411.1682, found 411.1658.
2-Methylquinoline-4-carboxylic Acid (15). A mixture of isatin
14 (1.0 g, 6.8 mmol) and 85% aqueous potassium hydroxide (2.67 g,
47.6 mmol) was heated to 50 °C for 40 min. Acetone (5 mL, 56.7
mmol) was then added dropwise, maintaining the temperature at 50
°C. After being stirred for 15 h, the reaction mixture was cooled to
Ethyl 4-Hydroxy-3,5-bis((2-carbamothioylhydrazinyl)-
methylene)benzoate (22h). In the same manner as that described
for the preparation of 1, 22h was prepared from 7a and
thiosemicarbazide. Yield: 54%. Mp 174−179 °C. 1H NMR (500
MHz, DMSO-d6) δ: 1.34 (t, J = 7.2 Hz, 6H), 4.33 (q, J1 = 7.2 Hz, J2 =
14.0 Hz, 2H), 8.16−8.28 (m, 5H), 8.42 (s, 2H), 11.56 (s, 2H). ESI-MS
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room temperature and acidified to pH 3 by addition of concentrated
HCl to obtain a heavy slurry which was filtered, washed, and dried to
afford 2-methylquinoline-4-carboxylic acid 15 (1.0 g, 79%) as a white
solid. Mp 248−250 °C. H NMR (400 MHz, DMSO-d6): δ 2.72 (s,
3H), 7.64 (t, J = 8.0 Hz, 1H), 7.78 (t, J = 8.0 Hz, 1H), 7.84 (s, 1H),
8.02 (d, J = 8.4 Hz, 1H), 8.63 (d, J = 8.4 Hz, 1H).
the hydrazine hydrate (2.5 mL). The reaction mixture was heated to
120 °C for 20 h. After being cooled to about 20 °C, it was poured into
ice−water. The resulting crystals were collected after filtration and
washed with water and dried to afford compound 19a (22 mg, 90%) as
a yellow solid. EI-MS m/z 339.1 (M+).
2-((2-(4-(Aminosulfonyl)phenyl)hydrazinylidene)methyl)-
quinoline-4-carbohydrazide (19b). In the same manner as that
described for the preparation of 19a, 19b was prepared from 18b and
hydrazine hydrate. Yield: 95%. 1H NMR (400 MHz, DMSO-d6) δ 4.73
(br, 2H), 7.21 (br, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.62 (s, 1H), 7.72−
7.80 (m, 4H), 7.92 (t, J = 7.2 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 8.44
(d, J = 8.4 Hz, 1H), 10.00 (br, 1H), 14.77 (s, 1H).
2-((2-(3-Bromophenyl)hydrazinylidene)methyl)quinoline-4-
carbohydrazide (19c). In the same manner as that described for the
preparation of 19a, 19c was prepared from 18c and hydrazine hydrate.
Yield: 88%. EI-MS m/z 383.0 (M+).
2-((2-(3-Nitrophenyl)hydrazinylidene)methyl)quinoline-4-
carbohydrazide (19d). In the same manner as that described for the
preparation of 19a, 19d was prepared from 18d and hydrazine hydrate.
Yield: 98%. 1H NMR (400 MHz, DMSO-d6) δ 4.71 (br, 2H), 6.35 (d,
J = 8.0 Hz, 1H), 6.61 (d, J = 8.0 Hz, 1H), 7.75 (s, 1H), 7.07 (t, J = 8.0
Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.77 (t, J = 7.6 Hz, 1H), 7.98−8.08
(m, 4H), 8.30 (br, 2H), 10.00 (br, 1H), 10.91 (s, 1H).
1
Ethyl 2-Methylquinoline-4-carboxylate (16). In the same
manner as that described for the preparation of 5, 16 was prepared
1
from 15. Yield: 80%. H NMR (400 MHz, CDCl3) δ 1.50 (t, J = 7.2
Hz, 3H), 2.83 (s, 3H), 4.53 (q, J1 = 7.2 Hz, J2 = 14.0 Hz, 2H), 7.61 (t, J
= 8.0 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.82 (s, 1H), 8.10 (d, J = 8.4
Hz, 1H), 8.71 (d, J = 8.4 Hz, 1H).
Ethyl 2-Formylquinoline-4-carboxylate (17). To a solution of
16 (0.5 g) in 10 mL of dioxane, selenium dioxide (SeO2, 1.3 g) was
added. The resulting reaction mixture was reflux for 4 h. Solvent was
removed under reduced pressure, and the residue was extracted with
EtOAc/H2O. The organic layer was washed with water and brine,
dried over anhydrous Na2SO4, filtered, and condensed. The residue
was purified by chromatography on silica gel, eluting with a mixture of
EtOAc/petroleum ether (1:4, v/v) to give 17 as a pale yellow solid.
