Design, synthesis, and biological evaluation of inhibitors of the NADPH oxidase, Nox4

Article abstract of DOI:10.1016/j.bmc.2017.12.023

NADPH oxidases (Nox enzymes) are critical mediators of both physiologic and pathophysiologic processes. Nox enzymes catalyze NADPH-dependent generation of reactive oxygen species (ROS), including superoxide and hydrogen peroxide. Until recently, Nox4 was proposed to be involved exclusively in normal physiologic functions. Compelling evidence, however, suggests that Nox4 plays a critical role in fibrosis, as well as a host of pathologies and diseases. These considerations led to a search for novel, small molecule inhibitors of this important enzyme. Ultimately, a series of novel tertiary sulfonylureas (23–25) was designed using pharmacophore modeling, synthesized, and evaluated for inhibition of Nox4-dependent signaling.

Full text of DOI:10.1016/j.bmc.2017.12.023

Accepted Manuscript
Design, synthesis, and biological evaluation of inhibitors of the NADPH oxi-
dase, Nox4
Qian Xu, Amol A. Kulkarni, Sajith Meleveetil, Dilbi Hussein, David Brown,
Osman F. Güner, M. Damoder Reddy, E. Blake Watkins, Bernard Lassègue,
Kathy K. Griendling, J. Phillip Bowen
PII:
S0968-0896(17)32106-5
https://doi.org/10.1016/j.bmc.2017.12.023
BMC 14128
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 October 2017
7 December 2017
16 December 2017
Please cite this article as: Xu, Q., Kulkarni, A.A., Meleveetil, S., Hussein, D., Brown, D., Güner, O.F., Reddy, M.D.,
Watkins, E.B., Lassègue, B., Griendling, K.K., Bowen, J.P., Design, synthesis, and biological evaluation of
inhibitors of the NADPH oxidase, Nox4, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/
j.bmc.2017.12.023
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Design, Synthesis, and Biological Evaluation
of Inhibitors of the NADPH Oxidase, Nox4
Leave this area blank for abstract info.
Qian Xu, Amol A. Kulkarni, Sajith Meleveetil, Dilbi Hussein, David Brown, Osman F. Güner, M. Damoder
Reddy, E. Blake Watkins, Bernard Lassègue, Kathy K. Griendling, and J. Phillip Bowen

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Design, synthesis, and biological evaluation of inhibitors of the NADPH oxidase,
Nox4
Qian Xua, Amol A. Kulkarnib,*, Sajith Meleveetilb, Dilbi Husseinb, David Browna, Osman F. Günerc,e, M.
Damoder Reddyd, E. Blake Watkinsd, Bernard Lassèguea, Kathy K. Griendlinga, and J. Phillip Bowenc,*
aDivision of Cardiology, Department of Medicine, Emory University, Atlanta, GA 30322, USA.
bDepartment of Pharmaceutical Sciences, College of Pharmacy, Howard University, Washington, DC 20059, USA.
cDepartment of Pharmaceutical Sciences, College of Pharmacy, Mercer University, Atlanta, GA 30341, USA.
dDepartment of Pharmaceutical Sciences, College of Pharmacy, Union University, Jackson, TN 38305, USA.
eCurrent address: Department of Chemistry and Physics, Santa Rosa Junior College, Santa Rosa, CA 95401, USA.
*Co-corresponding authors.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
NADPH oxidases (Nox enzymes) are critical mediators of both physiologic and
pathophysiologic processes. Nox enzymes catalyze NADPH-dependent generation of reactive
oxygen species (ROS), including superoxide and hydrogen peroxide. Until recently, Nox4 was
proposed to be involved exclusively in normal physiologic functions. Compelling evidence,
however, suggests that Nox4 plays a critical role in fibrosis, as well as a host of pathologies and
diseases. These considerations led to a search for novel, small molecule inhibitors of this
important enzyme. Ultimately, a series of novel tertiary sulfonylureas (23-25) was designed
using pharmacophore modeling, synthesized, and evaluated for inhibition of Nox4-dependent
signaling.
Available online
Keywords:
NADPH oxidases
Nox4
reactive oxygen species (ROS)
pharmacophore development
molecular modeling
2009 Elsevier Ltd. All rights reserved
.
1. Introduction
phenylephrine and pressure overload.18 Compared to wild-type
mice, global Nox4 knockout animals showed attenuated liver
injury, inflammation, and fibrosis after injury.19 Cardiac-specific
Nox4 knockout mice have less apoptosis, hypertrophy, interstitial
fibrosis, and better cardiac function.10b Nox4 has been shown to
have important roles in pulmonary fibrosis as well.14 In addition,
Nox4 in the hypothalamic paraventricular nucleus contributes to
hypertension induced by aldosterone and salt in mice,20 and a
small isoform of Nox4 mediates TLR4-induced apoptosis during
The NADPH oxidase family of enzymes plays a role in a
variety of physiological and pathophysiological responses. It
consists of one single subunit (Nox5) and six multi-subunit
enzymes (Nox1, Nox2, Nox3, Nox4, Duoxl, and Duox2). Of
particular interest, Nox4 is widely distributed in a variety of
tissues including kidney,1 lung,2 liver,3 as well as heart and
vasculature.4 Nox4 influences multiple biological functions by
constitutively generating H2O2.5
renal ischemia/reperfusion injury.21
Other important
pathophysiological roles of Nox4 include promoting the loss of
bone mass in osteoporosis,22 contributing to lung vascular
permeability induced by pseudomonas aeruginosa,23 promoting
glomerular lesions in a mouse model of diabetic nephropathy,24
and mediating fibrosis formation in response to TGF.25
Moreover, Nox4 plays an important role in abnormal
neuropharmacology by contributing to hypoxia-promoted tumor
progression in glioblastoma multiforme,26 as well as increasing
the severity of brain lesions in a model of ischemic stroke.27
Recent work using cultured cells and genetically modified
animals has shed new light on the biological functions of Nox4.
Nox4 favors vasodilation and thus lowers blood pressure,6
enhances capillary angiogenesis in ischemic limbs,7 and inhibits
angiotensin II-induced vascular inflammation and remodeling.8
On the other hand, a plethora of studies suggest that Nox4 also
contributes to disease development, especially in situations
involving ischemia or fibrosis. In fact, an increasing number of
human studies indicate that biosynthesis of Nox4 is upregulated
in various diseases including hypertension,9 cardiac
Thus, small-molecule pharmacologic inhibitors of Nox4 have
great potential for a wide spectrum of diseases. Developing
Nox4 inhibitors has been an active area of research for decades.28
Only a few molecules, however, are currently known to target
Nox4.29 GKT136901, which inhibits both Nox1 and Nox4,
protects against bleomycin-induced pulmonary fibrosis.30
VAS2870 inhibits both Nox2 and Nox4 activity in vascular
hypertrophy,10
atherosclerosis,11
diabetic
nephropathy,12
In
pulmonary hypertension,13 and pulmonary fibrosis.14
streptozotocin-induced diabetic rats, expression of Nox4 is
increased15 and deletion of Nox4 is reno-protective.16 Nox4 also
contributes to the atherosclerotic phenotype in smooth muscle17
and potentially mediates cardiac hypertrophy in response to

endothelial cells without scavenging superoxide.31 It has also
been applied in an ischemia-reperfusion animal model leading to
significantly reduced infarct size, improved neurological outcome
and mortality by intrathecal injection.27 Similarly, fulvene-5 has
been shown to inhibit Nox4 and Nox2 and to exert in vivo
effects.32 Because Nox2 participates in host defense, systemic
inhibition of Nox2 is not ideal. Another potential Nox4 inhibitor
is grindelic acid, which belongs to the diarylheptanoid family. It
is reported to be a Nox4 inhibitor with a low EC50 and no ROS
scavenging ability. Although it does not appear to inhibit Nox2,
its effect on other Nox enzymes and other flavoproteins remains
to be validated.33 Other proposed Nox4 inhibitors include
phenantridinones,34 but additional Nox4 inhibitors are desirable.
Following the biological testing of the selected lead
compounds from these models, we could identify compounds
that reduce ROS concentration, but apparently through
scavenging and not through Nox4 enzyme inhibition. Due to a
lack of evidence of enzyme inhibition, we abandoned further
development of the models based on these compounds.
Strategy II: The second set of models was developed
using existing patent data.39 Three representative compounds are
shown in Fig. 2.
The design of novel Nox4 inhibitors with computational
chemistry is appealing, and our approach has led to the discovery
of three sulfonylureas that have shown promising biological
activity. Our ultimate goal is to identify compounds with
potential therapeutic benefit for ischemic and fibrotic diseases.
2. Results and discussion
Fig. 2. Three representative compounds.39
2.1. Molecular modeling and chemistry
Based on the Nox4 inhibition structure activity
relationships (SAR) reported in the patent literature for the
compounds with a pyrazolo pyridine scaffold,39 the lead
pharmacophore model was developed through the alignment of
selected Nox4 inhibitors and subsequently used to retrieve
compounds from our in-house 3D-structural databases. The three
representative compounds are shown overlaid with the lead
pharmacophore model in Fig. 3.
