Discovery of potent and selective BRD4 inhibitors capable of blocking TLR3-induced acute airway inflammation

Article abstract of DOI:10.1016/j.ejmech.2018.04.006

A series of diverse small molecules have been designed and synthesized through structure-based drug design by taking advantage of fragment merging and elaboration approaches. Compounds ZL0420 (28) and ZL0454 (35) were identified as potent and selective BRD4 inhibitors with nanomolar binding affinities to bromodomains (BDs) of BRD4. Both of them can be well docked into the acetyl-lysine (KAc) binding pocket of BRD4, forming key interactions including the critical hydrogen bonds with Asn140 directly and Tyr97 indirectly via a H₂O molecule. Both compounds 28 and 35 exhibited submicromolar potency of inhibiting the TLR3-dependent innate immune gene program, including ISG54, ISG56, IL-8, and Groβ genes in cultured human small airway epithelial cells (hSAECs). More importantly, they also demonstrated potent efficacy reducing airway inflammation in a mouse model with low toxicity, indicating a proof of concept that BRD4 inhibitors may offer the therapeutic potential to block the viral-induced airway inflammation.

Full text of DOI:10.1016/j.ejmech.2018.04.006

Accepted Manuscript
Discovery of potent and selective BRD4 inhibitors capable of blocking TLR3-induced
acute airway inflammation
Zhiqing Liu, Bing Tian, Haiying Chen, Pingyuan Wang, Allan R. Brasier, Jia Zhou
PII:
S0223-5234(18)30333-7
10.1016/j.ejmech.2018.04.006
EJMECH 10355
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 January 2018
Revised Date: 30 March 2018
Accepted Date: 2 April 2018
Please cite this article as: Z. Liu, B. Tian, H. Chen, P. Wang, A.R. Brasier, J. Zhou, Discovery of potent
and selective BRD4 inhibitors capable of blocking TLR3-induced acute airway inflammation, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.04.006.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of Potent and Selective BRD4 Inhibitors Capable of Blocking
TLR3-Induced Acute Airway Inflammation
Zhiqing Liu,a,1 Bing Tian,b,c,1 Haiying Chen,a Pingyuan Wang,a
Allan R. Brasierb,c,d and Jia Zhoua,c,d,
*
aChemical Biology Program, Department of Pharmacology and Toxicology, bDepartment of
Internal Medicine, cSealy Center for Molecular Medicine, dInstitute for Translational Sciences,
University of Texas Medical Branch, Galveston, TX 77555, USA
1These authors contribute equally to this work.
Corresponding author:
*Jia Zhou, PhD
Chemical Biology Program
Department of Pharmacology and Toxicology
University of Texas Medical Branch
Galveston, Texas 77555, United States
Email: jizhou@utmb.edu

ACCEPTED MANUSCRIPT
Abstract
A series of diverse small molecules have been designed and synthesized through
structure-based drug design by taking advantage of fragment merging and elaboration
approaches. Compounds ZL0420 (28) and ZL0454 (35) were identified as potent and selective
BRD4 inhibitors with nanomolar binding affinities to bromodomains (BDs) of BRD4. Both of
them can be well docked into the acetyl-lysine (KAc) binding pocket of BRD4, forming key
interactions including the critical hydrogen bonds with Asn140 directly and Tyr97 indirectly via
a H2O molecule. Both compounds 28 and 35 exhibited submicromolar potency of inhibiting the
TLR3-dependent innate immune gene program, including ISG54, ISG56, IL-8, and Groβ genes
in cultured human small airway epithelial cells (hSAECs). More importantly, they also
demonstrated potent efficacy reducing airway inflammation in a mouse model with low toxicity,
indicating a proof of concept that BRD4 inhibitors may offer the therapeutic potential to block
the viral-induced airway inflammation.
Keywords: bromodomains; bromodomain-containing protein 4 (BRD4); structure-based drug
design; immune response genes; airway inflammation.
Abbreviations
BRD4, bromodomain-containing protein 4; BET, bromodomain and extra-terminal domain;
KAc, acetylated lysine; BCP, bromodomain-containing proteins; EMT, epithelial-mesenchymal
transition; DIPEA, N,N-diisopropylethylamine; HBTU, N,N,N’,N’-tetramethyl-O-(1H-
benzotriazol-1-yl)uronium hexafluorophosphate; Pd(OAc)2, palladium(II) acetate; hSAECs,

ACCEPTED MANUSCRIPT
human small airway epithelial cells; poly(I:C), polyinosinic:polycytidylic acid; TR-FRET, time
resolved-fluorescence resonance energy transfer. ISG, interferon stimulated gene; IL-8,
interleukin-8; Groβ, growth-regulated protein β; qRT-PCR, quantitative real-time polymerase
chain reaction; TLR3, toll-like receptor 3; CREB, cAMP responsive element binding protein;
CBP, CREB binding protein; CL, clearance; IV, intravenous; PO, per os; Vss, volume of
distribution at steady state; AUC, area under the curve; PEG, polyethylene glycol; i.p.,
intraperitoneal.

ACCEPTED MANUSCRIPT
1. Introduction
Bromodomain-containing protein 4 (BRD4) belongs to the family of bromodomain and extra-
terminal domain (BET) proteins which also includes BRD2, BRD3, and BRDT. Similar to other
BET protein members, BRD4 contains two bromodomains at its N-terminal that can recognize
acetylated lysine (KAc) residues [1]. Each bromodomain of BRD4 comprises a left-handed
bundle of four helices (αZ, αA, αB and αC) linked by the inter-helical ZA loop and BC loop which
constitute the active KAc-binding pocket [2]. Bromodomain-containing proteins (BCPs) act as
KAc readers of modified histones mediating signaling pathways of gene regulatory networks [3].
Disturbing the interaction between BET protein bromodomains and acetylated lysine represents a
very promising therapeutic target for human diseases including cancer and inflammations [4-6].
Discovery and development of BET inhibitors have attracted increasing attention since the BET
family is considered as the most druggable target proteins among BCPs for regulating cellular
epigenetics [7]. Based on the diverse structures, currently available BET inhibitors under the
investigation can be classified as azepines (e.g. (+)-JQ1, 1, Fig. 1) [8], quinazolin-4(3H)-one
(e.g. RVX-208, 2) [9], pyridones (e.g. ABBV-075, 3) [10], 3,5-dimethylisoxazoles (e.g. I-
BET151, 4) [11], tetrahydroquinolines (e.g. I-BET726, 5) [12], 4-acylpyrroles (e.g. XD14, 6)
[13], and others [3]. Among these compounds, compound 1 is one of the first reported BET
inhibitors that was most extensively used as a research tool in the field. Compound 2 is a well-
developed BET inhibitor that has been advanced into Phase III human clinical trials for
cardiovascular diseases [14]. In addition, more than a dozen of other BET inhibitors including
compound 3 are at the different phases of clinical trials [15,16]. Nevertheless, none of these
compounds have yet been approved by FDA. Meanwhile, most of them are pan-BET inhibitors
lacking selectivity for individual BET family members [17,18]. As the most heavily investigated