1
Yield: 99%. H NMR (400 MHz, CDCl3): δ 1.52 (t, J = 7.2 Hz, 3H),
4.56 (q, J1 = 7.2 Hz, J2 = 14.0 Hz, 2H), 7.83 (t, J = 8.0 Hz, 1H), 7.91
(t, J = 8.0 Hz, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.54 (s, 1H), 8.90 (d, J =
8.0 Hz, 1H), 10.29 (s, 1H).
2-((2-(3-(Trifluoromethyl)phenyl)hydrazinylidene)methyl)-
quinoline-4-carbohydrazide (19e). In the same manner as that
described for the preparation of 19a, 19e was prepared from 18e and
hydrazine hydrate. Yield: 87%. EI-MS m/z 373.1 (M+).
Ethyl 2-((2-(3-Chlorophenyl)hydrazinylidene)methyl)-
quinoline-4-carboxylate (18a). To a solution of 17 (23 mg, 0.1
mmol) in EtOH (5 mL) were added 3-chlorophenylhydrazine
hydrochloride (22 mg, 0.12 mmol) and AcOH (50 μL). The mixture
was refluxed for 2 h. The reaction mixture was allowed to cool to room
temperature and filtered. Then the precipitate was collected, washed
with H2O, and dried to afford 18a (26 mg, 75%) as a yellow solid. 1H
NMR (400 MHz, DMSO-d6) δ: 1.44 (t, J = 7.6 Hz, 3H), 4.53 (q, J1 =
7.2 Hz, J2 = 14.4 Hz, 2H), 6.96 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.0 Hz,
1H), 7.31−7.37 (m, 2H), 7.75 (t, J = 8.0 Hz, 1H), 7.91 (t, J = 7.6 Hz,
1H), 8.17 (d, J = 8.4 Hz, 1H), 8.25 (s, 1H), 8.52 (d, J = 8.4 Hz, 1H),
8.55 (s, 1H), 11.88 (br, 1H).
Ethyl 2-((2-(4-(Aminosulfonyl)phenyl)hydrazinylidene)-
methyl)quinoline-4-carboxylate (18b). In the same manner as
that described for the preparation of 18a, 18b was prepared from 17
and 4-sulfonamidophenylhydrazine hydrochloride. Yield: 75%. 1H
NMR (400 MHz, DMSO-d6) δ 1.45 (t, J = 7.2 Hz, 3H), 4.52 (q, J1 =
7.2 Hz, J2 = 14.0 Hz, 2H), 7.17 (s, 2H), 7.29 (d, J = 8.8 Hz, 2H), 7.70
(t, J = 8.0 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.85 (t, J = 8.0 Hz, 1H),
8.09 (d, J = 8.4 Hz, 1H), 8.15 (s, 1H), 8.48 (s, 1H), 8.52 (d, J = 8.4
Hz, 1H), 11.42 (s, 1H).
2-((2-(3-Chlorophenyl)hydrazinylidene)methyl)-N′-(3-
phenylpropylidene)quinoline-4-carbohydrazide (23a). To a
solution of 19a (80 mg, 0.23 mmol) in EtOH (5 mL) were added
phenylpropyl aldehyde (62 mg, 0.46 mmol) and AcOH (50 μL). The
mixture was refluxed for 2 h. The reaction mixture was allowed to cool
to room temperature and filtered. Then the precipitate was collected,
washed with H2O, and dried to afford 23a (78 mg, 75%) as a yellow
solid. Mp 266−268 °C. 1H NMR (400 MHz, DMSO-d6) δ: 2.62−2.67
(m, 2H), 2.88 (t, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 1H), 7.22−7.29
(m, 7H), 7.63 (t, J = 8.0 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.79 (t, J =
8.0 Hz, 1H), 8.03−8.08 (m, 4H), 8.19 (s, 1H), 11.17 (s, 1H), 11.90 (s,
1H). ESI-MS m/z 456.2 [M + H]+. HRMS (ESI) m/z calcd for
C26H22ClN5O [M + H]+ 456.1591, found 456.1586.
2-((2-(3-Chlorophenyl)hydrazinylidene)methyl)-N′-(2-
phenylethylidene)quinoline-4-carbohydrazide (23b). In the
same manner as that described for the preparation of 23a, 23b was
prepared from 19a and phenylacetaldehyde. Yield: 57%. Mp 255 °C.