There are currently no reported X-ray structures of the
NADPH oxidases. Without the advantage of 3D structural data
of the macromolecular target, we pursued a pharmacophore-
based approach35 that was coupled with energy-based
calculations, molecular overlays, and chemical informatics. This
combination in our hands over the years has yielded interesting
compounds with promising biological results.36 The first
iteration of pharmacophore modeling focused on lead
identification. Multiple models were generated using the Phase
module37 of Schrödinger Inc. software38 and scored in search of
novel biologically active chemotypes. The subsequent iterations
concentrated on lead optimization. The biological data obtained
from the leads identified in the first iteration were used to
improve the pharmacophore model which was then used to
identify more potent compounds.
Strategy I: Multiple pharmacophore models were pursued
using a series of compounds proposed by Borbély and co-
workers as represented by 1 in Figure 1.34 Some models utilized
diverse compounds, while others utilized representative
compounds as proposed by Borbély et al. Better quality hits
were obtained from the latter set of models. Some of the
prioritized hits were known antioxidants like robinetin, 2, and
catechin, 3 (Fig. 1).
Fig. 3. Compounds 4-6 overlaid with the pharmacophore model, AADHRR.9.
The six-feature pharmacophore model, AADHRR.9, is the
ninth in a series of models that contain two H-bond acceptors
(red), one H-bond donor (blue), one hydrophobic group (green),
and two aromatic groups (orange). Whereas the model retrieved
many compounds that contain a pyrazolo-pyridine scaffold (Fig.
4, left), we were interested in compounds possessing alternative
scaffolds, as a means to identify active compounds with a lower
molecular weight (Fig. 4, right).
Fig. 4. Sample of compounds retrieved by the lead pharmacophore model:
those that contain the pyrazolo pyridine scaffold (left), and those do not
contain the pyrazolo pyridine scaffold, but fit the pharmacophore model with
a high score (right).
Fig. 1. Two known antioxidants retrieved by the lead pharmacophore model
(top) based on (1): robinetin (2) and catechin (3). The six-feature
pharmacophore model included two hydrogen-bond acceptors (red), two
hydrogen-bond donors (blue) and two aromatic rings (orange).

O
N
a, b
O
14
HO
HO
OH
O
OMe
OH
N
MOMO
13
N
c, d
15
OH
Scheme 2. Synthesis of compounds 14-15. Reagents and conditions: (a) 2-
methylpyridine, LDA, THF, -78 °C, 2 h (52%); (b) 3N HCl (2 equiv.), EtOH,
80 °C, 3 h (81%); (c) 4-methylpyrimidine, LDA, THF, -78 °C, 2 h (42%); (d)
3N HCl (2 equiv.), EtOH, 45 °C, 3 h (52%).
Fig. 5. The commercially available lead compound (7) retrieved by the initial
pharmacophore model is displayed along with the pharmacophore model.
The first lead compound (7), retrieved from
a
commercially available chemical database, is displayed in Fig. 5.
To validate the model, we synthesized and tested a series of
related acetophenone derivatives (Fig. 6).
The final two acetophenone analogues were synthesized
by addition of lithiated 2-methylpyridine to the Weinreb amide
16 or ester 18 to give ketone 17 or resorcinol 19, following
hydrogenolysis under Pd/C conditions (Scheme 3). Biological
evaluation of the acetophenone derivatives (10-12, 14, 15, 17,
and 19) indicated peroxide scavenging activity, which prohibited
further analysis as specific Nox4 inhibitors.
Thus, we
determined to further refine our lead pharmacophore model in
order to eliminate peroxide scavenging capability.
Fig. 6. Acetophenone analogues derived from the lead compound (7) are
shown.
O
O
OMe
a
N
We initially envisioned gaining access to this series of
compounds through the Weinreb amide of suitably substituted
benzoic acids. Unfortunately, steric crowding around the amide
functionality precluded nucleophilic addition of the lithiated 2-
N
32%
MeO
OMe
17
MeO
OMe
O
16
O
and 4-methylheterocycles.
Therefore, we took a less
OMe
OBn
a (40%), b
N
conventional approach via the acid chloride, as all other means
failed. Thus, commercially available acid 8 was converted to the
corresponding acid chloride 9 via treatment with thionyl chloride
at 110°C for 4 hours (Scheme 1). The acid chloride was
subsequently treated with lithiated 2-methylpyridine, 4-
methylpyrimidine, or 3-(methoxymethoxy)-2,6-dimethylpyridine
at -78°C to give the corresponding derivatives 10, 11 and 12,
respectively.
BnO
19
HO
OH
18
Scheme 3. Synthesis of compounds 17 and 19. Reagents and conditions: (a)
2-methylpyridine, n-BuLi, THF, -20 °C, 2 h; (b) 10% Pd/C, EtOH, H2 (1
atm), overnight (48%).
Strategy III: Utilizing the biological assay results
obtained from the leads from Strategy II, we then focused on the
refinement of the lead pharmacophore model (Figs. 4 and 5).
Our general goal was to eliminate the inherent scavenging
behavior through structural modifications of the central region by
reducing conjugation and removal of the phenolic functionality.
By maintaining the aromatic pharmacophore features in the
periphery, we systematically made changes by eliminating the
central features one at a time. The features in the periphery were
maintained because they presumably bind to the Nox4 enzyme.
We envisioned utilizing sulfonylureas, which eliminated the
requirement for the central H-bond acceptor. This resulted in the
final model (Fig. 7). The modified version has the two aromatic
regions connected by a sulfonylurea moiety. Based on this
model, a subset of candidate sulfonylureas (Figs. 7 and 8) were
evaluated in biological assays (in vitro), in an effort to validate
the revised model and to find a novel lead compound for Nox4
inhibition. This model resulted in a series of sulfonylureas (Fig.
8) described below.
O
N
b
c
MeO
OMe
O
10
O
O
N
a
Cl
OMe
OH
OMe
N
MeO
MeO
11
MeO
MeO
OMe
O
9
8
OH
d, e
N
12
OMe
Scheme 1. Synthesis of compounds 10-12. Reagents and conditions: (a)
SOCl2, toluene, 110°C, 4 h; (b) 2-methylpyridine, TEA, THF, LDA, -78 °C, 2
h then rt-overnight (65%, 2 steps); (c) 4-methylpyrimidine, TEA, THF, LDA,
-78 °C, 2 h then rt-overnight (40%, 2 steps); (d) 3-(methoxymethoxy)-2,6-
dimethylpyridine, TEA, THF, LDA, -78 °C, 2 h then rt-overnight (45%, 2
steps); (e) 3N HCl (2 equiv.), EtOH, 80 °C, 2.5 h (80%).
Further acetophenone analogues were synthesized by
addition of lithiated 2-methylpyridine or lithiated 4-
methylpyrimidine to the MOM-protected ester 13 at -78 °C
followed by removal of the protecting group to give resorcinols
14 and 15 (Scheme 2).
Fig. 7. The molecular scaffold of the new sulfonylureas leads (purple) from
the third, modified pharmacophore modeling strategy is superimposed on the
original lead compound (green) and the pharmacophore in Spartan.

Fig. 8. Three sulfonylureas (23-25) were prepared and tested to determine
the validity of the pharmacophore model.
Fig. 10. Sulfonylurea compounds minimally scavenge ROS in vitro. ROS
scavenging by sulfonylurea compound 23, 24, or 25 was assessed in vitro. In
panel A, superoxide was generated with xanthine/xanthine oxidase and
measured using the cytochrome C assay. In panel B, 5 µM hydrogen peroxide
was added exogenously and measured using the Amplex Red assay. ROS
were measured in the presence of indicated concentrations of each compound
Synthesis of sulfonylurea compounds 23-25 is outlined in
Scheme 4. Treatment of 4-trifluoromethyl aniline 20 with 2-
chloroethyl isocyanate or with 3-chloropropyl isocyanate resulted
in the formation of chlorosubstituted acyclic ureas 21 and 22.
Sodium hydride (NaH)-mediated cyclization yielded cyclic urea
derivatives, which were subsequently converted into the desired
sulfonylurea products 23-25 upon reaction with 4-substituted
benzenesulfonyl chlorides.
or DMSO solvent. Bars represent means
SEM of 4 (superoxide) or 3
(hydrogen peroxide) independent experiments. Differences between DMSO
and sulfonylurea compounds are not significant.
To test the biological effectiveness of compounds that did
not scavenge ROS, we measured upregulation of vascular smooth
muscle alpha actin (α-SMA) by TGF-β treatment of human aortic
smooth muscle cells (HASMCs) in culture. This effect of TGF-β
has previously been shown to be Nox4-dependent.43 Cells were
pretreated with a candidate inhibitor 30 minutes before exposure
to TGF-β and harvested after 24 hours. Actin expression was
measured by western blotting with a specific antibody. As shown
in Fig. 11, compounds 23, 24 and 25 were capable of profoundly
inhibiting Nox4-dependent signaling. These results suggest that
sulfonylurea compounds may be direct Nox4 inhibitors.