ACCEPTED MANUSCRIPT
member of BET family, BRD4 represents a very promising therapeutic target. Developing potent
and highly selective BRD4 inhibitors for more applications is in an urgent need.
Fig. 1. Structures of representative BET inhibitors. Some privileged fragments that are important
for binding with BRD4 are highlighted in blue.
As part of our ongoing drug discovery efforts, we are interested in developing novel
potent and selective BRD4 inhibitors as potential therapeutics for acute airway inflammation and
chronic inflammation-associated airway remodeling as well as other inflammatory diseases. Our
team has observed the essential role of BRD4 in NF-κB mediated epithelial-mesenchymal
transition (EMT) in an in vitro model of airway epithelial cells and in a murine pulmonary
fibrosis model in vivo [19]. To identify new and more selective BRD4 inhibitors, we initiated our
structure-based drug design by analyzing the crystal structures of available BRD4 inhibitors with
BRD4 BD1 domain exemplified with co-complex of 1 as depicted in Fig. 2a. Most inhibitors
mimic acetyl-lysine, and occupy the central hydrophobic cavity and anchored by hydrogen bonds

ACCEPTED MANUSCRIPT
with Asn140 directly and Tyr97 indirectly via a water molecule [20]. BRD4 inhibitors can often
extend to the WPF shelf, a hydrophobic region of ZA loop that includes Trp/Pro/Phe motif, and
ZA channel (notably Pro82 to Leu91 in BRD4 BD1) via a substituted phenyl ring [21]. These
hydrophobic interactions are also crucial for BRD4 binding affinities. Thus, we have
summarized a pharmacophore model as two key aromatic rings attached with a proper linker.
The substituents or hetero atoms of the head aromatic ring form the critical hydrogen bonds with
Asn140 as well as Tyr97, and the tail aromatic ring with substituents interacts with the
hydrophobic WPF shelf and ZA channel (Fig. 2b). By utilizing a deconstruction and
reconstruction approach [22,23], we took advantage of druglike fragments from available
inhibitors and drug libraries as the critical head and tail moieties as well as the linker scaffold for
the structure and fragment-based drug design through fragment merging and elaboration [24,25].
For example, some privileged fragments of inhibitors 1, 4, 5 and 6 highlighted in Fig. 1 have
been considered in the design of the head and tail binding partners.
Fig. 2. a) Co-complex crystal structure of 1 with BRD4 BD1 (PDB code: 3MXF) as an example.
b) Proposed pharmacophore model and design of new BRD4 inhibitors in this effort.

ACCEPTED MANUSCRIPT
2. Results and discussion
2.1. Chemistry
New compounds 11~16 have been initially designed with benzenesulfonamide as the tail
pharmacophore inspired by a privileged scaffold of compound 6, and their synthetic route was
outlined in Scheme 1. With starting material 4-nitrobenzenesulfonyl chloride (7) and methyl L-
prolinate (8), key intermediate 9 was obtained in the presence of Hünig's base DIPEA. Reduction
of 9 with Zn dust and NH4Cl gave compound 10 in a quantitative yield. Intermediate 10 was
coupled with various acids in the presence of HBTU and DIPEA to generate compounds 11 and
12. Through acylation reaction of 10 with benzoyl chlorides, compounds 13-14 were readily
achieved. New compound 15 was produced via reductive amination reaction of 10 with the
treatment of 4-hydrobenzaldehyde in the presence of NaBH3CN. Intermediate 10 reacted with 5-
amino-2-methylphenol assisted by tert-butyl nitrite leading to compound 16 in a yield of 78%
[26].
Scheme 1. Synthetic route of newly designed compounds 11~16. Reagents and conditions: (a)
DIPEA, DCM, 0~5 ˚C, 89%; (b) Zn, NH4Cl, EtOH/H2O, 80 ˚C, quant.; (c) 5-propylisoxazole-3-
carboxylic acid (for 11) or 5-(furan-2-yl)isoxazole-3-carboxylic acid (for 12), HBTU, DIPEA,
DCM, 0~5 ˚C. 68% for 11; 86% for 12; (d) 4-fluorobenzoyl chloride, pyridine (for 13) or 4-

ACCEPTED MANUSCRIPT
trifluorobenzoyl chloride (for 14), DCM, 0~5 ˚C, 67% for 13, quant. for 14; (e) 4-
hydroxybenzaldehyde, NaBH3CN, MeOH, 80 ˚C, 85%; (f) 5-amino-2-methylphenol, tert-butyl
nitrite, 38% HCl (aq.), K2CO3, MeOH/CH3CN/H2O, 0 ˚C, 78%.
New compounds 19, 22 and 23 were designed by taking isoxazole ring as the head
moiety and its hetero atoms were expected to form the critical hydrogen bonds with Asn140 and
Tyr97. The synthetic route to access these compounds was out lined in Scheme 2. Starting
material ethyl 5-(tert-butyl)isoxazole-3-carboxylate (17) was hydrolyzed to the acid 18, followed
by the coupling with 1-(4-fluorophenyl)piperazine in the presence of HBTU and DIPEA to
produce compound 19 in a yield of 81%. 17 was also hydrazinolyzed with NH2NH2 leading to
intermediate 20 with subsequent coupling with various benzaldehydes to yield new compounds
21 and 22. Cyclization of compound 21 in the presence of I2 and K2CO3 in DMSO resulted in
compound 23 [27].
Scheme 2. Synthetic route of new compounds 19, 22 and 23. Reagents and conditions: (a)
LiOH·H2O, CH3OH/H2O, rt., 95%; (b) 1-(4-fluorophenyl) piperazine, HBTU, DIPEA, DCM,
81%; (c) NH2NH2, EtOH, reflux, used directly for the next step; (d) 3-chloro-5-
fluorobenzaldehyde (for 21) or 3,5-bis(trifluoromethyl)benzaldehyde (for 22), EtOH, rt. 44% for
21 (two steps). 59% for 22 (two steps); (e) I2, K2CO3, DMSO, 110 ˚C, 13%.

ACCEPTED MANUSCRIPT
3,4-Dihydroquinolin-2(1H)-one was also utilized to design new molecules 26~30 and
33~35 given that this moiety may play both roles of head and tail pharmacophores. The
according synthesis was outlined in Scheme 3. 6-Nitro-3,4-dihydroquinolin-2(1H)-one 24 was
reduced by Zn dust with NH4Cl to give the critical intermediate 6-amino-3,4-dihydroquinolin-
2(1H)-one 25. Building block 25 was sulfonized in the presence of the base of Et3N to give
compounds 26 and 27. Amine 25 was also treated with 5-amino-2-methylphenol in the presence
of tert-butyl nitrite to produce compound 28. C-N coupling of 25 with 5-bromo-N-
methylpicolinamide in the presence of the catalyst of Pd(OAc)2 led to compound 29. Compound
30 with a urea linker was obtained through the addition of amine 25 to 1-isocyanato-4-
(trifluoromethyl)benzene. 5-Amino-2-methylphenol 31 was then taken as the head moiety, and
reacted with several sulfonamide substituted aniline 32 in the presence of tert-butyl nitrite to
afford new compounds 33-35 in a yield of 67%-92%.
Scheme 3. Synthetic route of new compounds 26~30 and 33~35. Reagents and conditions: (a)
Zn, NH4Cl, MeOH/H2O, 80 ˚C, quant. (b) 5-chloro-2-methoxybenzenesulfonyl chloride (for 26)
or 4-nitrobenzenesulfonyl chloride (for 27), Et3N, DMF, rt., 29% for 26, 13% for 27; (c) 5-
amino-2-methylphenol, tert-butyl nitrite, 38% HCl (aq.), K2CO3, MeOH/CH3CN/H2O, 0˚C,