1H NMR (400 MHz, DMSO-d6) δ: 3.69 (d, J = 6.0 Hz, 2H), 6.88 (d, J
= 7.6 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.25−7.40 (m, 7H), 7.63 (t, J
= 7.6 Hz, 1H), 7.77−7.82 (m, 2H), 8.03−8.08 (m, 3H), 8.22 (s, 1H),
11.16 (s, 1H), 11.95 (s, 1H). ESI-MS m/z 442.2 [M + H]+. HRMS
(ESI) m/z calcd for C25H20ClN5O [M + H]+ 442.1435, found
442.1448.
Ethyl 2-((2-(3-Bromophenyl)hydrazinylidene)methyl)-
quinoline-4-carboxylate (18c). In the same manner as that
described for the preparation of 18a, 18c was prepared from 17 and
1
3-bromophenylhydrazine hydrochloride. Yield: 65%. H NMR (400
MHz, DMSO-d6) δ 1.45 (t, J = 7.2 Hz, 3H), 4.53 (q, J1 = 7.2 Hz, J2 =
14.0 Hz, 2H), 7.08 (d, J = 7.6 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 7.28
(t, J = 8.0 Hz, 1H), 7.44 (s, 1H), 7.75 (t, J = 7.6 Hz, 1H), 7.90 (t, J =
7.6 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.21 (s, 1H), 8.52−8.53 (m,
2H), 11.73 (br, 1H).
Ethyl 2-((2-(3-Nitrophenyl)hydrazinylidene)methyl)-
quinoline-4-carboxylate (18d). In the same manner as that
described for the preparation of 18a, 18d was prepared from 17 and
3-nitrophenylhydrazine hydrochloride. Yield: 60%. 1H NMR (400
MHz, DMSO-d6) δ 1.45 (t, J = 7.2 Hz, 3H), 4.52 (q, J1 = 7.2 Hz, J2 =
14.0 Hz, 2H), 7.18 (d, J = 7.6 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.28
(t, J = 8.0 Hz, 1H), 7.54 (s, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.94 (t, J =
7.6 Hz, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.34 (s, 1H), 8.72 (d, J = 8.4 Hz,
1H), 11.53 (br, 1H).
2-((2-(3-Chlorophenyl)hydrazinylidene)methyl)-N′-((E)-3-
phenylallylidene)quinoline-4-carbohydrazide (23c). In the same
manner as that described for the preparation of 23a, 23c was prepared
from 19a and cinnamaldehyde. Yield: 66%. Mp 272 °C. 1H NMR (400
MHz, DMSO-d6) δ: 6.88 (d, J = 8.0 Hz, 2H), 7.13−7.37 (m, 7H), 7.42
(t, J = 7.2 Hz, 1H), 7.66 (d, J = 7.6 Hz, 2H), 7.82 (t, J = 7.2 Hz, 1H),
8.05−8.11 (m, 3H), 8.17−8.18 (m, 1H), 8.28 (s, 1H), 11.18 (s, 1H),
12.17 (s, 1H). ESI-MS m/z 454.2 [M + H]+. HRMS (ESI) m/z calcd
for C26H20ClN5O [M + H]+ 454.1435, found 454.1448.
2-((2-(4-Aminosulfonylphenyl)hydrazinylidene)methyl)-N′-
(3-hydroxybenzylidene)quinoline-4-carbohydrazide (23d). In
the same manner as that described for the preparation of 23a, 23d was
prepared from 19b and 3-hydroxybenzaldehyde. Yield: 90%. Mp 252−
1
253 °C. H NMR (400 MHz, DMSO-d6) δ: 7.15 (t, J = 7.6 Hz, 3H),
7.29 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.66−7.85 (m, 4H),
8.06−8.15 (m, 2H), 8.17 (s, 1H), 8.29 (s, 1H), 8.32 (s, 1H), 9.69 (s,
1H), 11.39 (s, 1H), 12.24 (s, 1H). ESI-MS m/z 489.2 [M + H]+.
HRMS (ESI) m/z calcd for C24H20N6O4S [M + H]+ 489.1345, found
489.1356.
Ethyl 2-((2-(3-(Trifluoromethyl)phenyl)hydrazinylidene)-
methyl)quinoline-4-carboxylate (18e). In the same manner as
that described for the preparation of 18a, 18e was prepared from 17
and 3-(trifluoromethyl)phenylhydrazine. Yield: 65%. EI-MS m/z 387.2
(M+).