Scheme 4. Synthesis of sulfonylureas. Reagents and conditions: (a) 2-
chloroethyl isocyanate or 3-chloropropyl isocyanate, CH2Cl2, rt (87-89%); (b)
i. NaH, ii. 4-methoxy benzenesulfonyl chloride or 4-fluoro benzenesulfonyl
chloride (86-88%, 2 steps).
TGF-β is known to significantly upregulate Nox4
expression in smooth muscle cells by 24 hours.43b To ensure that
sulfonylurea compounds did not exert their effects by preventing
Nox4 upregulation, we measured Nox4 mRNA in cells treated as
in Fig. 11, using a qPCR assay described previously.44 In control
cells stimulated with TGF-β alone, Nox4 mRNA was
upregulated, as expected. However, preincubation with
compounds 23, 24 and 25 in the same range of concentrations as
in Fig. 11, did not affect Nox4 expression (results not shown).
Figure 9 summarizes the progression of structures from the initial
lead 7 to the sulfonylureas (23-25) possessing inhibitory activity
against Nox4 but lacking ROS scavenging properties.
Fig. 9. The three generations of potential Nox4 inhibitors beginning with
compound 7 and resulting in the active sulfonylureas (23-25) are displayed.
Each stage of the structural modifications corresponds to the iterative
refinement of the pharmacophore models coupled with elimination of any
molecular features responsible for hydrogen peroxide scavenging.
2.2. Biology
The primary products of the Nox enzymes are ROS. Nox
enzymes directly produce superoxide, which is quickly
dismutated to hydrogen peroxide. Many organic compounds are
able to scavenge superoxide and hydrogen peroxide without
affecting enzyme activity; thus, we first developed in vitro assays
to measure these undesirable non-specific effects. Previous
strategies for ROS detection using non-specific luminescent or
fluorescent probes in cells or in cell-free systems40 tend to result
in false positives. Recently, high-throughput screening
approaches30a, 41 for monitoring H2O2 and superoxide have been
developed that may represent an effective strategy to test
inhibitors of various Nox isoforms.42 Nevertheless, it remains
critical to rule out ROS scavenging and focus on the ability to
suppress Nox enzyme activity.28a
To assess ROS scavenging, we measured the consumption
of superoxide, generated by xanthine/xanthine oxidase, or
exogenous hydrogen peroxide, by increasing concentrations of
the compounds of interest. As shown in Fig. 10, compounds 23,
24, and 25 had no effect on superoxide and hydrogen peroxide in
vitro at concentrations up to 100 µM.
Fig. 11. Sulfonylurea compounds inhibit Nox4-dependent signaling in cells.
Human aortic smooth muscle cells in culture were incubated with indicated
concentrations of compound 23, 24, or 25 and stimulated with 2 ng/ml TGF-β
for 24 hours to induce a Nox4-dependent upregulation of smooth muscle
alpha-actin (α-SMA). Protein expression was measured by Western blotting
and normalized to β-tubulin. In the absence of inhibitor, α-SMA induction by
TGF-β was maximal (100%). All three sulfonylurea compounds reduced α-
SMA expression, allowing determination of each IC50 by non-linear
regression, as indicated. Data points represent means SEM from 3-4
independent experiments.

Finally, to rule out a possible toxic effect of these compounds
in cultured cells, we measured cell viability using a commercial
mammalian cell live/dead assay. Stimulation with TGF-β alone
had no effect on cell viability (99 0.61%, P=NS, n=4). However,
cell viability was slightly reduced by preincubation with the
highest concentration (100 µM) of compound 23 (92 3.09%
viability, P<0.05, n=6), but not 24 (100 µM, 100 0.97%
viability, P=NS, n=6) or 25 (10 µM, 97 2.50% viability, P=NS,
n=6). It is thus extremely unlikely that the slight cytotoxicity of
the highest concentration of compound 23 accounts for the
complete inhibition of α-SMA induction shown in Fig. 11.
4.2.1. 1-(2,4-Dimethoxy-6-methylphenyl)-2-(pyridin-2-yl)ethan-
1-one (10) and 1-(2,4-dimethoxy-6-methylphenyl)-2-(pyrimidin-
4-yl)ethan-1-one (11)
Thionyl chloride (79 mg, 0.663 mmol, 0.048 mL, 1.3 equiv.)
was added dropwise to 2,4-dimethoxy-6-methylbenzoic acid (8,
100 mg, 0.510 mmol) dissolved in anhydrous toluene (5 mL) at rt
in a two-neck, 10 mL RBF equipped with a condenser. The
solution was heated to reflux for 4 h. The solvent was evaporated
under vacuum, and the product (9) was used in the next step
without purification.
The acyl chloride (9) was dissolved in dry THF (3 mL),
cooled to -78 °C, and treated with triethylamine (0.142 mL, 1.02
mmol, 2 equiv.) with stirring. In a separate RBF, dry THF (3
mL) was added followed by diisopropylamine (0.16 mL, 1.121
mmol, 2.2 equiv.). This solution was cooled to -78 °C and
treated with n-BuLi (0.67 mL, 1.070 mmol, 2.1 equiv.) and
stirred for 10 mins. The 2-methylpyridine (104 mg, 0.111 mL,
1.12 mmol, 2.2 equiv.) was added dropwise. After 30 mins, the
solution of the acid chloride was added via syringe to the
lithiated 2-methylpyridine solution. The yellow solution was kept
at -78 °C and allowed to warm slowly to rt overnight. The
solvent was removed under vacuum. Dichloromethane (10 mL)
was added and washed with water (5 mL). The organic layer was
dried over MgSO4 and concentrated. The residue was purified on
silica (1% MeOH:DCM) to give the product.
3. Conclusions
In this study, we report a novel strategy for identifying
and synthesizing Nox4 inhibitors and demonstrate promising
results on three test compounds with respect to their ability to
inhibit Nox4 in a biological system. We took the novel approach
of applying a pharmacophore model to the development of
potential inhibitors. Our synthetic protocol is general, efficient,
and applicable to a wide array of functional groups.
As with other enzyme inhibitors, the ideal Nox4 inhibitor
would be selective, specific, efficacious, and lack toxicity. In this
study, we ruled out ROS scavenging, demonstrated efficacy of
inhibition of Nox4-dependent signaling using an in cellulo assay,
and showed a lack of cytotoxicity. Based on these results, we
identified three potential Nox4 inhibitors with IC50 values of 27
µM, 3.7 µM and 0.5 µM in cell-based assays. Additional work,
however, will be necessary to more fully characterize these
promising compounds. They will need to be tested against other
Nox enzymes to determine selectivity and tested in a direct assay
for Nox4 activity. Selectivity remains challenging due to
limitations in specific assays and the presence of multiple Nox
enzymes in most cell types.
10: Yield 65%, 2 steps, 77:23 mixture of keto:enol tautomers;
1H NMR (400 MHz, CDCl3, major isomer) δ 8.55 (dd, J = 4.0,
0.8 Hz, 1H), 7.65 (dt, J = 8.0, 2.0 Hz, 1H), 7.30 (d, J = 8.0 Hz,
1H), 7.17 (dt, J = 6.0, 0.8 Hz, 1H), 6.30 (s, 2H), 4.33 (s, 2H),
3.83 (s, 3H), 3.82 (s, 3H), 2.17 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 203.0, 161.4, 158.5, 155.4, 149.2, 144.1, 138.6, 136.4,
124.4, 121.7, 118.3, 107.3, 95.9, 55.6, 55.3, 53.8, 20.1; FTIR
(neat): 2934, 2839, 1688, 1594, 1459, 1320, 1199, 1150, 1093,
808, 751, 620 cm-1; MS (ESI) m/z 272 [M+H]+.
4. Experimental Section
11: Yield 40%, 2 steps, 55:45 mixture of keto:enol tautomers;
1H NMR (400 MHz, CDCl3, major isomer) δ 9.15 (bs, 1H), 8.67
(d, J = 4.8 Hz, 1H), 7.35 (d, J = 4.4 Hz, 1H), 6.31 (m, 1H), 4.29
(s, 2H), 3.82 (s, 6H), 2.24 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 200.9, 164.2, 161.8, 158.8, 156.6, 154.3, 139.3, 121.9, 119.7,
107.6, 95.9, 55.6, 55.4, 53.1, 20.3; FTIR (neat): 2997, 2921,
2842, 1629, 1577, 1466, 1316, 1200, 1000, 884, 744, 649 cm-1;
MS (ESI) m/z 273 [M+H]+.
4.1. General
Glassware was dried in an oven (120 °C), heated under
reduced pressure, and cooled under argon before use. Unless
otherwise noted, materials obtained from commercial suppliers
were used without further purification. Reactions were monitored
by thin-layer chromatography on Analtech silica gel plates using
UV-light and ceric sulfate or β-naphthol for visualization.