ACCEPTED MANUSCRIPT
81%. (d) 5-bromo-N-methylpicolinamide, Pd(OAc)2, xantphos, Cs2CO3, 1,4-dioxane, 110 ˚C,
63%. (e) 1-isocyanato-4-(trifluoromethyl)benzene, DCM, rt., 41%; (f) tert-butyl nitrite, 38% HCl
(aq.), K2CO3, MeOH/CH3CN/H2O, 0 ˚C, 67% for 33, 92% for 34, 81% for 35.
2.2. Biology
2.2.1. Evaluation of innate immune response genes in cellular assays
All the newly designed and synthesized compounds were evaluated in a cellular assay
system first (Table 1) given that a number of reported BRD4 inhibitors are lack of satisfactory
cellular activities due to limited drug properties, thereby hindering their further preclinical
development. This strategy allows us to prioritize our inhibitors and exclude those compounds
that are unable to penetrate cell membrane at the early evaluation stage. The in vitro efficacies of
all new molecules were first determined by polyinosinic:polycytidylic acid (poly(I:C))-induced
expression of innate immune response genes in human small airway epithelial cells (hSAECs)
which have many characteristics of representative primary lower-airway epithelial cells [28].
Administered in the extracellular medium, poly(I:C) is a potent and selective agonist of the Toll-
Like Receptor 3 (TLR3) pattern recognition receptor that mediates the innate response program
to RNA virus infection. The use of this synthetic viral pattern enables a robust and reproducible
assay to screen BRD4 inhibitors, and quantify their IC50 values. hSAECs were first preincubated
with 10 µM of test compounds overnight, followed by poly(I:C) addition into culture medium at
10 g/mL for another 4 h (the time when maximal innate immune gene expression is reached).
The total RNA was extracted and quantitative real-time polymerase chain reaction (Q-RT-PCR)
was performed to determine their inhibitory effect on poly(I:C) induced innate immune genes
including ISG54, ISG56, IL-8 and Groβ expression in hSAECs. Percentages of inhibition (%)
were calculated based on the control (poly(I:C) induced and without administration of

ACCEPTED MANUSCRIPT
compounds). Afterwards, IC50 values of most promising compounds were determined from their
inhibitory curves at 8 concentrations of each inhibitor using curve-fitting algorithms. Compound
(+)-JQ1 was also included as the positive control for comparison. It was found that among
compounds 11~16 with sulfonyl-L-prolinate attached to the tail moiety, compound 13 taking 4-
flurophenyl as the head moiety and substituted amide as the linker, showed the most promising
inhibitory activity. All the inhibitory rates of compound 13 regulating immune genes’
expression are around 90%. With an unsubstituted amide as a linker, compound 14 completely
lost its activity. Compound 16 with 2-amino-4-hydroxy-5-methylphenyl as the head and diazo as
a linker has a moderate inhibitory effect (60% to 64%). For compounds 19, 22 and 23 taking
tert-butyl isoxazole as the head, 19 and 23 with constrained linkers displayed better inhibitory
activities. The inhibitory effect on ISG54 and ISG56 expression of compound 23 reached to
99%. Compounds 26~30 employed 3,4-dihydroquinolin-2(1H)-one as their tail pharmacophore,
and diverse heads including substituted phenyl rings and pyridines and different linkers (e.g.
sulfonamide, diazo, NH and urea) all showed acceptable inhibitory activities. Interestingly,
compound 28, with 2-amino-4-hydroxy-5-methylphenyl as the head and diazo as a linker again,
exhibited very impressive inhibitory activity (94% to 95%). We kept the head and linker intact,
and modified several different tail pharmacophores (e.g. 33~35). Both 34 and 35, with a
substituent of sulfonamide attached to the tail phenyl ring, displayed promising inhibitory
activity, while 33 with a sulfone group in the ring was found inactive.
Based on the preliminary data, compounds 23, 28 and 35 were selected to calculate their
IC50 values (Table 2). Compounds 28 and 35 showed submicromolar IC50 values of 0.49~0.86
µM against all these innate immune genes, which are 15~20-fold more potent than 23. Also, both
of them are more potent than positive control compound (+)-JQ1, a widely used tool compound,

ACCEPTED MANUSCRIPT
and compound RVX-208, the most advanced Phase III clinical candidate, which inhibited the
production of immune genes with IC50 values of 1.38 to 3.85 µM.
Table 1. Effects of newly designed and synthesized molecules on TLR3 agonist-induced
expression of innate immune genes in vitro.
a
%
Compds
ISG54
91
ISG56
90
IL-8
90
Groβ
91
(+)-JQ1
11
-62
-114
92
-73
-93
92
-61
-86
88
-57
-98
88
12
13
3.6
-9.0
61
2.7
-17
64
-1.0
-5.0
60
5.7
-12
61
14
15
16
94
94
88
82
19
22
21
48
48
22
99
99
98
98
23
57
56
53
56
26
-21
95
-29
95
-32
94
-37
94
27
28
29
32
56
52
29
92
92
48
48
30
b
20
20
-
-
33
85
85
81
99
81
34
93
93
99
35
aConcentration of compounds: 10 µM. Inhibitory rates (%) are reported as the geometric mean
b
derived from three independent measurements except where indicated. Not tested.

ACCEPTED MANUSCRIPT
Table 2. IC50 values of selected compounds of inhibiting TLR3 agonist-induced expression of
innate immune genes in hSAECs.
IC50, µMa
Compds
ISG54
1.38
2.63
7.9
ISG56
1.63
2.74
7.7
IL-8
1.51
3.85
4.3
Groβ
1.49
3.73
3.9
(+)-JQ1
RVX-208
23
28
35
0.49
0.81
0.51
0.86
0.53
0.73
0.58
0.79
aIC50 values are reported as the mean derived from three independent measurements. Each one is
generated from at least 8 different concentrations.
2.2.2. Binding affinities and selectivity
Binding affinities of compounds 28 and 35 for BRD4 were evaluated using a
commercially available time resolved (TR)-fluorescence resonance energy transfer (FRET) assay
(Table 3) [8,29,30]. BRD4 specificity of both compounds was also confirmed through comparing
with close BET family members BRD2, BRD3 and BRDT as well as non-BET CREB binding
protein (CBP). Compound 28 exhibited potent BRD4 inhibitory activities, with IC50 values of 27
nM against BRD4 BD1 and 32 nM against BRD4 BD2, respectively. Compound 35 displayed
similar activity and did not show preference between BD1 and BD2 of BRD4 either. However,
both of them have 30~60-fold BRD4 selectivity over its close family member BRD2 (IC50 values
of 0.77~1.8 µM), 50~90-fold selectivity over BRD3 (IC50 values of 2.2 ~2.5 µM), and 70~120-
fold over BRDT (IC50 values of 2.8 ~3.3 µM) as well as more than 200-fold over non-BET
protein CBP (IC50 values of > 10 µM), while the positive control (+)-JQ1 is non-specific among
BET members and RVX-208 is BD2 selective. Off-target effects of compound 35 were also
evaluated through the Eurofins Cerep panel assays of various receptors and enzymes including

