Design and synthesis of small molecule RhoA inhibitors: A new promising therapy for cardiovascular diseases?

Article abstract of DOI:10.1021/jm200161c

RhoA is a member of Rho GTPases, a subgroup of the Ras superfamily of small GTP-binding proteins. RhoA, as an important regulator of diverse cellular signaling pathways, plays significant roles in cytoskeletal organization, transcription, and cell-cycle progression. The RhoA/ROCK inhibitors have emerged as a new promising treatment for cardiovascular diseases. However, to date, RhoA inhibitors are macromolecules, and to our knowledge, small molecular-based inhibitors have not been reported. In this study, a series of first-in-class small molecular RhoA inhibitors have been discovered by using structure-based virtual screening in conjunction with chemical synthesis and bioassay. Virtual screening of ～200,000 compounds, followed by SPR-based binding affinity assays resulted in three compounds with binding affinities to RhoA at the micromolar level (compounds 1-3). Compound 1 was selected for further structure modifications in considering binding activity and synthesis ease. Fourty-one new compounds (1, 12a-v, 13a-h, and 14a-j) were designed and synthesized accordingly. It was found that eight (12a, 12j, 14a, 14b, 14d, 14e, 14 g, and 14h) showed high RhoA inhibition activities with IC₅₀ values of 1.24 to 3.00 μM. A pharmacological assay indicated that two compounds (14g and 14 h) demonstrated noticeable vasorelaxation effects against PE-induced contraction in thoracic aorta artery rings and served as good leads for developing more potent cardiovascular agents.

Full text of DOI:10.1021/jm200161c

ARTICLE
pubs.acs.org/jmc
Design and Synthesis of Small Molecule RhoA Inhibitors:
A New Promising Therapy for Cardiovascular Diseases?
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Jing Deng,†, Enguang Feng,‡, Sheng Ma,§, Yan Zhang,§ Xiaofeng Liu,† Honglin Li,*,† Huang Huang,†
Jin Zhu,† Weiliang Zhu,‡ Xu Shen,†,‡ Liyan Miao,*,§ Hong Liu,‡ Hualiang Jiang,†,‡ and Jian Li*,†
†School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China
‡Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road,
Shanghai 201203, China
§Department of Clinical Pharmacology Research Lab, The First Affiliated Hospital of Soochow University, 188 Shi Zhi Street,
Suzhou 215006, China
S Supporting Information
b
ABSTRACT: RhoA is a member of Rho GTPases, a subgroup
of the Ras superfamily of small GTP-binding proteins. RhoA, as
an important regulator of diverse cellular signaling pathways,
plays significant roles in cytoskeletal organization, transcription,
and cell-cycle progression. The RhoA/ROCK inhibitors have
emerged as a new promising treatment for cardiovascular diseases. However, to date, RhoA inhibitors are macromolecules, and to
our knowledge, small molecular-based inhibitors have not been reported. In this study, a series of first-in-class small molecular RhoA
inhibitors have been discovered by using structure-based virtual screening in conjunction with chemical synthesis and bioassay.
Virtual screening of ∼200,000 compounds, followed by SPR-based binding affinity assays resulted in three compounds with binding
affinities to RhoA at the micromolar level (compounds 1ꢀ3). Compound 1 was selected for further structure modifications in
considering binding activity and synthesis ease. Fourty-one new compounds (1, 12aꢀv, 13aꢀh, and 14aꢀj) were designed and
synthesized accordingly. It was found that eight (12a, 12j, 14a, 14b, 14d, 14e, 14 g, and 14h) showed high RhoA inhibition activities
with IC50 values of 1.24 to 3.00 μM. A pharmacological assay indicated that two compounds (14g and 14 h) demonstrated
noticeable vasorelaxation effects against PE-induced contraction in thoracic aorta artery rings and served as good leads for
developing more potent cardiovascular agents.
1. INTRODUCTION
Rho family proteins are members of the main branches of the
Ras superfamily of small GTPases.1 Rho GTPases, including
Rho, Rac, and Cdc42, regulate the actin cytoskeleton2 and several
signaling pathways.3 RhoA, as one of the most well characterized
members, is essential for multiple cellular processes including
cytoskeletal rearrangement, gene expression, and membrane
trafficking, as well as cell adhesion, migration, differentiation,
proliferation, and apoptosis.4ꢀ6
RhoA, similar to all GTPases, functions as bimolecular switch
that cycles between active GTP-bound and inactive GDP-bound
forms. When activated, RhoA is able to interact with diverse
effectors to carry out its cellular functions. To date, the best-
characterized downstream target of RhoA is Rho kinase (ROCK).
Figure 1. Two regulatory pathways (Ca2þ-dependent and Ca2þ-in-
dependent) of vasoconstriction.
Within cardiovascular system, regulation of vascular tone is mainly
dependent on two pathways (Figure 1): the Ca2þ-dependent
increase of vascular tone (red arrows in Figure 1).9 Therefore,
vasoconstriction by activation of the myosin light chain kinase
(MLCK), and the Rho-ROCK-mediated Ca2þ sensitization, which
theconnectionbetweenRhoA/ROCKactivationandvasoconstric-
can occur independently of intracellular Ca2þ changes.7,8 In 1990s,
tion has made the inhibition of the RhoA pathway an appealing
target for pharmacological treatment of numerous cardiovascular
RhoA was identified as the upstream mediator of Ca2þ sensitiza-
tion, and RhoA/ROCK activation leads to inhibition of myosin
light chain phosphatase (MLCP). The event can also produce
directly phosphorylation of MLC, resulting in an synergistic
Received: January 28, 2011
Published: May 26, 2011
r
2011 American Chemical Society
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Figure 2. Structures of 3 binders (or hits) of RhoA selected from the candidates by virtual screening and the SPR-based binding affinity assay.
disorders, including hypertension, coronary and cerebral vasos-
pasm, atherosclerosis, vascular aneurisms, diabetes, stroke, and
heart failure.10ꢀ12 In addition, it is also a valuable target for the
treatment of cancer,13ꢀ16 nervous system disease,17 and asthma.18
Among ROCK inhibitors, Fasudil is an approved drug for
cerebral vasospasm after subarachnoid hemorrhage and a num-
ber of other ROCK inhibitors, such as Y-27632 and statins, have
also been reported in the literature.19ꢀ24 Despite the significant
progress, very limited efforts have been devoted toward the
development of direct RhoA inhibitors, especially small molec-
ular inhibitors have not been reported.
(Specs: Chemistry Solutions for Drug Discovery, 2008; http://
www.specs.net/ (accessed October 1, 2007)), which involves
∼200,000 compounds. All the compounds containing inorganic
atoms were removed prior to the screening. The remaining
molecules are prepared (including adding hydrogen atoms,
ionizing at the pH range from 6.0 to 9.0, generating stereo
isomers and valid single 3D conformers) by means of the Ligand
Preparation module in Discovery Studio 2.0 (Accelrys, Inc.).
A hierarchical virtual screening strategy was adopted. The
crystal structure of RhoA was retrieved from the Protein Data
Bank (PDB entry: 1KMQ). Amino acid residues located within
8.0 Å from the complexed ligand GNP were defined as part of the
binding site for docking studies. All crystallographic water
molecules were removed from the coordinate set. DOCK4.031,32
was used for the initial screening. Hydrogen atoms and Amber
charges of the receptor were added with Chimera (http://www.
cgl.ucsf.edu/chimera). The grid-enclosing box was generated
around the binding site defined previously. In the docking
process, the standard DOCK score was used to rank the result
list. All the parameters were set as the default values. The top
ranked 3000 candidates were reserved for detail docking and
reranking with Glide (Schr€odinger, Inc.) in standard precision
(SP) mode. The grid-enclosing box was centered on the centroid
of cocomplexed GNP in RhoA and defined so as to enclose
residues located within 14.0 Å around the GNP binding site, and
a scaling factor of 1.0 was set to van der Waals (VDW) radii of
those receptor atoms with the partial atomic charge less than
0.25. After visual observation of corresponding binding modes,
54 compounds were selected and purchased from the vendor for
the bioassay.
Clostridium botulinum C3 exoenzyme and the related transferases
have been used as a tool to inactivate RhoA and to analyze cellular
functions of Rho proteins.25 Nevertheless, C3 transferase with about
25ꢀ30 kDa, only consists of one enzyme domain and lacks a cell
binding and transport domain.26,27 Its extremely low efficiency of
cell entry and degradation in vivo has restricted the therapeutic
value. Instead, much effort has been paid for C3 transferase. Tan
et al. developed a membrane-permeating form of C3 transferase
(TAT-C3) and a biopolymer-based microsphere depot system for
sustainable controlled release of the protein to suit the pattern of
RhoA expression in injured central nervous.28 Lord-Fontaine et al.
reported an improved cell-permeable C3 variant (BA-210), ex-
pected to provide significant neuroprotective and neuroregenerative
benefits.29 In addition, Town et al. described a novel RhoA inhibitor
(a Gli3-regulated, transmembrane protein) that directed cell migra-
tion and actin polymerization and was implicated in lip and palate
formation.30 These results highlight the potential of RhoA as a
therapeutic target in the treatment of cardiovascular diseases.
Small molecules are highly valuable for developing clinically
useful agents. Toward this end, recently we initiated a program
aimed at seeking the first-in-class small molecule RhoA inhibitors.
By using a docking-based virtual screening approach in conjunc-
tion with surface plasmon resonance (SPR) determination, three
novel small molecule RhoA binders (hits) (compounds 1ꢀ3,
Figure 2) have been discovered. According to the binding potency
and syntheticcomplexity, compound 1 was selected as the starting
point for further structural optimization. Totally, 41 new com-
pounds have been designed, synthesized, and tested with biolo-
gical assays. Finally, eight compounds (12a, 12j, 14aꢀb, 14dꢀe,
and 14gꢀ4h) were found to show high RhoA inhibition activities,
and two compounds (14g and 14h) showed significant inhibitory
effects against phenylephrine (PE)-induced contraction in thor-
acic aorta artery rings. To our knowledge, the two compounds are
the first-in-class small molecular RhoA inhibitors.
2.1.2. Molecular Docking. The binding poses of compound 1
and the most potent synthesized compound 14h were modeled
by Glide in SP mode with the same parameter settings in virtual
screening.
2.2. Chemistry. 2.2.1. Design of Analogues of Compound 1.
Compounds 1ꢀ3 (Figure 2) showing high binding affinities to
RhoA were obtained by SPR determination (see section 3) from
the 120 candidate compounds selected by virtual screening.
Compound 1 with the potent binding affinity and easy synthetic
accessibility was used as lead compound for designing new RhoA
inhibitors. To provide expedient and significant structureꢀactiv-
ity relationship (SAR) information and improve inhibitory
activity of lead compound 1, chemical modifications were
performed in three cycles. First, we incorporated various steric,
electronic, and hydrophobic groups at positions 2, 3, and 6 of the
quinoxaline ring in compound 1, and 22 analogues (12aꢀv)
were designed (Table 1). Second, to simplify the structure, by
maintaining the 6-nitro-quinoxaline ring, we designed and
synthesized 8 monoamino-substituted analogues at 2 or 3 of
2. MATERIALS AND METHODS
2.1. Computation. 2.1.1. Virtual Screening. The virtual
screening was performed on the vendor database from SPECS
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Table 1. Chemical Structures of Compounds 1 and 12aꢀv and Their activities
a The inhibition rate of the exoenzyme C3 transferase(C3) as a positive control compound is 117% at 62.5 nM.
the quinoxaline ring (compounds 13aꢀh, Table 2) to determine
if double amino-substituents on quinoxaline ring are necessary
for ligandꢀenzyme interaction. Finally, 10 compounds 14aꢀj
(Table 3) were prepared to estimate if the type of heterocycles
and side-chain substituents on the heterocycle would affect
inhibitory activity.