Column chromatography was performed on silica gel (230–400
mesh) using hexanes and ethyl acetate as eluent. Evaporation of
solvents was conducted under reduced pressure at 50 °C. FTIR
spectra were recorded neat on a Perkin-Elmer Spectrum 65.
NMR spectra were recorded on a Bruker Avance III 400 NMR
spectrometer at 400 MHz (1H) and 100 MHz (13C), respectively.
Deuterated chloroform was used as the solvent, unless otherwise
noted, and spectra were calibrated against the residual solvent
peak (7.24 ppm for 1H and 77.0 ppm for 13C). Chemical shifts (δ)
and coupling constants (J) are given in ppm (parts per million)
and Hz (Hertz), respectively. The following abbreviations were
used to explain multiplicities: s = singlet, d = doublet, t = triplet,
q = quartet, m = multiplet, bs = broad singlet. Low resolution ESI
mass spectra were obtained on a Waters Acquity UPLC H-Class
with PDA and SQ mass detectors using a Waters BEH C18 1.7
µm column (2.1x50mm). High resolution mass spectra were
obtained on VG 70–70H or LC/MSD trapSL spectrometer
operating at 70 eV using a direct inlet system.
4.2.2.
1-(2,4-Dimethoxy-6-methylphenyl)-2-(5-hydroxy-6-
methylpyridin-2-yl)ethan-1-one (12)
To a stirred solution of the 2,6-dimethylpyridin-3-ol (500 mg,
4.06 mmol) in DMF (15 mL) in a 50 mL flame-dried RBF,
K2CO3 (0.617 g, 4.47 mmol) and MOM-Cl (0.37 mL, 4.87
mmol) were added successively. The solution was stirred at RT
overnight. Water (30 mL) was added and the reaction mixture
extracted with EtOAc (2 x 20 mL). The organic layer was
washed with cold water (2 x 30 mL) and brine (30 mL) and dried
over magnesium sulfate and concentrated under reduced
pressure.
Thionyl chloride (79 mg, 0.663 mmol, 0.048 mL, 1.3 equiv.)
was added dropwise to 2,4-dimethoxy-6-methylbenzoic acid (8,
100 mg, 0.510 mmol) dissolved in anhydrous toluene (5 mL) at rt
in a two-neck, 10 mL RBF equipped with a condenser. The
solution was heated to reflux for 4 h. The solvent was evaporated
under vacuum, and the product (9) was used in the next step
without purification.
4.2. Synthetic Procedures
The acyl chloride (9) was dissolved in dry THF (3 mL),
cooled to -78 °C, and treated with triethylamine (0.142 mL, 1.09

mmol, 2 equiv.) with stirring. In a separate RBF, dry THF (3
mL) was added followed by DIA (0.16 mL, 1.12 mmol, 2.2
equiv.). This solution was cooled to -78 °C and treated with n-
BuLi (0.73 mL, 1.121 mmol, 2.2 equiv.) and stirred for 10 mins.
The MOM-protected pyridine (179 mg, 1.07 mmol, 2.1 equiv.)
was added dropwise. After 30 mins, the acid chloride solution
was added dropwise. The yellow solution was kept at -78 °C and
allowed to warm slowly to rt overnight. The solvent was
removed under vacuum, and dichloromethane (10 mL) was added
and washed with water (10 mL). The organic layer was dried
over MgSO4 and concentrated. The residue was purified on silica
(2% MeOH: DCM) to give the MOM ether as a yellow solid.
The residue was purified on silica (2% MeOH: DCM) to give the
MOM-ether as a yellow solid.
A solution of the MOM-ether (60 mg, 0.209 mmol) and 3M
HCl (0.139 mL, 0.418 mmol) in EtOH (4 mL) was stirred at 80
οC. After the reaction was completed (monitored by TLC), the
solvent was removed under reduced pressure. The residue was
taken up in water (1 mL) and the pH adjusted to 6 using sodium
bicarbonate solution (1M), extracted with dichloromethane (3 x 5
mL), dried and concentrated. Further purification was achieved
on a flash column with 10 to 30% (1:3 ethanol in ethyl
acetate)/hexane.
1
14: Yield 81%; 80:20 mixture of keto-enol tautomers; H
A solution of the MOM ether (50 mg, 0.145 mmol), 3M HCl
NMR (400 MHz, DMSO-d6, major isomer) δ 10.16 (bs, 1H),
9.65 (s, 1H), 8.45 (d, J = 4.4 Hz, 1H), 7.71 (dt, J = 7.6, 2.0 Hz,
1H), 7.26-7.21 (m, 2H), 6.18 (d, J = 2.0 Hz, 1H), 6.06 (d, J = 2.0
Hz, 1H), 4.28 (s, 2H), 2.01 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 202.8, 159.8, 157.7, 156.5, 149.3, 138.9, 136.7, 124.7,
122.1, 120.2, 109.6, 100.6, 53.5, 20.5; FTIR (neat): 2923, 2763,
1592, 1570, 1442, 1375, 1214, 1189, 973, 844, 723, 622 cm-1;
MS (ESI) m/z 244 [M+H]+.
ο
(0.097 mL, 0.290 mmol) in EtOH (2 mL) was stirred at 80 C.
After the reaction was completed (monitored by TLC), the
organic solvent was directly removed under reduced pressure.
The residue was taken up in water (1 mL) and the pH adjusted to
6 using sodium bicarbonate solution (1M). The product was
extracted with dichloromethane (3 x 5 mL) and dried and
concentrated. Further purification was achieved on silica with
40% (3:1 EtOH in ethyl acetate)/hexane.
1
15: Yield 52%; 66:24 mixture of keto-enol tautomers; H
1
12: Yield 80%; 63:27 mixture of keto-enol tautomers; H
NMR (400 MHz, DMSO-d6, major isomer) δ 10.2 (s, 1H), 9.74
(s, 1H), 9.07 (d, J = 1.6 Hz, 1H), 8.71 (d, J = 5.2 Hz, 1H), 7.43
(dd, J = 4.8, 1.2 Hz, 1H), 6.20 (d, J = 2.0 Hz, 1H), 6.10 (d, J =
2.0 Hz, 1H), 4.32 (s, 2H), 2.08 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 201.0, 165.1, 160.2, 158.6, 158.2, 157.1, 139.4,
122.7, 119.6, 109.8, 100.6, 52.9, 20.8; FTIR (neat): 3082, 2923,
1605, 1548, 1434, 1350, 1248, 1159, 1051, 982, 820, 690 cm-1;
MS (ESI) m/z 245 [M+H]+.
NMR (400 MHz, DMSO-d6, major isomer) δ 9.54 (s, 1H), 7.00
(d, J = 8 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H), 6.44 (d, J = 2.0 Hz,
1H), 6.37 (d, J = 2.0 Hz, 1H), 4.10 (s, 2H), 3.80 (s, 3H), 3.75 (s,
3H), 2.29 (s, 3H), 2.16 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ 203.1, 161.1, 158.3, 149.7, 147.2, 143.3, 138.0, 123.8, 122.4,
122.3, 107.9, 96.3, 56.2, 55.6, 48.9, 23.3, 19.8; FTIR (neat):
2933, 2836, 1575, 1457, 1359, 1281, 1197, 1045, 949, 806, 618
cm-1; MS (ESI) m/z 302 [M+H]+.
4.2.4. 1-(2,4-Dimethoxyphenyl)-2-(pyridin-2-yl)ethan-1-one (17)
4.2.3. 1-(2,4-Dihydroxy-6-methylphenyl)-2-(pyridin-2-yl)ethan-1-
one (14) and 1-(2,4-dihydroxy-6-methylphenyl)-2-(pyrimidin-4-
yl)ethan-1-one (15)
In an oven dried 100 mL round-bottom flask was placed 2,4-
dimethoxybenzoic acid (1.0 g, 5.49 mmol) and anhydrous
dichloromethane (45 mL). The solution was stirred at room
temperature and treated with 1-methylpiperidine (3.35 mL, 27
mmol). After stirring for 10 minutes the solution was cooled to 0
°C and pivaloyl chloride (0.79 g, 0.81 mL, 6.59 mmol) was
added via syringe dropwise. The solution was stirred for 2 hours.
N,O-Dimethyl hydroxylamine hydrochloride (0.803 g, 8.23
mmol) was added, and the mixture was stirred at room
temperature for 24 h. The yellow solution was poured into HCl
(80 mL, 1M). The organic layer was sequentially washed with
saturated NaHCO3 and brine (80 mL) and dried over MgSO4.
After concentration, the yellow oil was dried under vacuum to
afford the amide (16) as a white solid.
To an ice-cooled solution of ethyl 2,4-dihydroxy-6-
methylbenzoate (300 mg, 1.529 mmol) in dichloromethane (10
mL), DIPEA (0.401 mL, 296 mg, 2.29 mmol) and MOM-Cl
(0.116 mL, 123 mg, 1.59 mmol) were added successively. The
solution was stirred at rt for 4 h. Saturated aqueous NH4Cl (15
mL) was added, and the reaction mixture was extracted with
dichloromethane (3 x 15 mL). The organic layer was washed
with water and brine and dried over magnesium sulfate and
concentrated under reduced pressure. The off-white, solid residue
was purified by flash chromatography to afford the MOM-ether
(13) as a viscous liquid (silica, 40% EtOAc: hexanes).