ACCEPTED MANUSCRIPT
kinases such as IKK (Supporting Information, Table S1). No significant off-target effects were
observed at the tested concentration of 10 µM.
Table 3. Binding affinities of compounds 28 and 35 with BRD4 and the selectivity over its BET
family members BRD2, BRD3, BRDT as well as non-BET protein CBP.a
BDs\Compds
(+)-JQ1
92
RVX-208
1,142
135
28
27
35
49
BRD4 BD1 (IC50, nM)
BRD4 BD2 (IC50, nM)
BRD2 BD1 (IC50, nM)
BRD2 BD2 (IC50, nM)
BRD3 BD1 (IC50, nM)
BRD3 BD2 (IC50, nM)
BRDT BD1 (IC50, nM)
BRDT BD2 (IC50, nM)
CBP (IC50, nM)
62
32
32
78
5,780
251
803
772
52
1,736
2,275
2,193
3,183
2,781
> 10,000
1,836
2,493
2,241
3,292
3,082
> 10,000
81
3,962
203
69
183
217
9,600
4,836
708
> 10,000
aBinding affinity was measured using an TR-FRET assay with the isolated bromodomain.
Reported as mean of at least two separate assay runs.
2.3. Predicted binding modes of compounds 28 and 35 with BRD4 BD1
Computational docking studies of compounds 28 and 35 with the first bromodomain of
human BRD4 protein complex (PDB code: 4NUD) as well as BRD2 BD1 (4A9M) using
Schrödinger Small-Molecule Drug Discovery Suite were conducted (Fig. 3 and Figure S1). Both
compounds 28 and 35 can well occupy the KAc binding pocket. The OH group of compound 35
forms critical hydrogen bonds with Asn140 directly and Tyr97 indirectly via a H2O molecule.
NH2 group of the head also interacts with Asn140 via a water molecule. Besides, the N atom of
diazo linker interacts with Pro82 through another water molecule. The fragment
cyclopentylbenzenesulfonamide extends to the hydrophobic WPF shelf and ZA channel, and

ACCEPTED MANUSCRIPT
importantly, it forms a T shape л-л interaction with Trp81. For the docking result of 35 with
BRD2 BD1, there are not that many critical interactions observed (Figure S1), and this may
explain their difference of binding affinities and the selectivity. The overlay analysis of 28 and
35 demonstrated that their binding poses are strikingly similar (Fig. 3b).
Fig. 3. a) Docking result of 35 with BRD4 BD1 (PDB code: 4NUD); b) Overlay analysis of 35
(pink) and 28 (green) docked into BRD4 BD1.
2.4. In vivo pharmacokinetic profiles of 28 and 35
On the basis of their combination of cellular potency and binding affinities, compounds 28 and
35 were further evaluated for their in vivo metabolic profile in rats. As shown in Table 4, a
relatively high clearance and low exposures were observed with oral bioavailability not favorable
for both compounds. In contrast, intravenous administration gave an excellent drug exposure
(high AUC value) and moderate half-life. Thus, in the subsequent in vivo efficacy study,
intraperitoneal (i.p.) injection was administered for mice instead of oral gavage. An alternative
drug delivery method through nanoparticles is currently under investigation to improve their
metabolic stability, and the findings will be reported in due course.

ACCEPTED MANUSCRIPT
Table 4. Pharmacokinetic parameters of compounds 28 and 35.a
Compounds
t1/2b (h)
28
1.2
35
0.83
11,400
1.125
14.7
36
AUC0-tc (ng·h/mL)
VSSd (L/kg)
14,700
0.864
11.5
80
IV
(10 mg/kg)
CLe (mL/min/kg)
Cmaxf (ng/mL)
PO
(20 mg/kg)
AUC0-t (ng·h/mL)
450
104
b
c
aCompounds were formulated in 10% DMSO/60% PEG-400/30% Saline. half-life. Exposure
d
e
f
over test time. Volume of distribution at steady state. Total clearance. Maximum plasma
concentration.
2.5. In vivo evaluations of compounds 28 and 35 in TLR3 mediated acute airway inflammatory
murine model
With two promising BRD4 inhibitors compound 28 and 35 available in hand, we then
performed in vivo efficacy evaluations in our established murine model of TLR3 mediated acute
airway inflammation [28]. Intranasal administration of poly(I:C)-induced a substantial increase
of total cells and neutrophils into the airway fluids, and cytokine expression in the lung tissue.
These changes were more effectively blocked by BRD4 inhibitors 28 and 35 than positive
control (+)-JQ1 or RVX-208 (Fig. 4a), at the dose of 10 mg/kg (via i.p.). Histology analyses of
lung sections in the poly(I:C)-treated mice with/without BRD4 inhibitors are also demonstrated
in Fig. 4b. Compounds 28 and 35 displayed higher efficacy and almost completely blocked the
profound accumulation of neutrophils around the small and medium sized airways induced by
poly(I:C) administration. In addition, daily administration of 35 from 1-50 mg/kg in groups of n
= 5 mice over one month each had no apparent toxic effects observed on body weight,
hematological measures (WBC, RBC, platelets), liver function (albumin, globulin, ALK, ALT),
and renal function (CRE, BUN) (Figure S2), indicating that they are much safer and better

ACCEPTED MANUSCRIPT
tolerated than pan-BET inhibitors (e.g. JQ1). No discernable effects on hepatic, renal or
pulmonary tissues derived from compound 35-treated mice were observed during histological
examination.
Fig. 4. BRD4 inhibitors 28 and 35 effectively blocked TLR3 agonist-induced acute airway
inflammation in vivo. Positive controls (+)-JQ1 and RVX-208 were also tested for comparison.

ACCEPTED MANUSCRIPT
3. Conclusions
In summary, we have identified two novel BRD4 inhibitors ZL0420 (28) and ZL0454
(35) via a structure-based drug design approach, which are more potent and selective than known
inhibitors (+)-JQ1 and RVX-208. These compounds are capable of significantly blocking the
TLR3-dependent expression of innate immune genes ISG54, ISG56, IL-8, and Groβ in hSAECs
and display nanomolar binding affinities for BDs of BRD4 protein with good selectivity over
that of the related BRD2 homolog. Molecular docking revealed their classical binding modes
with the critical interactions identified between the ligand and the target protein. Significant in
vivo efficacy in standardized murine model of TLR3 agonist-induced airway inflammation with
low toxicity further confirmed their therapeutic potential as a proof of concept for the treatment
of the viral-induced airway inflammation.
4. Experimental section
4.1. Chemistry
All commercially available starting materials and solvents were reagent grade, and used without
further purification. Reactions were performed under a nitrogen atmosphere in dry glassware
with magnetic stirring. Preparative column chromatography was performed using silica gel 60,
particle size 0.063-0.200 mm (70-230 mesh, flash). Analytical TLC was carried out employing
silica gel 60 F254 plates (Merck, Darmstadt). Visualization of the developed chromatograms was
performed with detection by UV (254 nm). NMR spectra were recorded on a Bruker-600 (1H,
600 MHz; 13C, 150 MHz) spectrometer or Bruker-300 (1H, 300 MHz; 13C, 75 MHz). 1H and 13C
NMR spectra were recorded with TMS as an internal reference. Chemical shifts were expressed
in ppm, and J values were given in Hz. High-resolution mass spectra (HRMS) were obtained