2-((2-(3-Chlorophenyl)hydrazinylidene)methyl)quinoline-4-
carbohydrazide (19a). 18a (25 mg, 0.07 mmol) was slowly added to
2-((2-(4-Aminosulfonylphenyl)hydrazinylidene)methyl)-N′-
(3-phenylpropylidene)quinoline-4-carbohydrazide (23e). In
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the same manner as that described for the preparation of 23a, 23e was
prepared from 19b and phenylpropyl aldehyde. Yield: 58%. Mp 260−
261 °C. 1H NMR (400 MHz, DMSO-d6) δ: 2.63−2.66 (m, 2H), 2.87
(t, J = 8.0 Hz, 2H), 7.24−7.33 (m, 7H), 7.56 (d, J = 8.0 Hz, 2H),
7.70−7.80 (m, 5H), 7.93−7.98 (m, 2H), 8.12 (d, J = 8.0 Hz, 1H), 8.47
(d, J = 8.0 Hz, 1H), 11.96 (s, 1H), 14.77 (s, 1H). ESI-MS m/z 501.2
[M + H]+. HRMS (ESI) m/z calcd for C26H24N6O3S [M + H]+
501.1709, found 501.1702.
2-((2-(4-Aminosulfonylphenyl)hydrazinylidene)methyl)-N′-
(2-phenylethylidene)quinoline-4-carbohydrazide (23f). In the
same manner as that described for the preparation of 23a, 23f was
prepared from 19b and phenylacetaldehyde. Yield: 67%. Mp 257 °C.
1H NMR (400 MHz, DMSO-d6) δ: 3.69 (d, J = 6.0 Hz, 2H), 7.21 (s,
2H), 7.27−7.40 (m, 5H), 7.55 (d, J = 8.8 Hz, 2H), 7.62 (s, 1H), 7.72−
7.84 (m, 5H), 7.92−7.95 (m, 2H), 8.45 (t, J = 8.0 Hz, 1H), 11.98 (s,
1H), 14.75 (s, 1H). ESI-MS m/z 487.2 [M + H]+. HRMS (ESI) m/z
calcd for C25H22N6O3S [M + H]+ 487.1552, found 487.1562.
2-((2-(4-Aminosulfonylphenyl)hydrazinylidene)methyl)-N′-
((E)-3-phenylallylidene)quinoline-4-carbohydrazide (23g). In
the same manner as that described for the preparation of 23a, 23g
was prepared from 19b and cinnamaldehyde. Yield: 90%. Mp 264 °C.
1H NMR (400 MHz, DMSO-d6) δ: 7.14 (d, J = 8.0 Hz, 1H), 7.22 (s,
2H), 7.27 (t, J = 8.0 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.42 (t, J = 7.6
Hz, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.66 (t, J = 8.0 Hz, 2H), 7.73−7.85
(m, 4H), 7.91−8.00 (m, 2H), 8.16 (t, J = 7.6 Hz, 1H), 8.47 (t, J = 7.6
Hz, 1H), 12.19 (s, 1H), 14.77 (s, 1H). ESI-MS m/z 499.2 [M + H]+.
HRMS (ESI) m/z calcd for C26H22N6O3S [M + H]+ 499.1552, found
499.1565.
2-((2-(3-Bromophenyl)hydrazinylidene)methyl)-N′-(3-
phenylpropylidene)quinoline-4-carbohydrazide (23h). In the
same manner as that described for the preparation of 23a, 23h was
prepared from 19c and phenylpropyl aldehyde. Yield: 78%. Mp 265−
267 °C. 1H NMR (400 MHz, DMSO-d6) δ: 2.62−2.67 (m, 2H), 2.88
(t, J = 8.0 Hz, 2H), 7.02 (d, J = 8.0 Hz, 1H), 7.18−7.33 (m, 7H), 7.63
(t, J = 8.0 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H),
8.02−8.08 (m, 4H), 8.18 (s, 1H), 11.15 (s, 1H), 11.91 (s, 1H). ESI-
MS m/z 500.2 [M + H]+. HRMS (ESI) m/z calcd for C26H22BrN5O
[M + H]+ 500.1086, found 500.1100.