2.2.2. Synthetic Procedures. Scheme 1 depicts the synthetic
route for the preparation of compounds 1 and 12aꢀv. Treatment
of 4-substituted-o-phenylenediamines 4 with diethyl oxalate
5 in hydrochloric acid solution under reflux quickly afforded
quinoxalinediones 6, followed by chlorination with POCl3 to get
2,3-dichloro-6-substituted quinoxaline 7. Compounds 7 were
converted to compounds 1 and 12aꢀt by SArN2 or CꢀN
coupling under microwave radiation. The catalytic hydrogena-
tion of 6-nitro-quinoxalines 12h and 12j in the presence of 10%
Pd/C in MeOH afforded the corresponding 6-aminoquinoxaline
derivatives 12u and 12v.
Compounds 13aꢀh were synthesized through the approach
outlined in Scheme 2. Quinoxalin-2-ol 8a was nitrified
using HNO3/AcOH and KNO3/H2SO4 at room temperature
to afford 7-nitroquinoxalin-2-ol 9a and 6-nitroquinoxalin-2-ol
9b, respectively, which was converted to the corresponding
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Table 2. Chemical Structures of Compounds 13aꢀh and
Table 3. Chemical Structures of Compounds 14aꢀj and
Their Activities
Their Activities
a The inhibition rate of the exoenzyme C3 transferase(C3) as a positive
control compound is 117% at 62.5 nM.
aryl chlorides 10a and 10b by refluxing with POCl3 and PCl5.
Then 10a and 10b were substituted by using different aromatic
amines, giving the target compounds 13cꢀd and 13fꢀg, and
esters of compounds 13aꢀb, 13e, and 13h, which were hydro-
lyzed using LiOH at 25 °C to give carboxylic acid products.
Compounds 14aꢀj were synthesized by using a process
similar to that for compounds 13aꢀh (Scheme 3).
a The inhibition rate of the exoenzyme C3 transferase(C3) as a positive
control compound is 117% at 62.5 nM.
2.3. Biological Assays. 2.3.1. SPR Assay. Interaction studies
between compounds and RhoA were performed with the SPR-
based biosensor instrument Biacore 3000 (Biacore AB, Uppsala,
Sweden). Each compound was dissolved in dimethyl sulfoxide
(DMSO) as a 10 mM stock solution for the Biacore experiments.
RhoA was immobilized on the sensor surface by the standard
primary amine coupling reaction to the carboxymethylated
matrix dextran of a sensor chip CM5 (Biacore AB, Uppsala,
Sweden). Equilibration of the baseline was completed by con-
tinuous flow of HBS-EP running buffer (10 mM Hepes, 150 mM
NaCl, 3 mM EDTA, and 0.005% (v/v) surfactant P20, pH 7.4)
through the chip overnight. One of the four serial flow cells was
activated for 7 min by injecting a 1:1 fresh mixture of 0.2 M N-
ethyl-N0-dimethylaminopropyl carbodiimide (EDC) and 50 mM
N-hydroxysuccinimide (NHS) at 25 °C. RhoA was diluted with
10 mM sodium acetate buffer at pH 4.2 to a concentration of
25 μg/mL and immobilized to the surface of sensor chip CM5.
Finally, the unreacted protein was blocked by injecting 1 M
ethanolamine-HCl at pH 8.5 for 7 min, resulting in immobilized
densities of 7000 response units (RU). After stabilizing the
baseline, Biacore data were collected at 25 °C with HBS-EP
(containing 0.4% DMSO) as the running buffer at a constant
flow of 30 μL/min. All the sensorgrams were processed by using
automatic correction for nonspecific bulk refractive index effects.
The equilibrium constants (KDs) evaluating the proteinꢀinhibi-
tor binding affinity were determined by the 1:1 Langmuir binding
fitting model using eq 1. The kinetic analysis of RhoA/inhibitors
binding by SPR were described in Supporting Information.
koff
KD
¼
ð1Þ
kon
where koff represents dissociation rate constant, kon represents
the association rate constant.
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Scheme 1a
a Reagents and conditions: (a) 4N HCl, reflux, 2 h; (b) POCl3, DMF, reflux, overnight; (c) Cs2CO3, dioxane, MW 160 °C, 30 min or Pb(Ph3P)4,
Cs2CO3, MW 160 °C, 30 min; (d) H2, 10% Pb/C, MeOH, rt, 3 h.
Scheme 2a
a Reagents and conditions: (a) HNO3, AcOH, 25 °C; (b) KNO3, H2SO4, 25 °C; (c) POCl3, PCl5, reflux, 4 h; (d) For 13aꢀb, 13e, and 13 h: (i) DMF,
reflux, 2ꢀ5 h, (ii) LiOH, MeOH, 25 °C, 2 h; (e) For 13cꢀd and 13f-g: DMF, reflux, 2ꢀ5 h.
2.3.2. RhoA Activation Assay. When the cell cultures reached
70% confluence (see Experimental Section), the culture media
was removed, and the cells were starved overnight in serum-free
DMEM (0% fetal bovine serum (FBS)). Then 1.25 μL of com-
pound or DMSO (final concentration of compound, 2.5 μM)
was added to the cells. After an additional 1 h of incubation at
37 °C, cells were treated with or without oleoyl-L-R-lysopho-
sphatidic acid (LPA, 5 μg/mL) for 3 min. Following treatment,
the culture medium was removed, and the cells were washed in
ice-cold PBS and lysed. Thereafter, RhoA activity was measured
from the cell lysates using the absorbance-based RhoA G-LISA
activation assay biochem kit (Cytoskeleton) according to the
manufacturer’s protocol. Briefly, cell lysates were isolated using
the provided cell lysis buffer and clarified by centrifugation at
10,000 rpm at 4 °C for 2 min before being snap-frozen. Active,
GTP-bound, RhoA in cell lysates would bind to the wells to
which a Rho-GTP-binding protein was linked but not the
inactive GDP-bound RhoA. Active RhoA was detected using
indirect immunodetection followed by a colorimetric reaction
measured by absorbance at 490 nm. The inhibitory rate (IR) was
calculated by using the follow eq 2.
mounted between two stainless steel clips in vertical 10-mL
organ baths filled with Modified K-H, maintained at 37 °C, and
equilibrated with 95% O2ꢀ5% CO2. The upper stainless steel
clip was connected to a force transducer. Isometric tension was
recorded by an analogue-to-digital system (ALCB10 MPA2000;
Shanghai Alcott Biotech Co. Ltd., China) connected to a desktop
computer. The rings were initially placed under the optimal
resting tension of 1.5 g and incubated for 90 min until a stable rest
tension was achieved, and the solution was changed every 20 min
to remove metabolites. The tissues were challenged with a K-rich
(80 mM) solution obtained by substituting an equimolar of KCl
for NaCl in the Modified K-H repeatedly before the experiment,
applied to obtain ring contractions similar in both amplitude and
kinetics. A cumulative concentrationꢀresponse curve to the R1-
agonist PE (10ꢀ9ꢀ10ꢀ5 M) was then constructed to determine
the concentration of agonist required to produce 70ꢀ80% of the
maximal contraction (EC70ꢀ80). Successful removal of endothe-
lium was confirmed by the inability of acetylcholine (1 μM) to
induce relaxation in PE-induced submaximal contraction. After
this run-up procedure, the ring was entered into an experimental
protocol. The pharmacological effect of the compound was
studied on rings submaximally contracted with PE. Once a steady
contraction was observed, one aorta ring was treated with the
compound (100 mM, dissolved in 100% DMSO) added in a
cumulative manner (final concentration was 30 μMꢀ180 μM),
whereas another ring of each vessel received only the same
volume of solvent (DMSO (3 μLꢀ18 μL)) and was used as a
paired time control. The concentration of compound was
increased once the maximal relaxant effect of the preceding
concentration had been recorded, i.e., after 5 min.
ODL ꢀ ODC
IR ¼
ð2Þ
ODL ꢀ ODD
where L represents DMSO þ LPA, C represents compound þ
LPA, and D is DMSO.
2.3.3. Isometric Contraction Measurement. Isometric con-
traction was measured in rings from the thoracic aortic rings
as previously described.33 Briefly, isolated aortic rings were
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Scheme 3a
a Reagents and conditions: (a) POCl3, PCl5, reflux, 4 h; (b) For 14a,
14dꢀf, and 14hꢀj: (i) DMF, reflux, 2ꢀ5 h, (ii) LiOH, MeOH, 25 °C,
2 h; (c) For 14bꢀc and 14g: DMF, reflux, 2ꢀ5 h.
3. RESULTS AND DISCUSSION
3.1. Identification of Binders (Hits) by Virtual Screening.
Targeting the crystal structure of RhoA, we searched the SPECS
database by molecular docking. Fifty-four candidates available
from the vendor were selected after manual observation of the
top 3000 ranked hits. Finally, three compounds (1, 2, and 3)
showing a great binding affinity to RhoA were selected according
to the binding affinity of compounds to RhoA in vitro using SPR
biosensor technology. The binding curves of compounds 1ꢀ3
with RhoA are shown in Figure 3. For kinetic analysis on the
Biacore 3000 instrument, various concentrations of these three
compounds were injected for 120 min at a flow rate of 30 μL/min
to allow for interaction with RhoA immobilized on the sensor
chip surface. For the RhoA, all these three candidate molecules
resulted in a significant and dose-dependent increase in SPR RU
and presented characteristic fast-binding and slow-dissociation
curves. The concentration series of 1, 2, and 3 were fitted by the
1:1 Langmuir binding model in the Biacore3000 evaluation
software for binding affinity determination. The collected data
indicated that these three compounds can bind to RhoA in vitro
and that the dissociation constant (KD) to RhoA is in the
micromolar range (KD = 4.47ꢀ13.1 μM). These compounds
could be designated as binders (or hits) of RhoA, and their
chemical structures and binding affinities are shown in Figure 2.
The kinetic data of three binders 1ꢀ3 are listed in Table 1S
(Supporting Information). The ranks of three hits (compounds
1ꢀ3) identified from the top 3000 docking candidates with Glide
are shown in Table 2S (Supporting Information). The Glide
GScore for the cocomplexed GNP in the crystal structure of
RohA was also calculated as a reference (Supporting Informa-
tion, Table 2S).
3.2. Analogue Design and Synthesis. In total, 41 com-
pounds (1, 12aꢀv, 13aꢀh, and 14aꢀj) were designed and syn-
thesized, and their chemical structures are shown in Tables 1ꢀ3.
These compounds were synthesized through the routes outlined
in Schemes 1ꢀ3, and the details for synthetic procedures and
structural characterizations are described in the Experimental
Section. All compounds (1, 12aꢀv, 13aꢀh, and 14aꢀj)
were confirmed to have g95% purity (Supporting Information,
Table 3S).
3.3. Biological Activities. 3.3.1. Inhibitory Activity of Com-
pounds 1, 12aꢀv, 13aꢀh, and 14aꢀj. For the primary assay,
using C3 transferase as a positive control, the percent inhibitions
of the compounds 1, 12aꢀv, 13aꢀh, and 14aꢀj at 2.5 μM were
measured. The results are summarized in Tables 1ꢀ3. Of the
Figure 3. Sensorgrams of compounds 1ꢀ3 binding to RhoA measured
by SPR. Representative sensorgrams obtained from the injection of
compounds 1ꢀ3 at concentrations of 1.68, 2.4, 3.43, 4.9, 7.0, and
10.0 μM over RhoA immobilized on the CM5 chip.
synthetic derivatives tested, 8 compounds (eg., 12a, 12j, 14aꢀb,
14dꢀe, and 14gꢀh) displayed good inhibitory activities against
RhoA (expressed as the inhibitory rate at 2.5 μM g 68%),
indicating that these are good candidate inhibitors of RhoA.
In further studies, we found that the inhibition of RhoA increased
in the concentration range from 0 to 10 μM of 8 inhibitors.