ACCEPTED MANUSCRIPT
from Thermo Fisher LTQ Orbitrap Elite mass spectrometer. Parameters include the following:
Nano ESI spray voltage was 1.8 kV; Capillary temperature was 275 ˚C and the resolution was
60,000; Ionization was achieved by positive mode. Melting points were determined on a hot
stage apparatus and are uncorrected. Purity of final compounds was determined by analytical
HPLC, which was carried out on a Shimadzu HPLC system (model: CBM-20A LC-20AD SPD-
20A UV/VIS). HPLC analysis conditions: Waters µBondapak C18 (300 × 3.9 mm); flow rate 0.5
mL/min; UV detection at 270 and 254 nm; linear gradient from 30% acetonitrile in water (0.1%
TFA) to 100% acetonitrile (0.1% TFA) in 20 min followed by 30 min of the last-named solvent.
All the new compounds have a purity of >95% prior to the submission for the biological studies.
4.1.1. Methyl ((4-(5-propylisoxazole-3-carboxamido)phenyl)sulfonyl)-L-prolinate (11).
To a solution of methyl L-prolinate (1.66 g, 10 mmol) and DIPEA (2.58 g, 20 mmol) in
40 mL of DCM, 4-nitrobenzenesulfonyl chloride (2.22 g, 10 mmol) was added at 0 ˚C. After
stirring at rt. for 2 h, the solution was extracted with DCM (50 mL×2). The organic layer was
washed with 1 N NaHSO4 (aq.), saturated NaHCO3 (aq.), brine and dried over anhydrous
Na2SO4. The resulting solution was evaporated, and the residue was purified by silica gel column
chromatography (hexane/EtOAc = 10/1 to 5/1) to give the intermediate 9 (2.8 g, 89%) as a white
solid. 1H NMR (300 MHz, CDCl3) δ 8.39 (d, J = 8.9 Hz, 2H), 8.10 (d, J = 8.9 Hz, 2H), 4.48 (dd,
J = 8.5 Hz, 3.7 Hz, 1H), 3.73 (s, 3H), 3.47 (dd, J = 7.0 Hz, 6.2 Hz, 2H), 2.26 – 1.87 (m, 4H). 13C
NMR (75 MHz, CDCl3) δ 172.14, 144.68, 128.67, 124.20, 60.57, 52.54, 48.32, 30.96, 24.74.
To a solution of 9 (2.68 g, 8.54 mmol) in EtOH (40 mL), NH4Cl (4.50 g, 85.4 mmol) in
H2O (20 mL) and Zn dust (3.33 g, 51.2 mmol) were added. After refluxing at 80 ˚C for 1 h, the
solution was filtered to remove Zn dust. After cooled to rt., the solution was extracted with
EtOAc (50 mL×2). The organic layer was washed with saturated NaHCO3 (aq.), brine and dried

ACCEPTED MANUSCRIPT
over anhydrous Na2SO4. The resulting solution was evaporated, and the residue was purified by
silica gel column chromatography (hexane/EtOAc = 3/1) to give 10 (2.2 g, quant.) as a white
1
solid. H NMR (300 MHz, CDCl3) δ 7.60 (d, J = 8.7 Hz, 2H), 6.68 (d, J = 8.7 Hz, 2H), 4.34 (s,
2H), 4.26 – 4.17 (m, 1H), 3.71 (s, 3H), 3.52 – 3.40 (m, 1H), 3.31 – 3.19 (m, 1H), 1.97 (dt, J = 9.2
Hz, 6.5 Hz, 3H), 1.72 (d, J = 4.9 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 172.93, 151.20, 129.57,
125.39, 113.98, 60.37, 52.41, 48.55, 30.90, 24.64.
To a solution of 5-propylisoxazole-3-carboxylic acid (58 mg, 0.375 mmol) and methyl
((4-aminophenyl)sulfonyl)-L-prolinate 10 (70 mg, 0.25 mmol) in 5 mL of DCM, HBTU (284
mg, 0.75 mmol) and DIPEA (220 µL, 1.25 mmol) were added. After stirring at rt. overnight, the
mixture was extracted with DCM (20 mL×3). The organic layer was washed with 1 N NaHSO4
(aq.), saturated NaHCO3 (aq.), brine and dried over anhydrous Na2SO4. The resulting solution
was evaporated, and the residue was purified by PTLC (DCM/MeOH = 40/1) to give the desired
1
product 11 (40 mg, 38%) as a pale yellow solid. m.p.: 142-143 ˚C. H NMR (300 MHz, CDCl3)
δ 8.83 (s, 1H), 7.87(dd, J = 8.9 Hz, 4H), 6.55 (s, 1H), 4.33 (dd, J = 7.6 Hz, 4.2 Hz, 1H), 3.73 (s,
3H), 3.51 (dd, J = 9.5 Hz, 6.9 Hz, 1H), 3.41 – 3.28 (m, 1H), 2.82 (t, J = 7.5 Hz, 2H), 2.10 – 1.95
(m, 3H), 1.79 (dd, J = 14.7 Hz, 7.3 Hz, 3H), 1.03 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3)
δ 176.36, 172.54, 158.35, 157.28, 141.12, 133.84, 128.89, 119.70, 100.75, 60.40, 52.45, 48.42,
30.91, 28.67, 24.66, 20.84, 13.56. HRMS (ESI) m/z calcd for C19H24N3O6S [M + H]+, 422.1386;
found, 422.1398.
4.1.2. Methyl ((4-(5-(furan-3-yl)isoxazole-3-carboxamido)phenyl)sulfonyl)-L-prolinate (12).
Compound 12 was prepared in 84% yield taking 2-(5-(furan-2-yl)isoxazol-3-yl)-2-
oxoacetic acid as a key intermediate by a procedure similar to that used to synthesize compound
11. The title compound was obtained as a pale yellow solid. m.p.: 186-188 ˚C. 1H NMR (300

ACCEPTED MANUSCRIPT
MHz, CDCl3) δ 8.83 (s, 1H), 7.98 – 7.83 (m, 4H), 7.62 (s, 1H), 7.02 (d, J = 3.4 Hz, 1H), 6.96 (s,
1H), 6.65 – 6.52 (m, 1H), 4.35 (dd, J = 7.7 Hz, 4.2 Hz, 1H), 3.74 (s, 3H), 3.52 (dd, J = 9.4 Hz,
13
7.0 Hz, 1H), 3.42 – 3.30 (m, 1H), 2.13 – 1.93 (m, 3H), 1.81 (dd, J = 6.8 Hz, 4.8 Hz, 1H). C
NMR (75 MHz, CDCl3) δ 172.54, 163.83, 158.62, 156.65, 145.05, 142.16, 140.96, 134.06,
128.93, 119.79, 112.20, 111.79, 98.64, 60.41, 52.47, 48.43, 30.93, 24.67. HRMS (ESI) m/z calcd
for C20H20N3O7S [M + H]+, 446.1022; found, 446.1032.
4.1.3. Methyl ((4-(4-fluoro-N-(4-fluorobenzoyl)benzamido)phenyl)sulfonyl)-L-prolinate (13).
To a solution of 10 (50 mg, 0.176 mmol) and pyridine (97 mg, 1.23 mmol) in 5 mL of
DCM, 4-fluorobenzoyl chloride (139 mg, 0.88 mmol) was added. After stirring at rt. overnight,
the mixture was extracted with DCM (30 mL×2). The organic layer was washed with 1 N
NaHSO4 (aq.), saturated NaHCO3 (aq.), brine and dried over anhydrous Na2SO4. The resulting
solution was evaporated, and the residue was purified by silica gel column chromatography
(DCM/MeOH = 100/1 to 50/1) to give the desired product (60 mg, 67%) as a pale yellow solid.
m.p.: 78-80 ˚C. 1H NMR (300 MHz, CDCl3) δ 7.89 (d, J = 8.7 Hz, 2H), 7.75 (dd, J = 8.9 Hz, 5.2
Hz, 4H), 7.30 (d, J = 8.7 Hz, 2H), 7.06 (t, J = 8.6 Hz, 4H), 4.35 (dd, J = 8.1 Hz, 3.9 Hz, 1H),
13
3.68 (s, 3H), 3.54 – 3.31 (m, 2H), 2.08 – 1.80 (m, 4H). C NMR (75 MHz, CDCl3) δ 172.31,
171.86, 167.05, 163.65, 143.97, 137.59, 132.03, 131.91, 130.12, 128.88, 127.96, 116.32, 116.02,
60.40, 52.42, 48.40, 30.91, 24.62. HRMS (ESI) m/z calcd for C26H23F2N2O6S [M + H]+,
529.1245; found, 529.1260.
4.1.4. Methyl ((4-(4-(trifluoromethyl)benzamido)phenyl)sulfonyl)-L-prolinate (14).
To a solution of 10 (50 mg, 0.176 mmol) and Et3N (53 mg, 0.528 mmol) in 5 mL of
DCM, 4-(trifluoromethyl)benzoyl chloride (73 mg, 0.352 mmol) was added. After stirring at rt.
overnight, the mixture was extracted with DCM (30 mL×2). The organic layer was washed with