°C. 1H NMR (400 MHz, DMSO-d6) δ: 3.69 (d, J = 5.6 Hz, 2H), 7.17
(d, J = 5.2 Hz, 2H), 7.31−7.38 (m, 4H), 7.44 (s, 1H), 7.51−7.54 (m,
2H), 7.64 (t, J = 7.2 Hz, 1H), 7.77−7.83 (m, 2H), 8.04−8.08 (m, 2H),
8.11 (s, 1H), 8.22 (s, 1H), 11.29 (s, 1H), 11.97 (s, 1H). ESI-MS m/z
476.2 [M + H]+. HRMS (ESI) m/z calcd for C26H20F3N5O [M + H]+
476.1698, found 476.1714.
2-((2-(3-(Trifluoromethyl)phenyl)hydrazinylidene)methyl)-
N′-((E)-3-phenylallylidene)quinoline-4-carbohydrazide (23m).
In the same manner as that described for the preparation of 23a,
23m was prepared from 19e and cinnamaldehyde. Yield: 86%. Mp 276
°C. 1H NMR (400 MHz, DMSO-d6) δ: 7.13 (t, J = 8.0 Hz, 2H), 7.40−
7.58 (m, 6H), 7.66 (d, J = 8.0 Hz, 2H), 7.76−7.85 (m, 2H), 8.06−8.18
(m, 5H), 8.28 (s, 1H), 11.32 (s, 1H), 12.19 (s, 1H). ESI-MS m/z
488.2 [M + H]+. HRMS (ESI) m/z calcd for C27H20F3N5O [M + H]+
488.1698, found 488.1714.
2-Formylquinoline-4-carboxylic Acid (20). To a solution of 15
(0.37 g) in 3 mL of dioxane, selenium dioxide (SeO2, 1.11 g) was
added. The resulting reaction mixture was heated to 40 °C for 15 h.
Solvent was removed under reduced pressure, and the residue was
extracted with EtOAc/H2O. The organic layer was washed with water
and brine, dried over anhydrous Na2SO4, filtered, and condensed. The
residue was purified by chromatography with DCM/MeOH (5:1, v/v)
to give 20 as a yellow solid. Yield: 56%. 1H NMR (400 MHz, DMSO-
d6): δ 7.93 (t, J = 8.0 Hz, 1H), 8.00 (t, J = 8.0 Hz, 1H), 8.32 (s, 1H),
8.33 (d, J = 8.0 Hz, 1H), 8.82 (d, J = 8.0 Hz, 1H), 10.16 (s, 1H).
2-Formyl-N-(3-phenylpropyl)quinoline-4-carboxamide
(21a). Into a solution of 20 (75 mg, 0.37 mmol) in methylene chloride
(DCM, 10 mL) were added EDCI (216 mg, 1.12 mmol) and HOBt
(203 mg, 1.5 mmol). After the mixture was stirred for 1 h at 0 °C, a
solution of the triethylamine (TEA, 400 μL) and 3-phenylpropan-1-
amine (78 mg, 0.56 mmol) in DCM (2 mL) was added dropwise. The
resulting mixture was stirred at room temperature overnight. The
solvent was removed under reduced pressure, and the residue was
extracted with EtOAc/H2O. The organic layer was washed with 2 N
aqueous HCl, saturated NaHCO3, brine and then dried over
anhydrous Na2SO4, filtered, and condensed. The residue was purified
by chromatography on silica gel, eluting with a mixture of EtOAc/
petroleum ether (1:4, v/v) to give 21a as a white solid. Yield: 70%. 1H
NMR (400 MHz, CDCl3): δ 2.07 (t, J = 7.2 Hz, 2H), 2.77−2.82 (m,
2H), 3.62 (t, J = 6.8 Hz, 2H), 7.23−7.32 (m, 5H), 7.77 (t, J = 7.2 Hz,
1H), 7.88 (t, J = 7.6 Hz, 1H), 7.96 (s, 1H), 8.29 (d, J = 8.4 Hz, 1H),
8.36 (d, J = 8.4 Hz, 1H), 10.23 (s, 1H).
2-((2-(3-Bromophenyl)hydrazinylidene)methyl)-N′-(2-
phenylethylidene)quinoline-4-carbohydrazide (23i). In the
same manner as that described for the preparation of 23a, 23i was
prepared from 19c and phenylacetaldehyde. Yield: 73%. Mp 258 °C.
1H NMR (400 MHz, DMSO-d6) δ: 3.69 (d, J = 6.0 Hz, 2H), 7.02 (br,
2-Formyl-N-(4-phenylbutyl)quinoline-4-carboxamide (21b).