The curve finally reached steady state in a concentration depen-
dent manner. The half maximal inhibitory concentration (IC50)
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Table 4. Inhibitory Activities and IC50 Values of the Compounds Tested
cmpd
inhibitory rate at 2.5 μMa (%)
IC50b (μM)
cmpd
inhibitory rate at 2.5 μM a (%)
IC50b (μM)
1
54.0
68.5
72.5
78.0
71.7
3.28 ( 0.30
2.58 ( 0.25
2.09 ( 0.34
1.51 ( 0.13
3.00 ( 0.31
14d
14e
14g
14h
76.1
68.0
81.8
90.3
2.05 ( 0.07
2.62 ( 0.59
1.68 ( 0.13
1.24 ( 0.08
12a
12j
14a
14b
a The inhibition rate of the exoenzyme C3 transferase (C3) as a positive control compound is 117% at 62.5 nM. b Data are the means of three
independent experiments.
values, ranging from 1.24 to 3.00 μM (Table 4), were calculated
by fitting the doseꢀresponse curve using a logistic derivative
equation in Origin 7.5. In order to do a direct comparison, the
IC50 of lead compound 1 was also given in the same methods
(Table 4).
Analyzing the data in Tables 1 and 2, we found that the 2,3-
amino- and 6-substituents on the quinoxaline ring markedly
affected the inhibitory activities (ranging from 11% to 72%).
Encouragingly, the inhibition rate of mono amino-substituted,
simplified derivatives 13aꢀb turned out to be good, 51% and
58%, respectively (Table 2), with little difference and in balance
with compound 1 (inhibition rate = 54%). It appeared that only
one amino-substituent (regardless of substituted position) was
sufficient for the interaction. To our surprise, compound 14a and
Figure 4. Effect of increasing concentration of 14h and DMSO on PE-
the non-nitro derivative of compound 13a displayed the highest
induced submaximal contraction in isolated arterial rings. Values are the
activity among the compounds probed (78%, Table 3), much
more potent than lead compound 1 (inhibition rate = 54%) and
means ( SE (*P < 0.01 ANOVA n = 6).
analogues 12aꢀv and 13aꢀh (inhibition rate e72%). Further-
more, the most potent derivative 14h (90%, Table 3), replacing
and ꢀNH2 (12j vs 12v) is not beneficial; (3) 6-nitro substitu-
the quinoxaline ring with the quinoline ring, was obtained in the
whole series. Remarkably, the inhibitory activity of the most
potent compound 14h (IC50 = 1.24 ( 0.08 μM, Table 4)
increased approximately 3 times than that of lead compound 1
(IC50 = 3.28 ( 0.30 μM, Table 4), demonstrating that the
chemical modification strategy employed in this study is efficient
and valuable for further structural modification.
tions on the monoamino quinoxaline ring substantially decreases
the inhibitory activity of the derivatives (14a vs 13a, 14d vs 13h,
and 14b vs 13d); (4) among the quinoxaline (14a), quinoline
(14h), benzimidazole (14i), and quinazoline (14j) frameworks,
the quinoline ring is optimal and significantly improves the
biological activities. Taken together, the SARs are mixed. A
subtle interplay between heterocyclic scaffolds, steric effects,
polarity, and hydrogen-bonding capability seemed to be critical
for high potency. Compounds 14g and 14h represented see-
mingly the best combination.
3.5. Binding Models. To understand the structural basis for
the binding affinities of the inhibitors for RhoA, we scrutinized
the binding poses of hit compound 1 and the most potent
derivate 14h by means of molecular docking. Figure 5 shows the
predicted binding poses of 1 (Figure 5B) and 14h (Figure 5C) in
the GNP-binding site. The binding mode of the cocomplexed
GNP of RhoA is shown in Figure 5A, which is featured by
the triphosphate chelating the magnesium cation and guanine
moiety anchored by Asp120 and Lys162. The triphosphate group
and the ribose ring also form complex hydrogen bonds with the
backbone of the surrounding residues as well as salt bridge with
Lys18, which further stabilized the binding pose of GNP. For hit
1, the quinoxaline moiety acts as a bridge between the pair of
acrylic acid groups, which extend to the two anchor binding sites
occupied by the guanine and triphosphate groups, respectively, in
GNP. The acrylic acid groups can be regarded as the payload and
form strong salt bridges with the basic residues such as Lys18 and
Lys118. Moreover, one of the carboxylates can coordinate with
the magnesium cation (Figure 5B). Although the anchor points
mentioned above contribute remarkably to the binding affinity of
compound 1, the hydrophobic quinoxaline moiety is predicted to
3.3.2. Effect of Compounds 14gꢀh against PE-Induced
Contraction in Thoracic Aorta Artery Rings. To test the vasor-
elaxation effects of the inhibitors against PE-induced contraction
in thoracic aorta artery rings, two of the most potent inhibitors of
RhoA (14g and 14h) have been evaluated. In all vessels
submaximally contracted with 100 nM PE (produce 70ꢀ80%
of the maximal contraction), compounds 14gꢀh (0ꢀ180 μM)
strongly inhibited the tension development in a dose dependent
fashion (Figure 4). The maximal inhibitions of compounds 14g
and 14h were 78.57 ( 2.47% and 55.92 ( 4.01%, respectively
(180 μM, Figure 4). The IC50 values of compounds 14g and 14h
were 135.9 μM and 161.3 μM, respectively. These results
indicated that compounds 14gꢀh showed partial influence on
the PE-induced contraction in thoracic aorta artery rings and
were good leads for designing more potent cardiovascular drugs.
3. 4. SAR. The SAR analysis of a set of 41 compounds provided
important insights of the essential structural requirements for
effective RhoA inhibition. An analysis of the data shown in
Tables 1ꢀ3 reveals some noteworthy observations of the SAR
for compounds 1, 12aꢀv, 13aꢀh, and 14aꢀj: (1) mono as well
as double amino-substituents on the quinoxaline ring can both
display high activities (1 vs 13a or 13b). The substituent effect is
limited; (2) the displacement of ꢀNO2 on the quinoxaline ring
with ꢀCF3 (12a vs 12r and 12b vs 12s), ꢀOMe (12b vs 12t),
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Figure 5. Two-dimensional (2D) and three-dimensional (3D) interaction schemes of cocomplexed GNP (A) of RhoA and docked poses of 1 (B) and
14h (C) in the GNP-binding site of RhoA. The figures were prepared using PoseViewWeb (http://poseview.zbh.uni-hamburg.de/poseview) and
PyMol (http://pymol.sourceforge.net/). The ligands are shown as sticks, and the noncarbon atoms are colored by atom types (receptor carbon in green
and ligand carbon in cyan). The magnesium ion in the phosphate binding site was rendered as gray spheres. Critical residues of the binding pocket are
labeled in the 2D interaction views. Hydrogen bonds are shown as dotted lines in both 2D and 3D views.
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be completely exposed to the solvent, rendering the binding pose
extremely unstable with respect to the remarkable entropy
penalty, which may explain its moderate inhibitory activity
against RhoA. Moreover, the gain of the binding affinity from
the hydrogen bonds between the carboxylate group and residues
in the guanine site such as Lys118 and Ala161 may be offset by
the electrostatic repellency with the nearby negatively charged
residue Asp120. The binding affinity for compound 1 with
Glide GScore also justifies the binding preference over the GNP
(as shown in Table 2S, Supporting Information) in the same
binding site and most likely in a competitive way. For compound
14h, the carboxylate can also form the hydrogen bond networks
and other strong interactions observed in the case of 1. The
quinoline ring occupies the guanine binding site and results in a
visible burying in the hydrophobic sandwiches consisting of
Cys20, Phe30, and Lys118 (Figure 5C), which may compensate
the binding affinity loss brought by truncation of the acrylic acid
group in 1. The bottom of the guanine binding site is spatially
constrained and relatively hydrophobic, and cannot accommo-
date larger polar groups such as nitro, which attributes to the
lower inhibitory activity of 13a and 13h compared with that of
their counterparts without the nitro groups such as 14a and 14d.
Information, Table 3S). The details for purity analyses of compounds 1,
12aꢀv, 13aꢀh, and 14aꢀj are described in the Supporting Information.
5.1.2. Biology. All solvents and reagents with reagent grade or
ultrapure quality were purchased commercially and were used without
further purification. The vector pRSET B and DMEM and FBS were
purchased from Invitrogen. The bacterial strain E.coli strain BL21 was
purchased from Qiagen. The chelating column and protein molecular
weight marker for SDSꢀPAGE were from Amersham Pharmacia
Biotech. Amicon Ultra-4 centrifugal filter devices were purchased from
Millipore. The Human brain vascular smooth muscle cells were pur-
chased from ScienCell. The PE, LPA, and DMSO were purchased from
Sigma. The RhoA G-LISA activation assay biochem kit and C3
transferase were purchased from Cytoskeleton.
5.1.2.1. 6-Nitro Quinoxaline-2,3(1H,4H)-dione (6a). Representa-
tive Procedure for 6aꢀ6c. 4-Nitrobenzene-1,2-diamine (15.3 g, 0.1
mol) and diethyl oxalate (0.15 mol) were added slowly to 40 mL of 4 M
HCl. After stirring for 5 min at room temperature, the reaction mixture
was heated to reflux for 2 h. Then the reaction mixture was moved to
room temperature for 10 min, the deposited crystals were obtained by
1
filtration and washed with water. Yield: 85%. H NMR (400 MHz,
DMSO-d6) δ: 7.21 (d, J = 8.8 Hz, 1H), 7.90ꢀ7.95 (m, 2H), 12.16
(s, 1H), 12.35 (s, 1H). LC-MS m/z 208 [M þ H]þ.
5.1.2.2. 6-Trifluoromethyl Quinoxaline-2,3(1H,4H)-dione (6b). In
the same manner as that described for the preparation of 6a, 6b was
prepared from 4-trifluoromethylbenzene-1,2-diamine and diethyl oxa-
late. Yield: 82%. LC-MS m/z 231 [M þ H]þ.
4. CONCLUSIONS
In summary, we have discovered first-in-class small molecular
RhoA inhibitors by using a structure-based virtual screening
approach in conjunction with chemical synthesis and bioassays.
On the basis of the structure of lead compound 1, chemical
modifications were performed in three cycles. In total, 41 new
compounds have been synthesized and tested with biological
assays. Finally, eight compounds (12a, 12j, 14aꢀb, 14dꢀe, and
14gꢀ4h) were found to show high RhoA inhibition activities,
and two compounds (14g and 14h) showed significant inhibi-
tory effects against PE-induced contraction in thoracic aorta
artery rings. Molecular binding models give rational explanations
about SARs, which are in good agreement with pharmacological
results. Notably, to our knowledge, compounds 14g and 14h
with IC50 values of 1.68 μM and 1.24 μM against RhoA activation
and IC50 values of 135.9 μM and 161.3 μM to the relaxation of
PE-induced contraction are the most potent small molecular
RhoA inhibitors reported up to date, which are likely to be
developed into new promising agents for cardiovascular diseases
and are currently being pursued in our laboratories.
5.1.2.3. 6-Methoxy Quinoxaline-2,3(1H,4H)-dione (6c). In the
same manner as that described for the preparation of 6a, 6c was prepared
from 4-methoxybenzene-1,2-diamine and diethyl oxalate. Yield: 83%.
LC-MS m/z 192 [M þ H]þ.
5.1.2.4. 2,3-Dichloro-6-nitroquinoxaline (7a). Representative Pro-
cedure for 7aꢀ7c. 6-Nitro quinoxaline-2,3(1H,4H)-dione 6a (0.1 mol)
was slowly added to POCl3 (30 mL), and then N,N-dimethylformamide
(DMF, 0.5 mL) was added. The reaction mixture was heated under
reflux and kept overnight. After being cooled to about 20 °C, it was
poured into 300 mL of ice water. The solid thus separated as shining
needles, was filtered, and washed with ice water to give 7a as a gray solid.
Yield: 90%. 1H NMR (400 MHz, DMSO-d6) δ: 8.19 (dd, J1 = 8.8 Hz,
J2 = 16 Hz, 1H), 8.29 (d, J = 8.8 Hz, 1H), 8.52 (s, 1H). LC-MS m/z 245
[M þ H]þ.
5.1.2.5. 2,3-Dichloro-6-trifluoromethyl Quinoxaline (7b). In the
same manner as that described for the preparation of 7a, 7b was
prepared from 6-trifluoromethyl quinoxaline-2,3(1H,4H)-dione (6b)
and POCl3. Yield: 85%. LC-MS m/z 268 [M þ H]þ.