ACCEPTED MANUSCRIPT
1 N NaHSO4 (aq.), saturated NaHCO3 (aq.), brine and dried over anhydrous Na2SO4. The
resulting solution was evaporated, and the residue was purified by silica gel column
chromatography (DCM/MeOH = 100/1 to 50/1) to provide the desired product (80 mg, quant.) as
1
a pale yellow solid. m.p.: 182-184 ˚C. HPLC purity 99.4% (tR = 19.27 min). H NMR (300
MHz, CDCl3) δ 8.03 (d, J = 7.9 Hz, 2H), 7.85 (dd, J = 23.2 Hz, 8.5 Hz, 4H), 7.73 (d, J = 8.0 Hz,
2H), 4.25 (d, J = 7.3 Hz, 1H), 3.70 (s, 3H), 3.47 (d, J = 6.0 Hz, 1H), 3.29 (t, J = 7.1 Hz, 1H),
1.88 (m, J = 68.6, 4.7 Hz, 4H). 13C NMR (75 MHz, CDCl3) δ 172.75, 165.37, 142.43, 137.69,
133.41, 132.94, 128.57, 127.99, 125.62, 120.24, 60.44, 52.46, 48.48, 30.88, 24.60. HRMS (ESI)
m/z calcd for C20H20F3N2O5S [M + H]+, 457.1045; found, 457.1053.
4.1.5. Methyl ((4-((4-hydroxybenzyl)amino)phenyl)sulfonyl)-L-prolinate (15).
To a solution of methyl ((4-aminophenyl)sulfonyl)-L-prolinate (10) (70 mg, 0.25 mmol)
and 4-hydroxybenzaldehyde (38 mg, 0.25 mmol) MeOH (5 mL), NaBH3CN (32 mg, 0.5 mmol)
was added. After refluxing for 30 min, the mixture was extracted with DCM (20 mL×3). The
organic layer was washed with saturated NaHCO3 (aq.), brine and dried over anhydrous Na2SO4.
The resulting solution was filtered and concentrated to give a crude solid, which was then
purified with PTLC to give the desired product (35 mg, 85% after recovery of starting material)
1
as a pale yellow solid with 40 mg of the starting material 10 recovered. m.p.: 151-153 ˚C. H
NMR (300 MHz, CDCl3) δ 7.60 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 8.3 Hz, 2H), 6.81 (d, J = 8.3
Hz, 2H), 6.61 (d, J = 8.7 Hz, 2H), 4.28 – 4.17 (m, 3H), 3.70 (s, 3H), 3.45 (dd, J = 9.5, 6.8 Hz,
1H), 3.24 (dd, J = 11.6 Hz, 4.7 Hz, 1H), 2.07 – 1.86 (m, 3H), 1.78 – 1.65 (m, 1H). 13C NMR (75
MHz, CDCl3) δ 173.15, 156.15, 151.75, 129.54, 129.05, 128.85, 124.10, 115.59, 111.81, 60.34,
52.43, 48.50, 47.00, 30.88, 24.63. HRMS (ESI) m/z calcd for C19H23N2O5S [M + H]+, 391.1328;
found, 391.1335.

ACCEPTED MANUSCRIPT
4.1.6. Methyl (E)-((4-((2-amino-4-hydroxy-5-methylphenyl)diazenyl)phenyl)sulfonyl)-L-prolinate
(16).
To a solution of 10 (114 mg, 0.4 mmol) and HCl (concentrated aq, 160 µL, 2.4 mmol) in
MeOH (3 mL) and CH3CN (3 mL) at 0 oC (pre-cooled for 15 min) under N2, isopentyl nitrite (48
µL, 0.4 mmol) was added over 15 min and then the mixture was stirred at 0 oC for 45 min to get
a solution. At the same time, 5-amino-2-methylphenol (50 mg, 0.4 mmol) and K2CO3 (276 mg,
2.0 mmol) were dissolved in MeOH (1 mL) and H2O (8 mL) and degassed for 15 min. To the
reaction mixture, the previous solution was added over 15 min and stirred at 0 ˚C for 1 h. After
the completion of the reaction, 10% HCl was added to adjust pH = 1 and then NaHCO3
(saturated aq.) was used to adjust pH = 8. The solution was extracted with DCM (30 mL×2). The
organic layer was washed with brine and dried over anhydrous Na2SO4. The resulting solution
was filtered and concentrated to give a crude solid, which was further purified by silica gel
column chromatography (DCM/CH3OH = 50/1 to 30/1) to give the desired product (130 mg,
1
78%) as a red solid. m.p.: 98-100 ˚C (decomposition). H NMR (300 MHz, CDCl3) δ 7.87 (s,
3H), 7.53 (s, 1H), 6.48 – 6.12 (m, 2H), 4.31 (t, J = 5.9 Hz, 1H), 3.74 (s, 3H), 3.52 (s, 1H), 3.38 –
13
3.22 (m, 1H), 2.14 (d, J = 7.6 Hz, 2H), 2.07 – 1.87 (m, 4H), 1.80 – 1.67 (m, 1H). C NMR (75
MHz, CDCl3) δ 173.01, 160.53, 156.10, 144.10, 136.04, 132.18, 131.67, 128.47, 122.06, 116.08,
101.13, 60.58, 52.68, 48.68, 30.96, 24.68, 15.05. HRMS (ESI) m/z calcd for C19H23N4O5S [M +
H]+, 419.1389; found, 419.1397.
4.1.7. (5-(tert-Butyl)isoxazol-3-yl)(4-(4-fluorophenyl)piperazin-1-yl)methanone (19).
Ethyl 5-(tert-butyl)isoxazole-3-carboxylate 17 (1,000 mg, 5.1 mmol) and LiOH (638 mg,
15.3 mmol) were dissolved in 30 mL MeOH and 10 mL H2O, respectively. Then the solution
was stirred at rt. for 1 hr. In an ice-cooled bath, 1 N Na2SO4 was added, and the mixture was