13: Yield 80%; 1H NMR (400 MHz, CDCl3) δ 11.76 (s, 1H),
6.49 (d, J = 2.4 Hz, 1H), 6.38 (dd, J = 2.4, 0.8 Hz, 1H), 5.17 (s,
2H), 4.41 (q, J = 7.2 Hz, 2H), 3.46 (s, 3H), 2.52 (s, 3H), 1.41 (t, J
= 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 171.6, 165.2,
161.3, 143.3, 111.8, 106.4, 101.5, 93.8, 61.32, 56.3, 24.5, 14.2;
FTIR (neat): 2985, 2938, 2828, 1613, 1575, 1398, 1263, 1217,
1022, 926, 866 cm-1.
16: Yield 80%; 1H NMR (400 MHz, CDCl3) δ 7.19 (d, J = 8.4
Hz, 1H), 6.47-6.43 (m, 2H), 3.78 (s, 6H), 3.55 (bs, 3H), 3.23 (bs,
3H); 13C NMR (100 MHz, CDCl3) δ 161.8, 157.3, 129.0, 117.7,
104.3, 98.6, 60.9, 55.7, 55.4; FTIR (neat): 2964, 2935, 1642,
1606, 1513, 1311, 1289, 1118, 988, 938 cm-1.
Dry THF (2 mL) and 2-methylpyridine (41 mg, 0.44 mmol)
were added to a 10 mL round-bottom flask and cooled to -78 οC.
To this, n-BuLi (277 µL, 0.444 mmol) was added dropwise. A
two-neck, round-bottom flask was charged with dry THF (2 mL)
and N-2,4-trimethoxy-N-methylbenzamide (16, 100 mg, 0.444
In a RBF, dry THF (3 mL) was added followed by DIA (0.158
mL, 0.397 mL, 2.78 mmol, 4.2 equiv.). This solution was cooled
to -78 °C and treated with n-BuLi (1.669 mL, 2.72 mmol, 4.1
equiv., 1.6 molar in hexane) and stirred for 10 mins. To this
solution, 2-methylpyridine (0.262 mL, 247 mg, 2.65 mmol, 4
equiv.) was added dropwise. After 30 mins, a solution of methyl
2-hydroxy-4-(methoxymethoxy)-6-methylbenzoate (13, 150 mg,
0.663 mmol) was added dropwise to the pyridine solution. The
yellow solution was kept at -78 °C and allowed to warm to rt
over 2 h. The solvent was removed under vacuum, and
dichloromethane (10 mL) was added and washed with water (5
mL). The organic layer was dried over MgSO4 and concentrated.
ο
mmol) was added. The solution was cooled to - 78 C with
stirring. After 30 min, the lithiated 2-methylpyridine solution was
added dropwise over 10 min via syringe pump. The reaction was
stirred at -78 οC for 2 h and warmed to 0 οC. Water was added to
the reaction, and the product was extracted with EtOAc (2 x 5
mL). The combined organic layers were washed with brine, dried
and concentrated. The residue was purified by flash

chromatography by eluting with 10 to 20% EtOAc in hexanes to
afford the ketone (17) in 32% yield.
isocyanate (10 mmol, 1.0 equiv.) was added, and the reaction was
stirred at rt for 72 h. The white precipitate was filtered, washed
with CH2Cl2 (10 mL), and dried under high vacuum. The
compound was used in subsequent steps without any further
purification.
1
17: Yield 32%; 88:12 mixture of keto: enol tautomers; H
NMR (400 MHz, CDCl3, major isomer) δ 8.48 (dd, J = 4.8 Hz,
0.8 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.56 (dt, J = 7.6 Hz, 2.0
Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H), 7.08 (ddd, J = 7.6, 4.8 Hz, 0.8
Hz, 1H), 6.46 (dd, J = 8.8, 2.4 Hz, 1H), 6.36 (d, J = 2.4 Hz, 1H),
4.43 (s, 2H), 3.83 (s, 3H), 3.78 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 196.4, 164.7, 161.0, 156.5, 149.3, 136.2, 133.2, 124.3,
121.5, 120.7, 105.3, 98.3, 55.6, 55.5, 52.8; FTIR (neat): 3006,
2940, 2838, 1661, 1592, 1502, 1435, 1258, 1110, 1024, 824, 750,
639 cm-1; MS (ESI) m/z 258 [M+H]+.
21: Compound 21 was synthesized as described in the general
procedure using 3-chloropropyl isocyanate (1.2 g, 10 mmol, 1.0
equiv). The desired product was isolated as a white solid (2.51 g,
89 % yield). The crude solid was used without further
1
purification. H NMR (400 MHz, DMSO-d6): δ 8.92 (s, 1H),
7.60-7.53 (m, 4H), 6.44-6.42 (t, J = 4.0 Hz, 1H), 3.68-3.65 (t, J1 =
8.0, 4.0 Hz, 2H), 3.25-3.20 (m, 2H), 1.93-1.86 (m, 2 H). 13C
NMR (100 MHz, DMSO-d6): 155.4, 144.7, 126.5, 126.3, 123.8,
121.6, 121.3, 117.7, 43.5, 37.1, 33.0. FTIR (neat): 3321, 2969,
1695, 1637, 1596, 1557, 1522, 1409, 1310, 1230, 1180, 1156,
1062, 1013 cm-1.
4.2.5. 1-(2,4-Dihydroxyphenyl)-2-(pyridin-2-yl)ethan-1-one (19)
Methyl 2,4-dihydroxybenzoate (3 g, 17.83 mmol),
benzylbromide (6.71 g, 4.67 mL, 39.3 mmol), and potassium
carbonate (5.4 g, 39.3 mmol) were added to acetonitrile (120 mL)
and were stirred under argon at 60 °C for 48 hours. After 24
hours an additional 0.5 equiv. BnBr and 1 equiv. K2CO3 were
added. The reaction mixture was filtered through Celite, and the
solvent removed under vacuum. The resulting oil was purified
by flash chromatography (silica: 10% EtOAc: hexanes) to give
the product (18) as a white solid (4.8 g, 77%).
18: Yield 77%; 1H NMR (400 MHz, CDCl3) δ 7.81 (d, J = 8.4
Hz, 1H), 7.43-7.41 (m, 2H), 7.33-7.23 (m, 8H), 6.53-6.49 (m,
2H), 5.06 (s, 2H), 4.99 (s, 2H), 3.79 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 136.6, 136.1, 133.9, 128.7, 128.5, 128.2, 127.7,
127.5, 126.7, 113.2, 113.1, 106.1, 106.0, 101.5, 101.4, 70.5, 70.2,
51.7; FTIR (neat): 3066, 3035, 2835, 1724, 1604, 1505, 1378,
1188, 1129, 1008, 811 cm-1.
22: Compound 22 was synthesized as described in the general
procedure using 2-chloroethyl isocyanate (1.05 g, 10 mmol, 1.0
equiv). The desired product was isolated as a white solid (2.32 g,
87 % yield). The crude solid was used without further
1
purification. H NMR (400 MHz, DMSO-d6): δ 9.11 (s, 1H),
7.61-7.55 (m, 4H), 6.56 (t, 1H), 3.68-3.65 (t, J = 8.0 Hz, 2H),
3.46-3.41 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 155.2,
144.5, 126.4, 126.4, 123.7, 121.8, 121.5, 117.8, 44.7, 41.7. FTIR
(neat): 3379, 2970, 2929, 1737, 1650, 1598, 1562, 1408, 1323,
1243, 1157, 1064, 1013 cm-1.
4.2.7.
phenyl)-tetrahydropyrimidin-2(1H)-one
Fluorophenyl)sulfonyl)-3-(4-(trifluoromethyl)phenyl)-
tetrahydropyrimidin-2(1H)-one (24), and
1-((4-Methoxyphenyl)sulfonyl)-3-(4-(trifluoromethyl)
(23),
1-((4-
1-((4-
Methoxyphenyl)sulfonyl)-3-(4-(trifluoromethyl)phenyl)-
imidazolidin-2-one (25)
Dry THF (9 mL) and 2-methylpyridine (167 mg, 0.178 mL,
1.79 mmol) were added to a 25 mL round-bottom flask and
cooled to -78 °C. To this, n-BuLi (1.12 mL, 1.79 mmol) was
added dropwise. A 50 mL, two-neck round-bottom flask was
charged with dry THF (9 mL) and methyl 2,4-
bis(benzyloxy)benzoate (18, 250 mg, 0.78 mmol) was added and
cooled to -78 °C with stirring. After 30 mins, the lithiated 2-
methylpyridine solution was added dropwise over 2 min. The
reaction was stirred at -78 °C for 10 min and water was added.
After warming to rt, the product was extracted with EtOAc and
the aqueous layer was back-extracted with EtOAc (2 x 10 mL).