5.1.2.6. 2,3-Dichloro-6-methoxy Quinoxaline (7c). In the same
manner as that described for the preparation of 7a, 7c was prepared
from 6-methoxy quinoxaline-2,3(1H,4H)-dione (6c) and POCl3. Yield:
81%. LC-MS m/z 230 [M þ H]þ.
5. EXPERIMENTAL SECTION
5.1.2.7. (2E,20E)-3,30-(3,30-(6-Nitroquinoxaline-2,3-diyl)bis(azanediyl)
bis(3,1-phenylene)diacrylic Acid (1). 2,3-Dichloro-6-nitroquinoxaline
(5a, 0.01 mol), (E)-ethyl 3-(3-aminophenyl) acrylate (0.05 mol),
Pb(Ph3P)4 (0.003 mol), and Cs2CO3 (0.05 mol) were added to a
solution of dioxane (5 mL) in a vial. The reaction mixture was then
irradiated for 30 min at 160 °C. After the reaction was cooled to ambient
temperature, it was poured into water and extracted with dichloro-
methane (DCM) three times, and the combined organic layer was
washed, dried over MgSO4, filtered, and concentrated in vacuo and then
purified by chromatography with EtOAc/petroleum ether to give the
precursor of product, which was then hydrolized with 10% NaOH. After
the reaction was completed, positive ion-exchange resin (Dowex 50w X
4-400) was added to the reaction mixture to neutralize the excess base,
filtered, and concentrated in vacuo to give 1 as a yellow solid. Yield: 35%;
mp >300 °C. 1H NMR (500 MHz, DMSO-d6) δ: 6.51 (d, J = 16.0 Hz,
2H), 7.49ꢀ7.51 (m, 4H), 7.60ꢀ7.70 (m, 3H), 7.95ꢀ8.04 (m, 2H),
5.1. General. 5.1.1. Chemistry. 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 performed on HSGF 254 (150ꢀ200 μm
thickness; Yantai Huiyou Co., China). 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) spec-
troscopy was performed on Bruker AMX-500, AMX-400, and AMX-300
NMR (IS as TMS). Chemical shifts were reported in parts per million
(ppm, δ) downfield from tetramethylsilane. Proton coupling patterns
were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), and broad (br). Low- and high-resolution mass spectra (LRMS and
HRMS) were given 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,
12aꢀv, 13aꢀh, and 14aꢀj were confirmed g95% purity (Supporting
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8.12ꢀ8.23 (m, 3H), 8.43 (s, 1H), 9.50 (s, 1H), 9.65 (s, 1H); HRMS
(ESI) m/z calcd C26H20N5O6 [M þ H]þ 498.1414; found, 498.1406.
5.1.2.8. (2E,20E)-3,30-(3,30-(6-Nitroquinoxaline-2,3-diyl)bis(azanediyl)
bis(4,1-phenylene)diacrylic Acid (12a). In the same manner as that
described for the preparation of 1, 12a was prepared from 2,3-dichloro-
6-nitroquinoxaline (5a) and (E)-ethyl 3-(4-aminophenyl) acrylate.
yellow solid. Yield: 83%. mp 135 °C. 1H NMR (400 MHz, DMSO-d6) δ:
1.49ꢀ1.69 (m, 4H), 1.79ꢀ1.99 (m, 4H), 2.57ꢀ3.13 (m, 5H),
3.86ꢀ4.33 (m, 4H), 7.68ꢀ7.72 (m, 1H), 8.10ꢀ8.14 (m, 1H),
8.33ꢀ8.35 (m, 1H); EI-MS m/z 429 (Mþ). HRMS (EI) m/z calcd
for Mþ 429.1648; found, 429.1656.
5.1.2.15. Diethyl 1,10-(6-nitroquinoxaline-2,3-diyl)bis(piperidine-
4-carboxylate) (12h). In the same manner as that described for the
preparation of 12c, 12h was prepared from 2,3-dichloro-6-nitroquinoxa-
line (5a) and ethyl piperidine-4-carboxylate. Yield: 89%. mp 97 °C. 1H
NMR (300 MHz, CDCl3) δ: 1.28 (t, J = 7.2 Hz, 6H), 1.80ꢀ1.91 (m,
4H), 2.06ꢀ2.12 (m, 4H), 2.53ꢀ2.58 (m, 2H), 2.88ꢀ3.04 (m, 4H),
4.14ꢀ4.21 (q, J = 7.2 Hz, 4H), 4.21ꢀ4.25 (m, 4H), 4.38ꢀ4.43 (m, 4H),
7.69 (d, J = 9 Hz, 1H), 8.16 (dd, J1 = 9 Hz, J2 = 2.4 Hz, 1H), 8.55 (d, J =
2.4 Hz, 1H); EI-MS m/z 485 (Mþ). HRMS (EI) m/z calcd for Mþ
485.2274; found, 485.2275.
5.1.2.16. Dimethyl 1,10-(6-nitroquinoxaline-2,3-diyl)bis(piperidine-4-
carboxylate) (12i). In the same manner as that described for the
preparation of 12c, 12i was prepared from 2,3-dichloro-6-nitroquinoxa-
line (5a) and methyl piperidine-4-carboxylate. Yield: 87%. mp 141 °C.
1H NMR (300 MHz, CDCl3) δ: 1.82ꢀ1.93 (m, 4H), 2.10 (m, 4H),
2.57ꢀ2.60 (m, 2H), 2.89ꢀ3.07 (m, 4H), 3.73 (s, 6H), 4.24 (d, J = 13.5
Hz, 2H), 4.42 (d, J = 13.5 Hz, 2H), 7.73 (d, J = 9.3 Hz, 1H), 8.18 (d, J =
9.3 Hz, 1H), 8.56 (s, 1H); EI-MS m/z 457 (Mþ). HRMS (EI) m/z calcd
for Mþ 457.1961; found, 457.1955.
5.1.2.17. 1,10-(6-Nitroquinoxaline-2,3-diyl)bis(piperidine-4-carboxylic
acid) (12j). Dimethyl 1,10-(6-nitroquinoxaline-2,3-diyl)bis(piperidine-
4-carboxylate) (12i) was hydrolized with 10% NaOH in MeOH. After
the reaction was completed, positive ion-exchange resin (Dowex 50w X
4ꢀ400) was added to the reaction mixture to neutralize the excess base,
filtered, and concentrated in vacuo to give 12j as a white solid. Yield:
81%. mp >300 °C. 1H NMR (400 MHz, CDCl3) δ: 1.63ꢀ1.73 (m, 4H),
1.97ꢀ2.00 (m, 4H), 2.87ꢀ3.02 (m, 4H), 3.40ꢀ3.49 (m, 1H), 4.14 (d,
J = 12.8 Hz, 2H), 4.33 (d, J = 12.8 Hz, 2H), 7.67 (d, J = 9.2 Hz, 1H), 8.11
(dd, J1 = 9.2 Hz, J2 = 2.8 Hz, 1H), 8.31 (d, J = 2.8 Hz, 1H), 12.34 (br,
2H); EI-MS m/z 429 (Mþ). HRMS (EI) m/z calcd for Mþ 429.1648;
found, 429.1646.
1
Yield: 25%; mp >300 °C. H NMR (300 MHz, DMSO-d6) δ: 6.46
(dd, J1 = 15.3 Hz, J2 = 3.6 Hz, 2H), 7.53ꢀ7.72 (m, 7H), 8.10ꢀ8.18 (m,
5H), 8.33 (d, J = 2.4 Hz, 1H). ESI-MS m/z 495.9 [M ꢀ H]ꢀ; HRMS
(ESI) m/z calcd C26H18N5O6 [M ꢀ H]ꢀ 496.1257; found, 496.1262.
5.1.2.9. (2E,20E)-Diethyl 3,30-(((6-nitroquinoxaline-2,3-diyl)bis-
(azanediyl))bis(4,1-phenylene))diacrylate (12b). In the same manner
as that described for the preparation of 1, 12b was prepared from 2,3-
dichloro-6-nitroquinoxaline (5a) and (E)-ethyl 3-(4-aminophenyl) ac-
rylate. Yield: 30%; mp 212 °C. 1H NMR (300 MHz, DMSO-d6) δ: 1.27
(t, J = 7.5 Hz, 6H), 4.20 (q, J = 7.5 Hz, 4H), 6.52ꢀ6.60 (m, 2H),
7.62ꢀ7.79 (m, 7H), 8.80ꢀ8.14 (m, 4H), 8.13 (d, J = 9 Hz, 1H), 8.34
(s, 1H), 9.63 (br, 1H), 9.77 (br, 1H); EI-MS m/z 553 (Mþ). HRMS (EI)
m/z calcd for Mþ 553.1961; found, 553.1946.
5.1.2.10. 2,3-Bis(4-(2-fluorophenyl)piperidin-1-yl)-6-nitroquinoxaline
(12c). 2,3-Dichloro-6-nitroquinoxaline (5a, 0.01 mol), 1-(2-fluorophe-
nyl)piperazine (0.05 mol), and Cs2CO3 (0.05 mol) were added to a
solution of dioxane (5 mL) in the vial. The reaction mixture was then
irradiated for 30 min at 160 °C. After the reaction was cooled to ambient
temperature, it was poured into water and extracted with DCM three
times, and the combined organic layer was washed, dried over MgSO4,
filtered, and concentrated in vacuo and then purified by chromatography
with DCM/MeOH to give 12c as a yellow solid. Yield: 80%. mp 189 °C.
1H NMR (300 MHz, CDCl3) δ: 3.28 (br, 8H), 3.82 (br, 4H), 3.93 (br,
4H), 6.98ꢀ7.04 (m, 8H), 7.76 (d, J = 9 Hz, 1H), 8.22 (dd, J1 = 9.3 Hz,
J2 = 2.4 Hz, 1H), 8.62 (d, J = 2.4 Hz, 1H); EI-MS m/z 531 (Mþ). HRMS
(EI) m/z calcd for Mþ 531.2194; found, 531.2194.
5.1.2.11. 2,3-Bis(4-(benzo[d][1,3]dioxol-5-yl)piperazin-1-yl)-6-ni-
troquinoxaline (12d). In the same manner as that described for the
preparation of 12c, 12d was prepared from 2,3-dichloro-6-nitroquinoxa-
line (5a) and 1-(benzo[d][1,3]dioxol-5-yl)piperazine. Yield: 75%. mp
93 °C. 1H NMR (300 MHz, CDCl3) δ: 2.57 (br, 8H), 3.58 (s, 4H), 3.69
(s, 4H), 5.96 (s, 4H), 6.77 (s, 4H), 6.89 (s, 2H), 7.67 (d, J = 6.3 Hz, 1H),
8.16 (dd, J1 = 9 Hz, J2 = 2.7 Hz, 1H), 8.54 (d, J = 2.7 Hz, 1H); EI-MS m/z
583 (Mþ). HRMS (EI) m/z calcd for Mþ 583.2179; found, 583.2183.
5.1.2.12. 8-(3-{1,4-Dioxa-8-azaspiro[4.5]decan-8-yl}-7-nitroqui-
noxalin-2-yl)-1,4-dioxa-8-azaspiro[4.5]decane (12e). In the same
manner as that described for the preparation of 12c, 12e was prepared
from 2,3-dichloro-6-nitroquinoxaline (5a) and 1,4-dioxa-8-azaspiro-
[4.5]decane. Yield: 88%. mp 182ꢀ183 °C. 1H NMR (300 MHz, CDCl3)
δ: 1.86 (br, 8H), 3.70 (br, 4H), 3.83 (br, 4H), 4.01 (s, 8H), 7.68(d, J = 9
Hz, 1H), 8.16 (dd, J1 = 9 Hz, J2 = 2.7 Hz, 1H), 8.54 (d, J = 2.7 Hz, 1H);
EI-MS m/z 457 (Mþ). HRMS (EI) m/z calcd for Mþ 457.1961; found,
457.1966.