ACCEPTED MANUSCRIPT
extracted by EtOAc. The organic layer was separated and washed with brine. After drying with
anhydrous Na2SO4, the solution was concentrated to give the intermediate 18 (818 mg, 95%) as a
light yellow oil. 1H NMR(300 MHz, CDCl3) δ 10.64 (s, 1H), 6.42 (s, 1H), 1.37 (s, 9H).
To a solution of 18 (70 mg, 0.41 mmol) and 1-(4-fluorophenyl)piperazine (74 mg, 0.41
mmol) in 5 mL DCM, HBTU (395 mg, 1.23 mmol) and DIEA (271 mg, 2.1 mmol) were added.
The mixture was stirred at rt. for 18 h. The mixture was washed with 1 N Na2SO4, saturated
NaHCO3 and brine. After drying over anhydrous Na2SO4, the solution was concentrated and
purified with silica gel column (hexane/EtOAc=10/1 to 5/1) to obtain the desired product (107
1
mg, 81%) as a white solid. m.p.: 93-95 ˚C. H NMR(300 MHz, CDCl3) δ 7.06 – 6.97 (m, 2H),
6.97 – 6.88 (m, 2H), 6.31 (s, 1H), 4.12 – 4.03 (m, 2H), 4.01 – 3.93 (m, 2H), 3.24 – 3.13 (m, 4H),
1.40 (s, 9H). 13C NMR (75 MHz, CDCl3) δ 181.83, 159.82, 159.25, 158.18, 118.72, 118.62,
115.87, 115.57, 99.92, 51.06, 50.52, 46.91, 42.56, 32.88, 28.80.
4.1.8.
(E)-N'-(3,5-Bis(trifluoromethyl)benzylidene)-5-(tert-butyl)isoxazole-3-carbohydrazide
(22).
Methyl 5-(tert-butyl)isoxazole-3-carboxylate (100 mg, 0.5 mmol) and NH2NH2 (76 mg,
1.5 mmol) were dissolved in 5 mL EtOH and refluxed for 2 h. 3-Chloro-5-fluorobenzaldehyde
(402 mg, 2.5 mmol) was then added and the solution was allowed to stir at rt. for 2 h. Then the
solution was concentrated and extracted with DCM. After drying over anhydrous Na2SO4, the
solution was concentrated and purified with silica gel column (hexane/EtOAc=20/1 to 10/1) to
1
obtain 21 (72 mg, 44% for two steps) as a white solid. H NMR (300 MHz, CDCl3) δ 9.98 (s,
1H), 8.26 (s, 1H), 7.57 (s, 1H), 7.43 (d, J = 7.9 Hz, 1H), 7.15 (d, J = 6.5 Hz, 1H), 6.57 (s, 1H),
1.40 (s, 9H). 13C NMR (75 MHz, CDCl3) δ 183.65, 164.37, 157.27, 155.56, 146.48, 136.54,
135.47, 123.77, 118.41, 118.08, 112.89, 112.58, 98.80, 33.06, 28.75.

ACCEPTED MANUSCRIPT
Methyl 5-(tert-butyl)isoxazole-3-carboxylate (100 mg, 0.5 mmol) and NH2NH2 (76 mg,
1.5 mmol) were dissolved in
5
mL EtOH and refluxed for
2
h. 3,5-
Bis(trifluoromethyl)benzaldehydebenzaldehyde (605 mg, 2.5 mmol) was then added and the
solution was allowed to stir at rt. for 2 h. Then the solution was concentrated and extracted with
DCM. After drying over anhydrous Na2SO4, the solution was concentrated and purified with
silica gel column (Hexane/EtOAc=20/1 to 10/1) to obtain 22 (120 mg, 59% for two steps) as a
1
white solid. m.p.: 169-171 ˚C. H NMR (300 MHz, CDCl3) δ 10.13 (s, 1H), 8.50 (s, 1H), 8.23 (s,
2H), 7.92 (s, 1H), 6.59 (s, 1H), 1.41 (s, 9H). 13C NMR(75 MHz, CDCl3) δ 183.79 , 157.19,
155.72, 145.91, 135.69, 133.04, 132.59, 132.14, 131.63, 127.46, 124.73, 123.84, 121.14, 98.82,
33.08, 28.73. HRMS (ESI) m/z calcd for C17H16F6N3O2 [M + H]+, 408.1147; found, 408.1142.
4.1.9. 2-(5-(tert-Butyl)isoxazol-3-yl)-5-(3-chloro-5-fluorophenyl)-1,3,4-oxadiazole (23).
Compound 21 (56 mg, 0.173 mmol), I2 (53 mg, 0.21 mmol) and K2CO3 (72 mg, 0.52
mmol) were dissolved in DMSO and stirred at 100 oC for 1 h. Then the solution was poured into
ice water and saturated Na2S2O3 was added. The mixture was extracted with DCM, and the
organic layer was dried over Na2SO4 and purified by silica gel column to obtain 23 (7 mg, 13%)
1
as a white solid. m.p.: 190-193 ˚C (decomposition). H NMR (300 MHz, CDCl3) δ 8.02 (s, 1H),
7.83 (ddd, J = 8.5, 2.3, 1.4 Hz, 1H), 7.38 – 7.31 (m, 1H), 6.70 (s, 1H), 1.46 (s, 9H). HRMS (ESI)
m/z calcd for C15H13N3FClNaO2 [M + Na]+, 344.0578; found, 344.0568.
4.1.10. 5-Chloro-2-methoxy-N-(2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)benzenesulfonamide (26).
6-Nitro-3,4-dihydroquinolin-2(1H)-one (1.0 g, 5.2 mmol) was dissolved in 40 mL of
EtOH, and then NH4Cl (2.76 g, 52 mmol) in 20 mL of H2O and Zn dust (2.37 g, 36.4 mmol)
were added. After refluxing at 80 ˚C for 1 h, the mixture was filtered to remove Zn dust. The
1
filtration was concentrated to give 25 (1.8 g, including partial NH4Cl) as a gray solid. H NMR

ACCEPTED MANUSCRIPT
(300 MHz, DMSO-d6) δ 9.67 (s, 1H), 6.55 (d, J = 8.2 Hz, 1H), 6.42 – 6.31 (m, 2H), 2.70 (t, J =
7.5 Hz, 2H), 2.33 (dd, J = 8.5 Hz, 6.5 Hz, 2H).
To a solution of 25 (70 mg, 0.43 mmol) and Et3N (87 mg, 0.86 mmol) in 5 mL DMF, 5-
chloro-2-methoxybenzenesulfonyl chloride (156 mg, 0.65 mmol) was added. After stirring at rt.
for 1 h, the mixture was poured into 20 mL of ice-water. The precipitate was filtered to get the
desired product (45 mg, 29%) as a white solid. m.p.: 253-255 ˚C (decomposition). 1H NMR
(300 MHz, DMSO-d6) δ 9.97 (s, 1H), 9.85 (s, 1H), 7.61 (s, 1H), 7.24 (dd, J = 10.2, 6.8 Hz, 2H),
6.94 – 6.79 (m, 2H), 6.67 (d, J = 8.4 Hz, 1H), 3.91 (s, 3H), 2.81 – 2.71 (m, 2H), 2.36 (t, J = 7.5
Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 170.38, 155.70, 135.72, 134.90, 133.70, 131.74,
129.59, 128.53, 127.45, 124.68, 124.05, 121.63, 120.67, 115.80, 115.36, 57.00, 30.58, 25.25.
HRMS (ESI) m/z calcd for C16H16N2O4SCl [M + H]+, 367.0519; found, 367.0506.
4.1.11. 4-Nitro-N-(2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)benzenesulfonamide (27).
Compound 27 was prepared from 4-nitrobenzenesulfonyl chloride in 13% yield by a
procedure similar to that used to prepare compound 26. The title compound was obtained as a
yellow solid. m.p.: 242-246 ˚C (decomposition). 1H NMR (300 MHz, DMSO-d6) δ 10.02 (s, 1H),
8.36 (d, J = 8.7 Hz, 2H), 7.94 (d, J = 8.8 Hz, 2H), 6.89 (s, 1H), 6.79 (s, 1H), 6.69 (d, J = 8.4 Hz,
13
1H), 2.78 (d, J = 7.1 Hz, 2H), 2.37 (dd, J = 8.5, 6.6 Hz, 2H). C NMR (75 MHz, DMSO-d6) δ
170.34, 149.99, 136.06, 128.73, 124.86, 115.84, 30.60, 25.23. HRMS (ESI) m/z calcd for
C15H14N3O5S [M + H]+, 348.0654; found, 348.0646.
4.1.12.
(E)-6-((2-Amino-4-hydroxy-5-methylphenyl)diazenyl)-3,4-dihydroquinolin-2(1H)-one
(28).
Compound 28 was prepared from 5-amino-2-methylphenol in 81% yield by a procedure
similar to that used to prepare compound 16. The title compound was obtained as a red solid.