The combined organic layers were dried and concentrated. The
bisbenzyl product was purified by flash chromatography (40%).
A round bottom flask (10 mL) equipped with a magnetic stir
bar was charged with a solution of the acyclic urea (0.25 mmol,
1.0 equiv.) in THF (2 mL). The solution was cooled to 0 °C for
15 mins. NaH (30 mg, 0.75 mmol, 3.0 equiv., 60% suspension in
oil) was slowly added. The reaction was stirred for 15 mins at 0
°C and allowed to warm to rt with stirring for 12 h. The
appropriate arylsulfonyl chloride (0.28 mmol, 1.1 equiv.) was
added, and the reaction was stirred at rt for an additional 4 h. The
reaction mixture was poured into a separatory funnel containing
ethyl acetate and 1 M HCl (10 mL each). The layers were
separated. The organic layer was washed with distilled H2O (2 x
10 mL) and dried over anhydrous Na2SO4. The solvent was
removed under vacuum. The crude product was purified using
silica gel flash column chromatography. Gradient elution from
20-40% ethyl acetate in hexanes furnished the purified products
as white crystalline solids.
The bisbenzyl ketone (120 mg, 0.293 mmol) was dissolved in
EtOH and 10% Pd/C (15.5 mg,) was added. The mixture was
stirred at rt under 1 atmosphere of H2 overnight. The reaction
mixture was filtered and purified by column chromatography by
eluting with 1-2% MeOH in dichloromethane to give the product
as a yellow solid (48%).
23: Compound 23 was synthesized as described in the general
procedure using acyclic urea 21 (70 mg, 0.25 mmol, 1.0 equiv.)
and 4-methoxybenzenesulfonyl chloride (58 mg, 0.28 mmol, 1.1
equiv). The desired cyclic sulfonylurea 23 was isolated as a white
crystalline solid (88 mg, 85% overall yield). 1H NMR (400 MHz,
CDCl3) δ 8.02–7.93 (m, 2H), 7.56 (t, J = 12.6 Hz, 2H), 7.36 (d, J
= 8.3 Hz, 2H), 6.99–6.93 (m, 2H), 4.14–4.04 (m, 2H), 3.86 (d, J
= 3.4 Hz, 3H), 3.70 (dd, J = 13.0, 7.0 Hz, 2H), 2.29–2.21 (m,
2H). 13C NMR (100 MHz, CDCl3): δ 163.6, 151.0, 145.4, 145.3,
131.2, 130.9, 126.2, 125.1, 122.3, 113.9, 55.7, 49.1, 45.4, 22.9.
FTIR (neat): 2924, 2855, 1739, 1667, 1595, 1578, 1519, 1497,
1476, 1422, 1324, 1283, 1265, 1175, 1107, 1092, 1065, 1019 cm-
1; HRESIMS m/z 415.0936 (M+H)+ (calcd for C18H18F3N2O4S,
415.0939).
1
19: Yield 48%; H NMR (400 MHz, DMSO-d6,) δ 12.4 (s,
1H), 10.14 (bs, 1H), 8.46 (m, 1H), 7.71 (dd, J = 9.2, 2.4 Hz, 1H),
7.64-7.59 (m, 1H), 7.25 (d, J = 7.6 Hz, 1H), 7.15-7.12 (m, 1H),
6.32-6.30 (m, 1H), 6.24-6.23 (m, 1H), 4.33 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) δ 200.8, 165.5, 165.4, 155.32, 149.5,
136.7, 133.2, 124.2, 122.0, 112.6, 108.6, 103.1, 47.7; FTIR
(neat): 2924, 2870, 2755, 1613, 1587, 1415, 1344, 1227, 1124,
945, 842, 761 cm-1; MS (ESI) m/z 230 [M+H]+.
4.2.6. 1-(2-Chloroethyl)-3-(4-(trifluoromethyl)phenyl)urea (21)
and 1-(3-Chloropropyl)-3-(4-(trifluoromethyl)phenyl)urea (22)
A 50 mL round-bottom flask was charged with a solution of 4-
trifluoromethyl aniline (20, 1.61 g, 10 mmol, 1.0 equiv.) in
CH2Cl2 (20 mL). 2-Chloroethyl isocyanate or 3-chloropropyl

24: Compound 24 was synthesized as described in the general
procedure using acyclic urea 22 (70 mg, 0.25 mmole, 1.0 equiv.)
and 4-fluorobenzenesulfonyl chloride (54 mg, 0.28 mmol, 1.1
equiv.). The desired cyclic sulfonylurea 24 was isolated as a
white crystalline solid (91 mg, 90 % overall yield). 1H NMR (400
MHz, CDCl3) δ 8.04 (ddd, J = 8.1, 5.1, 2.5 Hz, 2H), 7.58 (d, J =
8.5 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 7.20–7.12 (m, 2H), 4.15–
4.06 (m, 2H), 3.72 (dd, J = 11.6, 5.8 Hz, 2H), 2.32–2.20 (m, 2H).
13C NMR (100 MHz, CDCl3): δ 166.8, 164.3, 150.8, 145.0,
135.6, 131.6, 128.8, 126.2, 125.1, 122.4, 116.1, 49.0, 45.5, 22.8.
FTIR (neat): 2922, 2853, 1668, 1614, 1591, 1518, 1491, 1476,
1426, 1412, 1328, 1287, 1239, 1206, 1225, 1123, 1175, 1155,
1086, 1035, 1011 cm-1; HRESIMS m/z 403.0735 (M+H)+ (calcd
for C17H15F4N2O3S, 403.0740).
4.3.4 Western blotting
Potential Nox4 inhibitory activity of compounds under study
was measured with an indirect assay, based on the upregulation
of vascular smooth muscle alpha actin by TGF-β in cultured
HASMs. This effect of TGF-β was previously shown to be Nox4-
dependent.43b Cells were grown to 80% confluence and incubated
with low serum (0.1% fetal bovine serum) for 24h. Cells were
incubated with various concentrations (1–100 µM) of candidate
inhibitors for 30 min, before addition of TGF-β (2 ng/ml). Cells
exposed to TGF-β alone were used as positive controls. After 24
hours, cells were lysed in 1% triton buffer with protease
inhibitors. Protein lysates were separated using SDS-PAGE and
transferred to PVDF membranes. Blots were blocked, incubated
with mouse monoclonal primary antibodies against smooth
muscle alpha actin (A2547, Sigma) and beta tubulin (T4026,
Sigma), which served as a loading control. Detection was
performed using a horse radish peroxidase conjugated secondary
antibody (NA931, GE Healthcare Life Sciences) and enhanced
chemiluminescence.
25: Compound 25 was synthesized as described in the general
procedure from acyclic urea 22 (67 mg, 0.25 mmol, 1.0 equiv.)
and 4-methoxybenzenesulfonyl chloride (58.0 mg, 0.28 mmol,
1.1 equiv.). The desired cyclic sulfonylurea 25 was isolated as a
white crystalline solid (80 mg, 80 % overall yield). 1H NMR (400
MHz, CDCl3): δ 8.072 (d, J = 2.00, 8.00 Hz, 2H), 7.60 (m, 4H),
7.05-7.02 (m, 2H), 4.08-4.04 (m, 2H), 3.82 (m, 5H). 13C NMR
(100 MHz, CDCl3): δ 182.8, 164.2, 151.7, 141.5, 130.7, 129.1,
126.2, 122.6, 117.9, 114.3, 55.7, 42.1, 41.0, 30.9. FTIR (neat):
2908, 2849, 1719, 1617, 1593, 1576, 1524, 1496, 1467, 1397,
1358, 1313, 1296, 1260, 1178, 1153, 1089, 1069, 1022 cm-1;
HRESIMS m/z 423.0600 (M+Na)+ (calcd for C17H15F3N2O4SNa,
423.0602).
4.3.5 Reverse transcription and quantitative PCR
Total RNA samples (2 µg), purified from cultured MASMs,
using the RNeasy Plus kit (Qiagen, Chatsworth, CA), were used
to prepare cDNA. Reactions were carried out at 42 °C, using
random 15-mer primers and ProtoScript II reverse transcriptase
(New England Biolabs, Beverly, MA). Following enzyme
inactivation at 65 °C, cDNA samples were purified using the
QIAquick kit (Qiagen).
4.3 Biological Evaluation
Quantitative PCR was performed with
a LightCycler
4.3.1 Superoxide scavenging assay
instrument in glass capillaries (Roche Applied Science,
Indianapolis, IN), using Platinum Taq DNA polymerase and
SYBR green (Invitrogen, Carlsbad, CA). Nox4 was measured
using primers 5’- CTGGTCTGACGGGTGTC-TGCATGGTG-3’
and 5’-CTCCGCACAATAAAGGCACAAAGGTCCAG-3’ in
the presence of 4 mM MgCl2, and annealing at 65 °C. The
housekeeping 18S rRNA was used for normalization and
We modified an assay based on the measurement of
superoxide-dependent changes in cytochrome C absorbance at
550 nm.45 Briefly, superoxide was generated in vitro in phosphate
buffered saline solution, using hypoxanthine (100 µM)/xanthine
oxidase (6x103 U/mL) in the presence of catalase (200 U/mL) to
eliminate H2O2. Compounds under study were added at various
concentrations (1–100 µM) and remaining superoxide was
measured with cytochrome C (46 µM). Superoxide dismutase
(575 U/mL) was used as a positive control in separate samples
measured at the same time. Following a 1 min stabilization
period, absorbance was measured at 550 nm in a microplate
reader (Biotek), using a kinetic program (1 read/min) for 15 min.