5.1.2.18. 4,40-(6-Nitroquinoxaline-2,3-diyl)dimorpholine (12k). In
the same manner as that described for the preparation of 12c, 12k was
prepared from 2,3-dichloro-6-nitroquinoxaline (5a) and morpholine. Yield:
92%. mp 180 °C. 1H NMR (300 MHz, CDCl3) δ: 3.60ꢀ3.63 (m, 4H),
3.70ꢀ3.74 (m, 4H), 3.85ꢀ3.89 (m, 8H), 7.74 (d, J = 9.3 Hz, 1H), 8.22 (dd,
J1 = 9.3 Hz, J2 = 2.4 Hz, 1H), 8.60 (d, J = 2.7 Hz, 1H); EI-MS m/z 345
(Mþ). HRMS (EI) m/z calcd for Mþ 345.1437; found, 345.1437.
5.1.2.19. 2,3-Bis(4-methylpiperidin-1-yl)-6-nitroquinoxaline (12l). In
the same manner as that described for the preparation of 12c, 12l was
prepared from 2,3-dichloro-6-nitroquinoxaline (5a) and 4-methylpiperidine.
Yield: 89%. mp 159ꢀ161 °C. 1H NMR (300 MHz, CDCl3) δ: 0.99 (s, 3H),
1.01 (s, 3H), 1.25ꢀ1.39 (m, 5H), 1.68ꢀ1.86 (m, 3H), 1.80 (d, J = 12.3 Hz,
2H), 2.78 (dd, J1 = 27 Hz, J2 = 13.2 Hz, 4H), 4.27 (d, J = 12.6 Hz, 2H), 4.49
(d, J= 12.6 Hz, 2H), 7.62 (d, J= 8.7 Hz, 1H), 8.12 (dd, J1 =8.7Hz, J2 =3Hz,
1H), 8.51 (d, J=2.7Hz, 1H);EI-MSm/z369 (Mþ). HRMS(EI) m/zcalcd
for Mþ 369.2165; found, 369.2166.
5.1.2.20. 2,3-Bis(4-(2,4-difluorophenyl)piperazin-1-yl)-6-nitroqui-
noxaline (12m). In the same manner as that described for the
preparation of 12c, 12m was prepared from 2,3-dichloro-6-nitroqui-
noxaline (5a) and 1-(2,4-difluorophenyl)piperazine. Yield: 75%. mp
213-214 °C. 1H NMR (300 MHz, CDCl3) δ: 3.22 (br, 8H), 3.81 (br,
4H), 3.91 (br, 4H), 6.80ꢀ6.97 (m, 6H), 7.75 (d, J = 8.7 Hz, 1H), 8.21
(dd, J1 = 9 Hz, J2 = 3 Hz, 1H), 8.60 (d, J = 2.4 Hz,1H); EI-MS m/z 567
(Mþ). HRMS (EI) m/z calcd for Mþ 567.2006; found, 567.1997.
5.1.2.21. N2,N3-Bis(3,4-dimethoxyphenethyl)-6-nitroquinoxaline-
2,3-diamine (12n). In the same manner as that described for the
preparation of 12c, 12n was prepared from 2,3-dichloro-6-nitroquino-
xaline (5a) and 2-(3,4-dimethoxyphenyl)ethanamine. Yield: 71%. mp
5.1.2.13. Diethyl 1,10-(6-nitroquinoxaline-2,3-diyl)bis(piperidine-
3-carboxylate) (12f). In the same manner as that described for the
preparation of 12c, 12f was prepared from 2,3-dichloro-6-nitroquinoxa-
line (5a) and ethyl piperidine-3-carboxylate. Yield: 81%. mp <50 °C. 1H
NMR (300 MHz, CDCl3) δ: 1.24ꢀ1.31 (m, 6H), 1.61ꢀ2.11 (m, 8H),
2.66ꢀ3.22 (m, 6H), 3.96ꢀ4.29 (m, 6H), 4.33ꢀ4.53 (m, 2H), 7.70 (dd,
J1 = 9 Hz, J2 = 1.5 Hz, 1H), 8.17 (dd, J1 = 9 Hz, J2 = 2.4 Hz, 1H),
8.55ꢀ8.57(m, 1H); EI-MS m/z 485 (Mþ). HRMS (EI) m/z calcd for
M
þ 485.2274; found, 485.2262.
5.1.2.14. 1,10-(6-Nitroquinoxaline-2,3-diyl)bis(piperidine-3-car-
boxylic Acid) (12g). Diethyl 1,10-(6-nitroquinoxaline-2,3-diyl)bis-
(piperidine-3-carboxylate) (12f) was hydrolized with 10% NaOH in
MeOH. After the reaction was completed, positive ion-exchange resin
(Dowex 50w X 4ꢀ400) was added to the reaction mixture to neutralize
the excess base, filtered, and concentrated in vacuo to give 12g as a
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136ꢀ137 °C. 1H NMR (300 MHz, CDCl3) δ: 2.93 (t, J = 6.9 Hz, 4H),
3.78ꢀ3.86 (m, 16H), 6.72ꢀ6.81 (m, 6H), 7.61 (d, J = 9 Hz, 1H), 8.10
(dd, J1 = 9 Hz, J2 = 2.4 Hz, 1H), 8.49 (d, J = 2.7 Hz, 1H); EI-MS m/z 533
(Mþ). HRMS (EI) m/z calcd for Mþ 533.2274; found, 533.228.
5.1.2.22. 6-Nitro-N2,N3-bis(pyridin-4-ylmethyl)quinoxaline-2,3-
diamine (12o). In the same manner as that described for the prepara-
tion of 12c, 12o was prepared from 2,3-dichloro-6-nitroquinoxaline (5a)
and pyridin-4-yl-methanamine. Yield: 61%. mp 207ꢀ208 °C. 1H NMR
(300 MHz, DMSO-d6) δ: 4.79 (dd, J1 = 8.4 Hz, J2 = 2.1 Hz, 4H),
7.42ꢀ7.48 (m, 5H), 7.95 (dd, J1 = 8.4 Hz, J2 = 2.4 Hz, 1H), 8.11 (d, J =
2.7 Hz, 1H), 8.52ꢀ8.56 (m, 5H); EI-MS m/z 387 (Mþ). HRMS (EI)
m/z calcd for Mþ 387.1444; found, 387.1443.
(dd, J1 = 9 Hz, J2 = 2.7 Hz, 1H), 6.92 (d, J = 2.7 Hz, 1H), 7.51 (d, J = 9 Hz,
1H); EI-MS m/z 455 (Mþ). HRMS (EI) m/z calcd for Mþ 455.2533;
found, 455.2527.
5.1.2.29. 1,10-(6-Aminoquinoxaline-2,3-diyl)bis(piperidine-4-carboxylic
Acid) (12v). Diethyl 1,10-(6-aminoquinoxaline-2,3-diyl)dipiperidine-4-
carboxylate (12u) was hydrolized with 10% NaOH in MeOH. After the
reaction was completed, positive ion-exchange resin (Dowex 50w X
4ꢀ400) was added to the reaction mixture to neutralize the excess base,
filtered, and concentrated in vacuo to give the product (12v). Yield:
60%. mp >300 °C. 1H NMR (400 MHz, DMSO-d6) δ: 1.63ꢀ1.72 (m,
4H), 1.95ꢀ1.98 (m, 4H), 2.67ꢀ2.80 (m, 4H), 2.98 (d, J = 12.8 Hz, 2H),
4.19 (d, J = 12.8 Hz, 2H), 6.80 (d, J = 2.4 Hz, 1H), 6.87 (dd, J1 = 8.8 Hz,
J2 = 2.0 Hz, 1H), 7.38 (d, J = 8.8 Hz, 1H); EI-MS m/z 399 (Mþ). HRMS
(EI) m/z calcd for Mþ 399.1907; found, 399.1915.
1
1
2
2
0
5.1.2.23. N ,N -(6-Nitroquinoxaline-2,3-diyl)bis(N ,N -diethy-
lethane-1,2-diamine) (12p). In the same manner as that described
for the preparation of 12c, 12p was prepared from 2,3-dichloro-6-
nitroquinoxaline (5a) and N1,N1-diethylethane-1,2-diamine. Yield: 81%.
mp 161ꢀ163 °C. 1H NMR (300 MHz, DMSO-d6) δ: 1.05ꢀ1.12 (m,
12H), 2.80ꢀ2.94 (br, 12H), 3.66 (br, 4H), 7.48 (d, J = 9 Hz, 1H), 7.98
(dd, J1 = 9 Hz, J2 = 2.7 Hz, 1H), 8.15 (d, J = 2.7 Hz, 1H); EI-MS m/z 403
(Mþ). HRMS (EI) m/z calcd for Mþ 403.2696; found, 403.2701.
5.1.2.24. N2,N3-Bis(4-methylbenzyl)-6-nitroquinoxaline-2,3-dia-
mine (12q). In the same manner as that described for the preparation
of 12c, 12q was prepared from 2,3-dichloro-6-nitroquinoxaline (5a) and
p-tolylmethanamine. Yield: 83%. mp 210ꢀ212 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 2.26 (s, 6H), 4.66 (dd, J1 = 12.4 Hz, J2 = 4.8 Hz, 4H), 7.14
(d, J = 7.6 Hz, 4H), 7.27ꢀ7.30 (m, 4H), 7.49 (d, J = 9.2 Hz, 1H), 7.96
(dd, J1 = 8.8 Hz, J2 = 2.4 Hz, 1H), 8.14 (d, J = 2.1 Hz, 1H); EI-MS m/z
413 (Mþ). HRMS (EI) m/z calcd for Mþ 413.1852; found, 413.1856.
5.1.2.25. (2E,20E)-3,30-(((6-(Trifluoromethyl)quinoxaline-2,3-diyl)bis-
(azanediyl))bis(4,1-phenylene))diacrylic Acid (12r). In the same
manner as that described for the preparation of 1, 12r was prepared
from 2,3-dichloro-6-nitroquinoxaline (5a) and (E)-ethyl 3-(4-amino-
5.1.2.30. 7-Nitroquinoxalin-2-ol (9a). To a stirred solution of
quinoxalin-2-ol 8a (2.92 g, 20 mmol) in 85 mL of glacial acetic acid
was added the mixture of 0.88 mL of concentrated nitric acid and 5 mL of
acetic acid in small portions. The resulting solution was stirred at
ambient temperature overnight and evaporated, and the residue was
then poured into H2O (50 mL). After half an hour of stirring, the
mixture was filtered and washed with water to give compound 9a (3.22 g,
1
84%) as a pale yellow solid: mp 273ꢀ276 °C. H NMR (500 MHz,
DMSO-d6) δ: 8.00 (s, J = 8.8 Hz, 1H), 8.06 (dd, J1 = 2.4 Hz, J2 = 8.8 Hz,
1H), 8.12 (d, J = 2.4 Hz, 1H), 8.34 (s, 1H).
5.1.2.31. 6-Nitroquinoxalin-2-ol (9b). The powder of KNO3 (2.0 g, 20
mmol) was added quickly into a mixture of quinoxalin-2-ol 8a (2.92 g, 20
mmol) in H2SO4 (20 mL). The resulting solution was stirred at 0 °C. After
half an hour, the mixture was allowed to warm to ambient temperature,
stirred for 2 h, and poured into ice (500 mL). The resulting crystals were
collected after filtration and washed with water and dried to afford
compound 9b (3.55 g, 93%) as a pale yellow solid: mp 258ꢀ260 °C.
1H NMR (500 MHz, DMSO-d6) δ: 7.47 (d, J = 9.0 Hz, 2H), 8.33 (s, 1H),
8.39 (dd, J1 = 2.3 Hz, J2 = 9.0 Hz, 1H), 8.56 (d, J = 2.0 Hz, 1H).