ACCEPTED MANUSCRIPT
1
m.p.: 208-210 ˚C (decomposition). H NMR (300 MHz, DMSO-d6) δ 10.25 (s, 1H), 7.69 – 7.56
(m, 2H), 7.36 (s, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.25 (s, 1H), 2.95 (t, J = 7.4 Hz, 2H), 2.47 (s,
2H), 2.03 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 170.64, 160.39, 148.35, 145.73, 139.21,
130.98, 126.96, 124.67, 121.85, 120.73, 115.83, 114.51, 101.07, 30.75, 25.31, 15.66. HRMS
(ESI) m/z calcd for C16H17N4O2 [M + H]+, 297.1352; found, 297.1342.
4.1.13. 5-((4-(N-Cyclopentylsulfamoyl)phenyl)amino)-N-methylpicolinamide (29).
To a solution of 5-bromo-N-methylpicolinamide (80 mg, 0.37 mmol) and 6-amino-3,4-
dihydroquinolin-2(1H)-one (50 mg, 0.31 mmol) in 5 mL 1,4-dioxane, Pd(OAc)2 (18 g, 0.08
mmol), Cs2CO3 (202 mg, 0.62 mmol) and xantphos (90 mg, 0.16 mmol) were added. The
mixture was allowed to reflux at 110 ˚C overnight. Then the mixture was filtered, poured into
H2O and extracted by DCM. The organic layer was washed with saturated NaHCO3 (aq.), brine
and dried over anhydrous Na2SO4. The resulting solution was evaporated, and the residue was
purified by silica gel column (DCM/MeOH = 50:1) to give the desired product (58 mg, 63%) as
1
a white solid. m.p.: 276-278 ˚C (decomposition). H NMR (300 MHz, DMSO-d6) δ 10.02 (s,
1H), 8.58 (d, J = 2.5 Hz, 1H), 8.37 (s, 1H), 8.19 (s, 1H), 7.86 – 7.74 (m, 1H), 7.44 – 7.32 (m,
1H), 6.99 (d, J = 9.0 Hz, 2H), 6.84 (d, J = 3.6 Hz, 1H), 2.85 (s, 2H), 2.78 (s, 3H), 2.44 (s, 2H).
13C NMR (75 MHz, DMSO-d6) δ 170.31, 165.02, 144.05, 140.36, 135.99, 135.56, 134.07,
125.25, 123.17, 120.24, 119.83, 119.50, 116.31, 30.81, 26.25, 25.43. HRMS (ESI) m/z calcd for
C16H17N4O2 [M + H]+, 297.1352; found, 297.1345.
4.1.14. 1-(2-Oxo-1,2,3,4-tetrahydroquinolin-6-yl)-3-(4-(trifluoromethyl)phenyl)urea (30).
1-Isocyanato-4-(trifluoromethyl)benzene (131 mg, 0.7 mmol) and 6-amino-3,4-
dihydroquinolin-2(1H)-one (95 mg, 0.6 mmol) were mixed together and stirred at 50
̊C
overnight. Then the solution was concentrated and purified by silica gel column (DCM/CH3OH

ACCEPTED MANUSCRIPT
1
= 100/1 to 50/1) to give 30 (85 mg, 41%) as a white solid. m.p.: 258-260 ˚C (decomposition). H
NMR (300 MHz, DMSO-d6) δ 9.98 (s, 1H), 9.01 (s, 1H), 8.61 (s, 1H), 7.63 (q, J = 9.0 Hz, 4H),
7.31 (s, 1H), 7.18 (d, J = 7.2 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 2.85 (t, J = 7.4 Hz, 2H), 2.42 (t, J
= 7.4 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 170.36, 152.83, 144.04, 134.03, 133.79, 126.83,
126.51, 126.47, 126.42, 124.47, 123.24, 122.71, 122.28, 121.86, 121.44, 119.06, 118.21, 115.68,
30.86, 25.54. HRMS (ESI) m/z calcd for C17H15N3O2F3 [M + H]+, 350.1116; found, 350.1111.
4.1.15. (E)-4-(4-((2-Amino-4-hydroxy-5-methylphenyl)diazenyl)phenyl)thiomorpholine 1,1-
dioxide (33).
Compound 33 was prepared in 87% yield taking 4-(4-aminophenyl)thiomorpholine 1,1-
dioxide as the key intermediate by a procedure similar to that used to prepare compound 16. The
title compound was obtained as a yellow solid. m.p.: 214-216 ˚C (decomposition). 1H NMR (300
MHz, DMSO-d6) δ 7.70 (d, J = 8.9 Hz, 2H), 7.10 (d, J = 9.0 Hz, 2H), 6.63 (s, 2H), 6.24 (s, 1H),
3.88 (s, 4H), 3.15 (s, 4H), 2.03 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 159.79, 148.30, 146.13,
145.41, 130.95, 126.92, 123.36, 115.83, 113.92, 101.02, 50.46, 46.90, 15.66. HRMS (ESI) m/z
calcd for C17H21N4O3S [M + H]+, 361.1334; found, 361.1328.
4.1.16. (E)-5-Amino-2-methyl-4-((4-(pyrrolidin-1-ylsulfonyl)phenyl)diazenyl)phenol (34).
Compound 34 was prepared in 92% yield taking 4-(pyrrolidin-1-ylsulfonyl)aniline as the
key intermediate by a procedure similar to that used to prepare compound 16. The title
1
compound was obtained as a dark solid. m.p.: 177-179 ˚C (decomposition). H NMR (300 MHz,
DMSO-d6) δ 8.05 (d, J = 8.5 Hz, 2H), 7.84 (d, J = 8.7 Hz, 2H), 7.75 (t, J = 12.8 Hz, 1H), 6.38 (s,
13
1H), 3.16 (t, J = 6.6 Hz, 4H), 2.04 (s, 3H), 1.65 (dd, J = 8.1, 5.1 Hz, 4H). C NMR (75 MHz,
DMSO-d6) δ 166.60, 134.11, 131.06, 129.04, 120.43, 100.11, 48.31, 25.18, 16.02. HRMS (ESI)
m/z calcd for C17H21N4O3S [M + H]+, 361.1334; found, 361.1330.