The linear slope representing the rate of superoxide production
was used to calculate the percentage of superoxide scavenging.
measured
using
primers
5’-
GAATTGACGGAAGGGCACCACCAG-3’and 5’-GTGCAGC-
CCCGGACATCTAAGG-3’ in the presence of 3 mM MgCl2
with annealing at 60 °C. Data analysis was performed using the
mak3i module of the qpcR software in the R environment, as
described previously.44
4.3.6 Cell viability assay
HASMs were incubated with the compounds under study and
TGF-β for 24 hours, as described above, before measuring
viability using the Live/Dead Viability/Cytotoxicity kit
(Invitrogen), by fluorescence microscopy. This assay relies on
the uptake of calcein/AM (2 µM), hydrolyzed to green
fluorescent calcein in live cells, and labeling of nuclear DNA
with red fluorescent ethidium homodimer-1 (4 µM), due to loss
of plasma membrane integrity in dead cells. Cells were incubated
with both vital dyes for 10 min at room temperature before
measuring fluorescence at 485 10 nm and 530 12.5 nm for
calcein and ethidium, respectively. Untreated cells were used as
negative controls and cells incubated for 30 min with 70%
ethanol were used as positive controls for cytotoxicity.
4.3.2 Hydrogen peroxide scavenging assay
Hydrogen peroxide was measured in vitro, using an Amplex
Red assay kit (Invitrogen), according to the manufacturer’s
instructions. This assay measures the oxidation of Amplex Red
(10-acetyl-3,7-dihydroxyphenoxazine) to fluorescent resorufin in
the presence of horse radish peroxidase. Briefly, H2O2 (5 mM)
was added to various concentrations (1–100 µM) of the
compounds under study in reaction buffer. After an incubation of
5 min, Amplex Red was added to measure remaining H2O2.
Reactions were incubated for 30 min at room temperature,
protected from light. Resorufin fluorescence was measured in a
microplate reader (Biotek) using excitation at 530 nm and
emission at 590 nm.
4.3.7 Statistics
4.3.3 Cell culture
Prism software, version 7 (GraphPad) was used to perform
calculations. Reactive oxygen species scavenging data were
analyzed using one-way ANOVA, followed by Dunnett’s
multiple comparisons test. Inhibition of smooth muscle alpha
actin expression was analyzed using non-linear regression.
Human aortic smooth muscle cells (HASMs) were purchased
from Invitrogen, grown as recommended by the supplier and
used before passage 10 for experiments. Mouse aortic smooth
muscle cells (MASMs) were isolated from C57BL/6J mice and
grown as described previously46 for less than 10 passages.

4.4 Computational
the search retrieved more than 1,000 hits, the search was set to
stop. The search was then resubmitted with a smaller IDMT until
a hitlist of less than 1,000 was achieved. Follow-up searches
were pursued with reduced IDMT until only a handful of
compounds were retrieved. All searches with 1,000 hits and 0
hits were removed from the Project Table. This systematic
tightening of the IDMT is analogous to shrink-wrapping. The
typical starting IDMT value was 2.0 Å. Hit Treatment options
are left at the default values with the number of hits at 1,000 used
as a termination point.
4.4.1 Pharmacophore modeling and database searches
The details of pharmacophore model generation were
provided in the main text. The initial pharmacophore modeling
was carried using the Phase module in the Schrödinger software.
Additional pharmacophore modeling was carried out with
Spartan 10 (V.1.1.0).
The database searches were performed flexibly, with
conformations generated on-the-fly while keeping the initial
conformations stored in the database. The following settings
were used for searching:
Each pharmacophore model was initially subjected to searches
of databases with known drugs; first with the Drugs in the
Market database (from Zinc), which contains 4,356 compounds
that are either marketed drugs or natural products. Then the
search was followed with the Binding Database of 660,806
•
•
•
•
•
•
•
•
Generate conformations during search
Keep existing conformers
compounds with measured binding affinities.
Once the
Number of conformers per rotatable bond = 10
Maximum number of conformers per structure = 100
Sampling = Thorough
pharmacophore models were prioritized, based on the results
obtained by searching these two databases, those high priority
models were subjected to database searches with in-house built
databases including:
Amide bonds = Vary conformation
Relative Energy window = 10.0 kcal/mol
Skip conformer generation for structures with > 15
rotatable bonds
1. LeadsNow database of 2,819,898 compounds was
extracted from the Zinc database as “immediately
available” compounds.
For Matching options, Intersite Distance Matching Tolerance
(IDMT) was used to tighten or relax the fitting requirements. If
2. Aldrich Database of 279,000 commercially available
compounds.
Acknowledgments
This research was partially funded by Union University. The
authors are grateful for the HRESMS data provided by D. R.
Phillips and C.-W. Chou [Proteomics and Mass Spectrometry
(PAMS) Facility, NIH grant 1S10RR1028859] at the University
of Georgia, Department of Chemistry, Athens, Georgia.
References and notes
1. Geiszt M, Kopp JB, Várnai P, Leto TL, Proc. Natl. Acad. Sci. USA, 97
(2000) 8010.
2. van der Vliet A, Free Radical Biol. Med., 44 (2008) 938.
3. (a) Jiang JX, Török NJ, Advances in Hepatology, 2014 (2014) 742931; (b)
Liang S, Kisseleva T, Brenner DA, Frontiers in physiology, 7 (2016) 1.
4. Chen F, Haigh S, Barman S, Fulton DJR, Frontiers in physiology, 3 (2012)
412.
5. Nisimoto Y, Diebold BA, Cosentino-Gomes D, Lambeth JD, Lambeth JD,
Biochemistry, 53 (2014) 5111.
6. Ray R, Murdoch CE, Wang M, Santos CX, Zhang M, Alom-Ruiz S,
Anilkumar N, Ouattara A, Cave AC, Walker SJ, Grieve DJ, Charles RL,
Eaton P, Brewer AC, Shah AM, Atertio. Thromb. Vasc. Biol., 31 (2011)
1368.
7. Craige SM, Chen K, Pei Y, Li C, Huang X, Chen; C, Shibata R, Sato K,
Walsh K, Keaney JF, Circulation, 124 (2011) 731.
8. Schröder K, Zhang M, Benkhoff S, Mieth A, Pliquett R, Kosowski J,
Kruse C, Luedike P, Michaelis UR, Weissmann N, Dimmeler S, Shah AM,
Brandes RP, Circul. Res., 110 (2012) 1217.
9. Paravicini TM, Chrissobolis S, Drummond GR, Sobey CG, Stroke, 35
(2004) 584.
10. (a) Heymes C, Bendall JK, Ratajczak P, Cave AC, Samuel J-L,
Hasenfuss G, Shah AM, J. Am. Coll. Cardiol., 41 (2003) 2164; (b) Kuroda J,
Ago T, Matsushima S, Zhai P, Schneider MD, Sadoshima J, Proceedings of
the National Academy of Sciences of the United States of America, 107
(2010) 15565.
11. Sorescu D, Weiss D, Lassègue B, Clempus RE, Szöcs K, Sorescu GP,
Valppu L, Quinn MT, Lambeth JD, Vega JD, Taylor WR, Griendling KK,
Circulation, 105 (2002) 1429.
12. Shiose A, Kuroda J, Tsuruya K, Hirai M, Hirakata H, Naito S, Hattori M,
Sakaki Y, Sumimoto H, The Journal of biological chemistry, 276 (2001)
1417.
13. (a) Mittal M, Roth M, König P, Hofmann S, Dony E, Goyal P, Selbitz A-
C, Schermuly RT, Ghofrani HA, Kwapiszewska G, Kummer W, Klepetko W,
Hoda MAR, Fink L, Hänze J, Seeger W, Grimminger F, Schmidt HHHW,

Weissmann N, Circul. Res., 101 (2007) 258; (b) Sanders KA, Hoidal JR,
Circul. Res., 101 (2007) 224; (c) Barman SA, Chen F, Su Y, Dimitropoulou
C, Wang Y, Catravas JD, Han W, Orfi L, Szantai-Kis C, Keri G, Szabadkai I,
Barabutis N, Rafikova O, Rafikov R, Black SM, Jonigk D, Giannis A, Asmis
R, Stepp DW, Ramesh G, Fulton DJR, Atertio. Thromb. Vasc. Biol., 34
(2014) 1704.