5.1.2.32. 2-Chloro-7-nitroquinoxaline (10a). 7-Nitroquinoxalin-2-
ol 9a (0.67 g, 3.6 mmol) was slowly added to POCl3 (5 mL) and PCl5
(1.2 g). The reaction mixture was heated under reflux for 4 h. After being
cooled to about 20 °C, it was poured into ice water. The solid thus
separated as pale pink needles, was filtered, and washed with ice water to
give 10a (0.34 g, 46%) as a gray solid: mp 185ꢀ189 °C. 1H NMR (400
MHz, CDCl3) δ: 8.31 (d, J = 9.2 Hz, 1H), 8.57 (dd, J1 = 2.4 Hz, J2 = 9.2
Hz, 1H), 8.94 (br, 2H).
5.1.2.33. 2-Chloro-6-nitroquinoxaline (10b). In the same manner as
that described for the preparation of 10a, 10b was prepared from
6-nitroquinoxalin-2-ol (9b) and POCl3. Yield: 39%. mp 197ꢀ202 °C.
1H NMR (400 MHz, CDCl3) δ: 8.20 (d, J = 5.2 Hz, 1H), 8.52 (dd, J1 =
1.6 Hz, J2 = 8.8 Hz, 1H), 8.93 (s, 1H), 9.01 (d, J = 1.2 Hz, 1H).
5.1.2.34. 2-Chloroquinoxaline (11a). In the same manner as that
described for the preparation of 10a, 11a was prepared from quinoxalin-
2-ol (8a) and POCl3. Yield: 43%. mp 46ꢀ48 °C. 1H NMR (400 MHz,
CDCl3) δ: 7.78ꢀ7.85 (m, 2H), 8.04 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 7.2
Hz, 1H), 8.80 (s, 1H).
1
phenyl)acrylate. Yield: 35%. mp 155ꢀ157 °C. H NMR (300 MHz,
DMSO-d6) δ: 6.44ꢀ6.49 (m, 2H), 7.57ꢀ7.62 (m, 4H), 7.67ꢀ7.75 (m,
4), 7.89ꢀ7.90 (m, 1H), 8.03ꢀ8.05 (m, 4H), 9.70ꢀ9.74 (m, 2H); ESI-
MS m/z 518.9 [M-H]ꢀ. HRMS (ESI) m/z calcd for [M ꢀ H]ꢀ
519.1280; found, 519.1271.
5.1.2.26. (2E,20E)-Diethyl 3,30-(((6-(trifluoromethyl)quinoxaline-
2,3-diyl)bis(azanediyl))bis(4,1-phenylene)) Diacrylate (12s). In the
same manner as that described for the preparation of 1, 12s was prepared
from 2,3-dichloro-6-trifluoromethylquinoxaline (5a) and (E)-ethyl
1
3-(4-aminophenyl)acrylate. Yield: 35%. mp 193ꢀ194 °C. H NMR
(300 MHz, DMSO-d6) δ: 1.27 (t, J = 6.9 Hz, 6H), 4.19 (q, J = 7.2 Hz,
4H), 6.52ꢀ6.59 (m, 2H), 7.61ꢀ7.68 (m, 4H), 7.73ꢀ79 (m, 4H),
7.97ꢀ8.00 (m, 4H), 9.50 (d, J = 15.6 Hz, 2H); EI-MS m/z 576
(Mþ). HRMS (EI) m/z calcd for Mþ 576.1984; found, 576.1985.
5.1.2.27. (2E,20E)-Diethyl 3,30-(((6-methoxyquinoxaline-2,3-diyl)bis
(azanediyl))bis(4,1-phenylene))diacrylate (12t). In the same manner
as that described for the preparation of 1, 12t was prepared from 2,3-
dichloro-6-methoxyquinoxaline (5a) and (E)-ethyl 3-(4-aminophe-
nyl)acrylate. Yield: 37%. mp 103ꢀ105 °C. 1H NMR (300 MHz,
DMSO-d6) δ: 1.26 (t, J = 6.6 Hz, 6H), 4.19 (dd, J1 = 6.9 Hz, J2 = 1.2
Hz, 4H), 6.54 (dd, J1 = 15.6 Hz, J2 = 6.9 Hz, 2H), 7.08 (s, 2H), 7.54 (d,
J = 8.7 Hz, 1H), 7.52ꢀ7.62 (m, 1H), 7.66ꢀ7.77 (m, 4H), 7.89ꢀ7.99 (m,
4H), 9.17 (br,1H), 9.27 (br, 1H); EI-MS m/z 538 (Mþ). HRMS (EI)
m/z calcd for Mþ 538.2216; found, 538.2212.
5.1.2.35. 2-Chloroquinoline (11b). In the same manner as that
described for the preparation of 10a, 11b was prepared from quinoline-
1
2-ol (8b) and POCl3. Yield: 23%. H NMR (400 MHz, CDCl3) δ:
7.42 (d, J = 8.8 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.75 (t, J = 7.6 Hz,
1H), 7.85 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 8.14 (d,
J = 8.4 Hz, 1H).
5.1.2.28. Diethyl 1,10-(6-aminoquinoxaline-2,3-diyl)dipiperidine-
4-carboxylate (12u). Diethyl 1,10-(6-nitroquinoxaline-2,3-diyl)bis-
(piperidine-4-carboxylate) (12h) was treated with 10% Pb/C in MeOH
with H2. After the reaction was completed, it filtered and concentrated in
vacuo to give the product (12u).Yield: 91%. mp 185ꢀ186 °C. 1H NMR
(300 MHz, DMSO-d6) δ: 1.27 (t, J = 13.5 Hz, 6H), 1.83ꢀ1.91 (m, 4H),
2.09 (d, J = 7.2 Hz, 4H), 2.44ꢀ2.64 (m, 2H), 2.74ꢀ2.87 (m, 4H), 4.10
(d, J = 12 Hz, 2H), 4.17 (q, J = 13.5 Hz, 4H), 4.32 (d, J = 12 Hz, 2H), 6.84
5.1.2.36. 2-Chlorobenzimidazole (11c). In the same manner as that
described for the preparation of 10a, 11c was prepared from benzimi-
1
dazole-2-ol (8c) and POCl3. Yield: 45%. mp 178ꢀ180 °C. H NMR
(400 MHz, CDCl3) δ: 7.26 (d, J = 8.8 Hz, 1H), 7.70ꢀ7.83 (m, 2H).
5.1.2.37. 2-Chloroquinazoline (11d). Compound 11d was prepared
following the method reported by Ram et al.34 (see Scheme 1 in Sup-
porting Information for details). Yield: 50%. mp 101ꢀ109 °C. 1H NMR
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Journal of Medicinal Chemistry
ARTICLE
(400 MHz, CDCl3) δ: 7.71 (t, J = 8.0 Hz, 1H), 7.97ꢀ8.04 (m, 3H), 9.33
(s, 1H).
with a mixture of EtOAc/petroleum ether (1:6, v/v), to afford inter-
mediate ethyl 5-(6-nitroquinoxalin-3-ylamino)-2-chlorobenzoate (89
1
5.1.2.38. (E)-3-(3-(6-Nitroquinoxalin-3-ylamino)phenyl)acrylic Acid
(13a) and (E)-ethyl 3-(3-(6-nitroquinoxalin-3-ylamino)phenyl)acrylate
(13c). To a solution of 2-chloro-7-nitroquinoxaline 10a (209 mg, 1
mmol) in dry DMF (6 mL), (E)-ethyl 3-(3-aminophenyl)acrylate (229
mg, 1.2 mmol) was added. The mixture was refluxed gently for 5 h. DMF
was 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. The residue was purified by
flash chromatography on silica gel, eluted with a mixture of EtOAc/
petroleum ether (1:6, v/v), to afford (E)-ethyl 3-(3-(6-nitroquinoxalin-
3-ylamino)phenyl)acrylate 13c (182 mg, 57%) as a yellow solid: mp
183ꢀ193 °C. 1H NMR (400 MHz, DMSO-d6) δ: 1.29 (t, J = 7.2 Hz,
3H), 4.23 (q, 2H), 6.60 (d, J = 16.0 Hz, 1H), 7.46ꢀ7.47 (m, 2H), 7.71
(d, J = 16.0 Hz, 1H), 8.02ꢀ8.03 (m, 1H), 8.06 (d, J = 8.8 Hz, 1H), 8.18
(dd, J1 = 2.4 Hz, J2 = 8.8 Hz, 1H), 8.25 (s, 1H), 8.52 (d, J = 2.8 Hz, 1H),
8.70 (s, 1H), 10.37 (s, 1H); EI-MS m/z 364.1 (Mþ). HRMS (EI) m/z
calcd C19H16N4O4 (Mþ) 364.1172; found, 364.1171.
Amixtureof(E)-ethyl 3-(3-(6-nitroquinoxalin-3-ylamino)phenyl)acrylate
(45 mg, 0.12 mmol) and LiOH (100 mg, 4.2 mmol) in MeOH (3 mL)
was refluxed for 2 h to generate a yellow solution, which was acidified to
pH 5.5 after cooling to room temperature. Then the precipitate was
collected, washed with H2O, and dried to afford 13a (37 mg, 89%) as a
yellow solid: mp >300 °C. 1H NMR (400 MHz, DMSO-d6) δ: 6.52 (d,
J = 16.0 Hz, 1H), 7.41ꢀ7.48 (m, 2H), 7.64 (d, J = 16.0 Hz, 1H),
8.02ꢀ8.06 (m, 2H), 8.17 (dd, J1 = 2.4 Hz, J2 = 9.2 Hz, 1H), 8.23 (s, 1H),
8.51 (d, J = 2.0 Hz, 1H), 8.70 (s, 1H), 10.38 (s, 1H); EI-MS m/z 336.1
(Mþ). HRMS (ESI) m/z calcd C17H12N4O4 (Mþ) 337.0859; found,
337.0931.
5.1.2.39. (E)-3-(3-(6-Nitroquinoxalin-2-ylamino)phenyl)acrylic Acid
(13b). In the same manner as that described for the preparation of 13a,
13b was prepared from 10b and (E)-ethyl 3-(3-aminophenyl)acrylate.
Yield: 98%. mp >300 °C. 1H NMR (400 MHz, DMSO-d6) δ: 6.51 (d, J =
16.0 Hz, 1H), 7.43ꢀ7.49 (m, 2H), 7.65 (d, J = 16.0 Hz, 1H), 8.03ꢀ8.07
(m, 2H), 8.18 (dd, J1 = 2.4 Hz, J2 = 9.2 Hz, 1H), 8.24 (s, 1H), 8.53 (d, J =
2.0 Hz, 1H), 8.72 (s, 1H), 10.42 (s, 1H); EI-MS m/z 336.1 (Mþ). HRMS
(EI) m/z calcd C17H12N4O4 (Mþ) 336.0859; found, 336.0851.
5.1.2.40. N-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-7-nitroquinox-
alin-2-amine (13d). In the same manner as that described for the
preparation of 13c, 13d was prepared from 10a and 3,4-ethylenediox-
yaniline. Yield: 91%. mp 205ꢀ210 °C; 1H NMR (400 MHz, CDCl3) δ:
4.25ꢀ4.33 (m, 4H), 6.93 (d, J = 8.4 Hz, 1H), 7.06 (dd, J1 = 2.0 Hz, J2 =
8.4 Hz, 1H), 7.42 (d, J = 1.2 Hz, 1H), 8.03 (d, J = 9.2 Hz, 1H), 8.22 (dd,
J1 = 2.0 Hz, J2 = 9.2 Hz, 1H), 8.53 (s, 1H), 8.65 (d, J = 2.0 Hz, 1H); EI-
MS m/z 324.1 (Mþ). HRMS (EI) m/z calcd C16H12N4O4 (Mþ)
324.0859; found, 324.0854.
5.1.2.41. 5-(6-Nitroquinoxalin-3-ylamino)-2-chlorobenzoic acid
(13e). To a stirred solution of the 5-amino-2-chlorobenznoic acid
(500 mg) in dry C2H5OH (10 mL), cooled to 0 °C, SOCl2 (0.5 mL)
was added dropwise in 5 min. The resulting suspension was then allowed
to warm to room temperature with stirring for 1 h and then refluxed for 2
h. The mixture was poured onto sat. NaHCO3, the phases separated, and
the organic phase washed subsequently with sat. NaHCO3, H2O, and brine,
followed by drying to yield intermediate ethyl 5-amino-2-chlorobenzoate
(580 mg, 100%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ: 1.39 (t,
J = 7.2 Hz, 3H), 4.40 (q, J1 = 7.2 Hz, J2 = 14.4 Hz, 2H), 6.71 (dd,
J1 =2.8Hz,J2 = 8.4 Hz, 1H), 7.10 (d, J= 2.8 Hz, 1H), 7.19 (d, J= 8.8 Hz, 1H).