In the same manner as that described for the preparation of 21a, 21b
was prepared from 20 and 4-phenylbutan-1-amine. Yield: 86%. H
NMR (400 MHz, CDCl3) δ: 1.63−1.73 (m, 4H), 2.67 (t, J = 6.8 Hz,
2H), 3.41−3.45 (m, 2H), 7.23−7.32 (m, 5H), 7.76 (t, J = 7.2 Hz, 1H),
7.88 (t, J = 7.6 Hz, 1H), 7.94 (s, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.29 (d,
J = 8.4 Hz, 1H), 10.24 (s, 1H).
1H), 7.18−7.40 (m, 8H), 7.63 (t, J = 8.0 Hz, 1H), 7.77−7.83 (m, 2H),
8.03−8.07 (m, 3H), 8.21 (s, 1H), 11.14 (s, 1H), 11.95 (s, 1H). ESI-
MS m/z 486.1 [M + H]+. HRMS (ESI) m/z calcd for C25H20BrN5O
[M + H]+ 486.0929, found 486.0944.
1
2-((2-(3-Nitrophenyl)hydrazinylidene)methyl)-N′-((E)-3-
phenylallylidene)quinoline-4-carbohydrazide (23j). In the same
manner as that described for the preparation of 23a, 23j was prepared
2-((2-(4-(Aminosulfonyl)phenyl)hydrazinylidene)methyl)-N-
(3-phenylpropyl)quinoline-4-carboxamide (24a). To a solution
of 21a (40 mg, 0.13 mmol) in EtOH (5 mL) were added 4-
sulfonamidophenylhydrazine hydrochloride (34 mg, 0.15 mmol) and
AcOH (50 μL). The mixture was refluxed for 2 h. The reaction
mixture was allowed to cool to room temperature and filtered. Then
the precipitate was collected, washed with H2O, and dried to afford
1
from 19d and cinnamaldehyde. Yield: 56%. Mp 232 °C. H NMR
(400 MHz, DMSO-d6) δ: 7.11−7.14 (m, 1H), 7.25−7.43 (m, 8H),
7.62−7.82 (m, 6H), 8.05−8.19 (m, 3H), 11.35 (s, 1H), 12.24 (s, 1H).
ESI-MS m/z 465.2 [M + H]+. HRMS (ESI) m/z calcd for
C26H20N6O3 [M + H]+ 465.1675, found 465.1648.
2-((2-(3-(Trifluoromethyl)phenyl)hydrazinylidene)methyl)-
N′-(3-phenylpropylidene)quinoline-4-carbohydrazide (23k). In
the same manner as that described for the preparation of 23a, 23k was
prepared from 19e and phenylpropyl aldehyde. Yield: 93%. Mp 268−
275 °C. 1H NMR (400 MHz, DMSO-d6) δ: 2.62−2.67 (m, 2H), 2.88
(t, J = 8.0 Hz, 2H), 7.03−7.09 (m, 1H), 7.18−7.24 (m, 2H), 7.29−
7.34 (m, 3H), 7.42−7.53 (m, 3H), 7.64 (t, J = 8.0 Hz, 1H), 7.72 (t, J =
8.0 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 8.03−8.06 (m, 2H), 8.12 (s,
1H), 8.19 (s, 1H), 11.31 (s, 1H), 11.93 (s, 1H). ESI-MS m/z 490.2 [M
+ H]+. HRMS (ESI) m/z calcd for C27H22F3N5O [M + H]+ 490.1855,
found 490.1873.
1
24a (40 mg, 63%) as a red solid. Mp 259−261 °C. H NMR (400
MHz, DMSO-d6) δ: 1.93 (t, J = 8.0 Hz, 2H), 2.73 (t, J = 7.6 Hz, 2H),
3.40−3.44 (m, 2H), 7.20−7.39 (m, 7H), 7.70 (t, 1H), 7.75 (d, J = 8.4
Hz, 2H), 7.88 (t, J = 8.0 Hz, 1H), 8.11 (t, J = 8.0 Hz, 2H), 8.24 (d, J =
10.8 Hz, 2H), 9.01 (t, J = 5.2 Hz, 1H), 11.81 (s, 1H). ESI-MS m/z
488.2 [M + H]+. HRMS (ESI) m/z calcd for C26H25N5O3S [M + H]+
488.1756, found 488.1743.
2-((2-(4-(Aminosulfonyl)phenyl)hydrazinylidene)methyl)-N-
(4-phenylbutyl)quinoline-4-carboxamide (24b). In the same
manner as that described for the preparation of 24a, 24b was
prepared from 21b and 4-sulfonamidophenylhydrazine hydrochloride.