30. (a) Laleu Bt, Gaggini F, Orchard M, Fioraso-Cartier L, Cagnon Ln,
Houngninou-Molango S, Gradia A, Duboux G, Merlot Cd, Heitz F,
Szyndralewiez Cd, Page P, J. Med. Chem., 53 (2010) 7715; (b) Sedeek M,
Callera G, Montezano A, Gutsol A, Heitz F, Szyndralewiez C, Page P,
Kennedy CRJ, Burns KD, Touyz RM, Hebert RL, AJP: Renal Physiology,
299 (2010) F1348.
14. Hecker L, Vittal R, Jones T, Jagirdar R, Luckhardt TR, Horowitz JC,
Pennathur S, Martinez FJ, Thannickal VJ, Nat. Med., 15 (2009) 1077.
15. (a) Arozal W, Watanabe K, Veeraveedu PT, Ma M, Thandavarayan RA,
Suzuki K, Tachikawa H, Kodama M, Aizawa Y, Biol. Pharm. Bull., 32
(2009) 1411; (b) Liu Y, Lei S, Gao X, Mao X, Wang T, Wong GT,
Vanhoutte PM, Irwin MG, Xia Z, Clinical Science, 122 (2012) 161.
16. (a) Gorin Y, Block K, Hernandez J, Bhandari B, Wagner B, Barnes JL,
Abboud HE, J. Biol. Chem., 280 (2005) 39616; (b) Li J, Wang JJ, Yu Q,
Chen K, Mahadev K, Zhang SX, Diabetes, 59 (2010) 1528; (c) Thallas-
Bonke V, Jha JC, Gray SP, Barit D, Haller H, Schmidt HHHW, Coughlan
MT, Cooper ME, Forbes JM, Jandeleit-Dahm KAM, Physiological reports, 2
(2014) e12192.
31. (a) Stielow C, Catar RA, Muller G, Wingler K, Scheurer P, Schmidt
HHHW, Morawietz H, Biochem. Biophys. Res. Commun., 344 (2006) 200;
(b) Tenfreyhaus H, Huntgeburth M, Wingler K, Schnitker J, Baumer A,
Vantler M, Bekhite M, Wartenberg M, Sauer H, Rosenkranz S,
Cardiovascular Research, 71 (2006) 331.
32. Bhandarkar SS, Jaconi M, Fried LE, Bonner MY, Lefkove B,
Govindarajan B, Perry BN, Parhar R, Mackelfresh J, Sohn A, Stouffs M,
Knaus U, Yancopoulos G, Reiss Y, Benest AV, Augustin HG, Arbiser JL,
The Journal of clinical investigation, 119 (2009) 2359.
33. Kofler P, Pircher H, von Grafenstein S, Diener T, Höll M, Liedl K, Siems
K, Jansen-Dürr P, Planta Med., 79 (2013) 244.
34. Borbély Gb, Szabadkai In, Horváth Zn, Markó Pt, Varga Zn, Breza Nr,
Baska F, Vántus T, Huszár Mn, Geiszt Ms, Hunyady Ls, Buday Ls, Őrfi Ls,
Kéri Gr, J. Med. Chem., 53 (2010) 6758.
17. Xu S, Chamseddine AH, Carrell S, Miller FJ, Jr., Redox biology, 2
(2014) 642.
18. Matsushima S, Kuroda J, Ago T, Zhai P, Park JY, Xie L-H, Tian B,
Sadoshima J, Circul. Res., 112 (2013) 651.
35. Güner OF, Pharmacophore Perception, Development, and Use in Drug
Design, 1 ed., International University Line, La Jolla, CA2000.
36. (a) Phillips RS, Anderson AD, Gentry HG, Güner OF, Bowen JP,
Bioorganic and Medicinal Chemistry Letters, 27 (2017) 1705; (b) Zhong H,
Wees MA, Faure TD, Carrillo C, Arbiser J, Bowen JP, ACS Med. Chem.
Lett, 2 (2011) 455.
37. Dixon SL, Smondyrev AM, Knoll EH, Rao SN, Shaw DE, Friesner RA,
Journal of Computer-Aided Molecular Design, 20 (2006) 647.
38. Schrödinger, Small-Molecule Drug Discovery Suite 2014-1, Schrödinger,
LLC, New York, NY, 2014.
19. Lan T, Kisseleva T, Brenner DA, PLOS ONE, 10 (2015) e0129743.
20. Xue B, Beltz TG, Johnson RF, Guo F, Hay M, Johnson AK, American
journal of physiology. Heart and circulatory physiology, 302 (2012) H733.
21. Mkaddem SB, Bens M, Vandewalle A, Oncotarget, 1 (2010) 741.
22. Goettsch C, Babelova A, Trummer O, Erben RG, Rauner M, Rammelt S,
Weissmann N, Weinberger V, Benkhoff S, Kampschulte M, Obermayer-
Pietsch B, Hofbauer LC, Brandes RP, Schröder K, The Journal of clinical
investigation, 123 (2013) 4731.
23. Fu P, Mohan V, Mansoor S, Tiruppathi C, Sadikot RT, Natarajan V, Am.
J. Respir. Cell Mol. Biol., 48 (2013) 477.
39. Page P, Orchard M, Fioraso-Cartier L, Mottironi B, Pyrazolo pyridine
derivatives as NADPH oxidase inhibitors, 2013.
24. Jha JC, Gray SP, Barit D, Okabe J, El-Osta A, Namikoshi T, Thallas-
Bonke V, Wingler K, Szyndralewiez C, Heitz F, Touyz RM, Cooper ME,
Schmidt HHHW, Jandeleit-Dahm KA, Journal of the American Society of
Nephrology : JASN, 25 (2014) 1237.
25. Jiang F, Liu G-S, Dusting GJ, Chan EC, Redox biology, 2 (2014) 267.
26. Hsieh C-H, Shyu W-C, Chiang C-Y, Kuo J-W, Shen W-C, Liu R-S,
PLoS ONE, 6 (2011) e23945.
27. Kleinschnitz C, Grund H, Wingler K, Armitage ME, Jones E, Mittal M,
Barit D, Schwarz T, Geis C, Kraft P, Barthel K, Schuhmann MK, Herrmann
AM, Meuth SG, Stoll G, Meurer S, Schrewe A, Becker L, Gailus-Durner V,
Fuchs H, Klopstock T, de Angelis MH, Jandeleit-Dahm K, Shah AM,
Weissmann N, Schmidt HHHW, PLoS Biol., 8 (2010) e1000479.
28. (a) Jaquet V, Scapozza L, Clark RA, Krause K-H, Lambeth JD,
Antioxidants & Redox Signaling, 11 (2009) 2535; (b) Drummond GR,
Selemidis S, Griendling KK, Sobey CG, Nature Reviews Drug Discovery, 10
(2011) 453; (c) Kim J-A, Neupane GP, Lee ES, Jeong B-S, Park BC, Thapa
P, Expert Opinion on Therapeutic Patents, 21 (2011) 1147; (d) Kleniewska
P, Piechota A, Skibska B, Gorąca A, Archivum Immunologiae et Therapiae
Experimentalis, 60 (2012) 277.
40. (a) Liochev SI, Fridovich I, Arch. Biochem. Biophys., 337 (1997) 115;
(b) Zielonka J, Lambeth JD, Kalyanaraman B, Free Radical Biol. Med., 65
(2013) 1310.
41. Gaggini F, Laleu B, Orchard M, Fioraso-Cartier L, Cagnon L,
Houngninou-Molango S, Gradia A, Duboux G, Merlot C, Heitz F,
Szyndralewiez C, Page P, Biorg. Med. Chem., 19 (2011) 6989.
42. Zielonka J, Cheng G, Zielonka M, Ganesh T, Sun A, Joseph J, Michalski
R, O'Brien WJ, Lambeth JD, Kalyanaraman B, J. Biol. Chem., 289 (2014)
16176.
43. (a) Clempus RE, Sorescu D, Dikalova AE, Pounkova L, Jo P, Sorescu
GP, Schmidt HHH, Lassègue B, Griendling KK, Atertio. Thromb. Vasc.
Biol., 27 (2007) 42; (b) Martin-Garrido A, Brown DI, Lyle AN, Dikalova A,
Seidel-Rogol B, Lassègue B, Martín AS, Griendling KK, Free Radical Biol.
Med., 50 (2011) 354.
44. Sutliff RL, Hilenski LL, Amanso AM, Parastatidis I, Dikalova AE,
Hansen L, Datla SR, Long JS, El-Ali AM, Joseph G, Gleason RL, Jr., Taylor
WR, Hart CM, Griendling KK, Lassegue B, Arterioscler Thromb Vasc Biol,
33 (2013) 2154.
45. Quick KL, Hardt JI, Dugan LL, J. Neurosci. Methods, 97 (2000) 139.
46. Lee MY, San Martin A, Mehta PK, Dikalova AE, Garrido AM, Datla SR,
Lyons E, Krause KH, Banfi B, Lambeth JD, Lassegue B, Griendling KK,
Arterioscler Thromb Vasc Biol, 29 (2009) 480.
29. Cifuentes-Pagano E, Csanyi G, Pagano PJ, Cell. Mol. Life Sci., 69
(2012) 2315.