To a solution of the above intermediate (100 mg, 0.5 mmol) in dry
DMF (6 mL), 10a (125 mg, 0.6 mmol) was added. The mixture was
refluxed gently for 2 h. DMF was 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.
The residue was purified by flash chromatography on silica gel, eluted
mg, 48%) as a yellow solid; H NMR (400 MHz, CDCl3) δ: 1.35 (t,
J = 7.2 Hz, 3H), 4.06 (q, J1 = 7.2 Hz, J2 = 14.4 Hz, 2H), 7.13 (t, J = 8.0 Hz,
1H), 7.32 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.74 (dd, J1 = 2.8
Hz, J2 = 8.8 Hz, 1H), 7.94 (d, J = 2.4 Hz, 1H), 8.13ꢀ8.17 (m, 1H), 8.30
(d, J = 1.6 Hz, 1H).
A mixture of ethyl 5-(6-nitroquinoxalin-3-ylamino)-2-chlorobenzo-
ate (89 mg) and LiOH (100 mg, 4.2 mmol) in MeOH (3 mL) was
combined at 58 °C for 2 h to generate a yellow solution, which was
acidified to pH 5.5 after cooling to room temperature. Then the
precipitate was collected, washed with H2O, and dried to afford 13e
(22 mg, 27%) as an orange solid: mp 161ꢀ165 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 7.57 (d, J = 8.8 Hz, 1H), 8.10 (d, J = 8.8 Hz, 1H), 8.22 (dd,
J1 = 2.4 Hz, J2 = 8.8 Hz, 1H), 8.31 (dd, J1 = 2.8 Hz, J2 = 8.8 Hz, 1H), 8.41
(d, J = 6.8 Hz, 1H), 8.48 (d, J = 2.4 Hz, 1H), 8.71 (s, 1H); EI-MS m/z
344.1 (Mþ). HRMS (EI) m/z calcd C15H9ClN4O4 (Mþ) 344.0312;
found, 344.0309.
5.1.2.42. N-(3-Ethoxyphenyl)-7-nitroquinoxalin-2-amine (13f). In
the same manner as that described for the preparation of 13c, 13f was
prepared from 10a and 3-ethoxyaniline. Yield: 95%. mp 166ꢀ170 °C.
1H NMR (400 MHz, DMSO-d6) δ: 1.40 (t, J = 7.2 Hz, 3H), 4.08 (q,
2H), 6.70 (d, J = 7.6 Hz, 1H), 7.08ꢀ7.11 (m, 2H), 7.51 (s, 1H), 7.80 (t,
J = 8.0 Hz, 1H), 8.20 (s, 1H), 8.52 (s, 1H), 8.63 (t, J = 8.0 Hz, 1H); EI-
MS m/z 310.1 (Mþ). HRMS (EI) m/z calcd C16H14N4O3 (Mþ)
310.1066; found, 310.1050.
5.1.2.43. 7-Nitro-N-phenylquinoxalin-2-amine (13g). In the same
manner as that described for the preparation of 13c, 13g was prepared
from 10a and aniline. Yield: 68%. mp 230ꢀ233 °C. 1H NMR (400 MHz,
CDCl3) δ: 7.22 (t, J = 7.2 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.76 (d, J =
7.6 Hz, 2H), 8.05 (d, J = 9.2 Hz, 1H), 8.24 (dd, J1 = 2.8 Hz, J2 = 9.2 Hz,
1H), 8.56 (s, 1H), 8.67 (d, J = 2.4 Hz, 1H); EI-MS m/z 266.1 (Mþ).
HRMS (EI) m/z calcd C14H10N4O2 (Mþ) 266.0804; found, 266.0805.
5.1.2.44. 3-(3-(6-Nitroquinoxalin-3-ylamino)phenyl)propanoic
Acid (13h). To a solution of 3-(3-aminophenyl)propanoic acid (0.2 g,
1.2 mmol) in 15 mL of EtOH, 0.5 mL of concentrated H2SO4 was added.
The resulting reaction mixture was refluxed for 2 h. The solvent was
removedunder reducedpressure, and the residuewaspartitionedbetween
AcOEt and sat. NaHCO3. The organic layer was washed with water and
brine, dried over anhydrous Na2SO4, filtered, and condensed. The crude
material could be used in the next step without further purification. 1H
NMR (400 MHz, CDCl3) δ: 1.27 (t, J = 7.2 Hz, 3H), 2.58 (t, J = 7.6 Hz,
2H), 2.84 (t, J = 8.0 Hz, 2H), 4.18 (q, J1 = 7.2 Hz, J2 = 14.4 Hz, 2H), 6.54
(br, 2H), 6.61 (d, J = 7.6 Hz, 1H), 7.07 (t, J = 8.0 Hz, 1H).
To a solution of the above intermediate (100 mg, 0.5 mmol) in dry
DMF (3 mL), 10a (125 mg, 0.6 mmol) was added. The mixture was
refluxed gently for 2 h. DMF was then evaporated under reduced
pressure, and the residue was extracted with EtOAc/H2O 3 times.
The combined organic layer was washed, dried, filtered, and condensed.
The residue was purified by flash chromatography on silica gel, eluted
with a mixture of EtOAc/petroleum ether (1:2, v/v), to afford inter-
mediate ethyl 3-(3-(6-nitroquinoxalin-3-ylamino)phenyl)propanoate
1
(130 mg, 75%) as a yellow solid. H NMR (400 MHz, CDCl3) δ:
1.27 (t, J = 7.2 Hz, 3H), 2.71 (t, J = 7.6 Hz, 2H), 3.03 (t, J = 7.6 Hz, 2H),
4.18 (q, J1 = 7.2 Hz, J2 = 14.0 Hz,2H), 7.06 (d, J = 7.6 Hz, 1H), 7. 37 (t,
J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.67 (dd, J1 = 1.6 Hz, J2 = 8.0 Hz, 1H), 8.04
(d, J = 9.2 Hz, 1H), 8.24 (dd, J1 = 2.4 Hz, J2 = 8.8 Hz, 1H), 8.55 (s, 1H),
8.67 (d, J = 2.8 Hz, 1H).
A mixture of ethyl 3-(3-(6-nitroquinoxalin-3-ylamino)phenyl)-
propanoate (130 mg) and LiOH(100 mg, 4.2 mmol) in MeOH
(3 mL) was combined at 58 °C for 2 h to generate a yellow solution,
which was acidified to pH 5.5 after cooling to room temperature. Then
the precipitate was collected, washed with H2O, and dried to afford 13h
(59 mg, 49%) as an orange solid: mp 239ꢀ245 °C. 1H NMR (400 MHz,
4519
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Journal of Medicinal Chemistry
ARTICLE
DMSO-d6) δ: 2.59 (t, J = 7.6 Hz, 2H), 2.89 (d, J = 7.6 Hz, 2H), 6.97 (d,
J = 7.6 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.81 (s, 1H), 7.88 (d, J = 8.0 Hz,
1H), 8.05 (d, J = 9.2 Hz, 1H), 8.17 (dd, J1 = 2.4 Hz, J2 = 8.8 Hz, 1H), 8.50
(d, J = 2.4 Hz, 1H), 8.70 (s, 1H), 10.26 (s, 1H); EI-MS m/z 338.1 (Mþ).
HRMS (EI) m/z calcd C17H14N4O4 (Mþ) 338.1015; found, 338.1010.
5.1.2.45. (E)-Ethyl 3-(3-(quinoxalin-3-ylamino)phenyl)acrylate
(14c). In the same manner as that described for the preparation of
13c, 14c was prepared from 11a and (E)-ethyl 3-(3-aminophe-
nyl)acrylate. Yield: 90%. mp 110ꢀ120 °C. 1H NMR (400 MHz, CDCl3)
δ: 1.36 (t, J = 7.2 Hz, 3H), 4.30 (q, 2H), 6.48 (d, J = 16.0 Hz, 1H), 7.25
(d, J = 7.6 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.65
(t, J = 7.6 Hz, 1H), 7.71 (d, J = 16.0 Hz, 1H), 7.80ꢀ7.84 (m, 2H), 7.94
(d, J = 8.0 Hz, 1H), 8.02 (s, 1H), 8.45 (s, 1H); EI-MS m/z 319.2 (Mþ).
HRMS (EI) m/z calcd C19H17N3O2 (Mþ) 319.1321; found, 319.1316.
5.1.2.46. (E)-3-(3-(Quinoxalin-3-ylamino)phenyl)acrylic Acid
(14a). In the same manner as that described for the preparation of
13a, 14a was prepared from 14c and LiOH. Yield: 88%. mp
7.33 (d, J = 7.2 Hz, 1H), 7.45ꢀ7.50 (m, 3H), 7.61ꢀ7.74 (m, 3H), 7.78
(br, 1H), 8.10 (d, J = 8.0 Hz, 1H), 8.73 (s, 1H).
A mixture of the above intermediate (55 mg, 0.17 mmol) and LiOH
(60 mg, 1.43 mmol) was combined in 4 mL of THF and H2O mix
solvent (VTHF:VH2O = 3: 1) at 0 °C for 30 h, which was acidified to pH
5.5. Then the precipitate was collected, washed with H2O, and dried to
afford 14e (32 mg, 64%) as a white solid: mp 165ꢀ170 °C. 1H NMR
(400 MHz, DMSO-d6) δ: 6.58 (d, J = 16.0 Hz, 1H), 7.40 (d, J = 8.4 Hz,
1H), 7.54 (t, J = 8.0 Hz, 1H), 7.61 (d, J = 9.6 Hz, 1H), 7.64 (s, 1H),
7.72ꢀ7.75 (m, 4H), 8.09 (d, J = 8.0 Hz, 1H), 8.89 (s, 1H), 12.41 (s, 1H);
EI-MS m/z 292.1 (Mþ). HRMS (EI) m/z calcd C17H12N2O3 (Mþ)
292.0848; found, 292.0850.
5.1.2.50. (E)-2-Methyl-3-(3-(quinoxalin-3-ylamino)phenyl)acrylic
Acid (14f). In the same manner as that described for the preparation of
13a, 14f was prepared from 11a and (E)-ethyl 3-(3-aminophenyl)-2-
methylacrylate (see Scheme 2 in Supporting Information). Yield: 65%.
mp 235ꢀ245 °C. 1H NMR (400 MHz, DMSO-d6) δ: 2.19 (s, 3H), 7.12
(d, J = 8.0 Hz, 1H), 7.42ꢀ7.50 (m, 2H), 7.62ꢀ7.73 (m, 3H), 7.81 (d, J =
8.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 8.37 (s, 1H), 8.57 (s, 1H), 10.04 (s,
1H), 12.51 (s, 1H); EI-MS m/z 305.1 (Mþ). HRMS (EI) m/z calcd
C18H15N3O2 (Mþ) 305.1164; found, 305.1161.
5.1.2.51. (E)-3-(3-(Quinoxalin-3-ylamino)phenyl)prop-2-en-1-ol
(14g). To a solution of (E)-3-(3-aminophenyl)prop-2-en-1-ol (117
mg, 0.71 mmol) (see Scheme 3 in Supporting Information) in dry DMF
(5 mL), 11a (106 mg, 0.71 mmol) and anhydrous K2CO3 (300 mg)
were added. The mixture was refluxed for 3 h under N2. The solution was
poured into water and extracted with EtOAc. The combined organic
phases were washed with 1 N HCl, brine and water, dried, filtered,
and concentrated. The residue was purified by flash chromatography
on silica gel, eluted with a mixture of EtOAc/petroleum ether (1:4, v/v),
to afford 14g (30 mg, 16%) as a white solid: mp 90ꢀ98 °C. 1H NMR
(400 MHz, CDCl3) δ: 5.18 (d, J = 5.6 Hz, 2H), 6.48ꢀ6.55 (m, 1H), 6.77
(d, J = 16.0 Hz, 1H), 6.84ꢀ6.87 (m, 1H), 6.95ꢀ7.05 (m, 2H), 7.19ꢀ
7.24 (m, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H), 7.87 (d,
J = 8.8 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 8.53 (s, 1H); EI-MS m/z
277.1 (Mþ). HRMS (EI) m/z calcd C17H15N3O (Mþ) 277.1215;
found, 277.1216.