2-((2-(3-(Trifluoromethyl)phenyl)hydrazinylidene)methyl)-
N′-(2-phenylethylidene)quinoline-4-carbohydrazide (23l). In
the same manner as that described for the preparation of 23a, 23l
was prepared from 19e and phenylacetaldehyde. Yield: 56%. Mp 272
1
Yield: 76%. Mp 251−254 °C. H NMR (400 MHz, DMSO-d6) δ:
1.63−1.73 (m, 4H), 2.67 (t, J = 6.8 Hz, 2H), 3.41−3.45 (m, 2H), 7.02
(d, J = 8.4 Hz, 1H), 7.18−7.31 (m, 6H), 7.38 (d, J = 8.4 Hz, 1H),
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Journal of Medicinal Chemistry
Article
7.66−7.77 (m, 3H), 7.89 (t, J = 7.6 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H),
8.15 (d, J = 8.4 Hz, 1H), 8.21 (s, 1H), 8.27 (s, 1H), 9.00 (t, J = 8.8 Hz,
1H), 10.39 (br, 1H), 11.92 (br, 1H). ESI-MS m/z 502.3 [M + H]+.
HRMS (ESI) m/z calcd for C27H27N5O3S [M + H]+ 502.1913, found
502.1894.
ASSOCIATED CONTENT
* Supporting Information
■
S
General information for chemical agents and analytical
measurements; detailed synthetic procedures and related
spectroscopic data for starting materials of compounds 1 and
22l−n; HPLC reports for the purity check of the compounds 1,
22a−n, 23a−m, and 24a−h; and the materials and methods of
the reversed-phase HPLC analysis of protease inhibition assay.
This material is available free of charge via the Internet at
http://pubs.acs.org.
2-((2-(3-Bromophenyl)hydrazinylidene)methyl)-N-(3-
phenylpropyl)quinoline-4-carboxamide (24c). In the same
manner as that described for the preparation of 24a, 24c was prepared
from 21a and 3-bromophenylhydrazine hydrochloride. Yield: 79%. mp
1
237−240 °C. H NMR (400 MHz, DMSO-d6) δ: 1.93 (t, J = 7.6 Hz,
2H), 2.72 (t, J = 8.0 Hz, 2H), 3.40−3.45 (m, 2H), 7.11 (d, J = 7.2 Hz,
1H), 7.19−7.33 (m, 7H), 7.54 (s, 1H), 7.74 (t, J = 7.6 Hz, 1H), 7.94
(t, J = 7.6 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H),
8.31 (s, 2H), 9.10 (t, J = 5.2 Hz, 1H), 12.10 (s, 1H). ESI-MS m/z
487.2 [M + H]+. HRMS (EI) m/z calcd for C26H23BrN4O (M+)
486.1055, found 486.1036.
AUTHOR INFORMATION
Corresponding Author
*For W.T.: phone, +86-21-50806701; e-mail, tangwei@mail.
shcnc.ac.cn. For C.-G.Y.: phone, +86-21-50806029; e-mail,
yangcg@mail.shcnc.ac.cn. For J.L.: phone, +86-21-64252584; e-
mail, jianli@ecust.edu.cn. For W.Z.: phone, +86-21-50805020;
e-mail, wlzhu@mail.shcnc.ac.cn.
■
2-((2-(3-Bromophenyl)hydrazinylidene)methyl)-N-(4-
phenylbutyl)quinoline-4-carboxamide (24d). In the same man-
ner as that described for the preparation of 24a, 24d was prepared
from 21b and 3-bromophenylhydrazine hydrochloride. Yield: 60%. Mp
249−252 °C. 1H NMR (400 MHz, DMSO-d6) δ: 1.63−1.70 (m, 4H),
2.67 (t, J = 7.6 Hz, 2H), 3.42−3.44 (m, 2H), 7.17−7.28 (m, 8H), 7.54
(s, 1H), 7.72 (t, J = 8.0 Hz, 1H), 7.94 (t, J = 7.6 Hz, 1H), 8.10 (d, J =
8.0 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 8.28 (s, 2H), 9.07 (br, 1H),
12.09 (br, 1H). ESI-MS m/z 501.3 [M + H]+. HRMS (ESI) m/z calcd
for C27H25BrN4O [M + H]+ 501.1290, found 501.1276.
Author Contributions
⊥These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
2-((2-(3-Chlorophenyl)hydrazinylidene)methyl)-N-(3-
phenylpropyl)quinoline-4-carboxamide (24e). In the same
manner as that described for the preparation of 24a, 24e was
prepared from 21a and 3-chlorophenylhydrazine hydrochloride. Yield:
We thank Dr. C. Klein for the generous gift of NS2B-NS3
expression plasmid. We gratefully acknowledge the financial
support from the International S&T Cooperation Project
(Grant 2010DFB73280, W.Z.), National Natural Science
Foundation of China (Grant 21002028, J.Z.), National S&T
Major Project, China (Grant 2011ZX09102-005-02, J.L.), the
111 Project (Grant B07023, J.L.), the Shanghai Committee of
Science and Technology (Grant 11DZ2260600, J.L. and H.J.),
Hundred Talent Program of the Chinese Academy of Sciences
(C.-G.Y.), and the Fundamental Research Funds for the
Central Universities (J.L.).
1
71%. Mp 253−255 °C. H NMR (400 MHz, DMSO-d6) δ: 1.91 (m,
2H), 2.73 (t, J = 8.0 Hz, 2H), 3.40−3.45 (m, 2H), 6.97 (d, J = 8.0 Hz,
1H), 7.20−7.35 (m, 7H), 7.40 (s, 1H), 7.73 (t, J = 7.6 Hz, 1H), 7.92
(t, J = 7.6 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1H),
8.29 (d, J = 8.0 Hz, 2H), 9.08 (br, 1H), 11.98 (s, 1H). ESI-MS m/z
443.2 [M + H]+. HRMS (ESI) m/z calcd for C26H23ClN4O [M + H]+
443.1639, found 443.1624.
2-((2-(3-Chlorophenyl)hydrazinylidene)methyl)-N-(4-
phenylbutyl)quinoline-4-carboxamide (24f). In the same manner
as that described for the preparation of 24a, 24f was prepared from
21b and 3-chlorophenylhydrazine hydrochloride. Yield: 55%. Mp
225−226 °C. 1H NMR (400 MHz, DMSO-d6) δ: 1.64−1.71 (m, 4H),
2.67 (t, J = 7.6 Hz, 2H), 3.41−3.46 (m, 2H), 7.01 (d, J = 7.6 Hz, 1H),
7.15−7.36 (m, 7H), 7.44 (s, 1H), 7.74 (t, J = 8.0 Hz, 1H), 7.97 (t, J =
8.0 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.34 (s,
1H), 8.37 (s, 1H), 9.11 (t, J = 5.2 Hz, 1H), 12.35 (s, 1H). ESI-MS m/z
457.3 [M + H]+. HRMS (ESI) m/z calcd for C27H25ClN4O [M + H]+
457.1795, found 457.1782.
ABBREVIATIONS USED
■
DENV, dengue virus; NS, nonstructural; SI, selectivity index;
WNV, West Nile virus; YFV, yellow fever virus; JEV, Japanese
encephalitis virus; DF, dengue fever; DHF, dengue hemor-
rhagic fever; DSS, dengue shock syndrome; IC50, half-maximal
inhibitory concentration; EC50, half-maximal effective concen-
tration; SAR, structure−activity relationship; EDCI, 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;
CHAPS, 3-((3-cholamidopropyl)dimethylammonium)-1-pro-
panesulfonate; DMSO, dimethylsulfoxide; PCR, polymerase
chain reaction; BHK, baby hamster kidney; DMEM, Dulbecco’s
modified Eagle medium; FBS, fetal bovine serum; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CC50,
half-maximal cytotoxicity concentration; DCM, dichlorome-
thane; DMF, N,N-dimethylformamide
2-((2-(3-Carboxyphenyl)hydrazinylidene)methyl)-N-(3-
phenylpropyl)quinoline-4-carboxamide (24g). In the same
manner as that described for the preparation of 24a, 24g was
prepared from 21a and 3-hydrazinobenzoic acid. Yield: 57%. Mp 259−
1
262 °C. H NMR (400 MHz, DMSO-d6) δ: 1.92 (t, J = 7.6 Hz, 2H),
2.72 (t, J = 7.6 Hz, 2H), 3.38−3.43 (m, 2H), 7.21−7.50 (m, 8H), 7.62
(t, J = 7.2 Hz, 1H), 7.72 (s, 1H), 7.79 (t, J = 7.6 Hz, 1H), 8.01−8.09
(m, 4H), 8.95 (t, J = 5.2 Hz, 1H), 11.18 (s, 1H). ESI-MS m/z 453.2
[M + H]+. HRMS (ESI) m/z calcd for C27H24N4O3 [M + H]+
453.1927, found 453.1912.
REFERENCES
■
2-((2-(3-Carboxyphenyl)hydrazinylidene)methyl)-N-(4-
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