5.1.2.52. (E)-3-(3-(Quinolin-2-ylamino)phenyl)acrylic Acid (14h).
In the same manner as that described for the preparation of 13a, 14h was
prepared from 11b and (E)-ethyl 3-(3-aminophenyl)acrylate. Yield:
50%. mp >300 °C. 1H NMR (400 MHz, DMSO-d6) δ: 6.60 (d, J = 16.0
Hz, 1H), 7.34 (d, J = 8.8 Hz, 1H), 7.48ꢀ7.53 (m, 2H), 7.56 (d, J = 8.0
Hz, 1H), 7.62ꢀ7.66 (m, 3H), 7.76 (t, J = 7.6 Hz, 1H), 7.88 (d, J = 7.6 Hz,
1H), 7.94 (d, J = 7.6 Hz, 1H), 8.04 (s, 1H), 8.42 (d, J = 8.8 Hz, 1H),
11.29 (s, 1H); EI-MS m/z 289.1 (Mþ). HRMS (EI) m/z calcd
C18H14N2O2 (Mþ) 290.1055; found, 290.1048.
5.1.2.53. (E)-3-(3-(1H-Benzo[d]imidazol-2-ylamino)phenyl)acrylic
Acid (14i). In the same manner as that described for the preparation of
13a, 14i was prepared from 11c and (E)-ethyl 3-(3-aminophe-
nyl)acrylate. Yield: 48%. mp 271ꢀ278 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 6.46 (d, J = 8.0 Hz, 1H), 6.99ꢀ7.01 (m, 2H), 7.26 (d,
J = 7.6 Hz, 1H), 7.33ꢀ7.39 (m, 3H), 7.57 (d, J = 16.0 Hz, 1H), 7.84 (d,
J = 8.0 Hz, 1H), 7.97 (s, 1H), 9.54 (s, 1H), 11.05 (s, 1H), 12.38 (s, 1H);
EI-MS m/z 279.1 (Mþ). HRMS (EI) m/z calcd C16H13N3O2 (Mþ)
279.1008; found, 279.0999.
5.1.2.54. (E)-3-(3-(Quinazolin-2-ylamino)phenyl)acrylic Acid (14j).
In the same manner as that described for the preparation of 13a, 14j was
prepared from 11d and (E)-ethyl 3-(3-aminophenyl)acrylate. Yield:
58%. mp 225ꢀ230 °C. 1H NMR (400 MHz, DMSO-d6) δ: 6.47 (d, J =
16.0 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.36ꢀ7.43 (m, 2H), 7.56 (d, J =
16 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 8.34 (t, J = 7.2 Hz, 1H), 7.94 (d, J =
8.0 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H), 8.29 (s, 1H), 9.34 (s, 1H), 9.96 (s,
1H); EI-MS m/z 291.1 (Mþ). HRMS (EI) m/z calcd C17H13N3O2
(Mþ) 291.1008; found, 291.1007.
1
273ꢀ275 °C. H NMR (400 MHz, DMSO-d6) δ: 6.50 (d, J = 16.0
Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.50 (t, J = 8.0
Hz, 1H), 7.62 (d, J = 16.0 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.77 (t, J = 8.4
Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 8.30 (s, 1H),
8.59 (s, 1H), 10.08 (s, 1H), 12.44 (s, 1H); EI-MS m/z 291.1 (Mþ).
HRMS (EI) m/z calcd C17H13N3O2 (Mþ) 291.1008; found, 291.1010.
5.1.2.47. N-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)quinoxalin-2-
amine (14b). In the same manner as that described for the preparation
of 13c, 14b was prepared from 11a and 3,4-ethylenedioxyaniline. Yield:
33%. mp 133ꢀ138 °C. 1H NMR (400 MHz, CDCl3) δ: 4.29ꢀ4.30 (m,
4H), 6.89 (d, J = 8.4 Hz, 1H), 7.03 (dd, J1 = 1.6 Hz, J2 = 8.4 Hz, 1H), 7.27
(s, 1H), 7.39 (s, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H),
7.78 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 8.42 (s, 1H); EI-MS m/
z 279.1 (Mþ). HRMS (EI) m/z calcd C16H13N3O2 (Mþ) 279.1008;
found, 279.1006.
5.1.2.48. 3-(3-(Quinoxalin-3-ylamino)phenyl)propanoic Acid
(14d). In the same manner as that described for the preparation of
13h, 14d was prepared from 11a and 3-(3-aminophenyl)propanoic acid.
Yield: 30%. mp 198ꢀ201 °C; 1H NMR (400 MHz, DMSO-d6) δ: 2.59
(t, J = 7.2 Hz, 2H), 2.86 (d, J = 7.2 Hz, 2H), 6.91 (d, J = 6.8 Hz, 1H), 7.28
(t, J = 8.0 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.64 (t, J = 7.2 Hz, 1H), 7.74
(d, J = 8.4 Hz, 1H), 7.78 (s, 1H), 7.84ꢀ7.89 (m, 2H), 8.55 (s, 1H), 9.89
(s, 1H); EI-MS m/z 293.1 (Mþ). HRMS (EI) m/z calcd C17H15N3O2
(Mþ) 293.1164; found, 293.1168.
5.1.2.49. (E)-3-(3-(Qquinoxalin-3-yloxy)phenyl)acrylic Acid (14e).
To a solution of 3-hydroxycinnamic acid (1 g, 6.1 mmol) in 20 mL of
EtOH, 1 mL of concentrated H2SO4 was added. The resulting reaction
mixture was refluxed for 12 h. The solvent was removed under reduced
pressure, and the residue was partitioned between AcOEt and sat.
NaHCO3. The organic layer was washed with water and brine, dried
over anhydrous Na2SO4, filtered, and condensed to afford a white solid.
Yield: 100%. The crude material could be used in the next step without
further purification. 1H NMR (400 MHz, CDCl3) δ: 1.36 (t, J = 7.2 Hz,
3H), 4.29 (q, J1 = 7.2 Hz, J2 = 14.4 Hz, 2H), 6.42 (d, J = 16.0 Hz, 1H),
6.89 (d, J1 = 2.0 Hz, J2 = 8.0 Hz, 1H), 7.03 (s, 1H), 7.12 (d, 1H), 7.28 (t,
J = 7.6 Hz, 1H), 7.66 (br, 1H).
To a solution of the above intermediate (150 mg, 0.91 mmol) in dry
CCl4 (5 mL), 11a (128 mg, 0.78 mmol) and anhydrous K2CO3 (250
mg) was added. The mixture was refluxed for 12 h under N2. The
solution was poured into water and extracted with CH2Cl2. The
combined organic phases were washed with 1 N HCl, brine and water,
dried, filtered, and concentrated. The residue was purified by flash
chromatography on silica gel, eluted with a mixture of EtOAc/petroleum
ether (1:6, v/v), to afford intermediate compound (194 mg, 99%) as a
pale yellow solid, 1H NMR (400 MHz, CDCl3) δ: 1.35 (t, J = 7.2 Hz,
3H), 4.28 (q, J1 = 7.2 Hz, J2 = 14.4 Hz, 2H), 6.47 (d, J = 16.0 Hz, 1H),
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Journal of Medicinal Chemistry
ARTICLE
5.1.2.55. Protein Preparation. E. coli strain BL21 cells containing
pRSETB-RhoA were grown in 20 mL of LB medium containing
ampicillin (100 μg mLꢀ1) at 37 °C overnight and then inoculated into
1 L of LB supplemented with ampicillin (100 μg mLꢀ1). The expression
of RhoA was induced by the addition of 0.5 mM of isopropyl β-D-
thiogalactoside (IPTG). The cells were harvested by centrifugation at
8,000g, 4 °C for 10 min after induction for 6 h at 25 °C. The pellet was
washed, frozen, and then disrupted by sonication against the binding
buffer (20 mM TrisꢀHCl, 0.5 M NaCl, and 10 mM imidazole, pH 8.0).
The lysate was centrifuged at 18,000g for 30 min at 4 °C to pellet the
cellular debris. The supernatant was then added to a HiTrap Ni2þ
chelating column (1 mL) pre-equilibrated with the binding buffer,
followed by washing with 20 mL of washing buffer (20 mM TrisꢀHCl,
0.5 M NaCl, and 60 mM imidazole, pH 8.0). The protein of interest was
eluted with 10 mL of elution buffer (20 mM TrisꢀHCl, 0.5 M NaCl, and
300 mM imidazole, pH 8.0). The freshly prepared protein was dialyzed
against the appropriate buffer as required. The purified RhoA was
concentrated by Amicon Ultra-4 centrifugal filter devices (Millipore)
with the molecular cutoff at 10 kDa. The purity and molecular weights of
RhoA were verified by SDSꢀPAGE.
and 20902022), National S&T Major Project, China (Grant
2009ZX09501-001 and 2011ZX09102-005-02), the Natural Science
Foundation of Jiangsu Province (Grant BK2008172), the 111
Project (Grant B07023), and the Fundamental Research Funds
for the Central Universities.
’ ABBREVIATIONS USED
ROCK, Rho-associated, coiled-coil containing protein kinase;
MLCK, myosin light chain kinase; MLCP, myosin light chain
phosphatase; SPR, surface plasmon resonance;PE, phenylephrine;
PDB, protein data bank; SAR, structureꢀactivity relationship;
DMSO, dimethyl sulfoxide;EDC, N-ethyl-N0-dimethylamino-
propyl carbodiimide; NHS, N-hydroxysuccinimide; DMEM,
Dulbecco’s modified Eagle's medium; FBS, fetal bovine serum;
LPA, oleoyl-L-R-lysophosphatidic acid; RU, response units;
IC50, half maximal inhibitory concentration; DCM, dichloro-
methane; DMF, N,N-dimethylformamide; IPTG, isopropyl β-
D-thiogalactoside
5.1.2.56. Cell Culture. Human brain vascular smooth muscle cells
were maintained at 37 °C in a humidified atmosphere composed of 95%
air/5% CO2, grown in DMEM medium supplemented with 10% FBS,
100 U/mL penicillin, and 100 μg/mL streptomycin. The culture
medium was changed every other day until subculture. All of the
experiments were performed using the same primary cell cultures after
3 to 6 passages being seeded into T-25 culture flasks.
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Supporting Information. General information for
b
chemical agents and analytical measurements, kinetic analysis
of RhoA/inhibitor binding by surface plasmon resonance, the
ranks of the three hit compounds identified from the top 3000
docking candidates, the detailed synthetic procedures and related
spectroscopic data for compound 11d and starting material of
compounds 14f and 14g, and HPLC reports for the purity check
of the compounds 1, 12aꢀv, 13aꢀh, and 14aꢀj. This material is
available free of charge via the Internet at http://pubs.acs.org.
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(12) Rolfe, B. E.; Worth, N. F.; World, C. J.; Campbell, J. H.;
Campbell, G. R. Rho and vascular disease. Atherosclerosis 2005, 183,
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(13) Greenberg, D. L.; Mize, G. J.; Takayama, T. K. Protease-
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Corresponding Author
*E-mail: (L.M.) miaolysuzhou@163.com; (H.L.) hlli@ecust.edu.cn.
(J.L.) Phone: þ86-21-64252584. Fax: þ86-21-64252584. E-mail:
jianli@ecust.edu.cn.
Author Contributions
These authors contributed equally to this work.
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We gratefully acknowledge the financial supports from the
National Natural Science Foundation of China (Grants 90813005
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Journal of Medicinal Chemistry
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